Characterisation of the cell wall protein Pga29p in the human pathogenic fungus <i>Candida albicans</i>

Abstract

Candida albicans is an opportunistic fungal pathogen that is responsible for most of the Candida infections in humans. It can cause superficial infections on the mucosa, nails or skin and in severely immunocompromised individuals it is able to disseminate via the bloodstream and infect deep tissues, which can lead to life-threatening situations. In order to improve the treatment against C. albicans infections, a thorough understanding of the pathogenesis on a molecular level is needed. The last two decades, the cell wall of C. albicans has been an interesting object of research for investigators around the world, mainly because this structure is not present in mammalian cells and therefore it is a potential drug target. Moreover, the upper layer of the cell wall consists of mannoproteins that play an important role in the infection process of C. albicans. In this study we characterised Pga29p (Predicted GPI-anchored protein 29), a small yeast-specific protein, that is covalently linked to the beta-1,3-glucan framework of the cell wall via beta-1,6-glucan. The protein is abundantly present in the wall of yeast cells, but its covalent incorporation drastically decreases upon the yeast-to-hyphae transition. Deletion of PGA29 did not result in altered chitin or protein levels in the cell wall. However, the mannose/glucose ratio was significantly decreased, suggesting a reduced mannan level in in pga29/pga29 cell walls. The pga29/pga29 strain did not show differences in growth characteristics or sensitivity to cell wall stresses and antimycotics compared with the control strains. Subsequently the full cell wall proteome of the pga29/pga29 deletion mutant was analysed using liquid chromatography-tandem mass spectrometry (LC/MS/MS). The resulting data indicated that PGA29 deletion is not compensated by cell wall incorporation of the family members Pga30p and Pga31p or other CWPs, suggesting that Pga29p does not play a major role in maintaining the cell wall integrity of C. albicans. In two independent virulence models, a mouse model of systemic infection and the reconstituted human epithelium (RHE) oral model, the pga29/pga29 strain showed a reduction in virulence. As PGA29 is downregulated in the hyphal form, the protein seems to be involved in the very initial, yeast-morphology based stages of infection. However, the diminished virulence of the pga29/pga29 strain seemed not to be based on an altered adhesion capacity or a reduced ability to form hyphae. As cell wall mannans are known as important pathogen associated molecular patterns (PAMPs), further experiments were conducted to investigate whether the reduced mannan in the pga29/pga29 strain could be responsible for altered host cell interactions which may lead to the observed reduced virulence. O-mannosylation of Pga29p by protein-O-mannosyltransferase 1 (Pmt1p) was proven by analysis of CWP extracts from C. albicans mutants that are deficient in O-glycosylation. RHE infected with the pga29/pga29 strain showed a reduced expression of proinflammatory cytokines. This diminished immune response could be related with the decrease of mannan in the cell wall or the reduced tissue damage that was caused by pga29/pga29 strain. However, upon stimulation with pga29/pga29 mutants, murine primary dendritic cells (mDCs) and human polymorphonuclear leukocytes (PMNs) did not show altered cytokine or oxidative burst levels, respectively. These results indicate that the reduced virulence may not be related to an altered host-pathogen cross-talk via the mannans. We propose that Pga29p is involved in the initial stage of infection, prior to hyphae development, which designates this protein as the first yeast-specific CWP of C. albicans that directly plays a role in virulence.