Members 5 and 6 of the Candida albicans BMT family encode enzymes acting specifically on ß-mannosylation of the phospholipomannan cell-wall glycosphingolipid.

A family of nine genes encoding proteins involved in the synthesis of ß-1,2 mannose adhesins of Candida albicans has been identified. Four of these genes, BMT1-4, encode enzymes acting stepwise to add ß-mannoses on to cell-wall phosphopeptidomannan (PPM). None of these acts on phospholipomannan (PLM), a glycosphingolipid member of the mannose-inositol-phosphoceramide family, which contributes with PPM to ß-mannose surface expression. We show that deletion of BMT5 and BMT6 led to a dramatic reduction of PLM glycosylation and accumulation of PLM with a truncated ß-oligomannoside chain, respectively. Disruptions had no effect on sphingolipid biosynthesis and on PPM ß-mannosylation. ß-Mannose surface expression was not affected, confirming that ß-mannosylation is a process based on specificity of acceptor molecules, but liable to global regulation.

Beta-1,2 oligomannose adhesin epitopes are widely distributed over the different families of Candida albicans cell wall mannoproteins and are associated through both N- and O-glycosylation processes.

Beta-1,2-linked mannosides (beta-Mans) are believed to contribute to Candida albicans virulence. The presence of beta-Mans has been chemically established for two molecules (phosphopeptidomannan [PPM] and phospholipomannan) that are noncovalently linked to the cell wall, where they correspond to specific epitopes. However, a large number of cell wall mannoproteins (CWMPs) also express beta-Man epitopes, although their nature and mode of beta-mannosylation are unknown. We therefore used Western blotting to map beta-Man epitopes for the different families of mannoproteins gradually released from the cell wall according to their mode of anchorage (soluble, released by dithiothreitol, beta-1,3 glucan linked, and beta-1,6 glucan linked). Reduction of beta-Man epitope expression occurred after chemical and enzymatic deglycosylation of the different cell wall fractions, as well as in a secreted form of Hwp1, a representative of the CWMPs linked by glycosylphosphatidylinositol remnants. Enzyme-linked immunosorbent assay inhibition tests were performed to assess the presence of beta-Man epitopes in released oligomannosides. A comparison of the results obtained with CWMPs to the results obtained with PPM and the use of mutants with mutations affecting O and N glycosylation demonstrated that both O glycosylation and N glycosylation participate in the association of beta-Mans with the protein moieties of CWMPs. This process, which can alter the function of cell wall molecules and their recognition by the host, is therefore more important and more complex than originally thought, since it differs from the model established previously with PPM.

Biochemical and electron paramagnetic resonance study of the iron superoxide dismutase from Plasmodium falciparum.

Recombinant iron-containing superoxide dismutase (Fe-SOD) from Plasmodium falciparum was produced in a SOD-deficient strain of Escherichia coli, purified and characterised. The enzyme is a dimer, which contains 1.7 Fe equivalents and is sensitive to hydrogen peroxide (H(2)O(2)). Electron paramagnetic resonance (EPR) analysis showed two different signals, reflecting the presence of two different types of high-spin Fe sites with different symmetries. The role of the W71 residue during inactivation by H(2)O(2) of the P. falciparum Fe-SOD was studied by site-directed mutagenesis. First, the W71V mutation led to a change in the relative proportion of the two Fe-based EPR signals. Second, the mutant protein was almost as active as the wild-type (WT) protein but more sensitive to heat inactivation. Third, resistance to H(2)O(2) was only slightly increased indicating that W71 was marginally responsible for the sensitivity of Fe-SOD to H(2)O(2). A molecular model of the subunit was designed to assist in interpretation of the results. The fact that the parasite SOD does not belong to classes of SOD present in humans may provide a novel approach for the design of antimalarial drugs.