Nina R. Salama

Helicobacter pylori genetic diversity within the gastric niche of a single human host

Isolates of the gastric pathogen Helicobacter pylori harvested from different individuals are highly polymorphic. Strain variation also has been observed within a single host. To more fully ascertain the extent of H. pylori genetic diversity within the ecological niche of its natural host, we harvested additional isolates of the sequenced H. pylori strain J99 from its human source patient after a 6-year interval. Randomly amplified polymorphic DNA PCR and DNA sequencing of four unlinked loci indicated that these isolates were closely related to the original strain. In contrast, microarray analysis revealed differences in genetic content among all of the isolates that were not detected by randomly amplified polymorphic DNA PCR or sequence analysis. Several ORFs from loci scattered throughout the chromosome in the archival strain did not hybridize with DNA from the recent strains, including multiple ORFs within the J99 plasticity zone. In addition, DNA from the recent isolates hybridized with probes for ORFs specific for the other fully sequenced H. pylori strain 26695, including a putative traG homolog. Among the additional J99 isolates, patterns of genetic diversity were distinct both when compared with each other and to the original prototype isolate. These results indicate that within an apparently homogeneous population, as determined by macroscale comparison and nucleotide sequence analysis, remarkable genetic differences exist among single-colony isolates of H. pylori. Direct evidence that H. pylori has the capacity to lose and possibly acquire exogenous DNA is consistent with a model of continuous microevolution within its cognate host.

Helicobacter pylori strain-specific differences in genetic content, identified by microarray, influence host inflammatory responses

Helicobacter pylori enhances the risk for ulcer disease and gastric cancer, yet only a minority of H. pylori-colonized individuals develop disease. We examined the ability of two H. pylori isolates to induce differential host responses in vivo or in vitro, and then used an H. pylori whole genome microarray to identify bacterial determinants related to pathogenesis. Gastric ulcer strain B128 induced more severe gastritis, proliferation, and apoptosis in gerbil mucosa than did duodenal ulcer strain G1.1, and gastric ulceration and atrophy occurred only in B128+ gerbils. In vitro, gerbil-passaged B128 derivatives significantly increased IL-8 secretion and apoptosis compared with G1.1 strains. DNA hybridization to the microarray identified several strain-specific differences in gene composition including a large deletion of the cag pathogenicity island in strain G1.1. Partial and complete disruption of the cag island in strain B128 attenuated induction of IL-8 in vitro and significantly decreased gastric inflammation in vivo. These results indicate that the ability of H. pylori to regulate epithelial cell responses related to inflammation depends on the presence of an intact cag pathogenicity island. Use of an H pylori whole genome microarray is an effective method to identify differences in gene content between H. pylori strains that induce distinct pathological outcomes in a rodent model of H. pylori infection.

Global Transposon Mutagenesis and Essential Gene Analysis of Helicobacter pylori

We have constructed a genome-saturating mutant library of the human gastric pathogen Helicobacter pylori. Microarray tracking of transposon mutants (MATT) allowed us to map the position of 5,363 transposon mutants in our library. While we generally found insertions well distributed throughout the genome, 344 genes had no detectable transposon insertions, and this list is predicted to be highly enriched for essential genes. Comparison to the essential gene set of other bacteria revealed a surprisingly limited overlap with all organisms tested (11%), while 55% were essential in some organisms but not others. We independently verified the essentiality of several gene products, including an HtrA family serine protease, a hypothetical protein with putative phospholipase D activity, and a riboflavin specific deaminase. A limited screen for motility mutants allowed us to estimate that 4.5% of the genome is dedicated to this virulence-associated phenotype.

Modification of Helicobacter pylori outer membrane protein expression during experimental infection of rhesus macaques.

Clinical isolates of Helicobacter pylori show marked diversity, which may derive from genomic changes that occur during the often lifelong association of the bacterium with its human host. We used the rhesus macaque model, together with DNA microarrays, to examine genomic changes in H. pylori that occur early during experimental infection. Microarray analysis showed that H. pylori recovered from challenged macaques had deleted babA, a member of a large family of paralogous outer membrane proteins (OMPs) that mediates attachment of H. pylori to the Lewis B blood group antigen on gastric epithelium. In some cases the babA gene was replaced by babB, an uncharacterized OMP that is closely related to babA. In other cases the babA gene was present but was not expressed because of alteration in dinucleotide CT repeats in the 5′ coding region. In either case, strains lacking babA did not adhere to Lewis B, which is expressed on macaque gastric epithelium. Absence of babA and duplication of babB was also seen in H. pylori isolates derived from human clinical samples, suggesting that this gene conversion event is not unique to experimentally infected rhesus monkeys. These results demonstrate in real time with a relevant animal model that H. pylori regulates OMP expression in vivo by using both antigenic variation and phase variation. We suggest that changes in babA and babB after experimental infection of macaques represent a dynamic response in the H. pylori outer membrane that facilitates adherence to the gastric epithelium and promotes chronic infection.

Cag pathogenicity island-specific responses of gastric epithelial cells to Helicobacter pylori infection

Helicobacter pylori infects over half the worlds population and causes a wide range of diseases, including gastritis, peptic ulcer, and two forms of gastric cancer. H. pylori infection elicits a variety of phenotypic responses in cultured gastric epithelial cells, including the expression of proinflammatory genes and changes in the actin cytoskeleton. Both of these responses are mediated by the type IV secretion system (TFSS) encoded by the cag pathogenicity island (cag PAI). We used human cDNA microarrays to examine the temporal transcriptional profiles of gastric AGS cells infected with H. pylori strain G27 and a panel of isogenic mutants to dissect the contributions of various genes in the cag PAI. Infection with G27 induced expression of genes involved in the innate immune response, cell shape regulation, and signal transduction. A mutant lacking the cagA gene, which encodes an effector molecule secreted by the TFSS and required for the host cell cytoskeletal response, induced the expression of fewer cytoskeletal genes. A mutant lacking cagE, which encodes a structural component of the TFSS, failed to up-regulate a superset of host genes, including the cagA-dependent genes, and many of the immune response genes. A mutant lacking the entire cag PAI failed to induce both the cagE-dependent genes and several transiently expressed cagE independent genes. Host cell transcriptional profiling of infection with isogenic strains offered a detailed molecular picture of H. pylori infection and provided insight into potential targets of individual virulence determinants such as tyrosine kinase and Rho GTPase signaling molecules.

Vacuolating Cytotoxin of Helicobacter pylori Plays a Role during Colonization in a Mouse Model of Infection

ABSTRACT Helicobacter pylori, the causative agent of gastritis and ulcer disease in humans, secretes a toxin called VacA (vacuolating cytotoxin) into culture supernatants. VacA was initially characterized and purified on the basis of its ability to induce the formation of intracellular vacuoles in tissue culture cells. H. pyloristrains possessing different alleles of vacA differ in their ability to express active toxin. Those strains expressing higher toxin levels are correlated with more severe gastric disease. However, the specific role(s) played by VacA during the course of infection and disease is not clear. We have used a mouse model of H. pylori infection to begin to address this role. A null mutation of vacA compromises H. pylori in its ability to initially establish infection. If an infection by a vacAmutant is established, the bacterial load and degree of inflammation are similar to those associated with an isogenic wild-type strain. Thus, in this infection model, vacA plays a role in the initial colonization of the host, suggesting that strains of H. pylori expressing active alleles of vacA may be better adapted for host-to-host transmission.

Helicobacter pylori enter and survive within multivesicular vacuoles of epithelial cells

Although intracellular Helicobacter pylori have been described in biopsy specimens and in cultured epithelial cells, the fate of these bacteria is unknown. Using differential interference contrast (DIC) video and immunofluorescence microscopy, we document that a proportion of cell‐associated H. pylori enter large cytoplasmic vacuoles, where they remain viable and motile and can survive lethal concentrations of extracellular gentamicin. Entry into vacuoles occurs in multiple epithelial cell lines including AGS gastric adenocarcinoma, Caco‐2 colon adenocarcinoma and MDCK kidney cell line, and depends on the actin cytoskeleton. Time‐lapse microscopy over several hours was used to follow the movement of live H. pylori within vacuoles of a single cell. Pulsed, extracellular gentamicin treatments show that the half‐life of intravacuolar bacteria is on the order of 24 h. Viable H. pylori repopulate the extracellular environment in parallel with the disappearance of intravacuolar bacteria, suggesting release from the intravacuolar niche. Using electron microscopy and live fluorescent staining with endosomal dyes, we observe that H. pylori‐containing vacuoles are similar in morphology to late endosomal multivesicular bodies. VacA is not required for these events, as isogenic vacA– mutants still enter and survive within the intravacuolar niche. The exploitation of an intravacuolar niche is a new aspect of the biological life cycle of H. pylori that could explain the difficulties in eradicating this infection.

The Complete Genome Sequence of Helicobacter pylori Strain G27

Helicobacter pylori is a gram-negative pathogen that colonizes the stomachs of over half the worlds population and causes a spectrum of gastric diseases including gastritis, ulcers, and gastric carcinoma. The H. pylori species exhibits unusually high levels of genetic variation between strains. Here we announce the complete genome sequence of H. pylori strain G27, which has been used extensively in H. pylori research.

Peptidoglycan Crosslinking Relaxation Promotes Helicobacter pylori's Helical Shape and Stomach Colonization

The mechanisms by which bacterial cells generate helical cell shape and its functional role are poorly understood. Helical shape of the human pathogen Helicobacter pylori may facilitate penetration of the thick gastric mucus where it replicates. We identified four genes required for helical shape: three LytM peptidoglycan endopeptidase homologs (csd1-3) and a ccmA homolog. Surrounding the cytoplasmic membrane of most bacteria, the peptidoglycan (murein) sacculus is a meshwork of glycan strands joined by peptide crosslinks. Intact cells and isolated sacculi from mutants lacking any single csd gene or ccmA formed curved rods and showed increased peptidoglycan crosslinking. Quantitative morphological analyses of multiple-gene deletion mutants revealed each protein uniquely contributes to a shape-generating pathway. This pathway is required for robust colonization of the stomach in spite of normal directional motility. Our findings suggest that the coordinated action of multiple proteins relaxes peptidoglycan crosslinking, enabling helical cell curvature and twist.

Comparison of Genetic Divergence and Fitness between Two Subclones of Helicobacter pylori

ABSTRACT Helicobacter pylori has a very plastic genome, reflecting its high rate of recombination and point mutation. This plasticity promotes divergence of the population by the development of subclones and presumably enhances adaptation to host niches. We have investigated the genotypic and phenotypic characteristics of two such subclones isolated from one patient as well as the genetic evolution of these isolates during experimental infection. Whole-genome genotyping of the isolates using DNA microarrays revealed that they were more similar to each other than to a panel of other genotyped strains recovered from different hosts. Nonetheless, they still showed significant differences. For example, one isolate (67:21) contained the entire Cag pathogenicity island (PAI), whereas the other (67:20) had excised the PAI. Phenotypic studies disclosed that both isolates expressed adhesins that recognized human histo-blood group Lewisb glycan receptors produced by gastric pit and surface mucus cells. In addition, both isolates were able to colonize, to equivalent density and with similar efficiency, germ-free transgenic mice genetically engineered to synthesize Lewisb glycans in their pit cells (12 to 14 mice/isolate). Remarkably, the Cag PAI-negative isolate was unable to colonize conventionally raised Lewisb transgenic mice harboring a normal gastric microflora, whereas the Cag PAI-positive isolate colonized 74% of the animals (39 to 40 mice/isolate). The genomic evolution of both isolates during the infection of conventionally raised and germ-free mice was monitored over the course of 3 months. The Cag PAI-positive isolate was also surveyed after a 10 month colonization of conventionally raised transgenic animals (n = 9 mice). Microarray analysis of the Cag PAI and sequence analysis of the cagA,recA, and 16S rRNA genes disclosed no changes in recovered isolates. Together, these results reveal that the H. pyloripopulation infecting one individual can undergo significant divergence, creating stable subclones with substantial genotypic and phenotypic differences.