Summary: The fungal cell wall is a dynamic organelle required for cell shape, protection against the environment and, in pathogenic species, recognition by the innate immune system. The outer layer of the cell wall is comprised of glycosylated mannoproteins with the majority of these post‐translational modifications being the addition of O‐ and N‐linked mannosides. These polysaccharides are exposed on the outer surface of the fungal cell wall and are, therefore, the first point of contact between the fungus and the host immune system. This review focuses on O‐ and N‐linked mannan biosynthesis in the fungal pathogen Candida albicans and highlights new insights gained from the characterization of mannosylation mutants into the role of these cell wall components in host–fungus interactions. In addition, we discuss the use of fungal mannan as a diagnostic marker of fungal disease.

Candida albicans is able to proliferate in environments that vary dramatically in ambient pH, a trait required for colonising niches such as the stomach, vaginal mucosal and the GI tract. Here we show that growth in acidic environments involves cell wall remodelling which results in enhanced chitin and β-glucan exposure at the cell wall periphery. Unmasking of the underlying immuno-stimulatory β-glucan in acidic environments enhanced innate immune recognition of C. albicans by macrophages and neutrophils, and induced a stronger proinflammatory cytokine response, driven through the C-type lectin-like receptor, Dectin-1. This enhanced inflammatory response resulted in significant recruitment of neutrophils in an intraperitoneal model of infection, a hallmark of symptomatic vaginal colonisation. Enhanced chitin exposure resulted from reduced expression of the cell wall chitinase Cht2, via a Bcr1-Rim101 dependent signalling cascade, while increased β-glucan exposure was regulated via a non-canonical signalling pathway. We propose that this “unmasking” of the cell wall may induce non-protective hyper activation of the immune system during growth in acidic niches, and may attribute to symptomatic vaginal infection. To be able to colonise a host or cause infection, microbes must be able to adapt and respond to changes in their environment. One of the most important environmental signals for opportunistic pathogens is ambient pH, with changes in external pH resulting in phenotypic, metabolic and physical changes in fungi and bacteria (i.e. E. coli, Salmonella, Aspergillus, Candida). Candida albicans is an opportunistic fungal pathogen of humans that can colonise and infect niches of varying pH including the acidic mucosa of the vagina. Here we show that growth in an acidic environment results in structural modification of the fungal cell wall, a dynamic organelle key to immune recognition. These cell wall perturbations resulted in enhanced immune recognition of the fungal pathogen, a strong proinflammatory immune response and enhanced recruitment of neutrophils. Therefore, colonisation of acidic mucosa may result in the unmasking of cell wall components that trigger hyper-activation of the innate immune response and could contribute to immunopathology associated with vaginal candidiasis.

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. Candida albicans is an opportunistic fungal pathogen of humans, which can cause superficial infections of the oral and genital epithelium or fatal systemic disease in immune compromised individuals. The cell wall of C. albicans consists of an inner layer of chitin and β-glucan and an outer fibrillar layer of highly glycosylated mannoproteins. These mannose-rich carbohydrates play crucial roles in fungal immune recognition. Deletion of genes encoding the Mnn2 family of α1,2-mannosyltransferases, which are involved in the biosynthesis of N-mannan outer chains, demonstrated that the addition of mannan side chains is necessary for the stabilisation and extension of the N-mannan backbone. Mutants of the MNN2 gene family had marked alterations in mannan microfibril length. Reductions in the degree of N-mannan branching resulted in decreased immune recognition by monocytes, whilst complete removal of the branched outer chains increased β-glucan exposure, and correlated with an attenuated virulence. Therefore, the Mnn2 mannosyltransferase family is critical for microfibril structure, immune recognition and virulence.

Living as a commensal, Candida albicans must adapt and respond to environmental cues generated by the mammalian host and by microbes comprising the natural flora. These signals have opposing effects on C. albicans , with host cues promoting the yeast-to-hyphal transition and bacteria-derived quorum-sensing molecules inhibiting hyphal development. Hyphal development is regulated through modulation of the cyclic AMP (cAMP)/protein kinase A (PKA) signaling pathway, and it has been postulated that quorum-sensing molecules can affect filamentation by inhibiting the cAMP pathway. Here, we show that both farnesol and 3-oxo-C ₁₂ -homoserine lactone, a quorum-sensing molecule secreted by Pseudomonas aeruginosa , block hyphal development by affecting cAMP signaling; they both directly inhibited the activity of the Candida adenylyl cyclase, Cyr1p. In contrast, the 12-carbon alcohol dodecanol appeared to modulate hyphal development and the cAMP signaling pathway without directly affecting the activity of Cyr1p. Instead, we show that dodecanol exerted its effects through a mechanism involving the C. albicans hyphal repressor, Sfl1p. Deletion of SFL1 did not affect the response to farnesol but did interfere with the response to dodecanol. Therefore, quorum sensing in C. albicans is mediated via multiple mechanisms of action. Interestingly, our experiments raise the possibility that the Burkholderia cenocepacia diffusible signal factor, BDSF, also mediates its effects via Sfl1p, suggesting that dodecanol's mode of action, but not farnesol or 3-oxo-C ₁₂ -homoserine lactone, may be used by other quorum-sensing molecules.

When colonising host-niches or non-animated medical devices, individual cells of the fungal pathogen Candida albicans expand into significant biomasses. Here we show that within such biomasses, fungal metabolically generated CO₂ acts as a communication molecule promoting the switch from yeast to filamentous growth essential for C. albicans pathology. We find that CO₂-mediated intra-colony signalling involves the adenylyl cyclase protein (Cyr1p), a multi-sensor recently found to coordinate fungal responses to serum and bacterial peptidoglycan. We further identify Lys 1373 as essential for CO₂/bicarbonate regulation of Cyr1p. Disruption of the CO₂/bicarbonate receptor-site interferes selectively with C. albicans filamentation within fungal biomasses. Comparisons between the Drosophila melanogaster infection model and the mouse model of disseminated candidiasis, suggest that metabolic CO₂ sensing may be important for initial colonisation and epithelial invasion. Our results reveal the existence of a gaseous Candida signalling pathway and its molecular mechanism and provide insights into an evolutionary conserved CO₂-signalling system. Pathogenic microorganisms can produce a variety of secondary metabolites and signalling molecules which can affect the host, or provide them with a selective advantage against competing commensal organisms. We demonstrate that gaseous, metabolically generated CO₂ can serve as a signalling molecule to enhance the organism's virulence during infection establishment by using the fungal pathogen Candida albicans as a model. Furthermore, we identified a CO₂ receptor site within the catalytic domain of the soluble adenylyl cyclase, Cyr1p, which is critical for CO₂ sensing and hence virulence of the organism. CO₂ sensing is conserved in a variety of pathogenic species, and increased levels have been shown to suppress the host's immune system. Thus, CO₂ sensing may represent a mechanism to enhance C. albicans virulence when the host's immune system is suppressed.