Systematic genetic analysis of virulence in the human fungal pathogen Cryptococcus neoformans.

The fungus Cryptococcus neoformans is a leading cause of mortality and morbidity among HIV-infected individuals. We utilized the completed genome sequence and optimized methods for homologous DNA replacement using high-velocity particle bombardment to engineer 1201 gene knockout mutants. We screened this resource in vivo for proliferation in murine lung tissue and in vitro for three well-recognized virulence attributes-polysaccharide capsule formation, melanization, and growth at body temperature. We identified dozens of previously uncharacterized genes that affect these known attributes as well as 40 infectivity mutants without obvious defects in these traits. The latter mutants affect predicted regulatory factors, secreted proteins, and immune-related factors, and represent powerful tools for elucidating novel virulence mechanisms. In particular, we describe a GATA family transcription factor that inhibits phagocytosis by murine macrophages independently of the capsule, indicating a previously unknown mechanism of innate immune modulation.

A link between virulence and homeostatic responses to hypoxia during infection by the human fungal pathogen Cryptococcus neoformans.

Fungal pathogens of humans require molecular oxygen for several essential biochemical reactions, yet virtually nothing is known about how they adapt to the relatively hypoxic environment of infected tissues. We isolated mutants defective in growth under hypoxic conditions, but normal for growth in normoxic conditions, in Cryptococcus neoformans, the most common cause of fungal meningitis. Two regulatory pathways were identified: one homologous to the mammalian sterol-response element binding protein (SREBP) cholesterol biosynthesis regulatory pathway, and the other a two-component-like pathway involving a fungal-specific hybrid histidine kinase family member, Tco1. We show that cleavage of the SREBP precursor homolog Sre1-which is predicted to release its DNA-binding domain from the membrane-occurs in response to hypoxia, and that Sre1 is required for hypoxic induction of genes encoding for oxygen-dependent enzymes involved in ergosterol synthesis. Importantly, mutants in either the SREBP pathway or the Tco1 pathway display defects in their ability to proliferate in host tissues and to cause disease in infected mice, linking for the first time to our knowledge hypoxic adaptation and pathogenesis by a eukaryotic aerobe. SREBP pathway mutants were found to be a hundred times more sensitive than wild-type to fluconazole, a widely used antifungal agent that inhibits ergosterol synthesis, suggesting that inhibitors of SREBP processing could substantially enhance the potency of current therapies.

A major role for capsule-independent phagocytosis-inhibitory mechanisms in mammalian infection by Cryptococcus neoformans.

The antiphagocytic polysaccharide capsule of the human fungal pathogen Cryptococcus neoformans is a major virulence attribute. However, previous studies of the pleiotropic virulence determinant Gat201, a GATA family transcription factor, suggested that capsule-independent antiphagocytic mechanisms exist. We have determined that Gat201 controls the mRNA levels of ∼1100 genes (16% of the genome) and binds the upstream regions of ∼130 genes. Seven Gat201-bound genes encode for putative and known transcription factors--including two previously implicated in virulence--suggesting an extensive regulatory network. Systematic analysis pinpointed two critical Gat201-bound genes, GAT204 (a transcription factor) and BLP1, which account for much of the capsule-independent antiphagocytic function of Gat201. A strong correlation was observed between the quantitative effects of single and double mutants on phagocytosis in vitro and on host colonization in vivo. This genetic dissection provides evidence that capsule-independent antiphagocytic mechanisms are pivotal for successful mammalian infection by C. neoformans.

Ctr2 links copper homeostasis to polysaccharide capsule formation and phagocytosis inhibition in the human fungal pathogen Cryptococcus neoformans.

Cryptococcus neoformans is a human opportunistic fungal pathogen responsible for approximately 1/3 of HIV/AIDS deaths worldwide. This budding yeast expresses a polysaccharide capsule necessary for virulence. Capsule production inhibits phagocytosis by macrophages. Here we describe results that link copper homeostasis to capsule production and the inhibition of phagocytosis. Specifically, using Agrobacterium-mediated insertional mutagenesis, we identified an insertion in the promoter region of the putative copper transporter-encoding gene CTR2 that results in reduced expression of CTR2 and increased phagocytosis by murine RAW264.7 macrophages. The mutant also displayed sensitivity to copper starvation and defects in polysaccharide capsule production and melanization. These defects were all reversed by genetic correction of the promoter insertion by homologous targeting. Several melanization-defective mutants identified previously, those in the RIM20, RIM101, and VPS25 genes, also display sensitivity to copper starvation, reduced capsule production and increased phagocytosis. Together these results indicate a previously undescribed link between copper homeostasis to polysaccharide capsule production and phagocytosis inhibition in Cryptococcus neoformans.

Applying genetics and molecular biology to the study of the human pathogen Cryptococcus neoformans.

The basidiomycete yeast Crytococcus neoformans is a prominent human pathogen. It primarily infects immunocompromised individuals producing a meningoencephalitis that is lethal if untreated. Recent advances in its genetics and molecular biology have made it a model system for understanding both the Basidiomycota phylum and mechanisms of fungal pathogenesis. The relative ease of experimental manipulation coupled with the development of murine models for human disease allow for powerful studies in the mechanisms of virulence and host responses. This chapter introduces the organism and its life cycle and then provides detailed step-by-step protocols for culture, manipulation of the genome, analysis of nucleic acids and proteins, and assessment of virulence and expression of virulence factors.