Virulence Factors in C. neoformans/C. gattii Species Complexes

Virulence factors are defined as features that allow an organism to survive and cause disease within a susceptible host (Kozel 1995). There is much evidence supporting the hypothesis that cryptococcal virulence originated because of environmental selection pressure (Casadevall et al.

The pathogenesis of Cryptococcus is determined by three broad factors: the status of host defenses, the virulence of the strain, and the inoculum size (Mitchell and Perfect 1995). A summary of well-defined virulence factors of pathogenic members of C. neoformans and C. gattii species complexes is presented in Table 12.2.

The polysaccharide capsule plays an important role as a virulent factor in animal models of cryptococcosis, as well as in clinical settings (Schelenz et al. 1995). The main component of the polysaccharide capsule is glucuronoxylomannan. Capsule- free or poorly capsulated isolates have been found to elicit a strong immune response and less severe disease in humans (Levinson et al. 1974; Farmer and Komorowski 1973). However, the degree of virulence has been shown to be unrelated to the degree of encapsulation (Kwon-Chung and Bennett 1992). Four capsule's genes, namely, CAP64, CAP60, CAP59, and CAP10, are required for virulence in a murine model (Chang and Kwon-Chung 1998). The capsule appears to inhibit the ingestion of yeast cells by phagocytes in the absence of opsonins (Vecchiarelli 2000).

Table 12.2 Well-defined virulence factors of pathogenic members of C. neoformans/C. gattii species complexes

serum and severity of cryptococcosis provides circumstantial evidence for relationships among polysaccharide release, host damage, and disease progression (Vecchiarelli 2000).

An encapsulated melanin-forming isolate, which failed to grow at 37 °C, was found to be avirulent (Kwon-Chung et al. 2014). The generation time is known to contribute to virulence: isolates with a longer generation time were unable to cause fatal infection in mice even when high inoculum (10⁷ cells/mouse) was used (Kwon- Chung et al.

Melanin formation, which is used for accurate and rapid identification of this pathogen, has been identified as a second key virulence factor for C. neoformans (Rinaldi et al. 1986). In addition, melanin functions as an antioxidant and might protect C. neoformans from oxidative host defenses (Polak 1990; Jacobson and Tinnell 1993; Jacobson et al. 1994). Melanized cells of C. neoformans are less susceptible to antifungals (van Duin et al. 2002). The products of the laccase genes LAC1 and LAC2 are recognized as key enzymes of melanin biosynthesis (Torres- Guererro and Edman 1994; Zhu and Williamson 2004; Missall et al. 2005). Kronstad et al. (2011) identified 33 novel genes for melanization in Cryptococcus.

The ability to grow at 37 °C and above is an essential contributing factor to infection in warm-blooded animals and humans. Cryptococcus possesses the CNA1 gene, encoding the calcineurin A catalytic subunit, which confers the ability to survive at 37 °C (Odom et al. 1997). Numerous cryptococcal genes are known to be upregulated at 37 °C, including MGA2, although these genes are not essential for growth at 37 °C (Ma and May 2009). Proteinases and signal transduction pathways are also important for virulence. Both clinical and environmental isolates of C. neoformans possess protease activity, which has been shown to degrade host proteins including collagen, elastin, fibrinogen, immunoglobulin, and complement factors, and cause tissue damage, thereby providing nutrients to the pathogen and protecting it from the host (Chen et al.

Phenotypic switching has been shown to cause changes in virulence. Switching from smooth colonies to mucoid colonies has been observed in C. gattii during pulmonary infection (Jain et al. 2006). Similarly, the mucoid colony phenotype of C. neoformans triggers a macrophage- and neutrophil-dominated immune response, whereas the smooth-colony phenotype initiates a lymphocyte-dominated immune response (Pietrella et al. 2005).

C. neoformans and C. gattii are haploid yeasts that predominantly reproduce asexually (by budding). However, they also possess a bipolar mating system, with mating types MATa and MATα (Kwon-Chung 1975, 1976). Mating involves fusion between cells of opposite mating type (a and α), resulting in the conversion from a haploid budding yeast form to a dikaryotic mycelial form that produces basidia and basidiospores, which may serve as infectious propagules. Most of the clinical and environmental isolates are predominantly mating type α, which has been linked to virulence (Metin et al. 2010). The discovery that monokaryotic fruiting under laboratory conditions represents a novel type of sexual reproduction involving only one of the two mating types, most commonly MATα, demonstrated that same-sex or unisexual reproduction could profoundly influence the organism’s population structure. Haploid fruiting usually occurs in response to nitrogen starva­tion and/or desiccation. Recent population genetic studies provide robust evidence that both a-α opposite sex mating and α-α unisexual mating occur in nature in both C.

Three signal transduction pathways that regulate virulence and morphogenesis have been described in C. neoformans. These include the cAMP-PKA pathway—a pheromone-regulated MAP kinase pathway involved in mating—and a calcineurin- regulated pathway (Lengeler et al. 2002). These pathways act specifically on a variety of virulence attributes: the cAMP-PKA pathway regulates capsule produc­tion, melanin formation, mating, and virulence (Lengeler et al. 2002), whereas the calcineurin-regulated pathway is essential for growth at 37 °C, mating,

morphogenesis, and virulence (Steenbergen and Casadevall 2003). Moreover, the integrated signaling and regulatory pathways are especially important in the control of virulence in Cryptococcus yeasts (Ma and May 2009).

Mitochondrial tubular morphology has also been shown to protect Cryptococcus cells from cell death. The C. gattii strains, which can promote mitochondrial fusion to form long tubular mitochondria, are able to more efficiently repair mtDNA damage caused by the oxidative species and hypoxic conditions present within the macrophage phagosome (Ma et al. 2009; Springer et al. 2014). Mitochondrial tubularization is generally thought to result from mitochondrial fusion, a phenome­non that allows mitochondria within a cell to cooperate with each other, and protects cells from the detrimental effect of mtDNA mutations by allowing functional complementation of mtDNA gene products (Chen et al. 2003).

Recent studies also showed that C. neoformans produces large polyploid “titan cells” in response to the stress of the host environment (Okagaki et al. 2010). Typical cryptococcal cells are 5-7 μm in diameter and have a haploid (1C) genome, whereas titan cells can be five to ten times larger than normal cells and are predominantly tetraploid (4C) or octoploid (8C). Titan cell formation plays a key role in disease progression and enhances the virulence of C. neoformans to establish the initial pulmonary infection (Crabtree et al. 2012). Titan cells are shown to be more resistant to stress and antifungals, such as fluconazole, and produce populations of more- resistant aneuploids (Gerstein et al. 2015). Because titan cells cannot be phagocytosed, they may also prevent the phagocytosis of “normal” C. neoformans cells in their vicinity, especially at early stages of infection (Crabtree et al. 2012). Titan cells are also resistant to oxidative and nitrosative stresses; they hamper infection clearance and might play a key role in latent infection (Zaragoza et al. 2010).

12.6