The tools for virulence of Cryptococcus neoformans.

CHAPTER ONE

The Tools for Virulence of Cryptococcus neoformans Carolina Coelho*,†, Anamelia Lorenzetti Bocca}, Arturo Casadevall*,1

*Department of Microbiology and Immunology, Albert Einstein College of Medicine of Yeshiva University, New York, USA † Centre for Neuroscience and Cell Biology of Coimbra, Institute of Microbiology, Faculty of Medicine, University of Coimbra, Coimbra, Portugal } Department of Cellular Biology, Institute of Biological Sciences, University of Brası´lia, Brası´lia, Brazil 1 Corresponding author: e-mail address: [email protected]

Contents 1. Introduction to Cryptococcus neoformans and Cryptococcosis 2. What Tools Allow C. neoformans to Become a Pathogen? 2.1 The host–pathogen duo and virulence as an emergent property 2.2 Thermotolerance 2.3 Acquisition of nutrients 2.4 Capsule 2.5 Melanin and laccase 2.6 Urease 2.7 Phospholipase 2.8 Oxidative defenses 2.9 Antiphagocytic protein 1 2.10 Other virulence factors 2.11 Secreted vesicles 2.12 Morphological changes 3. How Does C. neoformans Survive Within a Host? 3.1 Intracellular survival of C. neoformans 3.2 Nonlytic exocytosis 3.3 Dissemination: Penetration blood–brain barrier 3.4 Subversion of host immune response 4. Why Is C. neoformans Successful as a Pathogen? 5. Future Directions References

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Abstract Cryptococcus neoformans is a fungal pathogen that causes almost half a million deaths each year. It is believed that most humans are infected with C. neoformans, possibly in a form that survives through latency in the lung and can reactivate to cause disease if the host becomes immunosuppressed. C. neoformans has a remarkably sophisticated

Advances in Applied Microbiology, Volume 87 ISSN 0065-2164 http://dx.doi.org/10.1016/B978-0-12-800261-2.00001-3

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Carolina Coelho et al.

intracellular survival capacities yet it is a free-living fungus with no requirement for mammalian virulence whatsoever. In this review, we discuss the tools that C. neoformans possesses to achieve survival, latency and virulence within its host. Some of these tools are mechanisms to withstand starvation and others aim to protect against microbicidal molecules produced by the immune system. Furthermore, we discuss how these tools were acquired through evolutionary pressures and perhaps accidental stochastic events, all of which combined to produce an organism with an unusual and unique intracellular pathogenic strategy.

1. INTRODUCTION TO CRYPTOCOCCUS NEOFORMANS AND CRYPTOCOCCOSIS Cryptococcus neoformans is the causative agent of cryptococcosis, an often fatal disease characterized by an initial pneumonia that can progress to fatal meningoenchepalitis. Although cryptococcosis was described over a century ago (Barnett, 2010) this disease rose to spotlight as an AIDSassociated opportunistic disease. It is now reported to cause at least half a million of AIDS-associated deaths each year (Park et al., 2009). While access to highly active antiretroviral therapy has dramatically reduced the prevalence of AIDS-related cryptococcosis, other risk factors have arisen in recent years. Any individual undergoing immunosupressive therapy, such as a transplant or a cancer patient, is at risk for cryptococcal disease, which can affect up to 20% of solid organ transplant recipients in the United States (Singh et al., 2008). C. neoformans is a ubiquitous organism with a worldwide distribution that includes both environmental and urban settings, particularly trees, soil, and avian guano (Barnett, 2010; Lin & Heitman, 2006). The genus Cryptococcus is remarkable for having a polysaccharide capsule (Barnett, 2010), which is unique among the pathogenic fungi. Although other species in the genus Cryptococcus are occasionally associated with human disease, the only consistently pathogenic species in this genus are C. neoformans and C. gattii. Originally with a geographical distribution restricted to subtropical regions, C. gattii has recently expanded to North America (Barnett, 2010; Lin & Heitman, 2006). An unexpected outbreak in the American Pacific Northwest has caused deaths in human hosts with no apparent immune defect (Hoang, Maguire, Doyle, Fyfe, & Roscoe, 2004). The events leading to cryptococcal disease can be roughly summarized as follows. An infectious particle becomes airborne from their environmental

Virulence Factors of C. neoformans

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Figure 1.1 Cryptococcus neoformans infectious cycle. C. neoformans has been found in the environment, associated with eucalyptus trees, soil, birds excreta and within amoeba. Infection occurs when spores and/or dissected yeasts are inhaled by human or mammalian hosts. In the lung, the inhaled fungi are deposited into the alveoli and establish a latent infection. Amoeba image courtesy of Lorena da S. Derengowski.

reservoirs, are inhaled by an unknowing host and deposited in the lungs (Giles, Dagenais, Botts, Keller, & Hull, 2009). Both spores and/or desiccated yeast cells possess the physical characteristics that allow inhalation and lung deposition. This was proved experimentally when spores were isolated and found to have the same virulence characteristics as yeast cells (Giles et al., 2009). Inhalation of fungi by humans is a common occurrence, since up to 80% of 5-year olds in an urban setting manifest serological reactivity to C. neoformans consistent with prior exposure (Davis et al., 2007; Goldman et al., 2001). While primary infection is believed to be asymptomatic or to pass unnoticed as one of the many illnesses of childhood (Goldman et al., 2001), the current prevailing thought is that in many individuals the infection is not completely cleared and instead it persists in a latent asymptomatic state (see Fig. 1.1). This is supported by both epidemiological data

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(Dromer, Ronin, & Dupont, 1992; Garcia-Hermoso, Janbon, &Dromer, 1999; Singh et al., 2008) and animal studies. In the rat model it is possible to establish latent infections that can be reactivated by subsequent immune suppression (Goldman, Lee, & Casadevall, 1994; Goldman, Lee, Mednick, Montella, & Casadevall, 2000). Latency occurs by persistence of intracellular fungal forms within the lung, site of primary infection (Lindell, Ballinger, McDonald, Toews, & Huffnagle, 2006), but the possibility of latency in other tissues, in particular the brain, has not been excluded. In the event of host immunosuppression, the latent form begins to proliferate in the lung and from here it can disseminate to extrapulmonary sites. Management of the resulting cryptococcosis is difficult and very aggressive treatment is required once the yeast crosses the blood–brain barrier (Longo et al., 2011). Despite adequate medical care, fatalities still amount to 10–25% of the cases (Hoang et al., 2004; Jarvis & Harrison, 2007; Longo et al., 2011; Park et al., 2009; Schwarz, Dromer, Lortholary, & Dannaoui, 2006; Singh et al., 2008). For individuals with reversible immune deficiencies, the best course of action is restoration of immune function (Longo et al., 2011; Schwarz et al., 2006). In individuals with severe immunodeficiency, therapy may not be curative, that is, eradication of fungus is not achieved, and the clinical goal is to reduce symptoms with prolonged, if not lifelong, antifungal therapy.

2. WHAT TOOLS ALLOW C. neoformans TO BECOME A PATHOGEN? 2.1. The host–pathogen duo and virulence as an emergent property C. neoformans survival and proliferation within a mammalian host is made possible by the combination of microbial virulence factors and host susceptibility (Casadevall & Pirofski, 1999, 2003; Steenbergen & Casadevall, 2003). Virulence factors include all the mechanisms that allow the fungus to efficiently divide and survive inside the host, factors that allow the fungus to resist host immune attack, and lastly, factors that eventually damage the host. The classical example of a condition predisposing individuals to cryptococcosis is a CD4þ T-cell deficiency, such as AIDS patients (Jarvis et al., 2013), but many others exist. There is a large body of literature showing that X-linked immunodeficiency (Szymczak et al., 2013) predisposes to cryptococcal disease. Equally Fc receptor (Rohatgi et al., 2013) and IL-4 (Mu¨ller et al., 2012) polymorphisms have recently been associated with altered susceptibilities to human cryptococcosis. When considering the


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phenomenon of virulence, it is important to remember that disease is an outcome of an interaction between a host and a microbe (Casadevall & Pirofski, 1999), since the host aims for effective control of the pathogen with minimization of tissue damage. Consequently, the phenomenon of virulence should not be viewed in isolation from the host. In this chapter, we attempted to frame the discussion of each virulence attribute with its host counterpart (Alanio, Desnos-Ollivier, & Dromer, 2011). However, we will focus primarily on the fungal attributes, the virulence factors, of C. neoformans, their hypothesized evolutionary origins, and the complex interactions that resulted in such a remarkable pathogen.

2.2. Thermotolerance Growth at host body temperature is an absolute requirement for virulence. C. neoformans isolates manifest considerable thermotolerance (Martinez, Garcia-Rivera, & Casadevall, 2001; Perfect, 2006; Robert & Casadevall, 2009), which might simultaneously explain its worldwide distribution and its capacity for growth at mammalian temperatures. Because thermotolerance is a prerequisite for virulence of any pathogen (Barnett, 2010; Garcia-Solache, Izquierdo-Garcia, Smith, Bergman, & Casadevall, 2013; Martinez et al., 2001; McClelland, Bernhardt, & Casadevall, 2006; Perfect, 2006; Robert & Casadevall, 2009), it was proposed that any enzyme that conferred fitness at high temperature would fit the definition of virulence factor (Robert & Casadevall, 2009). In fact, C. neoformans thermotolerance might have a disproportionate importance over any other virulence factors. When comparing C. neoformans with its phylogenetical relatives, many possess virulence factors, such as capsule and laccase, but are not known pathogens simply because they are incapable of growth at mammalian temperatures (Petter, Kang, Boekhout, Davis, & KwonChung, 2001). These findings have been reproduced in vertebrate and invertebrate models of infection (Garcia-Solache et al., 2013; McClelland et al., 2006) and it is likely that this is a common occurrence for pathogenic organisms.

2.3. Acquisition of nutrients The low pH of the phagosome is generally detrimental to microbes. However, members of the fungal kingdom are notorious for preferring an acidic environment for growth. In this regard, fungal growth media are commonly adjusted to acidic pH, which also inhibits bacterial growth. Preference for

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acidity extends to the cryptococcal phagosome, as blocking phagosomal acidification was associated with a lower replication rate (ArtavanisTsakonas, Love, Ploegh, & Vyas, 2006; Levitz, Harrison, Tabuni, & Liu, 1997; Qin et al., 2011). Consequently, acidification of the phagosomal compartment is favorable, not detrimental for C. neoformans growth. Phagosomes have limited availability of nutrients as a mechanism for interfering with microbial growth. Gene expression studies confirmed that C. neoformans (Derengowski Lda et al., 2013; Fan, Kraus, Boily, & Heitman, 2005; Hu, Cheng, Sham, Perfect, & Kronstad, 2008) and Candida albicans (Fernandez-Arenas et al., 2007) induce a starvation response upon phagocytosis by macrophages. When C. albicans is ingested by macrophages in vitro, it resorts to fatty acids as a carbon source by upregulating the glyoxylate cycle (Fernandez-Arenas et al., 2007). The importance of the glyoxylate cycle is assessed by mutation of its key enzyme isocitrate lyase. Deficiency of this enzyme results in an avirulent phenotype in C. albicans (Lorenz & Fink, 2001) and several bacterial pathogens (Appelberg, 2006). C. neoformans ingested by murine macrophages upregulate both the glyoxylate cycle and sugar transporters. However an isocitrate lyase mutant of C. neoformans did not show altered virulence (Rude, Toffaletti, Cox, & Perfect, 2002), suggesting that this organism has alternative mechanisms for carbon acquisition. Instead, C. neoformans might rely on gluconeogenesis and other lipids for its carbon sources (Derengowski Lda et al., 2013). In what regard nucleotide requirements, the yeast resorts to purine de novo synthesis, not the salvage pathway, for full virulence, which emerges as an attractive antimicrobial target (Morrow et al., 2012). To satisfy amino acid requirements, C. neoformans upregulates amino acid transporters during infection of macrophages and amoeba in vitro (Derengowski Lda et al., 2013; Fan et al., 2005) and during murine infection (Hu, Cheng, et al., 2008), suggesting that the uptake of amino acids is possible in the phagocytic compartment. It is also possible that C. neoformans does not suffer nutrient starvation in the phagosome for a prolonged period of time. C. neoformans is known to damage the phagosomal membrane (originating leaky phagosomes), and consequently to have access to cytoplasmic nutrients (Tucker & Casadevall, 2002). Another major difference of C. neoformans compared to other fungal pathogens concerns autophagy. In contrast to C. albicans and Aspergillus fumigatus (Palmer, Askew, & Williamson, 2008; Seider, Heyken, Luttich, Miramon, & Hube, 2010), C. neoformans is dependent on autophagic processes for virulence (Fan et al., 2005; Hu, Cheng, et al., 2008; Hu, Hacham, et al., 2008). For instance, Atg8 is upregulated


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during cryptococcal brain infection (Qin et al., 2011) and a vsp34 autophagy protein mutant is avirulent (Hu, Hacham, et al., 2008). Consequently it is possible that C. neoformans obtains part of its nutrients through autophagic recycling (Palmer et al., 2008). Metal starvation within the phagosome is achieved by the action of scavenging enzymes. One example is sequestration of iron by lysozyme (Bailao et al., 2006). However pathogens counteract host scavenger enzymes with an array of siderophore molecules. In the case of C. neoformans does not appear to depend on siderophores for iron acquisition. Instead, it depends on ferric reductases, of which laccase is an example. These enzymes catalyze the reaction of Fe3þ to Fe2þ and are required for full virulence in mice (Jung, Hu, Kuo, & Kronstad, 2009; Jung & Kronstad, 2008; Tangen, Jung, Sham, Lian, & Kronstad, 2007). Iron acquisition seems to be regulated by the Cir1 iron regulator and pH responsive factor Rim101 (Kronstad, Hu, & Jung, 2013). In addition, iron might be crucial for antifungal drug detoxification (Choi, Kim, Kim, Jung, & Lee, 2012; Kim, Cho, et al., 2012; Kim, Crary, Chang, Kwon-Chung, & Kim, 2012). To achieve inhibition of microbial enzyme activity within the host phagosome there is restricted access to other metallic cofactors. For example, zinc’s role in pathogenesis of C. neoformans is not well characterized, but deletion of zinc regulatory gene Zap1 in C. gattii resulted in attenuated virulence (Steen et al., 2003), thus adequate uptake of Zn is required for survival. Copper is the one element that the phagosome might not be deprived of, but instead enriched for. Although copper is required for virulenceassociated enzymes, such as laccase and superoxide dismutase, it has been recently suggested that toxic accumulation of copper in the phagosome functions as an antimicrobial mechanism. Fungal metalotheins bind copper near the cytosolic periphery, preventing copper from exerting toxic effects and are required for full virulence in murine models (Ding et al., 2013; Waterman et al., 2012). Additionally, it was found that C. neoformans prevents copper accumulation in the phagosome through an unknown immunomodulatory mechanism (Ding et al., 2013).

2.4. Capsule The prominence of the capsule in C. neoformans virulence is illustrated by the fact that acapsular mutants are avirulent (Chang & Kwon-Chung, 1994). Spores are initially unencapsulated but upon entering the host lung the transformation from spore to yeast cell is accompanied by the rapid synthesis of

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the polysaccharide capsule (Giles et al., 2009; Velagapudi, Hsueh, GeunesBoyer, Wright, & Heitman, 2009) and capsule size increases significantly during infection (Charlier et al., 2005; McFadden, Fries, Wang, & Casadevall, 2007; Zaragoza et al., 2009). Consequently, it is accepted that the capsule confers considerable advantages to C. neoformans survival within the host. The capsule is a complex structure that extends from the fungal cell wall and can reach dimensions that are several times the diameter of cell body. C. neoformans capsule is composed primarily of glucuronoxylomannan (GXM), a very large anionic heteropolymer. Historically, two other components, glucuronoxylomannogalactan (GalXM) and mannoproteins, were considered to be components of the capsule. Yet, studies of their relative localization have raised the possibility that GalXM and mannoproteins are instead associated with the cell wall and not the capsule (Jesus et al., 2010). Mannoproteins, a minor component of C. neoformans cell surface, are heavily mannosylated proteins which are shed during growth in vivo and in vitro and at least one of them (the product of Cig1 transcript) has iron acquisition functions at the cell surface (Cadieux et al., 2013). Mannoproteins are highly immunogenic and can elicit protective immune responses in mice (Mansour, Yauch, Rottman, & Levitz, 2004; Pietrella et al., 2001). Recently, it was suggested that the capsule has one additional polysaccharide component as b-glucans were found in aged yeast cells capsules (Cordero, Pontes, et al., 2011) and thus b-glucans might be components of the fungal capsule. Apart from the polysaccharide components, chitin is found associated with GXM (Fonseca et al., 2009; Ramos et al., 2012; Rodrigues, Alvarez, Fonseca, & Casadevall, 2008) with the proposed function of anchoring GXM polymers to the fungal cell wall. These chitin– GXM polymers have immunological properties distinct from isolated chitin or GXM and initiate an immunological pattern consistent with them being exposed to the immune system during fungal infection (Ramos et al., 2012), thus suggesting that this is a particle encountered by the immune system. Lastly, GXM has also been found associated with lipids (Oliveira et al., 2009) but the function of these components in capsular structure is not fully understood so far. Fungal polysaccharides, GXM and GalXM, are constitutively secreted to the medium and can be isolated from culture supernatants, even detected in the serum of patients (Feldmesser & Casadevall, 1997; Lee & Casadevall, 1996; Lee, Casadevall, & Dickson, 1996). Secretion into the media of the capsular polysaccharide GXM could be interpreted as a consequence of


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Figure 1.2 C. neoformans main virulence factors. (A) Schematic representation of C. neoformans virulence factors. The main capsule component is GXM. GXM can be secreted into extracellular milieu inside fungal vesicles. These vesicles contain major virulence-associated molecules like laccase, urease, and Plb1. C. neoformans is also remarkable for producing several antiphagocytic factors through an extensive Gat201-mediated regulatory network. GAT201 increases expression of GAT204 and BLP1. (B) India ink stain of C. neoformans cells. C. neoformans possesses a complex polysaccharide capsule that extends from the fungal cell wall with variable dimensions. BLP1, Barwin-like protein; Gat, Gata-family transcription factor; GXM, glucuronoxylomannan; Plb1, phospholipase B1. Panel (B) courtesy of Radamés J. B. Cordero.

extracellular assembly where some free pieces could escape to supernatants (see Fig. 1.2). When one considers that GXM polymers can have molecular masses of >1 MDa (McFadden, Zaragoza, & Casadevall, 2006), the synthesis and assembly of the fungal capsule becomes an intriguing problem as it is difficult to envision mechanisms by which such large molecules are exported through the rigid cell wall. Trans-cell wall transport of GXM was associated with vesicular structures (Kmetzsch et al., 2011; Oliveira et al., 2009; Panepinto et al., 2009; Rodrigues et al., 2007; Yoneda & Doering, 2006). Thus, GXM fragments would be synthesized intracellularly, exported via vesicle-mediated secretion and assembled into final polymers outside the cell wall (Garcia-Rivera, Chang, Kwon-Chung, & Casadevall, 2004; Rodrigues et al., 2007; Yoneda & Doering, 2006). In what concerns the problem of assembly after transcellular transport there is some evidence that assembly might be as simple as self-aggregation of polymers, as concentration of divalent cations in culture media influence capsule size (Nimrichter et al., 2007). Synthesis of GXM is not fully understood, however, gene mutagenesis studies identified proteins whose absence results in an acapsular mutant

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(Chang & Kwon-Chung, 1994, 1998, 1999). For example, cap59 deletion has a defect in GXM secretion suggesting that the mutated protein is involved in GXM secretion (Garcia-Rivera et al., 2004). In addition, capsular synthesis studies support the notion that GXM is the only capsular polysaccharide as these mutants lack GXM antibody reactivity but not GalXM antibody reactivity (Grijpstra, Gerwig, Wosten, Kamerling, & de Cock, 2009; Grijpstra, Tefsen, van Die, & de Cock, 2009). Assembly of the remarkable fungal capsule is then dependent on intracellular assembly, vesicular secretion, and extracellular self-aggregation of GXM fragments. C. neoformans capsule is remarkably immunomodulatory. GXM and GalXM polysaccharides extracted from fungal cell wall, capsule or secreted into extracellular media have similar molecular compositions and if isolated maintain the immunomodulatory properties of the capsule (Chang, Netski, Thorkildson, & Kozel, 2006; Chiapello, Baronetti, Garro, Spesso, & Masih, 2008; Murphy et al., 1988) and reviewed in Vecchiarelli et al. (2013). Cryptococcal polysaccharides can affect migration of immune cells (Ellerbroek, Ulfman, Hoepelman, & Coenjaerts, 2004; Ellerbroek, Walenkamp, Hoepelman, & Coenjaerts, 2004), inhibit cytokine secretion and interfere with MHC presentation (Siegemund & Alber, 2008; Villena et al., 2008). Secreted GXM induced expression of FasL in macrophages which in turn caused death in nearby T cells (Monari et al., 2005) and the presence of GXM can affect macrophage cellular proliferation and trigger apoptosis (Ben-Abdallah et al., 2012; Lupo et al., 2008; Vecchiarelli et al., 1994). Similarly, GalXM is capable of downregulation T-cell response (Vecchiarelli et al., 2011). Given their immunomodulatory properties, both polysaccharides have been investigated for potential therapeutic applications, such as treatment for endotoxic shock (Ellerbroek, Walenkamp, et al., 2004; Pericolini et al., 2013; Piccioni et al., 2013; Vecchiarelli et al., 2011). The most dramatic effect of the capsule is its capacity to interfere with phagocytosis in vitro (Shoham & Levitz, 2005), such that there is essentially no phagocytosis of encapsulated C. neoformans cells in the absence of opsonins. In vivo, complement-derived opsonins are probably responsible for ingestion of C. neoformans until an antibody response is elicited (Diamond, May, Kane, Frank, & Bennett, 1974; Kozel, 1993; MershonShier, Vasuthasawat, Takahashi, Morrison, & Beenhouwer, 2011, Rhodes, 1985; Shapiro et al., 2002). The fungal capsule inhibits the complement classical activation pathway and thus C3 deposition on the capsule proceeds through the alternative pathway (Mershon-Shier et al., 2011; Kozel, Wilson, & Murphy, 1991). It is hypothesized that the capsule is


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antiphagocytic because it conceals cell-surface pathogen-associated molecular patterns. In contrast to encapsulated cells, spores or acapsular C. neoformans are readily ingested through activation of mannose and b-glucan receptors (Cross & Bancroft, 1995; Giles et al., 2009). The capsule’s physical properties contribute to its antiphagocytic function. For example, capsular enlargement observed in vivo would pose a considerable challenge to phagocytosis due to pure physical impediments concerning ingestion of such large particles. Physical properties of phagocytic targets are known to influence their ingestion (Cross & Bancroft, 1995). Indeed, viscosity, zeta potential rigidity and size of a foreign particle can affect particle ingestion by Phagocytes (Tabata & Ikada, 1988; Underhill & Goodridge, 2012). Several studies have correlated physical properties of capsular polymers, such as stiffness and viscosity, to biological functions such as inhibition of phagocytosis and production of NO (Araujo Gde et al., 2012; Cordero, Frases, Guimaraes, Rivera, & Casadevall, 2011; Cordero et al., 2013; Fonseca et al., 2010; McFadden et al., 2007). For example, there was a correlation between the increase in stiffness caused by antibody binding and the facilitation of phagocytosis triggered by that antibody (Cordero et al., 2013) and an increase in the degree of polymer branching correlated with protection from H2O2 toxicity (Cordero, Frases, et al., 2011). In addition to its antiphagocytic capacities, the capsule has many other functions as a virulence factor. It can quench reactive oxygen species (ROS) and other microbicidal molecules (Feldmesser, Rivera, Kress, Kozel, & Casadevall, 2000; Zaragoza et al., 2009). The capsule is very poorly immunogenic, and infection seldom elicits strong antibody responses. Despite this lack of immunogenicity, several antibodies against capsular epitopes have been isolated (Casadevall et al., 1998; Shapiro et al., 2002) from mice immunized with polysaccharide–protein conjugate vaccines. These have been extensively studied for their immunization and therapeutic properties (reviewed in Casadevall & Pirofski, 2005). Despite being the subject of numerous studies, there are highly enigmatic aspects of the capsule as a virulence factor (Del Poeta & Casadevall, 2012). Furthermore these facts underscore that every virulence factor needs to be viewed in the context of the interaction. For example, the capsule is antiphagocytic in vitro but phagocytosis readily occurs in vivo. Consequently, it is not solely through the capsule’s antiphagocytic properties that we can explain its importance in infection (Del Poeta, 2004) and the many other virulence properties of the capsule need to be equated. Although the capsule is required for virulence in immunologically intact hosts, acapsular strains can be pathogenic in mice lacking cell-mediated immunity (Del Poeta, 2004).

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It is then conceivable that the capsule’s main role is to protect against a cell-mediated immunity-specific effector. In extremis, the capsule might be dispensable for virulence in cases of severe immunosupression (Salkowski & Balish, 1991). In contrast, other observations make a compelling argument for the evolution of virulence-specific properties in the capsule. It was found that the capsule of C. neoformans is more effective at preventing amoeba predation than capsules from nonpathogenic cryptococcal species (Araujo Gde et al., 2012). Thus, this enigmatic structure has accumulated physical and chemical characteristics which translate into a remarkable virulence gain for C. neoformans.

2.5. Melanin and laccase Laccase enzymes catalyze synthesis of melanin. Laccase-deficient, consequently melanin-deficient, mutants in C. neoformans have reduced virulence (Kwon-Chung, Polacheck, & Popkin, 1982; Rhodes, Polacheck, & Kwon-Chung, 1982). The association of melanin and virulence is not unique to C. neoformans and is conserved in numerous bacteria and fungi (Liu & Nizet, 2009). Albeit the molecular structure of the melanin pigment is unknown, the molecule is a stable-free radical, thus an efficient antioxidant (Wang & Casadevall, 1994a,1994b,1994c). Melanin can absorb electromagnetic radiation and thus protects organisms from radiation, such as the one originating from solar light or nuclear reactors (Dadachova et al., 2008). This protection is crucial for a free-living organism such as C. neoformans with the interesting consequence of defending against free radicals produced by the immune system. There is evidence that melanin has functions other than antioxidant activity. Melanized cells are more resistant to amphotericin B (Ikeda, Sugita, Jacobson, & Shinoda, 2003; van Duin, Casadevall, & Nosanchuk, 2002; Wang & Casadevall, 1994a,1994c), caspofungin (van Duin et al., 2002), and defensins (Doering, Nosanchuk, Roberts, & Casadevall, 1999; Wozniak & Levitz, 2008). Furthermore, melanin contributes to cell wall structure (Williamson, 1997) augmenting to the rigidity of the cell wall. Despite melanin’s amorphous structure, it can be recognized by antibodies and these antibodies are protective in murine infection models (Rosas, Nosanchuk, & Casadevall, 2001). C. neoformans possesses two laccase (Lac) isoforms, where Lac1 is more abundantly expressed in the cell wall while Lac2 is abundant in the cytoplasm (Missall, Moran, Corbett, & Lodge, 2005; Polacheck, Hearing, & KwonChung, 1982). Lac1 might play a predominant role in infection but both


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isoforms synergize for full virulence (Pukkila-Worley et al., 2005; Salas, Bennett, Kwon-Chung, Perfect, & Williamson, 1996). Laccase enzyme has iron oxidase activity and could contribute to increasing iron availability within the nutrient-deprived phagosome. Furthermore, laccase might be immunomodulatory through its capacity to catalyze production of prostaglandin E2 from extracellular arachidonic acid (Erb-Downward & Huffnagle, 2007). Production of a prostaglandin molecule would confer upon C. neoformans a remarkable virulence mechanism where increased prostaglandins at the site of infection interfere with control of infection. Thus, the duo laccase and melanin increases resistance of C. neoformans to chemical attack, aids fungal nutrition and with the added possibility of immunomodulatory capacity.

2.6. Urease In bacterial pathogens, urease and its accessory enzymes detoxify urea, resulting in the alkalinization of the surrounding media, a change that facilitates the acquisition of nitrogen. Urease plays an important role in bacterial pathogenesis and it was hypothesized that it would play an equivalent role in C. neoformans. Urease-deficient strains caused meningoenchephalitis but not pneumonitis (Cox, Mukherjee, Cole, Casadevall, & Perfect, 2000) and displayed a hypovirulent phenotype during intravenous murine infection murine (Cox et al., 2000). Urease appears to have a critical role for promoting fungal traversing of epithelial barriers and promotes brain invasion (Olszewski et al., 2004; Shi et al., 2010, Singh et al., 2013), but how urease contributes to traversing of tissue barriers is unknown. As the yeast prefers acidic environments, it is unlikely that alkalization is the ultimate goal of urease production. Similarly, the transverse of the epithelial barriers is not readily explained by urea detoxification and thus it is difficult to reconcile the classical functions of urease in with the observed role of urease in C. neoformans infection. However, alternative functions of urease have been discovered in Helicobacter pylori. Urease reaction products detoxify peroxynitrite, a highly reactive radical (Kuwahara et al., 2000), and urease is both antigenic and chemotactic for immune cells (Konieczna et al., 2012). It is likely that nonclassical roles of urease will be discovered in C. neoformans. It is noteworthy that occasional urease-negative strains have been recovered from clinical specimens (Varma et al., 2006), suggesting that urease is favorable for infection but not essential if other components of the virulence composite can compensate for its tasks within the host.

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2.7. Phospholipase Phospholipase are enzymes that degrade phospholipids. One isoform of phospholipase B (Plb1) is secreted during C. neoformans infection and enhances fungal survival within phagocytes (Cox et al., 2001). Addition of protozoan or mammalian phospholipids to C. neoformans cultures was a sufficient signal for induction of Plb1 and increase capsule growth, which led to the suggestion that phospholipids are sensed by the fungus as a danger signal or “phagocyte alert” signal. Degradation of host phospholipids by Plb1 could potentially mediate damage to phagosomal membranes, allow fungal access to cytoplasmatic components, and thus have a direct role in nutrient acquisition (Chrisman, Albuquerque, Guimaraes, Nieves, & Casadevall, 2011). Deletion of Plb1 decreases the frequency of nonlytic exocytosis (Chayakulkeeree et al., 2011) underscoring how this enzyme is crucial for yeast survival in the phagosome. Plb1 might also play a role in extrapulmonary dissemination. Plb1-deficient mutants have reduced virulence and decreased invasion of the brain (Cox et al., 2001) and, recently, Plb1 was described to interact with Rac1 and through host GTP–Rac1–STAT3 interaction promote brain invasion (Maruvada et al., 2012). Another isoform of phospholipases (Plc) was recently found to have function in virulence through a role in thermotolerance, capsule, and cell wall synthesis (Lev et al., 2013).

2.8. Oxidative defenses Immune cells secrete abundant ROS, powerful microbicides which through action of inducible Nitric Oxide Synthase (iNOS) can further augmented to nitrogen free radicals species (Novo & Parola, 2008). The role of ROS in a microbicidal agents against C. neoformans has proved difficult to decipher. This difficulty may be attributed to ROS widespread effects on host physiology and to the myriad of fungal oxidative defense mechanisms. These include fungal capsule, laccase and melanin, antioxidative enzymes and the sugar mannitol. When mice deficient in ROS as a result of gp91(pox) deletion were challenged with C. neoformans they had decreased lung and brain fungal burden. The detrimental role of ROS in infection might be interpreted as negating a role for ROS as antifungal molecules or alternatively these toxic metabolites mediate excessive host tissue damage. ROS are essential for cell signaling, strong activators of NLRP3 and consequently of inflammasome (Chen & Sun, 2013; Tschopp, 2011). In fact, biofilms of this pathogen activate the inflammasome in an ROS dependent manner and activation is inhibited


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by an ROS scavenger (Lei et al., 2013). However, the capsulated yeast form of C. neoformans is a poor activator of the inflammasome (Bocca, unpublished data) and thus the role of ROS and the inflammasome in Cn infection remains enigmatic. Chemically derived NO is fungicidal (Alspaugh & Granger, 1991) and in vivo NO is a major inducer of microbicidal mechanisms. Therefore, it is likely that in vivo direct and indirect functions of NO synergize for fungal killing (Goldman, Cho, Zhao, Casadevall, & Lee, 1996; VazquezTorres et al., 2008). This assumption is supported by mouse infection studies. C. neoformans with defective nitrosative defenses is less virulent than WT C. neoformans (de Jesus-Berrios et al., 2003), ascertaining that the yeast nitrosative defenses are important for survival within a mammalian host. Genes related to oxidative stress, such as superoxide dismutases and catalases, are upregulated by C. neoformans upon infection in both amoeba and macrophages (Derengowski Lda et al., 2013), consistent with the notion that the fungus is exposed to oxidative stress within the phagosome. Superoxide dismutase (SOD) enzymes are another commonly used oxidative defense enzymes and C. neoformans possesses two SOD isoforms (Seider et al., 2010). A SOD1-deficient mutant was more susceptible to macrophage killing but appeared to be only slightly attenuated in mice (Cox et al., 2003). However, the SOD2 isoform is vital for aerobic growth at 37 C, and deletion of SOD2 produces an avirulent phenotypes (Giles, Batinic-Haberle, Perfect, & Cox, 2005). Unfortunately, this result does not necessarily elucidate the direct role of SOD2 in defense against host-derived ROS (Cox et al., 2003). While catalase is crucial for oxidative defenses in other pathogens it seems dispensable for murine cryptococcosis. When a quadruple mutant for all catalase isoforms was obtained, no difference in virulence was observed (Chaturvedi, Wong, & Newman, 1996; Cox et al., 2003), Additional antioxidative enzymes, such as thiol peroxidases, are critical for cryptococcal virulence (Missall, Pusateri, & Lodge, 2004), defending against both oxidative and nitrosative stresses (Missall et al., 2006). The sugar mannitol might be another unique oxidative defense of C. neoformans. Mannitol protects against distal ROS (Chaturvedi et al., 1996) and this compound has been demonstrated in human cerebrospinal fluid of human patients (Megson, Stevens, Hamilton, & Denning, 1996), suggesting a role for mannitol in brain pathogenesis. Overall, redundancy in the oxidative stress defenses in C. neoformans suggests that the yeast is well adapted to an oxidative environment but it remains undetermined how oxidative stress and nitrosative stress synergize for C. neoformans control.

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2.9. Antiphagocytic protein 1 App1 (Stano et al., 2009) is an antiphagocytic secreted protein that protects C. neoformans from ingestion by macrophages independently of the antiphagocytic effects mediated by the fungal capsule. This protein interacts specifically with CD11b on the macrophages surface and prevents the binding of iC3B-opsonized C. neoformans cells (Stano et al., 2009). Deletion of App1 from fungal cells results in an interesting phenotype. This mutant is hypovirulent in complement deficient hosts, but hypervirulent in T and NK cell deficient mice (Luberto et al., 2003) leading to the conclusion that phagocytosis of fungi is beneficial for the fungi if T cells are not present.

2.10. Other virulence factors In addition to the major virulence factors described earlier, many other proteins and enzymes have been associated with virulence. Rim101 is crucial for virulence in C. neoformans, since it is involved in pH response, capsule assembly and many other signaling pathways (reviewed in Kozubowski, Lee, & Heitman, 2009). Another example is the recently discovered Virulenceassociated DEAD-box protein (VAD1) protein. Deficiency in this protein produces a strain that is rapidly cleared by the host immune system, but no function has yet been attributed to VAD1 (Qiu, Olszewski, & Williamson, 2013). Screens with mutant strains of C. neoformans have identified a myriad of proteins that contribute to differential lung/brain ratio (He et al., 2012). In addition to the aforementioned App1, capsule independent antiphagocytic mechanisms exist in C. neoformans which are mediated by an extensive regulatory network. A key player is the Gata-family transcription factor 201 (Gat201) (Chun, Brown, & Madhani, 2011, Liu et al., 2008). Genes regulated by Gat201 were as diverse as GAT204, another transcription factor, and Barwin-like protein 1 (Blp1) (Chun et al., 2011). Barwin-like proteins have been described as a family of fungal and plant glucanases and cellulases but its functions have yet to be elucidated in C. neoformans. While Blp1 mutant did not have a hypovirulent phenotype, Gat204 and Gat201 showed decreased lung colonization (Chun et al., 2011).

2.11. Secreted vesicles Mammalian cells secrete membrane bound vesicles that carry signaling molecules to the extracellular milieu. A similar phenomenon was first described in fungi for C. neoformans (Rodrigues, Alvarez, et al., 2008; Rodrigues et al., 2007). Functions of fungal vesicles, as well as the mechanisms by which vesicles


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traverse the cell wall and capsule are still under discussion (Rodrigues, Nimrichter, Oliveira, Nosanchuk, & Casadevall, 2008). In mammals, Sec6 is the enzyme responsible for fusion of post-Golgi secretory apparatus with the plasma membrane. Secretion of vesicles by C. neoformans was downregulated in a Sec6 mutant (Rodrigues, Nimrichter, et al., 2008) consistent with the view that fungal vesicles are comparable to mammalian exosomes. However vesicles are a heterogeneous population in morphology and size yeast mutant screens have failed to isolate a mutant completely devoid of vesicle secretion. Therefore it is plausible that this heterogeneity is due to multiple and distinct origins, i.e., multiple vesicle secretion pathways (Rodrigues, Nimrichter, et al., 2008). C. neoformans vesicles contain GXM (Rodrigues et al., 2007), urease and laccase (Rodrigues, Nakayasu, et al., 2008). A Sec6 mutant had decreased extracellular activity of laccase (Panepinto et al., 2009), implying Sec6 dependent vesicles as the secretion mechanism for laccase. Furthermore the presence of virulence enzymes within vesicles led to their labelling as “virulence bags” (Oliveira et al., 2010; Rodrigues, Nakayasu, et al., 2008), where vesicular transport could deliver concentrated loads of enzymes and virulence factors to target cells. Studies showed that these structures are readily disrupted in the presence of mammalian blood serum components (Wolf, Rivera, & Casadevall, 2012) but their components were taken up by murine macrophages (Oliveira et al., 2010). Therefore fungal vesicles are efficient transporters of virulence factors and are involved in immune response to yeast. Isolated vesicles carrying virulence enzymes and immunomodulatory GXM is sufficient to trigger NO and cytokine release in macrophages (Oliveira et al., 2009, 2010). Vesicles elicited fusion of brain endothelial cells (Huang et al., 2012) and thus vesicles could aid transverse of the blood–brain barrier. Overall in vitro evidence strongly supports the notion that vesicles play an important role in fungal pathogenesis.

2.12. Morphological changes Morphological changes result in a different fungal surface presented to the immune system and thus allow the fungus to escape immune recognition. Three main types of morphological changes have been described in C. neoformans: phenotypic switching, pseudohyphal forms, and titan cells. The process of phenotypic switching was first described by Goldman, Fries, Franzot, Montella, and Casadevall (1998) who described three types of colony morphologies in a strain of C. neoformans. These colony morphologies could revert into the other types but manifested distinct

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virulence in mouse model (Fries et al., 2005; Fries, Taborda, Serfass, & Casadevall, 2001; Guerrero, Jain, Goldman, & Fries, 2006; Jain, Guerrero, & Fries, 2006; Jain, Li, et al., 2006; Pietrella et al., 2003). A recent study reports that the interaction with amoeba resulted in an accumulation of microevolution characteristics that lead to a switch between yeast and pseudohyphal forms (Magditch, Liu, Xue, & Idnurm, 2012). These pseudohyphal forms are more resistant to amoeba attack but less virulent in a mouse model. However, spontaneous reversion to yeast form restored virulence in mice. Titan cells were simultaneously described by two groups (Okagaki et al., 2010; Zaragoza et al., 2010). These consist of C. neoformans cells that enlarge to such a large size that they cannot be ingested by the host cells and essentially precludes phagocytosis by the much smaller host phagocytes. Titan cell giant size is achieved by a combination of increased body size, cell wall thickening, and enlargement of the capsule. In addition, the yeast cells might undergo successive rounds of endoreplication without fission, as these cells have multiple copies of the genome (Okagaki et al., 2010; Zaragoza et al., 2010). Yet do not directly originate other Titan cells, they originate “yeast” size, diploid progeny by regular budding. The relevance of titan cells in infection was confirmed when mutants with a higher predominance of titan cells showed increased host mortality and fungal burden (Crabtree et al., 2012). Another relevant point is that morphological switching increases as C. neoformans cells undergo chronological aging. The phenotypic switching in turn allows the aged cell, that is, lung-resident latent fungal cell, to become more virulent (Bouklas et al., 2013; Jain, Li, et al., 2006). The importance of morphologic changes is underscored when one considers that every type of morphologic change in C. neoformans has directly influenced virulence of the isolate.

3. HOW DOES C. neoformans SURVIVE WITHIN A HOST? 3.1. Intracellular survival of C. neoformans A fascinating question involving C. neoformans is the mechanism by which it acquired an intracellular pathogenic strategy in animal cells. C. neoformans is an environmental organism that does not require animal passage for survival or completion of its life cycle. However, both animal models (Feldmesser, Kress, Novikoff, & Casadevall, 2000; Goldman et al., 1994; Nessa, Gross, Jarstrand, Johansson, & Camner, 1997) and human autopsies (Baker & Haugen, 1955; Schwartz, 1988) have regularly shown C. neoformans intracellularly associated with lung macrophages (see Fig. 1.3). Measurements of


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Figure 1.3 Interaction between C. neoformans and macrophages. (A) Differential interference contrast microscopy illustrating C. neoformans–macrophage interaction, showing adhered (white arrowhead) and ingested (black arrowhead) C. neoformans. (B) Schematic representation of C. neoformans–macrophage intracellular interactions. C. neoformans is opsonized by complement system proteins particularly C3bi which mediates internalization through several receptor interactions (not depicted). C. neoformans uses the fungal capsule, App1 and many other factors to avoid phagocytosis. App1 interacts with CD11b to decrease effective fungal internalization. After internalization, survival and proliferation of C. neoformans are facilitated by phagosome acidification. C. neoformans secretes Pbl1 might induces phagosomal membrane damage and subsequent increase in pH in the phagolysosome. C. neoformans now has access to the cytoplasm where it can interfere with host molecular machinery. However, phagosomal damage activates the NLRP3 inflammasome. The macrophage–C. neoformans interaction at the cell-surface results in production of ROS and through TLR-4/MyD88 cascade in NF-kB activation. Caspase1 is also activated through an unknown mechanism. The final result of receptor crosstalk is secretion of TNF-a, IL-10, and IL-1b. (C) Scanning electron micrographs showing the C. neoformans (white arrowhead)—macrophage (M) interaction. (D) Fluorescence microscopy of C. neoformans (yellow) internalized by macrophages (red). App1, antiphagocytic protein 1; IL-10, interleukin 10; IL-1b, interleukin 1 beta; MyD88, myeloid differentiation primary response 88; NF-kB, nuclear factor k-light-chain enhancer of activated B cells; NLRP3, Nod-like receptor family pyrin domain-containing; Plb1, Phospholipase b1; ROS, reactive oxygen species; TNF-a, tumor necrosis factor alpha; TLR-4, toll-like receptor 4. Panel (A) courtesy of Pedro H. M. Burgel. Panel (C) courtesy of Sabriya Stukes.

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budding index (as a marker of yeast replication) showed higher proliferation inside the host lung macrophages than outside phagocytic cells (Feldmesser, Kress, et al., 2000). The conclusion followed that C. neoformans is not an obligatory intracellular pathogen but can benefit from intracellular residence in mammalian macrophages. In fact, intracellular proliferation rates correlated with virulence of clinical isolates (Alanio et al., 2011; Tucker & Casadevall, 2002). The ultimate argument for an important role for the intracellular lifestyle in virulence comes from the Trojan horse hypothesis, which postulates that survival within macrophages allows fungal transport into the CNS (Charlier et al., 2009). Most intracellular pathogens have some type of phagosomal manipulation tool that is crucial for their survival within macrophages. Some microbes prevent adequate phagosomal maturation while others escape the phagosome. Proof of phagosome manipulation by C. neoformans has not been reported thus far. Instead the cryptococcal phagosome becomes leaky (Johnston & May, 2010; Tucker & Casadevall, 2002) after several hours of C. neoformans–macrophage interaction. Loss of phagosomal membrane integrity would provide the fungus with full access to the cytosolic nutrients and therefore simultaneously dissipate nutrient starvation and interfere with macrophage microbicidal mechanisms. Furthermore, phagosomal leakiness would eliminate the physical separation between pathogen and host, thus facilitating the manipulation of host machinery by the fungal pathogen. Molecular players that mediate phagosomal leakiness have not been described yet, but a likely candidate is Plb1 enzyme, which might damage phospholipid membranes. It is remarkable that C. neoformans is capable of thriving within the intracellular environment but has the capacity to avoid it whenever this intracellular environment becomes less hospitable. However, yeast survival in both intracellular and extracellular environment is required for full virulence. Several of the yeast’s virulence factors can be viewed as designed for avoiding phagocytosis or escaping the phagocytes, namely the capsule and its antiphagocytic proteins. A striking example is titan cell formation. These enormous cells cannot be ingested by macrophages and thus are effectively confined to in the extracellular environment (Crabtree et al., 2012). Another example is that acidification of the phagosome is beneficial for C. neoformans (Qin et al., 2011) and when acidification is blocked, yeast replication is less favorable but nonlytic exocytosis increases (Carnell et al., 2011; Nicola, Robertson, Albuquerque, Derengowski Lda, & Casadevall, 2011).


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3.2. Nonlytic exocytosis Nonlytic exocytosis is a process whereby C. neoformans cells escape from host cells without apparent damage to either host or microbial cells. The process has also been referred by the more colorful term “vomocytosis.” It is also remarkable that cell-to-cell spread has been observed (Alvarez, Burn, Luo, Pirofski, & Casadevall, 2009; Ma, Croudace, Lammas, & May, 2006), whereby yeast cells are passed from one phagosome to the one of a nearby cell. Nonlytic exocytosis was simultaneously described by two groups (Alvarez & Casadevall, 2006; Ma et al., 2006). It is inhibited by interference with phagosomal acidification and it requires viable fungal cells: it does not occur for heat-killed fungus or latex beads (Alvarez & Casadevall, 2006; Johnston & May, 2010; Ma et al., 2006; Nicola et al., 2011). This evidence leads to the conclusion that C. neoformans has an active role in exocytosis but little is known about how this event occurs. The most surprising feature of nonlytic exocytosis is an apparent lack of phagocyte damage. Curiously, host actin flashes inhibit nonlytic exocytosis and phagosomal permeability always precedes nonlytic exocytosis (Johnston & May, 2010). Consequently, it appears that the manipulation of the host cytoskeleton might be necessary for extrusion (Johnston & May, 2010). The existence of a complex process such as nonlytic exocytosis to hijack host intracellular processes (Alvarez & Casadevall, 2006; Carnell et al., 2011; Ma et al., 2006) suggests that C. neoformans suffered long years of evolutionary pressure to develop such a sophisticated escape to phagocytic predation in the environment.

3.3. Dissemination: Penetration blood–brain barrier Cryptococcal disease often results in a life threatening brain infection but the mechanism for the remarkable neurotropism of C. neoformans remains unexplained. The kinetics of brain dissemination from the primary infection site are similarly uncertain, that is, it is not known whether meningoenchephalitis is a result of reactivation of a latent brain infection or the result of dissemination after the onset of pneumonia. When extrapulmonary dissemination was investigated in a rat infection model, dissemination occurred to a very small degree only months after primary infection (Goldman et al., 2000). In this rat model, lung infection was never cleared, hypothetically forming a reservoir for brain dissemination. In mouse intravenous infection models yeast cells readily disappear from the bloodstream and are detected within the brain as early as 1 h (Chang et al.,

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2004; Charlier et al., 2005). These studies suggest that brain dissemination might occur at any point after primary infection of the lung and that once the fungus in the bloodstream it is very easy for the yeast to traverse to the brain. It is likely that C. neoformans establishes a latent infection in the brain in many aspects similar to what happens in the lung. The latent infection is controlled due to antifungal mechanisms of resident immune cells and microglia, the brain macrophage population, can control C. neoformans growth (Lee, Kress, Dickson, & Casadevall, 1995; Lee, Kress, Zhao, Dickson, & Casadevall, 1995). The mechanism of fungistasis by microglia is not dependent on ROS and, contrary to lung macrophages, is not enhanced by the addition of cytokines to influence microglia activation pattern (Lee, Dickson, Brosnan, & Casadevall, 1994; Lipovsky et al., 1998). Astrocytes, a non-hematopoietic brain cell can also prevent fungal growth (Lee et al., 1994), but this brain cell population uses distinct microbicidal effectors. While astrocytes mediated fungal killing through nitric oxide, signaling microglia did not require nitric oxide for fungal control (Lee et al., 1994; Lee, Kress, Dickson, et al., 1995). Similar to what has been reported in interactions with other phagocytes (Levitz et al., 1997), acidification of the phagosome is beneficial for the fungus as fungal control was improved by administration of chloroquine to mouse brain (Mazzolla et al., 1997). Another intriguing question in C. neoformans pathogenesis is how the rigid yeast cell can traverse the host lung epithelium and subsequent tissues to reach the CNS. When experiments are performed with airway epithelial cells, which are capable of binding C. neoformans, very little ingestion is detected. However in endothelial cell monolayers, penetration of yeast cells occurred without damage to the monolayer (Chang et al., 2004), explaining why tail vein injections of yeast suspensions lead to almost immediate fungal detection in the CNS (Charlier et al., 2005). Intravital microscopy has provided convincing data for the occurrence of a transcellular mechanism of brain penetration (Shi et al., 2010) and has shown that penetration of endothelial barriers by yeast cells is not a fortuitous event. Both polystyrene beads and yeast cells can be trapped within lung capillaries but only live yeast cells proceeded to penetrate the vasculature (Sabiiti & May, 2012; Shi et al., 2010). Not only do yeast cells have an active role in penetration but also they induce cytoskeletal changes in the endothelium with the probable goal of facilitating yeast traversing while inducing an after effect of cellular damage (Kim, Cho, et al., 2012; Kim, Crary, et al., 2012; Vu, Eigenheer, Phinney, & Gelli, 2013). Crossing of the endothelial barrier was decreased by deletion of


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the Plb1 (Maruvada et al., 2012) or urease (Shi et al., 2010), implying a role for these enzymes in transport across epithelial layers. The combination of several observations have allowed a tentative illustration of the gene networks required for brain invasion (Tseng et al., 2012; reviewed in Griffiths, Kretschmer, & Kronstad, 2012). The conclusion is that yeast cells possess machinery to quickly penetrate endothelial barriers. The Trojan horse hypothesis posits that a pathogen gains entry into the brain (or other organs) through dissemination within immune cells, in particular macrophage cells. This mechanism has been described for several viruses, such as HIV and simian immunodeficiency virus (Kim, Corey, Alvarez, & Williams, 2003). In C. neoformans, the Trojan horse hypothesis originated by the observation that cryptococci survive within host macrophages and that depletion of alveolar macrophages prevents brain dissemination (Charlier et al., 2009; Kawakami et al., 2002; Kechichian, Shea, & Del Poeta, 2007). In fact, tail vein injection of yeast-infected macrophages lead to a higher brain fungal burden than injection of yeast alone (Charlier et al., 2009). Similarly, an inositol phosphosphingolipid–phospholipase C mutant possesses such a large capsule that it cannot be ingested by lung macrophages. It was found that this mutant does not disseminate from the lungs, presumably because it cannot be carried within host macrophages (Shea, Kechichian, Luberto, & Del Poeta, 2006). Therefore, the evidence thus far supports the coexistence of a transcellular and a Trojan horse mechanism of C. neoformans to traverse into the brain (Casadevall, 2010), where the Trojan horse mechanism might explain the crossing of epithelial barriers necessary to exit the lung.

3.4. Subversion of host immune response C. neoformans, like many pathogenic organisms, manipulates the immune cascades to subvert host defense mechanisms and thus favors yeast survival. A classic example of this subversion is the immunosupression mediated by the capsule and isolated capsular components, as discussed earlier. Other processes have also been described in C. neoformans, such as instances of direct interference with host molecules. The fungal phosphatase Plb1 interacts with host cytoskeletal protein Rac1 facilitating brain invasion (Maruvada et al., 2012; see Fig. 1.3). Other fungal proteins have been described to be involved for capsule-independent antiphagocytic processes. For example, App1 might manipulate host CD11b for evasion of phagocytosis (Stano et al., 2009), while Gat201 transcription factor regulates gene

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expression of antiphagocytic proteins of unknown mechanism (Chun et al., 2011). Other examples of immunomodulation exist but for some studies it is hard to differentiate from a response that originated as a reaction to a more fragile pathogen or de facto immunomodulatory properties. For example, laccase might be immunomodulatory by catalyzing the formation of PGE2 (Erb-Downward & Huffnagle, 2007). In fact, studies with laccasedeficient yeasts (Huffnagle et al., 1995; Mednick, Nosanchuk, & Casadevall, 2005, Qiu et al., 2012) and/or urease-deficient yeasts (Osterholzer et al., 2009), among many other candidates (He et al., 2012) show that in the absence of certain enzymes there is an increased Th1-type immune response resulting in improved survival of the mouse host. However, these studies could not distinguish whether the effects were due to a direct interference with host immune system or an indirect role by hampering C. neoformans survival. There is reason to believe that these are only the first examples of fungal proteins who interact directly with host components and that these types of interactions will be crucial to explain C. neoformans survival within phagocytic cells.

4. WHY IS C. neoformans SUCCESSFUL AS A PATHOGEN? The capacity of C. neoformans for mammalian virulence is intriguing as infection of mammals is not required for completion of C. neoformans life cycle. This raises a fundamental question in cryptococcal pathogenesis: how does a soil organism with no requirement for mammalian hosts become such an important human pathogen? In C. neoformans life as an environmental fungus it must contend with constantly changing temperature and humidity, exposure to nutrient starvation and predation by phagocytic cells such as amoebae. Therefore, the yeast has adapted to nutrient-poor, stress-rich environments. For example, laccase is proposed to have a role in acquisition of nutrients from decaying wood and its product melanin protects against extreme temperatures and solar radiation. Thus free living in diverse and inhospitable environments has laid the groundwork for yeast survival to phagocytic attack. C. neoformans is subject to predation by amoeba (Ruiz, Neilson, Bulmer, 1982a,1982b). Amoebae are ubiquitous creatures that feed of other microorganisms that they encounter in their habitat (Steenbergen, Nosanchuk, Malliaris, & Casadevall, 2004). Serendipitous observations as early as midtwentieth century showed that amoeba could survive by ingesting C. neoformans. Analysis of the interactions of the Acanthamoebae castellani


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amoeba model with C. neoformans revealed numerous similarities between the amoeba intracellular environment and the macrophage intracellular environment (Bunting, Neilson, & Bulmer, 1979; Frager, Chrisman, Shakked, & Casadevall, 2010; Steenbergen, Shuman, & Casadevall, 2001). In both hosts, the yeast induces capsule growth and secretion of polysaccharide and instances of nonlytic exocytosis are detected. C. neoformans exposed to either amoeba or murine macrophage hosts responded with similar transcriptional profiles (Derengowski Lda et al., 2013). Comparable fungal responses have been observed after ingestion by Dictyostelium discoideum (Steenbergen, Nosanchuk, Malliaris, & Casadevall, 2003), Caenorhabditis elegans (Mylonakis, Ausubel, Perfect, Heitman, & Calderwood, 2002), Drosophila melanogaster (Apidianakis et al., 2004), and Galleria melonella (Mylonakis et al., 2005) and the ciliated protists of the Paramecium genus were remarkably efficient at predation and killing of yeast forms (Frager et al., 2010). On the basis of these observations, which establish the possibility for frequent environmental predation, it was proposed that the ubiquitous virulence of C. neoformans arose from selective pressures in the environment (Steenbergen & Casadevall, 2003; Steenbergen et al., 2001), where a combination of exposure to harsh environmental conditions and predation resulted in adaptation to phagocytic environments. Remarkably, compared to fungi subject to similar environmental pressures of the Filobasidiella clade, C. neoformans is by far the most prevalent pathogen (Garcia-Solache et al., 2013). It is curious that closely related fungi, possessing virulence factors such as capsule and laccase are not known pathogens. It suggests that thermotolerance has a disproportionate importance in virulence (Garcia-Solache et al., 2013; Petter, Kang, Boekhout, Davis & Kwon-Chung, 2001; McClelland et al., 2006; Robert & Casadevall, 2009). Thermotolerance will then synergize with adaptations that might have arisen specifically for virulence. For example, capsular polysaccharide from C. neoformans possesses a particular branched structure that might be more efficient in protecting from phagocyte attack than capsules from nonpathogenic species (Araujo Gde et al., 2012). It is possible that these divergent characteristics have resulted from adaptation to the intracellular niche which combined with wide thermotolerance resulted in a well-adapted pathogen. In conclusion, it is likely that the success of C. neoformans as a pathogen is explained by a remarkable combination of harsh environmental living, predation by amoeba, wide thermotolerance, and some degree of pathogen specific adaptation synergizing to greatly enhance intracellular survival skills.

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5. FUTURE DIRECTIONS The study of C. neoformans provides many interesting questions for microbial pathogenesis and for biology in general. Capsular enlargement, giant cell formation, nonlytic exocytosis, and vesicle secretion were first described for fungi in C. neoformans. Origin, assembly, and functions of the fungal capsule are still far from elucidation and will likely be an astonishing unique example of cellular biochemistry. Fungal vesicle secretion has posed new questions about cell to cell communication within a microbial population, within mixed microbial communities, and might even extend to the interaction with mammalian hosts. Another interesting aspect of cryptococcosis is the uniqueness of its virulence strategy. Despite sequencing of the fungal genome (Loftus et al., 2005) (other strains have been sequenced through a collaboration of the Broad Institute, Boston, MA, and the Duke Center for Genome Technology, Durham, NC), many fungal proteins have novel sequences and very few homologs from which to derive their function. For example, a study of protein kinase A signaling has unfolded a role for an OVA1 protein for which no function could be proposed. (Hu et al., 2007). Complementation of genomic studies with proteomic studies (Selvan et al., 2013) is crucial to permit the advancement of the field. Using a cross-species, genetic interaction profiling technique where C. neoformans genes were expressed in Saccharomyces cerevisiae resulted in the identification of two previously unknown virulence proteins, Liv6 and Liv7 (Brown & Madhani, 2012). Thus, study of this remarkable microbe has and will continue to unravel new biological phenomena. It is clear that cryptococcosis is associated with impaired cell-mediated responses. However the immune factor responsible for control of infection is under characterized, i.e., the immune factors that prevent disease in most infected individuals. It is likely that not one factor, but the cooperation of several factors, such as nutrient starvation, oxidative stress and other microbicidal molecules is responsible for control of infection in vivo. Recently, antimicrobicidal peptides, such as histatins, defensins, and cathepsins, were shown to be fungicidal for C. neoformans (Hole, Bui, Wormley, & Wozniak, 2012; Wozniak & Levitz, 2008). Additionally, within inflamed lungs and fungal granulomas, oxygen availability is restricted in comparison with the surrounding tissues. C. neoformans is a true obligate aerobe and thus hypoxia might be an effective microbicidal mechanism (Chun, Liu


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& Madhani, 2007). However, in the context of C. neoformans infection the integration of these microbicidal mechanisms remains a challenge for future studies. Another example of conundrums that need to be addressed is the intriguing phagosomal leakiness. Survival of C. neoformans within macrophages is facilitated by acidification of the phagosome (Kechichian et al., 2007). Nevertheless, during the course of infection, the phagolysosomal compartment becomes leaky and thus acidity is dissipated (Tucker & Casadevall, 2002). At the same time, phagolysosomal damage will result in strong activation of the NLRP3-inflammasone and activation of strong microbicidal mechanisms (Lamkanfi, 2011). The phagosomal damage thus becomes a double-edged sword where either pathogen or host could be conceivably favored. The outcome of infection is the result of a multifactorial interaction between host and pathogen. In fact, the emergence of this environmental yeast as a pathogen has challenged the view of what is a pathogenic organism, of how appropriate is the current definition of virulence factors (Garcia-Solache et al., 2013) and has emphasized the complexity of its virulence composite. For example, in an attempt to understand the virulence composite that resulted in the C. gattii outbreak in Vancouver Island, Canada, gene expression patterns were compared between virulent and nonvirulent strains (Ngamskulrungroj, Price, Sorrell, Perfect, & Meyer, 2011). Another approach compared C. neoformans with C. gattii after both were ingested by rat macrophages hoping to elucidate interspecies differential virulence (Goulart et al., 2010). Likewise, such factors have been investigated in the host side, and to name one variable, human host gender is known to influence susceptibility to cryptococcosis (Hajjeh et al., 1999; McClelland et al., 2013). To fully understand microbial pathogenesis one must bear in mind that numerous factors contribute to the virulence composite (Perfect, 2012; McClelland, Bernhardt & Casadevall, 2006). While there is some room for mammalian specific virulence adaptations in C. neoformans, it seems that the majority of C. neoformans extraordinary success as a pathogen is explained by stochastic evolution from free living in harsh environments, predation by other organisms, and within fungi subject to the same environmental pressures, and high thermotolerance. However, when considering the phenomenon of virulence, it is important to always remember that virulence is a microbial property expressed only in a susceptible host, and the outcome of this interaction is dependent on both players (Casadevall & Pirofski, 1999; Perfect, 2012).

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Catecholamines and virulence of Cryptococcus neoformans.

Virulence-Associated Enzymes of Cryptococcus neoformans.

Modulation of Replicative Lifespan in Cryptococcus neoformans: Implications for Virulence.

Capsule size of Cryptococcus neoformans: control and relationship to virulence.

The ecology of Cryptococcus neoformans.

Population genomics and the evolution of virulence in the fungal pathogen Cryptococcus neoformans.

The ZIP family zinc transporters support the virulence of Cryptococcus neoformans.

Galactoxylomannans of Cryptococcus neoformans.

Cryptococcus neoformans and Cryptococcus gattii, the etiologic agents of cryptococcosis.

Leu1 plays a role in iron metabolism and is required for virulence in Cryptococcus neoformans.

Impact of Protein Palmitoylation on the Virulence Potential of Cryptococcus neoformans.

Role of ferric reductases in iron acquisition and virulence in the fungal pathogen Cryptococcus neoformans.

The role of Aspartyl aminopeptidase (Ape4) in Cryptococcus neoformans virulence and authophagy.

Auxotrophic mutants of Cryptococcus neoformans.

The influence of bacterial interaction on the virulence of Cryptococcus neoformans.

A Small Protein Associated with Fungal Energy Metabolism Affects the Virulence of Cryptococcus neoformans in Mammals.

Heat shock protein 90 localizes to the surface and augments virulence factors of Cryptococcus neoformans.

Sirtuins in the phylum Basidiomycota: A role in virulence in Cryptococcus neoformans.

The lysine biosynthetic enzyme Lys4 influences iron metabolism, mitochondrial function and virulence in Cryptococcus neoformans.

Cryptococcus neoformans var gattii.

Characterization of the virulence of Cryptococcus neoformans strains in an insect model.

Corrigendum: Impact of Resistance to Fluconazole on Virulence and Morphological Aspects of Cryptococcus neoformans and Cryptococcus gattii Isolates.

Aging: an emergent phenotypic trait that contributes to the virulence of Cryptococcus neoformans.

Six-hour pigmentation test for the identification of Cryptococcus neoformans.