[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Galactofuranose (Galf) is the five-membered ring form of galactose. Although it is absent from mammalian glycans, it occurs as a structural and
antigenic component of important cell surface molecules in a variety of microbes, ranging from bacteria to parasites and fungi.
One such organism is Cryptococcus neoformans, a pathogenic yeast that causes lethal meningoencephalitis in immunocompromised individuals, particularly AIDS patients.
C. neoformans is unique among fungal pathogens in bearing a complex polysaccharide capsule, a critical virulence factor reported to include
Galf. Notably, how Galf modification contributes to the structure and function of the cryptococcal capsule is not known. We have determined that
Galf is β1,2-linked to an unusual tetrasubstituted galactopyranose of the glucuronoxylomannogalactan (GXMGal) capsule polysaccharide.
This discovery fills a longstanding gap in our understanding of a major polymer of the cryptococcal capsule. We also engineered
a C. neoformans strain that lacks UDP-galactopyranose mutase; this enzyme forms UDP-Galf, the nucleotide sugar donor required for Galf addition. Mutase activity was required for the incorporation of Galf into glucuronoxylomannogalactan but was dispensable for vegetative growth, cell integrity, and virulence in a mouse model.
Preview · Article · Feb 2013 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Cryptococcus neoformans is an opportunistic fungal pathogen that causes serious human disease in immunocompromised populations. Its polysaccharide capsule is a key virulence factor which is regulated in response to growth conditions, becoming enlarged in the context of infection. We used microarray analysis of cells stimulated to form capsule over a range of growth conditions to identify a transcriptional signature associated with capsule enlargement. The signature contains 880 genes, is enriched for genes encoding known capsule regulators, and includes many uncharacterized sequences. One uncharacterized sequence encodes a novel regulator of capsule and of fungal virulence. This factor is a homolog of the yeast protein Ada2, a member of the Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex that regulates transcription of stress response genes via histone acetylation. Consistent with this homology, the C. neoformans null mutant exhibits reduced histone H3 lysine 9 acetylation. It is also defective in response to a variety of stress conditions, demonstrating phenotypes that overlap with, but are not identical to, those of other fungi with altered SAGA complexes. The mutant also exhibits significant defects in sexual development and virulence. To establish the role of Ada2 in the broader network of capsule regulation we performed RNA-Seq on strains lacking either Ada2 or one of two other capsule regulators: Cir1 and Nrg1. Analysis of the results suggested that Ada2 functions downstream of both Cir1 and Nrg1 via components of the high osmolarity glycerol (HOG) pathway. To identify direct targets of Ada2, we performed ChIP-Seq analysis of histone acetylation in the Ada2 null mutant. These studies supported the role of Ada2 in the direct regulation of capsule and mating responses and suggested that it may also play a direct role in regulating capsule-independent antiphagocytic virulence factors. These results validate our experimental approach to dissecting capsule regulation and provide multiple targets for future investigation.
[Show abstract][Hide abstract] ABSTRACT: Cryptococcus neoformans causes serious disease in immunocompromised individuals, leading to over 600,000 deaths per year worldwide. Part of this impact is due to the organism's ability to thwart what should be the mammalian hosts' first line of defense against cryptococcal infection: internalization by macrophages. Even when C. neoformans is engulfed by host phagocytes, it can survive and replicate within them rather than being destroyed; this ability is central in cryptococcal virulence. It is therefore critical to elucidate the interactions of this facultative intracellular pathogen with phagocytic cells of its mammalian host.
To accurately assess initial interactions between human phagocytic cells and fungi, we have developed a method using high-throughput microscopy to efficiently distinguish adherent and engulfed cryptococci and quantitate each population. This method offers significant advantages over currently available means of assaying host-fungal cell interactions, and remains statistically robust when implemented in an automated fashion appropriate for screening. It was used to demonstrate the sensitivity of human phagocytes to subtle changes in the cryptococcal capsule, a major virulence factor of this pathogen.
Our high-throughput method for characterizing interactions between C. neoformans and mammalian phagocytic cells offers a powerful tool for elucidating the relationship between these cell types during pathogenesis. This approach will be useful for screens of this organism and has potentially broad applications for investigating host-pathogen interactions.
[Show abstract][Hide abstract] ABSTRACT: Cryptococcal meningoencephalitis is an AIDS-defining illness caused by the opportunistic pathogen Cryptococcus neoformans. This organism possesses an elaborate polysaccharide capsule that is unique among pathogenic fungi, and the glycobiology
of C. neoformans has been a focus of research in the field. The capsule and other cellular glycans and glycoconjugates have been described,
but the machinery responsible for their synthesis remains largely unexplored. We recently discovered Xpt1p, an enzyme with
the unexpected activity of generating a xylose-phosphate-mannose linkage. We now demonstrate that this novel activity is conserved
throughout the C. neoformans species complex, localized to the Golgi apparatus, and functions in the O-glycosylation of proteins. We also present the first survey of O-glycans from C. neoformans.
Full-text · Article · May 2011 · Journal of Biological Chemistry