Recombination-Based In Vivo Expression Technology Identifies Helicobacter Pylori Genes Important For Host Colonization

Here we undertook to identify colonization and gastric disease-promoting factors of the human gastric pathogen Helicobacter pylori as genes that were induced in response to the stomach environment. Using recombination-based in vivo expression technology (RIVET), we identified six promoters induced in the host compared to laboratory conditions. Three of these promoters, designated Pivi10, Pivi66, and Pivi77, regulate genes that H. pylori may use to interact with other microbes or the host. Pivi10 likely regulates the mobA, mobB, and mobD genes, which have potential roles in horizontal gene transfer through plasmid mobilization. Pivi66 occurs in the cytotoxin-associated gene pathogenicity island, a genomic region known to be associated with more severe disease outcomes, and likely regulates cagZ, virB11, and virD4. Pivi77 likely regulates HP0289, an uncharacterized paralogue of the vacA cytotoxin gene. We assessed the roles of a subset of these genes in colonization by creating deletion mutants and analyzing them in single-strain and coinfection experiments. We found that a mobABD mutant was defective for murine host colonization and that a cagZ mutant outcompeted the wild-type strain in a coinfection analysis. Our work supports the conclusion that RIVET is a valuable tool for identifying H. pylori factors with roles in host colonization

Conserved Transcriptional Unit Organization Of The Cag Pathogenicity Island Among Helicobacter Pylori Strains

The Helicobacter pylori cag pathogenicity island (cag PAI) encodes a type IV secretion system that is more commonly found in strains isolated from patients with gastroduodenal disease than from those with asymptomatic gastritis. Genome-wide organization of the transcriptional units in H. pylori strain 26695 was recently established using RNA sequence analysis (Sharma et al., 2010). Here we used quantitative reverse-transcription polymerase chain reaction of open reading frames and intergenic regions to identify putative cag PAI operons in H. pylori; these operons were analyzed further by transcript profiling after deletion of selected promoter regions. Additionally, we used a promoter-trap system to identify functional cag PAI promoters. The results demonstrated that expression of genes on the H. pylori cag PAI varies by nearly five orders of magnitude and that the organization of cag PAI genes into transcriptional units is conserved among several H. pylori strains, including, 26695, J99, G27, and J166. We found evidence for 20 transcripts within the cag PAI, many of which likely overlap. Our data suggests that there are at least 11 operons: cag1-4, cag3-4, cag10-9, cag8-7, cag6-5, cag11-12, cag16-17, cag19-18, cag21-20, cag23-22, and cag25-24, as well as five monocistronic genes (cag4, cag13, cag14, cag15, and cag26). Additionally, the location of four of our functionally identified promoters suggests they are directing expression of, in one case, a truncated version of cag26 and in the other three, transcripts that are antisense to cag7, cag17, and cag23. We verified expression of two of these antisense transcripts, those antisense to cag17 and cag23, by reverse-transcription polymerase chain reaction. Taken together, our results suggest that the cag PAI transcriptional profile is generally conserved among H. pylori strains, 26695, J99, G27, and J166, and is likely complex

CD44 Plays a Functional Role in Helicobacter pylori-induced Epithelial Cell Proliferation

The cytotoxin-associated gene (Cag) pathogenicity island is a strain-specific constituent of Helicobacter pylori (H. pylori) that augments cancer risk. CagA translocates into the cytoplasm where it stimulates cell signaling through the interaction with tyrosine kinase c-Met receptor, leading cellular proliferation. Identified as a potential gastric stem cell marker, cluster-of-differentiation (CD) CD44 also acts as a co-receptor for c-Met, but whether it plays a functional role in H. pylori-induced epithelial proliferation is unknown. We tested the hypothesis that CD44 plays a functional role in H. pylori-induced epithelial cell proliferation. To assay changes in gastric epithelial cell proliferation in relation to the direct interaction with H. pylori, human- and mouse-derived gastric organoids were infected with the G27 H. pylori strain or a mutant G27 strain bearing cagA deletion (ΔCagA::cat). Epithelial proliferation was quantified by EdU immunostaining. Phosphorylation of c-Met was analyzed by immunoprecipitation followed by Western blot analysis for expression of CD44 and CagA. H. pylori infection of both mouse- and human-derived gastric organoids induced epithelial proliferation that correlated with c-Met phosphorylation. CagA and CD44 co-immunoprecipitated with phosphorylated c-Met. The formation of this complex did not occur in organoids infected with ΔCagA::cat. Epithelial proliferation in response to H. pylori infection was lost in infected organoids derived from CD44-deficient mouse stomachs. Human-derived fundic gastric organoids exhibited an induction in proliferation when infected with H. pylori, that was not seen in organoids pre-treated with a peptide inhibitor specific to CD44. In the wellestablished Mongolian gerbil model of gastric cancer, animals treated with CD44 peptide inhibitor Pep1, resulted in the inhibition of H. pylori-induced proliferation and associated atrophic gastritis. The current study reports a unique approach to study H. pylori interaction with the human gastric epithelium. Here, we show that CD44 plays a functional role in H. pyloriinduced epithelial cell proliferation

H. pylori GPS: Modulating Host Metabolites for Location Sensing.

Almost 20 years ago, urea was described as a chemotaxis attractant for Helicobacter pylori. In this issue of Cell Host & Microbe, Huang et al. (2015) report that H. pylori employs its urease enzyme to destroy urea to bring the concentration into a range that provokes an attractant response

Methylation-Independent Chemotaxis Systems Are the Norm for Gastric-Colonizing Helicobacter Species

Many bacteria and archaea rely on chemotaxis signal transduction systems for optimal fitness. These complex, multiprotein signaling systems have core components found in all chemotactic microbes, as well as variable proteins found in only some species. We do not yet understand why these variations exist or whether there are specific niches that favor particular chemotaxis signaling organization. One variation is in the presence/absence of the chemotaxis methylation adaptation enzymes CheB and CheR. Genes for CheB and CheR are missing in the gastric pathogen Helicobacter pylori but present in related Helicobacter that colonize the liver or intestine. In this work, we asked whether there was a general pattern of CheB/CheR across multiple Helicobacter species. Helicobacter spp. all possess chemotactic behavior, based on the presence of genes for core signaling proteins CheA, CheW, and chemoreceptors. Genes for the CheB and CheR proteins, in contrast, were variably present. Niche mapping supported the idea that these genes were present in enterohepatic Helicobacter species and absent in gastric ones. We then analyzed whether there were differences between gastric and enterohepatic species in the CheB/CheR chemoreceptor target methylation sites. Indeed, these sites were less conserved in gastric species that lack CheB/CheR. Lastly, we determined that cheB and cheR could serve as markers to indicate whether an unknown Helicobacter species was of enterohepatic or gastric origin. Overall, these findings suggest the interesting idea that methylation-based adaptation is not required in specific environments, particularly the stomach. IMPORTANCE Chemotaxis signal transduction systems are common in the archaeal and bacterial world, but not all systems contain the same components. The rationale for this system variation remains unknown. In this report, comparative genomics analysis showed that the presence/absence of CheR and CheB is one main variation within the Helicobacter genus, and it is strongly associated with the niche of Helicobacter species: gastric Helicobacter species, which infect animal stomachs, have lost their CheB and CheR, while enterohepatic Helicobacter species, which infect the liver and intestine, retain them. This study not only provides an example that a chemotaxis system variant is associated with particular niches but also proposes that CheB and CheR are new markers distinguishing gastric from enterohepatic Helicobacter species

How Helicobacter pylori senses, targets and interacts with the gastric epithelium.

Helicobacter pylori is a human-specific pathogen that chronically infects about 50% of the world's population. After travelling through the harsh environment of the stomach lumen, H. pylori colonizes the mucosal surface and within the glands of the human stomach. During colonization, H. pylori uses motility and its chemotaxis signalling system to sense the environment to reach the gastric epithelium for colonization, where it is able to attach to the epithelial surface. The H. pylori population inside the stomach contains a subgroup of bacteria that are attached to the gastric epithelium and a larger subgroup of non-attached bacteria that are freely swimming. To establish a tight interaction between H. pylori and epithelial cells, the bacterium produces a variety of adhesins and delivers virulence factors. These lead to alterations in the host signalling pathways, inducing pro-inflammatory responses, apoptosis, uncontrolled cell proliferation, and eventually peptic ulcers and gastric cancer. To prevent disease and find a vaccine or better treatments, it is crucial to understand how H. pylori is able to sense its niche for chronic infection inside the stomach and how its virulence factors interact with the epithelial target cells

Control of bacterial colonization in the glands and crypts

The epithelial cell layer of the major organs of the mammalian gastrointestinal (GI) tract is extensively invaginated into thousands of gland and crypt structures. These are lined by distinct sets of epithelial cells and may comprise discrete niches. The host maximizes the distance between the epithelial cell layer and GI-inhabiting microbes to limit inflammation, and these strategies also likely keep bacteria out of the glands and crypts. We discuss here the specific host processes that have been shown to restrict bacterial presence in the glands and crypts, specifically the immune system, acid, mucin, oxygen, and reactive oxygen species. Not surprisingly, microbes have evolved sophisticated strategies to overcome these host factors and reside close to the epithelium in the glands and crypts. Bacterial properties important for gland and crypt colonization include bacterial immunomodulatory molecules, chemotaxis, and the use of certain metabolites. Overall, these as-yet limited studies suggest there are specific host and bacterial properties that control gland and crypt colonization, contributing to the overall microbial spatial organization of the GI tract. However, there remains much to be discovered in this area

Helicobacter pylori initiates successful gastric colonization by utilizing L-lactate to promote complement resistance

The complement system is a key protective response against infectious pathogens. Here, the authors show that Helicobacter pylori uses host L-lactate to generate a complement resistant state that promotes gastric colonisation

How Helicobacter pylori senses, targets and interacts with the gastric epithelium.

Helicobacter pylori is a human-specific pathogen that chronically infects about 50% of the world's population. After travelling through the harsh environment of the stomach lumen, H. pylori colonizes the mucosal surface and within the glands of the human stomach. During colonization, H. pylori uses motility and its chemotaxis signalling system to sense the environment to reach the gastric epithelium for colonization, where it is able to attach to the epithelial surface. The H. pylori population inside the stomach contains a subgroup of bacteria that are attached to the gastric epithelium and a larger subgroup of non-attached bacteria that are freely swimming. To establish a tight interaction between H. pylori and epithelial cells, the bacterium produces a variety of adhesins and delivers virulence factors. These lead to alterations in the host signalling pathways, inducing pro-inflammatory responses, apoptosis, uncontrolled cell proliferation, and eventually peptic ulcers and gastric cancer. To prevent disease and find a vaccine or better treatments, it is crucial to understand how H. pylori is able to sense its niche for chronic infection inside the stomach and how its virulence factors interact with the epithelial target cells