Flujo de Caja

Virulence is the relative (ie. compared against other genotypes of the species in question) capacity for a microorganism to cause disease in a host. Cryptococcus neoformans and C. gattii are opportunistic pathogens and human infections are accidental. Therefore, the human host is not the primary niche of the pathogen and does not contribute to the overall survival of the species. The arboreal fungus does not require an animal host to complete its replication. Soil and decaying matter is a complex ecological niche and the pathogenicity of the Cryptococcus neoformans/gattii species complex may have originated from environmental selective pressures. Infection of macrophages is similar to infection observed in amoeba and Steenbergen et al. (2001) hypothesized that the pathogenicity of the yeast in mammalian hosts evolved as a defence mechanism from environmental predators.

1.8.1

Cryptococcus fitness

Fitness describes the ability of an organism to survive and reproduce in a particular environ- ment. While many species of Cryptococcus are present in the environment, only two can invade and cause diseases in human. The Cryptococcus neoformans/gattii species complex is associated with different ecological niches which lead to potential fitness variations among Cryptococcus populations. During infection, the expression of virulence traits is central in determining the fitness of the pathogen. Panepinto et al. (2006) defined pathogenic fitness in the context of

Cryptococcus neoformans as ‘the capability to successfully propagate within the stressful envi- ronment of the host, causing disease by expression of virulence traits that damage the host.’The nature and the variety of the ecological niches occupied by the pathogen are still unknown but are excepted to be complex and to include bacteria, animals, plants or even other fungi (Steen- bergen and Casadevall, 2003). Each entity will impose a selective force upon cryptococcal cells in the environment, resulting in a range of genotypes that have been exposed to heterogeneous selective forces. These diverse selective regimes, and the resulting diversity in traits that are important for survival, are then expected to translate into variation in fitness amongst geno- types during human infection. Research in the study of host-pathogen interactions between cryptococcal cells and potential hosts using Next-Generation Sequencing (NGS) technologies are just beginning and will shed light on the evolution of cryptococcal pathogenicity.

1.8.2

How does an environmental yeast become a human pathogen?

The origin of virulence in pathogenic fungi is largely unknown and the interactions between the fungus and invertebrates remain abstract. Observations indicates that C. neoformans infection of amoeba resembles macrophage invasion (Steenbergen et al., 2001). For instance, Cng is able to escape from both amoeba and macrophages using non-lytic process (Chrisman Alvarez M, Casadevall A, 2010). Virulence in murine models are enhanced by serial passage in amoeba such as Hartmanella vermiformis, Willaertia magna or Acanthamoeba castellanii and exposure to amoeba leads to increases in the Cryptococcus hyphal form (Malliaris et al., 2004). Lastly, virulence factors observed in cryptococcal infection in mammals are similar to those observed in amoeba (Casadevall, 2012).

Non-vertebrates such as amoeba represent simple and inexpensive models to study the patho- genesis of Cryptococcus. Normally, amoeba feed on bacteria but there is an important diversity in this group of organism and some species seem to specialise in feeding on fungi (Coˆuteaux and Darbyshire, 1998). Known as mycophageous amoeba, the latter were discovered preying on Cryptococcus by accident in 1930 (Castellani, 1930), but the evidence of amoeba feeding on yeast was only established in the 1960s (Nero et al., 1964). The two species co-exist in many soil/woody environments and must interact in many different ways across microecological niches where they co-occur.

In the first study of the interactions between amoeba and C. neoformans, Steenbergen et al. (2001) showed that acapsular strains were killed in the presence of A. castellanii. The morphological switch from yeast to a pseudohyphal form, described by Magditch et al. (2012) seems to enable the survival of the fungal cell in amoeba. Melanisation and the enlargement of capsule size also promoted cell survival. The study showed that the fungal cell developed an intracellular survival strategy and was able to replicate inside a phagocytic vacuole. Malliaris et al. (2004) continued the investigation further and noted that despite variations between capsule structures, the different pathogenic Cryptococcus species had the ability to exploit amoeba for growth.

1.8.3

Virulence factors in C. neoformans

In the last decade, multiple virulence factors have been identified that are important in mam- malian infection (Table 1.1), each meeting the following requirements: 1) the property is as- sociated with the pathogen, 2) the inactivation of the gene leads to a decrease in the observed virulence, and 3) the restoration of the gene results in restoration of the virulence (Steenbergen and Casadevall, 2003).

Polysaccharide Capsule

The polysaccharide capsule is an important virulence factor. The main component is glu- curonoxylomannan (GXM) which allows the capsule to protect the cell against dehydration (Aksenov et al., 1973). The polysaccharide capsule has multiple functions both in the environ- ment and within the hosts 1.1 Interestingly, GXM is currently been targeted for the develop- ment of a cryptococcal vaccine (Chow and Casadevall, 2011). Capsule enlargement provides protection against microbicidal molecules released by the host during infection (Zaragoza et al., 2008). At least four genes (CAP10, CAP59, CAP60 and CAP64 ) have been identified and are required for virulence in mouse model (Kwon-Chung, 1998).

Melanin Production

Produced by laccase through the polymerisation of phenolic compounds, melanin is a critical virulence factor (Steenbergen and Casadevall, 2003). Melanised cryptococcal cells are less susceptible to antimicrobial activities and such as nitrogen and oxygen oxidants (Williamson, 1997). The fungal cells are resistant to high temperature, UV radiation, oxidant and heavy metals (Williamson, 1997). Laccase protects the fungus from oxidative damage of alveolar macrophages (Liu et al., 1999). Also associated with lignin degradation, the enzyme is thought to enhance the saprophyte ability to colonise decaying wood (Lazera et al., 1996).

Thermotolerance

To infect human, fungi must have the ability to grow at 37◦C. C. neoformans and C. gattii are the only Tremellales that are heat tolerant and can grow at temperatures above 30◦C (Bovers et al., 2008). Only 270 species of fungi over the 1.5 million estimated are able to cause disease in human (Hawksworth, 2001). Most of them are well adapted to growth in high temperatures, which is essential for mammalian infection. Mannitol production is associated with heat protection and resistance to osmotic stress. Previous studies revealed that the two thermotolerant species displayed elevated transcript levels of heat shock proteins and translation machinery when grown at 25 and 35◦C respectively (Steen et al., 2002).

Other Virulence Factors

Phospholipase can have nutritional roles and be involved in survival in the environment. The enzyme, encoded by PLB1 gene, helps in the degradation of cell membranes. The activity of PLB1 has been investigated in murine models by Ghannoum (2000) and a significant correlation was observed with virulence in mice. Many other enzymes are involved in C. neoformans virulence. For example, urease, a virulence factor in many bacteria, is present in the majority of clinical C. neoformans. Proteinases released by fungal cells can degrade host cell components and are thought to aid in escaping phagocytosis (Chen et al., 1996). Studies in murine models established the virulent character of the alpha mating type over a-mating type (Casadevall et al., 2003). Both in nature and in patients, the alpha mating type is present 30 times more commonly than MAT a.

Table 1.1: Virulence factors for C. neoformans - adapted from (Casadevall et al., 2003). Function

Virulence Factors In the Environment In Pathogenesis

Capsule

Prevents desiccation (Aksenov et al., 1973)

Immunomodulation (Chow and

Casadevall, 2011) Protection against amoeboid preda-

tors (Steenbergen et al., 2001) Antiphagocytic (Kozel et al., 1988) Intracellular agressin (Feldmesser et al., 2000)

Laccase Lignin degradation (Lazera et al.,

1996)

Interference with oxidative burst (Liu et al., 1999)

Melanin

Ultraviolet shielding (Wang and Casadevall, 1994)

Resistance to oxidative killing (Wang and Casadevall, 1994)

Heat and cold tolerance (Rosas and Casadevall, 1997)

Antiphagocytic (Wang and Casade- vall, 1994)

Reduced susceptibility to enzymatic degradation (Rosas and Casadevall, 2001)

Immunomodulator (Huffnagle et al., 1995)

Protection against heavy metals

(Garcia-Rivera and Casadevall,

2001)

Resistance to microbicidal peptides (Casadevall et al., 2000)

Protection against amoeboid preda- tors

Antifungal-drug resistance (van Duin et al., 2002)

Phospholipase

Nutritional function (Ghannoum, 2000)

Intracellular growth (Cox et al., 2000)

Protection against amoeboid preda- tors

Proteases Nutritional function (Chen et al.,

1996) Tissue damage (Chen et al., 1996)

Urease Nitrogen scavenging (Cox et al.,

2000)

Intracellular growth (Cox et al., 2000)

Other virulent functions Phenotypic switch-

ing

Generation of strain diversity to sur- vive environmental stress

Immune evasion (Goldman et al., 1998)

Mating type Sexual reproduction (Wickes et al.,

1996)

Virulence factor regulation (Wickes et al., 1996)

Calcineurin and

cAMP signalling

Development and reproduction

(D’Souza and Heitman, 2001)

Virulence factor regulation (D’Souza and Heitman, 2001)

Superoxide dismu- tase

Protection against oxygen-derived oxidants?

Intracellular growth (Cox et al., 2000)