Coincidence of antibodies against Hwp1 and ASCA, two distinct molecular targets of Candida albicans, reinforces the link between this fungal species and coeliac disease.

Candida albicans is an immunogen for anti-Saccharomyces cerevisiae antibodies (ASCA), a serological marker of Crohn's disease. ASCA has also been reported in other autoimmune diseases, including coeliac disease (CeD). A strong antibody response against Hwp1, a protein associated with invasive hyphal form of C. albicans which presents peptide sequence homologies with gliadin, has also been described in CeD. This observation supports the hypothesis that C. albicans hyphal transition in C. albicans may trigger CeD onset through a mechanism of molecular/antigenic mimicry. In this study, we assessed whether the anti-C. albicans oligomannose and anti-Hwp1 protein responses may be linked despite their different pathophysiological significance. The measurement of ASCA levels in a cohort of patients involved in our previous Hwp1 study showed a significant correlation between the two biomarkers. This new observation further reinforces the link between C. albicans and CeD.

From ASCA breakthrough in Crohn's disease and Candida albicans research to thirty years of investigations about their meaning in human health.

Anti-Saccharomyces cerevisiae antibodies (ASCA) are human antibodies that can be detected using an enzyme-linked immunosorbent assay involving a mannose polymer (mannan) extracted from the cell wall of the yeast S. cerevisiae. The ASCA test was developed in 1993 with the aim of differentiating the serological response in two forms of inflammatory bowel disease (IBD), Crohn's disease and ulcerative colitis. The test, which is based on the detection of anti-oligomannosidic antibodies, has been extensively performed worldwide and there have been hundreds of publications on ASCA. The earlier studies concerned the initial diagnostic indications of ASCA and investigations then extended to many human diseases, generally in association with studies on intestinal microorganisms and the interaction of the micro-mycobiome with the immune system. The more information accumulates, the more the mystery of the meaning of ASCA deepens. Many fundamental questions remain unanswered. These questions concern the heterogeneity of ASCA, the mechanisms of their generation and persistence, the existence of self-antigens, and the relationship between ASCA and inflammation and autoimmunity. This review aims to discuss the gray areas concerning the origin of ASCA from an analysis of the literature. Structured around glycobiology and the mannosylated antigens of S. cerevisiae and Candida albicans, this review will address these questions and will try to clarify some lines of thought. The importance of the questions relating to the pathophysiological significance of ASCA goes far beyond IBD, even though these diseases remain the preferred models for their understanding.

Antibodies as Models and Tools to Decipher Candida albicans Pathogenic Development: Review about a Unique Monoclonal Antibody Reacting with Immunomodulatory Adhesins.

Candidiasis, caused mainly by Candida albicans, a natural commensal of the human digestive tract and vagina, is the most common opportunistic fungal infection at the mucosal and systemic levels. Its high morbi-mortality rates have led to considerable research to identify the molecular mechanisms associated with the switch to pathogenic development and to diagnose this process as accurately as possible. Since the 1980s, the advent of monoclonal antibody (mAb) technology has led to significant progress in both interrelated fields. This linear review, intended to be didactic, was prompted by considering how, over several decades, a single mAb designated 5B2 contributed to the elucidation of the molecular mechanisms of pathogenesis based on ß-1,2-linked oligomannoside expression in Candida species. These contributions starting from the structural identification of the minimal epitope as a di-mannoside from the ß-1,2 series consisted then in the demonstration that it was shared by a large number of cell wall proteins differently anchored in the cell wall and the discovery of a cell wall glycoplipid shed by the yeast in contact of host cells, the phospholipomannan. Cytological analysis revealed an overall highly complex epitope expression at the cell surface concerning all growth phases and a patchy distribution resulting from the merging of cytoplasmic vesicles to plasmalema and further secretion through cell wall channels. On the host side, the mAb 5B2 led to identification of Galectin-3 as the human receptor dedicated to ß-mannosides and signal transduction pathways leading to cytokine secretion directing host immune responses. Clinical applications concerned in vivo imaging of Candida infectious foci, direct examination of clinical samples and detection of circulating serum antigens that complement the Platelia Ag test for an increased sensitivity of diagnosis. Finally, the most interesting character of mAb 5B2 is probably its ability to reveal C. albicans pathogenic behaviour in reacting specifically with vaginal secretions from women infected versus colonized by this species as well as to display higher reactivity with strains isolated in pathogenic circumstances or even linked to an unfavourable prognosis for systemic candidiasis. Together with a detailed referenced description of these studies, the review provides a complementary reading frame by listing the wide range of technologies involving mAb 5B2 over time, evidencing a practical robustness and versatility unique so far in the Candida field. Finally, the basic and clinical perspectives opened up by these studies are briefly discussed with regard to prospects for future applications of mAb 5B2 in current research challenges.

Identification of fungal trehalose for the diagnosis of invasive candidiasis by mass spectrometry.

The rapidity of the diagnosis of invasive candidiasis (IC) is crucial to allow the early introduction of antifungal therapy that dramatically increases the survival rate of patients. Early diagnosis is unfortunately often delayed because Candida blood culture, the gold standard diagnostic test, is positive in only 50% of cases of IC and takes several days to obtain this result. Complementary non-culture-based methods relying on the detection of Candida cell wall polysaccharides in the serum, ß-glucans and mannans, by enzymatic and immunological reagents have been successfully developed to allow a more efficient patients care. We have previously demonstrated that detection of circulating glycans by mass spectrometry could provide a reliable and cost-effective early diagnosis method called MS-DS for Mass Spectrometry of Di-Saccharide. Here, by comparing patient's sera and Candida albicans strains deficient in carbohydrates synthesis, we demonstrate that trehalose derived from fungal metabolism can be specifically targeted by MS-DS to allow early diagnosis. In particular, the use of C. albicans strains deficient in the synthesis of trehalose synthesizing enzymes Tps1 and Tps2 show that MS-DS results were correlated to the metabolism of trehalose. Finally, we demonstrate that the performance of the IC diagnosis can be significantly improved by using high resolution mass spectrometry, which opens new perspectives in the management of the disease.

Dissection of the anti-Candida albicans mannan immune response using synthetic oligomannosides reveals unique properties of ß-1,2 mannotriose protective epitopes.

Candida albicans mannan consists of a large repertoire of oligomannosides with different types of mannose linkages and chain lengths, which act as individual epitopes with more or less overlapping antibody specificities. Although anti-C. albicans mannan antibody levels are monitored for diagnostic purposes nothing is known about the qualitative distribution of these antibodies in terms of epitope specificity. We addressed this question using a bank of previously synthesized biotin sulfone tagged oligomannosides (BSTOs) of α and ß anomery complemented with a synthetic ß-mannotriose described as a protective epitope. The reactivity of these BSTOs was analyzed with IgM isotype monoclonal antibodies (MAbs) of known specificity, polyclonal sera from patients colonized or infected with C. albicans, and mannose binding lectin (MBL). Surface plasmon resonance (SPR) and multiple analyte profiling (MAP) were used. Both methods confirmed the usual reactivity of MAbs against either α or ß linkages, excepted for MAb B6.1 (protective epitope) reacting with ß-Man whereas the corresponding BSTO reacted with anti-α-Man. These results were confirmed in western blots with native C. albicans antigens. Using patients' sera in MAP, a significant correlation was observed between the detection of anti-mannan antibodies recognizing ß- and α-Man epitopes and detection of antibodies against ß-linked mannotriose suggesting that this epitope also reacts with human polyclonal antibodies of both specificities. By contrast, the reactivity of human sera with other α- and ß-linked BSTOs clearly differed according to their colonized or infected status. In these cases, the establishment of an α/ß ratio was extremely discriminant. Finally SPR with MBL, an important lectin of innate immunity to C. albicans, classically known to interact with α-mannose, also interacted in an unexpected way with the protective epitope. These cumulative data suggest that structure/activity investigations of the finely tuned C. albicans anti-mannose immune response are worthwhile to increase our basic knowledge and for translation in medicine.

A Pilot Clinical Study on Post-Operative Recurrence Provides Biological Clues for a Role of Candida Yeasts and Fluconazole in Crohn's Disease.

BACKGROUND AND AIMS: This study prompted by growing evidence of the relationship between the yeast Candida albicans and Crohn's disease (CD) was intended to assess the effect of a 6-month course of the antifungal fluconazole (FCZ) on post-operative recurrence of CD. METHODS: Mycological samples (mouth swabs and stools) and serum samples were collected from 28 CD patients randomized to receive either FCZ (n = 14) or placebo (n = 14) before surgical resection. Serological analysis focused on levels of calprotectin, anti-glycan antibodies, and antibody markers of C. albicans pathogenic transition. Levels of galectin-3 and mannose binding lectin (MBL) involved in C. albicans sensing and inflammation were also measured. RESULTS: 1, 2, 3, and 6 months after surgery, endoscopy revealed recurrence in 5/12 (41.7%) patients in the FCZ group and 5/9 (55.6%) in the placebo group, the small cohort preventing any clinical conclusions. In both groups, surgery was followed by a marked decrease in C. albicans colonization and biomarkers of C. albicans pathogenic transition decreased to non-significant levels. Anti-glycan antibodies also decreased but remained significant for CD. Galectin-3 and calprotectin also decreased. Conversely, MBL levels, which inversely correlated with anti-C. albicans antibodies before surgery, remained stable. Building biostatistical multivariate models to analyze he changes in antibody and lectin levels revealed a significant relationship between C. albicans and CD. CONCLUSION: Several combinations of biomarkers of adaptive and innate immunity targeting C. albicans were predictive of CD recurrence after surgery, with area under the curves (AUCs) as high as 0.86. FCZ had a positive effect on biomarkers evolution. ClinicalTrials.gov ID: NCT02997059, 19 December 2016. University Hospital Lille, Ministry of Health, France. Effect of Fluconazole on the Levels of Anti-Saccharomyces cerevisiae Antibodies (ASCA) After Surgical Resection for Crohn's Disease. Multicenter, Randomized, and Controlled in Two Parallel Groups Versus Placebo.

Risk factors for candidemia: a prospective matched case-control study.

BACKGROUND: Candidemia is an opportunistic infection associated with high morbidity and mortality in patients hospitalized both inside and outside intensive care units (ICUs). Identification of patients at risk is crucial to ensure prompt antifungal therapy. We sought to assess risk factors for candidemia and death, both outside and inside ICUs. METHODS: This prospective multicenter matched case-control study involved six teaching hospitals in Switzerland and France. Cases were defined by positive blood cultures for Candida sp. Controls were matched to cases using the following criteria: age, hospitalization ward, hospitalization duration, and, when applicable, type of surgery. One to three controls were enrolled by case. Risk factors were analyzed by univariate and multivariate conditional regression models, as a basis for a new scoring system to predict candidemia. RESULTS: One hundred ninety-two candidemic patients and 411 matched controls were included. Forty-four percent of included patients were hospitalized in ICUs, and 56% were hospitalized outside ICUs. Independent risk factors for candidemia in the ICU population included total parenteral nutrition, acute kidney injury, heart disease, prior septic shock, and exposure to aminoglycoside antibiotics. Independent risk factors for candidemia in the non-ICU population included central venous catheter, total parenteral nutrition, and exposure to glycopeptides and nitroimidazoles. The accuracy of the scores based on these risk factors is better in the ICU than in the non-ICU population. Independent risk factors for death in candidemic patients included septic shock, acute kidney injury, and the number of antibiotics to which patients were exposed before candidemia. DISCUSSION: While this study shows a role for known and novel risk factors for candidemia, it specifically highlights important differences in their distribution according to the hospital setting (ICU versus non-ICU). CONCLUSION: This study provides novel risk scores for candidemia accounting for the hospital setting and recent progress in patients' management strategies and fungal epidemiology.

Intravenous Immunoglobulin Therapy Eliminates Candida albicans and Maintains Intestinal Homeostasis in a Murine Model of Dextran Sulfate Sodium-Induced Colitis.

Intravenous immunoglobulin (IVIg) therapy has diverse anti-inflammatory and immunomodulatory effects and has been employed successfully in autoimmune and inflammatory diseases. The role of IVIg therapy in the modulation of intestinal inflammation and fungal elimination has not been yet investigated. We studied IVIg therapy in a murine model of dextran sulfate sodium (DSS)-induced colitis. Mice received a single oral inoculum of Candida albicans and were exposed to DSS treatment for 2 weeks to induce colitis. All mice received daily IVIg therapy starting on day 1 for 7 days. IVIg therapy not only prevented a loss of body weight caused by the development of colitis but also reduced the severity of intestinal inflammation, as determined by clinical and histological scores. IVIg treatment significantly reduced the Escherichia coli, Enterococcus faecalis, and C. albicans populations in mice. The beneficial effects of IVIg were associated with the suppression of inflammatory cytokine interleukin (IL)-6 and enhancement of IL-10 in the gut. IVIg therapy also led to an increased expression of peroxisome proliferator-activated receptor gamma (PPARγ), while toll-like receptor 4 (TLR-4) expression was reduced. IVIg treatment reduces intestinal inflammation in mice and eliminates C. albicans overgrowth from the gut in association with down-regulation of pro-inflammatory mediators combined with up-regulation of anti-inflammatory cytokines.

Evaluation of Mass Spectrometry-Based Detection of Panfungal Serum Disaccharide for Diagnosis of Invasive Fungal Infections: Results from a Collaborative Study Involving Six European Clinical Centers.

A mass spectrometry (MS) method that detects a serum disaccharide (DS) (MS-DS) was recently described for the diagnosis of invasive fungal infections (IFI). We carried out a European collaborative study to evaluate this assay. Patients with the following IFI were selected according to the availability of sera obtained at about the time that IFI was documented: invasive candidiasis (IC; n = 26 patients), invasive aspergillosis (IA; n = 19), and mucormycosis (MM; n = 23). Control sera originated from 20 neutropenic patients and 20 patients with bacteremia. MS-DS was carried out in blind manner for the diagnosis of IFI. A diagnosis of IC or IA was confirmed by detection of mannan (Man) or galactomannan (GM), respectively, associated with detection of (1,3)-ß-d-glucan (BDG) in both infections. MM was detected by quantitative real-time PCR (qPCR). All tests discriminated sera from patients with IC from sera from control subjects with bacteremia (P ≤ 0.0009). For IC, the MS-DS sensitivity and specificity were 51% and 87%, respectively. MS-DS complemented the high specificity of Man monitoring. All tests discriminated sera from IA patients from sera from neutropenic controls (P ≤ 0.0009). For IA, MS-DS sensitivity and specificity were 64% and 95%, respectively. Only 13/36 serum samples from patients with MM were concordant by MS-DS and qPCR (6 were positive, and 7 were negative); 14 were positive by MS-DS alone. qPCR and MS-DS made a similar contribution to the diagnosis of MM. In patients undergoing long-term monitoring, the persistent circulation of serum disaccharide was observed, whereas DNA was detected only for a short period after initiation of treatment. MS-DS has an important role to play in the early diagnosis of IFI. Its panfungal nature and complementarity with other tests may justify its use in the management of IFI.

Remodeling of the Candida glabrata cell wall in the gastrointestinal tract affects the gut microbiota and the immune response.

The gastrointestinal (GI) microbiota acts a natural barrier to the proliferation of opportunistic pathogens. Candida glabrata is an opportunistic yeast pathogen that has adapted to colonize all segments of the human GI tract. We observed an increase in Escherichia coli, Enterococcus faecalis, and Bacteroides vulgatus populations, and a decrease in Lactobacillus johnsonii, Bacteroides thetaiotaomicron, and Bifidobacterium animalis in mice with DSS-induced colitis. This reduction was more pronounced for L. johnsonii during C. glabrata overgrowth. In addition, C. glabrata overgrowth increased mouse mortality and inflammatory parameters, and modulated the expression of intestinal receptors and signaling pathways. The C. glabrata cell wall underwent various changes during the course of C. glabrata colonization, and showed a significant increase in chitin. C. glabrata deficient in chitin synthase-3 induced fewer inflammatory parameters than the parental strain during intestinal inflammation. Oral administration of chitin attenuated the impact of colitis, and reduced the number of aerobic bacteria and C. glabrata overgrowth, while chitinase-3-like protein-1 increased. This study provides evidence that inflammation of the gut alters the microbial balance and leads to C. glabrata cell wall remodeling through an increase in chitin, which is involved in promoting persistence of C. glabrata in the gut.

Evaluation of the (1,3)-ß-D-glucan assay for the diagnosis of neonatal invasive yeast infections.

Most newborns in the neonatal intensive care unit (NICU) are premature and at risk of invasive fungal infections (IFIs). Invasive yeast infections (IYIs) are the most common fungal infections in this population. These infections are difficult to diagnose because symptoms are nonspecific, and the sensitivity of blood cultures is low. The serum (1,3)-ß-D-glucan (BDG) assay provides a reliable marker for the diagnosis of IFIs in adults with haematological malignancies. We assessed the diagnostic performance of this test in neonatal IYIs and its contribution to the monitoring of antifungal treatment. A retrospective study was performed in the NICU of the French University Hospital of Amiens from February 2012 to February 2014. Forty-seven neonates (33 males, 14 females) with a median gestational age of 30 weeks (IQR: 27-31) and median birth weight of 1200 g (IQR: 968-1700) were included and divided into three groups: 21 control neonates (CTRL), 20 neonates with probable IYI (PB), and six with proven IYI (PV). Median BDG levels were significantly higher in the global IYI group (PB + PV): 149 pg/ml (IQR: 85-364) vs. CTRL group: 39 pg/ml (IQR: 20-94) (P < .001). The optimal cut-off was 106 pg/ml (sensitivity 61.5%; specificity 81%). BDG levels decreased with antifungal treatment. BDG was detectable in cerebrospinal fluid, but the interest of this for diagnostic purposes remains unclear. Our results suggest that the BDG assay may be useful for the early identification of IYIs in neonates and for monitoring antifungal therapy efficacy.

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.

Application of Mass Spectrometry Technology to Early Diagnosis of Invasive Fungal Infections.

We recently developed a mass spectrometry (MS) procedure based on the detection of a serum disaccharide (MS-DS) in patients with invasive candidiasis (IC). Here, we compare the performance of MS-DS for the diagnosis of IC, invasive aspergillosis (IA), and mucormycosis (MM) with those of commercially available antigen detection tests. This retrospective study included 48 patients (23 IC patients [74 serum samples], 15 IA patients [40 serum samples], and 10 MM patients [15 serum samples]) and 49 appropriate controls (102 serum samples). MS-DS, mannan (Mnn), galactomannan (GM), and (1,3)-ß-d-glucan (BDG) were detected by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) MS, Platelia, and Fungitell assays, respectively. For IC, the sensitivity and specificity of the MS-DS index, BDG detection, and Mnn detection were 62% and 84%, 82% and 60%, and 33% and 94% per serum sample and 83% and 69%, 96% and 31%, and 39% and 86% per patient, respectively. For IA, the corresponding values in comparison to BDG and GM detection were 83% and 81%, 62% and 95%, and 62% and 100% per serum sample and 93% and 76%, 87% and 90%, and 93% and 100% per patient, respectively. Nine of the 10 MM patients had a positive MS-DS result. MS-DS gave an early diagnosis in IC (73% positivity before blood culture), IA (positive before GM detection in six patients), and MM (positivity mainly preceded the date of diagnosis) patients. For IC, persisting MS-DS was associated with a poor prognosis. The different biomarkers were rarely detected simultaneously, suggesting different kinetics of release and clearance. For IA, MS-DS provided better complementation to GM monitoring than BDG monitoring. MS-DS detects panfungal molecules circulating during invasive fungal infections. The performance of MS-DS compared favorably with those of biological tests currently recommended for monitoring at-risk patients. Further validation of this test in multicenter studies is required.

Polymorphisms in the Mannose-Binding Lectin Gene are Associated with Defective Mannose-Binding Lectin Functional Activity in Crohn's Disease Patients.

Mannose-binding lectin, together with mannose-associated serine proteases, activates the lectin pathway of the complement system and subsequent inflammatory mechanisms. An association between mannose-binding lectin deficiency and anti-Saccharomyces cerevisiae antibody levels is observed in Crohn's disease and this deficiency is frequently associated with a severe Crohn's disease phenotype. In the present study, we assessed the relationship between serum concentrations of mannose-binding lectin, mannose-binding lectin functional activity, MBL2 and NOD2 polymorphisms, anti-S. cerevisiae antibody levels and clinical Crohn's disease phenotype in 69 Crohn's disease patients and 30 age- and sex-matched healthy controls. The results show that the MBL2 variant rs5030737 at codon 52 was associated with a low level of mannose-binding lectin and impaired mannose-binding lectin-mannose-associated serine protease (MBL-MASP) functional activity in Crohn's disease patients. This MBL2 variant was also associated with a higher level of anti-S. cerevisiae antibodies. In addition, the NOD2 variant rs2066844, which is associated with susceptibility to Crohn's disease, was significantly correlated with an impairment in MBL-MASP functional activity. These results provide evidence that Crohn's disease patients have an impairment in MBL-MASP functional activity and that this defect is associated with MBL2 and NOD2 variants.

Short fungal fractions of ß-1,3 glucans affect platelet activation.

Platelets are capable of binding, aggregating, and internalizing microorganisms, which enhances the elimination of pathogens from the blood. The yeast Candida albicans is a pathobiont causing life-threatening invasive infections. Its cell wall contains ß-1,3 glucans that are known to trigger a wide range of host cell activities and to circulate during infection. We studied the effect of ß-1,3 glucan fractions (BGFs) consisting of diglucosides (Glc2), tetraglucosides (Glc4), and pentaglucosides (Glc5) on human platelets, their mechanisms of action, and their possible impact on host defenses. The effect of BGFs on the coagulation process was determined by measuring thrombin generation. Platelets pretreated with BGFs were analyzed in terms of activation, receptor expression, aggregation, and adhesion to neutrophils and to C. albicans The results show that BGFs affected the endogenous thrombin potential in a concentration-dependent manner. For platelet activation, BGFs at a low concentration (2 µmol/l) reduced ATP release and prevented the phosphorylation of protein kinase C. BGFs diminished the expression of P-selectin and the activation of αIIbß3 BGFs decreased platelet aggregation and the interaction between thrombin-stimulated platelets and neutrophils, fibrinogen, and C. albicans GLc5 decreased ATP release and TGF-ß1 production in response to TLR4 upregulation in thrombin-stimulated platelets, but TLR4 blockage abolished the effect of BGFs on platelets. This study provides evidence that fungal pentaglucosides modulate platelet activity mediated via TLR4 stimulation and reduce platelet-neutrophil interaction.

Candida albicans ß-1,2 mannosyl transferase Bmt3: Preparation and evaluation of a ß (1,2), α (1,2)-tetramannosyl fluorescent substrate.

We describe for the first time the chemical synthesis of a tetramannoside, containing both α (1→2) and ß (1→2) linkages. Dodecylthio (lauryl) glycosides were prepared from odorless dodecyl thiol and used as donors for the glycosylation steps. This tetramannoside, was coupled to a mantyl group, and revealed to be a perfect substrate of ß-mannosyltransferase Bmt3, confirming the proposed specificity and allowing the preparation of a pentamannoside sequence (ß Man (1,2) ß Man (1,2) α Man (1,2) α Man (1,2) α Man) usable as a novel substrate for further elongation studies.

Candida albicans ß-1,2-mannosyltransferase Bmt3 prompts the elongation of the cell-wall phosphopeptidomannan.

ß-1,2-Linked mannosides are expressed on numerous cell-wall glycoconjugates of the opportunistic pathogen yeast Candida albicans. Several studies evidenced their implication in the host-pathogen interaction and virulence mechanisms. In the present study, we characterized the in vitro activity of CaBmt3, a ß-1,2-mannosyltransferase involved in the elongation of ß-1,2-oligomannosides oligomers onto the cell-wall polymannosylated N-glycans. A recombinant soluble enzyme Bmt3p was produced in Pichia pastoris and its enzyme activity was investigated using natural and synthetic oligomannosides as potential acceptor substrates. Bmt3p was shown to exhibit an exquisite enzymatic specificity by adding a single terminal ß-mannosyl residue to α-1,2-linked oligomannosides capped by a Manß1-2Man motif. Furthermore, we demonstrated that the previously identified CaBmt1 and CaBmt3 efficiently act together to generate Manß1-2Manß1-2[Manα1-2]n sequence from α-1,2-linked oligomannosides onto exogenous and endogenous substrates.