Systematic gene overexpression in Candida albicans identifies a regulator of early adaptation to the mammalian gut.

Candida albicans is part of the human gastrointestinal (GI) microbiota. To better understand how C. albicans efficiently establishes GI colonisation, we competitively challenged growth of 572 signature-tagged strains (~10% genome coverage), each conditionally overexpressing a single gene, in the murine gut. We identified CRZ2, a transcription factor whose overexpression and deletion respectively increased and decreased early GI colonisation. Using clues from genome-wide expression and gene-set enrichment analyses, we found that the optimal activity of Crz2p occurs under hypoxia at 37°C, as evidenced by both phenotypic and transcriptomic analyses following CRZ2 genetic perturbation. Consistent with early colonisation of the GI tract, we show that CRZ2 overexpression confers resistance to acidic pH and bile salts, suggesting an adaptation to the upper sections of the gut. Genome-wide location analyses revealed that Crz2p directly modulates the expression of many mannosyltransferase- and cell-wall protein-encoding genes, suggesting a link with cell-wall function. We show that CRZ2 overexpression alters cell-wall phosphomannan abundance and increases sensitivity to tunicamycin, suggesting a role in protein glycosylation. Our study reflects the powerful use of gene overexpression as a complementary approach to gene deletion to identify relevant biological pathways involved in C. albicans interaction with the host environment.

Citrulline and Monocyte-Derived Macrophage Reactivity before Conditioning Predict Acute Graft-versus-Host Disease.

During conditioning, intestinal damage induces microbial translocation which primes macrophage reactivity and leads to donor-derived T cell stimulation. Little is known about the role of intestinal health and macrophage reactivity before conditioning in the development of acute graft-versus-host disease (aGVHD) in patients undergoing allogeneic hematopoietic cell transplantation (allo-HCT). We assessed (1) citrulline, a surrogate marker of functional enterocyte mass and (2) circulating monocyte-derived macrophage reactivity, before allo-HCT. Forty-seven consecutive patients were prospectively included. Citrulline levels from blood samples withdrawn 30 days before transplantation were assessed using liquid chromatography combined with mass spectrometry. Monocyte-derived macrophages were isolated and incubated with 5 pathogen-associated molecular patterns: lipopolysaccharide, PamCSK4, flagellin, muramyl dipeptide, and curdlan. Multiplex fluorescent immunoassay on culture supernatant assessed levels of TNF-α, IL-1ß, IL-6, and IL-10 in each condition. Citrulline and cytokine levels were analyzed relatively to aGVHD onset within 100 days after transplantation. Citrulline levels were lower in the aGVHD group (n = 20) than in the no-aGVHD group (n = 27) (P = .005). Conversely, IL-6 and IL-10 were greater in aGVHD group, especially after curdlan stimulation (P = .005 and P = .012). Citrulline levels ≤20 µmol/L, IL-6 ≥ 332 pg/mL, and IL-10 ≥ 90 pg/mL were associated with aGVHD development (log-rank test, P = .002, P = .041, and P < .0001, respectively). In multivariate analysis, IL-10 ≥ 90 pg/mL, myeloablative conditioning, and citrulline ≤20 µmol/L remained independent factors of aGVHD development (hazard ratio [HR], 8.18, P = .0003; HR, 4.28, P = .006; and HR, 4.43, P = .01, respectively). Preconditioning citrulline and monocyte-derived macrophage reactivity are objective surrogate markers suitable to identify patients at risk of developing aGVHD. This work highlights the influence of preconditioning status in aGVHD development.

Role of TLR1, TLR2 and TLR6 in the modulation of intestinal inflammation and Candida albicans elimination.

BACKGROUND: Toll-like receptors (TLRs) are the major pattern recognition receptors that mediate sensing of a wide range of microorganisms. TLR2 forms heterodimers with either TLR1 or TLR6, broadening its ligand diversity against pathogens. TLR1, TLR2 and TLR6 have been implicated in the recognition of Candida albicans, an opportunistic fungal pathogen that colonizes the gastrointestinal tract. In this study, we explored whether the deficiency in TLR1, TLR2 or TLR6 impacts C. albicans colonization and inflammation-associated colonic injury in the dextran sulfate sodium (DSS)-induced colitis in mice. RESULTS: DSS treatment and C. albicans challenge induced greater weight loss, worse clinical signs of inflammation, higher histopathologic scores, and increased mortality rates in TLR1-/- and TLR2-/- mice when compared to TLR6-/- and wild-type mice. The number of C. albicans colonies in the stomach, colon and feces was decreased in TLR6-/- mice as compared to TLR2-/-, TLR1-/- and wild-type mice. Interestingly, the population of E. coli in colonic luminal contents, intestinal permeability to FITC-dextran and cytokine expression were significantly increased in TLR1-/- and TLR2-/- mice, while they were decreased in TLR6-/- mice. CONCLUSION: In contrast to TLR6, both TLR1 and TLR2 deficiencies increased intestinal inflammation, and the overgrowth of C. albicans and E. coli populations in the colitis model, suggesting the involvement of TLR1 and TLR2 in epithelial homeostasis, and a role of TLR6 in increasing intestinal inflammation in response to pathogen-sensing.

ß-1,2-Mannosyltransferases 1 and 3 Participate in Yeast and Hyphae O- and N-Linked Mannosylation and Alter Candida albicans Fitness During Infection.

ß-1,2-mannosylation of Candida albicans glycoconjugates has been investigated through the identification of enzymes involved in the addition of ß-1,2-oligomannosides (ß-Mans) to phosphopeptidomannan and phospholipomannan. ß-1,2-oligomannosides are supposed to have virulence properties that they confer to these glycoconjugates. In a previous study, we showed that cell wall mannoproteins (CWMPs) harbor ß-Mans in their O-mannosides; therefore, we analyzed their biosynthesis and impact on virulence. In this study, we demonstrate that O-mannans are heterogeneous and that α-mannosylated O-mannosides, which are biosynthesized by Mnt1 and Mnt2 α-1,2-mannosyltransferases, can be modified with ß-Mans but only at the nonreducing end of α-1,2-mannotriose. ß-1,2-mannosylation of this O-mannotriose depends on growth conditions, and it involves 2 ß-1,2-mannosyltransferases, Bmt1 and Bmt3. These Bmts are essential for ß-1,2-mannosylation of CWMPs and expression of ß-Mans on germ tubes. A bmt1Δ mutant and a mutant expressing no ß-Mans unexpectedly disseminated more in BALB/c mice, whereas they had neither attenuated nor enhanced virulence in C57BL/6 mice. In galectin (Gal)3 knockout mice, the reference strain was more virulent than in C57BL/6 mice, suggesting that the ß-Mans innate receptor Gal3 is involved in C. albicans fitness during infection.

Humoral immunity links Candida albicans infection and celiac disease.

OBJECTIVE: The protein Hwp1, expressed on the pathogenic phase of Candida albicans, presents sequence analogy with the gluten protein gliadin and is also a substrate for transglutaminase. This had led to the suggestion that C. albicans infection (CI) may be a triggering factor for Celiac disease (CeD) onset. We investigated cross-immune reactivity between CeD and CI. METHODS: Serum IgG levels against recombinant Hwp1 and serological markers of CeD were measured in 87 CeD patients, 41 CI patients, and 98 healthy controls (HC). IgA and IgG were also measured in 20 individuals from each of these groups using microchips sensitized with 38 peptides designed from the N-terminal of Hwp1. RESULTS: CI and CeD patients had higher levels of anti-Hwp1 (p=0.0005 and p=0.004) and anti-gliadin (p=0.002 and p=0.0009) antibodies than HC but there was no significant difference between CeD and CI patients. CeD and CI patients had higher levels of anti-transglutaminase IgA than HC (p=0.0001 and p=0.0039). During CI, the increase in anti-Hwp1 paralleled the increase in anti-gliadin antibodies. Microchip analysis showed that CeD patients were more reactive against some Hwp1 peptides than CI patients, and that some deamidated peptides were more reactive than their native analogs. Binding of IgG from CeD patients to Hwp1 peptides was inhibited by γIII gliadin peptides. CONCLUSIONS: Humoral cross-reactivity between Hwp1 and gliadin was observed during CeD and CI. Increased reactivity to Hwp1 deamidated peptide suggests that transglutaminase is involved in this interplay. These results support the hypothesis that CI may trigger CeD onset in genetically-susceptible individuals.

Candida albicans phospholipomannan: a sweet spot for controlling host response/inflammation.

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.

An immunological link between Candida albicans colonization and Crohn's disease.

The etiology of Crohn's disease (CD), an autoimmune, inflammatory bowel disease (IBD) which affects approximately one million people in Europe, is still unclear. Nevertheless, it is widely accepted that CD could result from an inappropriate inflammatory response to intestinal microorganisms in a genetically susceptible host. Most studies to date have concerned the involvement of bacteria in disease progression. In addition to bacteria, there appears to be a possible link between the commensal yeast Candida albicans and disease development. In this review, in an attempt to link the gut colonization process and the development of CD, we describe the different pathways that are involved in the progression of CD and in the host response to C. albicans, making the yeast a possible initiator of the inflammatory process observed in this IBD.

Deficient beta-mannosylation of Candida albicans phospholipomannan affects the proinflammatory response in macrophages.

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 ß-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 bmt6Δ 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-1ß 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-α 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-α production in macrophages. PLMs from C. albicans serotype B and from bmt6∆ mutant are not able to induce TNF-α 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.

Single-molecule analysis of the major glycopolymers of pathogenic and non-pathogenic yeast cells.

Most microbes are coated with carbohydrates that show remarkable structural variability and play a crucial role in mediating microbial-host interactions. Understanding the functions of cell wall glycoconjugates requires detailed knowledge of their molecular organization, diversity and heterogeneity. Here we use atomic force microscopy (AFM) with tips bearing specific probes (lectins, antibodies) to analyze the major glycopolymers of pathogenic and non-pathogenic yeast cells at molecular resolution. We show that non-ubiquitous ß-1,2-mannans are largely exposed on the surface of native cells from pathogenic Candida albicans and C. glabrata, the former species displaying the highest glycopolymer density and extensions. We also find that chitin, a major component of the inner layer of the yeast cell wall, is much more abundant in C. albicans. These differences in molecular properties, further supported by flow cytometry measurements, may play an important role in strengthening cell wall mechanics and immune interactions. This study demonstrates that single-molecule AFM, combined with immunological and fluorescence methods, is a powerful platform in fungal glycobiology for probing the density, distribution and extension of specific cell wall glycoconjugates. In nanomedicine, we anticipate that this new form of AFM-based nanoglycobiology will contribute to the development of sugar-based drugs, immunotherapeutics, vaccines and diagnostics.

Characterization of the recognition of Candida species by mannose-binding lectin using surface plasmon resonance.

The interaction of mannose-binding lectins (MBLs) with Candida albicans has been analyzed previously by microscopy and flow cytometry. We recently demonstrated that serum MBL levels vary during infection with Candida spp. and that serum MBLs are capable of interacting with yeast cell wall components. The aim of this study was to use, for the first time, surface plasmon resonance (SPR) technology to characterize the interaction between living label-free yeasts and non-mutated MBL purified from human serum. Our preliminary results demonstrate the robustness of this tool, which revealed specific and differential reactivities between the principal Candida spp. of medical interest. This model offers new perspectives as a tool for the characterization of yeast strains carrying mutations in gene coding for the mannosylation of fungal cell wall glycans and will enable better characterization of the interactions between C-lectins and glycan motifs expressed on the surface of yeasts.

The CARD8 p.C10X mutation associates with a low anti-glycans antibody response in patients with Crohn's disease.

BACKGROUND: Crohn's disease (CD) is associated with elevated anti-glycans antibody response in 60% of CD patients, and 25% of healthy first-degree relatives (HFDRs), suggesting a genetic influence for this humoral response. In mice, anti-glucan antibody response depends on the NLRP3 inflammasome. Here, we explored the effect of mutated CARD8, a component of the inflammasome, on anti-glycans antibody response in human. METHODS: The association between p.C10X mutation (rs2043211) of the CARD8 gene and the levels of anti-glycans antibody response was examined in 39 CD families. The family-based QTDT association test was used to test for the genetic association between CARD8 p.C10X mutation and anti-glycan antibodies in the pedigrees. The difference in antibody responses determined by ELISA was tested in a subgroup of CD probands (one per family) and in a subgroup of HFDRs using the Wilcoxon Kruskal Wallis non-parametric test. RESULTS: The QTDT familial transmission tests showed that the p.C10X mutation of CARD8 was significantly associated with lower levels of antibody to mannans and glucans but not chitin (p=0.024, p=0.0028 and p=0.577, for ASCA, ALCA and ACCA, respectively). These associations were independent of NOD2 and NOD1 genetic backgrounds. The p.C10X mutation significantly associated or displayed a trend toward lower ASCA and ALCA levels (p=0.038 and p=0.08, respectively) only in the subgroup of CD probands. Such associations were not significant for ACCA levels in both subgroups of CD probands and of HFDRs. CONCLUSION: Our results show that ASCA and ALCA but not ACCA levels are under the influence of CARD8 genotype. Alteration of CARD8, a component of inflammasome, is associated with lower levels of antibodies directed to mannans and glucans at least in CD patients.

Mannose-binding lectin levels and variation during invasive candidiasis.

The high morbi-mortality associated with invasive candidiasis (IC) is a persistent problem in hospitals. Mannose-binding lectin (MBL) plays a role in innate immunity through its interaction with mannosylated molecules of Candida albicans. A correlation between MBL deficiency and vulvovaginal candidiasis or peritonitis has been reported. We investigated circulating MBL levels and their evolution during the course of IC. Sixty-eight patients with proven IC, 82 hospitalized patients (HP) without evidence of infection, and 70 healthy subjects (HS) were studied in order to examine the relationship between serum MBL and IC. Serum MBL levels were measured by enzyme-linked immunosorbent assay (ELISA). MBL levels were significantly higher in IC patients than in HP and HS (p < 0.0001, p < 0.0055, respectively). A change in MBL concentrations was observed during the course of IC, with a dramatic decrease during the 2 days before positive blood culture sampling. This decrease was concomitant with the presence of high levels of circulating mannan (Mn). Like MBL levels, anti-mannan antibodies (AMn) increased after the mannanemia/blood culture period. These findings suggest a possible role of MBL during the early stage of IC. The mechanisms that regulate these observations in terms of effect and consequences on innate and adaptive immunity and the prognosis of IC require further investigation.

Members 5 and 6 of the Candida albicans BMT family encode enzymes acting specifically on ß-mannosylation of the phospholipomannan cell-wall glycosphingolipid.

A family of nine genes encoding proteins involved in the synthesis of ß-1,2 mannose adhesins of Candida albicans has been identified. Four of these genes, BMT1-4, encode enzymes acting stepwise to add ß-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 ß-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 ß-oligomannoside chain, respectively. Disruptions had no effect on sphingolipid biosynthesis and on PPM ß-mannosylation. ß-Mannose surface expression was not affected, confirming that ß-mannosylation is a process based on specificity of acceptor molecules, but liable to global regulation.

Variants of NOD1 and NOD2 genes display opposite associations with familial risk of Crohn's disease and anti-saccharomyces cerevisiae antibody levels.

BACKGROUND: NOD2 is involved in Crohn's disease (CD), but the role of NOD1 remains unclear. Anti-Saccharomyces cerevisiae antibodies (ASCA) are higher in CD patients and some of their relatives. Using family-based analyses we investigated the relationships between NOD2 mutations, NOD1 +32656 variant, and both the risk of CD and ASCA levels. We compared allelic frequencies between families with multiple CD cases (multiplex), those with one case of CD (simplex), and control families, searching for a gradient of at risk alleles according to the prevalence of the disease among families. METHODS: In all, 93 CD patients, 160 healthy relatives from 22 multiplex families, 22 CD patients and 81 healthy relatives from 22 simplex families, and 169 subjects from 27 control families were included in the study. ASCA levels were determined by enzyme-linked immunosorbent assay. NOD1 +32656, NOD2 R702W, G908R, and 1007fs were genotyped by polymerase chain reaction / restriction fragment length polymorphism. RESULTS: In family-based analyses NOD2 mutations and the NOD1 wildtype allele were associated with CD in multiplex families, with a synergetic effect when risk alleles of both genes were transmitted. Lower ASCA levels were strongly associated with the NOD1 variant allele. Simplex families had a lower frequency of the "at risk" +32656 allele than multiplex families. CONCLUSIONS: The +32656 variant was associated with low ASCA level and low risk of CD in multiplex families. NOD2 and NOD1 variants displayed antagonist effects on the risk of CD and ASCA level. A gradient of NOD1, NOD2 at-risk alleles was associated with the variable prevalence of CD in families.