Pathogenic basidiomycetous yeast, Cryptococcus neoformans, causes fatal meningitis in immunocompromised individuals. Fluconazole (FLC) is a fungistatic drug commonly administered to treat cryptococcosis. Unfortunately, FLC-resistant strains characterized by various degree of chromosomal instability were isolated from clinical patients. Importantly, the underlying mechanisms that lead to chromosomal instability in FLC-treated C. neoformans remain elusive. Previous studies in fungal and mammalian cells link chromosomal instability to the reactive oxygen species (ROS). This study provides the evidence that exposure of C. neoformans to FLC induces accumulation of intracellular ROS, which correlates with plasma membrane damage. FLC caused transcription changes of oxidative stress related genes encoding superoxide dismutase (SOD1), catalase (CAT3), and thioredoxin reductase (TRR1). Strikingly, FLC contributed to an increase of the DNA damage in vitro, when complexed with iron or copper in the presence of hydrogen peroxide. Strains with isogenic deletion of copper response protein metallothionein were more susceptible to FLC. Addition of ascorbic acid (AA), an anti-oxidant at 10 mM, reduced the inhibitory effects of FLC. Consistent with potential effects of FLC on DNA integrity and chromosomal segregation, FLC treatment led to elevated transcription of RAD54 and repression of cohesin-encoding gene SCC1. We propose that FLC forms complexes with metals and contributes to elevated ROS, which may lead to chromosomal instability in C. neoformans.

Members of the Cryptococcus species complex stand out by unique virulence factors that allowed evolutionary transition to pathogenesis. Among the factors contributing to cryptococcosis is a morphological transformation into giant (Titan) cells. It remains unclear whether species outside of the C. neoformans / C. gattii species complex are capable of titanization. We utilized two recently developed protocols that allow obtaining Titan cells in vitro to test if titanization occurs in non- C. neoformans / C. gattii species. We find that none of the tested strains, representing 10 species of basidiomycetous yeasts and the ascomycetous yeast Saccharomyces cerevisiae , undergo significant titanization under conditions that promote robust Titan cell formation in C. neoformans / C. gattii species complex. C. terreus formed occasional enlarged cells through a mechanism potentially similar to that of titanization. Our findings suggest that titanization is a rare phenomenon among basidiomycetous yeasts that occurs mostly in members of the C. neoformans/C. gattii species complex.

Opportunistic pathogens of the anamorphic genus Cryptococcus are unique considering their virulence factors that in the context of pathogenesis allowed them to achieve evolutionary success. Morphological transformation into giant (Titan) cells is one of the factors contributing to cryptococcosis. Recently established in vitro protocols demonstrate that 5 or 10% fetal bovine serum (FBS) combined with 5% CO2, 37 °C, and sufficiently low cell density, triggers cellular enlargement (Serum protocols). However, the FBS components that promote this morphological transition remain incompletely characterized. In search of minimal conditions necessary for stimulating the formation of Titan cells, we performed a study where we eliminated serum from the protocol (Serum-free protocol) and instead systematically adjusted the amount of glucose, source of nitrogen (ammonium sulfate), and the pH. We found that exposing cells to PBS with adjusted pH to 7.3, and supplemented with 0.05% glucose, 0.025% ammonium sulfate, 0.004% K2HPO4, 0.0035% MgSO4, in the presence of 5% CO2 at 37 °C triggers Titan-like cell formation to the same degree as the previously established protocol that utilized 10% FBS as the sole nutrient source. Titan-like cells obtained according to this Serum-free protocol were characterized by cell body size over ten microns, a single enlarged vacuole, thick cell wall, extensive polysaccharide capsule, and changes in the level of cell ploidy, all currently known hallmarks of Titan cells found in vivo. Strikingly, we found that in both, Serum and Serum-free protocols, an optimal pH for Titan-like cell development is ~7.3 whereas relatively acidic pH (5.5) prevents this morphological transition and promotes robust proliferation, while alkaline pH (~8.0) has a profound growth inhibitory effect. Our study demonstrates a critical role of pH response to the formation of Titan cells and indicates that conditions that allow restricted proliferation in the presence of 5% CO2 are sufficient for this morphological transition to form enlarged cells in Cryptococcus neoformans and Cryptococcus gattii species complex.

Dylag, Colon-Reyes, and Kozubowski 1 1 2 3 Fetal bovine serum-triggered Titan cell formation and growth inhibition are unique 4 to the Cryptococcus species complex 5 6 ABSTRACT 18In the genus Cryptococcus, C. neoformans and C. gattii stand out by the number of virulence 19 factors that allowed those fungi to achieve evolutionary success as pathogens. Among the factors 20 contributing to cryptococcosis is a peculiar morphological transition to form giant (Titan) cells. 21 Formation of Titans has been described in vitro. However, it remains unclear whether non-C. 22 neoformans/non-C. gattii species are capable of titanisation. Based on a survey of several 23 basidiomycetous yeasts, we propose that titanisation is unique to C. neoformans and C. gattii. In 24 addition, we find that under in vitro conditions that induce titanisation, fetal bovine serum (FBS) 25 possesses activity that inhibits growth of C. gattii and kills C. neoformans if incubation is 26 conducted in the absence of 5% CO 2 . Moreover, lowering of pH or addition of an antioxidant, 27 rescues growth in the presence of FBS and allows titanisation even in the absence of 5% CO 2 . 28Strikingly, acapsular mutants, cap10Δ and cap59Δ, show complete or partial reduction of 29 titanisation, respectively and a partial resistance to growth inhibition imposed by the FBS. Our 30 data implicate titanisation as a unique feature of C. neoformans and C. gattii and provide novel 31 insights about the nature of this unusual morphological transition. 32 33 34 35 36 37 42 Trichosporon 1 . In particular, two species belonging to the Cryptococcus genus (formerly 43 Filobasidiella), C. neoformans and C. gattii are the etiological agents of fatal systemic mycoses. 44 In recent years, nearly one million cases of cryptococcal meningitis occur annually resulting in 45 over 600,000 deaths globally 2 . Moreover, cryptococcosis is responsible for 15-17% AIDS-46 related deaths on a global scale 3,4 . While C. neoformans can cause mainly opportunistic 47 infections in immunocompromised patients, C. gattii is capable of infecting also 48 immunocompetent individuals 5-8 . Moreover, it is well established that among C. neoformans, 49 serotype A (varietas grubii) is responsible for the majority of cryptococcal infections, while 50 serotype D (varietas neoformans) is less common 7 . Among Cryptococcus species other than C. 51 neoformans and C. gattii, the following species have been described as causing occasional 52 infections in humans: C. laurentii 9-11 , C. albidus 9,12,13 , C. curvatus 14,15 , C. uniguttulatus 16 , and 53 C. adeliensis 17 . Such casuistic infections, reviewed in literature most recently by Smith et al. 18 54 can be also systemic in case of strains able to grow at 37°C. What makes those selected 55 Cryptococcus species capable of infecting humans is an important question, the answer to which 56 remains incomplete. Among the best-described cryptococcal characteristics necessary for 57 pathogenicity are the ability to proliferate at 37°C, melanisation, formati...