The fungal meningitis pathogen Cryptococcus neoformans is a central driver of mortality in HIV/AIDS. We report a genome-scale chemical genetic data map for this pathogen that quantifies the impact of 439 small-molecule challenges on 1,448 gene knockouts. We identified chemical phenotypes for 83% of mutants screened and at least one genetic response for each compound. C. neoformans chemical-genetic responses are largely distinct from orthologous published profiles of Saccharomyces cerevisiae, demonstrating the importance of pathogen-centered studies. We used the chemical-genetic matrix to predict novel pathogenicity genes, infer compound mode of action, and to develop an algorithm, O2M, that predicts antifungal synergies. These predictions were experimentally validated, thereby identifying virulence genes, a molecule that triggers G2/M arrest and inhibits the Cdc25 phosphatase, and many compounds that synergize with the antifungal drug fluconazole. Our work establishes a chemical-genetic foundation for approaching an infection responsible for greater than one-third of AIDS-related deaths.

Oropharyngeal candidiasis (OPC) is a common infection that complicates a wide range of medical conditions and can cause either mild or severe disease depending on the patient. The pathobiology of OPC shares many features with candidal biofilms of abiotic surfaces. The transcriptional regulation of C. albicans biofilm formation on abiotic surfaces has been extensively characterized and involves six key transcription factors (Efg1, Ndt80, Rob1, Bcr1, Brg1, and Tec1). To determine if the in vitro biofilm transcriptional regulatory network also plays a role in OPC, we carried out a systematic genetic interaction analysis in a mouse model of C. albicans OPC. Whereas each of the six transcription factors are required for in vitro biofilm formation, only three homozygous deletion mutants (tec1ΔΔ, bcr1ΔΔ, and rob1ΔΔ) and one heterozygous mutant (tec1Δ/TEC1) have reduced infectivity in the mouse model of OPC. Although single mutants (heterozygous or homozygous) of BRG1 and EFG1 have no effect on fungal burden, double heterozygous and homozygous mutants have dramatically reduced infectivity, indicating a critical genetic interaction between these two transcription factors during OPC. Using epistasis analysis, we have formulated a genetic circuit, [EFG1+BRG1]→TEC1→BCR1, that is required for OPC infectivity and oral epithelial cell endocytosis. Surprisingly, we also found transcription factor mutants with in vitro defects in filamentation, such as efg1ΔΔ, rob1ΔΔ, and brg1ΔΔ filament, during oral infection and that reduced filamentation does not correlate with infectivity. Taken together, these data indicate that key in vitro biofilm transcription factors are involved in OPC but that the network characteristics and functional connections during infection are distinct from those observed in vivo. IMPORTANCE The pathology of oral candidiasis has features of biofilm formation, a well-studied process in vitro. Based on that analogy, we hypothesized that the network of transcription factors that regulates in vitro biofilm formation has similarities and differences during oral infection. To test this, we employed the first systematic genetic interaction analysis of C. albicans in a mouse model of oropharyngeal infection. This revealed that the six regulators involved in in vitro biofilm formation played roles in vivo but that the functional connections between factors were quite distinct. Surprisingly, we also found that while many of the factors are required for filamentation in vitro, none of the transcription factor deletion mutants was deficient for this key virulence trait in vivo. These observations clearly demonstrate that C. albicans regulates key aspects of its biology differently in vitro and in vivo.

Candida albicans is a commensal fungus that colonizes the human oral cavity and gastrointestinal tract but also causes mucosal as well as invasive disease. The expression of virulence traits in C. albicans clinical isolates is heterogeneous and the genetic basis of this heterogeneity is of high interest. The C. albicans reference strain SC5314 is highly invasive and expresses robust filamentation and biofilm formation relative to many other clinical isolates. Here, we show that SC5314 derivatives are heterozygous for the transcription factor Rob1 and contain an allele with a rare gain-of-function SNP that drives filamentation, biofilm formation, and virulence in a model of oropharyngeal candidiasis. These findings explain, in part, the outlier phenotype of the reference strain and highlight the role heterozygosity plays in the strain-to-strain variation of diploid fungal pathogens.

The innate immune system is the first line of defense against invasive fungal infections. As a consequence, many successful fungal pathogens have evolved elegant strategies to interact with host immune cells. For example, Candida albicans undergoes a morphogenetic switch coupled to cell wall remodeling upon phagocytosis by macrophages and then induces macrophage pyroptosis, an inflammatory cell death program. To elucidate the genetic circuitry through which C. albicans orchestrates this host response, we performed the first large-scale analysis of C. albicans interactions with mammalian immune cells. We identified 98 C. albicans genes that enable macrophage pyroptosis without influencing fungal cell morphology in the macrophage, including specific determinants of cell wall biogenesis and the Hog1 signaling cascade. Using these mutated genes, we discovered that defects in the activation of pyroptosis affect immune cell recruitment during infection. Examining host circuitry required for pyroptosis in response to C. albicans infection, we discovered that inflammasome priming and activation can be decoupled. Finally, we observed that apoptosis-associated speck-like protein containing a CARD (ASC) oligomerization can occur prior to phagolysosomal rupture by C. albicans hyphae, demonstrating that phagolysosomal rupture is not the inflammasome activating signal. Taking the data together, this work defines genes that enable fungal cell wall remodeling and activation of macrophage pyroptosis independently of effects on morphogenesis and identifies macrophage signaling components that are required for pyroptosis in response to C. albicans infection.IMPORTANCECandida albicans is a natural member of the human mucosal microbiota that can also cause superficial infections and life-threatening systemic infections, both of which are characterized by inflammation. Host defense relies mainly on the ingestion and destruction of C. albicans by innate immune cells, such as macrophages and neutrophils. Although some C. albicans cells are killed by macrophages, most undergo a morphological change and escape by inducing macrophage pyroptosis. Here, we investigated the C. albicans genes and host factors that promote macrophage pyroptosis in response to intracellular fungi. This work provides a foundation for understanding how host immune cells interact with C. albicans and may lead to effective strategies to modulate inflammation induced by fungal infections.

Importance

Candida albicans is a commensal fungus that colonizes the human oral cavity and gastrointestinal tract but also causes mucosal as well as invasive disease. The expression of virulence traits in C. albicans clinical isolates is heterogeneous and the genetic basis of this heterogeneity is of high interest. The C. albicans reference strain SC5314 is highly invasive and expresses robust filamentation and biofilm formation relative to many other clinical isolates. Here, we show that SC5314 derivatives are heterozygous for the transcription factor Rob1 and contain an allele with a rare gain-of-function SNP that drives filamentation, biofilm formation, and virulence in a model of oropharyngeal candidiasis. These findings explain, in part, the outlier phenotype of the reference strain and highlight the role heterozygosity plays in the strain-to-strain variation of diploid fungal pathogens.