Analyses of the cag pathogenicity island of Helicobacter pylori

Most strains of Helicobacter pylori from patients with peptic ulcer disease or intestinal-type gastric cancer carry cagA, a gene that encodes an immunodominant protein of unknown function, whereas many of the strains from asymptomatically infected persons lack this gene. Recent studies showed that the cagA gene lies near the right end of a ≈37 kb DNA segment (a pathogenicity island, or PAI) that is unique to cagA⁺ strains and that the cag PAI was split in half by a transposable element insertion in the reference strain NCTC11638. In complementary experiments reported here, we also found the same cag PAI, and sequenced a 39 kb cosmid clone containing the left ‘cagII’ half of this PAI. Encoded in cagII were four proteins each with homology to four components of multiprotein complexes of Bordetella pertussis (‘Ptl’), Agrobacterium tumefaciens (‘Vir’), and conjugative plasmids (‘Tra’) that help deliver pertussis toxin and T (tumour inducing) and plasmid DNA, respectively, to target eukaryotic or prokaryotic cells, and also homologues of eukaryotic proteins that are involved in cytoskeletal structure. To the left of cagII in this cosmid were genes for homologues of HslU (heat-shock protein) and Era (essential GTPase); to the right of cagII were homologues of genes for a type I restriction endonuclease and ion transport functions. Deletion of the cag PAI had no effect on synthesis of the vacuolating cytotoxin, but this deletion and several cag insertion mutations blocked induction of synthesis of proinflammatory cytokine IL-8 in gastric epithelial cells. Comparisons among H. pylori strains indicated that cag PAI gene content and arrangement are rather well conserved. We also identified two genome rearrangements with end-points in the cag PAI. One, in reference strain NCTC11638, involved IS605, a recently described transposable element (as also found by others). Another rearrangement, in 3 of 10 strains tested (including type strain NCTC11637), separated the normally adjacent cagA and picA genes and did not involve IS605. Our results are discussed in terms of how cag-encoded proteins might help trigger the damaging inflammatory responses in the gastric epithelium and possible contributions of DNA rearrangements to genome evolution.     

幽门螺杆菌Ⅳ型分泌系统（T4SS）研究进展

Ⅳ型分泌系统（T4SS）广泛存在于革兰阴性菌中,细菌可通过该系统将生物大分子或毒力因子等运输至靶细胞中并发挥相应功能。目前在H. pylori中已发现了至少三种T4SS,其中研究较为透彻的是cag致病岛（cagPAI）编码的cagT4SS系统,此外可塑区编码的tfs3系统和comB系统也有相关的报道。H. pylori的T4SS作为其与致病相关的重要结构已受到很多学者关注,对该菌T4SS系统的研究有助于进一步明确H. pylori的致病机制,并为临床诊断和治疗相关胃十二指肠疾病提供新的靶点。本文将对H. pylori的T4SS相关研究进展作一简要综述。     

cag pathogenicity island of Helicobacter pylori in Korean children

Background. cag pathogenicity island is reported to be a major virulence factor of Helicobacter pylori. The aim of this study was to investigate the status of cag pathogenicity island genes and gastric histology in Korean children with H. pylori gastritis. Methods. Helicobacter pylori DNA was extracted from antral biopsy specimens from 25 children with H. pylori gastritis. Specific polymerase chain reaction assays were used for four genes of cag pathogenicity island. The features of gastritis were scored in accordance with the updated Sydney System. Results. cagA was present in 23 (92%) of 25 children, and cagE in 24 (96%). Twenty‐two (88%) children were cagT positive and 19 (76%) virD4 positive. All of the selected genes of the cag pathogenicity island were present in 17 (68%) children and completely deleted in one child. There were no differences in neutrophil activity and chronic inflammation between children infected with intact cag pathogenicity island strains and those with partially or totally deleted‐cag pathogenicity island strains. Conclusion. cag pathogenicity island is not a uniform, conserved entity in Korea. Completeness of cag pathogenicity island may not be the major factor to determine the severity of H. pylori gastritis in children.     

Characterization of peptidoglycan hydrolase in Cag pathogenicity island of Helicobacter pylori

The Cag Type IV secretion apparatus proteins in Helicobacter pylori can mediate the injection of effector CagA protein into eukaryotic target cells. Although this apparatus forms an important pathway for bacterium–host interaction, its assembly process in vivo is poorly understood, and the proteins which contribute to break the bacterial cell walls in Cag-PAI have not yet been identified. The cagγ gene in Cag-PAI is a unique member that contains a conserved SLT catalysis domain, which makes it an attracting question whether cagy gene has the capacity to digest the bacterial cell wall. In the current study, therefore, the cagγ gene was cloned from the H. pylori NCTC 11637 and expressed in Escherichia coli, and its lytic effect on cell walls in vitro was observed. Results indicated that Cagγ protein has a lytic activity against bacterial cell walls. An allelic-exchange mutant (Δcagγ) was further constructed to investigate the relationship between Cagγ and effector CagA translocation. These results suggested that Cagγ contributed to the assembly of Cag Type IV secretion apparatus by digesting the peptidoglycan meshwork of bacterial cell walls.     

Horizontal transfer of virulence genes encoded on the Enterococcus faecalis pathogenicity island

Enterococcus faecalis, a leading cause of nosocomial antibiotic resistant infections, frequently possesses a 150 kb pathogenicity island (PAI) that carries virulence determinants. The presence of excisionase and integrase genes, conjugative functions and multiple insertion sequence elements suggests that the PAI, or segments thereof, might be capable of horizontal transfer. In this report, the transfer of the E. faecalis PAI is demonstrated and a mechanism for transfer elucidated. In filter matings, chloramphenicol resistance was observed to transfer from strain MMH594b, a clinical isolate possessing the PAI tagged with a cat marker, to OG1RF (pCGC) with a frequency of 3.2 x 10(-10) per donor. Secondary transfer from primary transconjugant TCRFB1 to strain JH2SS in filter and broth matings occurred with a frequency of 1 and 2 x 10(-1) per donor respectively. Analysis of the transconjugants demonstrated that a 27,744 bp internal PAI segment was capable of excision and circularization in the donor, and is mobilized as a cointegrate with a pTEF1-like plasmid. High-frequency transfer also occurred from TCRFB1 to JH2SS during transient colonization of the mouse gastrointestinal tract. This is the first demonstration of the horizontal transfer of PAI-encoded virulence determinants in E. faecalis and has implications for genome evolution and diversity.     

The presence of the cag pathogenicity island is associated with increased superoxide anion radical scavenging activity by Helicobacter pylori

Reactive oxygen species (ROS) generated by Helicobacter pylori infection have been suggested to be important factors in induction of gastric malignancies. Utilizing electron spin resonance spectrometry, H. pylori-dependent radical formation and hydroxyl- and superoxide-anion radical scavenging activity was investigated. In contrast to previous reports, we found that H. pylori does not produce ROS, but displays superoxide scavenging activity. This scavenging activity was increased in cag-positive H. pylori strains when compared to strains lacking an intact cag pathogenicity island, and was dependent on enzyme activity. We hypothesize that the increased scavenging activity of cag-positive H. pylori strains is an adaptation to the increased inflammatory response associated with the cag-positive genotype of H. pylori.     

Quantitative detection of Helicobacter pylori in water samples by real-time PCR amplification of the cag pathogenicity island gene, cagE

Aims:  A new real-time PCR assay that simultaneously amplifies a 102-bp fragment of the cagE gene from Helicobacter pylori and a new internal positive control containing a specific sequence of the gyrB gene from Aeromonas hydrophila , was developed and validated for the detection of H. pylori in environmental samples.
Methods and Results:  The specificity, limits of detection and quantification, repeatability, reproducibility, and accuracy of the method were calculated. The resulting values confirmed the applicability of the method for the quantitative detection of H. pylori . The feasibility of the method was also evaluated by testing 13 pyloric antrum-positive biopsies and 69 water samples, including potable (10), surface (19) and wastewater (40) matrices. The results showed that all the biopsies and 3 of the 40 wastewater samples analysed were positive.
Conclusions:  This real-time PCR method provides a sensitive, specific, and accurate method for the rapid quantification of H. pylori in environmental samples.
Significance and Impact of the Study:  The PCR diagnostic system proposed in this work, provides a suitable tool for the quantitative detection of H. pylori in environmental samples and can be useful for verifying the role of water as a potential route of its transmission.     

The ShiA protein encoded by the Shigella flexneri SHI-2 pathogenicity island attenuates inflammation

Shigella spp. are the aetiologic agents of dysentery, a severe diarrhoeal syndrome characterized by acute inflammation in the colon. The inflammatory response, which includes recruitment of polymorphonuclear leukocytes (PMN), damages the colonic mucosa and exacerbates the infection. Shigella encodes a pathogenicity island (PAI), SHI-2, which is localized in a region of the chromosome linked to the induction of inflammation. Surprisingly, SHI-2 deletion mutants induce a stronger inflammatory response than wild-type Shigella as measured by increased villus blunting, increased PMN infiltration and induction of apoptosis in a rabbit ileal loop model of shigellosis. Mutational analysis mapped the hyper-inflammatory phenotype to a single gene, shiA. Similar to SHI-2 deletion mutants, infection with a shiA mutant strain induces dramatically elevated levels of inflammation when compared to the wild-type strain. Furthermore, infection with a wild-type strain containing multiple copies of shiA results in fewer infiltrating PMN and apoptotic cells, as well as preservation of a normal villus architecture at the site of infection, thus acting in a dominant fashion over the pro-inflammatory mechanisms of Shigella. The molecular mechanism of action of ShiA is independent of any in vitro phenotype associated with Shigella virulence. Our data suggest that ShiA allows Shigella to attenuate the host inflammatory response in a novel manner.     

Helicobacter pylori encoding the pathogenicity island activates matrix metalloproteinase 1 in gastric epithelial cells via JNK and ERK

Helicobacter pylori colonizes the human gastric epithelium and induces an inflammatory response that is a trigger for gastric carcinogenesis. Matrix metalloproteinases (MMPs) have recently been shown to be up-regulated in gastric epithelial cells infected with H. pylori and might contribute to the pathogenesis of peptic ulcer. The aim of this study was to extend the knowledge about the effect of H. pylori infection on MMP-1 expression by gastric epithelial cells, the kinetics of induction, the pathogenetic properties of the bacterium, and the intracellular signaling pathways required for MMP-1 up-regulation. Expression of MMP-1 was induced more than 10-fold by co-culture of AGS+cells with H. pylori strains carrying the pathogenicity island (PAI). H. pylori strains with mutations in the PAI and a defective type IV secretion system had no effect on MMP-1. Double immunofluorescence revealed strong MMP-1 staining in epithelial cells of gastric biopsies at sites of bacterial attachment. In vitro, MMP-1 is up-regulated by interleukin-1beta and tumor necrosis factor-alpha, but these regulatory mechanisms are not operating in H. pylori infection as shown by inhibitory antibodies. Specific inhibitors of JNK kinase and ERK1/2 kinase were found to suppress the H. pylori-induced MMP-1 expression and activity. AGS cells treated with antisense MMP-1 showed a significantly reduced potential to degrade reconstituted basement membrane. Our results suggest that in gastric epithelial cells, H. pylori up-regulates MMP-1 in a type IV secretion system-dependent manner via JNK and ERK1/2. Induction of MMP-1 is further implicated in complex processes induced by H. pylori, resulting in tissue degradation and remodeling of the gastric mucosa.