Evolution of Parasitism and Pathogenic Adaptations in Certain Medically Important Fungi

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.

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:

1. Obligate parasitism as seen in the case of certain anthropophilic dermatophytes and Malassezia species.

2. Parasitism to the extent of true pathogenesis as seen in the case of dimorphic systemic fungi, viz., Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis, and Paracoccidioides brasiliensis.

3. Parasitism is an accidental/opportunistic event, as seen in the case of C. neoformans and Candida species.

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.

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.

1. Geophilism of these fungi in the burrows of smaller mammals.

2. Colonization of the hair/fur of these small mammals.

3. Parasitism in these mammals leads to synanthropophization

4. Well-established anthropophization as seen in the case of T. rubrum by losing all the geophilic characteristics, such as conidial abundance and ornamentation, osmotolerance, heterothallic mating, etc. [4].

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:

1. Whether the lack of immune barrier in the host is the prime cause to elicit an infection (or)

2. The gradual development of parasitic adaptations in the course of evolution of Cryptococcus species have contributed to the parasitism?

3. Is there a changing face of this pathogen in the course of evolution? (opportunistic to truly pathogenic)

4. The adaptations to the parasitic life, which include the production of pigment (melanin), mannitol utilization, urease activity, and encapsulation in C. neoformans are unique and are seen only in certain fungi among the whole of the geophilic community. Do these characters provide an advantage for survival in the ecological niche for this pathogen?

5. Is the organism exhibiting a growing ecological niche with more and more newer habitats or reservoirs (droppings in different avifauna such as crows, water birds, etc.)

6. Better elucidation of such uniqueness in Cryptococcus species is essential for developing a better management strategy for fungal diseases.

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:

1. Geophilic: Free living organisms that exist as saprobes in soil subsisting on dead organic matter.

2. Zoophilic: Organisms that cannot constitutionally exist in soil as saprobes but evolved to live primarily on or in the bodies of animals other than humans.

3. Anthropophilic: Organisms that are unable to survive as free-living entities in soil but have evolved to be components of the microbiota of humans.

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

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

Organism	Virulence factors	Environmental advantage	Survival in Host/adaptability	Parasitic adaptation
C. neoformans	Capsule	Escape phagocytosis of amoebae	Escape phagocytosis by macrophages	Important virulence factors in human infection
	Melanin	Protection from Sunlight/UV; Escape predation from soil macroorganisms	Resistance to antifungals; Shield against immunologically active cells.	Able to use DOPA in CNS to cause meningitis
	Urease	Unknown. May contribute to the nutritional role in nitrogen acquisition.	Promotes nonlytic exocytosis	Survival advantage in a human host
	Phospholipase	Unknown	C. neoformans, calcineurin responds to stress caused by cell-wall-perturbing agents, physiological temperature of host, alkaline pH, high CO2, and high cation concentration [9]	Calcineurin activation is essential for; a. virulence and b. hyphal elongation during sexual reproduction (mating and monokaryotic fruiting) [9]
H. capsulatum	Ornamentation; Tuberculate macroconidia	May protect against predation by soil macroflora (mites, earthworms)	—	Spores disseminated by wind are inhaled
	Dimorphism	Survive as a mold at 25°C	Intracellular survival as yeast at 37°C; Resistance to microbiocidal products of neutrophils	Mold-to-yeast conversion is an important aspect of pathogenesis (similar phenomenon in other true pathogenic fungi; B. dermatitidis, C. immitis and P. brasiliensis).
Dermatophytes	Keratinase, collagenases	Nutrition (utilize keratin from dead animal issues)	Degrades scleroproteins in skin, hair, and nail
	Elastase	Nutrition (utilize keratin from dead animal issues)	Degrades elastin, scleroproteins	Enhances invasion of elastin containing tissue
	Pigment production		Resistance to antifungals.	Identification tool in diagnostic mycology
	Obligate parasitism	Loss of saprophytic survivability and soil association characteristics in certain anthropophilic dermatophytes	Enzyme moderation (protease).; Loss of hair perforation ability, enzymes (such as urease), osmotolerance, etc.; Exhibit slow growth rate.	Cause mild and chronic infections
Candida species	Cell wall glycoproteins	—	Adherence to epithelial surfaces
	Acid protease	—	Cleavage of IgA2
	Morphological variation; Ability to switch reversibly into different colony types at high frequencies.	—	Allows organisms to adapt to a different environment in the host.	Colonies of varying nature (smooth, rough, hat, fuzzy wrinkle, star, and stippled)
	Phospholipases	—	Hydrolyze ester linkages of glycophospholipids
	Secretory aspartic proteinases	—	Promote adherence and survival of the pathogen on mucosal surfaces.	Facilitate invasion of host tissues
	Biofilming	—	Resistance to antifungals
	Dimorphism	—	Ability to form hyphae helps in tissue invasion

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, 11, 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. tonsurans, 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 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

1. Did the inhibition by the soil/environmentally prevalent fungi forced M. furfur to adapt to an obligate parasitic/commensal existence?

2. Can this inhibition be considered as one of the early events in the course of evolution?? (But this hypothesis holds good if only there had been a past survival (existence) of Malassezia species or their related ancestors in soil).

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 (well-defined 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).

Test organisms	Number of isolates	Recovery in days/number of isolates
		10	20	30	40	50	60	90	120
T. rubrum	4	2+	—	—	—	—	—	—	—
T. mentagrophytes	4	2+	1+	—	—	—	—	—	—
T. tonsurans	4	—	—	—	—	—	—	—	—
T. violaceum	4	—	—	—	—	—	—	—	—
E. floccosum	4	—	—	—	—	—	—	—	—
N. gypsea	4	4+	4+	4+	4+	4+	4+	4+	4+
M. canis	4	4+	4+	3+	—	—	—	—	—

Table 1.

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

Note: + could be recovered, − could not be recovered.

Test organisms	Number of isolates	Recovery in days/number of isolates
		10	20	30	40	50	60	90	120
T. rubrum	4	2+	—	—	—	—	—	—	—
T. mentagrophytes	4	2+	1+	—	—	—	—	—	—
T. tonsurans	4	—	—	—	—	—	—	—	—
T. violaceum	4	—	—	—	—	—	—	—	—
E. floccosum	4	—	—	—	—	—	—	—	—
N. gypsea	4	4+	4+	4+	4+	4+	4+	4+	4+
M. canis	4	4+	4+	2+	—	—	—	—	—
M. furfur	4	—	—	—	—	—	—	—	—
C. neoformans	4	2+	—	—	—	—	—	—	—
H. capsulatum-Yeast form	2	—	—	—	—	—	—	—	—
H. capsulatum-Mold form	2	2+	2+	2+	2+	2+	2+	2+	2+

Table 2.

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

Note: + could be recovered, − could not be recovered.

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 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).

Clinical conditions	Enzyme activity (units)
	Mean	SD
Seborrheic dermatitis	188.57	91.51
Pityriasis versicolor	42.61	15.46
Dandruff	140.36	62.60
F-value	37.075
P-value	0.000

Table 3.

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

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. 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 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 non-pigment-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).

Figure 1.

Cryptococcus neoformans and its virulence factors.

Figure 2.

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

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?

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 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).

Figure 3.

Histoplasma capsulatum and its virulence factors.

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 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).

Figure 4.

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

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.