ABSTRACT
Cryptococcus neoformans (Cn) is a pathogenic yeast that is the leading cause of fungal meningitis in immunocompromised patients. Various Cn virulence factors, such as the enzyme laccase and its product melanin, phospholipase, and capsular polysaccharide have been identified. During a screen of knockout mutants, the gene resistance to aminocholesterol 1 (RTA1) was identified, the function of which is currently unknown in Cn. Rta1 homologs in S. cerevisiae belong to a lipid-translocating exporter family of fungal proteins with transmembrane regions and confer resistance to the antimicrobial agent 7-aminocholesterol when overexpressed. To determine the role of RTA1 in Cn, the knock-out (rta1Δ) and reconstituted (rta1Δ+RTA1) strains were created and phenotypically tested. RTA1 was involved in resistance to 7-aminocholesterol, and also in exocyst complex component 3 (Sec6)-mediated secretion of urease, laccase, and the major capsule component, glucuronoxylomannan (GXM), which coincided with significantly smaller capsules in the rta1Δ and rta1Δ+RTA1 strains compared to the wild-type H99 strain. Furthermore, RTA1 expression was reduced in a secretory 14 mutant (sec14Δ) and increased in an RNAi Sec6 mutant. Transmission electron microscopy demonstrated vesicle accumulation inside the rta1Δ strain, predominantly near the cell membrane. Given that Rta1 is likely to be a transmembrane protein located at the plasma membrane, these data suggest that Rta1 may be involved in both secretion of various fungal virulence factors and resistance to 7-aminocholesterol in Cn.
ABSTRACT
The pathogenic yeast Cryptococcus neoformans causes cryptococcosis, a life-threatening fungal disease. C. neoformans has multiple virulence mechanisms that are non-host specific, induce damage and interfere with immune clearance. Microarray analysis of C. neoformans strains serially passaged in mice associated a small gene (CNAG_02591) with virulence. This gene, hereafter identified as HVA1 (hypervirulence-associated protein 1), encodes a protein that has homologs of unknown function in plant and animal fungi, consistent with a conserved mechanism. Expression of HVA1 was negatively correlated with virulence and was reduced in vitro and in vivo in both mouse- and Galleria-passaged strains of C. neoformans. Phenotypic analysis in hva1Δ and hva1Δ+HVA1 strains revealed no significant differences in established virulence factors. Mice infected intravenously with the hva1Δ strain had higher fungal burden in the spleen and brain, but lower fungal burden in the lungs, and died faster than mice infected with H99W or the hva1Δ+HVA1 strain. Metabolomics analysis demonstrated a general increase in all amino acids measured in the disrupted strain and a block in the TCA cycle at isocitrate dehydrogenase, possibly due to alterations in the nicotinamide cofactor pool. Macrophage fungal burden experiments recapitulated the mouse hypervirulent phenotype of the hva1Δ strain only in the presence of exogenous NADPH. The crystal structure of the Hva1 protein was solved, and a comparison of structurally similar proteins correlated with the metabolomics data and potential interactions with NADPH. We report a new gene that modulates virulence through a mechanism associated with changes in fungal metabolism.
