**2.2 Enfermedad de Chagas-Phagocytosis of** *Trypanosoma cruzi*

Tissue-resident macrophages are the first host cells invaded by *T. cruzi* during in vivo infection. Trypomastigotes and epimastigotes are both readily absorbed by macrophages and detected within phagolysosomes. Only the trypomastigotes may escape the phagolysosome and grow in the cytosol, while the epimastigotes are killed. The plasma membrane of macrophages has been demonstrated to envelop the parasite by producing a tubular structure, also known as a coiled phagosome. Although this mechanism appears to be comparable to phagocytosis, data shows that, unlike noninfectious epimastigotes, trypomastigotes actively strive to route their own infection to macrophages. The escape of trypomastigotes to the cytosol is important because nitric oxide (NO) produced in the parasitophorous vacuole is the most potent agent in activated macrophages [5].

The parasite's primary target organ is the heart. Tissue damage in the heart is associated with severe parasitism of the myocardium during acute illness. To regulate parasite proliferation, monocytes migrate and extravasate from the circulation to the heart, where they develop into macrophages [6].

The surface receptor for sialodhesin can be expressed by macrophages (Sn). This receptor detects sialic acid, which is abundant on the parasite's surface and appears to play a significant role in the adhesion process during *T. cruzi* phagocytosis. TLR2 and TLR9 on the surface of macrophages have also been implicated in the identification of *T. cruzi* antigens: GPI (glycosylphosphatidylinositol) anchors, a dominating glycolipid dispersed on the surface of the *T. cruzi* membrane, and parasite DNA, respectively. Classical activation causes profound metabolic changes in macrophages, such as increased inducible nitric oxide synthase (iNOS or NOS2) activity and respiratory burst, as well as secretory responses, such as the production of proinflammatory cytokines and chemokines that lead to phagocytosis, intracellular pathogen destruction, antigen presentation, and costimulation. During experimental mouse infection, NO released by activated macrophages was thought to be a significant chemical for host defense against the parasite. The infection has also been demonstrated to enhance splenic but not peritoneal macrophage production of hydrogen peroxide (H2O2), indicating that in vivo production of antimicrobial compounds appears to be connected to certain kinds of macrophages and/or the parasite's capacity to activate these cells [6, 7].

*T. cruzi* amastigotes engage in phagocytic processes to invade both professional and non-professional phagocytic cells, depending significantly on the actin cytoskeleton of the host cell [21]. The GTPases of the Rho family of the host cell and their effector proteins were involved in the actin-dependent invasion [22].

#### **2.3** *Leishmania spp*

*Leishmania* promastigotes access macrophages after opsonization mainly through complement receptor 1 (CR1) or 3 (CR3), Other receptors have also been implicated such as the Toll-like receptor (TLR) family, the receptors for the Fc domain of immunoglobulins (FcR), mannose-fucose receptor (MR), and fibronectin receptors. In this regard, an important molecule is complement component 3 (C3), which mainly binds to gp63 and LPG (glycolipid lipophosphoglycan) *in vitro* after complement activation [23]. This is a RhoA-dependent phagocytosis process. RhoA is a small GTPase protein of the Rho family of GTPases that is primarily involved in the regulation of the cytoskeleton, specifically the formation of actin stress fibers and actomyosin contractility. Phagocytosis has been proposed to be the main mode of invasion of promastigotes since infection by macrophages is reduced in the absence of actin polymerization of the host cell [24]. Phagocytosis of promastigotes by macrophages appears to begin within 2 minutes of contact with the parasites *in vitro* [25]. It should be noted that, during the first few minutes of contact, 90% of promastigotes connect to macrophages with low affinity through their flagellar tip [25], implying a role for this structure in the formation of phagosome Caveolae-dependent phagocytosis is also activated by *Leishmania.* The entry of pathogenic metacyclic promastigotes into murine macrophages has been linked to caveolae, and this route is critical to prevent early lysosome fusion.

During the differentiation process, promastigotes arrest phagosome maturation and exhibit delayed or decreased recruitment of late endosomal lysosome markers such as rab7 and LAMP1. Arrested phagosomes are further distinguished by the presence of host actin coating, related polymerization factors, such as Arp 2, 3, Nck, and WASP, and the recruitment of a variety of host GTPases involved in actin

polymerization. Further phagosome remodeling is related to the breakdown of the lipid raft and reduced formation of the NADPH oxidase complex.

Amastigotes, like promastigotes, are taken up by a conventional phagocytic process that may be opsonic or non-opsonic. Uncoated parasites are taken up by Rho and Cdc42, but IgG-coated parasites are phagocytosed by a Rac1-dependent mechanism. The FcR and CR receptors are mostly involved in amastigotes invading macrophages. Vacuoles containing amastigotes are fusogenic and acquire markers associated with phagosome development into phagolysosomes. The vacuole contains hydrolytic enzymes and is positive for H+ ATPase. It also includes markers such as Rab7, LAMP1, and LAMP2. Amastigotes are resistant to hydrolysis and multiplying the acidic environment (pH 4.5–5.5) of the phagolysosome. The ability of *Leishmania* to control phagosome maturation depends on a surface-abundant glycolipid called lipophosphoglucan (LGP), which is a member of the phosphoglycan family. In addition, the parasite membrane contains a proton translocating ATPase, which presumably helps maintain pH homeostasis inside the parasite and contributes to lysosomal acidification. The proton gradient thus established drives the active transport of nutrients necessary for the growth of the parasite [26].

It has also been described that *Leishmania mexicana* induces an autophagy-like pathway in infected cells, redirecting cytosolic proteins for destruction and making them accessible to parasites within the phagolysosome for nutrition [27, 28].

#### **2.4** *Toxoplasma gondii*

Unlike *Leishmania*, *Toxoplasma gondii* infects by both phagocytic and non-phagocytic cells. The infection and subsequent demise of these cells following the parasite's rapid proliferation is a crucial event in the pathogenic course of this organism. The parasite may enter a cell as a macrophage using the well-known phagocytosis process without causing its own death within the cell.

Trophozoites may actively escape cells after phagocytosis, by reversion of the process of invasion. At the moment, it is considered that entrance into the host cell includes a complicated process that combines phagocytosis with aggressive invasion.

Macrophages can swallow the parasite, opsonized or not. *T. gondii* inhibits phagosome-lysosome fusion after phagocytosis [29, 30]. Toxoplasma phagocytosis occurs primarily via opsonins such as C3b and C3a, which are recognized by their corresponding receptors on macrophages [31].

### **3. The evasion mechanisms**

#### **3.1** *Plamodium* **can control the phagocytosis process through a variety of methods**

*Plasmodium spp.* can prevent phagocytosis by changing its interaction with host phagocytic receptors and controlling downstream signaling cascades.

*Plasmodium yoelii* parasites, for example, preferentially infect erythrocytes expressing large amounts of CD47, allowing them to evade phagocytosis by the red-pulp macrophages in the spleen. CD47 is a marker that inhibits phagocytosis; Therefore, CD47 depletion may enhance phagocytic clearance. Red cells infected with *Plasmodium falciparum* and *Plasmodium vivax* have been shown to display higher amounts of CD47 than uninfected red cells; however, the mechanism behind this increased expression remains unclear. Furthermore, parasites can avoid phagocytosis

by modifying complement regulatory proteins, which protect infected host cells from complement-mediated damage. They can, for example, inactivate C3b on the surface of infected erythrocytes, preventing complement-mediated phagocytic clearance of parasites. Moreover, monocytes and macrophages express less complement receptor 1 (CR1) during infection. Surprisingly, infected red blood cells preferentially bind CR1 produced by uninfected red blood cells to form rosettes, presumably isolating them from phagocyte detection.

Also, by removing superoxide and inhibiting ROS from neutrophils, mosquito salivary proteins can influence neutrophil activity. Ex vivo data demonstrate that neutrophils have a decreased ability to create ROS during malaria (**Figure 1**, Plasmodium spp.). In vitro evidence suggests that neutrophil phagocytosis of parasite products reduces their ability to engulf bacteria [1].

It was similarly shown that ex vivo monocytes from children with acute malaria had lower opsonic phagocytosis than their own monocytes 6 weeks later [2].

Finally, parasites in Kupffer cells during rodent malaria have been shown to directly trigger phagocyte death [4].

Humans are infected by parasite sporozoites, which enter hepatocytes and grow rapidly. *Plasmodium spp.* requires nutritional input to the parasitophorous vacuole to reproduce successfully, which implies the existence of host cell manipulation mechanisms. It has been shown that there are membrane connections of the parasitophorous vacuole to the Golgi membranes that were maintained throughout the growth stage in hepatocytes, which are believed to enhance the nutritional supply of hepatocytes. RAB11, a small GTPase, is important for organelle morphological changes during *Plasmodium berghei* infections, and functional alterations of this protein reduced this impact.

#### **Figure 1.**

*The image shows the molecules involved in the phagocytosis of pathogenic protists and the evasion mechanisms that evolve to resist in the host cell. Created with BioRender.com.*

Mature trophozoites within infected red blood cells can circulate to organs such as the brain, spleen, placenta, and lungs, where they can be sequestered as part of an immune evasion strategy [4].

#### **3.2 Resist the oxidative response, the smart strategy of** *T. cruzi*

*T. cruzi,* in vertebrate hosts, develops a variety of immune evasion strategies. Protection against direct cytotoxic effects of O2•/H2O2• on parasite mitochondria within the macrophage phagosome (**Figure 1**, *T. cruzi*); suppression of ONOO production in NO-exposed parasites, and regulation of NO-exposed parasites are among these methods. To resist host-derived oxidants, *T. cruzi* has an arsenal of detoxifying antioxidant defenses, as well as redox metabolism. Trypanothiol (T[SH]2), the main thiol used by the antioxidant system of trypanosomatids, is one of the most important. This system is considered an interesting target route for drug development.

Fe-dependent superoxide dismutases (Fe-SODs) from *T. cruzi* readily remove O2 • and may help to survive intracellularly [32].

TcAPxCcP, a type A hybrid peroxidase that employs ascorbate and cytochrome C as reducing substrates for H2O2 detoxification, has also been reported in *T. cruzi* [33]. TcAPxCcP is a membrane-bound peroxidase found in the endoplasmic reticulum and mitochondria throughout the parasite's life cycle, as well as in the plasma membrane during the infective stages of the *T. cruzi* life cycle [34]. Lastly, *T. cruzi* has two GSHlike peroxidases (GPX) that can metabolize fatty acids and phospholipid hydroperoxides despite the absence of selenium in the active site [35]. In the non-infectious epimastigote, GPX-I is found in the cytosol while GPX-II is found in the endoplasmic reticulum. In general, *T. cruzi*'s antioxidant arsenal works as a virulence factor by detoxifying reactive species in the phagosomal compartment.

Furthermore, it has been demonstrated in *T. cruzi* that peroxiredoxins, a family of proteins with antioxidant and redox signaling functions, were upregulated in the infective metacyclic trypomastigote stage and that their expression levels correlated with parasitemia in mice, implying that peroxiredoxin levels mediate *T. cruzi* virulence.

Another pathogen-encoded virulence strategy depends on repair mechanisms that restrict the potentially damaging oxidation of proteins and DNA. Methionine oxidation is mediated by a variety of reactive species such as H2O2, peroxynitrite, HOCl, and metal-catalyzed oxidation systems, yielding methionine-(S) and methionine- (R)-sulfoxide (Met-SO) epimers. Enzymatic pathways for methionine oxidation have also been identified. Methionine sulfoxide reductases (Msr) have been identified in a variety of pathogenic organisms, and these enzymes reduce Met-SO by using the reducing equivalents of Trx/TrxR and NADPH [36]. MsrA and MsrB, two distinct enzymes, catalyze the reduction of oxidized methionine diastereomers. MsrA action in proteins is confined to Met(S)-SO residues, whereas MsrB decreases Met(R)-SO. Another essential component for *T. cruzi* pathogenicity is the sanitization of oxidized bases in DNA. Guanine is highly oxidizable, and its most frequent oxidation product is 7,8-dihydro-8-oxo-29-deoxyguanosine (8-oxoG), which has the potential to be mutagenic owing to its structural similarities to thymine [37]. Trypanosomes have effective DNA repair mechanisms as well [38].

#### **3.3 Leishmania subversion of phagocytosis favors the infection**

After inoculation, *Leishmania* promastigotes are swiftly phagocytosed, but they can survive and change into immobile amastigote forms that can remain as intracellular parasites. The parasitophorous vacuole is an acidic intracellular compartment where *Leishmania* amastigotes proliferate. Although the amastigote cytoplasm is controlled to near-neutral pH by an active process of proton extrusion, pH plays an important role in the developmental changeover between the promastigote and amastigote phases. Amastigotes are metabolically more active when their environment is acidic. Endosomes, phagosomes, and autophagosomes can all fuse with the parasitophorous vacuole. *Leishmania* amastigotes have evolved to survive in the particular ecological niche of mammalian macrophage phagolysosomes. The parasitophorous vacuole contains a highly hydrolytic and acidic environment, which the parasite does not appear to mitigate. While the parasite's cytoplasm is deliberately kept at a neutral pH, the amastigote's surface membrane adapts to operate efficiently in an acidic mileu, allowing the parasite to collect nutrition while being exposed to extraordinarily high external proton concentrations [39].

It is remarkable how the parasite avoids this harmful surge of ROS generation: it may counteract endogenous ROS production via antioxidant systems or by actively lowering ROS production (**Figure 1**, Leishmania spp) [40].

Although promastigotes and amastigotes enter macrophages by phagocytosis, the oxidative burst that occurs is very different. After infection, both stages show a rise in O2• production of macrophages, although the reaction is significantly stronger in promastigotes than in amastigotes. The discrepancy can be attributed to a decrease in NADPH oxidase activity following amastigote infection. Only once the gp91phox precursor has matured to its full-length molecule, the NADPH oxidase complex can be successfully assembled. This stage of development is dependent on the availability of heme. Infection with *L. pifanoi* amastigotes causes the production of heme oxygenase-1, the rate-limiting enzyme for heme degradation, which inhibits the development of gp91phox and precludes the assembly of NADPH oxidase. *L. donovani* amastigotes also affected another component of the NADPH oxidase complex. Amastigotes caused barely detectable amounts of p47phox phosphorylation, which resulted in p67phox and p47phox phagosomal recruitment defects. Interestingly, protein kinase C (PKC) mediates p47phox phosphorylation, which is suppressed by *Leishmania* promastigotes and amastigotes. This action has been linked to the lipophosphoglycan (LPG) present in promastigotes; in amastigotes, the mechanism responsible for PKC inhibition is uncertain. Moreover, *L. donovani* amastigotes affect the phagosomal lipid raft integrity, which may lead to defective NADPH oxidase assembly [41].

Lastly, infection with *Leishmania* amastigotes can result in reduced O2• generation by inhibiting inositol phosphate buildup and calcium release in infected macrophages. While promastigotes have little effect on overall O2• generation in macrophages, they have been shown to locally impede the assembly of NADPH oxidase at the phagosomal membrane, a defensive system reliant on the presence of LPG repeat units. Moreover, LPG glycoconjugates can influence macrophage iNOS expression. When LPG is administered before IFN-ɣ, NO generation is decreased compared to control cells. LPG suppresses the production of NO in macrophages in a time and dose-dependent manner. It clearly shows that LPG may regulate iNOS expression in macrophages [42].

*Leishmania* has an antioxidant defense mechanism as well. Trypanothione/trypanothione reductase has been described in *L. major*, which is crucial for its antioxidant ability against H2O2, ONOO, and •NO. T(SH)2 was also discovered to be required for H2O2 elimination in trypanosomatids. T(SH)2 requires the proteins triperedoxin (TXN) and peroxiredoxin (PRX) (which has triperedoxin peroxidase activity) to

decrease H2O2. The presence of the enzyme ascorbate peroxidase has also been shown to reduce H2O2, this is also present in *T. cruzi.* Trypanothione S-transferase and 5,6,7,8-tetrahydrobiopterin superoxide dismutase are among the main antioxidant mechanisms [40].

In summary, the parasite protects itself from the macrophage's oxidative burst by expressing antioxidant enzymes and proteins and inhibiting the synthesis of O2• and •NO in the macrophage. Surprisingly, promastigotes and amastigotes have opposing inhibitory effects. Amastigotes produce a widespread drop in O2• levels in the macrophage, whereas promastigotes lower O2• production just locally in the phagosome. Amastigotes decrease the synthesis of IL-12, O2•, and •NO in addition to their impact on macrophage redox biology. Unlike promastigotes, where LPG was identified as a parasite effector, no chemical associated with amastigotes has been identified as being responsible for the drop in O2• levels. Finally, parasites of *Leishmania* have evolved to live and multiply within ROS-producing macrophages. They do this not just through the use of antioxidant mechanisms, but also by decreasing ROS generation in macrophages [43, 44].

*L. donovani* infection also activates nuclear translocation and (Nuclear factor erythroid 2-related factor 2) Nrf2 activity, which reduces oxidative stress, but there is no evidence of which molecular partners are required to trigger this signaling yet. What is known in particular is that Nrf2 expression and activation occur upon initial contact with the host cell by increasing the number of gene products related to an antioxidant profile and turning macrophages into an anti-inflammatory spectrum. Knockdown or inhibition of Nrf2 is also known to decrease parasitic infection. But despite the antioxidant effect on cells, continued Nrf2 activation can greatly decrease ROS levels, which is also essential for cellular homeostasis. One of Nrf2's targets is the ferritin gene, which sequesters Fe2+, reducing iron metabolism for parasite growth [41].

An acid phosphatase found in *Leishmania* has been shown to inhibit superoxide anion generation in chemoattractant-stimulated neutrophils. The parasite's LPG was also found to suppress protein kinase C (a regulator of macrophage oxidative metabolism). It has been proposed that *Leishmania* parasites could block lysosomal hydrolases by producing polyanionic compounds capable of forming complexes with positively modified hydrolases or binding to calcium ions.

#### **3.4** *T. gondii* **established a unique vacuole to avoid host cell defenses**

As previously observed, microorganisms avoid important host defense processes such as phagocytosis, allowing them to establish themselves in the host cell and growth. In mouse macrophages (where this parasite survives), the organelle containing *T. gondii* appears to be arrested, unable to fuse with lysosomes, unless the organism has been coated with antibodies prior to phagocytosis, in which case it is easily destroyed [29]. *T. gondii* also uses tiny Rab-family GTPases for nutrient delivery, demonstrating that intracellular pathogens use host pathways components to promote proliferation. In *T. gondii*-infected cells, for example, mitochondria are organelles that interact with the membrane of the parasitophorous vacuole. The parasites have a mitochondrial association factor 1 (MAF1) locus, which encodes numerous proteins involved in host cell mitochondrial association and immune evasion, with the MAF1b protein serving as the primary mediator. *T. gondii*'s interaction with host cell organelles is most likely due to a requirement for nutritional input, which allows the parasitophorous vacuole to spread. Pernas et al. discovered that *T. gondii* infection had an indirect effect on mitochondrial morphology (**Table 1**) [45].


#### **Table 1.**

*Summary of the phagocytosis of pathogenic protists and the evasion mechanisms that evolve to resist in the host cell.*
