Fungal cell walls contain several types of glycans, which play important roles in the pathogenesis of fungal infection and host immune response. Among them, glycosphingolipids have attracted much attention lately since they contribute actively to the fungi development and fungal-induced pathogenesis. Although glycosphingolipids are present in pathogenic and non-pathogenic fungi, pathogenic strains exhibit distinct glycan structures on their sphingolipids, which contribute to the regulatory processes engaged in inflammatory response. In Candida albicans, phospholipomannan (PLM) represents a prototype of these sphingolipids. Through its glycan and lipid moieties, PLM induces activation of host signaling pathways involved in the initial recognition of fungi, causing immune system disorder and persistent fungal disease. In this review, first we describe the general aspects of C. albicans sphingolipids synthesis with a special emphasize on PLM synthesis and its insertion into the cell wall. Then, we discuss the role of PLM glycosylation in regulating immune system activation and its contribution to the chronic persistent inflammation found in Candida infections and chronic inflammatory diseases.
The presence of ?-mannosides in their cell walls confers specific features on the pathogenic yeasts Candida albicans and Candida glabrata compared with non-pathogenic yeasts. In the present study, we investigated the enzymatic properties of Bmt1 (?-mannosyltransferase 1), a member of the recently identified ?-mannosyltransferase family, from C. albicans. A recombinant soluble enzyme lacking the N-terminal region was expressed as a secreted protein from the methylotrophic yeast Pichia pastoris. In parallel, functionalized natural oligosaccharides isolated from Saccharomyces cerevisiae and a C. albicans mutant strain, as well as synthetic ?-oligomannosides, were prepared and used as potential acceptor substrates. Bmt1p preferentially utilizes substrates containing linear chains of ?-1,2-linked mannotriose or mannotetraose. The recombinant enzyme consecuti-vely transfers two mannosyl units on to these acceptors, leading to the production of ?-mannosidase-resistant oligomannosides. NMR experiments further confirmed the presence of a terminal ?Man (?-1,2-linked mannose) unit in the first enzyme product. In the future, a better understanding of specific ?-1,2-mannosyltransferase molecular requirements will help the design of new potential antifungal drugs.
Glycosylation is a crucial step in the modification of proteins or sphingolipids that then play a prominent role in fungal biology. Glycosylation controls the structure and plasticity of the fungal cell wall and fungi-host interactions. Non-pathogenic and pathogenic yeasts, such as Saccharomyces cerevisiae and Candida albicans, respectively, have been useful models for analyzing the mannosylation of proteins and sphingolipids, which mainly takes place in the Golgi apparatus. Studies of these yeasts have identified different mannosyltransferases that belong to separate families of glycosyltransferases. The characterization of mannosyltransferases and their activities is essential for deciphering cell wall biogenesis, for identifying mannosides involved in virulence and for designing inhibitors that target specific mannosylation processes.
Pseudomonas aeruginosa and Candida albicans are two pathogens frequently encountered in the intensive care unit microbial community. We have demonstrated that C. albicans airway exposure protected against P. aeruginosa-induced lung injury. The goal of the present study was to characterize the cellular and molecular mechanisms associated with C. albicans-induced protection. Airway exposure by C. albicans led to the recruitment and activation of natural killer cells, innate lymphoid cells (ILCs), macrophages, and dendritic cells. This recruitment was associated with the secretion of interleukin-22 (IL-22), whose neutralization abolished C. albicans-induced protection. We identified, by flow cytometry, ILCs as the only cellular source of IL-22. Depletion of ILCs by anti-CD90.2 antibodies was associated with a decreased IL-22 secretion and impaired survival after P. aeruginosa challenge. Our results demonstrate that the production of IL-22, mainly by ILCs, is a major and inducible step in protection against P. aeruginosa-induced lung injury. This cytokine may represent a clinical target in Pseudomonas aeruginosa-induced lung injury.
Candida albicans produces a complex glycosphingolipid called phospholipomannan (PLM), which is present on the cell-wall surface of yeast and shed upon contact with host cells. The glycan moiety of PLM is composed of ?-mannosides with degrees of polymerization up to 19 in C. albicans serotype A. PLM from serotype B strains displays a twofold decrease in the length of the glycan chains. In this study we compared the proinflammatory activities of PLMs purified from C. albicans serotype A and serotype B strains and from a bmt6? mutant of C. albicans, whose PLM is composed of short truncated oligomannosidic chain. We found that PLMs activate caspase-1 in murine macrophage cell line J774 independent of the glycan chain length although IL-1? secretion is more intense with long glycan chain. None of the tested PLMs stimulate ROS production, indicating that caspase-1 activation may occur through a ROS-independent pathway. On the other hand, only long-chain oligomannosides present on PLM from serotype A strain (PLM-A) are able to induce TNF-? production in macrophages, a property that is not affect by blocking endocytosis through latrunculin A treatment. Finally, we demonstrate that soluble and not cell surface-bound galectin-3, is able to potentiate PLM-A-induced TNF-? production in macrophages. PLMs from C. albicans serotype B and from bmt6? mutant are not able to induce TNF-? production and galectin-3 pretreatment does not interfere with this result. In conclusion, we show here that PLMs are able to evoke a proinflammatory state in macrophage, which is in part dependent on their glycosylation status. Long-glycan chains favor interaction with soluble galectin-3 and help amplify inflammatory response.
Oral epithelial cells discriminate between the yeast and hyphal forms of Candida albicans via the mitogen-activated protein kinase (MAPK) signaling pathway. This occurs through phosphorylation of the MAPK phosphatase MKP1 and activation of the c-Fos transcription factor by the hyphal form. Given that fungal cell wall polysaccharides are critical in host recognition and immune activation in myeloid cells, we sought to determine whether ?-glucan and N- or O-glycosylation was important in activating the MAPK/MKP1/c-Fos hypha-mediated response mechanism and proinflammatory cytokines in oral epithelial cells. Using a series of ?-glucan and N- and O-mannan mutants, we found that N-mannosylation (via ?och1 and ?pmr1 mutants) and O-mannosylation (via ?pmt1 and ?mnt1 ?mnt2 mutants), but not phosphomannan (via a ?mnn4 mutant) or ?-1,2 mannosylation (via ?bmt1 to ?bmt6 mutants), were required for MKP1/c-Fos activation, proinflammatory cytokine production, and cell damage induction. However, the N- and O-mannan mutants showed reduced adhesion or lack of initial hypha formation at 2 h, resulting in little MKP1/c-Fos activation, or restricted hypha formation/pseudohyphal formation at 24 h, resulting in minimal proinflammatory cytokine production and cell damage. Further, the ?-1,6-mannose backbone of the N-linked outer chain (corresponding to a ?mnn9 mutant) may be required for epithelial adhesion, while the ?-1,2-mannose component of phospholipomannan (corresponding to a ?mit1 mutant) may contribute to epithelial cell damage. ?-Glucan appeared to play no role in adhesion, epithelial activation, or cell damage. In summary, N- and O-mannosylation defects affect the ability of C. albicans to induce proinflammatory cytokines and damage in oral epithelial cells, but this may be due to indirect effects on fungal pathogenicity rather than mannose residues being direct activators of the MAPK/MKP1/c-Fos hypha-mediated immune response.
Recent changes in the epidemiology of candidiasis highlighted an increase in non- Candida albicans species emphasizing the need for reliable identification methods. Molecular diagnostics in fungal infections may improve species characterization, particularly in cases of the closely related species in the Candida complexes. We developed two PCR/restriction fragment length polymorphism assays, targeting either a part of the intergenic spacer 2 or the entire intergenic spacer (IGS) of ribosomal DNA using a panel of 270 isolates. A part of the intergenic spacer was used for discrimination between C. albicans and C. dubliniensis and between species of the C. glabrata complex (C. glabrata/C. bracarensis/C. nivariensis). The whole IGS was applied to C. parapsilosis, C. metapsilosis, and C. orthopsilosis, and to separate C. famata (Debaryomyces hansenii) from C. guilliermondii (Pichia guilliermondii) and from the other species within this complex (ie, C. carpophila, C. fermentati and C. xestobii). Sharing similar biochemical patterns, Pichia norvegensis and C. inconspicua exhibited specific IGS profiles. Our study confirmed that isolates of C. guilliermondii were frequently mis-identified as C. famata. As much as 67% of the clinical isolates phenotypically determined as C. famata were recognized mostly as true P. guilliermondii. Conversely, 44% of the isolates initially identified as C. guilliermondii were corrected by the IGS fingerprints as C. parapsilosis, C. fermentati, or C. zeylanoides. These two PCR/restriction fragment length polymorphism methods may be used as reference tools [either alternatively or adjunctively to the existing ribosomal DNA (26S or ITS) sequence comparisons] for unambiguous determination of the Candida species for which phenotypic characterization remains problematic.
Almost 80 % of the dry weight of the yeast cell wall is composed of glycans including mannans, glucans and chitin. Within this variable and complex edifice, glycans play a major role in their relation with the environment. Experimental antibodies allowed to define the localization, the variability of expression and the biological role of numerous natural oligosaccharidic sequences. These glycans and their synthetic analogues were used to study the human humoral response during invasive candidiasis (IC) determined by Candida albicans and Crohns disease (CD) where antibodies against the dietary yeast Saccharomyces cerevisiae have been reported. On these bases, it was established experimentally and clinically that a large panel of CD biomarkers consisting in anti glycans antibodies were also generated during IC establishing a link never suspected between C. albicans and CD. We describe here the principle of this serological analysis and its perspectives related to the use of multianalyte profiling technology for a a better understanding of IC and CD pathophysiology. This may contribute to improve disease management in terms of diagnosis and therapy.
The human pathogenic yeast Candida albicans can cause an unusually broad range of infections reflecting a remarkable potential to adapt to various microniches within the human host. The exceptional adaptability of C. albicans is mediated by rapid alterations in gene expression in response to various environmental stimuli and this transcriptional flexibility can be monitored with tools such as microarrays. Using such technology it is possible to (1) capture a genome-wide portrait of the transcriptome that mirrors the environmental conditions, (2) identify known genes, signalling pathways and transcription factors involved in pathogenesis, (3) identify new patterns of gene expression and (4) identify previously uncharacterized genes that may be associated with infection. In this review, we describe the molecular dissection of three distinct stages of infections, covering both superficial and invasive disease, using in vitro, ex vivo and in vivo infection models and microarrays.
The molecular interactions between commensal microorganisms and their host are basically different from those triggered by pathogens since they involve tolerance. When the commensal is genetically equipped to become an opportunistic pathogen, as is the case with Candida albicans, the picture becomes more complex. In this case, the balance between protection and invasion depends on host reactivity to altered microbial expression of ligands interacting with innate immune sensors. Based on experimental evidence obtained with C. albicans, we discuss the different molecular processes involved in the sensing of this important opportunistic human pathogen by a panel of pattern recognition receptors (PRRs) according to the numerous pathogen-associated molecular patterns (PAMPs) that can be exposed at its surface. Beneficial or deleterious immune responses that either maintain a commensal state or favour damage by the yeast result from this dynamic interplay.
Current research on Crohns disease (CD) concerns molecular events related to loss of tolerance to microbes that could trigger or maintain inflammation in genetically susceptible individuals. CD is also associated with antimicrobial antibodies, including the antibodies we described against yeast oligomannosides (ASCA). This prompted us to investigate a role for another yeast, Candida albicans, a very common commensal of the human digestive tract and an important opportunistic pathogen.
A family of nine genes encoding proteins involved in the synthesis of ?-1,2 mannose adhesins of Candida albicans has been identified. Four of these genes, BMT1-4, encode enzymes acting stepwise to add ?-mannoses on to cell-wall phosphopeptidomannan (PPM). None of these acts on phospholipomannan (PLM), a glycosphingolipid member of the mannose-inositol-phosphoceramide family, which contributes with PPM to ?-mannose surface expression. We show that deletion of BMT5 and BMT6 led to a dramatic reduction of PLM glycosylation and accumulation of PLM with a truncated ?-oligomannoside chain, respectively. Disruptions had no effect on sphingolipid biosynthesis and on PPM ?-mannosylation. ?-Mannose surface expression was not affected, confirming that ?-mannosylation is a process based on specificity of acceptor molecules, but liable to global regulation.
Candida glabrata, like Candida albicans, is an opportunistic yeast pathogen that has adapted to colonize all segments of the human gastrointestinal tract and vagina. The C. albicans cell wall expresses ?-1,2-linked mannosides (?-Mans), promoting its adherence to host cells and tissues. Because ?-Mans are also present in C. glabrata, their role in C. glabrata colonization and virulence was investigated in a murine model of dextran sulfate sodium (DSS)-induced colitis. Five clustered genes of C. glabrata encoding ?-mannosyltransferases, BMT2-BMT6, were deleted simultaneously. ?-Man expression was studied by Western blotting, flow cytometry, and NMR analysis. Mortality, clinical, histologic, and colonization scores were determined in mice receiving DSS and different C. glabrata strains. The results show that C. glabrata bmt2-6 strains had a significant reduction in ?-1,2-Man expression and a disappearance of ?-1,2-mannobiose in the acid-stable domain. A single gavage of C. glabrata wild-type strain in mice with DSS-induced colitis caused a loss of body weight, colonic inflammation, and mortality. Mice receiving C. glabrata bmt2-6 mutant strains had normal body weight and reduced colonic inflammation. Lower numbers of colonies of C. glabrata bmt2-6 were recovered from stools and different parts of the gastrointestinal tract. Histopathologic examination revealed that the wild-type strain had a greater ability to colonize tissue and cause tissue damage. These results showed that C. glabrata has a high pathogenic potential in DSS-induced colitis, where ?-Mans contribute to colonization and virulence.
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