Characterization of Pro-Inflammatory Flagellin Proteins Produced by Lactobacillus ruminis and Related Motile Lactobacilli

peer-reviewedLactobacillus ruminis is one of at least twelve motile but poorly characterized species found in the genus Lactobacillus. Of these, only L. ruminis has been isolated from mammals, and this species may be considered as an autochthonous member of the gastrointestinal microbiota of humans, pigs and cows. Nine L. ruminis strains were investigated here to elucidate the biochemistry and genetics of Lactobacillus motility. Six strains isolated from humans were non-motile while three bovine isolates were motile. A complete set of flagellum biogenesis genes was annotated in the sequenced genomes of two strains, ATCC25644 (human isolate) and ATCC27782 (bovine isolate), but only the latter strain produced flagella. Comparison of the L. ruminis and L. mali DSM20444T motility loci showed that their genetic content and gene-order were broadly similar, although the L. mali motility locus was interrupted by an 11.8 Kb region encoding rhamnose utilization genes that is absent from the L. ruminis motility locus. Phylogenetic analysis of 39 motile bacteria indicated that Lactobacillus motility genes were most closely related to those of motile carnobacteria and enterococci. Transcriptome analysis revealed that motility genes were transcribed at a significantly higher level in motile L. ruminis ATCC27782 than in non-motile ATCC25644. Flagellin proteins were isolated from L. ruminis ATCC27782 and from three other Lactobacillus species, while recombinant flagellin of aflagellate L. ruminis ATCC25644 was expressed and purified from E. coli. These native and recombinant Lactobacillus flagellins, and also flagellate L. ruminis cells, triggered interleukin-8 production in cultured human intestinal epithelial cells in a manner suppressed by short interfering RNA directed against Toll-Like Receptor 5. This study provides genetic, transcriptomic, phylogenetic and immunological insights into the trait of flagellum-mediated motility in the lactobacilli.This work was supported by a Principal Investigator Award (07/IN.1/B1780) from Science Foundation Ireland to PWOT. BAN was the recipient of an Embark studentship from the Irish Research Council for Science Engineering and Technology. TD and KN were supported by the Alimentary Pharmabiotic Centre, funded by Science Foundation Ireland

Phylogenomics and comparative genomics of Lactobacillus salivarius, a mammalian gut commensal

The genus Lactobacillus is a diverse group with a combined species count of over 200. They are the largest group within the lactic acid bacteria and one of the most important bacterial groups involved in food microbiology and human nutrition because of their fermentative and probiotic properties. Lactobacillus salivarius, a species commonly isolated from the gastrointestinal tract of humans and animals, has been described as having potential probiotic properties and results of previous studies have revealed considerable functional diversity existing on both the chromosomes and plasmids. Our study consists of comparative genomic analyses of the functional and phylogenomic diversity of 42 genomes of strains of L . salivarius using bioinformatic techniques. The main aim of the study was to describe intra-species diversity and to determine how this diversity is spread across the replicons. We found that multiple phylogenomic and non-phylogenomic methods used for reconstructing trees all converge on similar tree topologies, showing that different metrics largely agree on the evolutionary history of the species. The greatest genomic variation lies on the small plasmids, followed by the repA-type circular megaplasmid, with the chromosome varying least of all. Additionally, the presence of extra linear and circular megaplasmids is noted in several strains, while small plasmids are not always present. Glycosyl hydrolases, bacteriocins and proteases vary considerably on all replicons while two exopolysaccharide clusters and several clustered regularly interspaced short palindromic repeats-associated systems show a lot of variation on the chromosome. Overall, despite its reputation as a mammalian gastrointestinal tract specialist, the intra-specific variation of L. salivarius reveals potential strain-dependant effects on human health

Pro-inflammatory flagellin proteins of prevalent motile commensal bacteria are variably abundant in the intestinal microbiome of elderly humans

Peer reviewedPublisher PD

On modeling and measuring viscoelasticity with dynamic Atomic Force Microscopy

The interaction between a rapidly oscillating atomic force microscope tip and a soft material surface is described using both elastic and viscous forces with a moving surface model. We derive the simplest form of this model, motivating it as a way to capture the impact dynamics of the tip and sample with an interaction consisting of two components: interfacial or surface force, and bulk or volumetric force. Analytic solutions to the piece-wise linear model identify characteristic time constants, providing a physical explanation of the hysteresis observed in the measured dynamic force quadrature curves. Numerical simulation is used to fit the model to experimental data and excellent agreement is found with a variety of different samples. The model parameters form a dimensionless impact-rheology factor, giving a quantitative physical number to characterize a viscoelastic surface that does not depend on the tip shape or cantilever frequency.Comment: 13 pages, 7 figure

Tumour-associated and non-tumour-associated microbiota in colorectal cancer

Objective: A signature that unifies the colorectal cancer (CRC) microbiota across multiple studies has not been identified. In addition to methodological variance, heterogeneity may be caused by both microbial and host response differences, which was addressed in this study. Design: We prospectively studied the colonic microbiota and the expression of specific host response genes using faecal and mucosal samples (‘ON’ and ‘OFF’ the tumour, proximal and distal) from 59 patients undergoing surgery for CRC, 21 individuals with polyps and 56 healthy controls. Microbiota composition was determined by 16S rRNA amplicon sequencing; expression of host genes involved in CRC progression and immune response was quantified by real-time quantitative PCR. Results: The microbiota of patients with CRC differed from that of controls, but alterations were not restricted to the cancerous tissue. Differences between distal and proximal cancers were detected and faecal microbiota only partially reflected mucosal microbiota in CRC. Patients with CRC can be stratified based on higher level structures of mucosal-associated bacterial co-abundance groups (CAGs) that resemble the previously formulated concept of enterotypes. Of these, Bacteroidetes Cluster 1 and Firmicutes Cluster 1 were in decreased abundance in CRC mucosa, whereas Bacteroidetes Cluster 2, Firmicutes Cluster 2, Pathogen Cluster and Prevotella Cluster showed increased abundance in CRC mucosa. CRC-associated CAGs were differentially correlated with the expression of host immunoinflammatory response genes. Conclusions: CRC-associated microbiota profiles differ from those in healthy subjects and are linked with distinct mucosal gene-expression profiles. Compositional alterations in the microbiota are not restricted to cancerous tissue and differ between distal and proximal cancers

T Cell Specific Adapter Protein (TSAd) Interacts with Tec Kinase ITK to Promote CXCL12 Induced Migration of Human and Murine T Cells

The chemokine CXCL12/SDF-1α interacts with its G-protein coupled receptor CXCR4 to induce migration of lymphoid and endothelial cells. T cell specific adapter protein (TSAd) has been found to promote migration of Jurkat T cells through interaction with the G protein β subunit. However, the molecular mechanisms for how TSAd influences cellular migration have not been characterized in detail. We show that TSAd is required for tyrosine phosphorylation of the Lck substrate IL2-inducible T cell kinase (Itk). Presence of Itk Y511 was necessary to boost TSAd\u27s effect on CXCL12 induced migration of Jurkat T cells. In addition, TSAd\u27s ability to promote CXCL12-induced actin polymerization and migration of Jurkat T lymphocytes was dependent on the Itk-interaction site in the proline-rich region of TSAd. Furthermore, TSAd-deficient murine thymocytes failed to respond to CXCL12 with increased Itk phosphorylation, and displayed reduced actin polymerization and cell migration responses. We propose that TSAd, through its interaction with both Itk and Lck, primes Itk for Lck mediated phosphorylation and thereby regulates CXCL12 induced T cell migration and actin cytoskeleton rearrangements

Measles vaccination in the presence or absence of maternal measles antibody: impact on child survival.

BACKGROUND: Measles vaccine (MV) has a greater effect on child survival when administered in early infancy, when maternal antibody may still be present. METHODS: To test whether MV has a greater effect on overall survival if given in the presence of maternal measles antibody, we reanalyzed data from 2 previously published randomized trials of a 2-dose schedule with MV given at 4-6 months and at 9 months of age. In both trials antibody levels had been measured before early measles vaccination. RESULTS: In trial I (1993-1995), the mortality rate was 0.0 per 1000 person-years among children vaccinated with MV in the presence of maternal antibody and 32.3 per 1000 person-years without maternal antibody (mortality rate ratio [MRR], 0.0; 95% confidence interval [CI], 0-.52). In trial II (2003-2007), the mortality rate was 4.2 per 1000 person-years among children vaccinated in presence of maternal measles antibody and 14.5 per 1000 person-years without measles antibody (MRR, 0.29; 95% CI, .09-.91). Possible confounding factors did not explain the difference. In a combined analysis, children who had measles antibody detected when they received their first dose of MV at 4-6 months of age had lower mortality than children with no maternal antibody, the MRR being 0.22 (95% CI, .07-.64) between 4-6 months and 5 years. CONCLUSIONS: Child mortality in low-income countries may be reduced by vaccinating against measles in the presence of maternal antibody, using a 2-dose schedule with the first dose at 4-6 months (earlier than currently recommended) and a booster dose at 9-12 months of age. CLINICAL TRIALS REGISTRATION: NCT00168558