A Changing Gastric Environment Leads to Adaptation of Lipopolysaccharide Variants in Helicobacter pylori Populations during Colonization

The human gastric pathogen Helicobacter pylori colonizes the stomachs of half of the human population, and causes development of peptic ulcer disease and gastric adenocarcinoma. H. pylori-associated chronic atrophic gastritis (ChAG) with loss of the acid-producing parietal cells, is correlated with an increased risk for development of gastric adenocarinoma. The majority of H. pylori isolates produce lipopolysaccharides (LPS) decorated with human-related Lewis epitopes, which have been shown to phase-vary in response to different environmental conditions. We have characterized the adaptations of H. pylori LPS and Lewis antigen expression to varying gastric conditions; in H. pylori isolates from mice with low or high gastric pH, respectively; in 482 clinical isolates from healthy individuals and from individuals with ChAG obtained at two time points with a four-year interval between endoscopies; and finally in isolates grown at different pH in vitro. Here we show that the gastric environment can contribute to a switch in Lewis phenotype in the two experimental mouse models. The clinical isolates from different human individuals showed that intra-individual isolates varied in Lewis antigen expression although the LPS diversity was relatively stable within each individual over time. Moreover, the isolates demonstrated considerable diversity in the levels of glycosylation and in the sizes of fucosylated O-antigen chains both within and between individuals. Thus our data suggest that different LPS variants exist in the colonizing H. pylori population, which can adapt to changes in the gastric environment and provide a means to regulate the inflammatory response of the host during disease progression

Performance of a 70-mer oligonucleotide microarray for genotyping of Campylobacter jejuni

<p>Abstract</p> <p>Background</p> <p><it>Campylobacter jejuni </it>is widespread in the environment and is the major cause of bacterial gastroenteritis in humans. In the present study we use microarray-based comparative genomic hybridizations (CGH), pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) to analyze closely related <it>C. jejuni </it>isolates from chicken and human infection.</p> <p>Results</p> <p>With the exception of one isolate, the microarray data clusters the isolates according to the five groups determined by PFGE. In contrast, MLST defines only three genotypes among the isolates, indicating a lower resolution. All methods show that there is no inherit difference between isolates infecting humans and chicken, suggesting a common underlying population of <it>C. jejuni</it>. We further identify regions that frequently differ between isolates, including both previously described and novel regions. Finally, we show that genes that belong to certain functional groups differ between isolates more often than expected by chance.</p> <p>Conclusion</p> <p>In this study we demonstrated the utility of 70-mer oligonucleotide microarrays for genotyping of <it>Campylobacter jejuni </it>isolates, with resolution outperforming MLST.</p

Intestinal Microbiota Regulate Xenobiotic Metabolism in the Liver

BACKGROUND: The liver is the central organ for xenobiotic metabolism (XM) and is regulated by nuclear receptors such as CAR and PXR, which control the metabolism of drugs. Here we report that gut microbiota influences liver gene expression and alters xenobiotic metabolism in animals exposed to barbiturates. PRINCIPAL FINDINGS: By comparing hepatic gene expression on microarrays from germfree (GF) and conventionally-raised mice (SPF), we identified a cluster of 112 differentially expressed target genes predominantly connected to xenobiotic metabolism and pathways inhibiting RXR function. These findings were functionally validated by exposing GF and SPF mice to pentobarbital which confirmed that xenobiotic metabolism in GF mice is significantly more efficient (shorter time of anesthesia) when compared to the SPF group. CONCLUSION: Our data demonstrate that gut microbiota modulates hepatic gene expression and function by altering its xenobiotic response to drugs without direct contact with the liver

Helicobacter pylori : Cellular interactions and pathogenesis

Helicobacter pylori colonizes the stomachs of about half of die world's population. It inevitably causes an inflammatory response in all its hosts, but in a subset of individuals the infection leads to severe gastric diseases such as peptic ulcers, gastric cancer or MALT lymphoma. The relative contributions of the host, microbe and environment to disease progression are not known. Host factors such as HLA-type, secretor status and Il-1beta allele type have been identified as contributing to the risk of disease. Microbial factors that have been recognized as virulence factors are, e.g., the vacuolating cytotoxin, and the Cag Pathogenicity Island (PAI) that encodes a type IV secretion system. The route of transmission of H. pylori is not known. Once colonization is established, it persists for life unless eradicated with antibiotics. Resistance to antibiotics in H. pylori is increasing; especially resistance to clarithromycin results in treatment failure. The genetic diversity of H. pylori strains is remarkable and it reflects a panmictic population structure with free recombination. This study aimed to characterize defined host, microbial and environmental factors that might impact on the outcome of the infection. These factors were: (i) production of specific receptors (Leb and NetiAcalpha2,3Galbeta1,4 glycans) and presence or absence of gastric acid; (ii) bacterial genotype; and (iii) presence or absence of a microflora competing with H. pylori colonization. The host factors were studied in genetically modified, transgenic, FVB/N mice. Bacterial genotypes were determined by microarray in clinical isolates obtained from patients with gastric adenocarcinoma and controls. The effect of the microflora was assessed by comparing germ-free and conventionally raised animals. After whole-genome genotyping of two subclones of one strain of H. pylori, we found that the isolates were more similar to each other than to any other strain that had been genotyped, but had undergone considerable divergence including excision of the Cag PAI. Both isolates colonized germfree Leb producing mice to the same density. The Cag PAI+ isolate colonized conventionally raised animals to the same extent as germ-free, while the Cag PAI- isolate was unable to colonize conventionally raised animals. Evolution of the isolates in ex-germ-free and conventional mice after three or ten month infections was limited. Among the factors that affect the appearance and spread of acquired antibiotic resistance, the mutation frequency and biological cost of resistance are of special importance. We used clinical pairs of H. pylori isolated before and after treatment with clarithromycin. The mutation frequency of a panel of H. pylori isolates was found to be higher than in enteric bacteria, (median 10-6). 1/4 of the isolates had a mutation frequency higher than Enterobacteriaceae mismatch repair defective mutants. Clarithromycin resistance was associated with a biological cost, as measured by decreased competitive ability of the resistant mutants compared to wildtype in colonization of mice. In clinical isolates, this cost could be reduced by compensatory mutations at extra-genie site(s), indicating that compensation is a clinically relevant phenomenon that could act to stabilize resistant bacteria in a population. We also studied the influence of specific bacterial binding to Le Leb and sialylated glycoconjugates, as well as the presence and absence of acid on the fecal-oral transmission of H. pylori between gnotobiotic transgenic mice. Transmission only occurred in transgenic mice without parietal cells, indicating that loss of acid production in the host facilitates transmission of H. pylori. Genotypes were compared among 13 strains that exhibited the ability to bind to Le b and/or NeuAcalpha2,3GaIbeta1,4 glycans and two strains that did not bind. Strains were selected from a panel of 96 strains from a case-control study of gastric cancer. Colonization ability and host response to the gentyped isolates were assessed in transgenic mice. H. pylori-induced expression of three genes were analyzed by quantitative real-time RT- PCR: heat shock protein 70, polymeric immunoglobulin receptor and interleukin-10. These markers were differentially induced (compared to uninfected controls) by the different isolates. We found that genes encoding specificity subunits of type 1 restriction modification systems (hsdS) in H. pylori were correlated with induction of a strong host response. The HsdS proteins are DNA binding proteins that confer sequence specificity for the type I R- M enzyme. We suggest that these regulate virulence gene expression. Our work describes part of the interplay between H. pylori and its host that lead to persistent infection. In addition, we have identified host genes that can potentially be used as molecular markers of a robust host response as well as genes in the bacterium that might regulate virulence of the isolate. This could help identify patients at risk for severe gastric disease due to H. pylori infection, to avoid massive treatment programs that could affect the total commensal and pathogenic microflora

Identification of novel sphingolipid-binding motifs in mammalian membrane proteins

AbstractSpecific interactions between transmembrane proteins and sphingolipids is a poorly understood phenomenon, and only a couple of instances have been identified. The best characterized example is the sphingolipid-binding motif VXXTLXXIY found in the transmembrane helix of the vesicular transport protein p24. Here, we have used a simple motif-probability algorithm (MOPRO) to identify proteins that contain putative sphingolipid-binding motifs in a dataset comprising proteomes from mammalian organisms. From these motif-containing candidate proteins, four with different numbers of transmembrane helices were selected for experimental study: i) major histocompatibility complex II Q alpha chain subtype (DQA1), ii) GPI-attachment protein 1 (GAA1), iii) tetraspanin-7 TSN7, and iv), metabotropic glutamate receptor 2 (GRM2). These candidates were subjected to photo-affinity labeling using radiolabeled sphingolipids, confirming all four candidate proteins as sphingolipid-binding proteins. The sphingolipid-binding motifs are enriched in the 7TM family of G-protein coupled receptors, predominantly in transmembrane helix 6. The ability of the motif-containing candidate proteins to bind sphingolipids with high specificity opens new perspectives on their respective regulation and function

Slow Genetic Divergence of Helicobacter pylori Strains during Long-Term Colonization

The genetic variability of Helicobacter pylori is known to be high compared to that of many other bacterial species. H. pylori is adapted to the human stomach, where it persists for decades, and adaptation to each host results in every individual harboring a distinctive bacterial population. Although clonal variants may exist within such a population, all isolates are generally genetically related and thus derived from a common ancestor. We sought to determine the rate of genetic change of H. pylori over 9 years in two asymptomatic adult patients. Arbitrary primed PCR confirmed the relatedness of individual subclones within a patient. Furthermore, sequencing of 10 loci (∼6,000 bp) in three subclones per time and patient revealed only two base pair changes among the subclones from patient I. All sequences were identical among the patient II subclones. However, PCR amplification of the highly divergent gene amiA revealed great variation in the size of the gene between the subclones within each patient. Thus, both patients harbored a single strain with clonal variants at both times. We also studied genetic changes in culture- and mouse-passaged strains, and under both conditions no genetic divergence was found. These results suggest that previous estimates of the rate of genetic change in H. pylori within an individual might be overestimates

Normal gut microbiota modulates brain development and behavior.

Microbial colonization of mammals is an evolution-driven process that modulate host physiology, many of which are associated with immunity and nutrient intake. Here, we report that colonization by gut microbiota impacts mammalian brain development and subsequent adult behavior. Using measures of motor activity and anxiety-like behavior, we demonstrate that germ free (GF) mice display increased motor activity and reduced anxiety, compared with specific pathogen free (SPF) mice with a normal gut microbiota. This behavioral phenotype is associated with altered expression of genes known to be involved in second messenger pathways and synaptic long-term potentiation in brain regions implicated in motor control and anxiety-like behavior. GF mice exposed to gut microbiota early in life display similar characteristics as SPF mice, including reduced expression of PSD-95 and synaptophysin in the striatum. Hence, our results suggest that the microbial colonization process initiates signaling mechanisms that affect neuronal circuits involved in motor control and anxiety behavior