<Emphasis Type="Italic">Helicobacter pylori</Emphasis> outer membrane vesicles involvement in the infection development and <Emphasis Type="Italic">Helicobacter pylori</Emphasis>-related diseases

Helicobacter pylori - (H. pylori) play a role in the pathogenesis of gastritis, gastric and duodenal ulcers as well as gastric cancer. A possible involvement of outer membrane vesicles (OMVs) produced by H. pylori in the distribution of bacterial antigens through the gastric epithelial barrier and their role in the development of local and systemic host inflammatory and immune responses has been suggested. OMVs contain various biologically active compounds, which internalize into host cells affecting signaling pathways and promoting apoptosis of gastric epithelial and immunocompetent cells. OMVs-associated H. pylori virulence factors may strengthen or downregulate the immune responses leading to disease development. This review describes the biological importance of H. pylori OMVs and their role in the course of H. pylori infections, as well as H. pylori related local and systemic effects.     

Lymphocyte proliferative response to <Emphasis Type="Italic">Helicobacter pylori</Emphasis> antigens in <Emphasis Type="Italic">H. pylori</Emphasis>-infected patients

Helicobacter pylori (Hp) contributes to the development of gastric and extra-gastric diseases such as autoimmune thyroiditis (AT), and causes persistent life-long infection despite local and systemic immune response. We determined the specific cellular immune response to Hp antigens and PWM (control mitogen) in two groups of Hp infected patients - group A (n = 21), involving patients with autoimmune thyroiditis and group B (n = 13) of patients without AT - using modified lymphocyte transformation test before and after eradication therapy in comparison with healthy controls (group C, n = 15). Immune reactivity to the majority of Hp antigens (aHp, hHp, HpAg, CagA) was significantly lower in group B before eradication therapy in comparison with healthy Hp negative controls. A significant increase in immune reactivity was observed in group B to certain Hp antigens after successful eradication. The same levels (but insignificant) of immune reactivity were shown in group A. Our results indicate that Hp can cause the inhibition of the specific cellular immune response in Hp infected patients with or without autoimmune diseases such as AT, which can be abrogated by successful eradication of Hp. Lymphocyte transformation test appears to be a good tool for detection of immune memory cellular response in patients with Hp infection.     

Overexpression of <Emphasis Type="Italic">spoT</Emphasis> gene in coccoid forms of clinical <Emphasis Type="Italic">Helicobacter pylori</Emphasis> isolates

Helicobacter pylori (H. pylori) can convert to coccoid form in unfavorable conditions or as a result of antibiotic treatment. In order to adapt to harsh environments, H. pylori requires a stringent response which, encoded by the spoT gene, has a bifunctional enzyme possessing both (p)ppGpp synthetic and degrading activity. Our goal in this study was to compare spoT gene expression in spiral and induced coccoid forms of H. pylori with use of amoxicillin. First, clinical isolate coccoid forms were induced with amoxicillin; then, the viability test was analyzed by flow cytometer. After RNA extraction, cDNA synthesis and designing a specific primer for spoT gene, evaluation of the desired gene expression in both forms were studied. Bacterial isolates exposed to amoxicillin at MIC and 1/2 MIC induced morphological conversion better and faster than other MIC concentration. The expression of spoT gene was significantly downregulated in spiral forms of H. pylori, while the gene expression was upregulated and + 30.3-fold changes was seen in coccoid forms of bacterium. To summarize, spoT gene is one of the key factors for antibiotic resistance and its enhanced expression in coccoid form can be a valuable diagnostic marker for recognition of H. pylori during morphological conversion.     

Characterization of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> adhesin thiol peroxidase (HP0390) purified from <Emphasis Type="Italic">Escherichia coli</Emphasis>

The antioxidant protein, adhesin thiol peroxidase (HpTpx or HP0390), plays an important role in enabling Helicobacter pylori to survive gastric oxidative stress. The bacterium colonizes the host stomach and produces gastric cancer. However, little information is available about the biochemical characteristics of HpTpx. We expressed recombinant HpTpx in Escherichia coli, purified to homogeneity, and characterized it. The results showed that HpTpx existed in a monomeric hydrodynamic form and the enzyme fully retained its peroxidase and antioxidant activities. The catalytic reaction of the enzyme was similar to an atypical 2-cysteine peroxiredoxin (Prx). The conformation of the enzyme was observed in the presence and absence of dithiothreitol (DTT); similar to other known thiol peroxidases, conformational change was observed in HpTpx by the addition of DTT.     

Association of <Emphasis Type="Italic">iceA</Emphasis> and <Emphasis Type="Italic">babA</Emphasis> genotypes in <Emphasis Type="Italic">Helicobacter pylori</Emphasis> strains with patient and strain characteristics

Data on the geographic prevalence of Helicobacter pylori iceA and babA alleles in Eastern Europe are still relatively scant. The aim of this study was to evaluate the prevalence of iceA and babA genotypes in Bulgarian symptomatic patients. The iceA and babA genotypes were evaluated by PCR with pure cultures in strains from 196 and 181 patients, respectively. Mixed infections were found in 10.2% of all 196 patients. Prevalence of H. pylori genotypes in patients with single-strain infections was 69.3% for iceA1, 30.7% for iceA2, 82.4% for cagA ⁺, 89.2% for vacA s1, 10.8% for vacA s2, 39.8% for vacA m1, 60.2% for vacA m2 and 48.8% for babA2. Within the iceA1 positive strains, 94.3% and 88.5% were also vacA s1a and cagA positive, respectively. Of the babA2 positive strains, 100.0%, 92.4% and 72.2% were also vacA s1a, cagA and iceA1 positive, respectively. Ulcer patients had more often strains with cagA positive status and vacA s1a allele. Although neither iceA1 nor babA2 were more common in ulcer patients, the combination of both alleles was more frequent (48.1%) in the ulcer patients than in the rest (28.7%). Clarithromycin susceptible strains had more often iceA1 allele (74.4%) than the resistant strains (55.3%). In conclusion, the results demonstrated a high prevalence of virulent H. pylori in Bulgaria. Both iceA1 and babA2 genotypes were associated with other virulence factors of H. pylori and, in addition, the iceA1 allele was associated with the strain susceptibility.     

Association between <Emphasis Type="Italic">cagA</Emphasis>, <Emphasis Type="Italic">vacAi</Emphasis>, and <Emphasis Type="Italic">dupA</Emphasis> genes of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> and gastroduodenal pathologies in Chilean patients

In addition to the already known cagA gene, novel genetic markers have been associated with Helicobacter pylori (H. pylori) virulence: the dupA and vacAi genes. These genes might play an important role as specific markers to determine the clinical outcome of the disease, especially the vacAi gene, which has been expected to be a good marker of severe pathologies like gastric adenocarcinoma. In the present study, the association of cagA, dupA, and vacAi genes with gastroduodenal pathologies in Chilean patients was studied. One hundred and thirty-two patients positive for H. pylori were divided into two groups—non-severe and severe gastric pathologies—and investigated for the presence of cagA, dupA, and vacAi H. pylori virulence genes by PCR. The cagA gene was detected in 20/132 patients (15.2%), the vacAi1 gene was detected in 54/132 patients (40.9%), the vacAi2 gene was detected in 26/132 patients (19.7%), and the dupA gene was detected in 50/132 (37.9%) patients. Logistic regression model analysis showed that the vacAi1 isoform gene in the infected strains and the severity of the diseases outcome were highly associated, causing severe gastric damage that may lead to gastric cancer (p < 0.0001; OR = 8.75; 95% CI 3.54–21.64). Conversely, cagA (p = 0.3507; OR = 1.62; 95% CI 0.59–4.45) and vacAi2 (p = 0.0114; OR = 3.09; 95% CI 1.26–7.60) genes were not associated with damage, while the dupA gene was associated significantly with non-severe clinical outcome (p = 0.0032; OR = 0.25; 95% CI 0.09–0.65). In addition, dupA gene exerts protection against severe gastric pathologies induced by vacAi1 by delaying the outcome of the disease by approximately 20 years.     

An Epidemiologic Study on the Correlation Between Oral <Emphasis Type="Italic">Helicobacter pylori</Emphasis> and Gastric <Emphasis Type="Italic">H. pylori</Emphasis>

The current study was conducted to determine the prevalence of Helicobacter pylori in the oral cavity of a Chinese population. Nested polymerase chain reaction (PCR) was used to investigate whether individuals with oral H. pylori show more possibility of gastric infection and to examine the relationship between gastric H. pylori infection and the presence of the bacteria in the oral cavity. A total of 443 dyspeptic patients participated in the study. Gastric H. pylori infection was detected by the rapid urease test and histology with Giemsa staining, PCR, or smear examination, whereas the presence of the bacteria in the oral cavity was observed by nested PCR from dental plaque. Relevant periodontal and dental parameters were recorded in the process of oral examination. Of the 443 study patients, oral H. pylori was found in the dental plaque of 263 (59.4%) and the stomach of 273 (61.6%). Additionally, in all four age groups, the prevalence of gastric infection was significantly higher among the patients with positive tests for H. pylori in their dental plaque than in the patients with no H. pylori in their dental plaque (P < 0.05). The oral cavity may be a potential reservoir for H. pylori, and the prevalence of oral H. pylori approximated that of gastric H. pylori in the studied population. Furthermore, a close relationship may exist between H. pylori in the oral cavity and bacteria in the stomach or gastric infection, and dyspeptic patients with gastric infection are more likely to harbor H. pylori in their oral cavity. Y. Liu and H. Yue contributed equally to the accomplishment of this study.     

Benefits of <Emphasis Type="Italic">Helicobacter pylori</Emphasis><Emphasis Type="Italic">cagE</Emphasis> genotyping in addition to <Emphasis Type="Italic">cagA</Emphasis> genotyping: a Bulgarian study

Associations of Helicobacter pylori cagE status with complex patient characteristics remain to be elucidated in Eastern Europe. The aim of this study was to assess the frequencies of cagE gene and cagA/cagE combinations in H. pylori strains from symptomatic Bulgarian patients and to improve cagA detection. cagA and cagE genotypes were evaluated in 219 patients with single-strain infections. In total, 84.9% of strains were cagA ⁺, while 68.5% were cagE ⁺. cagA ⁺, cagE ⁺, and cagA ⁺/cagE ⁺ strains were more prevalent in peptic ulcer (93.8%, 84.4%, and 84.4%) compared with nonulcer patients (81.3%, 61.9%, and 61.3%, respectively). In elderly patients, cagE ⁺ and cagA ⁺/cagE ⁺ strains were 1.9-fold more common than in the 12 children evaluated. Only 10% of the elderly subjects harbored low-virulence cagA ⁺/cagE ⁻ strains compared with 16.8% of adults and 41.7% of children. Intriguingly, prevalence of the cagA ⁺/cagE ⁻ genotype was 2.1-fold lower in men than in women, suggesting a higher frequency of more virulent strains in men. The presence of both cag genes and combinations was not linked to strain susceptibility to clarithromycin or metronidazole, place of residence, or prior therapy. Use of an extra primer pair increased cagA detection in 14.7% of 31 cagA ⁻ strains. In conclusion, use of a second primer pair for the cagA gene can be recommended in countries with common cagA ⁺ strains. Although both cag genes were linked to severe diseases in Bulgarian patients, the best discrimination of virulent strains was obtained by the cagA/cagE combination or by the cagE gene alone. cagE prevalence increased gradually with patient age, while the cagA ⁺/cagE ⁻ genotype, implying a disrupted cag pathogenicity island, was associated with both younger age and female gender.     

Food-Grade Expression of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> UreB Subunit in <Emphasis Type="Italic">Lactococcus lactis</Emphasis> and its Immunoreactivity

Helicobacter pylori is the principal cause of chronic active gastritis, peptic ulcer, and gastric cancer. To develop an oral vaccine against H. pylori infection, we had expressed the H. pylori ureB gene (Genbank accession no. FJ436980) in nisin-controlled expression vectors using Lactococcus lactis NZ3900 as host. The ureB gene was amplified by PCR from a H.pylori strain MEL-Hp27. Then the ureB gene was fused translationally downstream of the nisin-inducible promoter nisA in a L. lactis plasmid pNZ8149. Lactose utilization based on the complementation of the lacF gene was used as a dominant selection marker for the food-grade expression system employing L. lactis NZ3900. The conditions of UreB expression in this system were optimized by orthogonal experiment. The optimized conditions have been determined as follows: induction of expression was carried out at the cells density of OD₆₀₀ ≈ 0.4 with 25 ng/ml nisin, and harvest after 5 h. The maximum percentage of recombinant UreB was estimated to be 7% of total soluble cellular proteins and the yield was 12.9 μg/ml. Western blot demonstrated that the UreB protein was expressed in the L. lactis transformant and had favorable immunoreactivity. These results indicated that the lactococci-derived vaccines could be promising candidates as alternative vaccine strategies for preventing H. pylori infection.     

<Emphasis Type="Italic">Braf</Emphasis>, <Emphasis Type="Italic">Kras</Emphasis> and <Emphasis Type="Italic">Helicobacter pylori</Emphasis> epigenetic changes-associated chronic gastritis in Egyptian patients with and without gastric cancer

We aimed to study MLH1 and MGMT methylation status in Helicobacter pylori-associated chronic gastritis in Egyptian patients with and without gastric cancer. 39 patients were included in our study. They were divided into 2 groups; patients without (group I) and with gastric adenocarcinoma (group II). Patients were subjected to clinical examination, abdominal ultrasound and upper endoscopy for gastric biopsy. Biopsies were subjected to urease test, histological examination, and DNA purification. H. pylori, Braf, Kras, MLH1 and MGMT methylation were assessed by quantitative PCR. DNA sequencing was performed to assess Braf and Kras genes mutation. qPCR of H. pylori was significantly higher in patients with adenocarcinoma (group II) than those without adenocarcinoma (group I); with a p < 0.001 as well as in patients with age above 50 years with a p value = 0.008. By applying logistic regression analysis it was reported that the H. pylori qPCR is a significant predictor to the adenocarcinoma with OR = 1.025 (95 % CI: 1. 002–1.048), with sensitivity of 90 % and specificity of 100 %. Adenocarcinoma patients had a significantly higher mean age and levels of H. Pylori, Braf, K-ras, methylated MGMT and methylated MLH1 than those of gastritis patients. DNA sequence analysis of Braf (codon 12) and Kras (codon 600) had genes mutation in gastric adenocarcinoma versus chronic gastritis. Conclusion: H. pylori may cause epigenetic changes predisposing the patients to cancer stomach. Estimation of H. pylori by qPCR can be a good predictor to adenocarcinoma. Braf and Kras genes mutation were reveled in gastritis and adenocarcinoma patients.     

The <Emphasis Type="Italic">flhDC</Emphasis> gene affects motility and biofilm formation in <Emphasis Type="Italic">Yersinia pseudotuberculosis</Emphasis>

The flagella master regulatory gene flhDC of Yersinia pseudotuberculosis serotype III (YPIII) was mutated by deleting the middle region and replaced by a tetracycline resistant gene, and the subsequent mutant strain named YPIIIΔflhDC was obtained. Swimming assay showed that the swimming motility of the mutant strain was completely abolished. The promoter region of the flagella second-class regulatory gene fliA was fused with the lux box, and was conjugated with the mutant and the parent strains respectively for the first cross. LUCY assay result demonstrated that flhDC regulated the expression of fliA in YPIII as reported in E. coli. Biofilm formation of the mutant strain on abiotic and biotic surfaces was observed and quantified. The results showed that mutation of flhDC decreased biofilm formation on both abiotic and biotic surfaces, and abated the infection on Caenorhabdtis elegans. Our results suggest that mutation of the flagella master regulatory gene flhDC not only abolished the swimming motility, but also affected biofilm formation of YPIII on different surfaces. The new function of flhDC identified in this study provides a novel viewpoint for the control of bacterial biofilm formation.     

Core Genome Haplotype Diversity and <Emphasis Type="Italic">vacA</Emphasis> Allelic Heterogeneity of Chinese <Emphasis Type="Italic">Helicobacter pylori</Emphasis> Strains

The human gastric pathogen, Helicobacter pylori, has co-evolved with its host and established itself in the human stomach possibly millions of years ago. Therefore, the diversity of this bacterium is important in its clinical manifestations. Our aim has been to evaluate the genetic diversity of 40 H. pylori clinical isolates from four different parts of China. The methods of multi-locus sequence typing and vacA allele genotyping were used to assess their genetic diversity. To discriminate MLST, the vacA genotype method was used to identify strains. Patients from the northern, eastern, southern, and southwestern parts of China were recruited randomly from the cities of Beijing, Shanghai, Guangzhou, and Chongqing, respectively. Most of the sequence types are new and have never been reported in the database of the H. pylori multi-locus sequence typing system. The most prevalent vacA genotype in patients was s1a/m2 (80.0%), followed by s1b/m2 (17.5%). In contrast, the s1a/m1 genotype was scarcely represented (2.5%). The vacA genotype varied for each ST. These results showed that the MLST method offers high resolution of the H. pylori isolates in China when compared to vacA genotyping. The vacA allelic s1a has been correlated with the peptic ulcer. Because of the paucity of data on human isolates due to the absence of systematic investigations of H. pylori in China, the data provide useful information for understanding the epidemiology of H. pylori in China from the viewpoint of nucleotide sequence databases.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Population Identification of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> Isolates from Russia

Using multilocus sequence typing (MLST), 22 Helicobacter pylori isolates from Russia have been characterized. All of the Russian strains were assigned to a single population, hpEurope.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

Surface expression of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> urease subunit B gene E fragment on <Emphasis Type="Italic">Lactococcus lactis</Emphasis> by means of the cell wall anchor of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> protein A

Fragment E of ureB (ureBE) was cloned from a clinical isolate of Helicobacter pylori. A prokaryotic expression vector, pAMJ399, with the ureB fragment E and the Staphylococcus aureus protein A anchor fragment (spaX), was constructed. The fusion protein was expressed under the control of the P170 promoter in Lactococcus lactis. Western blot assay of lactococcal cell wall extracts with a polyclonal chicken antiserum confirmed the immunity of the expressed recombinant protein which was located on the cell surface. These results provide the first report of a surface display system in lactic acid bacteria for the delivery of oral vaccines against Helicobacter pylori.