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

PLoS Pathogens, Apr 2013

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.

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

Immune Recognition and Virulence of Candida albicans. PLoS Pathog 9(4): e1003276. doi:10.1371/journal.ppat.1003276 The Mnn2 Mannosyltransferase Family Modulates Mannoprotein Fibril Length, Immune Recognition and Virulence of Candida albicans Rebecca A. Hall 0 Steven Bates 0 Megan D. Lenardon 0 Donna M. MacCallum 0 Jeanette Wagener 0 Douglas W. Lowman 0 Michael D. Kruppa 0 David L. Williams 0 Frank C. Odds 0 Alistair J. P. Brown 0 Neil A. R. Gow 0 Marta Feldmesser, Albert Einstein College of Medicine, United States of America 0 1 Aberdeen Fungal Group, Institute of Medical Sciences, University of Aberdeen , Foresterhill, Aberdeen , United Kingdom , 2 Biosciences , College of Life and Environmental Sciences, University of Exeter , Exeter , United Kingdom , 3 Quillen College of Medicine, East Tennessee State University , Johnson City , Tennessee, United States of America , 4 AppRidge International , LLC , Telford, Tennessee , United States of America 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 Olinked 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 a1,2-mannose residue onto the a1,6-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 a1,2-mannose is required for stabilisation of the a1,6-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 a1,6-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, a1,2-mannose of N-mannan is important for both immune recognition and virulence of C. albicans. - Funding: This work was funded by the Wellcome Trust (080088, 086827, 075470 and 099215), and by a FP7-2007-2013 grant agreement (HEALTH-F2-2010260338ALLFUN). MDL was supported by an MRC New Investigator Award (MR/J008230/1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: I have read the journals policy and have the following conflicts: Douglas W. Lowman is an employee of AppRidge International, LLC. The noted receipt of funding from AppRidge does not alter our adherence to all PLoS Pathogens policies on sharing data and materials. Candida albicans is a polymorphic fungus that forms part of the natural human microflora. However, many adverse conditions result in predisposition to oral and vaginal infections and, under circumstances where the host immune system becomes severely compromised as a consequence of malignancy, trauma or chemotherapy, C. albicans can invade underlying epithelial cells and disseminate via the bloodstream and cause systemic disease. The associated mortality rate of systemic disease is approximately 3040%, which is higher than that observed for many bacterial systemic infections, making C. albicans a major pathogen of the immunocompromised and a significant global health burden [13]. The fungal cell wall is a highly dynamic structural organelle essential for maintaining cell shape and for protection against the environment. The cell wall is also the first point of contact between the fungus and the host, and as a result the cell wall is pivotal for fungus-host interactions and immune recognition. The cell wall is comprised of an inner skeletal layer of chitin and b-glucans (b1,3and b1,6-glucan) and an outer layer of highly glycosylated mannoproteins [46]. These proteins are decorated with linear O-linked mannan and highly branched N-linked mannan, which can be elaborated with additional mannan side chains attached via a phosphodiester linkage known as phosphomannan (PM). The mannan fraction of the cell wall is important for adhesion, cell wall integrity, immune recognition and comprises up to 40% of the cell wall dry weight [712]. All of the major cell wall carbohydrate components of fungal walls serve as pathogen associated molecular patterns (PAMPs), which are recognised by the innate immu (...truncated)


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Rebecca A. Hall, Steven Bates, Megan D. Lenardon, Donna M. MacCallum, Jeanette Wagener, Douglas W. Lowman, Michael D. Kruppa, David L. Williams, Frank C. Odds, Alistair J. P. Brown, Neil A. R. Gow. The Mnn2 Mannosyltransferase Family Modulates Mannoprotein Fibril Length, Immune Recognition and Virulence of Candida albicans, PLoS Pathogens, 2013, Volume 9, Issue 4, DOI: 10.1371/journal.ppat.1003276