Effect of beta-1,6-glucan inhibitors on the invasion process of Candida albicans: potential mechanism of their in vivo efficacy.
ABSTRACT: Beta-1,6-glucan is a fungus-specific cell wall component that is essential for the retention of many cell wall proteins. We recently reported the discovery of a small molecule inhibitor of beta-1,6-glucan biosynthesis in yeasts. In the course of our study of its derivatives, we found a unique feature in their antifungal profile. D21-6076, one of these compounds, exhibited potent in vitro and in vivo antifungal activities against Candida glabrata. Interestingly, although it only weakly reduced the growth of Candida albicans in conventional media, it significantly prolonged the survival of mice infected by the pathogen. Biochemical evaluation of D21-6076 indicated that it inhibited beta-1,6-glucan synthesis of C. albicans, leading the cell wall proteins, which play a critical role in its virulence, to be released from the cell. Correspondingly, adhesion of C. albicans cells to mammalian cells and their hyphal elongation were strongly reduced by the drug treatment. The results of the experiment using an in vitro model of vaginal candidiasis showed that D21-6076 strongly inhibited the invasion process of C. albicans without a significant reduction in its growth in the medium. These evidences suggested that D21-6076 probably exhibited in vivo efficacy against C. albicans by inhibiting its invasion process.
Project description:Cell wall beta-glucan in a pathogenic fungus, Candida albicans, is highly branched with beta-1,3 and beta-1,6 linkages. We have isolated the C. albicans cDNAs for KRE6 and SKN1, the genes required for beta-1,6-glucan synthesis in Saccharomyces cerevisiae. The results of Northern blot analysis revealed that C. albicans KRE6 was expressed at a higher level than SKN1 in the yeast phase, while SKN1 expression was strongly induced upon induction of hyphal formation. In addition, the C. albicans KRE6 and SKN1 mRNAs but not the actin mRNA were shortened during the yeast-hypha transition. Unlike S. cerevisiae, more than 50% of cell wall glucan was beta-1,6 linked in C. albicans. Neither beta-1,3-glucan nor beta-1,6-glucan was affected by the homozygous C. albicans skn1 delta null mutation. Although we never succeeded in generating the homozygous C. albicans kre6 delta null mutant, the hemizygous kre6 delta mutation decreased the KRE6 mRNA level by about 60% and also caused a more than 80% reduction of beta-1,6-glucan without affecting beta-1,3-glucan. The physiological function of KRE6 was further examined by studying gene regulation in C. albicans. When KRE6 transcription was suppressed by using the HEX1 promoter, C. albicans cells exhibited the partial defect in cell separation and increased susceptibility to Calcofluor White. These results demonstrate that KRE6 plays important roles in beta-1,6-glucan synthesis and budding in C. albicans.
Project description:The UDP-glucose:glycoprotein glucosyltransferase (UGGT) is an endoplasmic reticulum sensor for quality control of glycoprotein folding. Saccharomyces cerevisiae is the only eukaryotic organism so far described lacking UGGT-mediated transient reglucosylation of N-linked oligosaccharides. The only gene in S. cerevisiae with similarity to those encoding UGGTs is KRE5. S. cerevisiae KRE5 deletion strains show severely reduced levels of cell wall beta-1,6-glucan polymer, aberrant morphology, and extremely compromised growth or lethality, depending on the strain background. Deletion of both alleles of the Candida albicans KRE5 gene gives rise to viable cells that are larger than those of the wild type (WT), tend to aggregate, have enlarged vacuoles, and show major cell wall defects. C. albicans kre5/kre5 mutants have significantly reduced levels of beta-1,6-glucan and more chitin and beta-1,3-glucan and less mannoprotein than the WT. The remaining beta-1,6-glucan, about 20% of WT levels, exhibits a beta-1,6-endoglucanase digestion pattern, including a branch point-to-linear stretch ratio identical to that of WT strains, suggesting that Kre5p is not a beta-1,6-glucan synthase. C. albicans KRE5 is a functional homologue of S. cerevisiae KRE5; it partially complements both the growth defect and reduced cell wall beta-1,6-glucan content of S. cerevisiae kre5 viable mutants. C. albicans kre5/kre5 homozygous mutant strains are unable to form hyphae in several solid and liquid media, even in the presence of serum, a potent inducer of the dimorphic transition. Surprisingly the mutants do form hyphae in the presence of N-acetylglucosamine. Finally, C. albicans KRE5 homozygous mutant strains exhibit a 50% reduction in adhesion to human epithelial cells and are completely avirulent in a mouse model of systemic infection.
Project description:Innate immunity depends upon recognition of surface features common to broad groups of pathogens. The glucose polymer beta-glucan has been implicated in fungal immune recognition. Fungal walls have two kinds of beta-glucan: beta-1,3-glucan and beta-1,6-glucan. Predominance of beta-1,3-glucan has led to the presumption that it is the key immunological determinant for neutrophils. Examining various beta-glucans for their ability to stimulate human neutrophils, we find that the minor cell wall component beta-1,6-glucan mediates neutrophil activity more efficiently than beta-1,3-glucan, as measured by engulfment, production of reactive oxygen species, and expression of heat shock proteins. Neutrophils rapidly ingest beads coated with beta-1,6-glucan while ignoring those coated with beta-1,3-glucan. Complement factors C3b/C3d are deposited on beta-1,6-glucan more readily than on beta-1,3-glucan. Beta-1,6-glucan is also important for efficient engulfment of the human pathogen Candida albicans. These unique stimulatory effects offer potential for directed stimulation of neutrophils in a therapeutic context.
Project description:The cell wall is a dynamic structure that is important for the pathogenicity of Candida albicans. Mannan, which is located in the outermost layer of the cell wall, has been shown to contribute to the pathogenesis of C. albicans, however, the molecular mechanism by which this occurs remains unclear. Here we identified a novel ?-1,6-mannosyltransferase encoded by MNN10 in C. albicans. We found that Mnn10 is required for cell wall ?-1,6-mannose backbone biosynthesis and polysaccharides organization. Deletion of MNN10 resulted in significant attenuation of the pathogenesis of C. albicans in a murine systemic candidiasis model. Inhibition of ?-1,6-mannose backbone extension did not, however, impact the invasive ability of C. albicans in vitro. Notably, mnn10 mutant restored the invasive capacity in athymic nude mice, which further supports the notion of an enhanced host antifungal defense related to this backbone change. Mnn10 mutant induced enhanced Th1 and Th17 cell mediated antifungal immunity, and resulted in enhanced recruitment of neutrophils and monocytes for pathogen clearance in vivo. We also demonstrated that MNN10 could unmask the surface ?-(1,3)-glucan, a crucial pathogen-associated molecular pattern (PAMP) of C. albicans recognized by host Dectin-1. Our results demonstrate that mnn10 mutant could stimulate an enhanced Dectin-1 dependent immune response of macrophages in vitro, including the activation of nuclear factor-?B, mitogen-activated protein kinase pathways, and secretion of specific cytokines such as TNF-?, IL-6, IL-1? and IL-12p40. In summary, our study indicated that ?-1,6-mannose backbone is critical for the pathogenesis of C. albicans via shielding ?-glucan from recognition by host Dectin-1 mediated immune recognition. Moreover, our work suggests that inhibition of ?-1,6-mannose extension by Mnn10 may represent a novel modality to reduce the pathogenicity of C. albicans.
Project description:A biochemical pathway for the incorporation of cell wall protein into the cell wall of Neurospora crassa was recently proposed. In this pathway, the DFG-5 and DCW-1 endo-?-1,6-mannanases function to covalently cross-link cell wall protein-associated N-linked galactomannans, which are structurally related to the yeast outer chain mannans, into the cell wall glucan-chitin matrix. In this report, we demonstrate that the mannosyltransferase enzyme Och1p, which is needed for the synthesis of the N-linked outer chain mannan, is essential for the incorporation of cell wall glycoproteins into the Candida albicans cell wall. Using endoglycosidases, we show that C. albicans cell wall proteins are cross-linked into the cell wall via their N-linked outer chain mannans. We further demonstrate that the Dfg5p and Dcw1p ?-1,6-mannanases are needed for the incorporation of cell wall glycoproteins into the C. albicans cell wall. Our results support the hypothesis that the Dfg5p and Dcw1p ?-1,6-mannanases incorporate cell wall glycoproteins into the C. albicans cell wall by cross-linking outer chain mannans into the cell wall glucan-chitin matrix.
Project description:?-1,3-d-Glucan is a ubiquitous glucose polymer produced by plants, bacteria, and most fungi. It has been used as a diagnostic tool in patients with invasive mycoses via a highly-sensitive reagent consisting of the blood coagulation system of horseshoe crab. However, no method is currently available for measuring ?-1,6-glucan, another primary ?-glucan structure of fungal polysaccharides. Herein, we describe the development of an economical and highly-sensitive and specific assay for ?-1,6-glucan using a modified recombinant endo-?-1,6-glucanase having diminished glucan hydrolase activity. The purified ?-1,6-glucanase derivative bound to the ?-1,6-glucan pustulan with a KD of 16.4 nm We validated the specificity of this ?-1,6-glucan probe by demonstrating its ability to detect cell wall ?-1,6-glucan from both yeast and hyphal forms of the opportunistic fungal pathogen Candida albicans, without any detectable binding to glucan lacking the long ?-1,6-glucan branch. We developed a sandwich ELISA-like assay with a low limit of quantification for pustulan (1.5 pg/ml), and we successfully employed this assay in the quantification of extracellular ?-1,6-glucan released by >250 patient-derived strains of different Candida species (including Candida auris) in culture supernatant in vitro We also used this assay to measure ?-1,6-glucan in vivo in the serum and in several organs in a mouse model of systemic candidiasis. Our work describes a reliable method for ?-1,6-glucan detection, which may prove useful for the diagnosis of invasive fungal infections.
Project description:Cell wall mannans of Candida albicans mask ?-(1,3)-glucan from recognition by Dectin-1, contributing to innate immune evasion. Glucan exposures are predominantly single receptor-ligand interaction sites of nanoscale dimensions. Candida species vary in basal glucan exposure and molecular complexity of mannans. We used super-resolution fluorescence imaging and a series of protein mannosylation mutants in C. albicans and C. glabrata to investigate the role of specific N-mannan features in regulating the nanoscale geometry of glucan exposure. Decreasing acid labile mannan abundance and ?-(1,6)-mannan backbone length correlated most strongly with increased density and nanoscopic size of glucan exposures in C. albicans and C. glabrata, respectively. Additionally, a C. albicans clinical isolate with high glucan exposure produced similarly perturbed N-mannan structures and elevated glucan exposure geometry. Thus, acid labile mannan structure influences the nanoscale features of glucan exposure, impacting the nature of the pathogenic surface that triggers immunoreceptor engagement, aggregation, and signaling.
Project description:Sera from candidemic and non-candidemic subjects were examined for antibodies against the cell wall ?1,3- and ?1,6-glucans, as well as the ?-glucan-associated protein MP65 of Candida species. Although antibodies against each of the above components were detected in all subjects, candidemic patients had lower antibody titers against ?1,3-glucan, but higher antibody titers against ?1,6-glucan and MP65, than non-candidemic subjects. The elevated levels of anti-?1,6-glucan and -MP65 antibodies found in candidemic patients were independent on the patient risk category, APACHE II score, presence of co-morbidities, ?1,3-glucanemia level, Candida isolate, and antifungal treatment. Interestingly, however, the anti-MP65, but not the anti-?1,6-glucan antibodies, of candidemic patients had higher titers in survivors than in non-survivors, particularly in those subject categories with the highest mortality (>65-years old, diabetic, or septic shock patients). Thus, candidemic patients are capable of boosting anti-Candida immune responses upon infection, and some of these responses might be associated to the generation of protective immunity in patients with candidemia.
Project description:The cell wall of Candida albicans is composed largely of polysaccharides. Here we focus on ?-glucan, an immunogenic cell-wall polysaccharide whose surface exposure is often restricted, or "masked," from immune recognition by Dectin-1 on dendritic cells (DCs) and other innate immune cells. Previous research suggested that the physical presentation geometry of ?-glucan might determine whether it can be recognized by Dectin-1. We used direct stochastic optical reconstruction microscopy to explore the fine structure of ?-glucan exposed on C. albicans cell walls before and after treatment with the antimycotic drug caspofungin, which alters glucan exposure. Most surface-accessible glucan on C. albicans yeast and hyphae is limited to isolated Dectin-1-binding sites. Caspofungin-induced unmasking caused approximately fourfold to sevenfold increase in total glucan exposure, accompanied by increased phagocytosis efficiency of DCs for unmasked yeasts. Nanoscopic imaging of caspofungin-unmasked C. albicans cell walls revealed that the increase in glucan exposure is due to increased density of glucan exposures and increased multiglucan exposure sizes. These findings reveal that glucan exhibits significant nanostructure, which is a previously unknown physical component of the host-Candida interaction that might change during antifungal chemotherapy and affect innate immune activation.
Project description:The Saccharomyces cerevisiae RHO1 gene encodes a low-molecular-weight GTPase. One of its recently identified functions is the regulation of beta-1,3-glucan synthase, which synthesizes the main component of the fungal cell wall (J. Drgonova et al., Science 272:277-279, 1996; T. Mazur and W. Baginsky, J. Biol. Chem. 271:14604-14609, 1996; and H. Qadota et al., Science 272:279-281, 1996). From the opportunistic pathogenic fungus Candida albicans, we cloned the RHO1 gene by the PCR and cross-hybridization methods. Sequence analysis revealed that the Candida RHO1 gene has a 597-nucleotide region which encodes a putative 22.0-kDa peptide. The deduced amino acid sequence predicts that Candida albicans Rho1p is 82.9% identical to Saccharomyces Rho1p and contains all the domains conserved among Rho-type GTPases from other organisms. The Candida albicans RHO1 gene could rescue a S. cerevisiae strain containing a rho1 deletion. Furthermore, recombinant Candida albicans Rho1p could reactivate the beta-1,3-glucan synthesis activities of both C. albicans and S. cerevisiae membranes in which endogenous Rho1p had been depleted by Tergitol NP-40-NaCl treatment. Candida albicans Rho1p was copurified with the beta-1,3-glucan synthase putative catalytic subunit, Candida albicans Gsc1p, by product entrapment. Candida albicans Rho1p was shown to interact directly with Candida albicans Gsc1p in a ligand overlay assay and a cross-linking study. These results indicate that Candida albicans Rho1p acts in the same manner as Saccharomyces cerevisiae Rho1p to regulate beta-1,3-glucan synthesis.