The fungal pathogen Candida albicans has a multilayered cell wall composed of an outer layer of proteins glycosylated with N-or O-linked mannosyl residues and an inner skeletal layer of β-glucans and chitin. We demonstrate that cytokine production by human mononuclear cells or murine macrophages was markedly reduced when stimulated by C. albicans mutants defective in mannosylation. Recognition of mannosyl residues was mediated by mannose receptor binding to N-linked mannosyl residues and by TLR4 binding to O-linked mannosyl residues. Residual cytokine production was mediated by recognition of β-glucan by the dectin-1/ TLR2 receptor complex. C. albicans mutants with a cell wall defective in mannosyl residues were less virulent in experimental disseminated candidiasis and elicited reduced cytokine production in vivo. We concluded that recognition of C. albicans by monocytes/macrophages is mediated by 3 recognition systems of differing importance, each of which senses specific layers of the C. albicans cell wall.

The cell surface of Candida albicans is the immediate point of contact with the host. The outer layer of the cell wall is enriched in highly glycosylated mannoproteins that are implicated in many aspects of the host-fungus interaction. Glycosylation of cell wall proteins is initiated in the endoplasmic reticulum and then elaborated in the Golgi as the protein passes through the secretory pathway. Golgi-bound mannosyltransferases require Mn 2؉ as an essential cofactor. In Saccharomyces cerevisiae, the P-type ATPase Pmr1p transports Ca 2؉ and Mn 2؉ ions into the Golgi. To determine the effect of a gross defect in glycosylation on host-fungus interactions of C. albicans, we disrupted the PMR1 homolog, CaPMR1. This mutation would simultaneously inhibit many Golgi-located, Mn 2؉ -dependent mannosyltransferases. The Capmr1⌬ null mutant was viable in vitro and had no growth defect even on media containing low Ca 2؉ /Mn 2؉ ion concentrations. However, cells grown in these media progressively lost viability upon entering stationary phase. Phosphomannan was almost completely absent, and O-mannan was severely truncated in the null mutant. A defect in N-linked outer chain glycosylation was also apparent, demonstrated by the underglycosylation of surface acid phosphatase. Consistent with the glycosylation defect, the null mutant had a weakened cell wall, exemplified by hypersensitivity to Calcofluor white, Congo red, and hygromycin B and constitutive activation of the cell integrity pathway. In a murine model of systemic infection, the null mutant was severely attenuated in virulence. These results demonstrate the importance of glycosylation for cell wall structure and virulence of C. albicans.

Many drug candidates fail in clinical trials due to a lack of efficacy from limited target engagement or an insufficient therapeutic index. Minimizing off-target effects while retaining the desired pharmacodynamic (PD) response can be achieved by reduced exposure for drugs that display kinetic selectivity in which the drug:target complex has a longer half-life than off-target:drug complexes. However, while slow-binding inhibition kinetics are a key feature of many marketed drugs1,2, prospective tools that integrate drug-target residence time into predictions of drug efficacy are lacking, hindering the integration of drug-target kinetics into the drug discovery cascade. Here we describe a mechanistic PD model that includes drug-target kinetic parameters including the on- and off-rates for the formation and breakdown of the drug-target complex. We demonstrate the utility of this model by using it to predict dose response curves for inhibitors of the LpxC enzyme from Pseudomonas aeruginosa in an animal model of infection.

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 ...

ThecellwallproteinsoffungiaremodifiedbyN-andO-linkedmannosylation and phosphomannosylation, resulting in changes to the physicalandimmunologicalpropertiesofthecell.Glycosylationofcell wall proteins involves the activities of families of endoplasmic reticulum and Golgi-located glycosyl transferases whose activities are difficult to infer through bioinformatics. The Candida albicans MNT1/ KRE2 mannosyl transferase family is represented by five members. We showed previously that Mnt1 and Mnt2 are involved in O-linked mannosylation and are required for virulence. Here, the role of C. albicans MNT3, MNT4, and MNT5 was determined by generating single and multiple MnT⌬null mutants and by functional complementation experiments in Saccharomyces cerevisiae. CaMnt3, CaMnt4, and CaMnt5 did not participate in O-linked mannosylation, but CaMnt3 and CaMnt5 had redundant activities in phosphomannosylation and were responsible for attachment of approximately half of the phosphomannan attached to N-linked mannans. CaMnt4 and CaMnt5 participated in N-mannan branching. Deletion of CaMNT3, CaMNT4, and CaMNT5 affected the growth rate and virulence of C. albicans, affected the recognition of the yeast by human monocytes and cytokine stimulation, and led to increased cell wall chitin content and exposure of ␤-glucan at the cell wall surface. Therefore, the MNT1/KRE2 gene family participates in three types of protein mannosylation in C. albicans, and these modifications play vital roles in fungal cell wall structure and cell surface recognition by the innate immune system.The human pathogen Candida albicans is the most frequent cause of systemic candidosis, which is a common, life-threatening infection in immunocompromised patients (1). The C. albicans cell wall is a robust yet dynamic structure that protects the cell from changes in the extracellular environment. It is the immediate contact point with host cells and contains antigenic determinants, glycoproteins involved in the adhesion to host tissues, and most of the pathogen-associated molecular patterns that are recognized by host immune system (2). The wall is organized in an inner skeletal layer comprising chitin, ␤1,3-and ␤1,6-glucans, and an outer layer that is dominated by highly glycosylated proteins (3). These proteins are post-translationally modified with N-and/or O-linked mannans, both of which can be further elaborated with oligomannosides that are attached via phosphodiester linkages (phosphomannans). Mannans have important roles in cell wall integrity, adhesion to host cells and tissues, virulence, and the establishment of a response by immune cells (2, 4 -10). The O-and N-linked mannans, along with ␤-glucans, represent the main C. albicans pathogen-associated molecular patterns recognized by the innate immune system (2, 11-13).Mannan biosynthesis has been carefully characterized in Saccharomyces cerevisiae, and the main features of the pathways involved in the construction of these oligosaccharides are conserved in C. albicans. However, N-and O-linked mannans of C. al...