Functional analysis of iceA1, a CATG-recognizing restriction endonuclease gene in Helicobacter pylori

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.     

Helicobacter pylori iceA, clinical outcomes, and correlation with cagA: a meta-analysis

Background

Although the iceA (induced by contact with epithelium) allelic types of Helicobacter pylori have been reported to be associated with peptic ulcer, the importance of iceA on clinical outcomes based on subsequent studies is controversial. The aim of this study was to estimate the magnitude of the risk for clinical outcomes associated with iceA.

Methods

A literature search was performed using the PubMed and EMBASE databases for articles published through April 2011. Published case-control studies examining the relationship between iceA and clinical outcomes (gastritis, peptic ulcer, including gastric ulcer and duodenal ulcer, and gastric cancer) were included.

Results

Fifty studies with a total of 5,357 patients were identified in the search. Infection with iceA1-positive H. pylori increased the overall risk for peptic ulcer by 1.26-fold (95% confidence interval [CI], 1.09–1.45). However, the test for heterogeneity was significant among these studies. Sensitivity analysis showed that the presence of iceA1 was significantly associated with peptic ulcer (odds ratio [OR] = 1.25, 95% CI = 1.08–1.44). The presence of iceA2 was inversely associated with peptic ulcer (OR = 0.76, 95% CI = 0.65–0.89). The presence of iceA was not associated with gastric cancer. Most studies examined the cagA status; however, only 15 studies examined the correlation and only 2 showed a positive correlation between the presence of cagA and iceA1.

Conclusion

Our meta-analysis confirmed the importance of the presence of iceA for peptic ulcer, although the significance was marginal.     

Clinical relevance of the cagA,vacA and iceA genotypes of Helicobacter pylori in Brazilian clinical isolates

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.     

幽门螺杆菌毒力基因cagA、vacA和iceA与胃十二指肠疾病的关系

目的了解长春地区就医人群中幽门螺杆菌(Helicobacterpylori,H.pylori)毒力基因分布,分析本地区H.pylori主要毒力基因型与胃十二指肠疾病关系。方法将胃黏膜标本培养分离H.pylori并运用聚合酶链式反应(polymerase chain reaction,PCR)对H.pylori的三种毒力基因cagA、vacA(s1a、s1b、s1c、s2、m1a、m1b、m2、i1和i2)、iceA(iceA1和iceA2)及其亚型进行检测,分析H.pylori的毒力基因型与疾病之间的关系。结果分离H.pylori 60株,标本依据病理诊断结果分为4组:消化性溃疡组(peptic ulcer disease,PUD)25株、非溃疡性消化不良组(non-ulcer dyspepsia,NUD)9株、糜烂性胃炎组(gastritis erode,GE)15株和胃癌组(gastric cancer,GC)11株。cagA基因的阳性率为90.0%,与阴性相比在疾病中的分布差异有统计学意义(x~2=8.4440,P=0.0270)。vacA基因的s1和s2亚型的阳性率分别为86.7%和13.3%。在s1阳性菌株中,s1c等位基因检出率最高为33.3%,其次s1a检出率为23.3%,而s1b在NUD和GC中检出率为0.0%。vacA基因m区m1检出率为43.3%(m1a和m1b检出率均为21.7%)和m2检出率为56.7%。vacA基因i区等位基因i1和i2的检出率分别为78.3%和21.7%,i1明显高于i2,可能是胃肠道疾病的主要毒力因子,但与疾病的类型无明显相关性。iceA1和iceA2的阳性率分别为80.0%和16.7%,iceA1/iceA2混合型的阳性率为3.3%。三种毒力基因混合型以cagA~+/vacAs1m1i1/iceA1的菌株最多,占总数的31.7%,其次为cagA~+/vacAs1m2i1/iceA1,占总数的28.3%。结论本地区H.pylori毒力因子cagA、vacA和iceA都与胃十二肠疾病的发生密切相关,但与疾病的类型无关。H.pylori毒力基因混合型cagA+/vacAs1m1i1/iceA1和cagA~+/vacAs1m2i1/iceA1是胃十二肠疾病疾病中最主要的优势基因型,其分布也与疾病的类型无密切相关性。     

幽门螺杆菌临床分离株的REP-PCR分析

应用REP-PCR对来自20例单纯性胃炎和20例消化性溃疡病人的幽门螺杆菌菌株进行基因分型,并运用SAS软件进行聚类分析。结果显示这些菌株按照基因型被分为两大类,但两种来源的菌株在两大类中的比例无明显差异(P＞0.1),提示幽门螺杆菌临床分离株REP-PCR基因型与致病性之间不存在明显相关性。 REP-PCR Analysis of Helicobacter pylori Clinical Strains PENG Ying1,LU Jian-xin1,YE Si-ying2,HUANG Qing-hua2 1.Institute of Cellular and Molecular Medicine,Wenzhou Medical College,Wenzhou,Zhejiang 325027,China; 2.Department of Microbiology,Tongji Medical College,Wuhan 430030,China Abstract:Helicobacter pylori strains isolated from 20 gastritis patients and 20 peptic ulcer patients was genotyped by REP-PCR and was clustered with SAS software.These strains are devided into two categories according to their genotype.But the rate of the two sources of strain in the two categories shows no apparent difference(P＞0.1),indicating that there is no significant close relationship between the genotype and the pathogenicity. Key words：Helicobacter pylori;REP-PCR;genotype     

Comparative Genome Analysis of Helicobacter pylori Strains

DNA macroarrays were used to characterize 17 Helicobacter pylori strains isolated in four geographic regions of Russia (Moscow, St. Petersburg, Kazan, and Novosibirsk). Of all genes, 1272 (81%) proved to occur in all strains and to constitute a functional core of the genome, and 293 (18.7%) were strain-specific and greatly varied among the H. pylori strains. Most (71%) of the latter had unknown functions; the remainder included restriction–modification genes (3–9%), transposition genes (2–4%), and genes coding for outer membrane proteins (2–4%). The Russian H. pylori strains did not differ in genome organization or in the number and distribution of strain-specific genes from strains isolated in other countries.     

Analysis of Surface-Exposed Outer Membrane Proteins in Helicobacter pylori

More than 50 Helicobacter pylori genes are predicted to encode outer membrane proteins (OMPs), but there has been relatively little experimental investigation of the H. pylori cell surface proteome. In this study, we used selective biotinylation to label proteins localized to the surface of H. pylori, along with differential detergent extraction procedures to isolate proteins localized to the outer membrane. Proteins that met multiple criteria for surface-exposed outer membrane localization included known adhesins, as well as Cag proteins required for activity of the cag type IV secretion system, putative lipoproteins, and other proteins not previously recognized as cell surface components. We identified sites of nontryptic cleavage consistent with signal sequence cleavage, as well as C-terminal motifs that may be important for protein localization. A subset of surface-exposed proteins were highly susceptible to proteolysis when intact bacteria were treated with proteinase K. Most Hop and Hom OMPs were susceptible to proteolysis, whereas Hor and Hof proteins were relatively resistant. Most of the protease-susceptible OMPs contain a large protease-susceptible extracellular domain exported beyond the outer membrane and a protease-resistant domain at the C terminus with a predicted β-barrel structure. These features suggest that, similar to the secretion of the VacA passenger domain, the N-terminal domains of protease-susceptible OMPs are exported through an autotransporter pathway. Collectively, these results provide new insights into the repertoire of surface-exposed H. pylori proteins that may mediate bacterium-host interactions, as well as the cell surface topology of these proteins.     

Helicobacter pylori in pediatrics

This article reviewed the important publications on Helicobacter pylori research with children between April 2010 and March 2011. The most interesting studies in the last year lend further weight to the evidence for vertical transmission of H. pylori. The discovery of a potential role for jhp0562, the gene which encodes for the cell envelope protein glycosyltransferase, in the progression to peptic ulcer disease is also very interesting as it may provide a novel way to distinguish children at risk of peptic ulcer disease from those who are not, and so determine those who requires treatment to eradicate H. pylori. The rise in non-H. pylori-associated ulcers and erosions continues to be reported with no apparent risk factors for these ulcers identified to date. High levels of treatment failure continue to be reported, and there remains an urgent need for more effective treatment regimes for children.     

Helicobacter pylori in Pediatrics

This review summarizes important pediatric studies published from April 2011 up to March 2012. Proteomics profile of ulcerogenic Helicobacter pylori strains was defined in the most interesting study of the last year. The antigen stool test is becoming the "gold standard" in prevalence studies, and according to the last epidemiologic studies, the prevalence of H.?pylori infection in childhood is not decreasing any more in the developed world. The resistance rate of H.?pylori strains is high in children. Therefore, among other important issues concerning H.?pylori in pediatrics, guidelines published by ESPGHAN and NASPGHAN last year also recommended culture and susceptibility testing before first-line treatment in areas with high or unknown antibiotic resistance rates.     

Copper Promotes TFF1-Mediated Helicobacter pylori Colonization

The trefoil peptides (TFF1, TFF2 and TFF3) are a family of small highly conserved proteins that play an essential role in epithelial regeneration within the gastrointestinal tract, where they are mainly expressed. TFF1 expression is strongly induced after mucosal injury and it has been proposed that tff1 functions as a gastric tumor suppressor gene. Several studies confirm that tff1 expression is frequently lost in gastric cancer because of deletions, mutations or methylation of the tff1 promoter. Infection by Helicobacter pylori (H. pylori) results in chronic gastritis and it can lead to the development of gastric or duodenal ulcers. Moreover, it is known that there is a strong link to the development of gastric cancer. It has been shown that H. pylori interacts with the dimeric form of TFF1 and that the rough form of lipopolysaccharide mediates this interaction. We have previously reported that the carboxy-terminus of TFF1 is able to specifically bind copper ions (Cu) and that Cu binding favours the homodimerization of the peptide, thus enhancing its motogenic activity. Here, we report that the Cu-TFF1 cuprocomplex promotes adherence of H. pylori to epithelial cells. Adherence of H. pylori to gastric adenocarcinoma cells, AGS AC1 cells, induced to hyper-express TFF1 was enhanced compared to noninduced cells. Copper further promoted this interaction. A H. pylori mutant unable to bind TFF1 did not show enhanced infection of induced cells. Cu treatment induced a thickening of the mucus layer produced by the colorectal adenocarcinoma mucus secreting, goblet cells, HT29-E12 and promoted H. pylori colonisation. Finally, SPR analysis shows that the C-terminus of TFF1, involved in the binding of copper, is also able to selectively bind H. pylori RF-LPS.     

Helicobacter pylori motility

Motility is essential for Helicobacter pylori colonization. This review discusses the biochemistry, genetics and genomics of the H. pylori flagellum, and compares these features with well-characterized bacteria.