The yeasts Cryptococcus neoformans and Cryptococcus gattii are fungal pathogens that can be isolated from the environment, including the surfaces of many plants. Cryptococcus gattii caused an outbreak on Vancouver Island, British Columbia beginning in 1999 that has since spread to the Pacific Northwest of the United States. Coastal Douglas fir (Pseudotsuga menziesii) is an important lumber species and a major component of the ecosystems in this area. Previous research has explored Cryptococcus survival and mating on Douglas fir plants and plant-derived material, but no studies have been done on the production of cryptococcal virulence factors by cells grown on those media. Here, we investigated the effects of growth on Douglas fir-derived media on the production of the polysaccharide capsule and melanin, two of the most important cryptococcal virulence factors. We found that while the capsule was mostly unchanged by growth in Douglas fir media compared to cells grown in defined minimal media, Cryptococcus spp. can use substrates present in Douglas fir to synthesize functional and protective melanin. These results suggest mechanisms by which Cryptococcus species may survive in the environment and emphasize the need to explore how association with Douglas fir trees could affect its epidemiology for human cryptococcosis.
Cryptococcus gattii is a fungal pathogen that can be found in the environment. It is responsible for causing an outbreak in British Columbia, Canada, in the late 90s. In our study, we created media from Douglas fir, a tree commonly found in the affected areas. We examined the production of virulence factors by Cryptococcus cells grown in this media.

Cryptococcus neoformans is an environmentally acquired fungal pathogen that causes over 140,000 deaths per year. Cryptococcal infection occurs when infectious particles are deposited into the lung, where they encounter host phagocytic cells. C. neoformans may be engulfed by these phagocytes, an important step of infection that leads to outcomes ranging from termination of infection to cryptococcal dissemination. To study this critical process, we screened approximately 4,700 cryptococcal gene deletion mutants for altered uptake, using primary mouse and human phagocytic cells. Among the hits of these two screens, we identified 93 mutants with perturbed uptake in both systems, as well as others with differences in uptake by only one cell type. We further screened the hits for changes in thickness of the capsule, a protective polysaccharide layer around the cell which is an important cryptococcal virulence factor. The combination of our three screens yielded 45 mutants, including one lacking the phosphatidylinositol-4-phosphate phosphatase Sac1. In this work, we implicate Sac1 in both host cell uptake and capsule production. We found that sac1 mutants exhibit lipid trafficking defects, reductions in secretory system function, and changes in capsule size and composition. Many of these changes occur specifically in tissue culture media, highlighting the role of Sac1 phosphatase activity in responding to the stress of host-like conditions. Overall, these findings show how genome-scale screening can identify cellular factors that contribute to our understanding of cryptococcal biology and demonstrate the role of Sac1 in determining fungal virulence.IMPORTANCECryptococcus neoformans is a fungal pathogen with significant impact on global health. Cryptococcal cells inhaled from the environment are deposited into the lungs, where they first contact the human immune system. The interaction between C. neoformans and host cells is critical because this step of infection can determine whether the fungal cells die or proliferate within the human host. Despite the importance of this stage of infection, we have limited knowledge of cryptococcal factors that influence its outcome. In this study, we identify cryptococcal genes that affect uptake by both human and mouse cells. We also identify mutants with altered capsule, a protective coating that surrounds the cells to shield them from the host immune system. Finally, we characterize the role of one gene, SAC1, in these processes. Overall, this study contributes to our understanding of how C. neoformans interacts with and protects itself from host cells.

Dual-specificity LAMMER kinases are highly evolutionarily conserved in eukaryotes and play pivotal roles in diverse physiological processes, such as growth, differentiation, and stress responses. Although the functions of LAMMER kinase in fungal pathogens in pathogenicity and stress responses have been characterized, its role in Cryptococcus neoformans, a human fungal pathogen and a model yeast of basidiomycetes, remains elusive. In this study, we identified a LKH1 homologous gene and constructed a strain with a deleted LKH1 and a complemented strain. Similar to other fungi, the lkh1Δ mutant showed intrinsic growth defects. We observed that C. neoformans Lkh1 was involved in diverse stress responses, including oxidative stress and cell wall stress. Particularly, Lkh1 regulates DNA damage responses in Rad53-dependent and -independent manners. Furthermore, the absence of LKH1 reduced basidiospore formation. Our observations indicate that Lkh1 becomes hyperphosphorylated upon treatment with rapamycin, a TOR protein inhibitor. Notably, LKH1 deletion led to defects in melanin synthesis and capsule formation. Furthermore, we found that the deletion of LKH1 led to the avirulence of C. neoformans in a systemic cryptococcosis murine model. Taken together, Lkh1 is required for the stress response, sexual differentiation, and virulence of C. neoformans.

Glucuronoxylomannan (GXM) is the principal capsular component in the Cryptococcus genus. This complex polysaccharide participates in numerous events related to the physiology and pathogenesis of Cryptococcus, which highlights the importance of establishing methods for its isolation and analysis. Conventional methods for GXM isolation have been extensively discussed in the literature. In this chapter, we describe two fast methods for obtaining extracellular fractions enriched with cryptococcal GXM.

The polysaccharide capsule of Cryptococcus neoformans is the primary virulence factor and one of the most commonly studied aspects of this pathogenic yeast. Capsule size varies widely between strains, has the ability to grow rapidly when introduced to stressful or low-nutrient conditions, and has been positively correlated with strain virulence. For these reasons, the size of the capsule is of great interest to C. neoformans researchers. Inducing the growth of the C. neoformans capsule is used during phenotypic testing to help understand the effects of different treatments on the yeast or size differences between strains. Here, we describe one of the standard methods of capsule induction and detail two accepted methods of staining: (i) India ink, a negative stain, used in conjunction with conventional light microscopy and (ii) co-staining with fluorescent dyes of both the cell wall and capsule followed by confocal microscopy. Finally, we outline how to measure capsule diameter manually and offer a protocol for automated diameter measurement of India ink-stained samples using computational image analysis.

The polysaccharide capsule of fungal pathogen Cryptococcus neoformans is a critical virulence factor that has historically evaded complete characterization. Cryptococcal polysaccharides are known to either remain attached to the cell as capsular polysaccharides (CPSs) or to be shed into the extracellular space as exopolysaccharides (EPSs). While many studies have examined the properties of EPS, far less is known about CPS. In this work, we detail the development of new physical and enzymatic methods for the isolation of CPS which can be used to explore the architecture of the capsule and isolated capsular material. We show that sonication or Glucanex enzyme cocktail digestion yields soluble CPS preparations, while use of a French pressure cell press or Glucanex digestion followed by cell disruption removed the capsule and produced cell wall-associated polysaccharide aggregates that we call \"capsule ghosts\", implying an inherent organization that allows the CPS to exist independent of the cell wall surface. Since sonication and Glucanex digestion were noncytotoxic, it was also possible to observe the cryptococcal cells rebuilding their capsule, revealing the presence of reducing end glycans throughout the capsule. Finally, analysis of dimethyl sulfoxide-extracted and sonicated CPS preparations revealed the conservation of previously identified glucuronoxylomannan motifs only in the sonicated CPS. Together, these observations provide new insights into capsule architecture and synthesis, consistent with a model in which the capsule is assembled from the cell wall outward using smaller polymers, which are then compiled into larger ones.

Fungal infections impact over 25% of the global population. For the opportunistic fungal pathogen, Cryptococcus neoformans, infection leads to cryptococcosis. In the presence of the host, disease is enabled by elaboration of sophisticated virulence determinants, including polysaccharide capsule, melanin, thermotolerance, and extracellular enzymes. Conversely, the host protects itself from fungal invasion by regulating and sequestering transition metals (e.g., iron, zinc, copper) important for microbial growth and survival.
Here, we explore the intricate relationship between zinc availability and fungal virulence via mass spectrometry-based quantitative proteomics. We observe a core proteome along with a distinct zinc-regulated protein-level signature demonstrating a shift away from transport and ion binding under zinc-replete conditions towards transcription and metal acquisition under zinc-limited conditions. In addition, we revealed a novel connection among zinc availability, thermotolerance, as well as capsule and melanin production through the detection of a Wos2 ortholog in the secretome under replete conditions.
Overall, we provide new biological insight into cellular remodeling at the protein level of C. neoformans under regulated zinc conditions and uncover a novel connection between zinc homeostasis and fungal virulence determinants.

Cryptococcus gattii is a capsular pathogenic fungus causing life-threatening cryptococcosis. Although the capsular polysaccharides (CPs) of C. gattii are considered as virulence factors, the physiological significance of CP biosynthesis and of CPs themselves is not fully understood, with many conflicting data reported. First, we demonstrated that CAP gene deletant of C. gattii completely lacked capsule layer and its virulence, and that the strain was susceptible to host-related factors including oxidizing, hypoxic, and hypotrophic conditions in vitro. Extracellular CPs recovered from culture supernatant bound specifically to C. gattii acapsular strains, not to other fungi and immune cells, and rendered them the immune escape effects. In fact, dendritic cells (DCs) did not efficiently uptake the CP-treated acapsular strains, which possessed no visible capsule layer, and a decreased amount of phosphorylated proteins and cytokine levels after the stimulation. DCs recognized C. gattii acapuslar cells via an immune receptor CD11b- and Syk-related pathway; however, CD11b did not bind to CP-treated acapsular cells. These results suggested that CPs support immune evasion by coating antigens on C. gattii and blocking the interaction between CD11b and C. gattii cells. Here, we describe the importance of CPs in pathogenicity and immune evasion mechanisms of C. gattii.

Cryptococcus spp., in particular Cryptococcus neoformans and Cryptococcus gattii, have an enormous impact on human health worldwide. The global burden of cryptococcal meningitis is almost a quarter of a million cases and 181,000 deaths annually, with mortality rates of 100% if infections remain untreated. Despite these alarming statistics, treatment options for cryptococcosis remain limited, with only three major classes of drugs approved for clinical use. Exacerbating the public health burden is the fact that the only new class of antifungal drugs developed in decades, the echinocandins, displays negligible antifungal activity against Cryptococcus spp., and the efficacy of the remaining therapeutics is hampered by host toxicity and pathogen resistance. Here, we describe the current arsenal of antifungal agents and the treatment strategies employed to manage cryptococcal disease. We further elaborate on the recent advances in our understanding of the intrinsic and adaptive resistance mechanisms that are utilized by Cryptococcus spp. to evade therapeutic treatments. Finally, we review potential therapeutic strategies, including combination therapy, the targeting of virulence traits, impairing stress response pathways and modulating host immunity, to effectively treat infections caused by Cryptococcus spp. Overall, understanding of the mechanisms that regulate anti-cryptococcal drug resistance, coupled with advances in genomics technologies and high-throughput screening methodologies, will catalyse innovation and accelerate antifungal drug discovery.

Cryptococcus neoformans is an opportunistic fungal pathogen, which is a frequent cause of a life-threatening meningitis in immunocompromised individuals. We report the first total synthesis of the serotype B heptasaccharide repeating motif. The use of di- and trisaccharide building blocks enabled a concise convergent synthesis of the protected 6-O-acetylated repeating motif in three steps. Glycosylations gave total 1,2-trans selectivity, despite the absence of a neighboring participating group. Using our recently disclosed catalyst pre-tuning strategy global deprotection gave the desired 6-O-acetylated heptasaccharide with no saturation by-products, overall in four steps 31% yield. The serotype B glucuronoxylomannan (GXM) glycans accessed in this study will increase the structurally diversity of our GXM microarray, allowing further steps towards the development of semi-synthetic vaccines against cryptococcal infections.