The CRH family coding for cell wall glycosylphosphatidylinositol proteins with a predicted transglycosidase domain affects cell wall organization and virulence of Candida albicans

In Candida albicans UTR2 (CSF4), CRH11, and CRH12 are members of a gene family ( the CRH family) that encode glycosylphosphatidylinositol-dependent cell wall proteins with putative transglycosidase activity. Deletion of genes of this family resulted in additive sensitivity to compounds interfering with normal cell wall formation ( Congo red, calcofluor white, SDS, and high Ca2+ concentrations), suggesting that these genes contribute to cell wall organization. A triple mutant lacking UTR2, CRH11, and CRH12 produced a defective cell wall, as inferred from increased sensitivity to cell wall- degrading enzymes, decreased ability of protoplasts to regenerate a new wall, constitutive activation of Mkc1p, the mitogen-activated protein kinase of the cell wall integrity pathway, and an increased chitin content of the cell wall. Importantly, this was accompanied by a decrease in alkali- insoluble 1,3-beta-glucan but not total glucan content, suggesting that formation of the linkage between 1,3-beta- glucan and chitin might be affected. In support of this idea, localization of a Utr2p-GFP fusion protein largely coincided with areas of chitin incorporation in C. albicans. As UTR2 and CRH11 expression is regulated by calcineurin, a serine/ threonine protein phosphatase involved in tolerance to antifungal drugs, cell wall morphogenesis, and virulence, this points to a possible relationship between calcineurin and the CRH family. Deletion of UTR2, CRH11, and CRH12 resulted in only a partial overlap with calcineurin- dependent phenotypes, suggesting that calcineurin has additional targets. Interestingly, cells deleted for UTR2, CRH11, and CRH12 were, like a calcineurin mutant, avirulent in a mouse model of systemic infection but retained the capacity to colonize target organs ( kidneys) as the wild type. In conclusion, this work establishes the role of UTR2, CRH11, and CRH12 in cell wall organization and integrity.