Identification and characterization of glycoproteins on the spore surface of Clostridium difficile

In this study, we identify a major spore surface protein, BclA, and provide evidence that this protein is glycosylated. Following extraction of the spore surface, solubilized proteins were separated by one-dimensional PAGE and stained with glycostain to reveal a reactive high-molecular-mass region of approximately 600 kDa. Tandem mass spectrometry analysis of in-gel digests showed this band to contain peptides corresponding to a putative exosporangial glycoprotein (BclA3) and identified a number of glycopeptides modified with multiple N-acetyl hexosamine moieties and, in some cases, capped with novel glycans. In addition, we demonstrate that the glycosyltransferase gene sgtA (gene CD3350 in strain 630 and CDR3194 in strain R20291), which is located immediately upstream of the bclA3 homolog, is involved in the glycosylation of the spore surface, and is cotranscribed with bclA3. The presence of anti-β-O-GlcNAc-reactive material was demonstrated on the surface of spores by immunofluorescence and in surface extracts by Western blotting, although each strain produced a distinct pattern of reactivity. Reactivity of the spore surface with the anti-β-O-GlcNAc antibody was abolished in the 630 and R20291 glycosyltransferase mutant strains, while complementation with a wild-type copy of the gene restored the β-O-GlcNAc reactivity. Phenotypic testing of R20291 glycosyltransferase mutant spores revealed no significant change in sensitivity to ethanol or lysozyme. However, a change in the resistance to heat of R20291 glycosyltransferase mutant spores compared to R20291 spores was observed, as was the ability to adhere to and be internalized by macrophages

In vitro production and immunogenicity of a Clostridium difficile spore-specific BclA3 glycopeptide conjugate vaccine

The BclA3 glycoprotein is a major component of the exosporangial layer of Clostridium difficile spores and in this study we demonstrate that this glycoprotein is a major spore surface associated antigen. Here, we confirm the role of SgtA glycosyltransferase (SgtA GT) in BclA3 glycosylation and recapitulate this process by expressing and purifying SgtA GT fused to MalE, the maltose binding protein from Escherichia coli. In vitro assays using the recombinant enzyme and BclA3 synthetic peptides demonstrated that SgtA GT was responsible for the addition of β-O-linked GlcNAc to threonine residues of each synthetic peptide. These peptide sequences were selected from the central, collagen repeat region of the BclA3 protein. Following optimization of SgtA GT activity, we generated sufficient glycopeptide (10 mg) to allow conjugation to KLH (keyhole limpet hemocyanin) protein. Glycosylated and unglycosylated versions of these conjugates were then used as antigens to immunize rabbits and mice. Immune responses to each of the conjugates were examined by Enzyme Linked Immunosorbent Assay ELISA. Additionally, the BclA3 conjugated peptide and glycopeptide were used as antigens in an ELISA assay with serum raised against formalin-killed spores. Only the glycopeptide was recognized by anti-spore polyclonal immune serum demonstrating that the glycan moiety is a predominant spore-associated surface antigen. To determine whether antibodies to these peptides could modify persistence of spores within the gut, animals immunized intranasally with either the KLH-glycopeptide or KLH-peptide conjugate in the presence of cholera toxin, were challenged with R20291 spores. Although specific antibodies were raised to both antigens, immunization did not provide any protection against acute or recurrent disease

Targeted Metabolomics Analysis of Campylobacter coli VC167 Reveals Legionaminic Acid Derivatives as Novel Flagellar Glycans

Glycosylation of Campylobacter flagellin is required for the biogenesis of a functional flagella filament. Recently, we used a targeted metabolomics approach using mass spectrometry and NMR to identify changes in the metabolic profile of wild type and mutants in the flagellar glycosylation locus, characterize novel metabolites, and assign function to genes to define the pseudaminic acid biosynthetic pathway in Campylobacter jejuni 81-176 (McNally, D. J., Hui, J. P., Aubry, A. J., Mui, K. K., Guerry, P., Brisson, J. R., Logan, S. M., and Soo, E. C. (2006) J. Biol. Chem. 281, 18489-18498). In this study, we use a similar approach to further define the glycome and metabolomic complement of nucleotide-activated sugars in Campylobacter coli VC167. Herein we demonstrate that, in addition to CMP-pseudaminic acid, C. coli VC167 also produces two structurally distinct nucleotide-activated nonulosonate sugars that were observed as negative ions at m/z 637 and m/z 651 (CMP-315 and CMP-329). Hydrophilic interaction liquid chromatography-mass spectrometry yielded suitable amounts of the pure sugar nucleotides for NMR spectroscopy using a cold probe. Structural analysis in conjunction with molecular modeling identified the sugar moieties as acetamidino and N-methylacetimidoyl derivatives of legionaminic acid (Leg5Am7Ac and Leg5AmNMe7Ac). Targeted metabolomic analyses of isogenic mutants established a role for the ptmA-F genes and defined two new ptm genes in this locus as legionaminic acid biosynthetic enzymes. This is the first report of legionaminic acid in Campylobacter sp. and the first report of legionaminic acid derivatives as modifications on a protein

Candidaemia and antifungal therapy in a French University Hospital: rough trends over a decade and possible links

BACKGROUND: Evidence for an increased prevalence of candidaemia and for high associated mortality in the 1990s led to a number of different recommendations concerning the management of at risk patients as well as an increase in the availability and prescription of new antifungal agents. The aim of this study was to parallel in our hospital candidemia incidence with the nature of prescribed antifungal drugs between 1993 and 2003. METHODS: During this 10-year period we reviewed all cases of candidemia, and collected all the data about annual consumption of prescribed antifungal drugs RESULTS: Our centralised clinical mycology laboratory isolates and identifies all yeasts grown from blood cultures obtained from a 3300 bed teaching hospital. Between 1993 and 2003, 430 blood yeast isolates were identified. Examination of the trends in isolation revealed a clear decrease in number of yeast isolates recovered between 1995–2000, whereas the number of positive blood cultures in 2003 rose to 1993 levels. The relative prevalence of Candida albicans and C. glabrata was similar in 1993 and 2003 in contrast to the period 1995–2000 where an increased prevalence of C. glabrata was observed. When these quantitative and qualitative data were compared to the amount and type of antifungal agents prescribed during the same period (annual mean defined daily dose: 2662741; annual mean cost: 615629 €) a single correlation was found between the decrease in number of yeast isolates, the increased prevalence of C. glabrata and the high level of prescription of fluconazole at prophylactic doses between 1995–2000. CONCLUSION: Between 1993 and 2000, the number of cases of candidemia halved, with an increase of C. glabrata prevalence. These findings were probably linked to the use of Fluconazole prophylaxis. Although it is not possible to make any recommendations from this data the information is nevertheless interesting and may have considerable implications with the introduction of new antifungal drugs

Phenotypic spectrum of fetal Smith-Lemli-Opitz syndrome.

International audienceThe Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive multiple congenital malformation syndrome caused by dehydrocholesterol reductase deficiency. The diagnosis is confirmed by high 7- and secondarily 8-dehydrocholesterol levels in plasma and tissues and/or by detection of biallelic mutations in the DHCR7 gene. The phenotypic spectrum of SLOS is broad, ranging from a mild phenotype combining subtle physical anomalies with behavioral and learning problems, to a perinatally lethal multiple malformations syndrome. The fetal phenotype of SLOS has been poorly described in the literature. We report a series of 10 fetuses with molecularly proven SLOS. Even in young fetuses, the facial dysmorphism appears characteristic. Genital abnormalities are rare in 46,XX subjects. Gonadal differentiation appears histologically normal and in agreement with the chromosomal sex, contrary to what has been previously stated. We observed some previously unreported anomalies: ulnar hypoplasia, vertebral segmentation anomalies, congenital pulmonary adenomatoid malformation, fused lungs, gastroschisis, holomyelia and hypothalamic hamartoma. This latter malformation proves that SLOS phenotypically overlaps with Pallister-Hall syndrome which remains clinically a major differential diagnosis of SLOS

CAP-Gly proteins contribute to microtubule-dependent trafficking via interactions with the C-terminal aromatic residue of α-tubulin

International audienc

Occurrence of a nontypable Helicobacter pylori strain lacking Lewis blood group O antigens and DD-heptoglycan: evidence for the role of the core a 1,6-glucan chain in colonization.

Peer reviewed: YesNRC publication: Ye

Thermophysical and bionotox properties of solvo-surfactants based on ethylene oxide, propylene oxide and glycerol

Thermophys. and bionotox properties of a new class of natural solvo-surfactants, glycerol 1-monoethers, were investigated in comparison with widespread but harmful glycol ethers. Vapor pressures and heats of vaporization were measured between 25 °C and 50 °C, and calcd. thanks to two group contribution methods. Evapn. rates and Hansen parameters, evaluated from TGA measurements and group contributions resp., were compared as well. Bionotox properties, i.e. cytotoxicity, irritating power and biodegradability, were evaluated exptl. Glycerol 1-monoethers turned out to be less volatile than glycol derivs., but contrary to the latter they will not be considered as VOCs. Toxicities and irritating powers are equiv. and increase with increasing alkyl chain length, i.e. with increasing amphiphilicity. Glycerol ethers are degradable at lower concns. compared to glycol compds., which is related to their higher interfacial activity