Fungal Infectious Diseases

## **Chapter 3**

## *Talaromyces marneffei* Infection: Virulence Factors and Rapid Diagnostics

*Sirida Youngchim*

### **Abstract**

*Talaromyces* (*Penicillium*) *marneffei* is a thermally dimorphic fungus that causes talaromycosis, and the pathogen is found throughout tropical and subtropical Asia. *T. marneffei* has specifically emerged as an opportunistic fungal pathogen in individuals with advanced HIV disease and, to a lesser extent, other immunocompromised conditions, but more recently talaromycosis is increasingly described in immunocompetent people. Due to the high mortality rate of up to 50%, understanding *T. marneffei* interactions with host immune responses and diagnostic modalities is vital to the development of strategies to reduce morbidity and mortality. In this chapter, we describe *T. marneffei* virulence factors that enhance the fungus' capacity for survival and growth in the host to lead to disease. We also discuss approaches for early diagnosis, which are essential to reduce the mortality rate in talaromycosis. Talaromycosis remains a neglected disease, but advances in our understanding of host-pathogen dynamics as well as the ongoing development of new diagnostic approaches are poised to enhance our capacity to combat this disease.

**Keywords:** *Talaromyces* (*Penicillium*) *marneffei*, dimorphic fungus, endemic mycoses, virulence factors, rapid diagnosis

### **1. Introduction**

*Talaromyces* (*Penicillium*) *marneffei* is a thermally dimorphic fungus endemic in the tropical and subtropical regions of Asia (**Figure 1**) [1, 2]. It is by far the species that most commonly causes human illness in immunocompromised patients, especially those with AIDS over the last three decades, especially in endemic areas of Southeast Asia (Thailand, Vietnam, Myanmar), East Asia (southern mainland China, Hong Kong, and Taiwan area), and north-eastern India, resulting in a rapid increase in incidence [2, 3]. Talaromycosis is not only recognized in endemic areas, but it is also increasingly being recorded in travelers from non-endemic areas such as Australia, Belgium, France, Germany, Japan, the Netherlands, Oman, Sweden, Switzerland, Togo, the United Kingdom, and the United States [4–10]. Fever, weight loss, anemia, lymphadenopathy, hepatosplenomegaly, respiratory symptoms, and skin lesions were all common clinical manifestations of *T. marneffei*

### **Figure 1.**

*Geographic distribution of talaromycosis.*

infection. Furthermore, amphotericin B, itraconazole, and voriconazole were the most often used first-line treatments, either alone or in combination with other drugs. The majority of patients die if they are not treated.

Since 1994, talaromycosis is the fourth most general opportunistic infection, after tuberculosis, pneumocystis, and cryptococcosis in AIDS patients in Thailand [11, 12]. Currently, the number of *T. marneffei* infections has declined in the last few years because of a decreased incidence of HIV and widespread accessibility of antiretroviral therapy [13]. Talaromycosis is not limited to people living with HIV. It is becoming more common in non-HIV-infected people who have other immunosuppressive conditions, such as primary immunodeficiency, autoimmune diseases, cancer, and solid organ and bone marrow transplants [14]. Common clinical manifestations of infection caused by *T. marneffei* included fever, weight loss, anemia, lymphadenopathy, hepatosplenomegaly, respiratory signs, and skin lesions. Due to increased migration and global travel, talaromycosis is becoming more common outside of endemic areas [4]. Although most people with talaromycosis are immunocompromised, healthy people can be affected as well, although rarely [15]. Patients with advanced HIV disease (CD4 cell counts <100 cells/m3 ) are at high risk; they typically occur with disseminated disease affecting the lungs, liver, spleen, gastrointestinal system, bloodstream, skin, and bone marrow [15]. Individuals without HIV are less likely than individuals with HIV to have skin lesions and positive blood cultures. As a result, as compared to HIV-positive persons, diagnosis is delayed (180 days vs. 45 days) and death is greater (29% vs. 21%) [16]. **Table 1** summarizes the laboratory findings and clinical prognosis of talaromycosis in patients with and without HIV infection.

**Variable criteria HIV-infected related HIV-infected unrelated Reference** Positive blood culture (%) 76.7 47.1 [17] White blood cells (x 103 cells/mm3 ) 4.1 15.6 CD4 (%) 3 30 Lymphocytes (%) 11.8 16.4 [16] Neutrophils (%) 81.2 75.2 Skin lesions (%) 53.4 31.6 [16] Diagnosis delayed (days) 45 180 [17] Medium treatment duration (days) 84 180 [17] Death (%) 21 29 [17]

Talaromyces marneffei *Infection: Virulence Factors and Rapid Diagnostics DOI: http://dx.doi.org/10.5772/intechopen.108592*

### **Table 1.**

*The laboratory characteristics and clinical prognosis of talaromycosis in individuals with and without HIV infection.*

Talaromycosis can affect both immunocompetent and immunocompromised patients, and the disease can be localized or systemic [18].

The ecology and route of transmission of *T. marneffei* infection are unknown. The organism has been isolated from the internal organs of four species of bamboo rats (*Rhizomys sinensis*, *R. pruinosus*, *R. sumatranensis*, and the reddish-brown subspecies of *Cannomys badius*) as well as the soil environment in which they dwell [19, 20]. A recent occupational history or other exposures to fine soil dust during the rainy season were determined to be the most important risk factors for illness. Contact with or consumption of bamboo rats do not appear to be significant risk factors for *T. marneffei* infection [21].

### **2. Culture and morphological characteristics**

### **2.1 Macroscopic and microscopic appearances**

*T. marneffei* is classified as a thermally dimorphic fungus which grows as a saprophytic mold. The fungus produces abundant conidia at 25°C and converts to yeast cells at 37°C. Mycelial colonies grow relatively quickly on Potato dextrose agar (PDA) at 25°C, as do other *Penicillium* species, and appear as flat, powdery white colonies. With continued culture, the periphery of the colony becomes more rugose, with radial folds. The color of the fungal colony changes from white to light brown and becomes light green after 10 days of culture. The colony produces diffusible red pigments into the agar and the underside of the colony (**Figure 2A**). This pigment production is one of the most characteristic features of *T. marneffei*. The microscopic examination of the mycelia phase reveals typical morphology of *Penicillium* or *Talaromyces* species. The microscopic examination of the mycelial form of *T. marneffei* is recognized as dense brush-like, spore-bearing structures (**Figure 2B**). The conidiophores can be simple or branched structures with clusters of flask-shaped phialides at the ends. The conidiophores are hyaline, smooth-walled and have terminal verticils of 3 to 5 metulae, each with 3 to 7 phialides [1].

### **Figure 2.**

*Thermal dimorphism of Talaromyces marneffei: (A) at 25°C, T. marneffei was grown on PDA as a mold, producing greenish-yellow to yellow conidia and secreting a distinctive diffusible red pigment; (B) conidiophores have phialides and conidia chains that resemble those of Penicillium species; (C) at 37°C, T. marneffei grows as a yeast with a dark-brown colony on BHI agar, producing brown pigment; (D) yeast cells are divided by fission rather than budding. Bars, 5 μm.*

At 37°C on Brain heart infusion (BHI) agar, *T. marneffei* can convert to yeast phase growth. Macroscopically, yeast-like colonies appear cebriform, convoluted, or smooth. Colonies are glabrous and beige-colored and take up to 10–14 days to exhibit full growth. Pigment production is both decreased and altered; the pigment released from *T. marneffei* yeast cultures appears closer to brown in color in comparison with the red pigment released from *T. marneffei* mycelial cultures (**Figure 2C**). Microscopically, yeast cells of *T. marneffei* are spherical to ellipsoidal yeast like cells separating by single septum, measuring 2–3 to 2–6 μm (**Figure 2D**) [22].

### **3. Virulence attributes of** *T. marneffei*

It is generally believed that inhalation of *T. marneffei* conidia is the likely route for infection, in line with the mode of infection for other molds such as *Aspergillus fumigatus* [23, 24] and *Histoplasma capsulatum* [25]. Indeed, *T. marneffei* conidia are presumably small enough (2 μm in diameter) to reach the alveoli of the lung and then are subsequently phagocytized by pulmonary histiocytes. *T. marneffei*, however, is able to live and develop inside this hostile intracellular environment rather than being killed Talaromyces marneffei *Infection: Virulence Factors and Rapid Diagnostics DOI: http://dx.doi.org/10.5772/intechopen.108592*

**Figure 3.**

*A depiction of the acquisition process of mycelial T. marneffei from the environment to the lung with the transformation to fission yeast in macrophage after deposition in alveoli.*

by the action of these immune cells (**Figure 3**). Then, *T. marneffei* can be disseminated throughout the body once established within the phagocyte and cause systemic infection if the host's immunological state is impaired. Indeed, a better knowledge of how the virulence pathways of *T. marneffei* interact with the immunological response of the host has helped us in redefining the pathogenesis of this fungus.

### **3.1 Adherence to host tissues**

Adherence to host tissues by *T. marneffei* conidia may play an extremely important role in the establishment of talaromycosis. Although the infective propagule and route of entry have not been definitively confirmed, inhalation of fungal conidia is likely to be the proposed route of infection [1]. Indeed, *T. marneffei* conidia are tiny enough (2 μm in diameter) to reach the lung's alveoli, and the identify of a potential conidial laminin/fibronectin receptor in *T. marneffei* suggests a plausible mode of conidia attachment to the pulmonary epithelium [26, 27]. *T. marneffei* also bind extracellular matrix (ECM)-associated glycosaminoglycans, chondroitin sulfate B, heparin, and highly sulfated chitosan CP-3, which are major constituents of many tissues particularly the basal lamina [28]. These ECM may become exposed in the lung as a result of tissue damage facilitating conidia adhesion to the bronchoalveolar epithelium. Nonetheless, this hypothesis has yet to be confirmed in animal models. Following that, *T. marneffei* glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified and acted as an adherence protein to facilitate conidia attachment to the host's bronchoalveolar epithelium suggesting that this protein may play an important role in the establishment of disease [29]. Indeed, knowledge of the adherence mechanisms in *T. marneffei* is still limited. The development of proteomic tools and the availability of genomic data will be of great importance to elucidate the mechanisms of the hostfungus interplay, and particularly of its adherence to the host tissues.

### **3.2 Dimorphic switching**

Fungal morphogenesis appears to be a critical factor in infection establishment. Indeed, dimorphic switching between mycelial and yeast phases is regarded to be a significant virulence component in dimorphic pathogenic fungi including *H. capsulatum*, *Coccidioides immitis, Paracoccidioides brasiliensis*, *Blastomyces dermatitis*, and *T. marneffei* [30]. Conversion to the yeast form may provide protection against killing by neutrophils, monocytes, and macrophages. Thermal dimorphism of *T. marneffei* is essential for survival in host cells that are responsible for the host temperature change. During the past decade, significant progress has been made in the understanding of the phase transition to yeast forms. *T. marneffei* has the ability to change morphology from hyphal mold in the environment to pathogenic yeast cells once conidia are inhaled into the lung of a mammalian host. Within the human host, *T. marneffei* conidia are engulfed and destroyed by the host's phagocytes, particularly alveolar macrophages. After internalized conidia, *T. marneffei* can differentiate into yeast cells and proliferate within alveolar macrophages [1]. The conversion of conidia to the yeast phase is the first critical process that permits *T. marneffei* to establish an infection, which is supported by the deletion of genes involved in phase transition altering the host response. The dimorphic transition of *T. marneffei* is a complex process involved by a number of genetic factors [31, 32].

According to the study of Yang *et al*. [33], the transcription factor *madsA* gene, a member of the MADS-box gene family, functions as a global regulator involved in the conidiation and germination, especially in the dimorphic transition of *T. marneffei*. In addition, overexpression of *madsA* in *T*. *marneffei* induced mycelium growth at 37°C, indicating that *madsA* is involved in the control of the dimorphic transition from yeast to mold. The deletion mutant and a complemented mutant of *madsA* in *T. marneffei* were then constructed and to identify its involvement in morphogenesis, dimorphic transition, and stress response [34]. When compared to the wild type and complementary strains, the Δ*madsA* demonstrated a faster transition from yeast (37°C) to mycelium (25°C) with abnormal morphogenesis. This study suggested that *madsA* functions as a regulator of yeast-to-mycelium transition and is closely related to conidiation and germination in *T. marneffei* although its roles in the survival, pathogenicity, and transmission require more investigation.

Despite the fact that temperature is only established stimulus controlling dimorphism in fungi, little study has been performed on the cellular changes or intracellular processes between the mycelial and yeast forms of *T. marneffei*. Based on yeast-phase specific proteins involved in virulence, the expression of yeast antigens of *T. marneffei* during phase transition was recently studied using a yeast-specific monoclonal antibody (MAb) 4D1 [35]. The MAb 4D1, yeast phase-specific MAb against *T. marneffei*, was produced using a modification of standard hybridoma technology with incorporating of cyclophosphamide without cross-reactivity to a panel of dimorphic and common fungal antigens [36, 37]. In addition, the MAb 4D1 was reactive against a 50–180 kDa broad high-molecular-weight smear of yeast phase mannoprotein antigen *in T. marneffei*. Recently, the MAb 4D1 was used to track cellular events in *T. marneffei* during phase transition and demonstrated that conidia were directly converted to fission yeast cells, with the expression of the yeast-specific antigen occurring 12 hours after phagocytosis by human THP-1 macrophage. These phenomena were clearly exhibited by overlapping signals between the green color of fluorescence isothiocyanate (FITC)-labeled conidia and the red color of MAb4D1 specific to yeast antigens, resulting in a yellow co-localized signal 12 hours after macrophage internalization

Talaromyces marneffei *Infection: Virulence Factors and Rapid Diagnostics DOI: http://dx.doi.org/10.5772/intechopen.108592*

#### **Figure 4.**

*The overlapping signals between the green of FITC-labeled conidia and the red of MAb4D1 which gives the co-localized signal as a yellow at 12 hours after internalization (white arrows). T. marneffei yeast cells were labeled with MAb 4D1 and Alexaflor 555-conjugated goat anti-mouse IgG antibody. A: Light microscopic image; B: Fluorescence image showing the green channel (FITC-labeled conidia); C: Fluorescence image of the red channel (MAb 4D1-positive yeast cells); D: THP-1 nuclei were stained with DAPI (blue) a merged channel showing the overlapping of images. Bars, 5 μm.*

(**Figure 4**). When compared to the results in artificial cultivation media, this experiment demonstrated that the phase transitional ability of *T. marneffei* conidia in culture medium was converted to yeast cells at a slower rate than in the host macrophage THP-1 environment. Thus, MAb 4D1 can be applied as a biomolecular tool for understanding the phase transition of *T. marneffei* and provides strong evidence for this fungal shift from an environmental saprophyte to a pathogenic fungus.

The readily reversible nature of the mycelial to yeast and yeast to mycelial transformation processes in *T. marneffei* indicates that they are genetically controlled. A number of molecular biology studies have focused on the genetic factor that influences dimorphic switching in *T. marneffei*. Indeed, *abaA* expression is significantly upregulated during hyphal to yeast transformation in fungi [38]. The *abaA* deletion mutant also displays aberrant yeast morphology as both the developing transitorystate arthroconidial filaments and the yeast cells fail to couple nuclear and cellular division, resulting in multiple nuclei in both the arthroconidial compartments and yeast cells. Furthermore, during the yeast to mycelia transition, transient upregulation of expression of *cflA* [39] and *cflB* genes [40] was observed. However, mutations in these genes resulting in altered function to do not block the dimorphic property of *T. marneffei*. Further study has employed two-dimensional difference gel electrophoresis to investigate proteins expressed differently in the yeast and mycelial phases, as well as peptide mass fingerprinting to identify these *T. marneffei* differentially expressed proteins [41]. These two enzymes are required for *T. marneffei* to survive as yeast inside phagocytes, where it is protected from the host defense system. Isocitratelyase, in particular, is the key rate-limiting enzyme in the glyoxylate bypass, a metabolic pathway that supplements the tricarboxylic acid cycle and is required for the survival of some intracellular pathogenic fungi such as *P. brasiliensis* and *Cryptococcus neoformans* [42–44]. This demonstrates the requirement of *T. marneffei* in sustaining the glyoxylate cycle under the host's severe nutrient-depleted environment.

Transition to the yeast phase may provide protection from phagocyte destruction. Thermal dimorphism of this fungus plays an important role for survival in host phagocytes. However, the phenomenon that regulates this transit has remained an enigma. A number of molecular biology studies have concentrated upon the genetic element influenced in the dimorphic switching in *T. marneffei*. Many of those previously investigated including *stuA*, *stlA*, *gasA*, *gasC*, and *cflB* have no role in yeast cell development or the dimorphic switch. However, Borneman and his colleagues (2000) had found that *abaA* deletion mutant displayed aberrant mold to yeast conversion as both the developing transitory state arthroconidial filament and the yeast cells fail to couple nuclear and cell division, where multiple nuclei were observed within either arthroconidia or yeast cells. However, once conidia began to develop yeasts, a second series of genes appeared to take over the coupling of cell division events [38].

### **3.3 Oxidative stress response and heat-induced fungal adaptation proteins**

Oxidative stress is one of the native defenses produced by the phagocytes to kill parasitic microorganisms. The phagocytes play a crucial role in eliminating fungal pathogens by producing reactive oxygen or nitrogen species, including superoxide radical anion (O2−), hydrogen peroxide (H2O2), hydroxyl radicals (OH− ), and nitric oxide (NO) [45]. The reactive oxygen species (ROS) can damage pathogens by readily altering or inactivating proteins, membrane, nucleic acid, and they have potent immunoregulatory effects on the host immune system that affect the efficacy of the host response [46].

### **3.4 Catalase**

Catalase peroxidase is capable of either reducing H2O2 with an external reductant or exchanging it to water and oxygen. The enzyme has been shown to be a virulence factor of *Mycobacterium tuberculosis* and *A. fumigatus* [47]. The catalase-peroxidase encoding gene (*CpeA*) in *T. marneffei* is associated with the upregulated expression of *CpeA* transcript both in yeast phase and under macrophage environment [48]. In recent years, Pongpom and her collaborators showed that *CpeA* controlled the fungus tolerance to H2O2 but not to heat stress response. H2O2 treatments induced high expression of this gene in both mold and yeast phase. It is therefore proposed that the *CpeA* of *T. marneffei* is utilized to protect the conidia and yeast cells from oxidative stress in the host macrophage environment [49].

### **3.5 Superoxide dismutase (SOD)**

Superoxide dismutase (SOD) is an enzyme that alternately catalyzes the dismutation of the superoxide radical (O2 − ) into either ordinary molecular oxygen (O2) or hydrogen

peroxide (H2O2). *T. marneffei* has been shown to survive and replicate as yeast inside the macrophage phagosome. Previously, Thirach and her colleagues investigated the fungal superoxide dismutase encoding gene (*sodA*) and found that the putative SodA peptide consisted of 154 amino acid residues and shared identity to fungal copper, zinc superoxide dismutase. The results suggested that *sodA* might play a role in stress response and in the adaptation of *T. marneffei* inside the macrophage [50].

### **3.6 High-temperature-induced fungal adaptation proteins**

Since the pathogenic phase of *T. marneffei* is closely linked with the higher temperature for normal growth in the environment, the heat-shock proteins (HSPs) are proposed as potential virulence factors. HSP always serve as a molecular chaperone, control protein folding, and transport intracellular proteins, as well as repair or destroy proteins. HSPs are a group of proteins produced by eukaryotic cells in response to exposure to stressful conditions, as they could be upregulated upon infection to prevent misfolding of damaged proteins [51]. The Hsp 70 of *T. marneffei* was first isolated and identified by Kummasook and colleagues (2007) [52]. The results showed that the *hsp70* transcription was upregulated during the mycelium to yeast transition. Upregulation was also observed when mycelial or yeast cells confronted to a heat stress environment at 39°C. It has been suggested that Hsp 70 may play an important role to prevent the yeast proteins from damage during temperature increase. Subsequently, Vanittanakom and her colleagues investigated the *hsp30* of this fungus and showed high transcription degree in yeast phase grown at 37°C, but undetectable transcript level was observed in mycelium phase at 25°C. These researchers suggested that Hsp 30 may play an important role in heat-shock response and in cellular adaptation during infection [53]. Based on the role of HSPs in temperature adaptation, the Hsp70 and Hsp 30 have definite functions in the host intracellular response; therefore, further study in *T. marneffei* is necessary.

### **3.7 Fungal melanin**

Melanin is a high-molecular-weight dark brown or black pigment produced by oxidative polymerization of phenolic or indolic compounds. Melanins are produced by a wide range of organisms, including bacteria, fungi, plants, and animals. Although different types of melanins can be produced by fungal organisms, the majority of fungal melanins are 1,8-dihydroxynaph thalene (DHN) melanins and L-3,4-dihydroxyphenylalanine (DOPA) melanins [54]. In the DHN pathway, 1,3,6,8-tetrahydroxynaphthalene (1,3,6,8-THN), which is derived from acetyl-CoA or propionyl-CoA with malonyl-CoA or methylmalonyl-CoA, is the first product of a polyketide synthases (PKS) pathway. This compound is then sequentially converted to scytalone, 1,3,8-trihydroxynaphthalene (1,3,8-THN), vermelone, and lastly 1,8- DHN as demonstrated in **Figure 5** [55]. Finally, oxidative polymerization produces the end product, DHN-melanin [55]. The DHN melanin biosynthesis gene cluster of *T. marneffei* was studied [57]. A cluster of six-genes, *alb1*, *arp2*, *arp1*, *abr1*, *abr2*, and *ayg1* are associated with conidial pigment synthesis in this organism. The genes *alb1* (*pks4*), *arp1*, and *arp2* encode for a polyketide synthase (PKS), a scytalone dehydratase, and a 1,3,6,8-tetrahydroxynaphthalene reductase, while *abr1* and *abr2* appear to encode two oxidases, respectively. Furthermore, all of these genes are phylogenetically linked to the *A. fumigatus* counterparts, and the production of DHN-melanin in *T*. *marneffei* is thought to be comparable to that of *A. fumigatus* [58]. Tricyclazole

### **Figure 5.**

*Biosynthesis pathway of DHN melanin in fungi. Scheme adapted from [54–56].*

which inhibits two hydroxynaphthalene reductases in the DHN-melanin synthesis pathway was used to confirm the DHN melanin of *T. marneffei* [59].

In *T. marneffei*, DOPA melanin is produced by yeast cells and composed of spherical granular particles in a beaded arrangement in the innermost cell wall [60, 61]. Bell and Wheeler [62] proposed a biosynthesis pathway for fungal DOPA melanin. There is experimental evidence for some of the proposed intermediates of that pathway [62–64]. In brief, laccase or tyrosinase catalyzes the hydroxylation of L-tyrosine to dopaquinone or the oxidation of L-DOPA to dopaquinone. Dopaquinone is a highly reactive intermediate which then forms leucodopachrome, which is oxidized to dopachrome. Hydroxylation (and decarboxylation) yields dihydroxyindoles that can polymerize to form DOPA melanin (**Figure 6**).

### **Figure 6.**

*The biosynthesis pathway of the dihydroxyphenylalanine (DOPA) melanin in fungi. Scheme adapted from [54, 56].*

Talaromyces marneffei *Infection: Virulence Factors and Rapid Diagnostics DOI: http://dx.doi.org/10.5772/intechopen.108592*

Both *T. marneffei* mycelial grown on culture medium at 25°C and yeast cells cultivated in a defined liquid minimal medium (MM) with L-DOPA were reactive with anti-melanin MAb 8D6, a melanin-binding MAb generated against *A. fumigatus* conidial melanin [60]. In mycelial phase, conidia, phialides, and hyphae were all positive with the anti-melanin MAb 8D6 (**Figure 7**). Thus, the melanization of *T. marneffei* was confirmed in both the mold and yeast forms.

Melanins have been influenced in virulence in many pathogenic fungi including *H. capsulatum*, *P*. *brasiliensis*, *C. neoformans*, *A. fumigatus* and *Sporothrix schenckii*. Melanin synthesis can help fungi survive in a variety of environments [65] and increase their resistance to host immune responses, such as reducing macrophage oxidative burst capacity [66], inhibiting apoptosis in macrophages [67], and inhibiting cytokine production in the host [68]. Youngchim and colleagues (2005) were the first to describe melanin in *T*. *marneffei*. The study demonstrated that melanins were produced both in *vitro* and during infection by presenting the melanization of yeast cells inside skin tissue from talaromycosis. Furthermore, sera from *T. marneffei* inoculated mice produced a significant antibody response against melanin, suggesting that melanin can act as an immunologically active molecule recognized by the immune

#### **Figure 7.**

*The melanin production of* T. marneffei *was detected in both mycelial and yeast forms using anti-melanin MAb 8D6. Corresponding immunofluorescence (A,C) and bright field (B,D) microscopy images demonstrating the labeling of mycelial phase (A,B) and yeast cells (C,D) of T. marneffei by anti-melanin MAb 8D6. Bar, 5 μm.*

system [69]. Melanin extracted from *T. marneffei* was also effective in inhibiting the production of TNF-α by the human monocyte cell line THP-1 indicating that melanin could conceal the organism from initial recognition by the immune system. In this respect, melanin is thought to contribute to *T. marneffei* virulence by allowing the organism to survive and grow within host tissue. The study of Woo *et al*. [70] confirmed that knocking down the melanin-biosynthesis gene cluster, *alb1*, in *T. marneffei* resulted in a loss of virulence in mice when compared to the wild type. The mutant also had a 50% reduction in conidial survival when exposed to hydrogen peroxide compared to the wild type. In fact, melanin has been named "an antifungal resistance factor" due to its ability to make melanized cells less susceptible to antifungal drugs [71, 72]. It was confirmed from our previous study that melanin appears to protect *T. marneffei* by making it more resistant to antifungal drugs including amphotericin B, clotrimazole, fluconazole, itraconazole, and ketoconazole [73].

The melanin produced by *T. marneffei* may play some roles in the virulence factor of this fungus. Melanin acts as a power protector for *T. marneffei in vitro* by decreasing phagocytosis and increasing resistance to macrophage intracellular digestion [61]. Interestingly, heat-shock proteins (HSPs), particularly HSP90 in *T. Marneffei*, were found to be significantly more expressed in DOPA melanin yeast cells compared to non-melanized yeast cells using proteomic analysis [74]. By making further analysis in the proteomic pathway, heat-shock proteins were enriched in multiple important metabolic pathways, including stress response pathway and phagosome development indicating that HSP90 plays an important role in melanin synthesis pathway of *T. marneffei*.

### **3.8 Fungal laccase**

*P*-diphenol dioxygen oxidoreductases or laccases are multi-copper containing oxidoreductase that catalyzed the oxidation of organic and inorganic substances including phenol containing amino acid, methoxy phenol, and aromatics amine, with the concomitant 4 electrons reduction of oxygen to water [75]. The essential properties of fungal laccases have been investigated and were shown to influence fungal development, to control phenotype and morphogenesis, to detoxify toxins, and to control pathogenesis in pathogenic fungi and stress response adaptation [76]. Laccases have been associated as contributors to virulence in many fungal pathogens such as *A. fumigatus* and *C. neoformans.* In *C. neoformans*, this enzyme can also promote the pathogenicity of *C. neoformans* by catalyzing the formation of melanin precursors. Melanized *C. neoformans* cell were more negatively charged on the cell wall, and this phenomenon could interfere with the phagocytosis mechanism [71]. The role of laccases in the virulence factor and pathogenesis in *T. marneffei* were characterized by Sapmak and colleagues [77]. It was found that quadruple deletions of laccase encoding genes (*lac1, lac2, lac3*, and *arb2*) in *T. marneffei* mutant were more sensitive to oxidative stressor, cell wall stressor, and antifungal agents including itraconazole, fluconazole, and clotrimazole. Subsequently, the results showed that the mutant strain of *T. marneffei* was more susceptible to killing by human macrophages, THP-1, than the wild-type *T. marneffei*. Moreover, the observation on the pro-inflammatory cytokine production in THP-1 human macrophages showed that the mutant *T. marneffei* stimulated a significantly higher production of TNF-α, IL-1β, and IL-6 compared to the wild type. Altogether, these results defined the role of laccases that influenced *T. marneffei* resistance to the host immune response [77].

### **3.9 Fungal cell wall, mannoproteins Mp1p**

The fungal cell wall is a critical structure with high flexibility that is important for cellular integrity and vitality. Mannoproteins are one of the most important structural components of the fungal cell wall. In fact, substantial study with yeast has demonstrated that mannoproteins perform a variety of biological functions, including defining cell shape, stimulating cell growth and morphological change, functioning as a protective factor, aiding sex agglutination, and regulating cell wall porosity [78–81]. Mp1p is an antigenic cell wall mannoprotein found in yeast, hyphae, and conidia of *T*. *marneffei* and has been effectively employed in serodiagnosis and infection prevention [57, 82–85]. Mp1p is a 462-amino-acid protein having three domains: ligand-binding domain 1 (LBD1), ligand-binding domain 2 (LBD2), and a serine and threonine-rich domain near the C terminus [84]. Mp1p is a new virulence factor of *T. marneffei* through knockout and knockdown research employing an intracellular survival assay with murine macrophage cells and mice challenge models [86]. For a mouse model, the mice could live for up to 60 days without talaromycosis after being challenged with a Mp-knockout strain of *T. marneffei*, but the wild-type strain killed the mice within 21 days. In addition, the organ fungal burden and inflammatory response in mice infected with the MP1 knockout mutant were significantly reduced compared to the wild type.

Based on the structure of Mp1p, Mp1p-LBD2, a ligand-binding domain, is a strong arachidonic acid (AA) binder by forming a five-helix bundle monomeric structure with a long hydrophobic central cavity for high-affinity encapsulation of cellular AA [87]. AA is a key pro-inflammatory mediator because it is produced as a main eicosanoid precursor in response to microbial infection, which can generate many downstream prostaglandins and common markers of pro-inflammatory responses, including TNF-α and IL-6 [88]. Subsequently, Lam *et al*. [89] demonstrated that not only Mp1p-LBD2, but Mp1p-LBD1 is also a strong AA-binding domain in Mp1p. Thus, Mp1p is an effective AA-capturing protein that uses two AA-binding domains, Mp1p-LBD1 and Mp1p-LBD2, to capture released AA during the early stages of pro-inflammatory reactions. According to the crystal structure, Mp1p-LBD1-LBD2 are likely to function independently and equally important in terms of AA capturing, with each domain capable of accommodating two AA molecules. Taken together, Mp1p represents a novel class of fatty acid-binding proteins with the function of targeting key pro-inflammatory signaling lipid to suppress the host innate immune response.

### **3.10 Iron and calcium are essential cations required for growth and virulence**

Ca2+ signaling plays an essential role in various processes, including cation homeostasis, pH adaptation, glucose metabolism, morphogenesis, and virulence in fungi [30, 90]. The Ca2+-binding protein calmodulin and the Ca2+/calmodulin-dependent phosphatase calcineurin are two major mediators of calcium signals in eukaryotic cells [91]. Calcineurin is a serine/threonine phosphatase that composed of two subunits of catalytic (CnaA) and regulatory (CnaB) that is activated through the binding of Ca2+-calmodulin (CaM) [92].

Calcineurin plays a crucial role in fungal virulence such as *A. fumigatus* [93, 94], *C*. *neoformans* [95], *Candida* spp. [96–98] and *Pacoccidiodes brasiliensis* [99]. Recent studies reveal a role of calcineurin in growth and virulence of *T. marneffei* [100]. In *T. marneffei*, deletion of the *cnaA* gene resulted in substantial defects in conidiation, germination, morphogenesis, cell wall integrity, and tolerance to several stresses.

The importance of calcineurin functions in cell wall integrity of *T. marneffei* was supported by the study of MICs against caspofungin and micafungin, which revealed lower MICs in the *cnaA* mutant when compared to wild type. These two antifungal agents belong to the echinocandins that inhibit fungal cell wall biosynthesis by inhibiting cell wall β-(1,3)-D glucan synthesis [101]. In addition, the *cnaA* mutant conidia were not only more susceptible to salt, H2O2, and osmotic stress *in vitro*, but they also rarely germinated or processed yeast morphogenesis after being phagocytosed by macrophages. Calcineurin is also required for full virulence in a murine model of invasive *T. marneffei* infection. Thus, calcineurin homolog (*cnaA*) regulates fungal morphogenesis and the response of *T. marneffei* to external stressors, as well as the host immunological response and fungal pathogenicity.

Iron is an important trace element that is often limited for pathogens during infection; hence, adaptability to iron deficiency is critical for virulence [102, 103]. Indeed, iron has been demonstrated to be essential for *T. marneffei* development and pathogenicity. Iron overload also significantly decreased the antifungal activity of macrophages [104]. As *T. marneffei* lacks an iron excretion mechanism, controlling iron uptake, metabolism, and regulation can play an important role in iron homeostasis. Fungi have developed two methods to get iron in iron-limited environments: reductive iron assimilation (RIA) and siderophore-mediated iron acquisition [105, 106]. RIA begins with the reduction of ferric iron sources to more soluble ferrous iron by plasma membrane-localized ferrireductases [107]. Then, the ferrous iron is re-oxidized and imported by a protein complex composed of the ferroxidase, FetC, and the iron permease, FtrA. Both *fetC* and *ftrA* gene expression was higher in yeast cells (37°C) than in hyphal cells (25°C) with a clear upregulation in response to iron limitation [108]. Deletion of *ftrA* results in a defective RIA system, which reduces the growth of yeast cells but not hyphal cells under low iron conditions [109]. For siderophore biosynthesis pathway, *sidD* and *sidF* genes involved in the biosynthesis of extracellular siderophores of *T. marneffei* were upregulated early during yeast morphogenesis switching from 25 to 37°C and late during yeast cell growth [108].

Based on the functions of *sidA* and *sidX*, these two genes encoded the enzyme ornithine N5-oxygenase, which catalyzed ornithine to hydroxyornithine in an early step of the siderophore biosynthesis pathway. SidA is involved in extracellular siderophore formation in the mycelial phase, whereas SidX is involved in both intracellular and extracellular siderophore production in the yeast phase [109]. Mutant analysis revealed that *T. marneffei* yeast cells can utilize RIA for iron acquisition, providing another system in this cell type that varies extensively from hyphal cells. For example, the expression of *fetC* (involved in RIA) was significantly elevated in Δ*sidA* and Δ*sidX* yeast cells but not in hyphal cells.

Furthermore, *T. marneffei* has recently been studied for the expression of *acuM* and *acuK*, which have been found to be involved in gluconeogenesis and iron metabolism [110]. In fact, *AcuM* and *AcuK* are homologous Zn2Cys6 transcription factors previously identified as gluconeogenesis and iron metabolism regulators in other pathogenic fungi such as *A. nidulans* [111] and *A. fumigatus* [112]. *T. marneffei* transcript levels of *acuM* and *acuK* were sequentially downregulated when the fungus was grown in increasing iron concentrations [110]. As a result, the transcription factors AcuM and AcuK may play a role in iron metabolism by either reducing iron uptake or alleviating iron toxicity. In contrast, the *acuM* transcript was upregulated in the gluconeogenic condition, but the *acuK* transcript was only elevated in the acetate medium during the yeast phase. Taken together, the genes *acuM* and *acuK* have been linked to iron homeostasis and gluconeogenesis in *T. marneffei*. Deletion of *acuK* gene

Talaromyces marneffei *Infection: Virulence Factors and Rapid Diagnostics DOI: http://dx.doi.org/10.5772/intechopen.108592*

in *T. marneffei* resulted in a growth defect under iron-deficient conditions since the mutant produced fewer siderophores [113]. The *fetC* transcript in ΔacuK was significantly increased than in the wild type, indicating that *acuK* may be a negative regulator of *fetC* expression, the gene encoding an RIA enzyme in *T. marneffei*. In contrast, the *sidA* and *sidX* transcripts involved in the first step of siderophore biosynthesis in Δ*acuK* were relatively low. This finding implied that *sidA* and *sidX* may be controlled by Acuk, and the detailed mechanism has yet to be investigated. As iron assimilation is the complex system, more studies are required to completely understand its regulation mechanism.

### **3.11 Extracellular vesicles (EVs)**

Extracellular vesicles (EVs), a type of nanoscale lipid bilayer membrane structure, play a function in transporting molecules to the extracellular space and are referred to as "virulence bags" [114, 115]. The characteristics and potential roles of these vesicles in virulence have been studied in a number of pathogenic fungi such as *C. neoformans*, *C. albicans*, *H. capsulatum*, and *P. brasiliensis* [116–119].

In *T. marneffei*, EVs had a typical spherical shape with a diameter of 30 to 300 nm under the nanoparticle tracking analysis (NTA) and TEM [120]. The functions of EVs released by *T. marneffei* could promote the expression levels of reactive oxygen species (ROS), nitric oxide, and some inflammatory factors including interleukin-1β, interleukin-6, interleukin-10, and tumor necrosis factor in RAW 264.7 macrophage cells. It was also reported that *T. marneffei* could secrete EVs loaded with some active molecules including heat-shock protein, mannoprotein 1 (MP1p), and peroxidase. As an important carrier containing a variety of molecules, EVs play a crucial role in intercellular communication with host immune responses.

### **4. Laboratory diagnosis of talaromycosis**

### **4.1 Staining and culture methods**

Microbiological culture and histological staining are commonly used for diagnosis of *T. marneffei* infection. Clinical specimens including bone marrows aspirates, lymph node biopsies, blood, sputum, pleural fluid, cerebrospinal fluid (CSF), urine, and liver biopsies were used for diagnosis of *T. marneffei* infection [11, 121]. In addition, Wright's staining of bone marrows aspirates and touch smear of skin biopsy or lymph node biopsies is a rapid diagnostic method [122] (**Figure 8**). The fungus can be seen in histological sections stained with Grocott methenamine silver (GMS) or periodic acid–Schiff (PAS). In contrast, *T. marneffei* yeast cells may result in the false impression that a capsule mimics to *H. capsulatum* when staining with hematoxylin and eosin (H&E) [123].

Microbiological cultivation is a gold standard for diagnosis of talaromycosis. The bone marrows gave the highest yield for culture positive, approaching 100%, followed by culture of other specimens obtained from skin biopsy (90%) and hemoculture (76%) [11]. However, most of the fungal isolates in microbiological laboratory screening are usually obtained from hemoculture of HIV-infected patients and need to confirm the dimorphic transition of this fungus. *T. marneffei* was confirmed by morphology and thermal dimorphism. *T. marneffei* was confirmed by macroscopic and microscopic examination. However, a limitation of the culture method is time-consuming, taking about 1 to 2 weeks. Given that ineffective

### **Figure 8.**

*Typical disfiguring central-umbilicated skin lesions on the face of a patient with advanced HIV and disseminated talaromycosis in Thailand (A), calcofluor white stained touch skin smear, showing fission yeast cells (B) and sausage-shaped yeasts with binary fission outside macrophage (C). The arrow heads highlight the midline septum in a dividing yeast cell characteristic of T. marneffei. Scale bar represents 5 μm.*

fungal therapy of *T. marneffei* is associated with poor prognosis and can be fatal, more rapid diagnosis of infection is preferable.

### **4.2 Serodiagnosis**

Many serodiagnostic assays have been developed for the detection of *T. marneffei* antigen from various clinical specimens, as shown in **Table 2**. Based on a potent immunogenic protein known as Mp1p, the protein is made up of galactomannoprotein which located throughout the cell wall of *T*. *marneffei* yeast [85, 123] have developed monoclonal antibodies (MAbs) and polyclonal antibodies (PAbs) against cell wall mannoprotein Mp1p expressed in *Pichia pastoris*. These antibodies were applied to detect antigens by using antigen capture ELISA. The method exhibited the sensitivities and specificities of 55% and 99.6% for the MAbs-MAbs based method and 75% and 99.4% for the MAbs-PAbs based method. There was no cross-reactivity found in 11 common pathogenic fungi, including *Cryptococcus*, *Candida*, *Aspergillus*, and *Histoplasma*. The Mp1p EIA was then applied to plasma samples of 372 patients who had culture-proven talaromycosis from blood and 517 individuals without talaromycosis (338 healthy volunteers and 179 with other infections) in Vietnam, demonstrating 98.1% specificity and 86.3% sensitivity [124]. In addition, paired plasma and urine testing in the same patients (n = 269) significantly improved sensitivity when compared to testing plasma or urine alone.

The most recent antigen detection assays developed for the diagnosis of *T. marneffei* infection have demonstrated the potential diagnostic application of MAb 4D1 [36]. In addition, MAb 4D1 (an IgG1) recognizes a 50–180 kDa manoproteins and the MAb shows specificity without cross-reactivity to a panel of dimorphic and common fungal antigens. Then, a new inhibitory ELISA using MAb 4D1 was designed to determine the antigenic concentration of *T. marneffei* in patient sera. The test identified antigenemia in all 45 (100%) talaromycosis, with a mean antigen concentration of 4.32 μg/ml. No cross-reactivity in this assay was found in patients with other fungal or bacterial infections, and healthy controls. This result showed that the detection of circulating antigens in talaromycosis was beneficially useful not only for diagnostic purposes but also as a tool to evaluate the clearance of fungal burden during treatment.

Currently, there is no commercially available diagnostic kit for talaromycosis. The available alternative serodiagnostic method for talaromycosis in routine laboratory

Talaromyces marneffei *Infection: Virulence Factors and Rapid Diagnostics DOI: http://dx.doi.org/10.5772/intechopen.108592*


*N/A: No data or inconclusive data*

*\* Compared with culture confirmed talaromycosis*

*\*\*Compared with other microorganism infection, healthy individual living in endemic area and asymptomatic HIV infection living in endemic area*

### **Table 2.**

*Summarized immunological methods for detection of T. marneffei specific antibodies.*

is based on Platelia *Aspergillus* EIA which is designed for the detection of circulating *A. fumigatus* GM antigens. It has been reported that the GM antigens of *T*. *marneffei and Aspergillus* are very similar, and the EIA test could therefore give high degree of diagnostic sensitivity for talaromycosis [20, 125]. As a result, the intimate concordance rate between Mp1p antigen detection and the GM antigen assay in antigenemia of talaromycosis was demonstrated, which is extremely important. Several studies have revealed significant false-positive due to the cross-reaction of the MAb against GM (Rat MAb EB-A2) with GM antigen from non-*Aspergillus* spp*.,* e.g. *Geotrichum capitatum*, *H. capsulatum*, *P. brasiliensis*, *B. dermatitidis*, *Mycobacterium tuberculosis*, galactoxylomannan from *C. neoformans*, and *C. gattii* and serum of patient treated with piperacillin-tazobactam or amoxicillin-clavulanic acid. It is not surprising that GM antigen is a "Pan-specific" marker for the fungal infection. Similarity between the (1–3)-β-D-glucan (BG) or "Fungitell" has been used for the diagnosis of filamentous fungal infections [126]. However, the lack of specificity means was unable to discriminate between *Aspergillus* spp. and other pathogenic fungi.

### **4.3 Rapid lateral flow immunochromatographic assay (ICA)**

Recently, the rapid lateral flow ICAs have been developed for immunodiagnosis of the infection due to the clinically important fungi, e.g. polysaccharide antigen detection for *C. neoformans* [127], detection of specific IgG against *Pythium insidiosum* [128], hyphal-specific antigen detection of *Candida* species [129], and MAb against secreted glycoprotein of *A. fumigatus* for the diagnosis of invasive aspergillosis [130].

A "point-of-care" diagnosis of talaromycosis is urgently needed [2, 131]. Recently, we demonstrated a novel inhibition format of an ICT strip for rapid detection of the *T. marneffei* antigen from clinical urine samples. In this study, *T. marneffei* cytoplasmic yeast antigens (TM CYA) and the corresponding MAb 4D1 conjugated with nanoparticles of gold colloid were used. The inhibition (inh)-ICT strip was evaluated for its diagnostic performance in urine samples from both talaromycosis patients and a control group. The inh-ICT was highly specific against antigenuria from *T. marneffei* only, and it did not detect antigenuria of other clinically important microorganisms. The limit of the detection was 3.12 μg/mL of fungal antigen. The inh-ICT was used to test urine samples from 66 patients with confirmed *T. marneffei* infection, 40 patients with other microbial infections, and 72 healthy individuals from endemic area. The test exhibited diagnostic sensitivity, specificity, and accuracy of 87.87%, 100%, and 95.50%, respectively, for *T. marneffei* [132]. However, the Inh-ICT has some limitations on the relatively low diagnostic sensitivity and occasional ambiguity in the reading and interpretation of the observed results. The innovative sandwich ICT strip was created to improve the diagnostic efficiency of ICT strips by using a mannose binding lectin, which recognizes mannose residue called *Galanthus nivalis* agglutinin (GNA) or snowdrop lectin, in conjunction with MAb 4D1 [133]. The MAb4D1-GNA-based ICT showed specific binding activity with yeast phase antigen of *T*. *marneffei*, and it did not react with other common pathogenic fungal antigens. The diagnostic performance of the ICT was validated using 341 urine samples from patents with cultureconfirmed *T. marneffei* infection and from a control group of healthy individuals and patients with other infections in an endemic area resulting 89.47% sensitivity, 100% specificity, and 97.65% accuracy. As a result, the *T. marneffei* ICT should be evaluated for clinical use in the context of rapid and affordable point-of-care diagnostic test to reduce the burden of talaromycosis mortality in patients in low-income countries.

### **4.4 Molecular diagnosis**

The polymerase chain reaction (PCR) has been utilized effectively for the specific detection of many pathogenic fungi. The nucleotide primer PM2 and PM4 have been

Talaromyces marneffei *Infection: Virulence Factors and Rapid Diagnostics DOI: http://dx.doi.org/10.5772/intechopen.108592*

developed to amplify a 347 bp fragment of the internal transcribed spacer (ITS) element between 18 s rRNA and 5.8 s rRNA [134]. Novel oligonucleotide probes RRF1 and RRH1 were used in PCR southern hybridization format for the amplification of a 631 bp. fragment of the 18 s rRNA and then hybridized with a *T. marneffei* specific 15 oligonucleotide probes [135]. Further molecular diagnosis method was described; a one tube seminested PCR assay based on the 18 s rRNA region was developed to identify *T. marneffei* genome [136]. This method was useful and can detect *T*. *marneffei* DNA both from culture and clinical samples. An additional nested PCR test and a real-time PCR assay were used to detect *T. marneffei* in whole blood samples. Given the high sensitivity of nested PCR (82%) and real-time PCR (91%), the combination of these two PCR methods provides an interesting alternative for identifying *T. marneffei* DNA in whole blood samples [137]. However, these methods need the clinician establishing a hypothesis before to examination. *T*. *marneffei* is an uncommon pathogen in non-HIV individuals, particularly in areas outside of Southeast Asia where detection and therapy of talaromycosis may be restricted. Next-generation sequencing (NGS) based on metagenomics has recently been used to successfully diagnose disseminated *T. marneffei* infection. Under these conditions, the use of mNGS enabled for the rapid and accurate diagnosis of *T. marneffei* without the requirement for specified problematic pathogens, which is a proven advantage in talaromycosis diagnosis, resulting in improved individual patient treatment [138, 139]. At the time, the cost is the major hindrance to its wide usage. If the cost of NGS is reduced further and expertise is made more widely available, it will be an effective instrument in the repertoire for laboratory diagnosis of *T. marneffei* infection.

### **5. Conclusion**

*Talaromyces* (previously *Penicillium*) marneffei causes a life-threatening invasive mycosis both in immunocompromised and immunocompetent individuals living in tropical and subtropical Asia. *T*. *marneffei* can adapt and express many virulence factors to survive inside hosts and then infect in those patients. The current understanding of the dynamic interaction between *T. marneffei* and its mammalian hosts emphasizes the role of virulence factors, such as adhesion to host tissue, dimorphic switching, oxidative responses, heat-shock protein, and melanin, which allowed the pathogen to evade host immune cells. Diagnosis of talaromycosis is frequently delayed which can result in unnecessary antibiotic use, unnecessary hospital admissions, and increased morbidity and mortality. Conventional methods of diagnosis have relied on the culture or examination of fungi; however, the time required to obtain results from culture and the lack of sensitivity of visual inspection tests can make them inconvenient. Thus, rapid diagnosis frequently based on antigen testing can help with the identification of talaromycosis.

### **Conflict of interest**

The author declares no conflict of interest.

*Infectious Diseases Annual Volume 2022*

## **Author details**

Sirida Youngchim Department of Microbiology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand

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

© 2022 The Author(s). Licensee IntechOpen. 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.

Talaromyces marneffei *Infection: Virulence Factors and Rapid Diagnostics DOI: http://dx.doi.org/10.5772/intechopen.108592*

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### *Infectious Diseases Annual Volume 2022*

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**Chapter 4**

## Evolution of Parasitism and Pathogenic Adaptations in Certain Medically Important Fungi

*Gokul Shankar Sabesan, Ranjit Singh Aja, Ranjith Mehenderkar and Basanta Kumar Mohanty*

### **Abstract**

Fungi are eukaryotes designated as a separate kingdom because of their unique characteristics different from both animals and plants. Fungi are mainly classified into two major types as "saprobes" and "parasites" depending on their type of nutrition and existence. It is postulated that the present-day parasites also once existed as saprophytes in the soil. It is also curious to find the reasons on what early events could have been responsible for the evolution of the saprobes into human parasites? During this process of evolution, some of the anthropophilic organisms have totally lost all their soil-inhabiting traits and the ability for saprophytic survival, while few others have successfully retained their ability to survive in two different ecological niches (soil and animal/human host). The various possible reasons, such as predation, antagonism, and other factors contributing to the emergence of parasitic adaptations, are discussed using examples of dermatophytes, *Cryptococcus neoformans,* and *Histoplasma capsulatum.*

**Keywords:** fungal parasitism, fungal virulence factors, adaptations, fungal evolution, *Cryptococcus neoformans*, *Histoplasma capsulatum*, dermatophytes, dimorphism, anthropophization, fungal pathogenesis

### **1. Introduction**

Fungi are a group of eukaryotic organisms existing in the ecosystem as chemoheterotrophs, as they are dependent mostly upon the secretary exoenzymes to harvest energy from the organic substrates. Based on the heterotrophic nutrition and their dependency for survival, fungi are mainly classified into two major types: (a) saprobes and (b) parasites. Interestingly, there is difficulty to have a clear distinction between the human parasites and saprophytes as the natural habitats of most of these pathogenic fungi that cause systemic mycoses are only the dead and decaying organic matter. These fungi mostly dwell in soils enriched by droppings of birds or other organic wastes. Fungal parasitism is considered to be one of the largest areas in medical mycology that has attracted so many researchers all over the globe over the years. Enormous research work had been carried out in plant-parasitic fungi, but the role of fungi in veterinary and human medicine until recent years remained the

most neglected area, the reason being that most of the medically important fungi are opportunistic pathogens and that the person-to-person transmission of fungal diseases is not as common as the bacterial/viral infections.

In recent years, fungal diseases have reached epidemic proportions in causing morbidity and mortality all over the world as it is regarded that it may be just the tip of the iceberg. Increasing immunocompromised status in human beings due to the advent of human immunodeficiency virus/AIDS, chemotherapy, radiotherapy, debilitating illness such as cancer, COVID-19, prolonged steroid treatment, organ transplantation, and chronic diseases perhaps are the conditions that would have promoted these opportunistic pathogenic fungi into "Champion parasites" in causing human diseases [1]. Thus, these so-called low virulent saprophytic fungi are capable of causing diseases given the opportunity and availability of susceptible hosts. It is always of immense interest and curiosity to know why these fungi would have adapted themselves or equipped to develop virulence factors to emerge as human pathogens/parasites. It would always be necessary to understand what early events or environmental factors in their original habitats would have compelled/prompted/ facilitated certain groups of soil-inhabiting fungi to emerge as human pathogens [2].

## **2. Fungal parasitism**

Fungi exhibit three types of parasitism [3] in human beings:


## **3. Obligate parasitism**

Obligate parasitism is seen in the case of anthropophilic dermatophytes, such as *Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton tonsurans, Trichophyton violaceum*, and *Epidermophyton floccosum* [4]. It is also seen in the case of the lipophilic yeast, *Malassezia species*, viz., *Malassezia furfur and Malassezia globosa.* The existence of these organisms in "ex-anthropophilic" conditions for a prolonged period has not been established yet [5]. Several experimental studies [5] conducted on the saprophytic survivability of these organisms in the soil also reveal that these organisms, if at all, can exist in the soil only for a transient period. However, their counterparts, related groups of dermatophytes such as *Nannizzia gypsea* (previously named *Microsporum gypseum*)*, Microsporum nanum, Microsporum distortum,* and *Trichophyton ajelloi*, can exist in soil popularly as the geophilic group. Interestingly, the genetic variation between the anthropophilic and geophilic groups of dermatophytes is calculated less.

### *Evolution of Parasitism and Pathogenic Adaptations in Certain Medically Important Fungi DOI: http://dx.doi.org/10.5772/intechopen.105206*

The possible theory [3, 6] by which certain dermatophytes would have evolved as obligate parasites in human beings would have started and progressed in different phases.


Although the above theory clearly suggests the means of the development of parasitism in dermatophytes, it drastically fails to explain why it had occurred in certain species of dermatophytes. Furthermore, what was the sequence of events that occurred in their original habitat that would have compelled/promoted these fungi to become obligate pathogens beg an answer. Furthermore, it is also important to understand how these organisms adapt themselves to lead an obligate anthropophilic existence. In *Malassezia* spp., a similar lineage is seen, as the members of the species exist only as obligate parasites (commensals) in animals. There are no reports of the existence of *Malassezia* spp. in the soil so far.

### **4. Parasitism and true pathogenesis**

In the case of obligate parasitic fungi, such as *T. rubrum*, their existence is exclusively limited to the human habitat; hence, the diseases caused by these organisms are not debilitating or life-threatening, whereas parasitism is truly severe in true pathogenic fungi, such *as C. immitis, H. capsulatum, P. brasiliensis*, and *B. dermatitidis.* These systemic dimorphic fungi exist as saprophytes in the soil, droppings/guano of bats, and pellets of various avifauna, but these organisms accidentally encounter the human habitat. They exhibit true pathogenic potential even in the immunocompetent host. This ability to cause life-threatening infections irrespective of the immune status of the host is intriguing, and it is really amazing to know how these fungi evolved the super specialty of existing in the saprophytic form and yet cause diseases in "immunocompetent" people. The dimorphic mode of existence of these fungi is largely considered to be one of the predominant selective advantages [7] for their successful geophilism and anthropophization. One of the most intriguing aspects of its biology is the dimorphism exhibited by *H. capsulatum. H. capsulatum* produces mycelium at environmental temperatures less than 30°C, but reproduces as a budding yeast when growing intracellularly in patients with histoplasmosis. It has been estimated that in the endemic areas of the United States (histoplasmosis surveillance data of 2011–2014), the average incidence all over the country ranges even up to 39 cases per 100,000 population [8]. Though histoplasmosis is endemic to certain places and limited to certain geographical locations, *H. capsulatum* is found throughout the world [3].

## **5. Opportunistic parasitism**

Opportunistic pathogens are fungi that strike and cause infections under certain predisposing host conditions, such as severe immunodeficiency. These fungi are also widely present in soil bird droppings as saprophytes. When a host is available in an immunocompromised state, these fungi cause moderate to life-threatening diseases, as seen in the case of cryptococcal meningitis caused by *C. neoformans*. Several studies have shown that cryptococcal infection poses a major threat to the life of AIDS patients and other immunocompromised people all over the world. It would be interesting to unknot the mystery of how *Cryptococcus* species, originally a geophilic saprophytic yeast, can "spontaneously specialize" by developing several mechanisms/virulence factors to invade, colonize, and manifest life-threatening disease in the human host. Serious uncertainties exist in finding plausible answers to these questions:


For practical purposes, in the realm of Medical Mycology, the infectious microorganisms have been grouped into three ecological categories based on their natural histories. These entities, in a broad sense, have been traditionally designated as being geophilic, zoophilic, and anthropophilic and are designed as follows:


Interestingly, some geophilic and zoophilic organisms also cause human infections.

*Evolution of Parasitism and Pathogenic Adaptations in Certain Medically Important Fungi DOI: http://dx.doi.org/10.5772/intechopen.105206*

It is also curious to find the reasons for what early events could have been responsible for the evolution of the "Saprobes" into human "Parasites"? The line of demarcation between the saprophytes and parasites is neither here nor there.


## **6. Fungal evolution: pathogens and their parasitic adaptations**


### **7. Antibiosis by other soil fungi**

It has long been speculated and later had been confirmed using modern phylogenetic studies that the parasitic dermatophytes probably arose from the geophilic (soil-borne) nonpathogenic ancestors. The existence of today's nonpathogenic dermatophytoids in the same habitat is the exemplification for this hypothesis (e.g., *T. ajelloi* and *Trichophyton terrestre*) [10–12].

The studies by Gokulshankar et al. [13] revealed that the Secretory substances (SS) released by *Chrysosporium keratinophillum* possess significant inhibitory (antidermatophytic) activity against *T. tonsurans*, *T. rubrum*, *T. violaceum, T. mentagrophytes*, and *E. floccosum*. The SS of *C. keratinophillum* released on the 15th day inhibited all the isolates of *T. rubrum,* while the SS released on the 10th day inhibited all the isolates of *T. tonsurans*, *T. violaceum*, and *E. floccosum* [13].

The Secretory substances of *C. keratinophillum* further failed to inhibit the growth of the geophilic *N. gypsea* (previously named *M. gypseum*) and zoophilic *Microsporum canis*. This experiment should be correlated with the global prevalence of *N. gypsea* in soil. The selective ability of *N. gypsea* to counter the antagonistic activity of the SS of *C. keratinophillum* may be one of the reasons for the worldwide distribution of this fungus in soil [14].

It is also interesting to note that in the co-inoculation studies, when *C. keratinophillum* and anthropophilic dermatophytes were co-inoculated on Sabouraud Dextrose Agar (SDA), *C. keratinophillum* failed to inhibit the mycelial growth of *T. ton*surans, *T. rubrum*, *T. mentagrophytes*, and *E*. *floccosum*. However, conidia formation did not occur on the organisms (*T. rubrum*, *T. tonsurans,* and *E. floccosum)* when they were grown near *C. keratinophillum*. It is presumed that (a) the nature and (b) the quantity of the SS released by *C. keratinophillum* may affect the growth of these pathogenic dermatophytes. It may be because the SS produced by *C. keratinophillum* during its early

*Evolution of Parasitism and Pathogenic Adaptations in Certain Medically Important Fungi DOI: http://dx.doi.org/10.5772/intechopen.105206*

growth phase might not be very active to inhibit. Furthermore, when both *C. keratinophillum* and an anthropophilic species of dermatophytes were co-inoculated at the same time, the growth of dermatophytes may also start much before the actual release of SS (10–15 days) by *C. keratinophillum*. The absence of conidia formation in *E. floccosum, T. rubrum,* and *T. tonsurans* when grown near the *C. keratinophillum* establishes the fact that SS of this organism possesses definite antidermatophytic characteristics.

Gokulshankar et al. [14] and Gokulshankar et al. [13] performed co-inoculation studies of different individual species of pathogenic dermatophytes along with *C. keratinophillum* in both sterilized and unsterilized soil to study their compatibility in the near natural environment. Recovery of the dermatophytes was attempted at different time intervals. The results showed that none of the anthropophilic dermatophytes could be recovered after 15 days of incubation by either plating or hair baiting techniques. However, the dermatophytes could be recovered up to 40 days from sterilized soil when inoculated alone.

Interestingly, the isolation of geophilic *N. gypsea* (previously named *M. gypseum*) was not affected in these co-inoculation studies. The authors also found that whenever the baiting technique is employed for the isolation of dermatophytes from the soil, *Chrysosporium* species are the predominant fungi to be isolated [14]. *Chrysosporium* and allied genera accounted for 53.8% distribution, with *C. indicum* being the dominant species among the keratinophilic fungi in soil [15]. The attribution of *Chrysosporium* spp. as a principal contributor to the evolutionary divergence of some geophilic archi-dermatophyte to obligate parasitic dermatophyte species, such as *T rubrum and E. floccosum* [13].

The antibiosis of other soil-inhabiting microbes (bacteria, protozoans, etc.) on dermatophytes also cannot be ruled out or underestimated for their probable role in the evolution of parasitism in anthropophilic dermatophytes. Gokulshankar [16] tested several other soil-inhabiting fungi, such as *Aspergillus fumigatus, Aspergillus niger, Aspergillus flavus, Rhizopus oryzae*, *Penicillium sp,* and *Curvularia lunata* for antidermatophyte activity. Among the tested fungi, the SS and intracellular substances (ICS) of *C. lunata* were found to have a definite role in inhibition.

It was reported that *C. keratinophillum, A. flavus, A. niger, A. fumigatus, R. oryzae, Penicillium sp,* and *C. lunata* showed inhibitory effects on the lipophilic fungus*, M. furfur* on co-inoculation. Furthermore, the ICS and SS of *C. keratinophillum, A. flavus, A. niger, A. fumigatus, R. oryzae, Penicillium sp,* and *C. lunata* tested were also found to inhibit *M. furfur*. This clearly proves *M. furfur's* inability to co-exist with any of the tested environmentally prevalent fungi. Furthermore, *Malassezia* species are found only as obligate commensal/parasite on the human and/or animal hosts. The saprophytic existence of *M. furfur* or any other species of *Malassezia* is also not known. All these correlations helped Gokulshankar et al. [2] to contemplate and propose a hypothesis


The study has been done only with *C. keratinophillum,* but the possible role of other species of *Chrysosporium* group could have also contributed to the evolution process.

Several other environmental factors in combination with the antagonism/inhibition of other soil protozoa/fungi/bacteria could have compelled the dermatophytes to evolve parasitic adaptations. The reported low incidence of the anthropophilic dermatophytes in soil may also be due to the gradual weaning off soil-inhabiting characters (welldefined anthropophization) in these pathogenic dermatophytes.

### **8. Saprophytic survivability of obligate parasitic dermatophytes in soil**

Several studies suggest that saprophytic survivability for parasitic dermatophytes in soil may not be possible due to their well-defined anthropophization. However, the viability of the fungal elements (chlamydospores and arthroconidia) in soil for a shorter period cannot be ruled out. Likewise, the recovery of *M. canis,* a zoophilic dermatophyte, even from sterile soil by either hair baiting or plating technique was also not possible after 60 days, which suggested that this organism is not capable of soil existence. Did the organism lose the ability to survive even as a spore in soil for a prolonged period?

The recovery of *N. gypsea* (previously named *M. gypseum*) was possible from both unsterile and sterile soil for up to 120 days. The recovery was possible by hair bating and plating methods, substantiating the saprophytic surviving ability of *N. gypsea* [13, 14, 16].

Hair baiting was found to be a superior method for isolating all keratinophilic fungi from soil, especially the dermatophytes. The study suggests that saprophytic survival even without the interference of other micro- or macroorganisms may not be possible for the obligate anthropophilic dermatophytes, such as *E. floccosum, T. tonsurans, T. rubrum,* and *T. violaceum* (**Tables 1** and **2**).


**Table 1.**

*Saprophytic survivability of test organisms in unsterile soil by hair baiting technique.*


*Evolution of Parasitism and Pathogenic Adaptations in Certain Medically Important Fungi DOI: http://dx.doi.org/10.5772/intechopen.105206*


#### **Table 2.**

*Saprophytic survivability of test organisms in unsterile soil by a soil plating technique.*

### **9. Earthworms as predators of anthropophilic dermatophytes and** *M. furfur*

Results of the feeding studies of earthworms with dermatophytes [17] have revealed that either the anthropophilic (*T. rubrum, T. mentagrophytes, T. tonsurans, T. violaceum*, and *E. floccosum)* or zoophilic dermatophyte species (*M. canis*) were unable to survive in the gut of the earthworm. The recovery in culture from the gut of earthworms or the worm cast was not possible. However, all the species were recovered from control plates in the absence of earthworms (plates containing just soil admixed with milk powder and egg). This experiment portrayed that the earthworm gut may not be an ideal environment for the survival of these parasitic dermatophytes. However, all the tested strains of *N. gypsea* were recovered from the gut and the worm cast.

Four earthworm species were used in the study (*Amynthas alexandri, Lampito mauritii, Aporrectodea tuberculate,* and *Lumbricus terrestris*). The gut extracts of all the four species of earthworms showed a similar band pattern at the retention factor in Thin layer Chromatography (Rf) value of 0.32. This pattern probably may represent some inhibiting enzymes/antidermatophytic factors.

Further recovery of *M. furfur* from the gut of earthworms/worm cast after feeding assay with earthworms was also not possible.

### **10. Saprophytic existence of** *C. neoformans* **and** *H. capsulatum* **in soil**

Saprophytic survival of *C. neoformans* was recorded in sterile soil for up to 120 days, but it was not possible in unsterile soil. Steenbergen and Casadevall [18] and others have reported predation of *C. neoformans* by soil organisms such as nematodes and amoebae, as reported by earlier workers. The soil predators would have been eliminated in the process of sterilization, which could be the reason for their survival and recovery in sterile soil, whereas their survivability was affected in unsterile soil. Interestingly, both the melanin-producing and non-melanin-producing isolates could be isolated from sterile soil.

Saprophytic survivability for *H. capsulatum* was reported for up to 120 days only for the mold suspension of the organism. However, the yeast suspension could not

survive both in sterile and unsterile soil (10–20 days). This clearly illustrates that yeast morphogenesis is an important adaptation developed by the *C. neoformans,* which is required for survival in the host (for pathogenic intracellular state), while the mold form is mandatory for the existence in soil as saprophyte (**Table 2**).

### **11. Protease moderation in dermatophytes and pathogenesis**

The roles of protease in the pathogenesis of many microorganisms have been described [19]. Associations of protease in infections caused by *Candida* spp. and *Pseudomonas aeruginosa* have been documented [20, 21]. For the hydrolysis of structural proteins of skin, hair, and nails, dermatophytes require and, therefore, elaborate certain protein hydrolyzing enzymes. Lu [22] has reported that the hair perforation of *T. mentagrophytes* was due to certain enzymes. The roles of these enzymes in the pathogenesis of the disease have been established [23].

High enzyme activity was seen during the vegetative growth phase of all the species of anthropophilic dermatophytes studied. The enzyme activity of *N. gypsea* (previously named *M. gypseum*) and *M. canis* was found to be high during both the vegetative and sporulation phases by Ranganathan et al. [24]. Zoophilic and geophilic species usually evoke a severe inflammatory response in humans [25] on infection and is almost and always severe. Whether the ability to produce high levels of protease during the sporulation phase by *N. gypsea* and *M. canis* is the cause of the severe nature of infection when they clinically manifest in their unusual host (man) warrants a detailed study. However, a possible correlation between the abilities to produce high levels of proteolytic enzymes during both sporulation and vegetative phases of growth to the severity of infection may not be ruled out. The lowest enzyme activity among the anthropophilic group was recorded in all the strains of *T. rubrum* during the sporulation phase compared to the vegetative growth phase in all the isolates. Rippon [3] has reported enhanced sporulation during parasitism. The low level of enzyme production during sporulation in *T. rubrum* might be the reason for the mild lesions produced in the host. The severity of the lesions produced by *T. rubrum* is less compared to other dermatophytes species. As Gokulshankar [16] reported, it is strikingly evident that all the clinical isolates of *T. rubrum* were from chronic cases, and the case history of three isolates indicates the persistence of lesions for more than two years. In general, it is understandable that the noninflammatory mild lesions would be neglected by the patients and, therefore, are untreated. Rippon [3] also noted that the protease production is highly host specific, and the organism showed reduced physiological activity when growing on their preferred host (animal/man). This is a clear exemplification of the well-established anthropophization of the parasitic dermatophyte species.

The medium used to study the enzyme activity during sporulation was Takashio broth (1/10 diluted Sabouraud's dextrose broth with KH2PO4 and MgSO4). The spores obtained in Takashio broth were asexual conidia, but during parasitism, the organism produces more arthroconidia. The study of the enzyme activity of *T. rubrum* during arthroconidia formation (produced during parasitism) is not possible because of the nonavailability of techniques to induce arthroconidial formation *in vitro*. Therefore, the low levels of protease activity of *T. rubrum* during sporulation phase cannot be directly correlated with pathogenesis.

Nevertheless, it is really intriguing to know the reason for low protease production during sporulation in all the anthropophilic groups of dermatophytes when the geophilic and zoophilic organisms showed almost statistically comparable levels of

*Evolution of Parasitism and Pathogenic Adaptations in Certain Medically Important Fungi DOI: http://dx.doi.org/10.5772/intechopen.105206*

protease production during both the phases of growth [26]. Is this moderation of enzyme activity during sporulation an adaptation of well-defined anthropophization?

### **12. Protease in** *M. furfur* **and pathogenesis**

Enzyme secretion is regarded as one of the prominent virulence factors that are exhibited by many pathogens. Protease is an important virulence factor in several yeasts, including infections caused by *Candida* species in humans [27, 28] and keratinolytic proteases of *Candida albicans* is involved in the invasion and digestion of human stratum corneum *in vitro* [29]. Lipases produced by *Malassezia* are generally considered to be potential pathogenic factors. However, Coutinho and Paula [30] had reported that all the strains of *Malassezia pachydermatis* isolated from dogs showed protease activity. Protease released by *Malassezia* species was proposed as the mediator of itch at free nerve endings in the skin and a contributor to the prominent pruritus seen in affected dogs [31]. Members of the genus *Malassezia* are reported to have a role in inflammatory to mild scalp conditions such as seborrheic dermatitis and dandruff, besides being implicated in pityriasis versicolor.

Seborrheic dermatitis is characterized by inflammation and desquamation in areas that are rich in sebaceous glands, such as the scalp, face, and upper trunk. Dandruff is a major cosmetic concern with noninflammatory scaling conditions of the scalp [32, 33]. The importance of *Malassezia* organisms in these scalp conditions has been supported by studies demonstrating parallel decreases in the number of organisms and the severity of the diseases [34, 35]. *Malassezia* organisms produce lipases, which can alter sebum production in the host and can produce break-out products such as free fatty acids on the skin surface, which is responsible for the clinical conditions [36]. *M. pachydermatis* strains are known to produce proteases that are linked to its parasitic mode of life [37].

The protease activity of the isolates of *M. furfur* from different clinical conditions, such as pityriasis versicolor, dandruff, and seborrheic dermatitis, showed varied activity. The protease production is mild from isolates of pityriasis versicolor, high in dandruff, and very high in seborrheic dermatitis (**Table 3**).

It is interesting to note that the low protease activity of *M. furfur* isolates corresponds to the chronicity of pityriasis infection, which is in a similar line to that of *T. rubrum* isolates from chronic cases of dermatophytosis. Ranganathan et al. [24] reported a similar finding on the relationship between chronicity and the low protease profile of *T. rubrum* isolates. The protease activity is high in isolates of seborrheic dermatitis, which again corresponds to the high level of inflammation in the patients.


#### **Table 3.**

*Comparative enzyme activity in M. furfur isolates from different clinical conditions.*

The role of protease in pathogenesis or severity of infection caused by *M. furfur* is not clearly known; however, studies of Gokulshankar [16] throw light on the possible role. However, in the earlier study conducted by Chen et al. [38], the culture extracts of *Malassezia* sp. with and without proteases failed to stimulate canine keratinocytes *in vitro.* Probably, the combined activity of lipases and proteases is responsible for the clinical condition caused by *M. furfur.*

### **13. Pigment production in** *T. rubrum*

*T. rubrum* is a typical example of an anthropophilic dermatophyte that is globally prevalent. Several studies from different geographical locations have documented that *T. rubrum* is one among the predominant dermatophyte species, which is the most frequent cause of human dermatophyte infections. As early as 1982, [39] have reported the ability of this species to cause persistent infection, which is often found to be refractory to treatment.

The unique feature, which differentiates *T. rubrum* from other species of dermatophyte, is the cherry red pigment produced by the organism. It is a useful tool for the identification of this species in conventional diagnostic mycology. The question that remains unanswered is: what is the role of the pigments in pathogenesis? Rippon [3] has reported that *T. rubrum* var. *nigricans* cause a severe lesion in humans when compared to the usual variety of *T. rubrum*. However, the nature of the pigment produced by *T. rubrum* and the external factors that influence the pigment production in *T. rubrum* are not clearly understood. Interestingly, during pigment production, the enhanced sporulation of the fungus has been noted [5]. Further enhanced sporulation is seen during active parasitism [3].

Gokulshankar [16] found that the color and nature of the pigment released by *T. rubrum* during sporulation and vegetative phase were different. However, both the pigments were highly soluble in methanol:chloroform (1:1) solution. The single band of pigment released during the sporulation phase was similar to that of one of the bands of the two fractions of the pigment released during the vegetative phase when run on thin-layer chromatography (TLC) plate. Furthermore, these two bands showed fluorescence under UV light. However, earlier studies [40] indicate that bands produced by *T. rubrum* in the different phases have a different spectral pattern, suggesting that the chemical nature of the pigment released by the organism during the sporulation phase is totally different from the vegetative phase. However, a detailed study on the chemistry of the pigment may establish its probable role in pathogenesis.

Gokulshankar [16] also employed the susceptibility pattern of pigment-producing and non-pigment-producing isolates of *T. rubrum* against the antifungals, such as griseofulvin and miconazole, as a measure to correlate pigment production to pathogenesis. It is interesting to note that the pigment-producing variants are less susceptible when compared to the non-pigment-producing isolates. However, how these pigments interfere with the antifungals to promote resistance in *T. rubrum* isolates is not clearly known and warrants a detailed study.

### **14. Role of melanin in** *C. neoformans*

One important characteristic of *C. neoformans* that differentiates its pathogenic isolates from the nonpathogenic isolates and other *Cryptococcus* species is its ability

### *Evolution of Parasitism and Pathogenic Adaptations in Certain Medically Important Fungi DOI: http://dx.doi.org/10.5772/intechopen.105206*

to form a brown to black pigment (melanin) on any medium that contains diphenolic compounds (such as cowitchseed/niger seed/bird seed or caffeic acid agar) [7, 41]. This pigment was first described by Staib [42]. The importance of melanin production in *C. neoformans* virulence was first demonstrated by several workers. Rhodes et al. [43] reported that naturally occurring *C. neoformans* mutants lacking melanin (Mel) were less virulent in the mice model than the strains that produce melanin. Other researchers further established this fact [44, 45].

*C. neoformans* melanogenesis is capable of conversion of dihydroxyphenols (DOPA to dopaquinone). This conversion is catalyzed by a phenoloxidase, which is present in *C. neoformans*, and this conversion is a rate-limited step because subsequent steps in the melanin pathway are spontaneous, such as (a) dopaquinone rearranging to dopachrome and (b) subsequent autoxidation to melanin [46]. However, *C. neoformans* does not possess the tyrosinase enzyme. The absence of tyrosinase makes *C. neoformans* incapable of endogenous production of DOPA [45]. Therefore, the organism has to be grown in a medium containing diphenolic compounds (such as bird seed agar) to produce melanin. In the environment, if *C. neoformans* isolates are able to acquire diphenolic compounds, it is possible for the organism to produce melanin with phenoloxidase. The human brain is usually rich in catecholamines (such as DOPA) and, therefore, becomes an ideal (or is it favorite?) target site for infection (cryptococcal meningitis) by *C. neoformans.* However, the deterrent factor is that *C. neoformans* cannot use catecholamines as sole the carbon source of living. Hence, it is to be understood that the brain is not a preferred "nutritional niche" for the growth of *C. neoformans* [47]; rather, it may be rightly called a "survival niche." Melanin production is, thus, a virulence factor in the pathogenesis providing a survival advantage in meningeal infections. *C. neoformans* is most likely able to use catecholamines in the brain to become melanized, thereby capable of protecting itself from oxidative damage.

Melanin-producing isolates have several advantages as they were resistant to damage by an *in vitro* epinephrine oxidative system [47] and were found to be protected from damage by hypochlorite and permanganate [48]. Thus, *C. neoformans* mutants lacking phenoloxidase enzyme are highly susceptible both in the natural habitat and in the host tissue when the environment is hostile (decreased chance of survival).

Wang and Casadevall [49] added to the knowledge by testing the survival of *C. neoformans* in the presence of reactive nitrogen intermediates, nitric oxide, and the epinephrine oxidative system. Wang and Casadevall [50, 51] experimentally proved the survival advantage by culturing *C. neoformans* cells in a medium containing L-DOPA to allow them to produce melanin. Melanized *Cryptococci* survived damage in the test systems, which was significantly better than nonmelanized cells of the same strain.

Furthermore, in the human host, melanin production makes *C. neoformans* less susceptible to amphotericin B than nonmelanized yeast cells, and this may contribute to the challenge in the management of cryptococcal infections in immunocompromised hosts [50, 51].

Gokulshankar [16] demonstrated that melanin-producing clinical variants are more resistant to antifungals, viz., flucytosine and amphotericin B, than the nonpigment-producing environmental isolates. The clinical isolates were basically from AIDS cases, and their ability to produce melanin in defined minimal media containing DOPA and in caffeic acid agar combined with less susceptibility to standard antifungals is of greater significance.

The co-inoculation studies of *C. neoformans* with *C. lunata* also gave interesting results [16]. The colony of *C. lunata* was found to overgrow and inhibit the colony of *C. neoformans*. However, this phenomenon was observed only when the environmental isolates of *C. neoformans* were co-inoculated with *C. lunata.* The clinical isolates of *C. neoformans* showed a pattern of co-dominance (mutual inhibition of colonies at a distance) when grown on SDA with *C. lunata*. This pattern was unique as the environmental isolates used in the study were nonpigment producers (mel-) and the clinical isolates were pigment producing (mel+). *C. lunata* also produces a black pigment (similar to melanin?). It could be that the pigment produced by *C. lunata* is the inhibiting factor for nonmelanized cells of *C. neoformans*, whereas the melanin-producing *C. neoformans* shows co-dominance with the *C. lunata* because there is a mutual inhibition among the organisms at a distance. This clearly suggests that melanin production in *C. neoformans* is a key factor to survive the competition of the other fungi/bacteria in its environmental niche. *C. neoformans* is usually found in its natural environment, viz., pigeon coups in the melanized state [52].

The feeding assay of *C. neoformans* by earthworms showed that both the non-pigment-producing environmental isolates got digested in the gut of all the four species of earthworms, while the recovery of the pigment-producing clinical isolates was possible from all four species of earthworms. It can be, therefore, presumed that the melanization of *C. neoformans* may help not only in the UV protection as reported by earlier workers but also in escaping the predation by soil organisms such as earthworms.

In summary, melanin production in *C. neoformans* may have multiple functions. It is not only essential for protecting this opportunistic pathogen from host defenses but also provides a survival advantage in the environment (**Figures 1** and **2**).

It makes sense if the clinical isolates of *C. neoformans* are virulent, but Casadevall and Perfect (1998) found that isolates from soil samples are virulent. The environmental isolates are found to have two important traits: capsule and melanin production. It stimulated the interest of several researchers who put forth the following questions: (i) why does a soil/environment dwelling organism, such as *C. neoformans*, need to possess virulence characteristics and (ii) how did this organism acquire the ability to cause infections in animals and humans when the passage through them (as intermediate host/vector) is not required for their replication or survival?

**Figure 1.** *Cryptococcus neoformans and its virulence factors.*

*Evolution of Parasitism and Pathogenic Adaptations in Certain Medically Important Fungi DOI: http://dx.doi.org/10.5772/intechopen.105206*

**Figure 2.**

*Cryptococcus neoformans: Plausible factors that lead to the development and maintenance of virulence in the environment.*

Bunting et al. [53] suggested that certain types of amoebae, *Acanthamobae polyphage*, can predate on *C. neoformans.* Steenbergen et al. [54] proposed that *C. neoformans* cells are phagocytosed by *A. castellani* and demonstrated the intracellular replication of yeast cells by the process of budding inside the phagosomes of the amoebae. They also did immunofluorescence microscopy and immunogold transmission electron microscopy to prove that the formation of polysaccharide-containing vesicles is associated with the intracellular growth of *C. neoformans* in amoebae. The phenomenon is similar to the one that is observed during the growth of *C. neoformans* in macrophages.

Melanin production, capsule synthesis, and phospholipase secretion were, therefore, required to escape the predation by amoebae. Therefore, the soil amoebae influence the survival traits of *C. neoformans*, which helps the organism for maintenance of the fungal virulence in the environment. Gokulshankar [16] demonstrated that the predation and digestion of nonmelanized cells are possible by four species of earthworms. Therefore, it is scientifically possible to consider the role of earthworms in maintaining of the virulence of *C. neoformans* in soil as well.

Further experiments of Rosas and Casadevall [55] confirmed that *in vitro* melanization makes *C. neoformans* less susceptible to hydrolytic enzymes. A feeding assay of C. *neoformans* with four different species of earthworms was carried out, and a similar kind of protection from the digestive enzymes of the earthworms could be the plausible reason for the recovery of only melanized cells of *C. neoformans* from the worm cast [16].

Melanin may also play a role in the protection of *C. neoformans* from the digestive enzymes released by the antagonistic microbes (soil fungi and bacteria) and provides a survival advantage during the constant and complex interactions of *C. neoformans* with other soil micro and macroflora.

## **15. Dimorphism and** *H. capsulatum*

The ability of the fungus to have a morphologic transition from a geophilic (saprobe lifestyle) multicellular mold form to a parasitic (pathogenic/infective form) unicellular yeast form is called dimorphism. This phenomenon is governed by temperature in *H. capsulatum.* This MY shift (mold-to-yeast conversion) is an important virulence factor, and isolates that are incapable of this shift are avirulent [3].

This process can be replicated in the microbiology laboratory by just changing the incubation temperatures from room temperature 25°C (saprobic phase) to 37°C (parasitic phase), and the shift is usually reversible.

*H. capsulatum* is able to produce a defect in macrophages by shutting down the respiratory burst activity, which is key microbicidal activity to address the intracellular pathogens. Histoplasmosis, in general, considered the "fungal equivalent" (homolog) to the bacterial infection (tuberculosis) caused by *Mycobacterium tuberculosis.* Interestingly, both *H. capsulatum* and *M. tuberculosis* are capable of exploiting the immune cells of the host (macrophage) and using them as a vehicle for causing infections (acute or persistent pulmonary and its dissemination to the organ system [56].

Gokulshankar [16] wanted to find whether yeast form is more pathogenic than mold form. However, we have demonstrated experimentally that intracellular growth inside macrophages is not only possible for yeast form of *H. capsulatum* but also for the mold form. The yeast cells get converted to mold form (observed as formation of hyphae) in the macrophages at an incubation temperature of 25°C. This shift from yeast to mold form inside macrophages indicates that the mold form could also be equally infective. However, due to the constant body temperature of 37°C of the human host, which favors yeast growth, mold form is seldom encountered in the human tissues during infection. The infectivity of the mold form of *H. capsulatum* is further confirmed by infection assay in garden lizard (poikilothermic animal model), where experimental lesions are possible by injecting mold/yeast form and incubating the animal at both 25°C and 37°C. The saprophytic survival of the yeast form is not possible for *H. capsulatum.* The existence of the yeast form of *H. capsulatum* in

**Figure 3.** *Histoplasma capsulatum and its virulence factors.*

### *Evolution of Parasitism and Pathogenic Adaptations in Certain Medically Important Fungi DOI: http://dx.doi.org/10.5772/intechopen.105206*

the soil is challenged by predating organisms such as earthworms (may be also by soil amoebae as in the case of *C. neoformans*). The yeast cells of *H. capsulatum* get digested inside the gut of earthworms, and their recovery was not possible. The mold form of *H. capsulatum* survives predation by earthworms. This could be attributed to the formation of ornamented macroconidia (tuberculate) by the mold culture of *H. capsulatum.* Probably, in the course of evolution, *H. capsulatum* managed to adapt itself for intracellular growth (inside phagocytic cells) by developing intracellular yeast form, while a mold form is inevitable for existence as a saprophyte in soil. The compact yeast form may be more resistant to the enzymatic degradation in the intracellular state than a mold form. Eissenberg et al. [57] proposed that yeast cells of *C. neoformans* adapt the mechanism of increasing the pH of the phagolysosome to manage and survive in the otherwise extremely hostile environment is by increasing the pH of the phagolysosome. Thus, dimorphism gives the advantage to *H. capsulatum* for two modes of survival: parasitic (as yeast) and saprophytic (as mold) (**Figure 3**).

### **16. Conclusion**

The ecological niche of different groups of dermatophytes varies from species to species. For example, in the genus *Microsporum*, *N. gypsea* is geophilic, while *M. canis* is zoophilic despite the fact that both of these organisms are basically keratinophilic in nature. For a long time, no clear-cut answer was previously given for how and why such a unique divergence in their habitat preference has emerged. Several studies have established the existence of homology at the genetic level between the geophilic

### **Figure 4.**

*Proposed probabilities of the evolution of saprophytic fungi into pathogenic fungi.*

dermatophytes and the highly evolved "present-day" obligate parasitic dermatophytes. Mycologists strongly believe that these anthropophilic parasites might have existed in the soil before and gradually lost all these soil-inhabiting characteristics in their process of evolution as obligate parasites.

In the light of the findings of Gokulshankar et al, it is presumed that the inability of anthropophilic dermatophytes to escape the predation or manage to survive in the earthworm gut might have also contributed to the shrinkage of their prevalence in earthworm-rich soils, and therefore, these dermatophytes would have been forced to select a new line of adaptation. The ubiquitous prevalence of different species of earthworms in the majority of soil types all over the world is known, and this fact makes it possible to hypothesize that these once saprophytic dermatophytes would have the chance to pass through the gut of earthworms during their existence in soil. Probably, their inability to escape the predation (unable to survive successfully in the gut during this passage) could have also contributed to their elimination from the natural habitat. Other contributing factors could be the antidermatophytic activity of the predominant keratinophilic fungi in soil (such as *C. keratinophillum)*. This inhibition coupled with antibiosis by other soil fungi (such as *Aspergillus* species) could also have played a role in the parasitic divergence of these dermatophytes. The role of other soil macroflora, such as mycophagous insects and mites, cannot be ruled out.

Similarly, the predation by earthworms (also by other nematodes and amoebae) may help in the maintenance of virulence in saprophytically existing pathogens such as *C. neoformans* and *H. capsulatum* (**Figure 4**).

### **Conflict of interest**

None to declare.

The major part of this chapter is an extract from the PhD work of the first author.

### **Note**

At the time of conduction of this experiment, *M. furfur* was commonly implicated with dandruff, but according to the new classification method of molecular biology, currently, *M. globosa* and *M. restricta* are both attributed to dandruff and other scalp conditions caused by these lipophilic yeasts.

*Evolution of Parasitism and Pathogenic Adaptations in Certain Medically Important Fungi DOI: http://dx.doi.org/10.5772/intechopen.105206*

## **Author details**

Gokul Shankar Sabesan1 \*, Ranjit Singh Aja2 , Ranjith Mehenderkar3 and Basanta Kumar Mohanty4

1 Faculty of Medicine, Department of Microbiology, Manipal University College Malaysia, Melaka, Malaysia

2 Department of Biotechnology, Prathyusha Engineering College Chennai, India

3 Faculty of Medicine, Deputy Dean- Preclinical, Quest International University Perak, Malaysia

4 Faculty of Medicine, Department of Pharmacology, Manipal University College Malaysia, Melaka, Malaysia

\*Address all correspondence to: gokul.shankar@manipal.edu.my; gokkavi@gmail.com.

© 2022 The Author(s). Licensee IntechOpen. 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.

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Section 3
