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.

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.

Presence of Candida cell wall derived polysaccharides in the sera of intensive care unit patients: relation with candidaemia and Candida colonisation.

INTRODUCTION: Prompt diagnosis of candidaemia and invasive candidosis is crucial to the early initiation of antifungal therapy. The poor sensitivity of blood cultures (BCs) has led to the development of fungal glycan tests as a diagnostic adjunct. We analysed the performance of tests for the detection of circulating ß-D-1,3-glucan (BDG) and mannan in the intensive care unit (ICU) setting. METHODS: This retrospective, case-control study included 43 ICU patients with candidaemia and 67 controls, hospitalised on the same ward and assessed weekly for yeast colonisation with simultaneous serum sampling; 340 sera taken before and after positive BCs were available for the cases group and 203 for the controls. BDG and mannan levels were determined using the Fungitell® and Platelia™ Candida Ag tests, respectively. RESULTS: BDG was detected early in sera from cases patients but was also present in several sera from controls. Increasing the cut-off from 80 pg/mL to 350 pg/mL and 800 pg/mL resulted in sensitivity/specificity ratios of 0.97/0.31, 0.65/0.74, 0.30/0.86, respectively. Detection of mannan was more specific but lacked sensitivity. No obvious correlation was found between BDG and colonisation, but a trend existed between high colonisation and high BDG. Candidaemia relapses were associated with a rise in BDG and mannan but, in contrast to the transient nature of mannan, BDG persisted up to 7 weeks after positive BCs. CONCLUSION: A combination of mannan and BDG tests could be used to guide pre-emptive therapeutic decisions in ICU patients.

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.

Evaluation of MALDI-TOF mass spectrometry for the identification of medically-important yeasts in the clinical laboratories of Dijon and Lille hospitals.

Conventional identification (CI) of yeasts is based on morphological, biochemical and/or immunological methods. Matrix-assisted laser desorption/ionization - time of flight (MALDI-TOF or MT-MS) mass spectrometry has been proposed as a new method for the identification of microorganisms. This prospective study compared the performance of MT-MS and CI for the identification of yeasts isolated from clinical samples. Sequencing of the internal transcribed spacer (ITS) regions of ribosomal DNA was used as the reference method in the analysis of a total of 1207 yeast isolates. Concordance between MT-MS and CI was observed for 1105 isolates (91.5%), while 74 isolates (6.1%) were misidentified. Molecular identification revealed that 73 of these 74 isolates were identified correctly by MT-MS and CI correctly identified the last one. Concordance between the two techniques was excellent for the medically-important species (98-100%), including the identification of closely-related species (Candida albicans/C. dubliniensis; C. inconspicua/C. norvegensis; C. parapsilosis/C. metapsilosis/C. orthopsilosis). Only 2.3% of isolates belonging to C. famata, C. lambica and C. magnoliae or to Geotrichum spp. and Trichosporon spp. were not identified by MT-MS. This investigation highlights the potential of MT-MS-based yeast identification as a reliable, time and cost-efficient alternative to CI.

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.