Opportunistic Pathogens of the Genus Cryptococcus in Louis Pasteur Days and in 200th Anniversary of his Birth

As this year we are celebrating the 200th anniversary of the birth of Louis Pasteur (www.pasteur2022.com), microbiologists probably wonder what was the state of knowledge about the subject of their studies during times of Pasteur. This question also arose in my mind, namely, what was known about the fungi of the genus Cryptococcus in Louis Pasteur days? Long before the role of bacteria as the etiological agents of diseases was described [104] and before the germ theory of diseases was announced by Louis Pasteur [102, 103, 105], the pathogenicity of fungi was noticed [117]. Just during this period, before Pasteur disproved the theory of spontaneous generation [103], and long before the definition of „infection” was formulated, Robert Remak made a significant discovery in 1837 demonstrating the presence of fungal hyphae in yellow cup-shaped crusts (scutula). Indeed the description of Trichophyton schöenleinii (then known as Achorion schöenleinii) initiated the development of modern medical mycology [117] and, most importantly, gave rise to the perception of microscopic fungi, and thus other microorganisms, as potential etiological agents of infectious diseases. It wasn’t long before other etiological agents of infectious diseases were discovered. Soon after the discovery of Remak, in 1843, David Gruby, working in Paris, proved that Microsporum audouinii is the etiological agent of tinea microsporica [1]. In turn, in 1848 the Swedish microbiologist Pehr Henrik Malmsten isolated and described Trichophyton tonsurans as an etiological agent of tinea trichophytica [1], which was an extraordinary achievement in the pre-Pasteur period.

Slightly before the original descriptions of dermatophyte species appeared, the genus Cryptococcus was established and first described in 1833 by Kützing, who isolated this microorganism as saprophytic from a dirty window [74]. According to his assumption and later suggestions of Benham [6] and Vuillemin [133], this genus was to gather yeast-like fungi that did not produce hyphae, are not capable of undergoing ascospore formation and showing inability to ferment carbon sources. A year before Louis Pasteur’s death and 10 years after announcing the Koch’s postulates [1, 5], Cryptococcus neoformans was discovered as an etiological agent of cryptococcosis, a disease entity, which in many parts of the world more often leads to death in the group of HIV/AIDS patients than tuberculosis [101]. This yeast-like fungus was first isolated in 1894 by Busse [14, 15] and Buschke [13], from sarcoma-like lesion of the tibia from a 31-year-old woman and skin eruptions of the patient suspected of coccidioidomycosis, respectively. While it remains unclear whether these two scientists were aware of Koch’s postulates and Pasteur’s discoveries, it is apparent that Koch’s postulates and Pasteur’s assumptions were fully met by both researchers. Importantly, Busse and Buschke isolated the same etiological agent from the examined lesions, ultimately obtaining pure cultures of C. neoformans. It can be easily noticed that Koch's postulates and Pasteur’s assumptions were fully met by both researchers. In the same year Francesco Sanfelice isolated mentioned fungus as saprophytic microorganism from peach juice in Italy incorrectly classifying it as Saccharomyces neoformans, but species epithet „neoformans” is accepted till these days [115]. Later, in 1896 Sanfelice for the second time isolated this fungus from lymph node of a cattle indicating on its pathogenic nature [116]. All these findings did not escape the attention of Vuillemin, who transferred both Cryptococcus hominis and S. neoformans, respectively described by Busse and Sanfelice, to the genus Cryptococcus, which comprises significantly different species than members of the Saccharomyces genus – fermenting yeasts [133]. The first discoveries by Busse and Buschke and the later Sanfelice and Frothingham [41] allowed to establish that C. neoformans causes infection in both humans and animals. As it is currently known C. neoformans is not the only etiological agent of cryptococcosis. A year after descriptions of cryptococcosis by Busse and Buschke, Ferdinand Curtis postulated that Cryptococcus gattii, described then by him as Saccharomyces subcutaneous tumefaciens may also be an etiological agent of this disease entity [24]. The first cases of cryptococcal meningitis, caused respectively by C. neoformans and C. gattii, were noticed in 1914 by Verse in a dead woman [129] and in 1970 by Gatti and Eeckels in a 7-year-old leukemic child in Republic of Congo [45]. The significant progress in immunology in the 20th century and the availability of new serological techniques allowed Evans in 1949 to distinguish 3 different serotypes in the pool of pathogenic strains of Cryptococcus spp., which were isolated from humans [35]. Nearly 20 years later, Ralph Vogel added serotype D to previously described by Evans serotypes A, B and C. The new serotype introduced by Vogel was detected by using the indirect fluorescent antibody test (IFA) [132]. A growing awareness among clinicians and the availability of new diagnostic tools led to an increase of the new strains of Cryptococcus spp. isolated from sick people and animals. In the second half of the twentieth century, the name C. neoformans had 40 synonyms [112] and only in 1989 Jack W. Fell proposed conserving the genus name as Cryptococcus Vuillemin [37]. Still in 2010, in the pages of the 5th edition of the extensive monograph “The yeasts: a taxonomic study” [38], the genus Cryptococcus included 70 species of both pathogenic and nonpathogenic yeasts. In the clinical and practical context, the classification of strains isolated from humans and animals, based on determination of their serotype, was particularly important. Since the discovery of the teleomorphs Filobasidiella neoformans and F. bacillispora [64,65,66], serotypes A and D as well as B and C have been assigned to them, respectively [67, 71]. According to the newest nomenclature modifications and the recommendations established during the XVIII International Botanical Congress, (Melbourne, July 18–22, 2011) the species names of basidiomycetous yeasts have also undergone changes [55, 56]. In case of F. bacillispora, the initial change was to C. bacillisporus, then to C. neoformans var. gattii, and finally to C. gattii [68]. In turn, F. neoformans was changed to C. neoformans, for which two varieties came to be distinguished, namely C. neoformans var. neoformans (representing serotype D) and C. neoformans var. grubii (representing serotype A) [39]. Only strains representing serotype D were classified in the first varietas, and isolates of serotype A in the second [39]. The presence of four distinct serotypes (A, B, C, D) was also confirmed by Wilson in 1968 [135], who showed significant differences on the level of antigens present in polysaccharide capsule. Currently, based on advanced phylogenetic analyzes and studies at the level of genotypes and phenotypes, the Cryptococcus species complex includes seven haploid and four hybrid species [54]. Thus, there are currently two acceptable species within the Cryptococcus neoformans species complex: C. neoformans (serotype A) and C. deneoformans (serotype D). Strains that are hybrids of serotypes A and D – Cryptococcus neoformans × Cryptococcus deneoformans were also isolated from the natural environment. In turn, additional molecular characteristics of the strains currently included in the Cryptococcus gattii species complex revealed the existence of five separate species: C. gattii, C. deuterogattii, C. decagattii (all represent serotype B), C. bacillisporus and C. tetragattii (represent serotype C). The existence of three interspecific hybrids has also been demonstrated: Cryptococcus neoformans × Cryptococcus gattii (AB serotype), Cryptococcus neoformans × Cryptococcus deuterogattii (AB serotype) and Cryptococcus deneoformans × Cryptococcus gattii (DB serotype) [54]. All the mentioned species were isolated several times as aetiological agents of different disease entities in humans and animals. The nomenclature adopted in 2015 was the result of earlier efforts to systematize the naming of these pathogenic species. Earlier attempts to organize the terminology were based on the Amplified Fragment Length Polymorphism, AFLP [8]. There have also been attempts to introduce genotype nomenclature based on the Restriction Fragment Length Polymorphism, RFLP [84]. Both of these approaches provided a solid base to propose new solution by Hagen et al. [54]. Phylogenetic analyzes carried out in recent years shed new light on the proposed new systematics of the genus Cryptococcus. Based on four genes, namely LAC, URA5, mtLrRNA and ITS, it was estimated that the C. neoformans species complex split from the C. gattii species complex about 37 million years ago, and C. neoformans and C. deneoformans species evolved from a common ancestor approximately 18.5 million years ago. In turn, C. gattii and C. bacillisporus differentiated into separate species about 9.5 million years ago [138]. Recent calculations based on the analysis of the genomes suggest that the divergence between species of the C. neoformans and Cryptococcus gattii complexes could have occurred 80 to 100 million years ago [17]. Considering this finding, it is striking that the participation of individual species in causing various forms of cryptococcosis is unequal. Specifically, about 95% of cryptococcal infections are caused by strains of the species C. neoformans (serotype A), and the remaining 4% to 5% of infections are caused by C. deneoformans (serotype D) or strains of the C. gattii species complex (serotypes B and C) [81].

Virulence/pathogenicity has been defined as the relative ability of a microorganism to enter, cause tissue damage, and proliferate in the host body. Mortality is a commonly used measure of virulence/pathogenicity in human populations [18, 106]. Most pathogenic microorganisms have several virulence factors that can damage the host’s tissues, which determines the overall net virulence phenotype [82]. Members of the Cryptococcus species complex present a particularly rich spectrum of virulence factors (recently presented during the webinar organized by Polish Mycological Society [https://tinyurl.com/bdhhurrv]). Members of the Cryptococcus species complex are well known because they produce two virulence factors that significantly interfere with the host’s immune system, namely the polysaccharide capsule and melanin. These factors not only effectively disrupt the functions of the immune system, but also protect against phagocytosis and reactive oxygen species (ROS) released by elements of the immune system [141].

The virulence factor of pathogenic Cryptococcus species that is of considerable clinical importance is the polysaccharide capsule [82]. The synthesis of the capsule material begins in the early stages of infection and is important to the development of the disease to such an extent that acapsular strains are avirulent in murine models of cryptococcosis [20]. The capsule provides resistance to stress factors such as dehydration, to which fungal cells are particularly exposed in the natural environment [143]. During infection, the capsule helps to reduce the risk of phagocytosis and increases the chance of survival of engulfed cryptococci through several mechanisms. For instance, capsule impairs the recognition of fungal cell surface antigens by macrophages, contributing to the avoidance of phagocytosis [88]. Moreover, capsular polysaccharides have antioxidant properties, protecting fungal cells against the toxic effects of ROS produced during intracellular parasitism inside the phagolysosome [143]. Capsule enlargement can also confer resistance to antimicrobial peptides and amphotericin B, but increases susceptibility to fluconazole [34, 143]. Chemically capsule consists mainly of glucuronoxylomannan (GXM, approx. 90%), galactoxylomannan (GXMGal, 5–8%), and mannoproteins (∼ 1%). Variation at the level of this structure and in the proportions of the individual components of the capsule changes its antigenic properties, which are the basis for distinguishing between serotypes[16, 135]. Fluctuations in the size of the polysaccharide capsule impact the chances of yeast cell survival inside the macrophages [98].

There are several factors which can induce capsule production in vitro, such as CO₂, iron and other nutrient deficiency, and contact with mammalian serum [34]. Quantitative and qualitative changes at the capsule level result in the formation of a very heterogeneous population of Cryptococcus cells, differing in epitope composition, which reduces the effectiveness of a normal immune response [44]. Moreover, the structure and composition of the capsule can undergo microevolution even during the in vitro conditions, thanks to which the microbial population is phenotypically and antigenically variable depending on the growth conditions of the culture [83]. It is well known that the increase in capsule density makes it less permeable to elements of the immune response, such as antibodies, the complement system or antimicrobial peptides [34]. Capsule growth in thickness and increase in its density can be induced by various environmental factors. The in vitro studies showed that polysaccharides contained in the capsule are not only bound to the cell wall, but also released into the culture medium (exopolysaccharides). During infection, they can be found in tissues, cerebrospinal fluid, and blood [40]. Exopolysaccharides promote disease development through multiple mechanisms. Both GXM and GXMGal induce apoptosis of immune cells by activating membrane FasL/Fas proteins leading to the formation of complexes (trimerization) which initiates apoptosis in helper lymphocytes [130]. The secreted exopolysaccharides may also disturb the production of antibodies, induce conditions of complement deficiency [142], inhibit leukocyte migration [33], reduce the penetration of immune cells across the blood-brain barrier [26], or stimulate the production of cytokines and chemokines [128]. Thus, the polysaccharide capsule protects Cryptococcal cells in various ways, both against specific and non-specific mechanisms of host immune system defense, as well as against many environmental physico-chemical factors.

Another virulence factor of key importance for members of Cryptococcus species complex is the accumulation of dark pigment melanin in the cell wall, which may also be released extracellularly [32]. Unlike some fungi which constitutively synthesize dihydroxynaphthalene (DHN) melanin, Cryptococcus spp. produces DOPA-melanin only under induction conditions, in the presence of exogenous substrates such as 3,4-dihydroxyphenylalanine (DOPA) and other di/polyphenol compounds such as epinephrine and norepinephrine [12]. It is commonly known that the concentration of catecholamines, including adrenaline and noradrenaline, which are well known neurotransmitters, is higher in the cerebrospinal fluid than in the blood. This explains the neurotropism manifested by the cells of pathogenic Cryptococcus spp. [34]. Melanin synthesis depends on the enzyme diphenol oxidase, encoded by two genes: LAC1 and LAC2, but only LAC1 is significantly expressed under most conditions and its deletion results in a reduction in virulence [107]. Notably, the ability to synthesize melanin, and the presence of polysaccharide capsule, is not restricted to members of pathogenic Cryptococcus species complex. The presence of these virulence factors even in saprophytic species indicates the importance of these natural defense mechanisms for functioning in a changing natural environment [109]. Melanin plays an important role in changing environmental conditions, i.e. it protects against enzymatic degradation by soil microorganisms (amoebas), UV and gamma radiation [19, 62], heavy metals, and high temperature [90]. Melanin along with the capsule is described as one of the most important virulence factors for members of the Cryptococcus species complex [69]. Mutants incapable of melanization show much lower virulence [72]. Melanin has been shown to play a key role in the spread of pathogen cells from the lungs to the CNS [91], and to influence the host cytokine production [28]. This natural pigment may also directly bind and reduce efficacy of selected antifungal drugs, as has been shown for amphotericin B [27]. Laccase enzymes are mainly responsible for the synthesis of melanin which interfere with the massive release of ROS from cells, in part through iron sequestration and oxidation during the infection [139]. Moreover, it has also been shown that enzyme proteins of the laccase group are not only involved in the synthesis of melanin, but also play a role in the process of non-lytic exocytosis, allowing fungal cells to escape from macrophages, which in fact resembles the process of vomocytosis [96].

Urease is yet another important virulence factor common to fungi of the genus Cryptococcus. Urease catalyzes the decomposition of urea into carbon dioxide and ammonia, what is also manifested by an increase in pH. Urease enables yeast cells to use urea as a sole nitrogen source [141]. During infection, urease alkalizes the internal environment of the phagolysosome, limiting damage caused by the macrophage, and prolonging intracellular parasitism [42]. Relatively recently, it was shown that urease also influences non-lytic macrophage exocytosis, known as the vomocytosis phenomenon [42]. Urease has been shown to be required for CNS colonization as it promotes the sequestration of yeast cells in the microcapillary vessels of the brain. It has also been hypothesized that ammonia promotes the adhesion of C. neoformans / C. gattii cells to the surface of vascular endothelial cells, either by increasing the expression of adhesins on the endothelium, or by a direct toxic effect on the integrity of the tight connections of the blood-brain barrier, which facilitates invasion into the CNS [97]. Thus, urease contributes to virulence by allowing the pathogen to survive and spread in macrophages and invade the CNS. Interestingly, inhibition of urease activity does not block the development of infection, as mutants deficient in urease activity are still pathogenic in animal models and disseminated infections in humans caused by urease negative strains have been described [127].

One of the most important virulence determinants which enables the development of infection in the mammalian body is the ability to grow and proliferate at 37°C. It is a feature common to all pathogenic species clustered in the Cryptococcus species complex, although it is also found in some saprophytic strains of the genus Cryptococcus. This ability is believed to have allowed the members of Cryptococcus species complex to evolve into successful human and animal pathogens (reviewed in) [29]. Several proteins important for high temperature tolerance have been characterized [123]. Proteins important for high temperature growth include manganese-containing mitochondrial superoxide dismutase, which plays an important role in the response to this type of stress [50]. The importance of trechalose, a sugar specifically synthesized in fungal cells, that protects against various types of stress, including heat, was also described. Components of the trehalose biosynthetic pathway have been shown to be required for high temperature growth and thus determine the virulence of pathogen cells [134]. Various signal transduction pathways also play a role in detecting and responding to heat stress. These include the calcineurin pathway [63, 92], MAP kinase pathways, including the protein kinase C (PKC) cell integrity signaling pathway [47] and the high-osmolarity glycerol (HOG) mitogen-activated protein kinase pathway [140] and the Ras signaling pathway [3]. The pathway with the best documented influence on the ability to grow in mammal body temperature is the calcineurin A pathway. C. neoformans mutants with a deletion of the CNA1 gene (encoding calcineurin catalytic subunit) are not able to grow at temperatures above 35°C and are avirulent. Apart from the involvement of the calcineurin pathway in thermotolerance, this pathway participates in the response to other types of stress, and thus significantly influences the virulence [34].

An indispensable ability for the survival of yeast cells in the environment and in the host is the adaptation of the pathogen to the changing pH. It is commonly known that the transcription factor Rim101 is involved in the alkaline pH response and is the major protein involved in this adaptation [99]. This protein indirectly influences also virulence factors, such as polysaccharide capsule, and Titan cell formation, and impact the integrity of the cell wall [100].

Despite a general strategy to avoiding phagocytosis, Cryptococcus spp cells require macrophages to efficiently leave the lung environment and spread to other host organs, including crossing the blood-brain barrier and penetration into the CNS. This strategy, known as the Trojan horse tactic, allows the infection to spread effectively. It has been shown that fungal cells inside macrophages remain viable and replicate efficiently in the acidic environment of the phagolysosome [23]. It is therefore puzzling that, on the one hand, Cryptococcus spp cells have developed mechanisms to avoid phagocytosis, and on the other hand, proliferation inside macrophages promotes the infection into other organs. These apparently contradictory mechanisms are, in fact, two complementary strategies that account for the enormous plasticity of the pathogen’s cells depending on the prevailing conditions. In the context of antiphagocytic activity, the App1 protein, discovered by the Luberto et al. [76], plays an important role. This protein was first identified in the serum of AIDS patients and it has been shown that its release effectively inhibits the macrophage phagocytosis process [76]. In turn, proliferation inside macrophage phagolysosomes and transition to the CNS may be considered profitable for the pathogen’s cells. Supporting this view, is an evolutionary mechanism called vomocytosis that allows the fungal cells to leave the interior of macrophages without any negative effect on the pathogen and host cells. The mechanism of release from macrophages without damaging the host cells and thus without inducing inflammation, so far has been described only for members of the Cryptococcus species complex [77]. Vomocytosis, also called non-lytic exocytosis, is the fusion of the phagolysosome membrane with the macrophage plasma membrane, as a result of which the fungi are pushed out of the phagocyte. The urease catalyzes the hydrolysis of urea to form ammonia, which raises the phagosomal pH [118]. The alkalization of the phagolysosome environment causes the yeast to enter a resting state in which intracellular replication is delayed. This prolongs the intracellular parasitism of cells and increases the probability that fungal cells will have time to move from the lungs to the CNS before the yeasts will be released in the latent phase in an environment unfavorable for them [42].

An interesting and relatively recently discovered virulence factor are tubular mitochondria, the presence of which has so far been described only in members of the Cryptococcus gattii species complex [11, 131]. Tubular mitochondria form as a result of fusion, in a reversable process. The presence of this type of mitochondria in a subset of the population leads to the so-called „division of labor” [131]. The formation of cells with this phenotype is stimulated by host ROS, which are released in large quantities. As a result, some cells in the population that developed tubular mitochondria stop proliferating and are directly involved in neutralizing oxidative stress, allowing the remaining cells to proliferate intensively. It is currently known that C. deuterogattii (R265) strain that emerged recently on Vancouver Island, Canada due to its ability to form tubular mitochondria is characterized by a particularly hypervirulent phenotype [78, 89]. This is supported by the fact that mitochondria can be the source of the rapid evolution of pathogen virulence as their genomes are present in high copy numbers and show a much higher mutation rate than nuclear DNA [53].

The relatively recently described extracellular vesicles (EVs) seem to play an important role in cryptococcosis. Cells of the genus Cryptococcus produce EVs with a diameter of 80 to 500 nm, which cross the cell wall and are released into the environment. It is considered that fungal EVs participate in many biological processes related to virulence including induction of antifungal resistance and biofilm formation or transfer of virulence-associated molecules and modulation of host immune cells [111]. It has been shown that EVs are directly involved in the transport of the main component of the polysaccharide capsule – glucuronoxylomannan (GXM), but also such lipids as glucosylceramide and some sterols. These vesicles are also observed in acapsular strains, which suggests that Cryptococcus spp. may use vesicular transport to secrete very different compounds. EVs are also released during the proliferation of cryptococcal cells inside macrophages, suggesting a role in pathogenesis [75]. This is confirmed by the fact that the glucosylceramide released in this way induces the production of anticryptococcal antibodies during human infection [113]. It is currently known that glucosylceramide regulates the virulence of Cryptococcus spp. cells and is essential for fungal growth at neutral/alkaline pH in vitro and in vivo [110].

It has been shown that most of the virulence factors described herein can be regulated by both environmental factors and host environment. This means that the expression of virulence factors may be different in the natural environment compared to the environment of the host. A change in the expression level of individual virulence factors may affect the host-pathogen interaction, the host immune response, and finally the susceptibility of the pathogen’s cells to antifungal drugs [85].

Morphological transformation, called in some cases phenotype switching, is a reversible phenomenon that occurs both in vitro as in vivo in case of cultivable fungal species. It is defined as the spontaneous appearance of cells with altered morphology accompanied by a rate faster than that known for somatic mutations [59, 122]. Morphological transformation involves a series of changes at the cellular level, but is a reversible, controlled process, which distinguishes it from random changes caused by point mutations in the genome. For this reason, in vivo morphological transitions of the pathogen often impact the host-pathogen interaction diametrically. For instance, entirely different virulence factors are associated with blastoconidia than with other mycelial forms [59, 60]. Fungi have developed multiple regulatory pathways that recognize several environmental changes in response to which they react by switching phenotypes. The environmental signals inducing phenotype switching include the presence of blood serum, changes in temperature, pH, concentration of CO₂ and specific micronutrients. Those signals can trigger a transformation of yeast cells into mycelia or vice versa, or lead to an increase in the thickness of the capsule and initiate titanization process [60, 145]. Thanks to the ability to respond to signals from the environment, pathogenic fungi can dynamically adapt to changing conditions both in the environment and in the infected host. It is well known that the ability to switch phenotypes is one of the mechanisms to avert the host immune response [52, 60]. Microorganisms must constantly adapt to changing environments. In clonal populations this can be achieved by generating diversity through phenotype switching [73].

The phenotype switching phenomenon was first described in Candida albicans almost 40 years ago [120, 121]. In many pathogenic fungi, the ability to alter cell morphology is an integral part of the infection cycle. Dimorphic fungi of the genera Histoplasma, Blastomyces, Paracoccidioides, Emergomyces, Sporothrix and Talaromyces marneffei develop in the environment mycelial form, while in the host undergo transformation into the yeast form, which allows dissemination [125]. C. albicans and Candida dubliniensis, on the other hand, under appropriate conditions, both in vitro and in vivo, can switch between yeast and mycelial form. In the case of C. albicans and C. dubliniensis, blastoconidia play an important role in the spread of the pathogen cells within body fluids, while mycelial form plays an important role at the stage of tissue and organ colonization [80]. The recently described Goliath cells represent another manifestation of the morphological transformation specific only to some Candida species. Goliath cells are formed primarily under zinc starvation conditions what is related with immune system’s strategy of limiting the development of infection [79, 136]. Phylogenetic and phenotypic analyses showed that the cell enlargement as response to zinc starvation was species-specific and developed in the common ancestor of C. albicans, C. dubliniensis and C. tropicalis and was not observed in other Candida species studied so far [2, 79]. It is well known that many other species of the genus Candida can form pseudohyphae, however these structures do not display the virulence characteristics known to true hyphae [119].

Some filamentous fungi also have an ability to induce a transformation that is not associated with changing the shape of the cell, but with a significant increase in size and in the thickness of the cell wall. [125]. One example are pathogens of the genus Emmonsia that cause adiaspiromycosis. In the lung tissue, the conidia (typically 2–4 μm in diameter) of some Emmonsia species grow in size, reaching a diameter of 40–500 μm and increasing in volume up to a million times [9] transforming into adiaspores. These structures are de facto non-endosporulating spherules. Species of the genus Emmonsia are phylogenetically closely related with Coccidioides but the last one has endosporulating spherules unique among fungal pathogens, which within the infected tissue can achieve 30–80 μm in diameter [58, 125].

Pathogenic species of the genus Cryptococcus developed the richest spectrum of virulence factors which have contributed to their evolutionary success as pathogens [7, 29]. One of the key virulence factors is the ability to undergo morphological transformation into giant (Titan) cells. Some literature sources questioned that Titan cells are necessary for infection development as some clinical isolates of C. neoformans are presumably not capable of undergoing this morphological transition [25, 57, 124]. Such cells, due to their ten to twenty times larger volume, can effectively avoid macrophage phagocytosis. The unique phenotype of these cells consists not only of their size (diameter > 10 μm), but also significantly enlarged capsule, a thick layer of the cell wall, the presence of a large central vacuole and one polyploid nucleus [48, 94, 146]. Titan cells are not a final phenotype and are still capable of proliferation, producing both haploids, diploids and aneuploids daughter cells. In addition, Titan cells, as well as daughter cells, especially those with an aneuploid set of chromosomes, exhibit resistance to a number of physical and chemical factors [48]. The protocols for the induction of Titan cells in the presence of serum (10% or 5% fetal bovine serum, FBS), published simultaneously in 2018 [25, 124], make it possible to conduct large-scale in vitro studies on the titanization process. It has been shown that this ability to undergo such specific morphological transformation is unique for pathogenic yeasts of the Cryptococcus species complex [30]. It was confirmed then by two independent research centers that, as in the case of members of the C. neoformans species complex, species of the complex C. gattii, were able to form Titan cells, respectively according to commonly known in vitro serum protocol [31] and the newest protocol based on RPMI medium [114]. Moreover, recently it has been shown that strategies for titanization in case of members of C. gattii and C. neoformans species complexes are significantly different. While for C. neoformans typical is synchronous occurrence of cells enlargement and polyploidization, in the case of C. gattii we can observe delay in DNA endoreduplication and cell growth to around 30 μm as a haploid cell. Only after ∼ 3 days incubation under titanization conditions DNA endoreduplication can be observed, which finally leads to uninucleate polyploid cells [114]. Moreover, this process was equally effective. At the same time, it has been shown that the incubation conditions provided according to the protocol described by Hommel [57] yield typical Titan cells only for members of the C. neoformans species complex [31]. None of the tested strains belonging to C. gattii species complex was able to form Titan cells under such conditions [31]. More recent studies allowed to identify the minimum conditions sufficient to induce Titan cell formation in case of all the members of Cryptococcus species complex. Thus, it has been shown that, contrary to the assumptions made so far, serum is not a necessary and irreplaceable factor inducing the titanization process. This formed the basis for the development of a new serum-independent Titan cell induction protocols [31, 114]. In addition to determining the minimum quantitative and qualitative composition of the medium conditioning the formation of giant cells, the key role of the slightly alkaline pH (7.2–7.3) of the liquid medium used in the induction of Titan cells formation has been demonstrated [31]. Moreover, it was showed that Titan cells can be formed under lower temperature than typical for mammal body [30]. Such a new findings allow to conclude that the titanization process has been known to a limited extent only. It is commonly known, that formation of Titan cells allows averting the host immune attack [145] and it is likely a unique feature of human and animal fungal pathogens belonging to Cryptococcus neoformans/C. gattii species complex [30]. Moreover, the ability to form enlarged polyploid Titan cells seems to be important for dissemination of cryptococcosis yet it remains poorly described [145].

The qualitative changes that occur at the cell surface as a result of titanization have also been described [4, 86]. C. neoformans population present in the patient’s body is dominated by typical cells (5–7 μm in diameter) with a capsule consisting mainly of GXM and GalXM and a cell wall containing chitin, chitosan, α-glucan, melanin and β-glucan. In addition to typical blastoconidia, a significant percentage (∼ 10–20%) may constitute Titan cells with a diameter above 10 μm, which show thickened cell wall resulting from the deposition of additional chitin layers at the expense of β-1,3-glucan [4]. Additionally, in the cell walls of Titan cells there is a more exposed mannan layer [86]. It is now known that the population of C. neoformans found in body fluids is much more diverse. The so-called microcells with diameter below 1 μm and having thickened cell walls [36] and the so-called titanides (2–4 μm in diameter) with an oval shape and thinner cell walls have been identified. Titanides are associated with the induction of Titan cells in vitro, but surprisingly have not yet been observed in vivo [25]. Undoubtedly, the differentiation of cell sizes in the population present in the infected organism significantly influences the host-pathogen interactions. Some studies suggest that cell size varies depending on where the infection is located. Cells taken from the lungs of mice had thicker capsules and were larger, while cells isolated from the brain were smaller with thinner capsules [21]. This organ-dependent variation in cell size of Cryptococcus spp. has been also reported in human infections [137]. Different cell sizes may be advantageous at various stages of infection. For example, smaller cells may aid in proliferation and survival in macrophages, while larger cells may facilitate pathogen survival in lung tissue under conditions of increased oxidative stress. It has been shown, inter alia, that during the infection, a significant proportion of the yeast in the lungs has a volume of up to 900 times greater than that of cells grown under standard laboratory conditions [144]. This hypothesis seems to be the most correct since the highest percentage of Titan cells and also those with the largest size have been found so far in lungs (up to 100 μm including the capsule) [94]. It has also been shown that these cells promote pathogenesis by reducing the rate of phagocytosis and increasing stress tolerance [48, 93]. In the context of pathogenesis, the faster rate of budding (∼ 60 min/per bud) and a higher degree of melanization, demonstrated for Titan cells, also seem to be important [48]. It has also been shown that Titan cells grow from senescent cells and that the daughter cells they produce are of typical size [57].

It is currently known that the induction of titanization is mediated by Gpr5 and Ste3 G-protein coupled receptors which induce the protein kinase A cycle (cAMP/PKA) via the α subunit of Gpa1 [95]. Cyclic AMP also regulates the formation of polysaccharide capsule, in particular by acting on the ubiquitin-proteasome pathway [46]. It has been shown that adequate intracellular concentration of cAMP is required for titanization thus cells deficient in adenylate cyclase do not form Titan cells [25]. Carbon dioxide is also required to induce the titanization process. The carbonic anhydrase enzyme converts CO₂ into HCO₃⁻, which activates adenylate cyclase [49], so a higher concentration of CO₂ (optimally 5%) induces cell growth via the cAMP pathway [124]. In the context of the information provided here, it is also worth noting that the appropriate concentration of HCO₃⁻ in the blood is extremely important for maintaining the proper pH [51]. The role of Titan cells in cryptococcosis has not been fully elucidated, but they have been shown to be involved in several processes that allow to evade the immune system and persist in the host organism for a long time. The number of Titan cells is especially high during asymptomatic infections, which led to the conclusion that this type of cells can survive in the host organism for a long time as a latent form without causing disease [144]. Titan cells cannot be phagocytosed due to their size. Interestingly, they are also able to confer resistance to phagocytosis to smaller, neighboring yeast cells, what may be caused by extracellular vesicles produced by the pathogen [34, 93]. Given how large their capsule is, it is also likely that the exopolysaccharide secreted may have a strong influence on the surrounding environment and phagocytic cells. The number of formed Titan cells is highly dependent on the host environment, and therefore the percentage of these cells observed in vivo can vary considerably, as shown in experimentally infected mice. In mice that develop a Th1-type immune response (interferon-γ and TNF-α dependent), the percentage of Titan cells is low (approximately 15%). In contrast, mice that induce a Th2-type response (related with interleukines 4, 5, 10, and 13) have a very high percentage of Titan cells, even above 50% of the total population of Cryptococcus spp. cells [43]. Currently, it is impossible to clearly explain this relationship, however, Th2-type immune response which is de facto anti-inflammatory response is associated with a less aggressive environment, which, unlike the environment generated by Th1 lymphocytes and the associated with them proinflammatory response, significantly favors the formation of Titan cells [34].

At the end of the 19th century, exactly 128 years ago, Busse and Buschke almost simultaneously described the first two cases of cryptococcosis [13,14,15]. Certainly no one assumed that at the end of the next century, this disease entity in many places around the world would collect a greater death toll than tuberculosis [101], which was one of the most significant health problems in the nineteenth century. Observations performed during the last two to three decades demonstrate that members of the Cryptococcus species complex cause infection in approximately 1 million people per year, with more than 600 thousand mortalities, contributing to death toll in one-third AIDS patients [101, 108]. In recent years, thanks to the antiretroviral therapy (ART) implemented in many countries, the number of deaths caused by cryptococcosis in the group of AIDS patients has started to decline [10, 87]. For comparison, only in 2014 and 2020 on a global scale 223,100 and 152,000 people suffered from cryptococcosis of the central nervous system (CNS), of which 181,100 and 112,000 died, respectively [61, 108]. Currently, thanks to the increasingly better knowledge of the virulence factors of pathogenic fungi of the Cryptococcus species complex and thanks to the sequenced genomes of these fungi, there is a hope for more effective and less toxic anticryptococcal therapy. Furthermore, promising is also an ongoing intensive work on the anticryptococcal vaccine [22, 126] and the cyclical International Conferences on Cryptococcus and Cryptococcosis (ICCC) every three years [70]. Future work to delineate a set of unsolved problems in the cryptococcal virulence factors field will provide fertile ground for future improvements of therapies against cryptococcosis. It is impossible not to notice that the real need for research on pathogenic fungi of the genus Cryptococcus and the disease entities caused by them means that from the day of discovering this life-threatening etiological agent we have to deal with a never ending story.