Fusion-expressed CtxB-TcpA-C-CPE improves both systemic and mucosal humoral and T-cell responses against cholera in mice.

BACKGROUND: Development of an effective oral vaccine against Cholera, a life-threatening dehydrating diarrheal disease, proved to be a challenging task. To improve oral subunit vaccine immunogenicity and to prevent the state of oral tolerance, application of mucosal adjuvants might be a promising approach. In the present study, the CtxB-TcpA-C-CPE fusion was constructed in which CtxB and C-CPE were used as mucosal adjuvants and vaccine delivery system, respectively, to induce mucosal immune responses, and to improve the anti-toxin and anti-colonizing immunity against V. cholerae. MATERIALS & METHODS: The fusion construct was synthesized, sub-cloned in pQE30 and expressed in E. coli. The three antigen, making the fusion protein, were also separately expressed in E. coli. The recombinant proteins were purified by affinity chromatography using Ni-NTA agarose. Western blot analysis using anti-His antibody was applied to confirm identity of the purified proteins. BALB/c mice were subcutaneously immunized with CtxB, TcpA, C-CPE and the fusion protein CtxB-TcpA-C-CPE separately. The mice were orally immunized (in 3 boosts) by the same vaccine. Mucosal immune response stimulation was evaluated by measuring the levels of intestinal IgA. Systemic immune response was evaluated by measuring total serum IgG, IgG1, IgG2a, IgG2b subclasses, and also IL-4, IL-5, IL-10 and IFN-γ cytokines in spleen cell culture. RESULTS: The recombinant proteins CtxB, TcpA, C-CPE and the fusion protein CtxB-TcpA-C-CPE were expressed in E. coli and highly purified in a single step of chromatography. BALB/c mice immunized with the fusion protein had highest levels of intestinal IgA, serum IgG and IgG subclasses, compared to each of the three proteins making the fusion. Moreover, stimulated splenocytes of mice immunized with the fusion protein displayed significantly higher amounts of IL-5 and IFN-ɣ cytokines. Th2 dominance of the immune response was more evident in mice receiving the fusion protein. CONCLUSION: Inclusion of CtxB, as the mucosal adjuvant, and C-CPE, as the vaccine delivery system, in the fusion protein CtxB-TcpA-C-CPE significantly enhanced the elicited mucosal and systemic immune responses, compared to TcpA alone. Of note, significant production of intestinal IgA in mice immunized with the fusion protein is presumably capable of neutralizing TcpA, CtxB and C-CPE antigens, preventing V. cholera colonization, and toxic function of CtxB and C-CPE. Challenge infection of the immunized mice is required to evaluate protective potential of the fusion protein against V. cholera.

Computational evaluation of a fusion protein consisted of pertussis toxin and filamentous hemagglutinin from Bordetella pertussis to target Claudin-4 using C-terminal fragment of Clostridium perfringens enterotoxin.

Pertussis, caused by Bordetella pertussis is still one of the controversial diseases worldwide due to its high prevalence in both the developed and the developing countries, especially among young children. As currently approved vaccines are not protective enough and provide Th2-type immune responses, there is an urgent need to develop new vaccines. In the current study, we applied the C-terminal fragment of Clostridium perferingens enterotoxin (C-CPE) as a delivery system and F1S1 fragment (Filamentous hemagglutinin (F1) and subunit 1 of pertussis toxin (S1) of B. pertussis to design a novel chimeric protein in silico, to target Claudin-4 receptors in mice lung cells. To achieve this goal, the primary, secondary and tertiary structures of the fusion protein were evaluated and the interaction of this protein with Claudin-4 receptors was studied. Molecular dynamic (MD) simulation analysis was performed to investigate the physical movement of atoms in a fixed period. According to the results; the full-length fusion protein has consisted of 807 amino acid residues which could be classified as a stable protein. There was a convenient consistency between the 3D predicted structure and the secondary structure prediction. An acceptable percentage of the residues were also detected in the most favored and allowed regions for the model. Based on HADDOCK results, there were no considerable differences between the interactions and MD simulation analysis, indicating that the predicted structures were stable during the simulation. Altogether, the data reported in this study represents the first step toward developing a nasal vaccine candidate against B. pertussis infection. Communicated by Ramaswamy H. Sarma.

Milk of livestock as a possible transmission route of Helicobacter pylori infection.

AIM: The current investigation aimed to evaluate ruminant raw milk as a reservoir source of Helicobacter pylori and analyze the diversity of cagA and vacA genotypes as H. pylori virulence factors to find any relationship between these genotypes in human and animal H. pylori strains. BACKGROUND: The way of transmission of Helicobacter pylori as one of the most controversial bacteria in the world, which colonizes the human gastric tissue and is responsible for several gastric diseases is still unknown. The possibility of zoonotic transmission of H. pylori is feasible, but is not proven in ruminant reservoirs. METHODS: Overall 210 cows, sheep, goats, camels and buffalos' raw milk samples and 100 human gastric biopsies were collected in this survey. We applied PCR assays to identify H. pylori, vacA and cagA genes. Statistical tests were applied for data analysis. RESULTS: Totally 12(16%) cow, 8(13.79%) sheep, 2 (4.76%) goat, 2(13.33%), buffalo 4(20%) and 82 (82%) of human specimens were confirmed to be H. pylori positive. Among which s1a/m2 genotype was more frequent in isolated H. pylori strains and statistically significant between strains. Based on statistical analyses the s1b allele of sheep had a significant association with human strains. CONCLUSION: The current survey was prompted by our previous report. According to both results we can conclude that sheep may act as a reservoir for H. pylori and transmit this bacterium to human via its milk. Extended assessments in other geographical regions and other animals are recommended.

The study of the oipA and dupA genes in Helicobacter pylori strains and their relationship with different gastroduodenal diseases.

AIM: The purpose of this investigation was to determine the oipA and dupA genes of Helicobacter pylori isolates from west of Iran; Chaharmahalo Bakhtiyari region and find their relationship with the severity of the gastroduodenal diseases. BACKGROUND: Helicobacter pylori is an organism responsible for many gastroduodenal diseases. Many studies suggest that genetic diversity in H . pylori virulence factors such as oipA and dupA genes is high among isolates of different geographic regions and may cause more severe diseases. PATIENTS AND METHODS: In this cross-sectional study, gastric biopsy specimens were taken from 150 patients suffering from gastroduodenal diseases. The presence of ureC, dupA and oipA genes was tested by polymerase chain reaction (PCR). RESULTS: Overall, 123 (82%) H. pylori strains were isolated from 150 specimens. dupA gene was detected in 41 (33.33%) H.pylori-positive specimens. There was a reverse correlation between this gene and gastric cancer. The oipA gene was found in 88 (71.54%) samples and statistically there was no association between this gene and gastric disorders. As statistical analyses revealed, the presence of the dupA was more common in isolates with the oipA negative. CONCLUSION: Based on our findings, the presence of dupA gene can be considered as a marker for the onset of severe diseases. However, the oipA gene cannot be regarded for prediction of gastroenterology diseases. Meanwhile, extended molecular epidemiology researches in other populations are recommended.

Study of Helicobacter pylori genotype status in cows, sheep, goats and human beings.

BACKGROUND: Helicobacter pylori is one of the most controversial bacteria in the world causing diverse gastrointestinal diseases. The transmission way of this bacterium still remains unknown. The possibility of zoonotic transmission of H. pylori has been suggested, but is not proven in nonprimate reservoirs. In the current survey, we investigate the presence of H. pylori in cow, sheep and goat stomach, determine the bacterium virulence factors and finally compare the human H. pylori virulence factors and animals in order to examine whether H. pylori might be transmitted from these animals to human beings. METHODS: This cross- sectional study was performed on 800 gastric biopsy specimens of cows, sheep, goats and human beings. The PCR assays was performed to detection of H. pylori, vacA and cagA genes. The PCR products of Ruminant's samples with positive H. pylori were subjected to DNA sequencing analysis. Statistical tests were applied for data analysis. RESULTS: Overall 6 (3%) cows, 32 (16%) sheep and 164 (82%) human beings specimens were confirmed to be H. pylori positive; however we were not able to detect this bacterium in all 200 goat samples. The vacA s1a/m1a was the predominant H. pylori genotype in all three kinds of studied population. There was 3.4-8.4% variability and 92.9-98.5% homology between sheep and human samples. CONCLUSIONS: Considering the high sequence homology among DNA of H. pylori isolated from sheep and human, our data suggest that sheep may act as a reservoir for H. pylori and in the some extent share the ancestral host for the bacteria with human.

Genetic Analysis of cagA and vacA Genes in Helicobacter Pylori Isolates and Their Relationship with Gastroduodenal Diseases in the West of Iran.

BACKGROUND: Helicobacter pylori have different virulence factors which are associated with several gastroduodenal diseases; however, this association is variable in different geographical regions. Data of genotypes of Iranian H. pylori isolates are few. OBJECTIVES: The aim of the current study was to investigate the cagA/vacA genotypes of Helicobacter pylori isolates and determine the relationship between these genotypes with respect to different gastric disorders in patients of Chaharmahalo Bakhtiarian. MATERIALS AND METHODS: In this cross-sectional study, gastric biopsies were taken from 200 patients with gastrodoudenal diseases. Histopathological features were recognized by specialist. The samples were subjected to PCR for detection and identification of ureC, cagA and vacA genes. RESULTS: The frequency of the vacA genotypes, sa1/m1, s1a/m1b, s1a/m2, s1b/m1a, s1b/m1b, s1b/m2, s1c/m1a, s1c/m1b, s1c/m2, s2/m1a, s2/m1b and s2/m2 were 27(6.6%), 8(4.3%), 45(28.04%), 7(3.7%), 5(2.5%), 10 (6.1%), 12 (7.4%), 4 (2.5%), 18(11%), 6(3.7%), 0 and 22(13.5%) respectively. The cagA gene was detected in 92% of strains. Based on our findings, it seemed that cagPAI and vacA s1 genotypes were associated with some gastric disorders in patients with H. pylori. In this region, the isolates carrying s1a/m2 were the most prevalent. CONCLUSIONS: We found considerable relationship between s1a/m1a, s1a/m2, s2/m2 and s1c/m1a and some gastric disorders. Further studies about the role of H. pylori virulence factors and gastric disorders were recommended.

Uropathogenic Escherichia coli in Iran: serogroup distributions, virulence factors and antimicrobial resistance properties.

BACKGROUND: Urinary tract infections (UTIs) are one of the most common bacterial infections with global expansion. These infections are predominantly caused by uropathogenic Escherichia coli (UPEC). METHODS: Totally, 123 strains of Escherichia coli isolated from UTIs patients, using bacterial culture method were subjected to polymerase chain reactions for detection of various O- serogroups, some urovirulence factors, antibiotic resistance genes and resistance to 13 different antibiotics. RESULTS: According to data, the distribution of O1, O2, O6, O7 and O16 serogroups were 2.43%, besides O22, O75 and O83 serogroups were 1.62%. Furthermore, the distribution of O4, O8, O15, O21 and O25 serogroups were 5.69%, 3.25%, 21.13%, 4.06% and 26.01%, respectively. Overall, the fim virulence gene had the highest (86.17%) while the usp virulence gene had the lowest distributions of virulence genes in UPEC strains isolated from UTIs patients. The vat and sen virulence genes were not detected in any UPEC strains. Totally, aadA1 (52.84%), and qnr (46.34%) were the most prevalent antibiotic resistance genes while the distribution of cat1 (15.44%), cmlA (15.44%) and dfrA1 (21.95%) were the least. Resistance to penicillin (100%) and tetracycline (73.98%) had the highest while resistance to nitrofurantoin (5.69%) and trimethoprim (16.26%) had the lowest frequencies. CONCLUSIONS: This study indicated that the UPEC strains which harbored the high numbers of virulence and antibiotic resistance genes had the high ability to cause diseases that are resistant to most antibiotics. In the current situation, it seems that the administration of penicillin and tetracycline for the treatment of UTIs is vain.

Molecular characterization of Shiga toxin-producing Escherichia coli isolated from ruminant and donkey raw milk samples and traditional dairy products in Iran.

The aims of the current study were to detect the virulence factors and antibiotic resistance of Shiga toxin-producing E. coli, in animal milk and dairy products in Iran. After E. coli dentification with culture method, PCR assay were developed for detection of pathogenic genes, serotypes and antibiotic resistance genes of E. coli. Results showed that out of 719 samples, 102 (14.18%) were confirmed to be positive for E. coli and out of 102 positive samples, 17.64% were O26 and 13.72% were O157 and 1.96% were O91 and 1.96% were O145 serotypes. Totally, the prevalence of stx1 and papA genes were the highest while the prevalence of sfaS and fyuA were the lowest in the positive samples. PCR results showed that tetA, tetB were the highest (64.70%) and aac(3)-IV were the lowest (27.45%) antibiotic resistant genes in E. coli positive samples. Our study indicated that the isolated E. coli trains in these regions had a highest antibiotic resistance to tetracycline (58.82%) and the lowest to nitrofurantoin (3.92%). tetA gene and E. coli O157 serotype had highest and aac(3)-IV gene, and E. coli O145 serotype had a lowest frequency rates of antibiotics resistance genes, in the region.

Study of Helicobacter pylori genotype status in saliva, dental plaques, stool and gastric biopsy samples.

AIM: To compare genotype of Helicobacter pylori (H. pylori) isolated from saliva, dental plaques, gastric biopsy, and stool of each patient in order to evaluate the mode of transmission of H. pylori infection. METHODS: This cross-sectional descriptive study was performed on 300 antral gastric biopsy, saliva, dental plaque and stool samples which were obtained from patients undergoing upper gastrointestinal tract endoscopy referred to endoscopy centre of Hajar hospital of Shahrekord, Iran from March 2010 to February 2011. Initially, H. pylori strains were identified by rapid urease test (RUT) and polymerase chain reaction (PCR) were applied to determine the presence of H. pylori (ureC) and for genotyping of voculating cytotoxin gene A (vacA) and cytotoxin associated gene A (cagA) genes in each specimen. Finally the data were analyzed by using statistical formulas such as Chi-square and Fisher's exact tests to find any significant relationship between these genes and patient's diseases. P < 0.05 was considered statistically significant. RESULTS: Of 300 gastric biopsy samples, 77.66% were confirmed to be H. pylori positive by PCR assay while this bacterium were detected in 10.72% of saliva, 71.67% of stool samples. We were not able to find it in dental plaque specimens. The prevalence of H. pylori was 90.47% among patients with peptic ulcer disease (PUD), 80% among patients with gastric cancer, and 74.13% among patients with none ulcer dyspepsia (NUD) by PCR assay. The evaluation of vacA and cagA genes showed 6 differences between gastric biopsy and saliva specimens and 11 differences between gastric and stool specimens. 94.42% of H. pylori positive specimens were cagA positive and all samples had amplified band both for vacA s and m regions. There was significant relationship between vacA s1a/m1a and PUD diseases (P = 0.04), s2/m2 genotype and NUD diseases (P = 0.05). No statically significant relationship was found between cagA status with clinical outcomes and vacA genotypes (P = 0.65). The evaluation of vacA and cagA genes showed 6 differences between gastric biopsy and saliva specimens and 11 differences between gastric and stool specimens. CONCLUSION: Regard to high similarity in genotype of H. pylori isolates from saliva, stomach and stool, this study support the idea which fecal- oral is the main route of H. pylori transmission and oral cavity may serve as a reservoir for H. pylori, however, remarkable genotype diversity among stomach, saliva and stool samples showed that more than one H. pylori genotype may exist in a same patient.

Real-time PCR for Helicobacter pylori quantification and detection of clarithromycin resistance in gastric tissue from patients with gastrointestinal disorders.

Helicobacter pylori gram-negative bacteria commonly infect the human gastrointestinal (GI) tract and are readily diagnosed by endoscopy. H. pylori infection causes a broad range of host symptoms from discomfort to significant GI disorders (GIDs). Severity of the clinical manifestations depends mainly upon bacterial load. In this cross-sectional study, we investigated the affects of 23S rRNA point mutations on H. pylori count in naturally infected human GI tissues. Two-hundred H. pylori patients with suspected GIDs were evaluated to determine bacteria concentration and presence of four known 23S rRNA point mutations, causing clarithromycin resistance. Gastric biopsy specimens were examined by rapid urease test and 16S rRNA-targeted PCR to identify H. pylori; then bacterial load was quantified by real-time PCR targeting wild type and known 23S rRNA mutations. Eighty-two percent of the samples were confirmed as H. pylori-positive, having 10(4)-10(12) colony-forming units (CFU)/ml. The 10(6) load was most strongly associated with peptidyltransferase point mutations of the 23S rRNA gene A2144G (p = 0.033), A2143G (p = 0.005), A2143C (p = 0.005), and A2142G (p = 0.015). Thus, our findings indicated that dominant 23S rRNA mutated H. pylori strains have the same growth rate as the wild type in a gastric environment.

Comparison of the virulence factors of Helicobacter pylori isolated in stomach and saliva in Iran.

INTRODUCTION: Helicobacter pylori is a microaerophilic, spiral-shaped motile bacterium that is strongly associated with gastroduodenal diseases, but recently, dental plaque and saliva have been implicated as possible sources of H pylori infection. Two virulence factors that are expressed by the alleles of the cytotoxin genes, cagA and vacA, have been identified. The aim of this study was to compare cagA and vacA genotypes of H pylori between stomach and saliva in a same patient. METHODS: This study was performed on antrum gastric biopsy specimens and saliva samples, which were obtained from 250 patients undergoing upper gastrointestinal tract endoscopy in Hagar Hospital of Shahrekord Township in Iran. Initially, H pylori strains were identified by rapid urease test; then, we applied polymerase chain reaction assay to analyze cagA and vacA genotypes of H pylori from both gastric and saliva specimens. RESULTS: One hundred eighty-nine (75.6%) and 36 (14.4%) samples were H pylori positive in gastric and saliva samples, respectively. Evaluation of virulence factors in the 36 patients whose saliva and gastric samples were both positive for H pylori showed a great deal of cytotoxin genotypic diversity between stomach and saliva in the same patient; in fact, 14 patients (38.8%) had different H pylori strains in their saliva and gastric samples. CONCLUSIONS: The data suggest that >1 H pylori strain may exist in stomach and saliva in the same patient.