**Between Armour and Weapons — Cell Death Mechanisms in Trypanosomatid Parasites**

Rubem Figueiredo Sadok Menna-Barreto and Solange Lisboa de Castro

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61196

#### **Abstract**

Among the pathogenic protozoa, trypanosomatids stand out due to their medical and economic impact, especially for low-income populations in tropical countries. Togeth‐ er, sleeping sickness, Chagas disease and leishmaniasis affect millions of humans and animals worldwide, yet are neglected by the pharmaceutical industry. The current drugs for trypanosomatid infections are limited and unsatisfactory, with severe side effects leading to reduced quality of life and, in several instances, to the abandonment of treatment. An intense search for alternative compounds has been performed, aim‐ ing at specific parasite targets by cellular, molecular and biochemical approaches. One interesting strategy could be interference with the protozoan cell death pathways. However, these pathways are poorly understood in unicellular eukaryotes, with the controversial existence and uncertain biological relevance of programmed cell death (PCD). This chapter will discuss apoptosis-like and autophagic cell death and necrosis in *Trypanosoma brucei*, *Trypanosoma cruzi* and *Leishmania* sp. and the possible implica‐ tions of these pathways for the parasite life cycle and infection persistence. It will also revisit the genomic and proteomic metadata of these trypanosomatids in the literature to rebuild the map of cell death proteins expressed under different conditions. The in‐ teraction of leading candidates with parasite-specific molecules, especially with en‐ zymes that regulate key steps in the cell death process, is a rational and attractive alternative for drug development for these neglected diseases.

**Keywords:** Cell death, apoptosis-like, autophagy, necrosis, *Leishmania* sp, *T. cruzi*, *T. brucei*

### **1. Introduction**

Neglected tropical diseases (NTDs) are a group of the seventeen mostly life-threatening infections, which affect more than a billion people worldwide. They affect poor populations,

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

often in underdeveloped and developing countries (low-income countries) [1]. Among NTDs, infections caused by the so-called "protozoan" parasites, such as African trypanosomiasis, Chagas disease and leishmaniasis, are responsible for a high annual death toll among the poor populations of tropical countries. New safe and affordable medicines are urgently needed. These diseases all present therapeutic difficulties by developing resistance to existing therapies and/or by toxic side effects.

### **1.1. Neglected tropical diseases and trypanosomatids**

#### *1.1.1. Sleeping sickness*

Human African trypanosomiasis (HAT), or sleeping sickness, is caused by extracellular protozoa belonging to the genus *Trypanosoma* and the species *T. brucei*. Two subspecies of *T. brucei* cause diseases with different epidemiological and clinical patterns: *T. b. gambiense*, a chronic disease present in western and central Africa accounting for 98% of the cases, and *T. b. rhodesiense*, an acute zoonosis located in eastern and southern Africa that occasionally infects humans. In 2001, WHO launched a major initiative to reinforce disease control and surveil‐ lance. After 10 years, the number of new cases of HAT decreased by 73.4%. Presently, the estimated the number of cases is 30, 000, and 70 million people are at risk [2, 3]. HAT clinically evolves in two stages. In the first stage, parasites are found in the lymphatic system and bloodstream. After a variable period of time, which is much shorter for the rhodesiense form, the second stage begins, with the parasites penetrating the blood-brain barrier and invading the central nervous system, leading to progressive neurological damage [4]. HAT is usually fatal if left untreated. Rhodesiense HAT usually progresses to death within six months, while gambiense HAT has a more chronic progressive course with an average duration of almost three years [5].

*T. brucei* is transmitted by the tsetse fly Glossina spp when it takes a blood meal. Non-dividing metacyclic forms enter the bloodstream of the mammalian host and differentiate into a rapidly dividing slender form able to evade antibody responses through antigenic variation [6]. Most of these forms undergo cell cycle arrest and develop into short-stumpy forms. When the tsetse fly bites an infected host, only the short-stumpy parasites survive in the insect's midgut and develop into a procyclic form, which undergoes multiple developmental phases on its way to the salivary gland, finally culminating in the infective metacyclic form [7, 8].

The drug of choice for treatment depends on the infecting species and the stage of infection. In early stages, *T. b. gambiense* and *T. b. rhodesiense* infections can be treated with pentamidine and suramin, respectively [9]. If the disease has progressed, treatment relies on melarsoprol or eflornithine. Melarsoprol, an arsenical drug, is extremely toxic. Eflornithine is less toxic, but is expensive, and has a difficult administration than melarsoprol and lacks efficacy against *T. b. rhodesiense* [2]. Since 2001, this drug has been combined with nifurtimox (NECT) for firstline treatment for CNS-stage *T. b. gambiense* HAT. It is the most recent breakthrough in antitrypanosomiasis drug research and was added to the World Health Organisation's list of essential medicines in 2009. A major problem related to the treatment of HAT is the develop‐ ment of resistance to melarsoprol and the other drugs [10].

### *1.1.2. Chagas disease*

often in underdeveloped and developing countries (low-income countries) [1]. Among NTDs, infections caused by the so-called "protozoan" parasites, such as African trypanosomiasis, Chagas disease and leishmaniasis, are responsible for a high annual death toll among the poor populations of tropical countries. New safe and affordable medicines are urgently needed. These diseases all present therapeutic difficulties by developing resistance to existing therapies

Human African trypanosomiasis (HAT), or sleeping sickness, is caused by extracellular protozoa belonging to the genus *Trypanosoma* and the species *T. brucei*. Two subspecies of *T. brucei* cause diseases with different epidemiological and clinical patterns: *T. b. gambiense*, a chronic disease present in western and central Africa accounting for 98% of the cases, and *T. b. rhodesiense*, an acute zoonosis located in eastern and southern Africa that occasionally infects humans. In 2001, WHO launched a major initiative to reinforce disease control and surveil‐ lance. After 10 years, the number of new cases of HAT decreased by 73.4%. Presently, the estimated the number of cases is 30, 000, and 70 million people are at risk [2, 3]. HAT clinically evolves in two stages. In the first stage, parasites are found in the lymphatic system and bloodstream. After a variable period of time, which is much shorter for the rhodesiense form, the second stage begins, with the parasites penetrating the blood-brain barrier and invading the central nervous system, leading to progressive neurological damage [4]. HAT is usually fatal if left untreated. Rhodesiense HAT usually progresses to death within six months, while gambiense HAT has a more chronic progressive course with an average duration of almost

*T. brucei* is transmitted by the tsetse fly Glossina spp when it takes a blood meal. Non-dividing metacyclic forms enter the bloodstream of the mammalian host and differentiate into a rapidly dividing slender form able to evade antibody responses through antigenic variation [6]. Most of these forms undergo cell cycle arrest and develop into short-stumpy forms. When the tsetse fly bites an infected host, only the short-stumpy parasites survive in the insect's midgut and develop into a procyclic form, which undergoes multiple developmental phases on its way to

The drug of choice for treatment depends on the infecting species and the stage of infection. In early stages, *T. b. gambiense* and *T. b. rhodesiense* infections can be treated with pentamidine and suramin, respectively [9]. If the disease has progressed, treatment relies on melarsoprol or eflornithine. Melarsoprol, an arsenical drug, is extremely toxic. Eflornithine is less toxic, but is expensive, and has a difficult administration than melarsoprol and lacks efficacy against *T. b. rhodesiense* [2]. Since 2001, this drug has been combined with nifurtimox (NECT) for firstline treatment for CNS-stage *T. b. gambiense* HAT. It is the most recent breakthrough in antitrypanosomiasis drug research and was added to the World Health Organisation's list of essential medicines in 2009. A major problem related to the treatment of HAT is the develop‐

the salivary gland, finally culminating in the infective metacyclic form [7, 8].

ment of resistance to melarsoprol and the other drugs [10].

and/or by toxic side effects.

196 Cell Death - Autophagy, Apoptosis and Necrosis

*1.1.1. Sleeping sickness*

three years [5].

**1.1. Neglected tropical diseases and trypanosomatids**

Chagas disease is caused by the intracellular obligatory parasite *Trypanosoma cruzi* and affects approximately eight million individuals in Latin America [11]. The transmission of this disease occurs through the faeces of sucking triatominae insects, blood transfusions, organ transplantation, oral contamination, laboratory accidents and congenital routes [12, 13]. Current major concerns are the outbreaks of acute Chagas disease associated with the ingestion of contaminated food and its emergence in non-endemic areas, such as North America and Europe, due to the immigration of infected individuals [14-16]. This disease is characterised by two clinical phases. The acute phase appears shortly after infection and is defined by patent parasitaemia. If left untreated, symptomatic chronic disease develops in about one-third of individuals after a long latent period (10-30 years), which is known as the indeterminate form. The main clinical manifestations of Chagas disease include digestive and/or cardiac alterations. The chronic cardiac form of the disease is the most significant clinical manifestation. Consequences include dilated cardiomyopathy, congestive heart failure, arrhythmias, cardioembolism and stroke [17].

The life cycle of *T. cruzi* involves four major developmental stages during its passage through vertebrate and invertebrate hosts [18]. The infective stage of the parasite, the metacyclic trypomastigote, enters the mammalian host from insect faeces through wound openings or mucous membranes. In the mammalian host, the metacyclic trypomastigote differentiates into the amastigote form. After several rounds of replication in the host cells, the amastigote differentiates into the bloodstream trypomastigote, which can enter new cells and perpetuate the infection. When the insect bites an infected host, the bloodstream trypomastigote differ‐ entiates into the replicative epimastigote that lives in the insect's gut. Finally, in the rectum of the insect, the epimastigote differentiates into the infective metacyclic trypomastigote, which is ready to infect its host again.

The available chemotherapy for this illness includes two nitroheterocyclic agents, nifurtimox and benznidazole, which are effective against acute infections, but show poor activity in the late chronic phase, with severe collateral effects and limited efficacy against different parasitic isolates. These drawbacks justify the urgent need to identify better drugs to treat chagasic patients, and several new compounds are currently in preclinical development involving *in vitro* parasite phenotype screens and target-based drug discovery [19-21]. Recently, clinical trials with the azoles posaconazole and E1224 (ravuconazole prodrug) led to higher percen‐ tages of treatment failure in chronic patients than benznidazole [22, 23], suggesting their potential use in combination therapy [24].

#### *1.1.3. Leishmaniasis*

Leishmaniasis, which is caused by different species of *Leishmania,* is a vector-borne disease, with an estimated 12 million cases worldwide. Infection is caused by the bite of infected female sand flies of the genera *Phlebotomus* (Europe, Asia, Africa) and *Lutzomyia* (America) [25]. *Leishmania* parasites live a digenetic life cycle as either a promastigote flagellar or an amastigote form. The type of clinical manifestation depends on the infecting species and host factors, such as general health and genetic and immune constitution [26]. It is a disease complex with three clinical manifestations, visceral (VL, kala-azar), cutaneous (CL) and muco-cutaneous (MCL), which arise from parasite replication in the mononuclear phagocyte system, dermis and nasooropharyngeal mucosa, respectively [27]. Some post-treated *L. donovani*-infected patients develop the diffuse cutaneous form named post-kala-azar dermal leishmaniasis (PKDL) [28, 29]. VL, after initial skin lesions, takes 2-8 months to develop gross inflammatory reactions within the viscera (liver and spleen in particular) and is usually fatal unless treated. CL manifests as an open sore at the site of the insect bite and will frequently self-heal, leaving a scar. The diffuse form of CL is more problematic, causing lepromatous type lesions dissemi‐ nated across the skin that can be difficult to heal. The MCL form, endemic in parts of Latin America, starts with skin sores that spread to the mucosal membranes of the face. Profound inflammatory damage can lead to the erosion of the nostrils and mouth in particular [29].

In the *Leishmania* life cycle, there are two principal parasite forms: amastigotes and motile promastigotes. In the alimentary tract of the insect vector, the parasite exists as multiplicative, non-infective procyclic promastigotes and non-multiplicative, infective metacyclic promasti‐ gotes [30]. Upon injection into the mammalian host, promastigotes are taken up by macro‐ phages where the metacyclic forms differentiate into small multiplicative, non-motile amastigotes that live in a lysosomal compartment known as the parasitophorous vacuole [31]. These developmental forms are distinguished by their nutritional requirements, their growth rate and ability to divide, the regulated expression of their surface molecules, and their morphology. Metacyclic promastigotes are pre-adapted for survival in the mammalian host, as they are complement-resistant. Amastigotes are intracellular, non-motile forms that have adapted to the low pH of this compartment and have an adapted energy metabolism.

The current drugs are highly toxic, resistance is common and compliance of patients to treatment is low, as the treatment is long and the drug price is high. Although recent initiatives have improved the antileishmanial drug arsenal by combining current medicines or using new formulations of old ones, none are ideal for treatment due to their high toxicity, resistance issues, prohibitive prices, long treatment length and need of intravenous administration [32-34]. Pentavalent antimonials (glucantime and pentostan) are first-line drugs for both VL and CL. However, they present several limitations, including variable efficacy, need for daily injectable administration for approximately one month, and severe side effects. Many patients are unable to complete the treatment, increasing the risk of drug resistance development. Amphotericin B is a systemic antifungal that is used as a second-line drug for VL. It is highly toxic, requiring careful and slow intravenous administration. Lipid formulations of ampho‐ tericin B have been developed to improve its bioavailability and pharmacokinetic properties, reducing toxicity [35]. Miltefosine is the most recent antileishmanial drug on the market and the first effective oral treatment against VL [36]. However, it has common gastrointestinal side effects and is also limited by its relatively high cost [34], potential teratogenicity and growing concerns in relation to increases in clinical isolate susceptibility [37]. Paromomycin is an aminoglycoside antibiotic that is used in topical treatment for CL and as a parenteral drug for VL. Pentamidine was used as a second-line drug in antimony-resistant VL treatment. How‐ ever, its high toxicity combined with decreased efficacy led to the abandonment of this drug to treat VL in India, but it is valuable for combined therapies [38].

### **2. Cell death: State of art**

clinical manifestations, visceral (VL, kala-azar), cutaneous (CL) and muco-cutaneous (MCL), which arise from parasite replication in the mononuclear phagocyte system, dermis and nasooropharyngeal mucosa, respectively [27]. Some post-treated *L. donovani*-infected patients develop the diffuse cutaneous form named post-kala-azar dermal leishmaniasis (PKDL) [28, 29]. VL, after initial skin lesions, takes 2-8 months to develop gross inflammatory reactions within the viscera (liver and spleen in particular) and is usually fatal unless treated. CL manifests as an open sore at the site of the insect bite and will frequently self-heal, leaving a scar. The diffuse form of CL is more problematic, causing lepromatous type lesions dissemi‐ nated across the skin that can be difficult to heal. The MCL form, endemic in parts of Latin America, starts with skin sores that spread to the mucosal membranes of the face. Profound inflammatory damage can lead to the erosion of the nostrils and mouth in particular [29].

198 Cell Death - Autophagy, Apoptosis and Necrosis

In the *Leishmania* life cycle, there are two principal parasite forms: amastigotes and motile promastigotes. In the alimentary tract of the insect vector, the parasite exists as multiplicative, non-infective procyclic promastigotes and non-multiplicative, infective metacyclic promasti‐ gotes [30]. Upon injection into the mammalian host, promastigotes are taken up by macro‐ phages where the metacyclic forms differentiate into small multiplicative, non-motile amastigotes that live in a lysosomal compartment known as the parasitophorous vacuole [31]. These developmental forms are distinguished by their nutritional requirements, their growth rate and ability to divide, the regulated expression of their surface molecules, and their morphology. Metacyclic promastigotes are pre-adapted for survival in the mammalian host, as they are complement-resistant. Amastigotes are intracellular, non-motile forms that have

adapted to the low pH of this compartment and have an adapted energy metabolism.

to treat VL in India, but it is valuable for combined therapies [38].

The current drugs are highly toxic, resistance is common and compliance of patients to treatment is low, as the treatment is long and the drug price is high. Although recent initiatives have improved the antileishmanial drug arsenal by combining current medicines or using new formulations of old ones, none are ideal for treatment due to their high toxicity, resistance issues, prohibitive prices, long treatment length and need of intravenous administration [32-34]. Pentavalent antimonials (glucantime and pentostan) are first-line drugs for both VL and CL. However, they present several limitations, including variable efficacy, need for daily injectable administration for approximately one month, and severe side effects. Many patients are unable to complete the treatment, increasing the risk of drug resistance development. Amphotericin B is a systemic antifungal that is used as a second-line drug for VL. It is highly toxic, requiring careful and slow intravenous administration. Lipid formulations of ampho‐ tericin B have been developed to improve its bioavailability and pharmacokinetic properties, reducing toxicity [35]. Miltefosine is the most recent antileishmanial drug on the market and the first effective oral treatment against VL [36]. However, it has common gastrointestinal side effects and is also limited by its relatively high cost [34], potential teratogenicity and growing concerns in relation to increases in clinical isolate susceptibility [37]. Paromomycin is an aminoglycoside antibiotic that is used in topical treatment for CL and as a parenteral drug for VL. Pentamidine was used as a second-line drug in antimony-resistant VL treatment. How‐ ever, its high toxicity combined with decreased efficacy led to the abandonment of this drug

As used for whole organisms, the term death is employed to describe a sequence of events culminating in the breakdown of all biological functions. However, more than one century after the first citation [39], cell death still represents a crucial gap in our understanding of cellular physiology. It can be triggered by natural processes or induced by extrinsic factors (exposure to chemicals or physical stresses). The consequent tissue injury usually leads to a state of disease [40]. On the other hand, many studies pointed to cell death playing a funda‐ mental role in the physiology of multicellular organisms, especially in processes such as metamorphosis and embryogenesis [41]. In this context, in 1964, the term programmed cell death (PCD) was created, proposing a sequence of well-controlled steps regulating a nonaccidental cell death process in the absence of an inflammatory response [42]. Currently, it is known that distinct death mechanisms and phenotypes participate in PCD, with apoptosis and autophagy being the most prominent [43].

### **2.1. Apoptosis**

The apoptotic pathway was first described in the early 1970s as a fundamental step for proper embryo development [44]. This process is crucial during tissue development, especially in immune response regulation and removal of infected or damaged cells [45, 46]. Apoptosis is involved not only in growth regulation in multicellular organisms [47, 48] but also in their defence against viral, bacterial or parasitic infections [49-53] and even against cancer devel‐ opment [54-57]. The removal of non-functional cells by the apoptotic pathway is efficient and prevents the inflammatory response [58].

During apoptosis in multicellular organisms, the cell activates death machinery that culmi‐ nates in chromosomal condensation and nuclear DNA fragmentation [59, 60]. Biochemically, apoptosis is orchestrated by the activation of a family of cysteine proteases, named caspases, that are activated by extrinsic and intrinsic factors [45, 46]. The extrinsic pathway is activated by the interaction of death ligands with their respective cell surface receptor (i.e., FasL/Fas, TNF-α/TNFR) [61-63]. Such binding triggers the cleavage of procaspase 8 into active caspase 8, which cleaves procaspase 3. Executioner caspase 3 activates endonuclease G (EndoG), starting the characteristic DNA fragmentation, a distinctive marker of apoptosis [63-65]. On the other hand, the intrinsic pathway can be triggered by two distinct mechanisms with mitochondrion or endoplasmic reticulum (ER) dependency. In the mitochondrial pathway, activation occurs by membrane permeabilization, releasing cytochrome c, apoptosis induction factor (AIF), EndoG and regulators of the B-cell lymphoma 2 (Bcl2) protein family into the cytosol. In the cytosol, the apoptosome is formed by the interaction of released cytochrome c with apoptotic protease activating factor 1 (APAF-1) and procaspase 9, activating caspase 9, which subsequently activates the effector caspase 3 [66-70]. The ER pathway is mainly caspase 12-dependent and occurs in this organelle during stress conditions. Because this pathway was described in the mouse and humans lack functional caspase 12, the relevance of ER-mediated apoptosis is still debatable [71-73].

Undoubtedly, the caspase cascade represents a central point in the apoptotic process. Its regulation is well-controlled by pro- and anti-apoptotic molecules from the Bcl-2 family [74]. The apoptotic morphological and biochemical phenotypes include cell shrinkage, membrane blebbing (formation of apoptotic bodies), chromatin condensation and typical internucleoso‐ mal DNA fragmentation, externalization of phosphatidylserine (PS), loss of mitochondrial membrane potential (ΔΨm), and target protein degradation by caspase activation [75-79]. The characterization of apoptosis is experimentally based on the detection of apoptotic markers. The loss of ΔΨm (labelling with rhodamine 123 derivatives, such as TMRE), PS exposure (binding to labelled annexin V), chromatin condensation (DAPI labelling) and DNA fragmen‐ tation (TUNEL technique) are usually quantified by fluorescence microscopy or flow cytom‐ etry. DNA fragmentation can also be assessed by agarose gel electrophoresis, presenting a laddering pattern that represents internucleosomal cleavage. Analysis of caspase activity using labelled specific substrates and/or inhibitors can be performed by immunotechniques such as ELISA [80].

#### **2.2. Autophagy**

In the 1950s, acidic organelles involved in the intracellular degradation of macromolecules were described and termed lysosomes by Dr. Christian de Duve. In a subsequent study [81], he proposed the term autophagy for a self-degrading process [82]. Currently, the autophagic pathway is considered to be the main cellular mechanism for the degradation of non-functional organelles and/or macromolecules and is fundamental for homeostasis in eukaryotic cells [83]. In other words, autophagy is a housekeeping self-digestion mechanism that is crucial for cellular turnover and recycling and occurs by the engulfment of cytosolic portions containing material that should be degraded. Degradation starts immediately after the fusion of auto‐ phagosomes to lysosomes in an organelle named the autophagolysosome [84, 85].

In multicellular organisms, autophagy is involved in many physiological situations, including development, cell growth and cell differentiation. Autophagy sustains cell survival under 'extracellular stress', such as nutrient starvation, hypoxia, acidic pH and high temperature. It acts as a housekeeping device under 'intracellular stress' by removing damaged or redundant cytoplasmic components, including organelles [86]. Increased autophagic activity is observed in pathological states and in host defences against pathogens [87-92]. Despite the relevant role of autophagy for the maintenance of the regular cell cycle, prolonged starvation periods or other strong autophagic stimuli induce a cellular misbalance and promote autophagic cell death [93, 94].

The autophagic molecular machinery was first assessed in the yeast model *Saccharomyces cerevisiae*, and 30 proteins, called Atgs (AuTophaGy-related), were described and associated with different steps of the pathway [95]. Atg orthologues were identified in all eukaryotes, with Atg8 (LC3 in mammals) being one of the most studied [82]. Autophagy can be a selective or non-selective process, degrading specific or random cellular components. Examples of selective routes are mitophagy, pexophagy or reticulophagy, in which mitochondria (or part of the organelle), peroxisomes and ER are degraded, respectively [82].

Additionally, there are three types of autophagy: macroautophagy, microautophagy and chaperone-mediated autophagy (CMA). The most common is macroautophagy, a process that involves the engulfment of cytosolic portions by a double membrane structure called the phagophore. The double-membrane vesicle formed from phagophore engulfment is named the autophagosome and is directed to lysosomes for degradation by lysosomal hydrolases. These steps are regulated by Atgs [92, 96-98]. The chronological events related to macroau‐ tophagy are (a) autophagic induction; (b) cargo selection; (c) phagophore elongation; (d) autophagosome formation; (e) fusion to lysosomes; and (f) cargo degradation [99]. The early steps in this process depend on the serine/threonine protein kinase TOR (target of rapamycin), which is essential for autophagic regulation. TOR complexes 1 and 2 work as sensors of nutritional availability (especially amino acids). The autophagic enzyme Atg6 (Beclin 1 in mammals) is a phosphatidylinositol 3-kinase (PI-3K) and shares its signalling function with other cellular pathways. For autophagy, these kinases present a critical role for autophagosome formation [82].

In contrast, there are no autophagosomes in the microautophagic pathway. Invagination of the lysosomal membrane occurs, resulting in a single-membrane small vesicle inside the lysosomes that will be degraded. Interestingly, both macro- and microautophagy could be selective or non-selective processes. Indeed, CMA appears to be the most selective type of autophagy. The proteins that will be degraded contain pentapeptide motifs (KFERQ, QREFK or VDKFQ), the binding sites of a cytosolic chaperone. Such a chaperone-substrate complex binds to a LAMP-2A receptor in the lysosomal membrane, promoting receptor dimerization. A membrane channel is formed, and the specific protein reaches the lysosomal lumen to be degraded [82, 100].

For many years, electron microscopy was the only tool available for the identification of autophagic morphological features, especially the presence of double-membrane vesicles (autophagosomes). In the last 20 years, advances in the molecular description of autophagy allowed the detection, localization and quantification of Atgs by molecular, biochemical and morphological approaches. Currently, the gold-standard method to monitor autophagy is Atg8/LC3 detection by different techniques: (a) Western blotting (presence of two isoforms); (b) confocal or fluorescence microscopy (identification of LC3 puncta); (c) knock down or knock out (deletion and analysis of the phenotype); and (d) pharmacological induction/ inhibition (rapamycin and/or PI-3K inhibitors). These techniques can also be employed *in vitro* or *in vivo* for other Atgs, indicating autophagic activity [101].

#### **2.3. Necrosis**

Undoubtedly, the caspase cascade represents a central point in the apoptotic process. Its regulation is well-controlled by pro- and anti-apoptotic molecules from the Bcl-2 family [74]. The apoptotic morphological and biochemical phenotypes include cell shrinkage, membrane blebbing (formation of apoptotic bodies), chromatin condensation and typical internucleoso‐ mal DNA fragmentation, externalization of phosphatidylserine (PS), loss of mitochondrial membrane potential (ΔΨm), and target protein degradation by caspase activation [75-79]. The characterization of apoptosis is experimentally based on the detection of apoptotic markers. The loss of ΔΨm (labelling with rhodamine 123 derivatives, such as TMRE), PS exposure (binding to labelled annexin V), chromatin condensation (DAPI labelling) and DNA fragmen‐ tation (TUNEL technique) are usually quantified by fluorescence microscopy or flow cytom‐ etry. DNA fragmentation can also be assessed by agarose gel electrophoresis, presenting a laddering pattern that represents internucleosomal cleavage. Analysis of caspase activity using labelled specific substrates and/or inhibitors can be performed by immunotechniques such as

In the 1950s, acidic organelles involved in the intracellular degradation of macromolecules were described and termed lysosomes by Dr. Christian de Duve. In a subsequent study [81], he proposed the term autophagy for a self-degrading process [82]. Currently, the autophagic pathway is considered to be the main cellular mechanism for the degradation of non-functional organelles and/or macromolecules and is fundamental for homeostasis in eukaryotic cells [83]. In other words, autophagy is a housekeeping self-digestion mechanism that is crucial for cellular turnover and recycling and occurs by the engulfment of cytosolic portions containing material that should be degraded. Degradation starts immediately after the fusion of auto‐

In multicellular organisms, autophagy is involved in many physiological situations, including development, cell growth and cell differentiation. Autophagy sustains cell survival under 'extracellular stress', such as nutrient starvation, hypoxia, acidic pH and high temperature. It acts as a housekeeping device under 'intracellular stress' by removing damaged or redundant cytoplasmic components, including organelles [86]. Increased autophagic activity is observed in pathological states and in host defences against pathogens [87-92]. Despite the relevant role of autophagy for the maintenance of the regular cell cycle, prolonged starvation periods or other strong autophagic stimuli induce a cellular misbalance and promote autophagic cell

The autophagic molecular machinery was first assessed in the yeast model *Saccharomyces cerevisiae*, and 30 proteins, called Atgs (AuTophaGy-related), were described and associated with different steps of the pathway [95]. Atg orthologues were identified in all eukaryotes, with Atg8 (LC3 in mammals) being one of the most studied [82]. Autophagy can be a selective or non-selective process, degrading specific or random cellular components. Examples of selective routes are mitophagy, pexophagy or reticulophagy, in which mitochondria (or part

of the organelle), peroxisomes and ER are degraded, respectively [82].

phagosomes to lysosomes in an organelle named the autophagolysosome [84, 85].

ELISA [80].

**2.2. Autophagy**

200 Cell Death - Autophagy, Apoptosis and Necrosis

death [93, 94].

Necrosis is a term that is extensively employed as synonymous with cell death. In the Greek aetiology, it signifies the "stage of dying". In this death type, strong cellular damage occurs caused by external stimuli (drugs, infection, mechanical trauma), promoting the random degradation of the whole cell, with plasma membrane disruption. Necrosis is defined as an accidental cell death process, differing from PCD (especially apoptosis) [102]. One of the main differences between apoptosis and necrosis is the induction of the inflammatory response in the latter. The release of intracellular material into the extracellular environment during necrotic cell death triggers intense inflammation in the surrounding cells and tissues [103]. Classical necrotic features are the loss of plasma membrane integrity, cytosolic vacuolization, disruption of calcium homeostasis, general degradation by lysosomal hydrolases and induc‐ tion of the inflammatory response.

Necrosis can also be a regulated process. Necroptosis is a programmed and non-accidental death pathway. Surprisingly, the activation of this pathway can occur by TNF-α or FasL, classical apoptotic ligands. Necroptosis depends on the participation of the receptor-interact‐ ing protein kinases 1 and 3 (RIPK1 and RIPK3), which are kinases that regulate this pathway. RIPK1 is pharmacologically inhibited by a small molecule named necrostatin-1 (Nec-1) [104-106].

### **2.4. Others**

In addition to apoptosis, autophagy and necrosis (accidental or not), other non-canonical death styles can take place in eukaryotic cells. In an inflammatory context, pyroptosis and NETosis are prominent. Pyroptosis, primarily observed in macrophages after bacterial infection, is caspase 1-dependent. This caspase promotes an increase in the inflammatory cytokine levels (IL-1β and IL-18) and the formation of plasma membrane pores, leading to the release of cellular material to the extracellular matrix. The main difference between pyroptosis and apoptosis is the participation of caspase 1, which is only involved in the pyroptotic death pathway, a proinflammatory PCD [106-108]. Another type of cell death that plays a crucial role in the innate immune response is the neutrophil extracellular trap (NETosis), where neutrophilic death leads to the release of a neutrophil DNA network coated with histones and elastase to the extracellular environment to capture pathogens. However, the direct antimi‐ crobial effect of the NETs is still controversial [109, 110]. Currently, DNA release has also been described in other immune cells, such as eosinophils, basophils, macrophages and mast cells, but its precise role deserves further analysis [110-114].

Other cell death types not involved in inflammation have been characterized. Ferroptosis is iron-dependent cell death that has been identified in some mammalian cells and involves oxidative stress induced by a small molecule named erastin, which is inhibited by ferrostatin 1. Despite that lack of complete understanding of the erastin mechanism, the XC−Cys/Glu antiporter system is inhibited in ferroptosis, leading to a misbalance of these amino acids inside the cell [106, 115]. Additionally, there is another non-canonical cell death pathway in cancer cells (*in vitro* and *in vivo* models) called autoschizis, which involves oxidative stress induced by treatment with ascorbate and menadione. Autoschizic cell death presents remarkable morphological evidence, with electron microscopy as the best technique for its identification. Among the autoschizic features are cell shrinkage, extrusion of large portions of the cytosol (without any organelles), random DNA fragmentation and the subsequent deterioration of all cellular structures [116, 117]. Interestingly, annexin V (AV) and propidium iodide (PI) assays (gold standards for apoptosis detection in mammals) of cells treated with ascorbate and menadione demonstrate high percentages of AV-/PI+ cells [117], which are not discussed in almost all apoptotic studies, suggesting that these membrane shedding events could occur in a large variety of cell models. Table 1 summarizes the main types of cell death discussed herein.


**Table 1.** Types of cell death

necrotic cell death triggers intense inflammation in the surrounding cells and tissues [103]. Classical necrotic features are the loss of plasma membrane integrity, cytosolic vacuolization, disruption of calcium homeostasis, general degradation by lysosomal hydrolases and induc‐

Necrosis can also be a regulated process. Necroptosis is a programmed and non-accidental death pathway. Surprisingly, the activation of this pathway can occur by TNF-α or FasL, classical apoptotic ligands. Necroptosis depends on the participation of the receptor-interact‐ ing protein kinases 1 and 3 (RIPK1 and RIPK3), which are kinases that regulate this pathway. RIPK1 is pharmacologically inhibited by a small molecule named necrostatin-1 (Nec-1)

In addition to apoptosis, autophagy and necrosis (accidental or not), other non-canonical death styles can take place in eukaryotic cells. In an inflammatory context, pyroptosis and NETosis are prominent. Pyroptosis, primarily observed in macrophages after bacterial infection, is caspase 1-dependent. This caspase promotes an increase in the inflammatory cytokine levels (IL-1β and IL-18) and the formation of plasma membrane pores, leading to the release of cellular material to the extracellular matrix. The main difference between pyroptosis and apoptosis is the participation of caspase 1, which is only involved in the pyroptotic death pathway, a proinflammatory PCD [106-108]. Another type of cell death that plays a crucial role in the innate immune response is the neutrophil extracellular trap (NETosis), where neutrophilic death leads to the release of a neutrophil DNA network coated with histones and elastase to the extracellular environment to capture pathogens. However, the direct antimi‐ crobial effect of the NETs is still controversial [109, 110]. Currently, DNA release has also been described in other immune cells, such as eosinophils, basophils, macrophages and mast cells,

Other cell death types not involved in inflammation have been characterized. Ferroptosis is iron-dependent cell death that has been identified in some mammalian cells and involves oxidative stress induced by a small molecule named erastin, which is inhibited by ferrostatin 1. Despite that lack of complete understanding of the erastin mechanism, the XC−Cys/Glu antiporter system is inhibited in ferroptosis, leading to a misbalance of these amino acids inside the cell [106, 115]. Additionally, there is another non-canonical cell death pathway in cancer cells (*in vitro* and *in vivo* models) called autoschizis, which involves oxidative stress induced by treatment with ascorbate and menadione. Autoschizic cell death presents remarkable morphological evidence, with electron microscopy as the best technique for its identification. Among the autoschizic features are cell shrinkage, extrusion of large portions of the cytosol (without any organelles), random DNA fragmentation and the subsequent deterioration of all cellular structures [116, 117]. Interestingly, annexin V (AV) and propidium iodide (PI) assays (gold standards for apoptosis detection in mammals) of cells treated with ascorbate and menadione demonstrate high percentages of AV-/PI+ cells [117], which are not discussed in almost all apoptotic studies, suggesting that these membrane shedding events could occur in a large variety of cell models. Table 1 summarizes the main types of cell death discussed herein.

tion of the inflammatory response.

202 Cell Death - Autophagy, Apoptosis and Necrosis

but its precise role deserves further analysis [110-114].

[104-106].

**2.4. Others**

### **3. Cell death in trypanosomatids: An overview**

The term PCD was employed for decades to exclusively describe cell death in metazoans and its involvement in embryogenesis and maintenance of homeostasis. Indeed, the relevance of PCD for lower eukaryotes is unclear. In an evolutionary scenario, these regulated processes could allow clonal selection in the parasite population, guaranteeing the propagation of identical genetic information even in adverse environmental conditions. However, differences in the cell death mechanisms observed between metazoans and protozoans must be considered [78, 118]. In the following sections, we will discuss the role of different death styles described in pathogenic trypanosomatids.

#### **3.1. Apoptosis-like**

In trypanosomatids, the first PCD report was published in 1995 by Ameisen and coworkers describing apoptotic characteristics (DNA fragmentation and cytoplasmic and nuclear morphological alterations) in *T. cruzi* epimastigotes during differentiation to trypomastigotes [119]. In the last two decades, a variety of stimuli were reported to induce the appearance of the apoptotic phenotype in this parasite, including exposure to fresh human serum (FHS), heat shock and drugs [76, 119-128]. Curiously, the apoptosis-like phenotype was also associated with the regulation of the *T. cruzi* life cycle [129]. These cell death phenotypes in pathogenic trypanosomatids have been characterized by the use of classical apoptotic markers (see item 2.1) [76, 79, 118, 123, 130-133]. Among the apoptotic hallmarks identified, we found (a) loss of ΔΨm, (b) cytochrome c release, (c) PS externalization, and (d) abnormal DNA condensation and fragmentation [76, 119, 129, 130, 134] (Table 3, Figure 1).

In *Leishmania* sp., apoptotic features (nuclear condensation, DNA fragmentation, cell shrink‐ age, loss of ΔΨm, and release of cytochrome c) were also observed in stress conditions induced by heat, starvation, oxidative agents and drugs [118, 134, 136-139, 135]. *L. donovani, L. major* and *L. mexicana* stationary phase promastigotes and axenic amastigotes exhibited DNA fragmentation with a laddering electrophoretic profile, suggesting oligonucleosomal cleavage. These data were corroborated by the description of a non-canonical, Ca2+- and Mg2+-inde‐ pendent 45-59 kDa endonuclease [76, 136, 140].

As in other pathogenic trypanosomatids, apoptotic features were also identified in *T. brucei* under non-physiological conditions, such as incubation with drugs, cytokines or ROS [129, 133, 141-143]. Interestingly, the gene for prohibitin and the receptor for activated protein kinase C have been correlated with the apoptotic process, suggesting convergence between these pathways in protozoa and mammals (Table 2) [129]. Despite several reports about caspase-like activity in trypanosomatids [75, 134, 136, 144], the exact role of these proteases in protozoa is not clear. Metacaspases are structurally similar to mammalian orthologues, but their catalytic activity on caspase substrates is quite controversial [145-147]. Despite their presence in *T. cruzi*, *T. brucei* and *Leishmania* sp., only *L. major* metacaspase shows *in vitro* self-proteolytic activity (Table 2) [146]. In fact, the participation of metacaspases cleaving vital substrates in the cell death cascade has not yet been described [148, 149]. Surprisingly, experimental

**3. Cell death in trypanosomatids: An overview**

and fragmentation [76, 119, 129, 130, 134] (Table 3, Figure 1).

pendent 45-59 kDa endonuclease [76, 136, 140].

in pathogenic trypanosomatids.

204 Cell Death - Autophagy, Apoptosis and Necrosis

**3.1. Apoptosis-like**

The term PCD was employed for decades to exclusively describe cell death in metazoans and its involvement in embryogenesis and maintenance of homeostasis. Indeed, the relevance of PCD for lower eukaryotes is unclear. In an evolutionary scenario, these regulated processes could allow clonal selection in the parasite population, guaranteeing the propagation of identical genetic information even in adverse environmental conditions. However, differences in the cell death mechanisms observed between metazoans and protozoans must be considered [78, 118]. In the following sections, we will discuss the role of different death styles described

In trypanosomatids, the first PCD report was published in 1995 by Ameisen and coworkers describing apoptotic characteristics (DNA fragmentation and cytoplasmic and nuclear morphological alterations) in *T. cruzi* epimastigotes during differentiation to trypomastigotes [119]. In the last two decades, a variety of stimuli were reported to induce the appearance of the apoptotic phenotype in this parasite, including exposure to fresh human serum (FHS), heat shock and drugs [76, 119-128]. Curiously, the apoptosis-like phenotype was also associated with the regulation of the *T. cruzi* life cycle [129]. These cell death phenotypes in pathogenic trypanosomatids have been characterized by the use of classical apoptotic markers (see item 2.1) [76, 79, 118, 123, 130-133]. Among the apoptotic hallmarks identified, we found (a) loss of ΔΨm, (b) cytochrome c release, (c) PS externalization, and (d) abnormal DNA condensation

In *Leishmania* sp., apoptotic features (nuclear condensation, DNA fragmentation, cell shrink‐ age, loss of ΔΨm, and release of cytochrome c) were also observed in stress conditions induced by heat, starvation, oxidative agents and drugs [118, 134, 136-139, 135]. *L. donovani, L. major* and *L. mexicana* stationary phase promastigotes and axenic amastigotes exhibited DNA fragmentation with a laddering electrophoretic profile, suggesting oligonucleosomal cleavage. These data were corroborated by the description of a non-canonical, Ca2+- and Mg2+-inde‐

As in other pathogenic trypanosomatids, apoptotic features were also identified in *T. brucei* under non-physiological conditions, such as incubation with drugs, cytokines or ROS [129, 133, 141-143]. Interestingly, the gene for prohibitin and the receptor for activated protein kinase C have been correlated with the apoptotic process, suggesting convergence between these pathways in protozoa and mammals (Table 2) [129]. Despite several reports about caspase-like activity in trypanosomatids [75, 134, 136, 144], the exact role of these proteases in protozoa is not clear. Metacaspases are structurally similar to mammalian orthologues, but their catalytic activity on caspase substrates is quite controversial [145-147]. Despite their presence in *T. cruzi*, *T. brucei* and *Leishmania* sp., only *L. major* metacaspase shows *in vitro* self-proteolytic activity (Table 2) [146]. In fact, the participation of metacaspases cleaving vital substrates in the cell death cascade has not yet been described [148, 149]. Surprisingly, experimental

**Figure 1.** Schematic representation of apoptosis-like PCD in pathogenic trypanosomatids. Apoptotic stimuli induce loss of ΔΨm, release of mitochondrial cytochrome c to the cytosol, PS externalization and DNA fragmentation by En‐ doG activity. Apoptotic regulators from the Bcl-2 family were not found until now, and the role of metacaspases is controversial, suggesting that apoptosis-like PCD in trypanosomatids is a caspase-like- and Bcl-2-independent path‐ way. N: nucleus; M: mitochondrion; K: kinetoplast; F: flagellum.

evidence pointed to the involvement of these proteases in cell cycle control and metacyclo‐ genesis, not in death [145, 150-153].

In unicellular organisms, the mitochondrion is a central organelle in cell death pathways, leading to ROS production [125]. In *T. brucei* procyclic forms, mitochondrial Ca2+ influx misbalance culminates in ROS generation [154]. Additionally, prostaglandin D2-induced ROS production in both the bloodstream and procyclic forms led to the labelling of different apoptotic markers, with the death phenotype reverted by oxidative scavengers, such as Nacetyl cysteine [130, 155, 156]. In *L. donovani*, hydrogen peroxide induced classical apoptotic features (DNA fragmentation, loss of ΔΨm and caspase-like activity). This phenotype was partially reverted by caspase inhibitors [134, 137]. Oxidative stress plays a crucial role not only in apoptosis-like PCD but also in autophagy and necrosis, as we will discuss later [78, 157].


**Table 2.** Apoptotic molecules described in pathogenic trypanosomatids

The participation of EndoG-like in mitochondrial-mediated cell death has been reported, but the process is metacaspase-independent (Table 2) [132, 158, 159]. *L. infantum* submitted to heat stress also presents an apoptotic pattern, but without caspase-like activity, which was partially reversed by the expression of the anti-apoptotic mammalian gene Bcl-XL [160]. On the other hand, the overexpression of mammalian anti-apoptotic Bcl-2 in *T. brucei* caused no reversion of the mitochondrial damage induced by ROS [154]. However, members of the Bcl-2 protein family have not been described in trypanosomatids [129]. More studies regarding the regula‐ tion steps of apoptosis-like processes in trypanosomatids need to be performed.

#### **3.2. Autophagy**

Almost forty years ago, the first morphological autophagic evidence was described in trypa‐ nosomatids by electron microscopy of *T. brucei* [170]. In the last four decades, many studies have described recurrent autophagosome formation (initially named autophagic vacuoles), multivesicular bodies as well as myelin-like structures in pathogenic trypanosomatids treated with different classes of drugs (Figure 2) [169, 168, 171-178]. Such autophagosomes showed distinct levels of degradation depending on the degree of cellular structure damage inside the organelle. Myelin-like structures are one of the most frequent ultrastructural alterations detected in drug-treated parasites and are suggestive of the cellular recycling of damaged structures. Currently, it is postulated that myelin-like structures are phagophores (or preautophagosomal structures, PAS), an early step in the formation of doubled-membrane autophagosomes (Table 3). In *T. cruzi*, ER profiles were reported as the main origin of phago‐ phores (Figure 2). These profiles usually surround a pre-lysosomal compartment, named the reservosome, suggesting the participation of this organelle in autophagolysosome formation in epimastigote forms [82, 178].

**Molecule Organism References**

*L. donovani*

*L. donovani*

*T. cruzi*

*T. cruzi T. brucei L. major*

*T. cruzi*

*L. major T. brucei L. infantum L. donovani*

tion steps of apoptosis-like processes in trypanosomatids need to be performed.

**Table 2.** Apoptotic molecules described in pathogenic trypanosomatids

Metacaspases 4 *T. brucei* [162, 164]

Elongation factor 1 ∝ *T. cruzi* [161]

*T. brucei* [129]

*T. brucei* [147, 162, 163]

*T. brucei* [145, 147, 162]

*L. donovani* [124, 134, 136]

*L. donovani* [158]

The participation of EndoG-like in mitochondrial-mediated cell death has been reported, but the process is metacaspase-independent (Table 2) [132, 158, 159]. *L. infantum* submitted to heat stress also presents an apoptotic pattern, but without caspase-like activity, which was partially reversed by the expression of the anti-apoptotic mammalian gene Bcl-XL [160]. On the other hand, the overexpression of mammalian anti-apoptotic Bcl-2 in *T. brucei* caused no reversion of the mitochondrial damage induced by ROS [154]. However, members of the Bcl-2 protein family have not been described in trypanosomatids [129]. More studies regarding the regula‐

Almost forty years ago, the first morphological autophagic evidence was described in trypa‐ nosomatids by electron microscopy of *T. brucei* [170]. In the last four decades, many studies have described recurrent autophagosome formation (initially named autophagic vacuoles), multivesicular bodies as well as myelin-like structures in pathogenic trypanosomatids treated

*T. brucei* [145, 150, 153, 162]

[145, 150, 162, 165]

[132, 158, 166]

Prohibitin RACK

Metacaspases 1

Metacaspases 2

Metacaspases 3

Metacaspases 5

Endonuclease G

LdTatD-like nuclease

**3.2. Autophagy**

LdFEN-1

Metacaspase Z-DEVD-FMK -sensitive

206 Cell Death - Autophagy, Apoptosis and Necrosis

**Figure 2.** Schematic representation of autophagy in pathogenic trypanosomatids. Autophagic stimuli induce the for‐ mation of phagophores from ER profiles. The phagophore engulfs organelles and molecules, generating autophago‐ somes. Targeting and engulfment are Atg-dependent processes. These autophagosomes fused with lysosomes generate autophagolysosomes. Continuous autophagic stimuli lead to autophagic cell death, which is inhibited by the pre-treat‐ ment of the parasite with autophagic inhibitors (wortmannin or 3-methyladenine).


**Table 3.** Types of cell death described in pathogenic trypanosomatids

In the last few years, a functional autophagic pathway was characterized in trypanosomatids and ATG homologues were identified. However, almost half of the yeast Atgs are lacking in these protozoa [167, 179-181]. Currently, in trypanosomatids, twenty autophagic genes have been found to be involved in all of the steps, from vesicle expansion and completion to degradation (Figure 2) [167]. Bioinformatic approaches revealed all four genes of the Atg8 conjugation system (Atg3, Atg4, Atg7 and Atg8). Atg8 is well-characterized in *T. cruzi*, *T. brucei* and *Leishmania* sp. and is located in autophagosomes, as observed in yeast and mammals. Atg8 has four isoforms (Atg8, Atg8A, Atg8B and Atg8C) that are processed by two isoforms of Atg4 (Atg4.1 and Atg4.2) [92, 181-185] (Table 3). On the other hand, the Atg12 conjugation system has poor sequence similarity in trypanosomatids, and Atg5, Atg10 and Atg12 sequen‐ ces are lacking [167, 180]. Pathogenic trypanosomatids have two TOR kinases (TOR1 and TOR2) that form their respective complexes, TORC1 and TORC2 (Table 4). These two com‐ plexes show a distinct molecular behaviour, subcellular localization and susceptibility to rapamycin [186, 187]. The treatment of *T. brucei* bloodstream forms with rapamycin led to cell cycle arrest and an increase in the number of autophagosomes due to TORC2 inhibition. However, rapamycin had no effect on the parasite TORC1, suggesting another function for this complex [186, 188].

In addition to the recycling function, autophagy plays a fundamental role in parasite differ‐ entiation and survival, mitochondrial function and homeostasis of phospholipids [92, 182, 189, 190]. In metacyclogenesis, the autophagic pathway is triggered by nutritional deprivation, playing an important function in both the infectivity and virulence to the vertebrate host [182]. During the *T. cruzi* life cycle, epimastigotes are submitted to starvation in the insect rectum, a crucial event for protozoa differentiation. Starved epimastigotes express Atg8.1, but such expression is decreased in metacyclic forms [82, 92]. Reservosomes disappeared during


**Table 4.** Autophagic molecules described in pathogenic trypanosomatids

**Cell death Features Organism References**

*T. cruzi T. brucei Leishmania* sp.

*T. cruzi T. brucei Leishmania* sp.

*T. cruzi T. brucei Leishmania* sp.

In the last few years, a functional autophagic pathway was characterized in trypanosomatids and ATG homologues were identified. However, almost half of the yeast Atgs are lacking in these protozoa [167, 179-181]. Currently, in trypanosomatids, twenty autophagic genes have been found to be involved in all of the steps, from vesicle expansion and completion to degradation (Figure 2) [167]. Bioinformatic approaches revealed all four genes of the Atg8 conjugation system (Atg3, Atg4, Atg7 and Atg8). Atg8 is well-characterized in *T. cruzi*, *T. brucei* and *Leishmania* sp. and is located in autophagosomes, as observed in yeast and mammals. Atg8 has four isoforms (Atg8, Atg8A, Atg8B and Atg8C) that are processed by two isoforms of Atg4 (Atg4.1 and Atg4.2) [92, 181-185] (Table 3). On the other hand, the Atg12 conjugation system has poor sequence similarity in trypanosomatids, and Atg5, Atg10 and Atg12 sequen‐ ces are lacking [167, 180]. Pathogenic trypanosomatids have two TOR kinases (TOR1 and TOR2) that form their respective complexes, TORC1 and TORC2 (Table 4). These two com‐ plexes show a distinct molecular behaviour, subcellular localization and susceptibility to rapamycin [186, 187]. The treatment of *T. brucei* bloodstream forms with rapamycin led to cell cycle arrest and an increase in the number of autophagosomes due to TORC2 inhibition. However, rapamycin had no effect on the parasite TORC1, suggesting another function for

In addition to the recycling function, autophagy plays a fundamental role in parasite differ‐ entiation and survival, mitochondrial function and homeostasis of phospholipids [92, 182, 189, 190]. In metacyclogenesis, the autophagic pathway is triggered by nutritional deprivation, playing an important function in both the infectivity and virulence to the vertebrate host [182]. During the *T. cruzi* life cycle, epimastigotes are submitted to starvation in the insect rectum, a crucial event for protozoa differentiation. Starved epimastigotes express Atg8.1, but such expression is decreased in metacyclic forms [82, 92]. Reservosomes disappeared during

[76, 118, 119, 130]

[82, 92, 167]

[149, 168, 169]

apoptosis-like cell shrinkage

208 Cell Death - Autophagy, Apoptosis and Necrosis

membrane blebbing DNA fragmentation PS exposure loss of the ΔΨm release of cytochrome c

autophagy presence of autophagosomes-like

organelles

necrosis cytosolic vacuolization

this complex [186, 188].

Golgi and/or ER profiles surrounding

detection of Atg8 and Atg4

plasma membrane disruption

**Table 3.** Types of cell death described in pathogenic trypanosomatids

differentiation, most likely due to the cysteine proteinase activity, in particular, cruzipain [159, 191, 192].

In *Leishmania* sp., autophagy is essential for metacyclogenesis, with several observed auto‐ phagosomes during the process [193, 194]. The deletion of Atg4.2 led to an accumulation of Atg8 lipidated isoforms, compromising the autophagic activity. Subsequently, a reduction in the number of differentiating promastigotes was observed [194]. Interestingly, autophagy also participates in the differentiation of *L. mexicana* metacyclic promastigotes to amastigotes [193]. In the sandfly, the exposure of promastigotes to different stress stimuli, including higher temperature, low pH, and nutritional deprivation, acts as a crucial event for the success of the metacyclogenesis [189, 192]. *L. mexicana* shows that lysosome-like structures, called mega‐ somes, are involved in parasite differentiation, with the activity of two megasomal cysteine peptidases (CPA and CPB) associated with autophagy. The deletion of these proteases strongly impaired its differentiation into amastigotes, leading to an accumulation of autophagosomes containing multi-vesicular tubules (structures related to endocytosis) [180, 193].

A peculiar role for autophagy was observed in *T. brucei*. In a selective pathway, glycosomes are degraded during differentiation from bloodstream to procyclic forms. This organelle is a peroxisome-like structure that is also involved in the glycolytic pathway, and its degradation via autophagy led to important changes in the protozoa bioenergetics [195]. This evidence supported the existence of pexophagy in trypanosomes, an essential event for energy balance during the parasite life cycle. Depending on the environmental conditions (distinct hosts), the sources of energetic substrates vary, as does the ATP demand [180]. Recently, it was also reported that *T. brucei* acidocalcisomes (an acidic compartment that stores ions responsible for polyphosphate metabolism) regulate autophagy by the acidification of this organelle. More‐ over, the blockage of acidocalcisome biogenesis also inhibited the autophagic pathway without the impairment of lysosomal biogenesis or function, suggesting the relevance of acidocalci‐ somes as an autophagic regulator [196].

Autophagic cell death occurs when the homeostatic balance is broken [40]. To evaluate whether autophagy participates in the cell death process, the use of the PI-3K inhibitors wortmannin and 3-methyladenine (3-MA) before the autophagic stimulus is provided is an interesting experimental approach. Pre-treatment with these inhibitors totally abolished the trypanocidal activity of naphthoimidazoles in *T. cruzi* epimastigotes and trypomastigotes. Although the involvement of components of the Atg8 conjugation system was also demonstrated, the molecular mechanisms of cell death regulation in this parasite deserve further examination [82, 178].

### **3.3. Necrosis**

As described for higher eukaryotes, necrosis is poorly studied in protozoa, especially due to its conception as an accidental and uncontrolled process. The most typical necrotic feature is the plasma membrane rupture that leads to the loss of cellular homeostasis and consequent cell lysis as the consequence of a mechanical or chemical stimulus [103]. Necrosis is always the cell death endpoint, culminating in the generation of cellular debris. Thus, independent of the cell death mechanism that is induced, all parasites will lyse in a system without phagocytic cells to clean the microenvironment. In this context, a high percentage of anti-trypanosomatid natural or synthetic drugs present a mechanism of action with a lytic effect [29, 149, 202-205] (Table 3).

Another crucial stress condition that induces trypanosomatid disruption is the activation of the complement pathway. This cascade can be triggered by the binding of lectins to lipophos‐ phoglycans presented on the surface of *Leishmania* sp. promastigotes and of glycosylated molecules in the *T. cruzi* metacyclic form [206-209]. Indeed, pathogenic trypanosomatids show different mechanisms to evade the complement pathway. For example, *T. brucei* expresses a vast number of variant surface glycoproteins (VSG) that change the parasite coat to escape from the host immune system [210]. In relation to programmed necrosis, RIPK-like molecules have not yet been identified in unicellular organisms, and the direct effect of Nec-1 has not been evaluated, suggesting that an orchestrated pathway similar to necroptosis is absent in trypanosomatids.

#### **3.4. Others**

Curiously, no studies have been reported about non-canonical PCD pathways in trypanoso‐ matids. Pyroptosis and NETosis are processes that are characterized exclusively in mammalian cells, specifically during an inflammatory response. Such pathways involve the death of immune cells to block the progression of any infection by a well-regulated mechanism [106, 110]. The absence of these PCD types in unicellular organisms is not strange. On the other hand, the existence of specific oxidative stress-related cell death types in trypanosomatids would be reasonable. Continuous exposure of these parasites to ROS under distinct environ‐ mental conditions during their life cycles indicates the important role of oxidative stress in the control of protozoa populations. ROS involvement in trypanosomatid apoptosis-like processes and autophagy has been described in different experimental conditions [130, 155, 156, 211, 212], but ferroptosis has not yet been investigated. Further studies about the effect of erastin as well as the inhibition by ferrostatin 1 should be performed in these parasites. Autoschizis was only observed in cancer cells under very specific conditions, but interestingly, an auto‐ schizic phenotype (high percentages of AV-/PI+ cells) was detected in *T. cruzi* treated with naphthoimidazoles [178]. The AV-/PI+ population is ignored in the majority of the studies, including in pathogenic trypanosomatids [213-215]. A better characterization of this parasite population must be performed to exclude the existence of autoschizis in protozoa.

#### **3.5. Cell death and evasion of host immune response**

the impairment of lysosomal biogenesis or function, suggesting the relevance of acidocalci‐

Autophagic cell death occurs when the homeostatic balance is broken [40]. To evaluate whether autophagy participates in the cell death process, the use of the PI-3K inhibitors wortmannin and 3-methyladenine (3-MA) before the autophagic stimulus is provided is an interesting experimental approach. Pre-treatment with these inhibitors totally abolished the trypanocidal activity of naphthoimidazoles in *T. cruzi* epimastigotes and trypomastigotes. Although the involvement of components of the Atg8 conjugation system was also demonstrated, the molecular mechanisms of cell death regulation in this parasite deserve further examination

As described for higher eukaryotes, necrosis is poorly studied in protozoa, especially due to its conception as an accidental and uncontrolled process. The most typical necrotic feature is the plasma membrane rupture that leads to the loss of cellular homeostasis and consequent cell lysis as the consequence of a mechanical or chemical stimulus [103]. Necrosis is always the cell death endpoint, culminating in the generation of cellular debris. Thus, independent of the cell death mechanism that is induced, all parasites will lyse in a system without phagocytic cells to clean the microenvironment. In this context, a high percentage of anti-trypanosomatid natural or synthetic drugs present a mechanism of action with a lytic effect [29, 149, 202-205]

Another crucial stress condition that induces trypanosomatid disruption is the activation of the complement pathway. This cascade can be triggered by the binding of lectins to lipophos‐ phoglycans presented on the surface of *Leishmania* sp. promastigotes and of glycosylated molecules in the *T. cruzi* metacyclic form [206-209]. Indeed, pathogenic trypanosomatids show different mechanisms to evade the complement pathway. For example, *T. brucei* expresses a vast number of variant surface glycoproteins (VSG) that change the parasite coat to escape from the host immune system [210]. In relation to programmed necrosis, RIPK-like molecules have not yet been identified in unicellular organisms, and the direct effect of Nec-1 has not been evaluated, suggesting that an orchestrated pathway similar to necroptosis is absent in

Curiously, no studies have been reported about non-canonical PCD pathways in trypanoso‐ matids. Pyroptosis and NETosis are processes that are characterized exclusively in mammalian cells, specifically during an inflammatory response. Such pathways involve the death of immune cells to block the progression of any infection by a well-regulated mechanism [106, 110]. The absence of these PCD types in unicellular organisms is not strange. On the other hand, the existence of specific oxidative stress-related cell death types in trypanosomatids would be reasonable. Continuous exposure of these parasites to ROS under distinct environ‐ mental conditions during their life cycles indicates the important role of oxidative stress in the

somes as an autophagic regulator [196].

210 Cell Death - Autophagy, Apoptosis and Necrosis

[82, 178].

(Table 3).

trypanosomatids.

**3.4. Others**

**3.3. Necrosis**

Trypanosomatids presented a highly sophisticated repertoire to evade mammalian immune systems, including the capacity to prevent the cell death pathways of the infected host cells [188]. This efficient strategy allows host PCD modulation by the parasites to establish the infection. Depending on the protozoan species and the host cell type, PCD exacerbation or inhibition fluctuates. For example, the induction of apoptosis in immune cells increases the parasite persistence and survival in immunocompetent hosts [78]. In *T. cruzi* infection, apoptosis of lymphocytes and macrophages is essential for the parasite to escape, promoting inflammation reduction by anti-inflammatory cytokines and also amastigote proliferation [78, 216, 217]. The *Leishmania* strategy is quite different. Promastigotes externalize PS to be recognized by phagocytic cells. The binding of PS to its receptor on the phagocyte surface triggers a signalling cascade that guides TGF-β production and the subsequent anti-inflam‐ matory response. This phenomenon, called apoptotic mimicry, facilitates parasite internaliza‐ tion and increases the success of the infection [218, 219]. Additionally, the intracellular cycle of *Leishmania* sp. also depends on the impairment of host cell apoptosis. This event is necessary to stop or delay the elimination of infected cells. For example, *L. major* uses the infected apoptotic granulocytes as "Trojan horses" to invade macrophages, the definitive host cells, avoiding the direct activation of phagocytes via the interaction between host receptors and protozoa [220].

Host autophagy also represents a valuable mechanism for both innate and adaptive responses to stop the infection. Its blockage is a crucial tactic for pathogenic trypanosomatids to evade host defences. Autophagy uses a process to eliminate pathogens, called xenophagy, directing microorganisms to be digested in lysosomes. This strategy is usually employed by protozoa living inside parasitophorous vacuoles to use the autophagic machinery to provide nutrients [82]. However, protozoa, such as *Leishmania* sp., change the autophagosomal pH and impair vesicular traffic, compromising the fusion to lysosomes. *L. amazonensis* amastigotes proliferate in starvation or even after treatment with rapamycin, but the proliferation is inhibited by incubation with the autophagic inhibitors wortmannin or 3-methyladenine [221]. The impor‐ tance of autophagy for the *Leishmania* infection was corroborated by the observation that this pathway is exacerbated in *L. amazonensis*-infected mice and in a natural *L. donovani* infection in humans [222, 223]. Similar data were observed in the *in vitro T. cruzi* infection, suggesting that the autophagic pathway favours the parasite during its interaction with the host cell [224, 225]. However, the role of host autophagy in this trypanosomatid is still controversial due to the autophagic participation in the control of *T. cruzi* infection [226-228]. Furthermore, differences among strains and host cells must be considered to clarify whether host autophagy kills *T. cruzi* or provides nutrients for its survival.

### **4. Concluding remarks**

In spite of the variety of studies about cell death in protozoans, including trypanosomatids, and the evidence of PCD, the detailed aspects of the molecular mechanisms and regulation remain unclear. The absence of key molecules together with the lack of an obvious role for this process in unicellular organisms makes the existence of PCD in these cells a debatable point, and the term "apoptosis-like" is more convenient [130, 172, 229]. In this context, the lack of a strong correlation between the proteolytic properties of caspases and their role in PCD should be highlighted. Currently, there is no description of the participation of trypanosomatid metacaspases in cell death processes, but these proteases have been postulated to function in proliferation and differentiation, which are important events for parasite survival [145, 148, 149, 153, 230]. In the post-genomic era, a rigorous search should be performed in proteomic databases of pathogenic trypanosomatids to correct misannotations in cell death proteins, validating the real role of these molecules for PCD processes.

Nevertheless, PCD was conserved during evolution, suggesting its essential function for the survival and maintenance of these species. However, it has been proposed that these pathways appeared in the phylogenetic tree in the multicellular organism branches, suggesting that the death molecular mechanisms identified in unicellular parasites came from a divergent evolutionary event [48]. This idea is supported by the replacement or complete absence of some PCD molecules, justifying the differences observed in protozoa mechanisms [79]. In addition to being an interesting evolutionary model for PCD, its physiological relevance for protozoa is still the most attractive question.

An altruistic hypothesis has been raised for unicellular organisms, especially for pathogenic trypanosomatids [130]. It was associated with the control of parasite populations, including protozoa density regulation, clonal selection and immune host system evasion, events related to the success of the infection [7, 76, 82, 136, 231]. Trypanosomatid cell death limits parasite colonization in insects in response to scarce resources of nutrients, avoiding invertebrate death [118, 130, 134]. On the other hand, PCD of *T. cruzi* or *L. amazonensis* insect forms under mammalian temperatures could evade host immune response derived from parasite lysis, facilitating the infection [76, 119, 135].

Autophagic cell death has been proposed as a PCD pathway, suggesting an active role of autophagy in death processes, but the precise mechanisms of regulation are not yet clear [174, 178, 232]. The majority of the autophagic studies were performed in yeast and mammal models. However, little is known about protozoan pathways. Autophagy is a regulated process that is directly involved in the preservation of cellular homeostasis and survival. Several hypotheses have been raised about the participation of this pathway in cell death in dying cells. The selective autophagic degradation of essential cellular factors, such as cell death regulators, triggers death events, including caspase activation [232, 233]. Another hypothesis suggested that autophagy is not a specific and regulated cell death process but is a consequence of extensive injury. Once such an injury compromises cellular physiology, the damaged structure needs to be degraded for cell survival. This hypothesis also explains the presence of similar phenotypes in parasites after treatment with different compounds with distinct mechanisms of action. Such autophagic phenotypes, detected independent of the stimuli, reinforced this pathway as a desperate attempt of the cells to stay alive [168, 212, 232]. The determination of the connection between the autophagic cell death of pathogens, such as trypanosomatids, could have crucial implications for human health, but further mechanistic studies should be addressed in this field.

225]. However, the role of host autophagy in this trypanosomatid is still controversial due to the autophagic participation in the control of *T. cruzi* infection [226-228]. Furthermore, differences among strains and host cells must be considered to clarify whether host autophagy

In spite of the variety of studies about cell death in protozoans, including trypanosomatids, and the evidence of PCD, the detailed aspects of the molecular mechanisms and regulation remain unclear. The absence of key molecules together with the lack of an obvious role for this process in unicellular organisms makes the existence of PCD in these cells a debatable point, and the term "apoptosis-like" is more convenient [130, 172, 229]. In this context, the lack of a strong correlation between the proteolytic properties of caspases and their role in PCD should be highlighted. Currently, there is no description of the participation of trypanosomatid metacaspases in cell death processes, but these proteases have been postulated to function in proliferation and differentiation, which are important events for parasite survival [145, 148, 149, 153, 230]. In the post-genomic era, a rigorous search should be performed in proteomic databases of pathogenic trypanosomatids to correct misannotations in cell death proteins,

Nevertheless, PCD was conserved during evolution, suggesting its essential function for the survival and maintenance of these species. However, it has been proposed that these pathways appeared in the phylogenetic tree in the multicellular organism branches, suggesting that the death molecular mechanisms identified in unicellular parasites came from a divergent evolutionary event [48]. This idea is supported by the replacement or complete absence of some PCD molecules, justifying the differences observed in protozoa mechanisms [79]. In addition to being an interesting evolutionary model for PCD, its physiological relevance for protozoa

An altruistic hypothesis has been raised for unicellular organisms, especially for pathogenic trypanosomatids [130]. It was associated with the control of parasite populations, including protozoa density regulation, clonal selection and immune host system evasion, events related to the success of the infection [7, 76, 82, 136, 231]. Trypanosomatid cell death limits parasite colonization in insects in response to scarce resources of nutrients, avoiding invertebrate death [118, 130, 134]. On the other hand, PCD of *T. cruzi* or *L. amazonensis* insect forms under mammalian temperatures could evade host immune response derived from parasite lysis,

Autophagic cell death has been proposed as a PCD pathway, suggesting an active role of autophagy in death processes, but the precise mechanisms of regulation are not yet clear [174, 178, 232]. The majority of the autophagic studies were performed in yeast and mammal models. However, little is known about protozoan pathways. Autophagy is a regulated process that is directly involved in the preservation of cellular homeostasis and survival. Several hypotheses have been raised about the participation of this pathway in cell death in dying cells. The selective autophagic degradation of essential cellular factors, such as cell death regulators,

kills *T. cruzi* or provides nutrients for its survival.

validating the real role of these molecules for PCD processes.

**4. Concluding remarks**

212 Cell Death - Autophagy, Apoptosis and Necrosis

is still the most attractive question.

facilitating the infection [76, 119, 135].

**Figure 3.** Different pathways of trypanosomatid death. The death stimulus triggers specific mechanisms of action de‐ pending on the environmental conditions, time of treatment and dose. Death signals lead to distinct well-known phe‐ notypes from each pathway. Cross-talk could also be observed between apoptosis-like processes, autophagy and necrosis, culminating in protozoa death. The existence of an alternative unknown process cannot be discarded (dashed arrow).

The existence of cross-talk among different cell death pathways, especially autophagy and apoptosis, has been proposed (Figure 3) [93, 234]. In unicellular parasites, different cell death types have been described to be induced by physical and/or chemical stress conditions (drugs, heat shock, and nutritional deprivation, among others), resulting in a non-classical cell death phenotype. The total absence of commercial typical PCD markers, such as antibodies and enzyme activity kits, for protozoa and of key autophagic and apoptotic-like molecules reinforce the hypothesis of an interplay of distinct death mechanisms, suggesting their convergence, leading to necrosis. Likewise, the possibility of the occurrence of other PCD forms cannot be excluded [74, 78, 168, 178]. A better molecular characterization of cell death in pathogenic trypanosomatids is essential for advances in novel alternatives for therapeutic intervention.

### **Author details**

Rubem Figueiredo Sadok Menna-Barreto\* and Solange Lisboa de Castro

\*Address all correspondence to: rubemsadok@gmail.com

Laboratory of Cell Biology, Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Manguinhos, Rio de Janeiro, Brazil

### **References**


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The existence of cross-talk among different cell death pathways, especially autophagy and apoptosis, has been proposed (Figure 3) [93, 234]. In unicellular parasites, different cell death types have been described to be induced by physical and/or chemical stress conditions (drugs, heat shock, and nutritional deprivation, among others), resulting in a non-classical cell death phenotype. The total absence of commercial typical PCD markers, such as antibodies and enzyme activity kits, for protozoa and of key autophagic and apoptotic-like molecules reinforce the hypothesis of an interplay of distinct death mechanisms, suggesting their convergence, leading to necrosis. Likewise, the possibility of the occurrence of other PCD forms cannot be excluded [74, 78, 168, 178]. A better molecular characterization of cell death in pathogenic trypanosomatids is essential for advances in novel alternatives for therapeutic

and Solange Lisboa de Castro

intervention.

**Author details**

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\*Address all correspondence to: rubemsadok@gmail.com

Laboratory of Cell Biology, Oswaldo Cruz Institute, Oswaldo Cruz Foundation,

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### **Chapter 12**

## **Apoptosis and Infections**

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Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61306

#### **Abstract**

Apoptosis is a process that plays a critical role in the elimination of infected cells. Infectious diseases modulate apoptosis, and this contributes to disease pathogene‐ sis. Apoptosis is initiated by various kinds of stimuli, including infections, radia‐ tion, etc. Increased apoptosis may assist the dissemination of intracellular pathogens or induce immunosuppression. However, apoptosis may also help eradi‐ cate pathogens from the host in many cases. Consequently, several viruses, bacteria, and parasites have evolved mechanisms to inhibit host cell by apoptosis as a strat‐ egy that may support intracellular survival and persistence of the pathogen. Bacte‐ ria are recognized by cellular receptors and elicit a multitude of signal transduction events that alter the cell's response toward apoptotic stimuli. The result of patho‐ genic bacteria entering into mammalian cells evokes variety of responses, including internalization or phagocytosis of the bacteria, release of cytokines, secretion of de‐ fensins, production of oxygen radicals and the triggering of apoptosis. Bacteria can trigger apoptosis through a large variety of mechanisms that include the secretion of protein synthesis inhibitors and pore forming proteins. They can also activate apoptotic proteins such as caspases, inactivate antiapoptotic proteins, or lead to up‐ regulation of the endogenous receptor/ligand system. However, new research has shown that many bacterial pathogens can in fact prevent apoptosis during infection. As in bacteria, many viral genomes encode proteins that repress apoptosis to escape from immune attack by the host or viruses promote apoptotic death of the host cells. Virus-host interactions may determine viral persistence, extent and severity of inflammation, and pathology associated with infectious disease. The elucidation of the signaling pathways, the cellular receptors, and/or the microbial factors involved in the induction or reduction of apoptosis could reveal new therapeutic targets for blocking microbial-induced apoptosis. This chapter will summarize the most recent research on microorganisms' apoptotic and antiapoptotic strategies and the mecha‐ nisms relating to disease.

**Keywords:** apoptosis, infection, bacteria, viruses, parasitis

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **1. Introduction**

#### **1.1. Apoptosis regulators**

Programmed cell death or apoptosis is an intrinsic death program that occurs in various physiological and pathological situations. Apoptosis is also a physiological process that is critical for tissue homeostasis. It is essential for the regulation of immune responses. The main regulators of apoptosis are caspases, Bcl-2 family, p53, tumor necrosis factor (TNF) family, and/or inhibitors of apoptosis proteins (IAPs).

### **2. Caspases**

Caspases are a family of proteins that are one of the main effectors of apoptosis. Their activation is a hallmark of apoptosis. Caspases are synthesized as inactive zymogens, the so-called procaspases. Upon maturation, the procaspases are proteolytically processed between the large and small subunit resulting in a small and a large subunit.

Based on their function, the caspases can be classified into three groups: (1) inflammatory caspases—this group includes caspases 1, 4, 5, 11, 12, 13, and 14, which are involved in inflammation instead of apoptosis; (2) apoptotic initiator caspases that possess long prodo‐ mains containing either a death effector domain (DED) (caspases 8 and 10) or a caspase activation and recruitment domain (CARD) (caspases 2 and 9), which mediate the interaction with upstream adaptor molecules; and (3) apoptotic effector caspases. This executioner class (caspases 3, 6, 7) is characterized by the presence of a short prodomain [1]. Apoptotic signals trigger the oligomerization of death adaptor proteins, while death adaptor oligomers in turn induce the aggregation of procaspases. It was previously believed that the initiator caspases are autoproteolytically activated when brought into close proximity of each other. This is called the "induced proximity" model. Effector procaspases are normally cleaved and activated by active initiator caspases. They then cleave various death substrates to induce cell death [2].

### **3. IAPs**

The IAPs represent a family of evolutionarily conserved apoptosis suppressors. Although IAP family proteins may possess other functions, several of them have been shown to bind and potently inhibit activated caspases. Among the caspases inhibited by human IAP family members, XIAP, cIAP1, and cIAP2 are the effector caspases 3 and 7 as well as the initiator caspase 9 [3]. IAP expression can be upregulated in response to survival signals such as those coming from growth factor receptors, e.g., by the activation of the transcription factor. Nuclear factor-kappa B (NF-κB), however, provides a means to suppress apoptosis signaling. IAP inhibitor SMAC/Diablo was recently described as those that bind multiple IAP family members and those that allow caspases to induce apoptosis [4].

### **4. Bcl-2 family**

**1. Introduction**

**2. Caspases**

**3. IAPs**

**1.1. Apoptosis regulators**

232 Cell Death - Autophagy, Apoptosis and Necrosis

and/or inhibitors of apoptosis proteins (IAPs).

large and small subunit resulting in a small and a large subunit.

members and those that allow caspases to induce apoptosis [4].

Programmed cell death or apoptosis is an intrinsic death program that occurs in various physiological and pathological situations. Apoptosis is also a physiological process that is critical for tissue homeostasis. It is essential for the regulation of immune responses. The main regulators of apoptosis are caspases, Bcl-2 family, p53, tumor necrosis factor (TNF) family,

Caspases are a family of proteins that are one of the main effectors of apoptosis. Their activation is a hallmark of apoptosis. Caspases are synthesized as inactive zymogens, the so-called procaspases. Upon maturation, the procaspases are proteolytically processed between the

Based on their function, the caspases can be classified into three groups: (1) inflammatory caspases—this group includes caspases 1, 4, 5, 11, 12, 13, and 14, which are involved in inflammation instead of apoptosis; (2) apoptotic initiator caspases that possess long prodo‐ mains containing either a death effector domain (DED) (caspases 8 and 10) or a caspase activation and recruitment domain (CARD) (caspases 2 and 9), which mediate the interaction with upstream adaptor molecules; and (3) apoptotic effector caspases. This executioner class (caspases 3, 6, 7) is characterized by the presence of a short prodomain [1]. Apoptotic signals trigger the oligomerization of death adaptor proteins, while death adaptor oligomers in turn induce the aggregation of procaspases. It was previously believed that the initiator caspases are autoproteolytically activated when brought into close proximity of each other. This is called the "induced proximity" model. Effector procaspases are normally cleaved and activated by active initiator caspases. They then cleave various death substrates to induce cell death [2].

The IAPs represent a family of evolutionarily conserved apoptosis suppressors. Although IAP family proteins may possess other functions, several of them have been shown to bind and potently inhibit activated caspases. Among the caspases inhibited by human IAP family members, XIAP, cIAP1, and cIAP2 are the effector caspases 3 and 7 as well as the initiator caspase 9 [3]. IAP expression can be upregulated in response to survival signals such as those coming from growth factor receptors, e.g., by the activation of the transcription factor. Nuclear factor-kappa B (NF-κB), however, provides a means to suppress apoptosis signaling. IAP inhibitor SMAC/Diablo was recently described as those that bind multiple IAP family

The Bcl-2 family consists of both antiapoptotic and proapoptotic proteins that share sequence homology within conserved regions known as Bcl-2 homology (BH) domains. All antiapop‐ totic members such as Bcl-2 and Bcl-XL and a subset of proapoptotic family members such as Bax and Bak are multidomain proteins sharing sequence homology within three to four BH domains [5].

### **5. p53**

One of the most important p53 functions is its ability to activate apoptosis. The disruption of this process can promote tumor progression and chemoresistance. p53 tumor suppressor protein blocks cell cycle progression allowing time to repair the damage or induces apoptosis largely through the upregulation of the Bcl-2 family BH3-only protein Puma (p53 upregulated modulator of apoptosis). Many apoptosis-related genes that are transcriptionally regulated by p53 have been identified. p53-dependent apoptosis is frequently the one induced following DNA damage caused by irradiation, UV or viral infections. p53-independent pathways are usually those resulting from growth factor deprivation. The activation of p53 by DNA damage induces either cell cycle arrest or apoptosis. p53 mediates apoptosis through a linear pathway that involves Bax transactivation, Bax translocation from the cytosol to membranes, cyto‐ chrome *c* release from mitochondria, and caspase 9 activation followed by the activation of caspases 3, 6, and 7 [6,7].

#### **5.1. Apoptosis signaling pathways**

Apoptosis can be induced in response to various signals from inside and outside the cell. Apoptosis process involves two pathways: (1) by the release of cytochrome *c* from mitochon‐ dria—intrinsic pathway and/or (2) by the activation of cell-surface death receptor—extrinsic pathway [8].

#### **5.2. Intrinsic pathway**

Mitochondria is a central regulator of intrinsic apoptotic pathways. Intrinsic apoptotic pathways are initiated inside cells. Numerous cytotoxic stimuli and proapoptotic signaltransducing molecules converge on mitochondria to induce outer mitochondrial membrane permeabilization. Mitochondria are known as an important intracellular organelle for pro‐ ducing energy. Mitochondria also play a key role in the modulation of Ca2+ homeostasis and oxidative stress. The dysfunction of mitochondria induced by DNA damage or other genotoxic factors leads to an irreversible event, apoptotic cell death. The intrinsic apoptotic pathway is also called "mitochondrial pathway." A pivotal event in the mitochondrial pathway is mitochondrial outer membrane permeabilization (MOMP). MOMP is mainly mediated and controlled by Bcl-2 family members [9]. Many proteins of the Bcl-2 family either with antia‐ poptotic (e.g., Bcl-2, BclXL, or Mac1) or proapoptotic (Bax, Bak, or Bik) functions reside in the outer membrane of the mitochondria. In healthy cells, a small proportion of Bak molecules are bound to voltage-dependent anionic channel (VDAC; part of the [permeability transition] [PT]). The antiapoptotic molecules Bcl-2 and BclXL prevent the translocation of cytochrome *c* from the mitochondria, while the induced expression or enforced dimerization of Bax results in dysfunction leading to cytochrome *c* release [10].

Specific stimuli such as oxidants, calcium overload, or ceramide cause a decrease in mito‐ chondrial inner transmembrane potential (∆Ψm) and result in the release of cytochrome *c* from the mitrochondrion. Active Bax/Bak causes the release of cytochrome *c*, which then binds to APAF-1 and causes its oligomerization. The release of cytochrome *c* from the mitochondrial intermembrane space to the cytosol contributes to the formation of the apoptosome that consists of cytochrome *c*, APAF-1, and dATP. Caspase 9 is recruited into the complex and activated in this process. The apoptosome activates caspase 9, which is another initiator caspase. Active caspase 9 cleaves and thereby activates effector caspases (most notably caspase 3), and active effector caspases cause the morphological signs of apoptosis by cleavage of other effector proteins [11]. During apoptosis, cells undergo several morphological and biochemical changes. Due to endonuclease activation, the chromatin is cleaved into oligonucleosomal fragments. Recently, it was shown that structural changes in the plasma membrane of the apoptotic cell are functional in signaling the process of cell death to the environment [12]. Active proteases, including caspases, calpains, cathepsins, and/or serine proteases, can promote the activation of DNases in different ways. Effector caspases cleave and inactivate DNA repair enzyme poly-ADP-ribose polymerase (PARP). Regulators of the cell cycle such as retinoblastoma protein and structural proteins of the nucleus and cytoskeleton such as lamins, growth arrest-specific protein 2, gelsolin, fodrin, and survival proteins such as protein kinase C-δ (PKC-δ) cause cell death [13].

Caspase 3 is responsible for degradation of the nuclear protein PARP, which is involved in DNA repair. Apoptosis inducing factor (AIF) is a proapoptotic factor in mitochondria. It triggers chromatin condensation and DNA degradation in a cell in order to induce apoptosis [14].

#### **5.3. Extrinsic pathway**

The extrinsic pathway is activated by ligand-bound death receptors, mainly including (a) TNF-TNFR1, (b) FasL-Fas, and (c) TNF-related apoptosis-inducing ligand (TRAIL) DR4 or DR5. Death receptors belong to the tumor necrosis factor receptor gene (TNFR) superfamily and can generally have several functions that include initiating apoptosis. The TNFR superfamily is characterized by the presence of cysteine-rich domains that mediate binding between ligands and these type I transmembrane domain receptors. Among them, the death receptors, including TNF-R1, Fas (or CD95), and the TRAIL receptors DR4 and DR5, are best character‐ ized for induction of apoptosis [15].

#### *5.3.1. TNF pathway*

TNF is a multifunctional proinflammatory cytokine mainly produced by macrophages. There are two major TNF receptors, TNF-R1 and TNF-R2. TNF-R1 is ubiquitously expressed in most tissues and is the major mediator of TNF signaling, whereas TNF-R2 is mainly found in the immune system and only can be fully activated by membrane bound TNF [16].

TNF-induced activation of NF-κB, JNK, and apoptosis has been intensively studied. NF-κB is a transcription factor that can be induced by a variety of signals. Inhibitor of КB (I-КB) binds to NF-κB and inactivates it by localizing NF-κB to the cytosol, where it is unable to regulate transcription. The phosphorylation of I-КB targets it for ubiquitination and degradation. In the absence of I-КB, NF-κB's nuclear localization signal is exposed, and NF-κB localizes to the nucleus where it is able to induce transcription. The role of NF-κB was originally described as a factor associated with apoptosis. This process is triggered by the phosphorylation of I-КB by the I-КB kinase (IKK) complex. Different activation pathways of NF-κB may cause the expression of proteins that promote apoptosis (e.g., Fas, c-myc, p53, TNF, DR, and caspase 11) or inhibit apoptosis (e.g., IAP proteins, Bcl-2-like proteins) [17]. It was subsequently shown that the inhibition of NF-κB activation potentiates apoptosis [18]. In addition, the inhibition of I-КB expression by oligonucleotides led to cell transformation. Consistent with this role, NFκB has been shown to induce transcription of antiapoptotic proteins. Akt is reported to phosphorylate and activate IKK. The activation of IKK causes phosphorylation and degrada‐ tion of I-КB, which leads to the localization of NF-κB to the nucleus where it can induce transcription of antiapoptotic genes. Thus, Akt can inhibit apoptosis by activating NF-κB [19].

TNF also induces the activation of the stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) pathway. Upon activation, JNK kinases translocate into the nucleus and enhance the transcriptional activity of transcription factors, for example, c-Jun and activating tran‐ scription factor-2 by the phosphorylation of their amino-terminal activation domains. c-Jun belongs to a group of basic region-leucine zipper proteins that dimerize to form transcription factors commonly designated as activator protein 1 (AP-1). The AP-1 proteins have an important role in a variety of cellular processes, including proliferation, differentiation, and induction as well as prevention of apoptosis [20].

#### *5.3.2. FAS pathway*

outer membrane of the mitochondria. In healthy cells, a small proportion of Bak molecules are bound to voltage-dependent anionic channel (VDAC; part of the [permeability transition] [PT]). The antiapoptotic molecules Bcl-2 and BclXL prevent the translocation of cytochrome *c* from the mitochondria, while the induced expression or enforced dimerization of Bax results

Specific stimuli such as oxidants, calcium overload, or ceramide cause a decrease in mito‐ chondrial inner transmembrane potential (∆Ψm) and result in the release of cytochrome *c* from the mitrochondrion. Active Bax/Bak causes the release of cytochrome *c*, which then binds to APAF-1 and causes its oligomerization. The release of cytochrome *c* from the mitochondrial intermembrane space to the cytosol contributes to the formation of the apoptosome that consists of cytochrome *c*, APAF-1, and dATP. Caspase 9 is recruited into the complex and activated in this process. The apoptosome activates caspase 9, which is another initiator caspase. Active caspase 9 cleaves and thereby activates effector caspases (most notably caspase 3), and active effector caspases cause the morphological signs of apoptosis by cleavage of other effector proteins [11]. During apoptosis, cells undergo several morphological and biochemical changes. Due to endonuclease activation, the chromatin is cleaved into oligonucleosomal fragments. Recently, it was shown that structural changes in the plasma membrane of the apoptotic cell are functional in signaling the process of cell death to the environment [12]. Active proteases, including caspases, calpains, cathepsins, and/or serine proteases, can promote the activation of DNases in different ways. Effector caspases cleave and inactivate DNA repair enzyme poly-ADP-ribose polymerase (PARP). Regulators of the cell cycle such as retinoblastoma protein and structural proteins of the nucleus and cytoskeleton such as lamins, growth arrest-specific protein 2, gelsolin, fodrin, and survival proteins such as protein kinase

Caspase 3 is responsible for degradation of the nuclear protein PARP, which is involved in DNA repair. Apoptosis inducing factor (AIF) is a proapoptotic factor in mitochondria. It triggers chromatin condensation and DNA degradation in a cell in order to induce

The extrinsic pathway is activated by ligand-bound death receptors, mainly including (a) TNF-TNFR1, (b) FasL-Fas, and (c) TNF-related apoptosis-inducing ligand (TRAIL) DR4 or DR5. Death receptors belong to the tumor necrosis factor receptor gene (TNFR) superfamily and can generally have several functions that include initiating apoptosis. The TNFR superfamily is characterized by the presence of cysteine-rich domains that mediate binding between ligands and these type I transmembrane domain receptors. Among them, the death receptors, including TNF-R1, Fas (or CD95), and the TRAIL receptors DR4 and DR5, are best character‐

TNF is a multifunctional proinflammatory cytokine mainly produced by macrophages. There are two major TNF receptors, TNF-R1 and TNF-R2. TNF-R1 is ubiquitously expressed in most

in dysfunction leading to cytochrome *c* release [10].

234 Cell Death - Autophagy, Apoptosis and Necrosis

C-δ (PKC-δ) cause cell death [13].

ized for induction of apoptosis [15].

apoptosis [14].

**5.3. Extrinsic pathway**

*5.3.1. TNF pathway*

Fas is involved in the cytotoxic T lymphocyte (CTL)-mediated killing of cells (e.g., CTLmediated killing of virus-infected cells), destruction of inflammatory and immune cells in immune-privileged sites, and deletion of self-reacting B cells and activated T cells at the end of an immune response [21]. Fas binding to Fas ligand results in intracellular clustering of death domains (DD) followed by its internalization into an endosomal pathway. This allows an adaptor protein called Fas-associated death domain (FADD) to associate with the receptor through an interaction between homologous death domains on both molecules. FADD also contains a DED that allows binding of procaspase 8 to the CD95-FADD complex. Procaspase 8 (also known as FLICE) associates with FADD through its own death effector domain. Caspase 8, the main initiator caspase in CD95 signaling, is expressed as two isoforms, caspases 8/a and 8/b, which are both recruited to the activated CD95 receptor. FasL-induced clustering of Fas, FADD, and caspase 8 within the death-inducing signaling complex leads to autoproteolytic processing of caspase 8 by induced proximity and dimerization followed by the release of the processed active proteases [22]. Cells can be divided into two types according to their require‐ ment for mitochondrial pathway in FAS-induced apoptosis. In type I cells, processed caspase 8 is sufficient to directly activate other members of the caspase family. In type II cells, the efficient activation of effector caspases by Fas depends on an amplification loop that relies on caspase-8-mediated cleavage of Bid and subsequent release of mitochondrial proapoptotic factors such as SMAC/Diablo or cytochrome *c* to drive the formation of the caspase-9-activating apoptosome. Active caspase 9 activates the executioner caspase 3, which in turn activates caspase 8 [23].

### *5.3.3. TRAIL pathway*

Like Fas, TRAIL may also be involved in the immune response and in tumor surveillance. Five distinct TRAIL receptors have been identified: death receptor 4 (TRAIL-R1), 125 KILLER/DR5 (TRAIL-R2, TRICK2), DcR1 (TRID, TRAIL-R3), DcR2 (TRUNDD OR TRAIL-R4), and osteo‐ protegerin. The apoptotic signaling induced by TRAIL is similar to that induced by FAS. Binding of TRAIL to its receptors DR4 or DR5 triggers the formation of a death-inducing signaling complex by recruiting FADD and/or caspases 8 and/or 10. TRAIL-induced apoptosis also involves the mitochondrial pathway, like Fas-induced apoptosis, in type II cells [24,25].

### **6. Apoptosis and bacterial infections**

During a microbial infection, organism faces the challenge of recognizing and combating the invading organism. The entrance of bacterial pathogens into human organism initiates the innate immune response characterized by the recruitment of leukocytes to sites of infection [26]. After phagocytosis by human macrophages, microorganisms are destroyed by reactive oxygen species (ROS) microbicidal products contained within granules [27]. Otherwise, bacterial pathogens that cause apoptosis target immune cells such as macrophages and neutrophils [28]. Bacteria can trigger apoptosis by different mechanisms, including the secretion of protein synthesis inhibitors, pore forming proteins, molecules activating the endogenous death machinery in the infected cell, or lipopolysaccharides and other superan‐ tigens [29]. These molecules might be either proapoptotic and activated by the bacteria or antiapoptotic and inhibited upon infection to trigger apoptotic death of the infected cell [30] (Table 1).



ment for mitochondrial pathway in FAS-induced apoptosis. In type I cells, processed caspase 8 is sufficient to directly activate other members of the caspase family. In type II cells, the efficient activation of effector caspases by Fas depends on an amplification loop that relies on caspase-8-mediated cleavage of Bid and subsequent release of mitochondrial proapoptotic factors such as SMAC/Diablo or cytochrome *c* to drive the formation of the caspase-9-activating apoptosome. Active caspase 9 activates the executioner caspase 3, which in turn activates

Like Fas, TRAIL may also be involved in the immune response and in tumor surveillance. Five distinct TRAIL receptors have been identified: death receptor 4 (TRAIL-R1), 125 KILLER/DR5 (TRAIL-R2, TRICK2), DcR1 (TRID, TRAIL-R3), DcR2 (TRUNDD OR TRAIL-R4), and osteo‐ protegerin. The apoptotic signaling induced by TRAIL is similar to that induced by FAS. Binding of TRAIL to its receptors DR4 or DR5 triggers the formation of a death-inducing signaling complex by recruiting FADD and/or caspases 8 and/or 10. TRAIL-induced apoptosis also involves the mitochondrial pathway, like Fas-induced apoptosis, in type II cells [24,25].

During a microbial infection, organism faces the challenge of recognizing and combating the invading organism. The entrance of bacterial pathogens into human organism initiates the innate immune response characterized by the recruitment of leukocytes to sites of infection [26]. After phagocytosis by human macrophages, microorganisms are destroyed by reactive oxygen species (ROS) microbicidal products contained within granules [27]. Otherwise, bacterial pathogens that cause apoptosis target immune cells such as macrophages and neutrophils [28]. Bacteria can trigger apoptosis by different mechanisms, including the secretion of protein synthesis inhibitors, pore forming proteins, molecules activating the endogenous death machinery in the infected cell, or lipopolysaccharides and other superan‐ tigens [29]. These molecules might be either proapoptotic and activated by the bacteria or antiapoptotic and inhibited upon infection to trigger apoptotic death of the infected cell [30]

**Bacteria Apoptosis Proposed or demonstrated mechanism Cell type**

*Pseudomonas aeruginosa* Induction Fas/Fas ligand system Endothelial cells

*Neisseria gonorrhoeae* Induction Increases mitochondrial permeability Epithelial cells

*Neisseria meningitidis* Inhibition Prevents cytochrome *c* release HeLa cells

Inhibition Increases antiapoptotic genes PMLs

Cytochrome *c* release Epithelial cells

Epithelial cells

caspase 8 [23].

(Table 1).

*5.3.3. TRAIL pathway*

236 Cell Death - Autophagy, Apoptosis and Necrosis

**6. Apoptosis and bacterial infections**



**Table 1.** Microorganisms that induce and/or inhibit host cell apoptosis

Inhibition Inhibits both Fas pathway and intrinsic

Rabies virus Induction Expression of Bax and caspase 1 Neuroblastoma cell

Activation of caspase 8 Upregulation of AIF

Induction Caspase gene Nedd-2 Neurons

Upregulation of antiapoptotic genes

Induction Extrinsic pathway Cervical carcinoma cells Inhibition Caspase inactivation fibroblasts, osteosarcoma

NF-κB pathway lymphoblastoid cell lines

cells

carcinoma cells

cell lines

Jurkat, CEM, and HuT78

T lymphoma cell line

Hepatocytes

pathway

HIV-1 gp120 protein Induction Fas pathway CD4+ T cells

Epstein-Barr virus Inhibition Bcl-2 proteins, BHRF1, and BALF1 B cells

Baculovirus Inhibition Antiapoptotic genes (p35 and IAP) Insect cells HPV E2 protein Induction p53 pathway HeLa cells HPV E6 protein Induction Degradation p53 pathway Epithelial cells

HPV E7 protein Induction Retinoblastoma gene Lens

E1A Induction p53 pathway REF52 cells E4orf4 Induction p53 pathway, caspase activation H1299, 293 cells E4orf6 Induction PARP-induced cell death U251 cells

E1B-55K Inhibition Inactivates p53 293 cells

surface

surface

E4 orf 6 Inhibition Blocks p53 H1299 cells

E3-6.7 Inhibition Blocks TRAIL-induced apoptosis Cytotoxic T cells E3 RID Inhibition Inhibits E1A or TNF-induced apoptosis HT29.14S cells

E3-14.7K Inhibition Inhibits TNF and TRAIL-induced apoptosis Fibroblast C3HA cell lines Inhibition inhibits MHC class I transport to the cell

Decreased presentation of Fas on the cell

E1B-19K Inhibition Inhibits extrinsic pathway HeLa cells ,A549 lung

HIV-1 proteins Env Induction p53-dependent genes Puma and Bax

HIV Nef Induction Extrinsic pathway

238 Cell Death - Autophagy, Apoptosis and Necrosis

Adenoviral proteins

Human cytomegalovirus

Apoptosis is induced by both intrinsic and extrinsic pathways. Due to their key role in cell survival, mitochondria represent attractive targets for pathogens. Several pathogens, includ‐ ing both viruses and bacteria, have been shown to target mitochondria in order to interfere with the host cell apoptotic machinery. For example, bacterial pore-forming toxins such as the neisserial porin PorB, which causes rapid Ca+2 influx into target cells and induces apoptosis, exhibit striking structural and functional homology with the mitochondrial anion channels that mediate mitochondrial permeability transition and apoptosis [31]. Apoptosis, which is executed by caspase activity, can be induced either by the ligation of death receptors or by the release of another proapoptotic factor cytochrome *c* from mitochondria. Apoptotic stimuli (either death ligands, binding to death receptors, or any of the multitude of agents that induce apoptosis) are received by some cellular receptor. In the case of death receptors, this directly causes caspase activity [32].

New studies have shown that many bacterial pathogens can prevent apoptosis during infection. Bacteria inhibits apoptosis by the use of multiple mechanisms: the protection of the mitochondria and prevention of cytochrome *c* release (i.e., *Chylamidia* sp. and/or *Neisseria* sp.), the activation of cell survival pathways (i.e., *Salmonella* sp. and/or *Rickettsia* sp.), the inhibition of caspases, and the activation of phosphoinositide 3-kinase (PI3K)-Akt/protein kinase B (PKB) pathway and interaction with cellular caspases (i.e., *Shigella* sp. and/or *Legionella* sp.) [33]. The PI3K/Akt pathway is also a strong activator of cyclin D1, a critical player in cell cycle progres‐ sion. Cyclin D1 protein levels are also regulated by glycogen synthase kinase 3 (GSK-3). GSK-3 is the primary kinase that phosphorylates cyclin D1 at this residue. Rapid cyclin D1 degrada‐ tion induced by GSK-3 is inhibited by the activation of PI3K/Akt pathway because Akt directly phosphorylates and inactivates GSK-3 [34]. c-Fos is a transcriptional regulator that can elevate the expression of many proliferatory genes. PI3K/Akt pathway executes some of its antiapop‐ totic effects through transcription factors such as Elk-1 and c-Fos. The activation of the Raf/MEK/ERK pathway is associated with the increased expression of antiapoptotic proteins. The Raf/MEK/ERK cascade can also activate the PI3K/Akt pathway. Raf has been shown to directly phosphorylate Bad and Bcl-2 to exert antiapoptotic effects. Depending on cell type as well as apoptotic stimulus, Raf can inhibit or promote apoptosis [35]. Toll-like receptors (TLR) have been described to have both apoptosis-inducing and inhibiting capacity. Early reports have linked the activation of TLR2 to the induction of apoptosis through the adapter MyD88 [36]. At the same time, TLR-signaling has clearly an antiapoptotic activity via NF-κB and the PI3Kpathways. NF-κB induces antiapoptotic gene expression [37].

### **6.1.** *Pseudomonas*

*Pseudomonas aeruginosa* can cause disease in animals, including humans. The symptoms of such infections are generalized inflammation and sepsis. If such colonizations occur in critical body organs such as the lungs, the urinary tract and kidneys, the results can be fatal [38]. *P. aeruginosa* kills mammalian cells by an activation of the endogenous CD95 (Fas)/CD95 ligand (Fas-ligand) system. An upregulation of cell surface CD95 and CD95 ligand resulting in the activation of this death receptor has been recently shown to be pivotal for the induction of apoptosis by several *P. aeruginosa* strains. The upregulation of CD95 and the CD95 ligand on cells infected with *P. aeruginosa* depends on the function of the type III secretion system (T3SS). The ligation of the receptor stimulates caspases, mitochondrial changes, and finally execution of apoptosis *in vitro* and *in vivo* [30]. The binding of CD95 by the CD95 ligand upon upregu‐ lation induces the activation of caspases and JNK. In addition, reactive oxygen intermediates in the induction of *P. aeruginosa* triggered death [39].

#### **6.2.** *Neisseria*

*Neisseria gonorrhoeae* is the etiological agent of the sexually transmitted disease gonorrhea. It penetrates the mucosa, enters phagocytes and epithelial cells, and causes a massive inflam‐ matory response in the subepithelial tissue [40]. Several factors play a role in infection, for example, the pili, which mediates primary adherence; the Opa proteins, which mediate adhesion and invasion; and the PorB porin [41,42]. There is conflicting information regarding the effects of neisserial porins on apoptosis. These discrepancies may be due to the specific responses of different cell types, culture conditions, and bacterial strains. *N. gonorrhoeae* porin PorB1B interacts with HeLa cell mitochondria and induces calcium eflux and apoptosis [43]. Massari *et al*. [44] showed that Neisserial PorB is translocated to the mitochondria of HeLa cells infected with *Neisseria meningitidis* and prevent apoptosis by the inhibition of cytochrome *c* release. Massari *et al.* [44] speculated that differences in cell types or porin purification explain the discrepancies between the results. *N. gonorrhoeae* also increases the transcription of host antiapoptotic genes. These genes include bfl-1, cox-2 and c-IAP-2, each coding for a product that acts to inhibit apoptosis.

Bfl-1 is a member of the Bcl-2 family of apoptotic regulators and has been characterized to have a protective effect on host cells when overexpressed. Anti-apoptotic Bcl-2/Bcl-XL interact with the mitochondrial porin VDAC, thus blocking the opening of the PT and/or mitochondrial membrane depolarization and inhibiting cytochrome *c* release [45]. *N. gonorrhoeae* can inhibit apoptosis induced by the intrinsic and extrinsic apoptosis inducers staurosporine (STS) and TRAIL in HL-60 cells and primary polymorphonuclear leukocytes (PMLs) [46]. In addition Follows *et al.* [47] showed that *N. gonorrhoeae* infection in human endocervical epithelial cells induced NF-κB activation and resulted in the increased gene expression of the NF-κBregulated antiapoptotic genes Bfl-1 and/or cIAP-2.

#### **6.3.** *Shigellas*

tion induced by GSK-3 is inhibited by the activation of PI3K/Akt pathway because Akt directly phosphorylates and inactivates GSK-3 [34]. c-Fos is a transcriptional regulator that can elevate the expression of many proliferatory genes. PI3K/Akt pathway executes some of its antiapop‐ totic effects through transcription factors such as Elk-1 and c-Fos. The activation of the Raf/MEK/ERK pathway is associated with the increased expression of antiapoptotic proteins. The Raf/MEK/ERK cascade can also activate the PI3K/Akt pathway. Raf has been shown to directly phosphorylate Bad and Bcl-2 to exert antiapoptotic effects. Depending on cell type as well as apoptotic stimulus, Raf can inhibit or promote apoptosis [35]. Toll-like receptors (TLR) have been described to have both apoptosis-inducing and inhibiting capacity. Early reports have linked the activation of TLR2 to the induction of apoptosis through the adapter MyD88 [36]. At the same time, TLR-signaling has clearly an antiapoptotic activity via NF-κB and the

*Pseudomonas aeruginosa* can cause disease in animals, including humans. The symptoms of such infections are generalized inflammation and sepsis. If such colonizations occur in critical body organs such as the lungs, the urinary tract and kidneys, the results can be fatal [38]. *P. aeruginosa* kills mammalian cells by an activation of the endogenous CD95 (Fas)/CD95 ligand (Fas-ligand) system. An upregulation of cell surface CD95 and CD95 ligand resulting in the activation of this death receptor has been recently shown to be pivotal for the induction of apoptosis by several *P. aeruginosa* strains. The upregulation of CD95 and the CD95 ligand on cells infected with *P. aeruginosa* depends on the function of the type III secretion system (T3SS). The ligation of the receptor stimulates caspases, mitochondrial changes, and finally execution of apoptosis *in vitro* and *in vivo* [30]. The binding of CD95 by the CD95 ligand upon upregu‐ lation induces the activation of caspases and JNK. In addition, reactive oxygen intermediates

*Neisseria gonorrhoeae* is the etiological agent of the sexually transmitted disease gonorrhea. It penetrates the mucosa, enters phagocytes and epithelial cells, and causes a massive inflam‐ matory response in the subepithelial tissue [40]. Several factors play a role in infection, for example, the pili, which mediates primary adherence; the Opa proteins, which mediate adhesion and invasion; and the PorB porin [41,42]. There is conflicting information regarding the effects of neisserial porins on apoptosis. These discrepancies may be due to the specific responses of different cell types, culture conditions, and bacterial strains. *N. gonorrhoeae* porin PorB1B interacts with HeLa cell mitochondria and induces calcium eflux and apoptosis [43]. Massari *et al*. [44] showed that Neisserial PorB is translocated to the mitochondria of HeLa cells infected with *Neisseria meningitidis* and prevent apoptosis by the inhibition of cytochrome *c* release. Massari *et al.* [44] speculated that differences in cell types or porin purification explain the discrepancies between the results. *N. gonorrhoeae* also increases the transcription of host antiapoptotic genes. These genes include bfl-1, cox-2 and c-IAP-2, each coding for a product

PI3Kpathways. NF-κB induces antiapoptotic gene expression [37].

in the induction of *P. aeruginosa* triggered death [39].

**6.1.** *Pseudomonas*

240 Cell Death - Autophagy, Apoptosis and Necrosis

**6.2.** *Neisseria*

that acts to inhibit apoptosis.

The genus *Shigella* consists of four pathogenic "species": *S. dysenteriae*, *S. flexneri*, *S. sonnei*, and *S. boydii. S. flexneri* causes dysentery (shigellosis) by invading the human colonic mucosa. It directly activates proapoptotic signaling pathways to initiate apoptosis in macrophages. It crosses epithelium and goes to lamina propria of intestinal villi. The three proposed effectors of *S. flexneri* internalization are invasion plasmid antigens (Ipa) IpaB, IpaC, and IpaD, all of which are encoded on the pathogen's 230-kb virulence plasmid. These effectors cause caspase 1 activation. Activated caspase 1 then cleaves and activates prointerleukin (proIL)-1β and proIL-18, which are proinflammatory cytokines involved in host inflammatory [48,49]. The secretion of Ipa proteins is dependent on T3SS, which is encoded by 20 genes in the mxi-spa locus of the virulence plasmid. Additional T3SS effector proteins are secreted through the T3 needle when the bacteria are inside the cytoplasm of the host cell [50]. *S. flexneri* kills more of macrophages and promotes the spreading of the bacteria because of the release of interleukin 1β (IL-1β). IL-1β recruits PMLs to the infection sites. The PMLs cross the intestinal epithelium altering the integrity of this epithelial barrier. This promotes massive secondary invasion of the bacteria and acute inflammation [51]. *S. flexneri* infects enterocytes from the basolateral side. The ultrastructural morphology of infected macrophages includes condensation of chromatin at the nuclear boundary blebbing at the cell surface, dilation of the endoplasmic reticulum, and cytoplasmic vacuolization. This is identical to the morphology of cells under‐ going apoptosis [52]. *Shigella* does not induce apoptosis in epithelial cells because these intestinal cells are the primary sites for intracellular bacterial proliferation during shigellosis. It was shown that in the presence of STS, *S. flexneri* inhibits apoptosis by preventing the activation of caspase 3. This happens because of both cytochrome *c* release from the mito‐ chondria and caspase 9 activation [53]. Faherty *et al.* [54] suggested that *Shigella* is protected from apoptosis in epithelial cells by various mechanisms. The bacteria prevent cytochrome *c* release from the mitochondria through the upregulation of Bcl-2 proteins. Second, the extrinsic pathway of apoptosis is inhibited from *in vivo* stimuli such as TNF and FasL. Third, infection leads to the induced expression of JNK and NF-κB, which has many prosurvival effects, including the increased expression of the IAPs (BIRC4, BIRC1, BIRC5, and BIRC7), the Bcl-2 family, and the caspase 8 inhibitors. Finally, the bacteria prevent caspase 3 activation to provide downstream protection in the presence of strong apoptosis inducers. Through the use of T3SS effector proteins, the bacteria could directly generate mitochondrial protection, extrinsic pathway inhibition, and caspase 3 inhibition [54].

Shiga toxins comprise a family of structurally and functionally related protein toxins expressed by *S. dysenteriae* serotype 1 and multiple serotypes of *Escherichia coli.* Shiga toxins cause bloody diarrheal diseases, which may progress to life-threatening extraintestinal complications. The kidneys and central nervous system are the organs most frequently involved [55]. Shiga toxins induce apoptosis in human epithelial, endothelial, myeloid, and lymphoid cells *in vitro* and appear to induce apoptosis in rabbit neurons *in vivo*. Apoptosis induction involves the activation of both extrinsic (caspases 8, 6, and 3 activation) and intrinsic (Bid generation, cytochrome *c* release, and/or caspase 9 activation) pathways. Alterations in the balanced expression of pro- and antiapoptotic Bcl-2 family members also contribute to apoptosis induction [56].

#### **6.4.** *Salmonellas*

*Salmonella* cause an acute localized inflammation in the intestine. *Salmonella enterica* serovar *Typhimurium*, one of the most common food-borne pathogens, causes self-limiting gastroen‐ teritis in humans and a similar diarrheal disease in calves and pigs. *Salmonella typhimurium* can access systemic tissue (mainly spleen and liver) via the lymphatic system and the Peyer's patches. *Salmonella* uses specific virulence factors to invade other cell types such as T3SS. *S. typhimurium* directly activate apoptosis in macrophages [57]. *Salmonella* invasion protein (Sip)B activates ICE (IL-1β converting enzyme). Caspase 1 activation in *Salmonella*-infected macro‐ phages results in the production of active IL-1β and IL-18 and rapid cell lysis with the release of proinflammatory intracellular contents [58]. Knodler *et al.* [59] showed that *S. enterica* serovar *typhimurium* effector protein SopB protects epithelial cells from apoptosis by sustained the activation of Akt. SopB has antiapoptotic activity in infected epithelial cells, which is dependent on the phosphatase activity of SopB and the presence of Akt. PI3K-Akt/PKBpathway protect against apoptosis by the phosphorylation of the proapoptotic Bad. This pathway prevents cytochrome *c* release.

#### **6.5.** *Listeria monocytogenes*

*Listeria monocytogenes*, a facultative anaerobe and/or intracellular bacterium, is the causative agent of listeriosis. *L. monocytogenes*, similar to *Shigella*, lyses the phagosomal membrane and escapes into the cytosol to initiate an intracellular infection, a process that is mediated by a secreted pore-forming toxin, listeriolysin O (LlyO). *L. monocytogenes* induces LlyO-dependent apoptosis in a variety of cell types, including hepatocytes, lymphocytes, and dendritic cells. LlyO might insert into the mitochondrial membrane causing the release of cytochrome *c*. In addition, the insertion of LlyO into the mitochondrial and/or endoplasmic reticulum mem‐ brane may stimulate calcium efflux, thereby activating the calpain and/or caspases [60,61]. Stavru and Cossart [62] showed that LlyO is responsible for mitochondrial network disruption along with a decrease in mitochondrial membrane potential and intracellular ATP levels. However, *L. monocytogenes* does not induce apoptosis in macrophages but causes LlyOmediated necrosis [63].

#### **6.6.** *Chlamydiae*

Shiga toxins comprise a family of structurally and functionally related protein toxins expressed by *S. dysenteriae* serotype 1 and multiple serotypes of *Escherichia coli.* Shiga toxins cause bloody diarrheal diseases, which may progress to life-threatening extraintestinal complications. The kidneys and central nervous system are the organs most frequently involved [55]. Shiga toxins induce apoptosis in human epithelial, endothelial, myeloid, and lymphoid cells *in vitro* and appear to induce apoptosis in rabbit neurons *in vivo*. Apoptosis induction involves the activation of both extrinsic (caspases 8, 6, and 3 activation) and intrinsic (Bid generation, cytochrome *c* release, and/or caspase 9 activation) pathways. Alterations in the balanced expression of pro- and antiapoptotic Bcl-2 family members also contribute to apoptosis

*Salmonella* cause an acute localized inflammation in the intestine. *Salmonella enterica* serovar *Typhimurium*, one of the most common food-borne pathogens, causes self-limiting gastroen‐ teritis in humans and a similar diarrheal disease in calves and pigs. *Salmonella typhimurium* can access systemic tissue (mainly spleen and liver) via the lymphatic system and the Peyer's patches. *Salmonella* uses specific virulence factors to invade other cell types such as T3SS. *S. typhimurium* directly activate apoptosis in macrophages [57]. *Salmonella* invasion protein (Sip)B activates ICE (IL-1β converting enzyme). Caspase 1 activation in *Salmonella*-infected macro‐ phages results in the production of active IL-1β and IL-18 and rapid cell lysis with the release of proinflammatory intracellular contents [58]. Knodler *et al.* [59] showed that *S. enterica* serovar *typhimurium* effector protein SopB protects epithelial cells from apoptosis by sustained the activation of Akt. SopB has antiapoptotic activity in infected epithelial cells, which is dependent on the phosphatase activity of SopB and the presence of Akt. PI3K-Akt/PKBpathway protect against apoptosis by the phosphorylation of the proapoptotic Bad. This

*Listeria monocytogenes*, a facultative anaerobe and/or intracellular bacterium, is the causative agent of listeriosis. *L. monocytogenes*, similar to *Shigella*, lyses the phagosomal membrane and escapes into the cytosol to initiate an intracellular infection, a process that is mediated by a secreted pore-forming toxin, listeriolysin O (LlyO). *L. monocytogenes* induces LlyO-dependent apoptosis in a variety of cell types, including hepatocytes, lymphocytes, and dendritic cells. LlyO might insert into the mitochondrial membrane causing the release of cytochrome *c*. In addition, the insertion of LlyO into the mitochondrial and/or endoplasmic reticulum mem‐ brane may stimulate calcium efflux, thereby activating the calpain and/or caspases [60,61]. Stavru and Cossart [62] showed that LlyO is responsible for mitochondrial network disruption along with a decrease in mitochondrial membrane potential and intracellular ATP levels. However, *L. monocytogenes* does not induce apoptosis in macrophages but causes LlyO-

induction [56].

242 Cell Death - Autophagy, Apoptosis and Necrosis

**6.4.** *Salmonellas*

pathway prevents cytochrome *c* release.

**6.5.** *Listeria monocytogenes*

mediated necrosis [63].

Two species of chlamydiae commonly infect humans, *Chlamydia trachomatis* and *Chlamydia pneumoniae. C. trachomatis* causes trachoma, a scarring eye infection in developing countries. *C. pneumoniae* causes pneumonia [64]. *Chlamydia* species require differentiation to produce sufficient infectious elementary bodies to spread to adjacent cells. In an infection, elementary bodies are taken up by a host cell and begin their cycle inside a membrane-bound vacuole in the host cell cytosol. During the early stages of infection, *Chlamydia* has antiapoptotic effect that helps to maintain the metabolic activities of the infected cell. It was shown that *C. trachomatis* has antiapoptotic effect in epithelial cells and macrophages by blocking the release of cytochrome *c* [65]. *C. pneumoniae* also inhibited apoptosis through an additional activity that was described as blocking caspase activation by cytochrome *c* in a cell-free system [66]. The activation of the host cell apoptotic pathways in the late stages of infection may facilitate dispersal of the bacteria and initiate a host inflammatory response. The various subfamilies of the Bcl-2-family of proteins play a decisive role in this event. The trigger of cytochrome *c*release is the activation of one or several BH3-only proteins. Bcl-2 and its antiapoptotic homologues can bind active BH3-only proteins and probably by this interaction block apoptosis [67]. Du *et al.* [68] demonstrated another way of that inhibiting apoptosis involves the activation of Raf/MEK/ERK survival pathway.

#### **6.7.** *Yersinia*

*Yersinia* invades epithelial cells, fibroblasts, and M cells in mammalian cells *in vitro* and *in vivo* [69,70]. *Yersinia enterocolitica* and *Yersinia pseudotuberculosis* are transmitted by fecal-oral route and cause gastrointestinal syndromes lymphadenitis and septicemia. *Yersinia pestis* is typically transmitted through the bite of an infected flea and causes bubonic and/or septicemic plague. Yersinia virulence plasmid encodes T3SS and 6 known effector proteins termed Yersinia outer proteins (Yops) [71]. The translocation of the effector molecule YopJ (*Y. pseudotuberculosis*), YopP (*Y. enterocolitica*), or YopJKIM (*Y. pestis*) into the cells has been shown to rapidly activate apoptosis in macrophages and dendritic cells but not in human epithelial cells [72,73]. Yersinia YopJ/P represses the activation of ERK and the NFКB. More recent studies indicate that YopJ has acetyltransferase activity, acetylating Ser and Thr residues critical for the activation of IKK-B and ERK kinases in *Yersinia*-infected macrophages [74,75]. Also, the TLR4 has been shown to act as a potent inducer of apoptosis in macrophage [76]. YopJKIM has two amino acid changes that give it an enhanced ability to inhibit survival signals in macro‐ phages. The increased apoptosis may cause membrane permeability resulting in the efflux of K+ and activation of caspase 1. It was suggested that caspase 1 activation is a normal outcome of a type of apoptosis that is triggered in naive macrophages by TLR4 signaling combination with pathogen interference with ERK and NF-κB pathways [77]. YopJ and/or YopP did not induce pronounced apoptosis in human PMLs. It was suggested that Yersinia inhibition of PMLS ROS production plays a role in evasion of the human innate immune response in part by limiting PMLs apoptosis [78].

### **6.8.** *Legionella*

*Legionella pneumophila* invades and replicates within alveolar macrophages, monocytes, and possibly alveolar epithelial cells and causes Legionnaires disease. The expression and/or export of apoptosis-inducing factor(s) in *L. pneumophila* is regulated by the Dot/Icm type IVlike secretion system [79,80].

*L. pneumophila* utilizes several strategies to ensure intracellular replication and protect itself against the host immune system. These are the following: (1) upon entry into a human phagocyte, *L. pneumophila* becomes contained in a vacuole called *Legionella*-containing phagosome that avoids the typical fusion with the lysosome. *L. pneumophila* promotes the cleavage of Rabaptin-5 by caspase 3, thus preventing the default phagosome-lysosome fusion. (2) *L. pneumophila* promotes the cleavage of Rabaptin-5 by caspase 3, thus preventing the default phagosome-lysosome fusion. (3) *L. pneumophila* does not activate caspases 1 and 7 in human monocytes consequently aborting the phagosome-lysosome fusion. (4) *L. pneumophi‐ la* inhibits host cell apoptosis by upregulating antiapoptotic genes. (5) *L. pneumophila* controls the local balance of activating cytokines (IFN-γ and/or TNF-α) that inhibit its replication and inhibiting cytokines (IL-10) that allow its survival. (6) *L. pneumophila* activates the NF-κB pathway to maintain host cell survival and/or 7-*L. pneumophila* modulates other innate immune responses to establish a replicative niche [81].

### **6.9.** *Escherichia coli*

*E. coli* K1 is a leading causative agent of neonatal meningitis. OmpA of *E. coli* can directly interact with monocytes and macrophages for entry. Some *E. coli* serotypes produce a Shigalike toxin that can bind to human intestinal epithelium and produce Shiga-like toxins, which are associated with hemorrhagic colitis and the hemolytic-uremic syndrome [82]. One pathway involving apoptosis is mediated by death receptors such as CD95 and TNF-R. Intrinsic apoptotic pathway is involved in the Shiga-like toxin-mediated apoptosis of epithelial cells [83]. *E. coli K1* induces the expression of the antiapoptotic protein BclXL for its own survival and that of host macrophages. In addition, the bacteria may also block the activation of caspases. Besides upregulating BclXL, OmpA+ *E. coli* interaction with macrophages may alter the signaling pathways of the host cell to use it as a protected reservoir for the time required to reach septic levels [84,85].

#### **6.10.** *Rickettsia*

*Rickettsia* is a genus of nonmotile, Gram-negative, non-spore-forming, and/or highly pleomor‐ phic bacteria. *Rickettsia rickettsii* is a unicellular Gram-negative coccobacillus. *R. rickettsii is* most commonly known as the causative agent of Rocky Mountain spotted fever [86]. *R. rickettsii* prevents apoptosis by the activation of cell survival pathways. *R. rickettsii* induces NF-κB activity and the upregulation of prosurvival proteins, the downregulation of proapoptotic proteins, and a lack of cytochrome *c* release in endothelial cells. Bechelli *et al.* [87] performed a screening of pro- and antiapoptotic genes that were differentially expressed in human microvascular endothelial cells during *R. rickettsii* infection and after staurosporine challenge. A total of 14 genes were significantly upregulated of which 8 (TRAF1, BNIP2, BCL2L1, TRAF3, BIRC2, BNIP3L, AKT1, and BIRC5) are known apoptosis suppressors while 6 are known to promote apoptosis (BOK, BCL2L13, DAPK2, TP53, ABL1, and BAK1). However, *R. rickettsii* efficiently infects neuronal cells and that the infection causes apoptotic death of neuron [88].

### **6.11.** *Mycobacterium*

**6.8.** *Legionella*

like secretion system [79,80].

244 Cell Death - Autophagy, Apoptosis and Necrosis

**6.9.** *Escherichia coli*

to reach septic levels [84,85].

**6.10.** *Rickettsia*

responses to establish a replicative niche [81].

*Legionella pneumophila* invades and replicates within alveolar macrophages, monocytes, and possibly alveolar epithelial cells and causes Legionnaires disease. The expression and/or export of apoptosis-inducing factor(s) in *L. pneumophila* is regulated by the Dot/Icm type IV-

*L. pneumophila* utilizes several strategies to ensure intracellular replication and protect itself against the host immune system. These are the following: (1) upon entry into a human phagocyte, *L. pneumophila* becomes contained in a vacuole called *Legionella*-containing phagosome that avoids the typical fusion with the lysosome. *L. pneumophila* promotes the cleavage of Rabaptin-5 by caspase 3, thus preventing the default phagosome-lysosome fusion. (2) *L. pneumophila* promotes the cleavage of Rabaptin-5 by caspase 3, thus preventing the default phagosome-lysosome fusion. (3) *L. pneumophila* does not activate caspases 1 and 7 in human monocytes consequently aborting the phagosome-lysosome fusion. (4) *L. pneumophi‐ la* inhibits host cell apoptosis by upregulating antiapoptotic genes. (5) *L. pneumophila* controls the local balance of activating cytokines (IFN-γ and/or TNF-α) that inhibit its replication and inhibiting cytokines (IL-10) that allow its survival. (6) *L. pneumophila* activates the NF-κB pathway to maintain host cell survival and/or 7-*L. pneumophila* modulates other innate immune

*E. coli* K1 is a leading causative agent of neonatal meningitis. OmpA of *E. coli* can directly interact with monocytes and macrophages for entry. Some *E. coli* serotypes produce a Shigalike toxin that can bind to human intestinal epithelium and produce Shiga-like toxins, which are associated with hemorrhagic colitis and the hemolytic-uremic syndrome [82]. One pathway involving apoptosis is mediated by death receptors such as CD95 and TNF-R. Intrinsic apoptotic pathway is involved in the Shiga-like toxin-mediated apoptosis of epithelial cells [83]. *E. coli K1* induces the expression of the antiapoptotic protein BclXL for its own survival and that of host macrophages. In addition, the bacteria may also block the activation of caspases. Besides upregulating BclXL, OmpA+ *E. coli* interaction with macrophages may alter the signaling pathways of the host cell to use it as a protected reservoir for the time required

*Rickettsia* is a genus of nonmotile, Gram-negative, non-spore-forming, and/or highly pleomor‐ phic bacteria. *Rickettsia rickettsii* is a unicellular Gram-negative coccobacillus. *R. rickettsii is* most commonly known as the causative agent of Rocky Mountain spotted fever [86]. *R. rickettsii* prevents apoptosis by the activation of cell survival pathways. *R. rickettsii* induces NF-κB activity and the upregulation of prosurvival proteins, the downregulation of proapoptotic proteins, and a lack of cytochrome *c* release in endothelial cells. Bechelli *et al.* [87] performed a screening of pro- and antiapoptotic genes that were differentially expressed in human microvascular endothelial cells during *R. rickettsii* infection and after staurosporine challenge. *Mycobacterium tuberculosis* is the causative agent of most cases of tuberculosis. *M. tuberculo‐ sis* infected macrophages die by (a) necrosis, a death modality defined by cell lysis, or (b) apoptosis, a form of death that maintains an intact plasma membrane. Necrosis is a mechanism used by bacteria to exit the macrophage, evade host defenses, and spread. *M. tuberculosis* is complex and includes both the induction of cell-death and cell-survival signals. It induces apoptosis in macrophages *in vitro* and *in vivo* [89]. Apoptosis occurs by TNF pathway. In addition, MOMP leading to the activation of the intrinsic apoptotic pathway is required. Both pathways lead to caspase 3 activation which then results in apoptosis [90]. Bacteria cell wall components connect TLR-2 molecules. *M. tuberculosis* also protects cells against apoptosis via two key pathways: first, through the induction of TLR-2 dependent activation of the NF-κB cell survival pathway, and second, by enhancing the production of soluble TNFR-2 (sTNFR2), which neutralizes the proapoptotic activity of TNFα. *M. tuberculosis* can prevent apoptosis in alveolar epithelial cells. Danelishvili *et al.* [91] showed that *M. tuberculosis* infection of macrophages results in the downregulation of the Bcl-2 gene and the upregulation of Bax and Bad proapoptotic genes. In contrast, the increased expression of Bcl-2 and the inhibition of Bax and Bad genes were observed in alveolar epithelial cells. *M. tuberculosis* infection was associated with the repression of the Bcl-2 gene and the induction of p53 in human macrophages. In alveolar epithelial cells, the expression of p53 was unchanged during *M. tuberculosis* infection [92].

### **7. Viral infections**

After infecting target cells, viruses replicate to produce large number of progeny virions and spread the progeny to initiate the next round of infection. Some viruses encode specific proteins to optimize their replication. Infection by viruses, however, triggers the apoptosis of the infected cell to restrict virus infection. This is done by reprogramming of the host cell apoptotic pathway to effect death of the infected host cell before the release of progeny viruses. In order to ablate host defense mechanisms, viruses have evolved proteins that are able to inhibit or delay the host protective actions by targeting strategic points in the apoptotic pathways [93]. Apoptosis can be induced by intrinsic or extrinsic signals (Table 1). Intrinsic signals may result from viral infection and include stress, cell cycle arrest, cytoplasmic calcium perturbation, and DNA damage. Extrinsic signals arise as a result of the host immune response through TNFreceptor or Fas activation or via delivery of proteases by cytotoxic lymphocytes. Once induced, apoptosis may eliminate infected cells prior to release of viral progeny [94, 95]. Many viruses have been shown to induce apoptosis, either as a mechanism for the release and dissemination of progeny virions or as a defense strategy of multicellular host organisms for the destruction of infected cells and therefore preventing the spread of the virus [96]. Innate and the acquired immune system induce apoptosis as a host defense against viral infections. The innate immune system directly activates inflammatory cells such as macrophages (e.g., granulocytes, Kupffer cells in the liver) and natural killer (NK) cells, which may directly cause death of the infected cells. On the other hand, viral RNA or proteins can bind to intracellular molecules that modulate or directly induce cell death [97]. In this immune cell-independent, virus-induced apoptosis of the host cell protein kinase R (PKR) and the cytoplasmic RNA helicase RIG-I play important roles [98]. PKR acts via the downstream transcription factor eIF-2α [99]. At the same time, acquired immune system works to eliminate the virus and the recognition of viral antigens presented by specific cells (e.g., dendritic cells). The antigen-primed CD8+ -T-lym‐ phocytes cytotoxic T lymphocytes directly kill infected cells via direct cell-cell contact and release of cytotoxic and/or antiviral cytokines (e.g., interferons IFNs and/or TNF-α), whereas IFN-γ and IFN-α are also able to eliminate the virus without killing the host cell [100].

The IFNs are considered to play a critical role in innate immunity to viral infection and aside from effectively preventing intracellular viral replication can also mediate the activation and recruitment of the adaptive immune response. The IFNs can be induced by a number of stimuli, including viruses and dsRNA through mechanisms involving the activation of interferon regulatory factor (IRF-3), NF-κB, and perhaps the dsRNA-dependent PKR and the JNK2 pathway, all of which have been reported to be mediators of cell death [101,102].

### **7.1. Hepatitis C virus**

Hepatitis C virus (HCV) causes liver cirrhosis and hepatocellular carcinoma [103]. In hepato‐ cytes, apoptosis induction via cytotoxic T lymphocytes and macrophages largely occurs via extrinsic pathway. Ligand binding activates caspase 8 signaling cascade [104]. Another mechanism of apoptosis involves viral protein and their interactions. HCV core protein has been shown to be proapoptotic and antiapoptotic effects [105]. Machida *et al*. [106] showed that the expression of HCV proteins may directly or indirectly inhibit Fas-mediated apoptosis and death in mice by repressing the release of cytochrome *c* from mitochondria.

#### **7.2. HIV**

Human immunodeficiency virus type 1 (HIV-1)-infected individuals often suffer from neurological complications such as memory loss, mental slowing, and gait disturbance [107]. HIV infection is associated with a progressive decrease in and/or loss of CD4<sup>+</sup> and the decline of CD8+ T cells and viral replication [108]. Inappropriate signaling through the binding of the HIV-1 envelope to the CD4 may induce abnormal programmed CD4<sup>+</sup> T-cell death. Viral proteins such as HIV-1 gp120 have an important role development in HIV-associated apop‐ tosis. HIV proteins implicated in the induction of apoptosis *in vitro* include tat, nef, vpr, and protease. Cross-linking of bound gp120 on human CD4<sup>+</sup> T cells followed by signaling through the T-cell receptor for antigen was found to increase susceptibility to Fas and result in apoptosis [109,110]. In addition, deregulation in cytokine production occurs during HIV infection, perturbing the immune response. The overproduction of IL-4 and/or IL-10 cytokines is known to increase susceptibility to activation-induced cell death. IFN-α produced by HIV-1-infected dendritic cells contributes to CD4 T-cell apoptosis by the TRAIL/DR5 pathway [111]. HIV-1 protein Env triggers apoptosis by the transactivation of the p53-dependent genes Puma and Bax [112]. HIV Nef is able to induce apoptosis by extrinsic pathway [113,114].

### **7.3. Rabies virus**


of infected cells and therefore preventing the spread of the virus [96]. Innate and the acquired immune system induce apoptosis as a host defense against viral infections. The innate immune system directly activates inflammatory cells such as macrophages (e.g., granulocytes, Kupffer cells in the liver) and natural killer (NK) cells, which may directly cause death of the infected cells. On the other hand, viral RNA or proteins can bind to intracellular molecules that modulate or directly induce cell death [97]. In this immune cell-independent, virus-induced apoptosis of the host cell protein kinase R (PKR) and the cytoplasmic RNA helicase RIG-I play important roles [98]. PKR acts via the downstream transcription factor eIF-2α [99]. At the same time, acquired immune system works to eliminate the virus and the recognition of viral

antigens presented by specific cells (e.g., dendritic cells). The antigen-primed CD8+

pathway, all of which have been reported to be mediators of cell death [101,102].

and death in mice by repressing the release of cytochrome *c* from mitochondria.

protease. Cross-linking of bound gp120 on human CD4<sup>+</sup>

**7.1. Hepatitis C virus**

246 Cell Death - Autophagy, Apoptosis and Necrosis

**7.2. HIV**

of CD8+

phocytes cytotoxic T lymphocytes directly kill infected cells via direct cell-cell contact and release of cytotoxic and/or antiviral cytokines (e.g., interferons IFNs and/or TNF-α), whereas IFN-γ and IFN-α are also able to eliminate the virus without killing the host cell [100].

The IFNs are considered to play a critical role in innate immunity to viral infection and aside from effectively preventing intracellular viral replication can also mediate the activation and recruitment of the adaptive immune response. The IFNs can be induced by a number of stimuli, including viruses and dsRNA through mechanisms involving the activation of interferon regulatory factor (IRF-3), NF-κB, and perhaps the dsRNA-dependent PKR and the JNK2

Hepatitis C virus (HCV) causes liver cirrhosis and hepatocellular carcinoma [103]. In hepato‐ cytes, apoptosis induction via cytotoxic T lymphocytes and macrophages largely occurs via extrinsic pathway. Ligand binding activates caspase 8 signaling cascade [104]. Another mechanism of apoptosis involves viral protein and their interactions. HCV core protein has been shown to be proapoptotic and antiapoptotic effects [105]. Machida *et al*. [106] showed that the expression of HCV proteins may directly or indirectly inhibit Fas-mediated apoptosis

Human immunodeficiency virus type 1 (HIV-1)-infected individuals often suffer from neurological complications such as memory loss, mental slowing, and gait disturbance [107]. HIV infection is associated with a progressive decrease in and/or loss of CD4<sup>+</sup> and the decline

HIV-1 envelope to the CD4 may induce abnormal programmed CD4<sup>+</sup> T-cell death. Viral proteins such as HIV-1 gp120 have an important role development in HIV-associated apop‐ tosis. HIV proteins implicated in the induction of apoptosis *in vitro* include tat, nef, vpr, and

the T-cell receptor for antigen was found to increase susceptibility to Fas and result in apoptosis [109,110]. In addition, deregulation in cytokine production occurs during HIV infection, perturbing the immune response. The overproduction of IL-4 and/or IL-10 cytokines is known to increase susceptibility to activation-induced cell death. IFN-α produced by HIV-1-infected

T cells and viral replication [108]. Inappropriate signaling through the binding of the

T cells followed by signaling through

Rabies virus (RV) is a neurotropic virus and travels to the brain by following the peripheral nerves. RV, a member of the genus *Lyssavirus* of the family Rhabdoviridae, is known to cause fatal encephalomyelitis in many mammalian species. RV has developed two main mechanisms to escape the host defenses: (1) its ability to kill protective migrating T cells and (2) its ability to sneak into the nervous system without triggering the apoptosis of the infected neurons and preserving the integrity of neurites [115]. In one of the studies, Ubol *et al.* [116] showed the expression of Bax and caspase 1 activation in RV-infected neuroblastoma cells. In another study, the expression of caspase gene Nedd-2 was significantly upregulated in infected adult and suckling mice [117]. Thoulouze *et al.* [118] showed that apoptosis induced by rabies virus involves the activation of caspase 8 and disappearance of procaspases 9 and 3. In addition, AIF translocated from the cytoplasm to the nucleus, suggesting that caspase-independent pathway is also involved in RV-induced apoptosis. Sarmento *et al.* [119] showed that AIF, a caspaseindependent apoptotic protein, was upregulated and translocated from the cytoplasm to the nucleus postinfection, suggesting that apoptosis induced by RV induces apoptosis by both the caspase-dependent and caspase-independent pathways.

#### **7.4. Epstein-Barr virus**

Infection with Epstein-Barr virus (EBV) is very common and usually occurs in childhood or early adulthood. In fact, up to 95% of people in the U.S. have been infected with EBV. EBV is the cause of infectious mononucleosis (also termed "mono"), an illness associated with fever, sore throat, swollen lymph nodes in the neck, and sometimes enlarged spleen. Less commonly, EBV can cause more serious disease. To establish a persistent latent infection, EBV must access the memory B-cell compartment and reside within long-lived peripheral B cells where few viral gene products are expressed in order to escape immune detection [120]. EBV encodes two viral Bcl-2 proteins, BHRF1 and BALF1, with apparently redundant functions. The viral BHRF1 gene expresses a Bcl-2 homologue protein that resembles Bcl-2 in its subcellular localization and capacity to enhance B-cell survival [121]. *In vitro*, EBV infects resting human B lymphocytes and transforms them into lymphoblastoid cell lines (LCLs). In LCLs, 11 so-called latent genes are consistently expressed. These are the EBV nuclear antigens EBNA1, EBNA2, EBNA-LP, EBNA3A, B, and/or C and the latent membrane proteins LMP1, LMP2A, and B and two noncoding RNAs [122]. LMP1 indirectly inhibits apoptosis by upregulating several cellular antiapoptotic genes presumably through the induction of the NF-κB pathway [123].

### **7.5. Baculovirus**

Baculovirus antiapoptotic genes include p35, which encodes the most broadly acting caspase inhibitor protein known and IAP genes [124]. The baculovirus IAP blocks apoptosis induced by caspase activation. All viral IAPs (vIAPs) contain a carboxyl ring finger and a variable number of highly conserved Cys⁄His motifs known as baculoviral IAP repeats (BIRs). The BIR domains bind directly to caspases and inhibit their proteolytic activity and molecules that contain an IAP-binding motif (second mitochondrial-derived activator of caspases and Omi) antagonize IAP function via binding to BIR motifs displacing IAP binding to caspases or by promoting their degradation. Therefore, vIAPs act downstream of mitochondria, inhibiting the activity of procaspase 9 and effector caspases 3 and 7 [125]. The antiapoptotic protein p35 from baculovirus is thought to prevent the suicidal response of infected insect cells by inhibiting caspases [126]. Zhou *et al.* [127] showed that purified recombinant p35 inhibits human caspases 1, 3, 6, 7, 8, and 10. There may be interaction of the baculovirus antiapoptotic protein p35 with caspases. Sah *et al.* [128] demonstrated the ability of the p35 gene to inhibit oxidative stress-induced apoptosis. Oxidative damage to cellular macromolecules such as nuclear and mitochondrial DNA and proteins caused by reactive oxygen species is considered to be of key importance in the aging process. The chain of oxidative reactions initiated by ROS eventually knocks down the crucial biomolecules, thereby driving the cellular machinery to undergo apoptosis via the activation of caspases, which ultimately brings about the execution of cell death. p35 is able to directly mop out free radicals and prevent cell death by also acting in an oxidant-dependent pathway at a very upstream step in the cascade of events associated with oxidative stress-induced apoptosis [129].

### **7.6. Human papillomavirus (HPV)**

HPVs are small DNA viruses that are known to be the most common etiological agents in cervical cancer. HPVs are implicated in the mucosal and epithelial infections that may range from a benign lesion to a malignant carcinoma [130]. Recent studies have shown that 13 different types of HPV are associated with carcinogenesis. HPVs are DNA tumor viruses whose genome is organized in three regions: the early gene (E1 to E7), the late gene (L1 and L2) regions, and the upper regulatory region (URR). The late region units, L1 and L2, encode for viral capsid proteins during the late stages of virion assembly. E1 and E2 encode proteins that are vital for extrachromosomal DNA replication and the completion of the viral life cycle. The E4 protein plays an important role for the maturation and replication of the virus. The E5 in open reading frame (ORF) interacts with various transmembrane proteins like the receptors of the epidermal growth factor, platelet-derived growth factor β, and colony stimulating factor-1. E6 and E7 ORF encode for oncoproteins that allow replication of the virus and the immortalization and transformation of the cell that hosts the HPV DNA [131]. The HPV E2 regulates the transcription of E6/E7 and facilitates apoptosis via p53-dependent pathway in HeLa cells [132]. HPV E7 is involved with cell cycling and binds to the retinoblastoma tumor suppressor protein and related proteins and induces apoptosis in mouse lens [133]. The inactivation of p53 by E6 should lead to a reduction in cellular apoptosis. Numerous studies showed that E6 could in fact sensitize cells to apoptosis. HPV E6 induce the degradation of p53. E6 expression correlated with the prolonged expression of Bcl-2 reduces the elevation of Bax and loss of p53. Several studies have shown that E2 could also induce apoptosis inde‐ pendent of its effects on transcription of E6 and E7 [134-136]. Tan *et al.*[137] showed that HPV16 E6 RNA interference enhances cisplatin and death receptor-mediated apoptosis in human cervical carcinoma cells. Moreover, HPV-16 E6 was shown to bind TNF R1 and protect cells from TNF-induced apoptosis in mouse fibroblasts and human histiocyte/monocyte and osteosarcoma cells. Caspase 3 and caspase 8 activation were significantly reduced in E6 expressing cells [138].

### **7.7. Adenovirus**

number of highly conserved Cys⁄His motifs known as baculoviral IAP repeats (BIRs). The BIR domains bind directly to caspases and inhibit their proteolytic activity and molecules that contain an IAP-binding motif (second mitochondrial-derived activator of caspases and Omi) antagonize IAP function via binding to BIR motifs displacing IAP binding to caspases or by promoting their degradation. Therefore, vIAPs act downstream of mitochondria, inhibiting the activity of procaspase 9 and effector caspases 3 and 7 [125]. The antiapoptotic protein p35 from baculovirus is thought to prevent the suicidal response of infected insect cells by inhibiting caspases [126]. Zhou *et al.* [127] showed that purified recombinant p35 inhibits human caspases 1, 3, 6, 7, 8, and 10. There may be interaction of the baculovirus antiapoptotic protein p35 with caspases. Sah *et al.* [128] demonstrated the ability of the p35 gene to inhibit oxidative stress-induced apoptosis. Oxidative damage to cellular macromolecules such as nuclear and mitochondrial DNA and proteins caused by reactive oxygen species is considered to be of key importance in the aging process. The chain of oxidative reactions initiated by ROS eventually knocks down the crucial biomolecules, thereby driving the cellular machinery to undergo apoptosis via the activation of caspases, which ultimately brings about the execution of cell death. p35 is able to directly mop out free radicals and prevent cell death by also acting in an oxidant-dependent pathway at a very upstream step in the cascade of events associated

HPVs are small DNA viruses that are known to be the most common etiological agents in cervical cancer. HPVs are implicated in the mucosal and epithelial infections that may range from a benign lesion to a malignant carcinoma [130]. Recent studies have shown that 13 different types of HPV are associated with carcinogenesis. HPVs are DNA tumor viruses whose genome is organized in three regions: the early gene (E1 to E7), the late gene (L1 and L2) regions, and the upper regulatory region (URR). The late region units, L1 and L2, encode for viral capsid proteins during the late stages of virion assembly. E1 and E2 encode proteins that are vital for extrachromosomal DNA replication and the completion of the viral life cycle. The E4 protein plays an important role for the maturation and replication of the virus. The E5 in open reading frame (ORF) interacts with various transmembrane proteins like the receptors of the epidermal growth factor, platelet-derived growth factor β, and colony stimulating factor-1. E6 and E7 ORF encode for oncoproteins that allow replication of the virus and the immortalization and transformation of the cell that hosts the HPV DNA [131]. The HPV E2 regulates the transcription of E6/E7 and facilitates apoptosis via p53-dependent pathway in HeLa cells [132]. HPV E7 is involved with cell cycling and binds to the retinoblastoma tumor suppressor protein and related proteins and induces apoptosis in mouse lens [133]. The inactivation of p53 by E6 should lead to a reduction in cellular apoptosis. Numerous studies showed that E6 could in fact sensitize cells to apoptosis. HPV E6 induce the degradation of p53. E6 expression correlated with the prolonged expression of Bcl-2 reduces the elevation of Bax and loss of p53. Several studies have shown that E2 could also induce apoptosis inde‐ pendent of its effects on transcription of E6 and E7 [134-136]. Tan *et al.*[137] showed that HPV16 E6 RNA interference enhances cisplatin and death receptor-mediated apoptosis in human cervical carcinoma cells. Moreover, HPV-16 E6 was shown to bind TNF R1 and protect cells

with oxidative stress-induced apoptosis [129].

**7.6. Human papillomavirus (HPV)**

248 Cell Death - Autophagy, Apoptosis and Necrosis

Adenoviruses (Ads) were first described as the etiological agents isolated from human adenoids and respiratory secretions that cause spontaneous cytopathic effects in cultures of human cells. Adenovirus has evolved ways to commandeer host cell machinery for successful entry, viral DNA replication, and propagation of progeny virions. Adenoviral proteins interact with host-cell proteins to either exploit or inhibit cellular functions for the purpose of viral propagation. The Ad genome is a 36 kbp linear double-stranded DNA molecule that encodes five early transcription units (E1A, E1B, E2, E3, and E4), two delayed early units, and one major late unit that is processed to make five families of late mRNAs. The early genes are transcribed before viral DNA replication begins, and the late proteins are made following the onset of replication [139]. At least 51 serotypes have been distinguished based on resistance to neu‐ tralization by antibodies specific to other known serotypes. These are divided into six sub‐ groups (A-F) based on hemagglutination patterns, oncogenicity, and genome homologies. The common subgroup C Ads, which include Ad serotypes 1, 2, 5, and 6, are endemic virtually all over the world. They cause mild upper respiratory tract infections in young children [140]. Early in infection, the expression of E1A drives the host cell into the S phase of the cell cycle in order to induce DNA synthesis that is required for viral replication. The genes in the E3 region of Ad encode several proteins that function to protect the virus-infected cell from host immune responses [141]. Table 1 describes the adenovirus immunoregulatory proteins and how they function to block or induce apoptosis of infected cells [142-150].

#### **7.8. Human cytomegalovirus**

Human cytomegalovirus (CMV), a beta herpes virus with a widespread distribution, is a major cause of morbidity and mortality in immunocompromised individuals such as organ trans‐ plant recipients and patients with AIDS. During pregnancy, CMV is a major cause of congenital disease [151]. CMV genes UL36 and UL37 encode viral inhibitor of caspase-8-induced apoptosis (vICA) and viral mitochondria inhibitor of apoptosis (vMIA), respectively. Skalet‐ skaya *et al.* [152] identified a human cytomegalovirus cell-death suppressor denoted vICA and encoded by the viral UL36 gene. vICA inhibits Fas-mediated apoptosis by binding to the prodomain of caspase 8 and preventing its activation. vMIA blocks cytochrome *c* release and activation of downstream effector caspases in a manner analogous to Bcl-2 homologues. Like Bcl-2, vMIA localizes to mitochondria and inhibits mitochondrial permeabilization induced by apoptotic signals [153]. vMIA also counteracts serine protease HtrA2⁄Omi (high tempera‐ ture requirement protein A2⁄Omi stress-regulated endonuclease)-dependent cell death and allows infected cells to survive and continuously produce a virus for several days [154]. Additional human CMV gene products, including IE1 and IE2, as well as the murine CMV UL45 homologue may influence cell susceptibility to apoptosis [155]. Transient transfection assays indicate that the IE1 and IE2 proteins regulate transcription. The IE1 and IE2 proteins each inhibit the induction of apoptosis by TNF or by the E1B 19-kDa-protein-deficient adenovirus. IE1 and IE2 proteins inhibit apoptosis in part by modulating the activity of p53[156]. In addition, IE1 and IE2 and the viral RNA beta 2.7, which bind to the mitochondrial respiratory complex I, maintain ATP production late in infection and prevent death induced by mitochondrial poison [157].

### **8. Apoptosis and parasitic infections**

Apoptosis plays crucial roles in the interaction between the host and the parasite. This includes innate and adaptive defense mechanisms to restrict intracellular parasite replication as well as regulatory functions to modulate the host's immune response. During their evolution, parasites have developed mechanisms to induce or avoid host cell apoptosis in order to be able to survive and complete their life cycle (Table 1). Among the factors involved in that balance in infected organisms, the time of apoptosis (early or late occurrence), the cell type, and the type of parasitism (intracellular or not) are the major modulators. For example, the early apoptosis of host cells could contribute toward their fight against infection by intracellular parasites; equally, early apoptosis could favor the penetration of the parasite. The late apoptosis of cells of the defense system could be beneficial to the host clearing excess cells, thereby avoiding the detrimental effects of excessive inflammatory response in the tissue that they would cause [158].

#### **8.1.** *Toxoplasma gondii*

*Toxoplasma gondii* is a species of parasitic protozoa in the genus *Toxoplasma*. Humans can become infected with *T. gondii*, either through contact with soil contaminated by cat feces or by eating infected meat. Toxoplasmosis is usually asymptomatic because our immune system keeps the parasite from causing illness. The disease is more problematic for pregnant women and people who have weakened immune systems. Some results indicate a strong correlation between schizophrenia, brain cancer, and toxoplasmosis [159]. Toxoplasma promote or inhibit apoptosis. Begum-Haque *et al.* [160] demonstrated marked difference in the death of activated T cells between early (day 3 post infection) and acute (day 6 post infection) stage of *T. gondii*. The decreased production of IL-2 and augmented synthesis of IL-10 during acute stage of *T. gondii* infection may have a role in the enhanced level of apoptosis. It has been suggested that the apoptosis of T lymphocytes in *T. gondii* infection is associated with the virulence and density of the parasite in the host. In *T. gondii* infection, IFN-γ locally produced in Peyer's patches contributes to the induction of apoptosis in Peyer's patch T cells [160]. *T. gondii* inhibits the apoptosis of host cells by indirect and direct mechanisms. Granulocyte colony-stimulating factor and granulocyte-macrophage cerebrospinal fluid secreted by *T. gondii*-infected human fibroblasts increased the expression of antiapoptotic Bcl-2 family member Mcl-1 and abolished apoptosis in neutrophils *in vitro* indirectly [161]. Many studies have shown that *T. gondii* has evolved strategies to directly inhibit cell apoptosis by various mechanisms: (a) increased expression of antiapoptotic members of the Bcl-2 protein family; (b) inhibition of the cyto‐ chrome *c* release; (c) upregulation of IAPs; (d) activation of NF-κB by *T. gondii* in distinct cell types or under distinct conditions thereby inducing the transcription of genes encoding antiapoptotic molecules, including Bfl-1 and IAPs; and/or (e) degradation of the PARP as described is involved in the inhibition of apoptosis. Although direct evidence is still lacking, it appears plausible that diminished PARP levels in *Toxoplasma*-infected cells may inhibit apoptosis in a caspase-independent fashion [162].

### **8.2.** *Plasmodium falciparum*

each inhibit the induction of apoptosis by TNF or by the E1B 19-kDa-protein-deficient adenovirus. IE1 and IE2 proteins inhibit apoptosis in part by modulating the activity of p53[156]. In addition, IE1 and IE2 and the viral RNA beta 2.7, which bind to the mitochondrial respiratory complex I, maintain ATP production late in infection and prevent death induced

Apoptosis plays crucial roles in the interaction between the host and the parasite. This includes innate and adaptive defense mechanisms to restrict intracellular parasite replication as well as regulatory functions to modulate the host's immune response. During their evolution, parasites have developed mechanisms to induce or avoid host cell apoptosis in order to be able to survive and complete their life cycle (Table 1). Among the factors involved in that balance in infected organisms, the time of apoptosis (early or late occurrence), the cell type, and the type of parasitism (intracellular or not) are the major modulators. For example, the early apoptosis of host cells could contribute toward their fight against infection by intracellular parasites; equally, early apoptosis could favor the penetration of the parasite. The late apoptosis of cells of the defense system could be beneficial to the host clearing excess cells, thereby avoiding the detrimental effects of excessive inflammatory response in the tissue that

*Toxoplasma gondii* is a species of parasitic protozoa in the genus *Toxoplasma*. Humans can become infected with *T. gondii*, either through contact with soil contaminated by cat feces or by eating infected meat. Toxoplasmosis is usually asymptomatic because our immune system keeps the parasite from causing illness. The disease is more problematic for pregnant women and people who have weakened immune systems. Some results indicate a strong correlation between schizophrenia, brain cancer, and toxoplasmosis [159]. Toxoplasma promote or inhibit apoptosis. Begum-Haque *et al.* [160] demonstrated marked difference in the death of activated T cells between early (day 3 post infection) and acute (day 6 post infection) stage of *T. gondii*. The decreased production of IL-2 and augmented synthesis of IL-10 during acute stage of *T. gondii* infection may have a role in the enhanced level of apoptosis. It has been suggested that the apoptosis of T lymphocytes in *T. gondii* infection is associated with the virulence and density of the parasite in the host. In *T. gondii* infection, IFN-γ locally produced in Peyer's patches contributes to the induction of apoptosis in Peyer's patch T cells [160]. *T. gondii* inhibits the apoptosis of host cells by indirect and direct mechanisms. Granulocyte colony-stimulating factor and granulocyte-macrophage cerebrospinal fluid secreted by *T. gondii*-infected human fibroblasts increased the expression of antiapoptotic Bcl-2 family member Mcl-1 and abolished apoptosis in neutrophils *in vitro* indirectly [161]. Many studies have shown that *T. gondii* has evolved strategies to directly inhibit cell apoptosis by various mechanisms: (a) increased expression of antiapoptotic members of the Bcl-2 protein family; (b) inhibition of the cyto‐ chrome *c* release; (c) upregulation of IAPs; (d) activation of NF-κB by *T. gondii* in distinct cell

by mitochondrial poison [157].

250 Cell Death - Autophagy, Apoptosis and Necrosis

they would cause [158].

**8.1.** *Toxoplasma gondii*

**8. Apoptosis and parasitic infections**

*Plasmodium falciparum* is the agent of malaria. Enhanced levels of RBC apoptosis have been observed in clinical disorders in which anemia is a common feature such as iron and renal insufficiency, thalassemia, sickle-cell disease, and apoptosis has been associated to cerebral malaria, thrombocytopenia, and lymphocytopenia in malaria infection [163].

*P. falciparum* induces oxidative stress, which in turn activates the Ca+2 permeable cation channels followed by Ca+2 entry, and the stimulation of eryptosis has been coined to describe the suicidal erythrocyte death. The Ca+2 uptake, however, eventually triggers eryptosis of the parasitized erythrocyte, and thus the parasitized erythrocytes is doomed to be phagocytosed by macrophages [164].

*P. falciparum* firstly enter red blood cells. Second, parasitized red blood cell sticks endothelial cells, inducing the expression of iNOS in brain cells. The activation of caspases 8 and 9 results in apoptosis and blood-brain barrier disruption [165].

### **8.3. Trypanosomatids**

Trypanosomatids are the causative agents of diseases such as the Chagas disease and the African sleeping sickness [166]. Trypanosomatids lack some of the key molecules contributing to apoptosis in metazoans like caspase genes, Bcl-2 family genes, and the TNF-related family of receptors. Apoptosis triggered in response to heat shock, prostaglandins, antibodies, and mutations in cell cycle regulates genes [167]. These stimuli result in loss of ΔΨm, generation of ROS, lipid peroxidation, and increase in cytosolic Ca2+. This also potentiates the release of cytochrome *c* and EndoG into the cytoplasm and the activation of proteases and nucleases to dismantle the parasites in an ordered fashion. Upon release from the mitochondrion, EndoG translocates to the nucleus to degrade DNA. These events finally lead to the execution of apoptosis [168]. *Trypanosoma brucei* causes neuronal demyelination and apoptosis after bloodbrain barrier damage. This leads to apoptosis in cells of the cerebellum and brain stem. Welburn *et al.* [170] described cytoplasmic vacuolization and marginalization, extensive membrane blebbing, and condensation of nuclear chromatin in *Trypanosoma cruzi* and *T. brucei* respectively. Lectins such as ConA were among the first compounds shown to induce the expression of apoptotic markers in *T. brucei* [169]. *T. cruzi* is the etiological agent of Chagas disease. It also inhibits apoptosis through the action of parasite-derived neurotrophic factor, a parasite-derived protein in neuronal and glial cells. The parasite-derived neurotrophic factor is both a substrate and an activator of the serine-threonine kinase Akt and an antiapoptotic molecule binding to the neurotrophic surface receptor TrkA (neurotrophic tyrosine kinase receptor type 1) triggering the PI3-K/PKB pathway resulting in increased Bcl-2 expression. This results in protection of Schwann cells from apoptosis induced by H2O2 and TNF-α/TGFβ (transforming growth factor b) [170-172].

### **8.4.** *Leishmania*

Leishmanias are agents of ulcerative skin lesions (cutaneous leishmaniasis) and disseminated visceral infection (visceral leishmaniasis or kala-azar). Leishmania is able to inhibit the spontaneous apoptosis of short-lived neutrophils, increasing their life span and providing a safe place for the parasites during the first days of the infection [173]. With most apoptosis inducing stimuli, *Leishmania donovani* shows typical features of apoptotic death like cell shrinkage, nuclear condensation, and DNA fragmentation. Ca2+ appears to be a vital ion involved in Leishmania apoptosis. Extracellular or intracellular Ca2+ during oxidative stress results in the significant rescue of the fall of the mitochondrial membrane potential and consequently apoptosis [174].

### **9. Concluding remarks**


### **10. Abbreviations**

**AIF:** apoptosis inducing factor

**AP-1:** activator protein 1

**BH:** Bcl-2 homology

This results in protection of Schwann cells from apoptosis induced by H2O2 and TNF-α/TGF-

Leishmanias are agents of ulcerative skin lesions (cutaneous leishmaniasis) and disseminated visceral infection (visceral leishmaniasis or kala-azar). Leishmania is able to inhibit the spontaneous apoptosis of short-lived neutrophils, increasing their life span and providing a safe place for the parasites during the first days of the infection [173]. With most apoptosis inducing stimuli, *Leishmania donovani* shows typical features of apoptotic death like cell shrinkage, nuclear condensation, and DNA fragmentation. Ca2+ appears to be a vital ion involved in Leishmania apoptosis. Extracellular or intracellular Ca2+ during oxidative stress results in the significant rescue of the fall of the mitochondrial membrane potential and

**•** Apoptosis is a genetically programmed process of cellular destruction that is indispensable

**•** T3SS effectors have also been shown to tamper with the host's cell cycle, and some of them are able to induce apoptosis bacteria such as *Pseudomonas*, *Shigella*, *Salmonella*, and *Yersinia*.

**•** Microorganisms inhibit apoptosis by multiple mechanisms: protection of the mitochondria and prevention of cytochrome *c* release (i.e., *Chylamidia* sp. and/or *Neisseria* sp.), activation of cell survival pathways (i.e., *Salmonella* sp. and/or *Rickettsia* sp.), inhibition of caspases, activation of phosphoinositide 3-kinase (PI3K)-Akt/protein kinase B (PKB) pathway, and

**•** A clear understanding of the molecular basis of apoptosis inhibition or induction is needed.

**•** Elucidation of the mechanisms, the cellular receptors, and/or the microbial factors involved in modulating of apoptosis could reveal insights into the host-pathogen relationship and

**•** Microorganisms induce apoptosis by intrinsic and extrinsic pathway in the host cell.

for the normal development and homeostasis of multicellular organisms.

interaction with cellular caspases (i.e., *Shigella* sp. and/or *Legionella* sp.)

**•** Prevention of apoptosis enables microorganisms to replicate and survive in host.

β (transforming growth factor b) [170-172].

252 Cell Death - Autophagy, Apoptosis and Necrosis

**8.4.** *Leishmania*

consequently apoptosis [174].

**9. Concluding remarks**

new therapeutic targets.

**AIF:** apoptosis inducing factor

**10. Abbreviations**

**AP-1:** activator protein 1

**CARD:** caspase activation and recruitment domain

**CMV:** cytomegalovirus

**CTL:** cytotoxic T lymphocyte

**DD:** death domains

**DED:** death effector domain

**∆Ψm:** transmembrane potential

**EBV:** Epstein-Barr virus

**FADD:** Fas-associated death domain

**GSK-3:** Glycogen synthase kinase 3

**JNK:** c-Jun N-terminal kinase

**HIV-1:** human immunodeficiency virus type 1

**IAPs:** inhibitors of apoptosis proteins

**IL-1β:** interleukin 1β

**ICE:** IL-1β converting enzyme

**I-КB:** inhibitor of КB

**IKK:** I-КB kinase

**IPA:** invasion plasmid antigens

**LlyO:** listeriolysin O

**MOMP:** mitochondrial outer membrane permeabilization

**NF-κB:** nuclear factor-kappa B

**TLR:** toll-like receptors

**TNF:** tumor necrosis factor

**TNFR:** tumor necrosis factor receptor gene

**TRAIL:** tumor necrosis factor (TNF)-related apoptosis-inducing ligand

**T3SS :** type III secretion system

**PI3K:** phosphoinositide 3-kinase

**PKB:** protein Kinase B

**PMLs:** polymorphonuclear leukocytes

**proIL:** prointerleukin

**PT:** permeability transition **ROS:** reactive oxygen species **RV:** Rabies virus **SAPK:** stress-activated protein kinase **STS:** staurosporine **VDAC:** voltage-dependent anionic channel

### **Acknowledgements**

I thank Halic University and Emine Kurt for contributions.

### **Author details**

Yorulmaz Hatice

Address all correspondence to: haticeyorulmaz@halic.edu.tr

Department of Physiology, Medical Faculty, Halic University, Istanbul, Turkey

### **References**


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**PT:** permeability transition **ROS:** reactive oxygen species

254 Cell Death - Autophagy, Apoptosis and Necrosis

**SAPK:** stress-activated protein kinase

**VDAC:** voltage-dependent anionic channel

I thank Halic University and Emine Kurt for contributions.

Address all correspondence to: haticeyorulmaz@halic.edu.tr

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Department of Physiology, Medical Faculty, Halic University, Istanbul, Turkey

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