Christine Josenhans

THE ROLE OF MOTILITY AS A VIRULENCE FACTOR IN BACTERIA

Many bacteria that cause diseases of humans, animals and plants use flagella to move. This review summarises recent studies that have analysed the role of motility and chemotaxis in the host-parasite relationship of pathogenic bacteria. These studies have shown that for many pathogens, motility is essential in some phases of their life cycle and that virulence and motility are often intimately linked by complex regulatory networks. Possibilities to exploit bacterial motility as a specific therapeutic antibacterial target to cure or prevent disease are discussed.

The impact of the microbiota on the pathogenesis of IBD: lessons from mouse infection models

Inflammatory bowel disease (IBD), including Crohns disease and ulcerative colitis, is a major human health problem. The bacteria that live in the gut play an important part in the pathogenesis of IBD. However, owing to the complexity of the gut microbiota, our understanding of the roles of commensal and pathogenic bacteria in establishing a healthy intestinal barrier and in its disruption is evolving only slowly. In recent years, mouse models of intestinal inflammatory disorders based on defined bacterial infections have been used intensively to dissect the roles of individual bacterial species and specific bacterial components in the pathogenesis of IBD. In this Review, we focus on the impact of pathogenic and commensal bacteria on IBD-like pathogenesis in mouse infection models and summarize important recent developments.

The complete genome sequence of the carcinogenic bacterium Helicobacter hepaticus

Helicobacter hepaticus causes chronic hepatitis and liver cancer in mice. It is the prototype enterohepatic Helicobacter species and a close relative of Helicobacter pylori, also a recognized carcinogen. Here we report the complete genome sequence of H. hepaticus ATCC51449. H. hepaticus has a circular chromosome of 1,799,146 base pairs, predicted to encode 1,875 proteins. A total of 938, 953, and 821 proteins have orthologs in H. pylori, Campylobacter jejuni, and both pathogens, respectively. H. hepaticus lacks orthologs of most known H. pylori virulence factors, including adhesins, the VacA cytotoxin, and almost all cag pathogenicity island proteins, but has orthologs of the C. jejuni adhesin PEB1 and the cytolethal distending toxin (CDT). The genome contains a 71-kb genomic island (HHGI1) and several genomic islets whose G+C content differs from the rest of the genome. HHGI1 encodes three basic components of a type IV secretion system and other virulence protein homologs, suggesting a role of HHGI1 in pathogenicity. The genomic variability of H. hepaticus was assessed by comparing the genomes of 12 H. hepaticus strains with the sequenced genome by microarray hybridization. Although five strains, including all those known to have caused liver disease, were indistinguishable from ATCC51449, other strains lacked between 85 and 229 genes, including large parts of HHGI1, demonstrating extensive variation of genome content within the species.

Helicobacter pylori genome evolution during human infection

High genetic diversity is a hallmark of the gastric pathogen Helicobacter pylori. We used 454 sequencing technology to perform whole-genome comparisons for five sets of H. pylori strains that had been sequentially cultured from four chronically infected Colombians (isolation intervals = 3–16 y) and one human volunteer experimentally infected with H. pylori as part of a vaccine trial. The four sets of genomes from Colombian H. pylori differed by 27–232 isolated SNPs and 16–441 imported clusters of polymorphisms resulting from recombination. Imports (mean length = 394 bp) were distributed nonrandomly over the chromosome and frequently occurred in groups, suggesting that H. pylori first takes up long DNA fragments, which subsequently become partially integrated in multiple shorter pieces. Imports were present at significantly increased frequency in members of the hop family of outer membrane gene paralogues, some of which are involved in bacterial adhesion, suggesting diversifying selection. No evidence of recombination and few other differences were identified in the strain pair from an infected volunteer, indicating that the H. pylori genome is stable in the absence of mixed infection. Among these few differences was an OFF/ON switch in the phase-variable adhesin gene hopZ, suggesting strong in vivo selection for this putative adhesin during early colonization.

Genome-wide analysis of transcriptional hierarchy and feedback regulation in the flagellar system of Helicobacter pylori

Summary The flagellar system of Helicobacter pylori, which comprises more than 40 mostly unclustered genes, is essential for colonization of the human stomach mucosa. In order to elucidate the complex transcriptional circuitry of flagellar biosynthesis in H. pylori and its link to other cell functions, mutants in regulatory genes governing flagellar biosynthesis (rpoN, flgR, flhA, flhF, HP0244) and whole‐genome microarray technology were used in this study. The regulon controlled by RpoN, its activator FlgR (FleR) and the cognate histidine kinase HP0244 (FleS) was characterized on a genome‐wide scale for the first time. Seven novel genes (HP1076, HP1233, HP1154/1155, HP0366/367, HP0869) were identified as belonging to RpoN‐associated flagellar regulons. The hydrogenase accessory gene HP0869 was the only annotated non‐flagellar gene in the RpoN regulon. Flagellar basal body components FlhA and FlhF were characterized as functional equivalents to master regulators in H. pylori, as their absence led to a general reduction of transcripts in the RpoN (class 2) and FliA (class 3) regulons, and of 24 genes newly attributed to intermediate regulons, under the control of two or more promoters. FlhA‐ and FlhF‐dependent regulons comprised flagellar and non‐flagellar genes. Transcriptome analysis revealed that negative feedback regulation of the FliA regulon was dependent on the antisigma factor FlgM. FlgM was also involved in FlhA‐ but not FlhF‐dependent feedback control of the RpoN regulon. In contrast to other bacteria, chemotaxis and flagellar motor genes were not controlled by FliA or RpoN. A true master regulator of flagellar biosynthesis is absent in H. pylori, consistent with the essential role of flagellar motility and chemotaxis for this organism.

A Global Overview of the Genetic and Functional Diversity in the Helicobacter pylori cag Pathogenicity Island

The Helicobacter pylori cag pathogenicity island (cagPAI) encodes a type IV secretion system. Humans infected with cagPAI–carrying H. pylori are at increased risk for sequelae such as gastric cancer. Housekeeping genes in H. pylori show considerable genetic diversity; but the diversity of virulence factors such as the cagPAI, which transports the bacterial oncogene CagA into host cells, has not been systematically investigated. Here we compared the complete cagPAI sequences for 38 representative isolates from all known H. pylori biogeographic populations. Their gene content and gene order were highly conserved. The phylogeny of most cagPAI genes was similar to that of housekeeping genes, indicating that the cagPAI was probably acquired only once by H. pylori, and its genetic diversity reflects the isolation by distance that has shaped this bacterial species since modern humans migrated out of Africa. Most isolates induced IL-8 release in gastric epithelial cells, indicating that the function of the Cag secretion system has been conserved despite some genetic rearrangements. More than one third of cagPAI genes, in particular those encoding cell-surface exposed proteins, showed signatures of diversifying (Darwinian) selection at more than 5% of codons. Several unknown gene products predicted to be under Darwinian selection are also likely to be secreted proteins (e.g. HP0522, HP0535). One of these, HP0535, is predicted to code for either a new secreted candidate effector protein or a protein which interacts with CagA because it contains two genetic lineages, similar to cagA. Our study provides a resource that can guide future research on the biological roles and host interactions of cagPAI proteins, including several whose function is still unknown.

Switching of Flagellar Motility in Helicobacter pylori by Reversible Length Variation of a Short Homopolymeric Sequence Repeat in fliP, a Gene Encoding a Basal Body Protein

ABSTRACT The genome of Helicobacter pylori contains numerous simple nucleotide repeats that have been proposed to have regulatory functions and to compensate for the conspicuous dearth of master regulatory pathways in this highly host-adapted bacterium. H. pylori strain 26695, whose genomic sequence was determined by The Institute for Genomic Research (TIGR), contains a repeat of nine cytidines in the fliP flagellar basal body gene that splits the open reading frame in two parts. In this work, we demonstrate that the 26695C9 strain with a split fliP gene as sequenced by TIGR was nonflagellated and nonmotile. In contrast, earlier isolates of strain 26695 selected by positive motility testing as well as pig-passaged derivatives of 26695 were all flagellated and highly motile. All of these motile strains had a C8 repeat and consequently a contiguous fliP reading frame. By screening approximately 50,000 colonies of 26695C9 for motility in soft agar, a motile revertant with a C8 repeat could be isolated, proving that the described switch is reversible. ThefliP genes of 20 motile clinical H. pyloriisolates from different geographic regions possessed intactfliP genes with repeats of eight cytidines or the sequence CCCCACCC in its place. Isogenic fliP mutants of a motile, C8 repeat isolate of strain 26695 were constructed by allelic exchange mutagenesis and found to be defective in flagellum biogenesis. Mutants produced only small amounts of flagellins, while the transcription of flagellin genes appeared unchanged. These results strongly suggest a unique mechanism regulating motility in H. pylori which relies on slipped-strand mispairing-mediated mutagenesis of fliP.

Functional characterization of the antagonistic flagellar late regulators FliA and FlgM of Helicobacter pylori and their effects on the H. pylori transcriptome

Helicobacter pylori is thought to regulate gene ex‐pression with a very small set of regulatory genes. We identified a previously unannotated open reading frame (ORF) in the H. pylori 26695 genome (HP1122) as a putative H. pylori flgM gene (σ28 factor antagonist) by a motif‐based bioinformatic approach. Deletion of HP1122 resulted in a fourfold increase in transcription of the σ28‐dependent major flagellin gene flaA, supporting the function of HP1122 as H. pylori FlgM. Helicobacter pylori FlgM lacks a conserved 20‐amino‐acid N‐terminal domain of enterobacterial FlgM proteins, but was able to interact with the Salmonella typhimuriumσ28 (FliA) and inhibit the expression of FliA‐dependent genes in Salmonella. Helicobacter pylori FlgM inhibited FliA to the same extent in a Salmonella strain with an intact flagellar export system and in an export‐deficient strain. Helicobacter pylori FliA was able to drive transcription of FliA‐dependent genes in Salmonella. The effects of mutations in the H. pylori flgM and fliA genes on the H. pylori transcriptome were analysed using whole genome DNA microarrays. The antagonistic roles of FlgM and FliA in controlling the transcription of the major flagellin gene flaA were confirmed, and two additional FliA/FlgM dependent operons (HP472 and HP1051/HP1052) were identified. None of the three genes contained in these operons has a known function in flagellar biogenesis in other bacteria. Like other motile bacteria, H. pylori has a FliA/FlgM pair of sigma and anti‐sigma factors, but the genes controlled by these differ markedly from the Salmonella/Escherichia coli paradigm.

Closely related Campylobacter jejuni strains from different sources reveal a generalist rather than a specialist lifestyle

BackgroundCampylobacter jejuni and Campylobacter coli are human intestinal pathogens of global importance. Zoonotic transmission from livestock animals or animal-derived food is the likely cause for most of these infections. However, little is known about their general and host-specific mechanisms of colonization, or virulence and pathogenicity factors. In certain hosts, Campylobacter species colonize persistently and do not cause disease, while they cause acute intestinal disease in humans.ResultsHere, we investigate putative host-specificity using phenotypic characterization and genome-wide analysis of genetically closely related C. jejuni strains from different sources. A collection of 473 fresh Campylobacter isolates from Germany was assembled between 2006 and 2010 and characterized using MLST. A subset of closely related C. jejuni strains of the highly prevalent sequence type ST-21 was selected from different hosts and isolation sources. PCR typing of strain-variable genes provided evidence that some genes differed between these strains. Furthermore, phenotypic variation of these strains was tested using the following criteria: metabolic variation, protein expression patterns, and eukaryotic cell interaction. The results demonstrated remarkable phenotypic diversity within the ST-21 group, which however did not correlate with isolation source. Whole genome sequencing was performed for five ST-21 strains from chicken, human, bovine, and food sources, in order to gain insight into ST-21 genome diversity. The comparisons showed extensive genomic diversity, primarily due to recombination and gain of phage-related genes. By contrast, no genomic features associated with isolation source or host were identified.ConclusionsThe genome information and phenotypic data obtained in vitro and in a chicken infection model provided little evidence of fixed adaptation to a specific host. Instead, the dominant C. jejuni ST-21 appeared to be characterized by phenotypic flexibility and high genetic microdiversity, revealing properties of a generalist. High genetic flexibility might allow generalist variants of C. jejuni to reversibly express diverse fitness factors in changing environments.

Genomic Changes during Chronic Helicobacter pylori Infection

The gastric pathogen Helicobacter pylori shows tremendous genetic variability within human populations, both in gene content and at the sequence level. We investigated how this variability arises by comparing the genome content of 21 closely related pairs of isolates taken from the same patient at different time points. The comparisons were performed by hybridization with whole-genome DNA microarrays. All loci where microarrays indicated a genomic change were sequenced to confirm the events. The number of genomic changes was compared to the number of homologous replacement events without loss or gain of genes that we had previously determined by multilocus sequence analysis and mathematical modeling based on the sequence data. Our analysis showed that the great majority of genetic changes were due to homologous recombination, with 1/650 events leading to a net gain or loss of genes. These results suggest that adaptation of H. pylori to the host individual may principally occur through sequence changes rather than loss or gain of genes.