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1.
Analysis of iceA1 transcription in Helicobacter pylori   总被引:7,自引:0,他引:7  
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iceA1 in Helicobacter pylori is a homolog of nlaIIIR, which encodes the CATG-specific restriction endonuclease NlaIII in Neisseria lactamica. Analysis of iceA1 sequences from 49 H.pylori strains shows that a full-length NlaIII-like ORF is present in 10 strains, including CH4, but in other strains, including strain 60190, the ORFs are truncated due to a variety of mutations. Our goal was to determine whether iceA1 can encode a NlaIII-like endonuclease. Overexpression in Escherichia coli of iceA1 from CH4, but not from 60190, yielded NlaIII-like activity, indicating that the full-length iceA1 is a functional endonuclease gene. Repair of the iceA1 frameshift mutation in strain 60190 and its expression in E.coli yielded functional NlaIII-like activity. We conclude that iceA1 in CH4 is a functional restriction endonuclease gene, while iceA1 in 60190 is not, due to a frameshift mutation, but that its repair restores its restriction endonuclease activity.  相似文献   

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Yu X  McBride JW  Zhang X  Walker DH 《Gene》2000,246(1-2):59-68
The genetic organization and sequence heterogeneity of the iceA locus of Helicobacter pylori was studied, and the existence of two distinct gene families, iceA1 and iceA2, at this locus was confirmed. iceA1 has significant sequence homology to nlaIIIR, encoding an endonuclease in Neisseria lactamica, but the similarity at the protein level is limited, due to frameshift mutations of iceA1 in most H. pylori strains. In only five of the 19 iceA1 strains studied, a full-length open reading frame (ORF), capable of encoding a 228aa protein, with 52% homology to NlaIII was observed. The region upstream of iceA2 is highly variable in length, containing up to 15 copies of 8bp tandem repeats. iceA2 can encode proteins of 24, 59, 94, or 129 amino acids, consisting of 14 and 10aa domains, conserved in all iceA2 strains, flanking 0, 1, 2, or 3 copies of a 35aa cassette. This 35aa cassette consists of domains of 13, 16 and 6aa, respectively. The 13aa and 6aa domains are highly conserved, but the 16aa domain exists in two variants. In total, five distinct iceA2 subtypes were defined. Database searches did not reveal any homologous sequences. Recombinant IceA1 and IceA2 proteins were expressed in Escherichia coli, confirming the predicted ORFs. Genotype-specific PCR primers permitted iceA genotyping in 318 (99. 1%) of a worldwide collection of 321 H. pylori strains. The conserved sizes of the amplification products confirmed the worldwide distribution of discrete variants of iceA1 and iceA2.  相似文献   

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A recently identified Helicobacter pylori gene, hrgA, was previously reported to be present in 70 (33%) of 208 strains examined (T. Ando, T. M. Wassenaar, R. M. Peek, R. A. Aras, A. I. Tschumi, L.-J. Van Doorn, K. Kusugami, and M. J. Blaser, Cancer Res. 62:2385-2389, 2002). Sequence analysis of nine such strains indicated that in each strain hrgA replaced hpyIIIR, which encodes a restriction endonuclease and which, together with the gene for its cognate methyltransferase, constitutes the hpyIII locus. As a consequence of either the hrgA insertion or independent mutations, hpyIIIM function was lost in 11 (5%) of the 208 strains examined, rendering chromosomal DNA sensitive to MboI digestion. The evolutionary history of the locus containing either hpyIII or hrgA was reconstructed. By homologous recombination involving flanking sequences, hrgA and hpyIIIR can replace one another in the hpyIII locus, and there is simultaneous replacement of several flanking genes. These findings, combined with the hpyIM/iceA2 locus discovered previously, suggest that the two most strongly conserved methylase genes of H. pylori, hpyIIIM and hpyIM, are both preceded by alternative genes that compete for presence at their loci.  相似文献   

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Infection with Helicobacter pylori strains harboring determinants of pathogenicity may lead to a strong inflammatory response in gastric mucosa. In this work, we examined the frequency of the cagA, vacA and iceA genotypes in H. pylori strains isolated from Brazilian patients and correlated these with the clinical manifestations. H. pylori was isolated from 165 patients [30 with non-ulcer dyspepsia cases (NUD); 93 peptic ulcer disease (PUD): 31 gastric ulcers (GU) and 62 duodenal ulcer disease (DU); 18 with erosive gastritis (EG); and 24 gastroesophageal reflux disease (GERD)]. Allelic variants of cagA, vacA and iceA were identified using the polymerase chain reaction. More than one H. pylori strain was detected in 28 cases (17%), and these were excluded from the statistical analysis. We were unable to confirm an association between iceA status and clinical outcome. There was a strong association between the genotype cagA-positive vacA s1 and PUD. However, logistic regression analysis showed that vacA s1 was the only predictive factor for PUD (OR=4.19; 95% CI 1.95-8.98). The presence of the less virulent strain vacA s2 was related to GERD (OR=8.59; 95% CI 2.85-25.91). Our results support the hypothesis that virulent strains may protect against the development of GERD.  相似文献   

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We have isolated a novel restriction endonuclease, Hpy188I, from Helicobacter pylori strain J188. Hpy188I recognizes the unique sequence, TCNGA, and cleaves the DNA between nucleotides N and G in its recognition sequence to generate a one-base 3' overhang. Cloning and sequence analysis of the Hpy188I modification gene in strain J188 reveal that hpy188IM has a 1299-base pair (bp) open reading frame (ORF) encoding a 432-amino acid product. The predicted protein sequence of M.Hpy188I contains conserved motifs typical of aminomethyltransferases, and Western blotting indicates that it is an N-6 adenine methyltransferase. Downstream of hpy188IM is a 513-bp ORF encoding a 170-amino acid product, that has a 41-bp overlap with hpy188IM. The predicted protein sequence from this ORF matches the amino acid sequence obtained from purified Hpy188I, indicating that it encodes the endonuclease. The Hpy188I R-M genes are not present in either strain of H. pylori that has been completely sequenced but are found in two of 11 H. pylori strains tested. The significantly lower G + C content of the Hpy188I R-M genes implies that they have been introduced relatively recently during the evolution of the H. pylori genome.  相似文献   

9.
The Helicobacter pylori CagA protein is translocated into gastric epithelial cells through a type IV secretion system (TFSS), and published studies suggest CagA is critical for H. pylori-associated carcinogenesis. CagA is thought to be necessary and sufficient to induce the motogenic response observed in response to CagA+ strains, as CagA interacts with proteins involved in adhesion and motility. We report that H. pylori strain 60190 stimulated AGS cell motility through a CagA- and TFSS-dependent mechanism, because strains 60190DeltacagA or 60190DeltacagE (TFSS-defective) did not increase motility. The JNK pathway is critical for H. pylori-dependent cell motility, as inhibition using SP600125 (JNK1/2/3 inhibitor) or a JNK2/3-specific inhibitor blocked motility. JNK mediates H. pylori-induced cell motility by activating paxillin, because JNK inhibition blocked paxillinTyr-118 phosphorylation, and paxillin expression knockdown completely abrogated bacteria-induced motility. Furthermore, JNK and paxillinTyr-118 were activated by 60190DeltacagA but not 60190DeltacagE, demonstrating CagA-independent signaling critical for cell motility. A beta1 integrin-blocking antibody significantly inhibited JNK and paxillinTyr-118 phosphorylation and cell scattering, demonstrating that CagA-independent signaling required for cell motility occurs through beta1. The requirement of both Src and focal adhesion kinase for signaling and motility further suggests the importance of integrin signaling in H. pylori-induced cell motility. Finally, we show that JNK activation occurs independent of known upstream kinases and signaling molecules, including Nod1, Cdc42, Rac1, MKK4, and MKK7, which demonstrates novel signaling leading to JNK activation. We report for the first time that H. pylori mediates CagA-independent signaling that promotes cell motility through the beta1 integrin pathway.  相似文献   

10.
The clinical outcome of Helicobacter pylori infection has been particularly associated with virulence genotypes. These genotypes are useful as molecular markers in the identification of patients that are infected and at high risk for developing more severe gastric pathologies. Our main objective was to determine the prevalence of virulence genotypes cagA, vacA, iceA and babA2 of H. pylori, in patients with functional dyspepsia who are infected with the bacteria. H. pylori genotypes babA2 and cagA as well as vacA and iceA allelic variants were identified by PCR in 122 isolates resulting from 79 patients with functional dyspepsia. A high prevalence of genes cagA+ (71%), vacAs1am1 (34%), babA2 (57%) and iceA1 (87%) was found. The most frequent combined genotype found were cagA+/vacAs1am1/babA2+/iceA1 and cagA-/vacAs1am1/babA2+/iceA1, regardless of any family history of gastric cancer or MALT lymphoma. The very virulent genotype cagA+/vacAs1am1/babA2+/iceA1 prevailed in the studied patients with functional dyspepsia. Our results provide information about the prevalence of four of the more important virulent factors and constitute new evidence on the prevalence of the most virulent H. pylori genotype in patients with functional dyspepsia.  相似文献   

11.
Distinctiveness of genotypes of Helicobacter pylori in Calcutta, India   总被引:13,自引:0,他引:13       下载免费PDF全文
The genotypes of 78 strains of Helicobacter pylori from Calcutta, India (55 from ulcer patients and 23 from more-benign infections), were studied, with a focus on putative virulence genes and neutral DNA markers that were likely to be phylogenetically informative. PCR tests indicated that 80 to 90% of Calcutta strains carried the cag pathogenicity island (PAI) and potentially toxigenic vacAs1 alleles of the vacuolating cytotoxin gene (vacA), independent of disease status. This was higher than in the West (where cag PAI(+) vacAs1 genotypes are disease associated) but lower than in east Asia. The iceA2 gene was weakly disease associated in Calcutta, whereas in the West the alternative but unrelated iceA1 gene at the same locus is weakly disease associated. DNA sequence motifs of vacAm1 (middle region) alleles formed a cluster that was distinct from those of east Asia and the West, whereas the cagA sequences of Calcutta and Western strains were closely related. An internal deletion found in 20% of Calcutta iceA1 genes was not seen in any of approximately 200 strains studied from other geographic regions and thus seemed to be unique to this H. pylori population. Two mobile DNAs that were rare in east Asian strains were also common in Calcutta. About 90% of Calcutta strains were metronidazole resistant. These findings support the idea that H. pylori gene pools differ regionally and emphasize the potential importance of studies of Indian and other non-Western H. pylori populations in developing a global understanding of this gastric pathogen and associated disease.  相似文献   

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Q Xu  R M Peek  Jr  G G Miller    M J Blaser 《Journal of bacteriology》1997,179(21):6807-6815
To understand mechanisms of DNA methylation in Helicobacter pylori, a human pathogen associated with peptic ulcer disease and gastric adenocarcinoma, we cloned a putative DNA methyltransferase gene, hpyIM. This gene contains a 990-bp open reading frame encoding a 329-amino-acid protein, M.HpyI. Sequence analysis revealed that M.HpyI was closely related to CATG-recognizing adenine DNA methyltransferases, including M.NlaIII in N. lactamica. hpyIM was present in all H. pylori strains tested. DNA from wild-type H. pylori strains was resistant to digestion by SphI and NlaIII, which recognize DNA at sites containing CATG, whereas their isogenic hpyIM mutants were susceptible, indicating lack of modification. Overexpression of hpyIM in Escherichia coli rendered DNA from these cells resistant to NlaIII digestion, confirming the role of hpyIM in modifying CATG sites. We conclude that hpyIM encodes a DNA methyltransferase, M.HpyI, that is well conserved among diverse H. pylori strains and that modifies H. pylori genomes at CATG sites.  相似文献   

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为探讨复旦大学附属华东医院(以下简称本院)分离培养的幽门螺杆菌(Helicobacter pylori,H. pylori)的耐药性、毒力和感染特征与其生物膜形成能力的相关性,收集2014年12月-2015年6月于本院消化内镜中心的胃活检组织标本及相应临床病例资料,分离培养获得幽门螺杆菌,分析菌株的耐药性、毒力基因型、临床病例特征。结果显示,从胃活检组织样本中共分离培养28株幽门螺杆菌,对左氧氟沙星(levofloxacin,LEV)、甲硝唑(metronidazole,MTZ)和克拉霉素(clarithromycin,CLA)的耐药率分别为32%、75%和11%,未发现阿莫西林(amoxicillin,AMX)耐药。单一药物耐药17株(17/28,61%),双重耐药10株(10/28,36%)。毒力基因cagA、oipA和vacAs1检出率为100%,未检出vacAs2。基因型vacAs1m1占39%(11/28),vacAs1m2占61%(17/28);iceA1占54%(15/28),iceA2占21%(6/28),iceA1A2占25%(7/28);dupA占36%(10/28)。28株菌株均能形成生物膜,但能力不尽相同。单因素及独立样本t检验分析显示,45~59岁、iceA1dupA基因型和甲硝唑敏感菌株形成生物膜的能力较强。结果提示,本院分离的幽门螺杆菌对甲硝唑耐药率最高,双重耐药不容忽视。菌株主要毒力基因型为cagA、oipA、vacAs1m2。幽门螺杆菌的生物膜形成能力与患者年龄有关,45~59岁组较强;携带毒力基因iceA1的菌株生物膜形成能力强;dupA基因型及甲硝唑耐药与菌株生物膜形成呈负相关。  相似文献   

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