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In the human body, fungi and bacteria share many niches where the close contact of these organisms maintains a balance among the microbial population. However, when this microbial balance is disrupted, as with antibiotic treatment, other bacteria or fungi can grow uninhibited. C. albicans is the most common opportunistic fungal pathogen affecting humans and can uniquely control its morphogenesis between yeast, pseudohyphal, and hyphal forms. Numerous studies have shown that C. albicans interactions with bacteria can impact its ability to undergo morphogenesis; however, the genetics that govern this morphological control via these bacterial interactions are still relatively unknown. To aid in the understanding of the cross-kingdom interactions of C. albicans with bacteria and the impact on morphology we utilized a haploinsufficiency based C. albicans mutant screen to test for the ability of C. albicans to produce hyphae in the presence of three bacterial species (Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus). Of the 18,144 mutant strains tested, 295 mutants produced hyphae in the presence of all three bacterial species. The 295 mutants identified 132 points of insertion, which included identified/predicted genes, major repeat sequences, and a number of non-coding/unannotated transcripts. One gene, CDR4, displayed increased expression when co-cultured with S. aureus, but not E. coli or P. aeruginosa. Our data demonstrates the ability to use a large scale library screen to identify genes involved in Candida-bacterial interactions and provides the foundation for comprehending the genetic pathways relating to bacterial control of C. albicans morphogenesis.

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.

β-Glucan is a (1→3)-β-linked glucose polymer with (1→6)-β-linked side chains and a major component of fungal cell walls. β-Glucans provide structural integrity to the fungal cell wall. The nature of the (1-6)-β-linked side chain structure of fungal (1→3,1→6)-β-D-glucans has been very difficult to elucidate. Herein, we report the first detailed structural characterization of the (1→6)-β-linked side chains of Candida glabrata using high-field NMR. The (1→6)-β-linked side chains have an average length of 4 to 5 repeat units spaced every 21 repeat units along the (1→3)-linked polymer backbone. Computer modeling suggests that the side chains have a bent curve structure that allows for a flexible interconnection with parallel (1→3)-β-D-glucan polymers, and/or as a point of attachment for proteins. Based on these observations we propose new approaches to how (1→6)-β-linked side chains interconnect with neighboring glucan polymers in a manner that maximizes fungal cell wall strength, while also allowing for flexibility, or plasticity.