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Unlabelled: Cryptococcus neoformans is a ubiquitous, opportunistic fungal pathogen that kills over 600,000 people annually. Here, we report integrated computational and experimental investigations of the role and mechanisms of transcriptional regulation in cryptococcal infection. Major cryptococcal virulence traits include melanin production and the development of a large polysaccharide capsule upon host entry; shed capsule polysaccharides also impair host defenses. We found that both transcription and translation are required for capsule growth and that Usv101 is a master regulator of pathogenesis, regulating melanin production, capsule growth, and capsule shedding. It does this by directly regulating genes encoding glycoactive enzymes and genes encoding three other transcription factors that are essential for capsule growth: GAT201, RIM101, and SP1. Murine infection with cryptococci lacking Usv101 significantly alters the kinetics and pathogenesis of disease, with extended survival and, unexpectedly, death by pneumonia rather than meningitis. Our approaches and findings will inform studies of other pathogenic microbes., Importance: Cryptococcus neoformans causes fatal meningitis in immunocompromised individuals, mainly HIV positive, killing over 600,000 each year. A unique feature of this yeast, which makes it particularly virulent, is its polysaccharide capsule; this structure impedes host efforts to combat infection. Capsule size and structure respond to environmental conditions, such as those encountered in an infected host. We have combined computational and experimental tools to elucidate capsule regulation, which we show primarily occurs at the transcriptional level. We also demonstrate that loss of a novel transcription factor alters virulence factor expression and host cell interactions, changing the lethal condition from meningitis to pneumonia with an exacerbated host response. We further demonstrate the relevant targets of regulation and kinetically map key regulatory and host interactions. Our work elucidates mechanisms of capsule regulation, provides methods and resources to the research community, and demonstrates an altered pathogenic outcome that resembles some human conditions., (Copyright © 2016 Gish et al.)

Cryptococcus neoformans is an opportunistic yeast responsible for lethal meningoencephalitis in humans. This pathogen elaborates a polysaccharide capsule, which is its major virulence factor. Mannose constitutes over one-half of the capsule mass and is also extensively utilized in cell wall synthesis and in glycosylation of proteins and lipids. The activated mannose donor for most biosynthetic reactions, GDP-mannose, is made in the cytosol, although it is primarily consumed in secretory organelles. This compartmentalization necessitates specific transmembrane transporters to make the donor available for glycan synthesis. We previously identified two cryptococcal GDP-mannose transporters, Gmt1 and Gmt2. Biochemical studies of each protein expressed in Saccharomyces cerevisiae showed that both are functional, with similar kinetics and substrate specificities in vitro. We have now examined these proteins in vivo and demonstrate that cells lacking Gmt1 show significant phenotypic differences from those lacking Gmt2 in terms of growth, colony morphology, protein glycosylation, and capsule phenotypes. Some of these observations may be explained by differential expression of the two genes, but others suggest that the two proteins play overlapping but nonidentical roles in cryptococcal biology. Furthermore, gmt1 gmt2 double mutant cells, which are unexpectedly viable, exhibit severe defects in capsule synthesis and protein glycosylation and are avirulent in mouse models of cryptococcosis., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)