Inactivation of CaMIT1 Inhibits Candida albicans Phospholipomannan β-Mannosylation, Reduces Virulence, and Alters Cell Wall Protein β-Mannosylation

Abstract

Studies on Candida albicans phospholipomannan have suggested a novel biosynthetic pathway for yeast glycosphingolipids. This pathway is thought to diverge from the usual pathway at the mannose-inositol-phospho-ceramide (MIPC) step. To confirm this hypothesis, a C. albicans gene homologue for the Saccharomyces cerevisiae SUR1 gene was identified and named MIT1 as it coded for GDP-mannose:inositol-phospho-ceramide mannose transferase. Two copies of this gene were disrupted. Western blots of cell extracts revealed that strain mit1⌬ contained no PLM. Thin layer chromatography and mass spectrometry confirmed that mit1⌬ did not synthesize MIPC, demonstrating a role of MIT1 in the mannosylation of C. albicans IPCs. As MIT1 disruption prevented downstream ␤-1,2 mannosylation, mit1⌬ represents a new C. albicans mutant affected in the expression of these specific virulence attributes, which act as adhesins/immunomodulators. mit1⌬ was less virulent during both the acute and chronic phases of systemic infection in mice (75 and 50% reduction in mortality, respectively). In vitro, mit1⌬ was not able to escape macrophage lysis through down-regulation of the ERK1/2 phosphorylation pathway previously shown to be triggered by PLM. Phenotypic analysis also revealed pleiotropic effects of MIT1 disruption. The most striking observation was a reduced ␤-mannosylation of phosphopeptidomannan. Increased ␤-mannosylation of mannoproteins was observed under growth conditions that prevented the association of ␤-oligomannosides with phosphopeptidomannan, but not with PLM. This suggests that C. albicans has strong regulatory mechanisms associating ␤-oligomannoses with different cell wall carrier molecules. These mechanisms and the impact of the different presentations of ␤-oligomannoses on the host response need to be defined.

Specific Recognition of Candida albicans by Macrophages Requires Galectin-3 to Discriminate Saccharomyces cerevisiae and Needs Association with TLR2 for Signaling

“…This hypothesis, which agrees with results obtained in other systems (14), may explain for one part the observations obtained by Hobson et al (51) who showed that disruption of the gene required for mannosyl phosphate transfer and, hence, the attachment of ␤-1,2 mannose oligosaccharides to mannan, did not alter the interaction between C. albicans and macrophages. An important drawback from the conclusions raised in this study is that, beside the fraction attached to mannan by mannosyl phosphate, a large amount of ␤-1,2 mannose oligosaccharides remained present at the cell wall surface, either attached to the mannan acidstable part, the PLM, as well as to a large number of mannoproteins (52).…”

Conformational Analysis of .BETA.-1,2-Linked Mannobiose to Mannoheptaose, Specific Antigen of Pathogenic Yeast Candida albicans

“…13) Deletion of a mannosyltransferase gene, CaMIT1, which is responsible for the synthesis of the oligomannosyl moieties of the phospholipomannan, reduce the virulence of the C. albicans cells. 14) These findings suggest that the b-1,2-linked oligomannosyl moiety has a specific structure compared to the common a-1,2-linked oligomannosyl moiety. Therefore, determination of the conformation of these oligosaccharides in an aqueous solution is very important for elucidation of the structure-activity relationship.…”

Activity and Stability of .ALPHA.- and .BETA.-Mannosyltransferases in Candida albicans Cells Cultured at High Temperature and at Low pH

“…It is reasonable to think that the specific mannan structure of C. albicans cells grown in a candidiasis patient's body acts as an important virulence factor. [1][2][3][4][5][8][9][10]25) Therefore, in the next stage, we need to study the mannan structure and MT activity of C. albicans grown in the body to perform an accurate diagnosis and determine effective therapy for candidiasis. The enzyme solution was incubated at various pH values.…”

“…[8][9][10] With the objective of clarifying the antigenicity of C. albicans cells grown in a patient's body, we first used body temperature (high temperature, 37°C) for Candida cell cultivation. It is known that the candidacidal activity of gamma interferon-activated peritoneal macrophages correlates well with the induction of highly activated phagolysosomes with low pH (Ͻ4.0).…”