We analyzed data on multilocus sequence typing (MLST), ABC typing, mating type-like locus (MAT) status, and antifungal susceptibility for a panel of 1,391 Candida albicans isolates. Almost all (96.7%) of the isolates could be assigned by MLST to one of 17 clades. eBURST analysis revealed 53 clonal clusters. Diploid sequence type 69 was the most common MLST strain type and the founder of the largest clonal cluster, and examples were found among isolates from all parts of the world. ABC types and geographical origins showed statistically significant variations among clades by univariate analysis of variance, but anatomical source and antifungal susceptibility data were not significantly associated. A separate analysis limited to European isolates, thereby minimizing geographical effects, showed significant differences in the proportions of isolates from blood, commensal carriage, and superficial infections among the five most populous clades. The proportion of isolates with low antifungal susceptibility was highest for MAT homozygous a/a types and then ␣/␣ types and was lowest for heterozygous a/␣ types. The tree of clades defined by MLST was not congruent with trees generated from the individual gene fragments sequenced, implying a separate evolutionary history for each fragment. Analysis of nucleic acid variation among loci and within loci supported recombination. Computational haplotype analysis showed a high frequency of recombination events, suggesting that isolates had mixed evolutionary histories resembling those of a sexually reproducing species.

Summary ParagraphAs they proliferate, fungi expose antigens at their cell surface that are potent stimulators of the innate immune response, and yet the commensal fungus Candida albicans is able to colonize immuno-competent individuals. We show that C. albicans may evade immune detection by presenting a moving immunological target. We report that the exposure of β-glucan, a key Pathogen Associated Molecular Pattern (PAMP) located at the cell surface of C. albicans and other pathogenic Candida species, is modulated in response to changes in carbon source. Exposure to lactate induces β-glucan masking in C. albicans via a signaling pathway that has recruited an evolutionarily conserved receptor (Gpr1) and transcriptional factor (Crz1) from other wellcharacterized pathways. In response to lactate, these regulators control the expression of cell wall related genes that contribute to β-glucan masking. This represents the first description of active PAMP masking by a Candida species, a process that reduces the visibility of the fungus to the immune system.

The pathogenicity of the opportunistic human fungal pathogen Candida albicans depends on its ability to escape destruction by the host immune system. Using mutant strains that are defective in cell surface glycosylation, cell wall protein synthesis, and yeast-hypha morphogenesis, we have investigated three important aspects of C. albicans innate immune interactions: phagocytosis by primary macrophages and macrophage cell lines, hyphal formation within macrophage phagosomes, and the ability to escape from and kill macrophages. We show that cell wall glycosylation is critically important for the recognition and ingestion of C. albicans by macrophages. Phagocytosis was significantly reduced for mutants deficient in phosphomannan biosynthesis (mmn4⌬, pmr1⌬, and mnt3 mnt5⌬), whereas O-and N-linked mannan defects (mnt1⌬ mnt2⌬ and mns1⌬) were associated with increased ingestion, compared to the parent wild-type strains and genetically complemented controls. In contrast, macrophage uptake of mutants deficient in cell wall proteins such as adhesins (ece1⌬, hwp1⌬, and als3⌬) and yeast-locked mutants (clb2⌬, hgc1⌬, cph1⌬, efg1⌬, and efg1⌬ cph1⌬), was similar to that observed for wild-type C. albicans. Killing of macrophages was abrogated in hypha-deficient strains, significantly reduced in all glycosylation mutants, and comparable to wild type in cell wall protein mutants. The diminished ability of glycosylation mutants to kill macrophages was not a consequence of impaired hyphal formation within macrophage phagosomes. Therefore, cell wall composition and the ability to undergo yeast-hypha morphogenesis are critical determinants of the macrophage's ability to ingest and process C. albicans.

The MNT1 gene of the human fungal pathogen Candida albicans is involved in O-glycosylation of cell wall and secreted proteins and is important for adherence of C. albicans to host surfaces and for virulence. Here we describe the molecular analysis of CaMNT2, a second member of the MNT1-like gene family in C. albicans. Candida albicans is the major fungal pathogen of humans. This opportunistic pathogen can cause irritating superficial infections of the mucosa and serious life threatening systemic infections in the immunocompromised patient (1, 2). Invasive candidosis in hospitals now represents the third or fourth most common form of septicaemia (3, 4). The cell surface of C. albicans is the immediate point of contact between the fungus and host and plays vital roles in adhesion and immunomodulation of host responses, and it is a source of antigens (5-8). The outer cell wall layer is enriched in mannoproteins, which are embedded in a matrix of structural polysaccharides consisting of ␤-1,3-and ␤-1,6-linked glucan and chitin (9). This layer is important in adhesion to host surfaces and their subsequent colonization (10 -12). Both the protein and carbohydrate components of mannoproteins have been implicated in adhesion to the host (10, 13-15), although details of the nature of the ligands and receptors are still lacking. Hence, glycosylation of cell wall proteins is critical for host-fungal interactions and pathogenicity. Mnt2p also functions inKnowledge of glycosylation in Saccharomyces cerevisiae (16 -28) and information from the C. albicans genome data base has provided significant resources for the identification and analysis of glycosylation genes in C. albicans. Mannoproteins of S. cerevisiae and C. albicans contain both N-and O-linked oligosaccharides. The N-linked glycans, attached to asparagine residues of proteins, contain a conserved core structure and an elaborate, highly branched outer mannose chain that is specific to fungi and contains both acid-stable and acid-labile components (17,29,30). Glycosylation in C. albicans has its own relevance in investigations of the role of specific oligosaccharide moieties in host-fungal interactions. The acid-labile mannosylphosphate component, containing ␤-1,2-linked mannose, has been implicated in adhesion and recognition of phagocytic leukocytes, although mutants lacking this component have been shown to have normal interactions with macrophages (31). Both ␤-1,2-and ␣-1,2-linked mannan oligosaccharides have been implicated directly in adhesion functions (12,32).In C. albicans, O-glycans are linear oligosaccharides of one to five ␣-1,2-linked mannose residues (32-34). In S. cerevisiae an ␣-1,2-linked O-linked glycan is capped with one or two ␣-1,3-linked mannose residues (27). O-Glycosylation in S. cerevisiae is initiated in the endoplasmic reticulum where at least four of the seven-membered PMT gene family act to transfer mannose from dolichyl phosphate-activated mannose to serine or threonine (18,35,36). Evidently this step is essential, as certain combinations ...

Candida albicans is a major life-threatening human fungal pathogen. Host defence against systemic Candida infection relies mainly on phagocytosis of fungal cells by cells of the innate immune system. In this study, we have employed video microscopy, coupled with sophisticated image analysis tools, to assess the contribution of distinct C. albicans cell wall components and yeast-hypha morphogenesis to specific stages of phagocytosis by macrophages. We show that macrophage migration towards C. albicans was dependent on the glycosylation status of the fungal cell wall, but not cell viability or morphogenic switching from yeast to hyphal forms. This was not a consequence of differences in maximal macrophage track velocity, but stems from a greater percentage of macrophages pursuing glycosylation deficient C. albicans during the first hour of the phagocytosis assay. The rate of engulfment of C. albicans attached to the macrophage surface was significantly delayed for glycosylation and yeast-locked morphogenetic mutant strains, but enhanced for non-viable cells. Hyphal cells were engulfed at a slower rate than yeast cells, especially those with hyphae in excess of 20 µm, but there was no correlation between hyphal length and the rate of engulfment below this threshold. We show that spatial orientation of the hypha and whether hyphal C. albicans attached to the macrophage via the yeast or hyphal end were also important determinants of the rate of engulfment. Breaking down the overall phagocytic process into its individual components revealed novel insights into what determines the speed and effectiveness of C. albicans phagocytosis by macrophages.