Parallel β-Helix Proteins Required for Accurate Capsule Polysaccharide Synthesis and Virulence in the Yeast Cryptococcus neoformans

Abstract

The principal capsular polysaccharide of the opportunistic fungal pathogen Cryptococcus neoformans consists of an ␣-1,3-linked mannose backbone decorated with a repeating pattern of glucuronyl and xylosyl side groups. This structure is critical for virulence, yet little is known about how the polymer, called glucuronoxylomannan (GXM), is faithfully synthesized and assembled. We have generated deletions in two genes encoding predicted parallel ␤-helix repeat proteins, which we have designated PBX1 and PBX2. Deletion of either gene results in a dry-colony morphology, clumpy cells, and decreased capsule integrity. Two-dimensional nuclear magnetic resonance spectroscopy of purified GXM from the mutants indicated that both the wild-type GXM structure and novel, aberrant linkages were present. Carbohydrate composition and linkage analysis determined that these aberrant structures are correlated with the incorporation of terminal glucose residues that are not found in wild-type capsule polysaccharide. We conclude that Pbx1 and Pbx2 are required for the fidelity of GXM synthesis and may be involved in editing incorrectly added glucose residues. PBX1 and PBX2 knockout mutants showed severely attenuated virulence in a murine inhalation model of cryptococcosis. Unlike acapsular strains, these mutant strains induced delayed symptoms of cryptococcosis, though the infected animals eventually contained the infection and recovered.

Pbx Proteins in Cryptococcus neoformans Cell Wall Remodeling and Capsule Assembly

“…On the basis of compositional analysis of GXM, it was previously proposed that Pbx proteins act to remove glucose residues that are aberrantly incorporated into capsule polysaccharides (32). Our analysis of the composition of GXM and GXMGal from wild-type and mutant cells and detailed determination of their structures by NMR do not indicate the presence of any glucose and therefore do not support this model.…”

“…In some scenarios, residence within host cells may benefit C. neoformans. However, in the context of mutant cells that have deformed cell walls and increased sensitivity to oxidative stress, this engulfment is likely a dead end, and the increased phagocytosis may contribute to the observed virulence defect of cells lacking Pbx proteins (32).…”

“…does not), but the chemical structure of the mutant polysaccharide was not determined (32). To pursue this observation, we purified GXM from pbx1⌬ mutant, pbx2⌬ mutant, and wild-type cells for NMR analysis (see Tables S2 and S3 in the supplemental material).…”

“…Strains lacking the genes PBX1 and PBX2 showed defects in both surface capsule and capsule transfer in our screen. It has previously been reported that these mutants have reduced virulence in mice (32), highlighting the importance of investigating the function of the encoded proteins. We found that these mutants additionally exhibit morphological defects, reduced capsule synthesis and association, and increased uptake by mammalian phagocytes.…”

The Cryptococcus neoformans Capsule: a Sword and a Shield

“…Pbx1 and Pbx2 are parallel ␤-helix proteins that potentially act as a complex to regulate the incorporation of glucose residues into the backbone (133). Mutations in these proteins do not prevent encapsulation, but the mutant capsule is easily detached from the cell by sonication.…”