Pbx Proteins in Cryptococcus neoformans Cell Wall Remodeling and Capsule Assembly

Abstract

The cryptococcal capsule is a critical virulence factor of an important pathogen, but little is known about how it is associated with the cell or released into the environment. Two mutants lacking PBX1 and PBX2 were found to shed reduced amounts of the capsule polysaccharide glucuronoxylomannan (GXM). Nuclear magnetic resonance, composition, and physical analyses showed that the shed material was of normal mass but was slightly enriched in xylose. In contrast to previous reports, this material contained no glucose. Notably, the capsule fibers of pbx⌬ mutant cells grown under capsule-inducing conditions were present at a lower than usual density and were loosely attached to the cell wall. Mutant cell walls were also defective, as indicated by phenotypes including abnormal cell morphology, reduced mating filamentation, and altered cell integrity. All observed phenotypes were shared between the two mutants and exacerbated in a double mutant. Consistent with a role in surface glycan synthesis, the Pbx proteins localized to detergent-resistant membrane domains. These results, together with the sequence motifs in the Pbx proteins, suggest that Pbx1 and Pbx2 are redundant proteins that act in remodeling the cell wall to maintain normal cell morphology and precursor availability for other glycan synthetic processes. Their absence results in aberrant cell wall growth and metabolic imbalance, which together impact cell wall and capsule synthesis, cell morphology, and capsule association. The surface changes also lead to increased engulfment by host phagocytes, consistent with the lack of virulence of pbx mutants in animal models.

Unraveling the Biology of a Fungal Meningitis Pathogen Using Chemical Genetics

“…S2D). These data are consistent with a recently published study on the role of Pbx1 in capsule attachment and assembly (Kumar et al , 2014). …”

“…4, S4). A recent study on Pbx1 and its ortholog, Pbx2, proposes that the two proteins act redundantly in capsule assembly (Kumar et al , 2014). pbx1 Δ and pbx2 Δ cells shed lower amounts of GXM into the culture medium but that the GXM functions in a capsule transfer assay.…”

Intracellular Action of a Secreted Peptide Required for Fungal Virulence

“…Of these, we identified six additional mutants with the dry colony phenotype (Table S3). These include three known acapsular mutants ( cap10Δ , cap60Δ , and cap64Δ ; Chang and Kwon-Chung, 1998, 1999; Chang et al, 1996) and one mutant defective in capsule attachment ( pbx1Δ ; Kumar et al, 2014; Liu et al, 2007). …”

A Single Protein S-acyl Transferase Acts through Diverse Substrates to Determine Cryptococcal Morphology, Stress Tolerance, and Pathogenic Outcome

“…An example data set from one plate of the mutant collection ( Fig 1B ) shows strikingly increased phagocytosis of three mutants, two of which, pka1 and rim101 , are known to have altered cell surface structures that would explain this phenotype [ 24 , 25 ]. Additionally, pbx1 , the top hit of the high uptake mutants ( Table 1 ), has defects in cell wall structure and capsule assembly that cause increased engulfment by macrophages [ 26 ]. These observations validated our strategy for probing the interactions between C .…”