The Mnn2 Mannosyltransferase Family Modulates Mannoprotein Fibril Length, Immune Recognition and Virulence of Candida albicans

Abstract

The fungal cell wall is the first point of interaction between an invading fungal pathogen and the host immune system. The outer layer of the cell wall is comprised of GPI anchored proteins, which are post-translationally modified by both N- and O-linked glycans. These glycans are important pathogen associated molecular patterns (PAMPs) recognised by the innate immune system. Glycan synthesis is mediated by a series of glycosyl transferases, located in the endoplasmic reticulum and Golgi apparatus. Mnn2 is responsible for the addition of the initial α1,2-mannose residue onto the α1,6-mannose backbone, forming the N-mannan outer chain branches. In Candida albicans, the MNN2 gene family is comprised of six members (MNN2, MNN21, MNN22, MNN23, MNN24 and MNN26). Using a series of single, double, triple, quintuple and sextuple mutants, we show, for the first time, that addition of α1,2-mannose is required for stabilisation of the α1,6-mannose backbone and hence regulates mannan fibril length. Sequential deletion of members of the MNN2 gene family resulted in the synthesis of lower molecular weight, less complex and more uniform N-glycans, with the sextuple mutant displaying only un-substituted α1,6-mannose. TEM images confirmed that the sextuple mutant was completely devoid of the outer mannan fibril layer, while deletion of two MNN2 orthologues resulted in short mannan fibrils. These changes in cell wall architecture correlated with decreased proinflammatory cytokine induction from monocytes and a decrease in fungal virulence in two animal models. Therefore, α1,2-mannose of N-mannan is important for both immune recognition and virulence of C. albicans.

Mannan Molecular Substructures Control Nanoscale Glucan Exposure in Candida

“…This suggests that glucan exposure and DC phagocytic uptake are not necessarily correlated with cell wall glucan abundance and led to the hypothesis that the molecular structure of mannan may be the major determinant of glucan exposure. Consistent with this hypothesis, previous studies reported the molecular weight of C. albicans N-mannans to be 2.6-fold greater than that of C. glabrata (Hall et al, 2013; West et al, 2013). In addition, the molecular structure of C. glabrata mannan is lower in structural complexity relative to C. albicans (Shibata et al, 2012).…”

“…We began with the CaMNN2 gene family mannosylation mutants previously characterized by Hall et al (2013). This gene family is responsible for normal abundance and complexity of N-mannan side chains (Hall et al, 2013; Rayner and Munro, 1998), and deletion mutant strains of the CaMNN2 family exhibit altered mannan structure. The C. albicans parental strain was compared to the mannosylation mutants mnn2Δ , mnn2Δ /mnn26Δ, and a sextuple mutant (all CaMNN2 gene family deleted).…”

“…There is no direct analog between Camnn2Δ and Camnn2Δ /mnn26Δ mutant’s mannans and the C. glabrata mannans investigated below as a result of their more extensive side-chain defects and more extreme diminution of molecular weight. Based on previous work (Hall et al, 2013), we hypothesized that the negatively charged acid labile mannan as well as the large complex side chains are important N-mannan features for regulating nanoscale features of β-glucan exposure.…”

“…Serotype A C. albicans may also possess side chain β-(1,2) or (1,3) linked oligomannosides. Additionally, acid-labile β-mannoside moieties of variable length may be appended to N-mannan side chains via phosphodiester linkages (Cutler, 2001; Hall and Gow, 2013; Hall et al, 2013; West et al, 2013). β-(1,2)- or (1,3)-linked oligomannosides have also been identified in C. albicans as part of the phospholipomannan moiety of the cell wall (Trinel et al, 2002).…”

The Metacaspase (Mca1p) Restricts O-glycosylation During Farnesol-induced Apoptosis in Candida albicans

“…Glycosylation mostly affects plasma membrane and secreted proteins, and it plays a critical role in regulating many cell-cell interactions (1,3). In pathogenic yeasts, glycosylation has been implicated in host/ pathogen interactions (3,4) and particularly in virulence and immune recognition (5). As protein glycosylation is essential for cell viability in yeast, the glycosylation pathways is a potential target for new anti-fungal drugs.…”