A novel mouse model of Campylobacter jejuni enteropathy and diarrhea

Campylobacter infections are among the leading bacterial causes of diarrhea and of ‘environmental enteropathy’ (EE) and growth failure worldwide. However, the lack of an inexpensive small animal model of enteric disease with Campylobacter has been a major limitation for understanding its pathogenesis, interventions or vaccine development. We describe a robust standard mouse model that can exhibit reproducible bloody diarrhea or growth failure, depending on the zinc or protein deficient diet and on antibiotic alteration of normal microbiota prior to infection. Zinc deficiency and the use of antibiotics create a niche for Campylobacter infection to establish by narrowing the metabolic flexibility of these mice for pathogen clearance and by promoting intestinal and systemic inflammation. Several biomarkers and intestinal pathology in this model also mimic those seen in human disease. This model provides a novel tool to test specific hypotheses regarding disease pathogenesis as well as vaccine development that is currently in progress

Strong-coupling induced damping of spin-echo modulations in magic-angle-spinning NMR:implications for J coupling measurements in disordered solids

Abstract In the context of improving J coupling measurements in disordered solids, strong coupling effects have been investigated in the spin-echo and refocused INADEQUATE spin-echo (REINE) modulations of three- and four-spin systems under magic-angle spinning (MAS), using density matrix simulations and solid-state NMR experiments on a cadmium phosphate glass. Analytical models are developed for the different modulation regimes, which are shown to be distinguishable in practice using Akaike’s information criterion. REINE modulations are shown to be free of the damping that occurs for spin-echo modulations when the observed spin has the same isotropic chemical shift as its neighbour. Damping also occurs when the observed spin is bonded to a strongly-coupled pair. For mid-chain units, the presence of both direct and relayed damping makes both REINE and spin-echo modulations impossible to interpret quantitatively. We nonetheless outline how a qualitative comparison of the modulation curves can provide valuable information on disordered networks, possibly also pertaining to dynamic effects therein

Human papillomavirus (HPV) contamination of gynaecological equipment.

OBJECTIVE: The gynaecological environment can become contaminated by human papillomavirus (HPV) from healthcare workers' hands and gloves. This study aimed to assess the presence of HPV on frequently used equipment in gynaecological practice. METHODS: In this cross-sectional study, 179 samples were taken from fomites (glove box, lamp of a gynaecological chair, gel tubes for ultrasound, colposcope and speculum) in two university hospitals and in four gynaecological private practices. Samples were collected with phosphate-buffered saline-humidified polyester swabs according to a standardised pattern, and conducted twice per day for 2 days. The samples were analysed by a semiquantitative real-time PCR. Statistical analysis was performed using Pearson's χ(2) test and multivariate regression analysis. RESULTS: Thirty-two (18%) HPV-positive samples were found. When centres were compared, there was a higher risk of HPV contamination in gynaecological private practices compared with hospitals (OR 2.69, 95% CI 1.06 to 6.86). Overall, there was no difference in the risk of contamination with respect to the time of day (OR 1.79, 95% CI 0.68 to 4.69). When objects were compared, the colposcope had the highest risk of contamination (OR 3.02, 95% CI 0.86 to 10.57). CONCLUSIONS: Gynaecological equipment and surfaces are contaminated by HPV despite routine cleaning. While there is no evidence that contaminated surfaces carry infectious viruses, our results demonstrate the need for strategies to prevent HPV contamination. These strategies, based on health providers' education, should lead to well-established cleaning protocols, adapted to gynaecological rooms, aimed at eliminating HPV material

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

Preparation, structural characterisation and antibacterial properties of Ga-doped sol-gel phosphate-based glass

A sol-gel preparation of Ga-doped phosphate-based glass with potential application in antimicrobial devices has been developed. Samples of composition (CaO)(0.30)(Na2O)(0.20-x) (Ga2O3) (x) (P2O5)(0.50) where x = 0 and 0.03 were prepared, and the structure and properties of the gallium-doped sample compared with those of the sample containing no gallium. Analysis of the P-31 MAS NMR data demonstrated that addition of gallium to the sol-gel reaction increases the connectivity of the phosphate network at the expense of hydroxyl groups. This premise is supported by the results of the elemental analysis, which showed that the gallium-free sample contains significantly more hydrogen and by FTIR spectroscopy, which revealed a higher concentration of -OH groups in that sample. Ga K-edge extended X-ray absorption fine structure and X-ray absorption near-edge structure data revealed that the gallium ions are coordinated by six oxygen atoms. In agreement with the X-ray absorption data, the high-energy XRD results also suggest that the Ga3+ ions are octahedrally coordinated with respect to oxygen. Antimicrobial studies demonstrated that the sample containing Ga3+ ions had significant activity against Staphylococcus aureus compared to the control

Sialylation of campylobacter jejuni lipo-oligosaccharides: impact on phagocytosis and cytokine production in mice

<p>Background: Guillain-Barré syndrome (GBS) is a post-infectious polyradiculoneuropathy, frequently associated with antecedent Campylobacter jejuni (C. jejuni) infection. The presence of sialic acid on C. jejuni lipo-oligosaccharide (LOS) is considered a risk factor for development of GBS as it crucially determines the structural homology between LOS and gangliosides, explaining the induction of cross-reactive neurotoxic antibodies. Sialylated C. jejuni are recognised by TLR4 and sialoadhesin; however, the functional implications of these interactions in vivo are unknown.</p> <p>Methodology/Principal Findings: In this study we investigated the effects of bacterial sialylation on phagocytosis and cytokine secretion by mouse myeloid cells in vitro and in vivo. Using fluorescently labelled GM1a/GD1a ganglioside-mimicking C. jejuni strains and corresponding (Cst-II-mutant) control strains lacking sialic acid, we show that sialylated C. jejuni was more efficiently phagocytosed in vitro by BM-MΦ, but not by BM-DC. In addition, LOS sialylation increased the production of IL-10, IL-6 and IFN-β by both BM-MΦ and BM-DC. Subsequent in vivo experiments revealed that sialylation augmented the deposition of fluorescent bacteria in splenic DC, but not macrophages. In addition, sialylation significantly amplified the production of type I interferons, which was independent of pDC.</p> <p>Conclusions/Significance: These results identify novel immune stimulatory effects of C. jejuni sialylation, which may be important in inducing cross-reactive humoral responses that cause GBS</p&gt

Analysis of glycoprotein processing in the endoplasmic reticulum using synthetic oligosaccharides

Protein quality control (QC) in the endoplasmic reticulum (ER) comprises many steps, including folding and transport of nascent proteins as well as degradation of misfolded proteins. Recent studies have revealed that high-mannose-type glycans play a pivotal role in the QC process. To gain knowledge about the molecular basis of this process with well-defined homogeneous compounds, we achieved a convergent synthesis of high-mannose-type glycans and their functionalized derivatives. We focused on analyses of UDP-Glc: glycoprotein glucosyltransferase (UGGT) and ER Glucosidase II, which play crucial roles in glycoprotein QC; however, their specificities remain unclear. In addition, we established an in vitro assay system mimicking the in vivo condition which is highly crowded because of the presence of various biomacromolecules

High throughput method for analysis of repeat number for 28 phase variable loci of C. jejuni strain NCTC11168

Mutations in simple sequence repeat tracts are a major mechanism of phase variation in several bacterial species including Campylobacter jejuni. Changes in repeat number of tracts located within the reading frame can produce a high frequency of reversible switches in gene expression between ON and OFF states. The genome of C. jejuni strain NCTC11168 contains 29 loci with polyG/polyC tracts of seven or more repeats. This protocol outlines a method for rapidly determining ON/OFF states of these 28 phase-variable loci in a large number of individual colonies. The method combines a series of multiplex PCR assays with a GeneScan assay and automated extraction of tract length, repeat number and expression state. This high throughput, multiplex assay has utility for detecting shifts in phase variation states within and between populations over time and for exploring the effects of phase variation on adaptation to differing selective pressures. An important output of this assay is combinatorial expression states that cannot be determined by other methods. This method can be adapted to analysis of phase variation in other C. jejuni strains and in a diverse range of bacterial species

Phylogenetic and Molecular Characterization of a 23S Ribosomal-Rna Gene Positions the Genus Campylobacter in the Epsilon-Subdivision of the Proteobacteria and Shows That the Presence of Transcribed Spacers Is Common in Campylobacter Spp

The nucleotide sequence of a 23S rRNA gene of Campylobacter coli VC167 was determined. The primary sequence of the C. coli 23S rRNA was deduced, and a secondary-structure model was constructed. Comparison with Escherichia coli 23S rRNA showed a major difference in the C. coli rRNA at approximately position 1170 (E. coli numbering) in the form of an extra sequence block approximately 147 bp long. PCR analysis of 31 other strains of C. coli and C. jejuni showed that 69% carried a transcribed spacer of either ca, 147 or ca. 37 bp. Comparison of all sequenced Campylobacter transcribed spacers showed that the Campylobacter inserts were related in sequence and percent G+C content. All Campylobacter strains carrying transcribed spacers in their 23S rRNA genes produced fragmented 23S rRNAs. Other strains which produced unfragmented 23S rRNAs did not appear to carry transcribed spacers at this position in their 23S rRNA genes. At the 1850 region (E. coli numbering), Campylobacter 23S rRNA displayed a base pairing signature most like that of the beta and gamma subdivisions of the class Proteobacteria, but in the 270 region, Campylobacter 23S rRNA displayed a helix signature which distinguished it from the alpha, beta, and gamma subdivisions. Phylogenetic analysis comparing C. coli VC167 23S rRNA and a C. jejuni TGH9011 (ATCC 43431) 23S rRNA with 53 other completely sequenced (eu)bacterial 23S rRNAs showed that the two campylobacters form a sister group to the alpha, beta, and gamma proteobacterial 23S rRNAs, a positioning consistent with the idea that the genus Campylobacter belongs to the epsilon subdivision of the class Proteobacteria