Evaluation of the Anti-East Asian CagA-Specific Antibody for CagA Phenotyping

The determination of the cagA genotype is generally based on sequencing the variable 3′ region of the cagA gene. In a previous study, we successfully generated an anti-East Asian CagA-specific antibody (anti-EAS Ab) immunoreactive only with the East Asian CagA and not with the Western CagA. In a small number of Japanese patients, anti-EAS Ab appeared to be a useful tool for phenotyping CagA immunohistochemically. The present study was conducted to validate the anti-EAS Ab immunohistochemistry method in a larger number of patients from Vietnam and Thailand. A total of 385 Vietnamese and Thais were recruited. Helicobacter pylori status was determined by a combination of three methods, including culture, histology, and immunohistochemistry with anti-H. pylori antibody. The sensitivity, specificity, and accuracy of the anti-EAS Ab immunohistochemistry method for the diagnosis of CagA phenotype were calculated based on the results of the cagA sequencing as the gold standard. The sensitivity, specificity, and accuracy of our immunohistochemistry method were 96.7%, 97.9%, and 97.1%, respectively. Moreover, anti-EAS Ab was not cross-reactive with noninfected gastric mucosa. In conclusion, immunohistochemistry with anti-EAS Ab appears to be a good method for determination of CagA phenotype.Helicobacter pylori is a spiral, gram-negative bacterium that chronically infects the human stomach and plays a causative role in the pathogenesis of gastritis, gastroduodenal ulcer, gastric cancer, and mucosa-associated lymphoid tissue lymphoma (19, 22). It is well recognized that H. pylori strains possessing an approximately 40-kb cluster of genes named the cag pathogenicity island (cag PAI) are more virulent and more strongly associated with severe clinical outcomes, such as peptic ulcer and gastric cancer (3, 5). The cag PAI consists of approximately 30 genes, several of which encode component proteins of the type IV secretion system and are essential for the induction of proinflammatory cytokines, such as interleukin-8, from gastric epithelial cells (3, 5). Moreover, the cag PAI contains cagA, the gene encoding the CagA protein, which is currently believed to have oncogenic potential (8, 9).CagA has several repeated 5-amino-acid sequences (glutamic acid-proline-isoleucine-tyrosine-alanine), named EPIYA motifs, located at the C terminus of the protein. The EPIYA motif is divided into EPIYA-A, EPIYA-B, EPIYA-C, and EPIYA-D stretches, based on the amino acid sequences flanking each of them. According to the combinations of these EPIYA motifs, two major types of CagA protein have been observed. The Western CagA has EPIYA-A and EPIYA-B followed by one to five repeats of the EPIYA-C sequence. The East Asian CagA also has EPIYA-A and EPIYA-B, but the third motif is EPIYA-D (8, 9).After H. pylori adheres to the gastric epithelium, CagA is translocated via the type IV secretion system into the host cell cytoplasm, where it undergoes phosphorylation by several Src family kinases at the tyrosine residues of all EPIYA motifs (10, 11). Phosphorylated CagA is then able to interact with and dysregulate several cellular transduction signal pathways. Notably, phosphorylated CagA binds to the Src homology 2 (SHP-2) domain containing tyrosine phosphatase via the EPIYA-C or EPIYA-D motif (10, 11, 14). Compared to the Western CagA, the East Asian type exhibits stronger binding affinity for SHP-2, due to a specific sequence overlying EPIYA-D (Y-A-T-I-D-F) that perfectly matches the consensus ligand binding motif for SHP-2 (Y-[V/T/A/I/S]-X-[L/I/V]-X-[F/W]) (10, 14). As a result, the East Asian CagA is considered to be more toxic than its Western homologue and more strongly associated with severe clinical outcomes, including gastric cancer (2, 8, 9). Therefore, the accurate diagnosis of the CagA phenotype would be useful, especially for molecular epidemiologic surveys.In a previous study, we successfully generated an anti-East Asian CagA-specific antibody (anti-EAS Ab) which was immunoreactive only with the East Asian CagA and not with the Western CagA (23). We have also shown that anti-EAS Ab might be a useful tool for genotyping CagA immunohistochemically (23). However, in the previous study, with only Japanese patients, most strains analyzed possessed East Asian CagA; therefore, the accuracy of the test when using anti-EAS Ab was not confirmed. Due to the geographical genomic diversity of H. pylori, the value of our method in other populations needs to be validated. Therefore, the present study was carried out using a large number of patients from countries other than Japan, including Thailand, where the presence of Western CagA would be expected.     

Diverse Characteristics of the cagA Gene of Helicobacter pylori Strains Collected from Patients from Southern Vietnam with Gastric Cancer and Peptic Ulcer

The pathogenesis of gastroduodenal diseases is related to the diversity of Helicobacter pylori strains. CagA-positive strains are more likely to cause gastric cancer than CagA-negative strains. Based on EPIYA (Glu-Pro-Ile-Tyr-Ala) motifs at the carboxyl terminus corresponding to phosphorylation sites, H. pylori CagA is divided into East Asian CagA and Western CagA. The former type prevails in East Asia and is more closely associated with gastric cancer. The present study used full sequences of the cagA gene and CagA protein of 22 H. pylori strains in gastric cancer and peptic ulcer patients from Southern Vietnam to make a comparison of genetic homology among Vietnamese strains and between them and other strains in East Asia. A phylogenetic tree was constructed based on full amino acid sequences of 22 Vietnamese strains in accordance with 54 references from around the world. The cagA gene was found in all Vietnamese H. pylori strains. Twenty-one of 22 (95.5%) strains belonged to the East Asian type and had similar characteristics of amino acid sequence at the carboxyl terminus to other strains from the East Asian region. From evidence of East Asian CagA and epidemiologic cancerous lesions in Vietnam, H. pylori-infected Vietnamese can be classified into a high-risk group for gastric cancer, but further studies on the interaction among environmental and virulence factors should be done. Finally, phylogenetic data support that there is a Japanese subtype in the Western CagA type.Helicobacter pylori is a gram-negative bacterium that infects about 50% of the world''s population. Infections with H. pylori can result in chronic active gastritis and are a risk factor for peptic ulcers, gastric cancer and gastric mucosa-associated lymphoid tissue lymphoma (40, 52, 53, 56). The prevalence of H. pylori infections is not the same in different parts of the world. Recent studies reported that humans actually acquired H. pylori in the early days of their history, long before the migration of modern humans out of Africa, and the diverse distribution of H. pylori today is associated with waves of human migration in the past (19, 32, 36, 61, 62). The rate of H. pylori infections is high in Africa, East Asia, and South Asia; however, the incidence of gastric cancer is high in East Asia but not in South Asia or Africa; this may be explained partly by the diversity of H. pylori strains in these regions (62).In cases of gastroduodenal diseases, especially gastric cancer, the pathogenesis involves three major factors: H. pylori virulence factors, host factors, and environmental factors (1, 10, 14, 16, 21, 34, 57). Two H. pylori virulence factors that have been focused on in many studies all around the world are VacA and CagA. VacA is encoded by the vacA gene and found in all H. pylori strains. The vacA gene is classified into three major genotypes: s1/m1, s1/m2, and s2/m2. s1/m1 strains produce higher levels of cytotoxin than s1/m2 or s2/m2 strains (2, 3). CagA is encoded by cagA, located in the cagPAI (pathogenicity island) region of the H. pylori genome, and the presence of this protein is a marker of cagPAI. Unlike the vacA gene, the cagA gene has been found in only 50% to 70% of H. pylori strains infecting Western populations: however, 80% to 100% of H. pylori strains from East Asia have the cagA gene, with the cagPAI region, in their genome (17, 49, 55, 61, 64, 65). Patients infected with H. pylori strains possessing CagA were at greater risk of developing gastric adenocarcinoma than those uninfected or infected with CagA-negative strains (11, 41). CagA is injected directly from H. pylori into intragastric epithelial cells via the type IV secretion system. The injected CagA mimics eukaryotic adaptor proteins that recruit multiple host signaling factors into protein complexes that target cellular junctions, cell proliferation, and actin-cytoskeletal rearrangements (54). The translocated CagA undergoes tyrosine phosphorylation by Src and Abl family kinases and binds to SHP-2 in the human gastric mucosa (45, 47, 48). The repeated EPIYA (Glu-Pro-Ile-Tyr-Ala) motifs at the carboxyl-terminal end of CagA are the targets of tyrosine phosphorylation (8, 22, 24, 38, 63). CagA multimerization plays an important role in the pathophysiological activity of CagA in disturbing host cell functions via SHP-2 deregulation, and EPIYA polymorphisms of CagA greatly influence the magnitude and duration of phosphorylation-dependent CagA activity (37, 42). SHP-2, like its Drosophila melanogaster homolog Corkscrew, is known to play an important role in the mitogenic signal transduction that connects receptor tyrosine kinases and ras (20). It is possible that deregulation of SHP-2 by the translocation of CagA plays a role in the acquisition of a cellular-transformed phenotype at a relatively early stage in the carcinogenesis of gastric carcinoma. A recent study on generating CagA in transgenic mice has provided the first direct evidence of the role of CagA as a bacterium-derived oncoprotein that acts in mammals and further indicates the importance of tyrosine phosphorylation, which enables CagA to deregulate SHP-2, in the development of H. pylori-associated neoplasms (39). Based on characteristics of the EPIYA motif, the H. pylori CagA protein could be divided into a Western type and an East Asian type. The East Asian CagA protein exhibits stronger SHP-2-binding activity and so is more pathogenic than the Western CagA protein in H. pylori-infected patients (4, 7, 23, 37). Clinical data from East Asia, Japan, and South Korea indicated that the East Asian form of CagA was more closely related to persistent active inflammation, atrophic gastritis, and a higher risk of gastric cancer than the Western form (5, 6, 26, 44). It is quite clear that by studying the characteristics of H. pylori strains, especially the cagA gene and CagA protein, one can categorize H. pylori-infected patients into those at high risk of developing gastric cancer and those not.Vietnam is a developing country located in Southeast Asia. However, according to historical and migrational evidence, the Vietnamese are more closely related to people from East Asia than people from South Asia. Gastric cancer is one of the five most common cancers in Vietnam, including lung, stomach, liver, recto-colon and naso-pharynx cancer in males. In females, it ranks third behind breast and cervical-uterine cancer. The prevalence of gastric cancer in Northern Vietnam is as high as that in China or Korea. Its prevalence in Southern Vietnam is lower but still higher than that in Thailand and South Asia (46, 50). A few studies reported that cagA was found in nearly 100% of H. pylori-infected Vietnamese (60, 61), but no studies have examined the type of CagA protein or the full sequence of cagA in Vietnamese patients. The present study reports the diverse characteristics of cagA and classification of CagA in H. pylori-infected patients from Southern Vietnam based on the full genomic cagA sequence.     

High Prevalence of Clarithromycin-Resistant Helicobacter pylori Strains and Risk Factors Associated with Resistance in Madrid,Spain

Clarithromycin is one of the antibiotics used for the treatment of Helicobacter pylori infections, and clarithromycin resistance is the most important factor when it comes to predicting eradication failure. The present study analyzed H. pylori isolates for the presence of 23S rRNA gene mutations and determined the risk factors associated with resistance among H. pylori isolates collected in Madrid, Spain, in 2008. We studied 118 H. pylori strains isolated from the same number of patients. A total of 76.3% of the patients were born in Spain, 52.7% were children, 20.3% had previously been treated, and 66.1% were female. Clarithromycin resistance was determined by Etest. H. pylori strains were considered resistant if the MIC was ≥1 mg/liter. DNA extraction was carried out by use of the NucliSens easyMAG platform with NucliSens magnetic extraction reagents (bioMérieux). The DNA sequences of the 23S rRNA genes of clarithromycin-resistant and -sensitive strains were determined to identify specific point mutations. The vacA genotype and cagA status were determined by PCR. We found that 42 (35.6%) strains were resistant to clarithromycin by Etest. Etest results were confirmed by detection of the presence of point mutations in 34 (88.1%) of these strains. Eight H. pylori strains were resistant to clarithromycin by Etest but did not have a point mutation in the 23S rRNA gene. Mutation at A2143G was found in 85.3% of the strains, mutation at A2142G in 8.8%, and mutation at T2182C in 5.9%. Dual mutations were found in 8.8% of the strains. H. pylori clarithromycin-resistant strains were strongly associated with pediatric patients, with patients born in Spain, and with patients who had previously been treated (P ≤ 0.02). In addition, H. pylori strains resistant to clarithromycin more frequently presented the vacA s2/m2 genotype and were more likely to be cagA negative than susceptible strains (39.1% and 11.2%, respectively; P value < 0.001). We concluded that, in the present study, H. pylori clarithromycin-resistant strains are more frequently found in children, in patients mostly born in Spain, and in individuals who were previously treated for H. pylori infection and that these individuals are more likely colonized with a less virulent H. pylori strain.Helicobacter pylori is a microaerobic, Gram-negative spiral bacterium that colonizes the human stomach and is found in more than half of the world''s population (32). Infections with H. pylori are closely associated with chronic gastritis, peptic ulcer disease, and the development of gastric cancer (8, 32).All consensus guidelines recommend eradication of H. pylori for patients with symptoms (9, 28). Standard therapy combines a proton pump inhibitor (PPI) or ranitidine bismuth citrate and two antibiotics, chosen from among amoxicillin, clarithromycin, and metronidazole (24, 25). However, this therapy has been questioned because of the increased eradication failure rates. Many factors have been implicated as causes of treatment failure, including ineffective penetration of antibiotics into the gastric mucosa, antibiotic inactivation by the low stomach pH, a lack of patient compliance, and the emergence of acquired resistance to antibiotics by H. pylori (26, 27).In many cases, clarithromycin is the key component of these combination therapies. However, resistance to clarithromycin has become one of the major reasons for treatment failure (13). The prevalence of H. pylori resistance to clarithromycin varies among different countries, such as 10.6% to 25% in North America, 16% in Japan, and 1.7% to 23.4% in Europe (14, 19, 21). Overall, resistance to clarithromycin has been detected more in patients living in the south than in those living in the north of Europe (21). Fewer studies have focused on the prevalence of clarithromycin resistance among H. pylori strains from children compared with that among strains from the adult population. These studies will be useful for estimating the rate of clarithromycin-resistant H. pylori isolates among children and adults in Spain in the future (1, 23).Clarithromycin acts by binding to the peptidyltransferase region of 23S rRNA and inhibits protein synthesis (36). The resistance to clarithromycin in H. pylori has been shown to be due to point mutations in the peptidyltransferase region of domain V of the 23S rRNA. Two copies of the 23S rRNA gene are present in H. pylori, and the most common mutation is an A-to-G transition at position 2143 (A2143G) (13, 36), but several point mutations, at positions A2142G, A2144G, and T2182C, have been described. Recent reports have indicated that other mutations, such as A2115G, G2141A, C2147G, T2190C, C2195T, A2223G, and C2694A, might also be associated with clarithromycin resistance (20, 31). Other mechanisms of resistance, such as methylase production, the actions of macrolide-inactivating enzymes, and active efflux, have been described in several bacteria. Active efflux has also recently been described in H. pylori (22).Since the worldwide increase in the rate of clarithromycin resistance represents a problem of relevance, some studies have been performed in order to identify its relationship with bacterial genetic factors (12, 35, 38).Two genes (cytotoxin-associated gene A [cagA] and vacuolation-associated gene A [vacA]) have been identified to be the main virulence factors. cagA is located in the cag pathogenicity island (PAI), which encodes a type IV secretion system, and the presence of cagA is closely associated with more severe gastric diseases (2, 15, 34). The VacA toxin induces vacuole formation in the host cells. There is considerable variation in vacuolation activity among H. pylori strains, primarily due to differences in the vacA gene structure in the signal region (s1 and s2) and the middle region (m1 and m2). vacA s1/m1 and s1/m2 produce high and moderate levels of VacA toxin, respectively, whereas s2/m2 produces little or no toxin (11). A strong association between clarithromycin susceptibility and these virulence factors has been reported (12, 38).The focus of the present study was to evaluate the distribution of clarithromycin-resistant H. pylori strains and their association with genotypic markers, such as the cagA gene and allelic variants of the vac gene. We also examined the distribution of H. pylori clarithromycin resistance in relation to the patient''s age, place of birth, and history of treatment. Our main goal is to determine potential host and bacterial factors that may help in predicting resistance to clarithromycin among H. pylori isolates.     

Pediatric Helicobacter pylori Isolates Display Distinct Gene Coding Capacities and Virulence Gene Marker Profiles

Helicobacter pylori strains display remarkable genetic diversity, and the presence of strains bearing the toxigenic vacA s1 allele, a complete cag pathogenicity island (PAI), cagA alleles containing multiple EPIYA phosphorylation sites, and expressing the BabA adhesin correlates with development of gastroduodenal disease in adults. To better understand the genetic variability present among pediatric strains and its relationship to disease, we characterized H. pylori strains infecting 47 pediatric North American patients. Prevalence of mixed infection was assessed by random amplified polymorphic DNA analysis of multiple H. pylori clones from each patient. Microarray-based comparative genomic hybridization was used to examine the genomic content of the pediatric strains. The cagA and vacA alleles were further characterized by allele-specific PCR. A range of EPIYA motif configurations were observed for the cagA gene, which was present in strains from 22 patients (47%), but only 19 (41%) patients contained a complete cag PAI. Thirty patients (64%) were infected with a strain having the vacA s1 allele, and 28 patients (60%) had the babA gene. The presence of a functional cag PAI was correlated with ulcer disease (P = 0.0095). In spite of declining rates of H. pylori infection in North America, at least 11% of patients had mixed infection. Pediatric strains differ in their spectrum of strain-variable genes and percentage of absent genes in comparison to adult strains. Most children were infected with H. pylori strains lacking the cag PAI, but the presence of a complete cag PAI, in contrast to other virulence markers, was associated with more severe gastroduodenal disease.It is estimated that >50% of the world''s population is colonized with Helicobacter pylori in the stomach, making it one of the most common bacterial pathogens of humans. H. pylori infection is generally acquired in childhood (24, 33) and can persist for life. Gastritis (inflammation of the gastric mucosa) results in all who are colonized with H. pylori, but some hosts remain asymptomatic, while others develop peptic ulcers, gastric adenocarcinomas, and mucosa-associated lymphoid tissue lymphoma. Gastric cancer is the second leading cause of cancer death worldwide, and 63% of gastric cancer cases in 2002 were attributable to H. pylori infection (38, 49). While severe disease most often presents in adulthood, children display H. pylori-associated gastritis and the incidence of ulcer disease among infected children was 6.8% in a European pediatric population (31). Many studies have examined bacterial, host, and environmental risk factors associated with development of H. pylori-associated diseases in adults, but similar studies in children have been limited.Genetic differences among H. pylori strains contribute to differences in disease outcome among infected individuals in adult populations. The gene encoding VacA, which induces vacuolation of host cells, is present in nearly all H. pylori strains, but a number of allele types have been defined. Strains having the type s1 vacA signal sequence and the m1 vacA middle region allele (vacA s1/m1) are associated with ulcer disease (9). The cag pathogenicity island (PAI) encodes a type IV secretion system (T4SS) (1, 15) that translocates the CagA protein effector, also encoded in the island, into host cells. Presence of the cag PAI is associated with increased inflammation, promoting host cell interleukin-8 (IL-8) production, and cagA-positive strains are associated with peptic ulcers (50) as well as gastric cancer (13). Inside the host cell, CagA protein becomes tyrosine phosphorylated at C-terminal EPIYA (Glu-Pro-Ile-Tyr-Ala) sites by src family kinases, deregulates SHP-2, and induces the hummingbird phenotype (26, 45). Strains having more C-type EPIYA motifs, the major phosphorylation site, induce stronger effects on host cells and are associated with gastric cancer (7, 12, 35). The presence of a functional allele of babA, a gene encoding an adhesin that mediates binding to Lewis B antigens expressed on gastric epithelial cells, is associated with duodenal ulcer and gastric adenocarcinoma (21).While these H. pylori genes and alleles have been associated with disease outcome in adults, studies in children have provided mixed results. A recent study identified two genes (jhp0562, coding for a putative glycosyltransferase, and jhp0870, coding for an outer membrane protein) associated with peptic ulcer disease in children, but not adults, suggesting a different spectrum of genetic risk factors in adults and children (37). Studies using a whole-genome microarray-based approach have been done to investigate the variability in genomic content of H. pylori strains, but these studies have included mostly strains from adult patients (25, 29, 41, 42). Studies of the genetic variability of pediatric H. pylori strains have largely been limited to genes previously associated with virulence in adult populations. To better understand the genetic variability present among pediatric strains, we used whole-genome microarray-based comparative genomic hybridization to examine the genomic content of H. pylori strains isolated from symptomatic North American children and compared the pediatric isolate genetic variability to that observed in adult strains. We then examined the frequency of known virulence genes and virulence alleles among the pediatric H. pylori strains and the associations of strain genotype with the clinical and histological characteristics of the patients.     

High Prevalence of Clarithromycin Resistance and cagA,vacA, iceA2, and babA2 Genotypes of Helicobacter pylori in Brazilian Children

We isolated 45 Helicobacter pylori strains from 217 child patients. Resistance to clarithromycin, metronidazole, amoxicillin, and tetracycline was detected in 27%, 13%, 4%, and 0% of strains, respectively. The A2143G mutation was the most prevalent (67%) among clarithromycin-resistant strains. In addition, strain genotyping revealed a significant association between gastritis severity and the simultaneous presence of cagA, vacA s1m1, iceA2, and babA2 genes.Helicobacter pylori infection is found worldwide and constitutes a public health concern in many countries. Previous epidemiological studies have shown a high prevalence of H. pylori infection in Brazil (2, 20, 24). H. pylori infection, generally acquired in childhood, persists asymptomatically for decades in most individuals.Amoxicillin, tetracycline, metronidazole, and clarithromycin are frequently used, combined with proton pump inhibitors or bismuth salts, for the treatment of H. pylori infections (25). However, antibiotic resistance is frequently associated with eradication failure (3, 16). Resistance to metronidazole and clarithromycin is population dependent, and several studies suggest that clarithromycin resistance is higher in strains isolated from children than in strains isolated from adults (10). In Brazil, the prevalence of clarithromycin-resistant strains in adults is reported to be from 7 to 10% (15, 18). However, little is known about the prevalence of clarithromycin-resistant H. pylori infection in Brazilian children.The primary aims of this study were to determine the prevalence of clarithromycin-resistant H. pylori strains in children, to identify those isolates via rapid methodology, and to examine the severity of gastritis caused by the antibiotic-resistant H. pylori isolates. Metronidazole, amoxicillin, and tetracycline resistance was also studied. Furthermore, the study aimed to genotype the vacA and iceA genes and to detect the cagA gene in gastric biopsy specimens, since recent studies found a high frequency of cagA-positive and iceA2-positive strains as well as a strain with the vacA signal region genotype s1 and middle region sequence m1 among pediatric H. pylori isolates in Brazil (6, 7, 11, 23). This is also the first investigation of babA2 gene prevalence in Brazilian children.A total of 217 consecutive child patients, aged 1 to 18 years (mean age, 10 years) (105 girls and 112 boys), who underwent upper gastrointestinal endoscopy for the evaluation of dyspeptic symptoms at the outpatient clinic of Pediatric Gastroenterology at the Instituto da Criança, Faculdade de Medicina da Universidade de São Paulo, during 2008 and 2009 were included. The study was approved by the Ethics Committee of the University Hospital. Patients previously treated for H. pylori infections were not included.Gastric biopsy specimens were processed for histological examination and evaluated according to the updated Sydney system of classification and grading of gastritis (4).Antral gastric specimens were transported in sodium thioglycolate broth (Difco, Detroit, MI) in an ice bath and ground before submission to DNA extraction and PCR-restriction fragment length polymorphism (PCR-RFLP) analysis with primers specific to the H. pylori 23S rRNA gene (17). The QIAmp tissue kit (Qiagen) was used for DNA extraction. Point mutations related to clarithromycin resistance in the 23S rRNA amplicon were investigated in all H. pylori isolates by PCR-RFLP using BsaI and MboII enzymes (27). The vacA, cagA, iceA, and babA2 genotypes were detected by PCR, as described elsewhere (1, 9, 21, 26, 28). In each experiment, H. pylori strain 26695 (ATCC 700392) was used as the positive-control strain.H. pylori strains were cultured on Belo Horizonte medium (22) under microaerophilic atmosphere at 37°C for 3 to 7 days, and the isolates were identified by Gram staining and biochemical tests for oxidase, catalase, and urease production. Resistance to clarithromycin, metronidazole, amoxicillin, and tetracycline was determined by the disc diffusion method (Oxoid), and MICs were determined by the Etest according to the manufacturer''s recommendations (AB Biodisk, Solna, Sweden). An isolate was considered resistant to clarithromycin or tetracycline if the MIC was >1 mg/liter and to metronidazole or amoxicillin if the MIC was >4 mg/liter (19).Data were analyzed by the two-tailed χ² test and Fisher exact test. P values of <0.05 were considered statistically significant.H. pylori was isolated in 45 (20.7%) of the 217 children; 12 (26.7%) of the 45 strains were clarithromycin resistant, 6 (13.3%) were metronidazole resistant, and 2 (4.4%) were amoxicillin resistant. All cultured H. pylori strains were susceptible to tetracycline (Fig. ​(Fig.1).1). No histological differences were observed between biopsy specimens with antibiotic-resistant strains and those with susceptible strains. PCR-RFLP was performed with all 12 clarithromycin-resistant isolates: 8 had the 23S rRNA A2143G point mutation, and 4 had the 23S rRNA A2142G mutation.Open in a separate windowFIG. 1.Distribution of MICs for the 45 H. pylori strains.Among the 45 H. pylori-infected children, 13 had mild chronic gastritis, 28 had moderate chronic gastritis, 2 had marked chronic gastritis, and 2 had normal gastric mucosa. The percentage of H. pylori-infected children with chronic gastritis was 95.5% (43 patients), while 4.4% of the children (2 patients) had normal mucosa (P < 0.001).vacA was detected in all 45 H. pylori-positive gastric biopsy specimens. The vacA genotypes s1m1, s2m2, and s1m2 or s2m1 were found in 57.7, 33.3, and 4.4% of the specimens, respectively. The iceA1 allele was detected in 9 (20%) and the iceA2 allele in 31 (68.9%) of the samples. Of the 45 H. pylori-positive biopsy specimens, 28 (62%) were cagA positive and 38 (84.4%) were babA2 positive. Correlation of histopathology results with vacA, cagA, and iceA genotypes showed that vacA s1m1-, cagA-, and iceA2-positive strains were more frequently found in patients with moderate and marked gastritis (77%) than in patients with mild gastritis (23%) (P < 0.001). Interestingly, in Slovenian children, vacA s1 and cagA were also shown to be associated with more pronounced chronic gastritis (12). In contrast, in Korean children, although vacA s1m1 cagA iceA1 was the predominant genotype, no association with gastritis severity was observed (14).In conclusion, we found a high incidence of clarithromycin-resistant H. pylori strains (27%) in Brazilian children. Furthermore, we found an association between clarithromycin resistance and either the vacA s1m1 (P = 0.007) or the iceA2 (P = 0.038) genotype. The high level of clarithromycin resistance among strains from children compared to adults (15, 18) suggests the importance of susceptibility testing, especially in Brazilian children. All together, these data stress the relevance of susceptibility testing and genotyping for establishing antibiotic treatment in pediatric H. pylori infection.In our study, PCR-RFLP proved to be a rapid and accurate method for the detection of clarithromycin resistance gene mutation directly in gastric biopsy samples. Only a few groups have studied mutations involved in clarithromycin resistance in strains obtained from children, and their results are similar to those obtained in our study (5, 13, 29).Our data also demonstrate an association between H. pylori infection and gastritis in Brazilian children. In addition, we confirmed the reported association of infection with vacA s1m1 cagA iceA2-positive H. pylori strains and gastritis severity (6, 11, 23). Furthermore, a high frequency of babA2 was found among H. pylori isolates. Previous studies of adults in Brazil reported a high prevalence of babA2-positive strains from patients with different upper gastrointestinal diseases (8). The high incidence of babA2 in H. pylori Brazilian isolates suggests that this gene could be a useful marker for identifying patients with a high risk of H. pylori infection in Brazil.     

Helicobacter pylori Exploits Cholesterol-Rich Microdomains for Induction of NF-κB-Dependent Responses and Peptidoglycan Delivery in Epithelial Cells

Infection with Helicobacter pylori cag pathogenicity island (cagPAI)-positive strains is associated with more destructive tissue damage and an increased risk of severe disease. The cagPAI encodes a type IV secretion system (TFSS) that delivers the bacterial effector molecules CagA and peptidoglycan into the host cell cytoplasm, thereby inducing responses in host cells. It was previously shown that interactions between CagL, present on the TFSS pilus, and host α₅β₁ integrin molecules were critical for CagA translocation and the induction of cytoskeletal rearrangements in epithelial cells. As the α₅β₁ integrin is found in cholesterol-rich microdomains (known as lipid rafts), we hypothesized that these domains may also be involved in the induction of proinflammatory responses mediated by NOD1 recognition of H. pylori peptidoglycan. Indeed, not only did methyl-β-cyclodextrin depletion of cholesterol from cultured epithelial cells have a significant effect on the levels of NF-κB and interleukin-8 (IL-8) responses induced by H. pylori bacteria with an intact TFSS (P < 0.05), but it also interfered with TFSS-mediated peptidoglycan delivery to cells. Both of these effects could be restored by cholesterol replenishment of the cells. Furthermore, we demonstrated for the first time the involvement of α₅β₁ integrin in the induction of proinflammatory responses by H. pylori. Taking the results together, we propose that α₅β₁ integrin, which is associated with cholesterol-rich microdomains at the host cell surface, is required for NOD1 recognition of peptidoglycan and subsequent induction of NF-κB-dependent responses to H. pylori. These data implicate cholesterol-rich microdomains as a novel platform for TFSS-dependent delivery of bacterial products to cytosolic pathogen recognition molecules.Helicobacter pylori strains harboring the cytotoxin-associated gene pathogenicity island (cagPAI) are associated with the production of higher levels of interleukin-8 (IL-8), more destructive tissue damage, and an increased risk of severe disease (8, 13, 15, 43, 44). The cytotoxin-associated gene A product (CagA) is translocated into eukaryotic epithelial cells by the cagPAI-encoded type IV secretion system (TFSS) (7, 40). Once intracellular, CagA localizes on the inner surface of the plasma membrane and becomes phosphorylated on tyrosine residues by Src family kinases (5). The phosphorylated CagA subsequently induces a signaling cascade, ultimately resulting in the development of a “cell-scattering” phenotype in gastric epithelial cells (5, 50).A large body of evidence from previous studies has suggested that CagA is not essential for IL-8 secretion from gastric epithelial cells, as ΔcagA mutants retained the ability to induce IL-8 production (11, 15, 18, 52). More recently, however, CagA was found to play a role in the potentiation of IL-8 induction upon extended coculture (>36 h) of epithelial cells with cagPAI-positive H. pylori strains (10). Another report recently described the rapid induction of nuclear factor kappa B (NF-κB) activation in epithelial cells via CagA (35). The reason for the lack of concordance of these more recent findings with those of previous works is not known, but it may be partly attributable to H. pylori strain differences.Notwithstanding the ambiguity regarding the role of CagA in the induction of proinflammatory responses in epithelial cells, it is broadly accepted in the field that the H. pylori TFSS is essential for induction of NF-κB-dependent responses in these cells. Our group previously reported that induction of such responses was dependent on TFSS delivery of cell wall peptidoglycan (PG) to host cells (57). Once intracellular, PG was proposed to be detected by a cytosolic pathogen recognition molecule (PRM), nucleotide-binding oligomerization domain protein (NOD1) (19, 57), resulting in activation of NF-κB and the induction of IL-8 secretion by epithelial cells (57). Nevertheless, the exact mechanism by which PG may enter the host cell via the actions of the TFSS, so as to initiate these NOD1-dependent responses, has remained elusive.It has recently been suggested that virulence factors of H. pylori associate with cholesterol-rich microdomains of the plasma membrane, commonly termed lipid rafts (34). These domains not only are enriched in cholesterol but also contain sphingolipids and proteins (53) and have been reported to be sites utilized by bacteria to interact with host cells (1) or as portals of entry by which bacteria enter these cells (29, 33). Wunder and colleagues demonstrated colocalization of H. pylori bacteria with G_M1 ganglioside, a characteristic component of cholesterol-rich microdomains, and established a role for cholesterol in the attraction of H. pylori to host cells (62). H. pylori was reported to migrate toward and acquire exogenous cholesterol from the plasma membranes of host epithelial cells (62). Furthermore, disruption of cholesterol-rich microdomains using cholesterol-depleting agents such as methyl-β-cyclodextrin (MβCD) was shown to significantly reduce the internalization of the vacuolating cytotoxin (VacA) into target cells (49) and to inhibit the ability of the toxin to induce cell vacuolation (31, 48). Moreover, Lai et al. reported that cholesterol-rich microdomains are also crucial for efficient TFSS-mediated CagA translocation by H. pylori, as CagA-induced cellular responses, including the cell-scattering phenotype and IL-8 production, were found to be reduced in MβCD-treated cells (34).The translocation and phosphorylation of CagA are triggered by direct interaction of the H. pylori TFSS with the α₅β₁ integrin, which is found on the surfaces of gastric epithelial cells (32). A number of viruses and bacteria are known to use integrin receptors to adhere to and invade host cells (9, 24). Indeed, Kwok et al. demonstrated that the H. pylori adhesin CagL is targeted to the surface of the secretory pilus encoded by the cagPAI, where it binds to and activates α₅β₁ integrin (32). This interaction triggers the delivery of CagA into the host as well as subsequent Src kinase activation (32). Given that α₅β₁ associates with cholesterol-rich microdomains (27, 37) and that these membrane microdomains have been shown to be important for CagA effects on host responses (34), we speculated that H. pylori strains may exploit a similar mechanism to induce proinflammatory responses in epithelial cells via delivery of PG to NOD1. We now show for the first time that H. pylori PG translocation into epithelial cells and the subsequent activation of NF-κB-dependent responses (a characteristic of NOD1 activation) are dependent on lipid rafts and more specifically on α₅β₁ integrin.     

Improved Performance of a Rapid Office-Based Stool Test for Detection of Helicobacter pylori in Children before and after Therapy

A modified version of a rapid office based one-step monoclonal immunoassay for detection of Helicobacter pylori antigen in stool samples from children was evaluated against biopsy specimen-based methods and compared to a monoclonal enzyme immunoassay using the same antigen. Blinded stool samples from 185 children (0.3 to 18.2 years) were investigated at the time of upper endoscopy prior to anti-H. pylori therapy; 62 children were H. pylori infected and 123 noninfected according to predefined reference standards. Samples obtained 6 to 8 weeks after anti-H. pylori therapy were available from 58 children (3.8 to 17.7 years) and were compared to results of the [¹³C]urea breath test (14/58 were positive). The rapid stool tests were performed by two independent readers. Of 243 rapid tests performed, 1 (0.4%) was invalid for technical reasons. Equivocal results (very weak line) were reported 16 times by reader 1 and 27 times by reader 2. When equivocal results were considered positive, the two observers agreed on 76 positive and 160 negative results and disagreed on 7 samples (2.9%). The sensitivity was 90.8% for reader 1 and 85.5% for reader 2, and the specificity was 91.0% and 93.4%, respectively. The monoclonal enzyme immunoassay revealed a sensitivity and specificity of 94.7% and 97.6%, respectively. The modified chromatographic immunoassay is a good alternative in settings or situations when the monoclonal enzyme immunoassay or the [¹³C]urea breath test are not available or feasible. In order to improve sensitivity, very weak lines should be considered positive test results.Several noninvasive methods are available for the diagnosis of H. pylori infection (5, 14). Serological tests are not appropriate, since they cannot distinguish between a present and previous infection and, in addition, they have a low sensitivity in children younger than 12 years of age (6, 13). The [¹³C]urea breath test (UBT) is the preferred noninvasive diagnostic tool and gives excellent performance for both adults and children, but specificity decreases in very young and mentally disabled children who are not able to cooperate with the test procedure (10, 11, 25). So far, tests for detection of H. pylori antigen in stool samples are the only noninvasive diagnostic tools which do not show an age dependence for the diagnostic accuracy (14, 15). This makes stool tests very attractive, particularly for young children and for epidemiological studies. Several tests have been developed, but validation studies showed differences in performance. An enzyme immunoassay (EIA) based on polyclonal antibodies that was developed by the Meridian Company has been validated in several studies, with controversial results (17, 20, 24). Lack of accuracy is obviously related to intertest variability (19). In contrast, EIA based on monoclonal antibodies showed consistently excellent results, with very high sensitivity and specificity in both children and adults (15, 21). A meta-analysis with head-to-head comparison has judged the monoclonal EIA superior to the polyclonal EIA (8).Recently, we reported on the performance of a one-step monoclonal chromatographic immunoassay for detection of H. pylori antigen in stool samples from symptomatic children compared to the results of a well-established monoclonal EIA using the same antigen, namely, the catalase of H. pylori (22). Evaluation against biopsy specimen-based diagnostic methods showed a moderate sensitivity but a good specificity. After publication of the data, the manufacturer modified the tests. The aim of this study was to evaluate this new version of the rapid office-based one-step stool test in symptomatic children against invasive diagnostic methods and to compare the results with those of the monoclonal EIA.     

Differences in Genome Content among Helicobacter pylori Isolates from Patients with Gastritis,Duodenal Ulcer,or Gastric Cancer Reveal Novel Disease-Associated Genes

Helicobacter pylori establishes a chronic infection in the human stomach, causing gastritis, peptic ulcer, or gastric cancer, and more severe diseases are associated with virulence genes such as the cag pathogenicity island (PAI). The aim of this work was to study gene content differences among H. pylori strains isolated from patients with different gastroduodenal diseases in a Mexican-Mestizo patient population. H. pylori isolates from 10 patients with nonatrophic gastritis, 10 patients with duodenal ulcer, and 9 patients with gastric cancer were studied. Multiple isolates from the same patient were analyzed by randomly amplified polymorphic DNA analysis, and strains with unique patterns were tested using whole-genome microarray-based comparative genomic hybridization (aCGH). We studied 42 isolates and found 1,319 genes present in all isolates, while 341 (20.5%) were variable genes. Among the variable genes, 127 (37%) were distributed within plasticity zones (PZs). The overall number of variable genes present in a given isolate was significantly lower for gastric cancer isolates. Thirty genes were significantly associated with nonatrophic gastritis, duodenal ulcer, or gastric cancer, 14 (46.6%) of which were within PZs and the cag PAI. Two genes (HP0674 and JHP0940) were absent in all gastric cancer isolates. Many of the disease-associated genes outside the PZs formed clusters, and some of these genes are regulated in response to acid or other environmental conditions. Validation of candidate genes identified by aCGH in a second patient cohort allowed the identification of novel H. pylori genes associated with gastric cancer or duodenal ulcer. These disease-associated genes may serve as biomarkers of the risk for severe gastroduodenal diseases.Infection with Helicobacter pylori is one of the most common bacterial infections in humans worldwide, and like the case for many other infections, rates are higher in developing countries (80 to 90%) than in developed countries (<50%) (20, 33, 39). The infection is associated with peptic ulcers, gastric carcinoma, and mucosa-associated lymphoma (32, 36, 39). Most infected people remain asymptomatic during their lifetime, and only about 15% develop gastroduodenal illness. Environmental, host, and bacterial factors all play a role in the outcome of the infection. A number of bacterial virulence factors associated with disease have been described for H. pylori, and the most consistently reported are the cag pathogenicity island (cag PAI) (4, 40, 52) and vacA (2, 43). Outer membrane proteins such as BabA2 (18), OipA (54), and SabB (11), as well as the iceA gene, have also been reported to be associated with disease. An outstanding characteristic of H. pylori is the high level of genetic diversity among isolates from different patients, even if they belong to the same ethnic population. Several molecular typing techniques have been used to genotype H. pylori strains from different populations, demonstrating genetic differences among populations and even among isolates from the same individual, suggesting the presence of mixed infection (3, 9, 19, 26, 35, 46). The ability of this bacterium to generate such genetic diversity is due to its natural competence, high recombination and mutation rates, or the occurrence of slipped-strand synthesis and phase variation (17, 24).H. pylori was the first bacterial species for which whole genome sequences of two independent strains were available (J99 and 26695). Their comparison showed that approximately 6 to 7% of the H. pylori genes present in one strain are absent in the other. These genes are called strain-specific genes, and almost half of them are located in hypervariable regions of the genome (1, 51). These regions contain a considerable number of restriction-modification genes and genes for transposases, topoisomerases, and outer membrane proteins. One of these regions is the cag PAI, whereas the others have been termed plasticity zones (PZs). Whole-genome DNA microarrays facilitated further analyses of the genomic contents of 15 H. pylori clinical isolates, revealing 362 genes (22% of all genes) that are not conserved among strains and represent variable or strain-specific genes (45). Similar microarray-based comparative genomic hybridization (aCGH) studies have been used to explore the genetic diversity in the H. pylori strain population colonizing the different regions of the stomach of a single host for both adults and children (46) and to correlate the genetic contents of H. pylori strains with pathogenesis in animal models (6, 25). These studies indicate that H. pylori strains gain or lose loci during chronic infection, suggesting a continuous genetic flux, mainly inside the PZs (14, 22, 26, 27, 28). The sequence of an H. pylori strain isolated from a patient with chronic atrophic gastritis was recently published, identifying additional strain-specific genes (38) and, by comparison with previous studies (22), suggesting that 121 genes are “chronic atrophic gastritis associated.”Other studies have reported that genes located in the PZs, such as jhp0947 and jhp0949, are associated with disease (12, 37, 47). The dupA gene was associated with an increased risk for duodenal ulcer (DU) and a low risk for gastric atrophy and cancer (31). The aim of the present study was to compare the genomic contents of H. pylori strains isolated from patients with nonatrophic gastritis (NAG), DU, or gastric cancer (GC) in a Mexican-Mestizo population in order to look for genes previously associated with severe gastroduodenal diseases and to identify novel disease biomarkers.     

CagA and VacA Polymorphisms Do Not Correlate with Severity of Histopathological Lesions in Helicobacter pylori-Infected Greek Children

The presence of various numbers of EPIYA tyrosine phosphorylation motifs in the CagA protein of Helicobacter pylori has been suggested to contribute to pathogenesis in adults. In this prospective study, we characterized H. pylori isolates from symptomatic children, with reference to the diversity of functional EPIYA motifs in the CagA protein and vacA isotypes, and assessed the potential correlation with the histopathological manifestations of the infection. We analyzed 105 H. pylori isolates from 98 children and determined the diversity of EPIYA motifs in CagA by amplification and sequencing of the 3′ variable region of the cagA gene as well as vacA isotypes for the signal, middle, and intermediate regions. CagA phosphorylation and levels of secreted IL-8 were determined following in vitro infection of AGS gastric epithelial cells. Histopathological evaluation of H. pylori colonization, activity, and severity of the associated gastritis was performed according to the updated Sydney criteria. EPIYA A (GLKN[ST]EPIYAKVNKKK), EPIYA B (Q[V/A]ASPEPIY[A/T]QVAKKVNAKI), and EPIYA C (RS[V/A]SPEPIYATIDDLG) motifs were detected in the ABC (46.6%) and ABCC (17.1%) combinations. No isolates harboring more than two EPIYA C motifs in CagA were found. The presence of isogenic strains with variable numbers of CagA EPIYA C motifs within the same patient was detected in seven cases. Occurrence of increasing numbers of EPIYA C motifs correlated strongly with presence of a high-vacuolation (s1 or s2/i1/m1) phenotype and age. A weak positive correlation was observed between vacuolating vacA genotypes and presence of nodular gastritis. However, CagA- and VacA-dependent pathogenicities were not found to contribute to severity of histopathology manifestations in H. pylori-infected children.Helicobacter pylori infects 50% of the world''s population, and wide differences in prevalence of infection appear to exist between countries with different levels of socioeconomic development. Infection usually occurs in childhood and in the majority of cases remains asymptomatic, although major reasons for endoscopy referral can include recurrent epigastric or abdominal pain, with or without vomiting, neither of which correlates with H. pylori infection (17). Antral nodularity is a well-described endoscopic feature of H. pylori-infected children, and histological observations usually include superficial chronic active gastritis with occasional infiltration of eosinophils; in far fewer cases, they include peptic ulcers; and very rarely, they include gastric atrophy and intestinal metaplasia (13, 27). If the infection is left untreated, it persists through adulthood, and although it can still remain asymptomatic in the vast majority of infected hosts, H. pylori infection is now regarded as the most important etiological risk factor for development of gastric cancer in developed countries (28). H. pylori pathogenesis is manifested through a combined effect of bacterial virulence factors, host genetics, and environmental factors, which orchestrate toward the development of distinct phenotypes in adults, namely, superficial asymptomatic gastritis, duodenal ulcer, and gastric cancer (3). The expression and translocation of cytotoxin-associated gene antigen (CagA), a putative H. pylori virulence factor, inside gastric epithelial cells by cagA-positive H. pylori strains harboring a functional type IV secretion system has been suggested to play an important role in H. pylori pathogenesis (22). Early epidemiological studies of adults associated the presence of the cagA gene with development of peptic ulcer disease (31); gastric cancer (14); and increased inflammation (35), cellular proliferation (36), and intestinal metaplasia (20) of the gastric mucosa. However, in infected children, neither cagA status nor any other putative H. pylori virulence factor has been found to correlate with clinical outcome or severity of histological manifestations. However, recent advances into the fascinating cellular biology of CagA inside the gastric epithelial cell have enhanced its reputation as a potential bacterial oncoprotein (22). Following its translocation inside the gastric epithelial cell via the type IV secretion system (32), CagA has been shown to become at least partly tyrosine phosphorylated (5, 11, 41) by Src family kinases (42, 44) on repeating 5-amino-acid glutamic-proline-isoleucine-tyrosine-alanine (EPIYA) motifs present at the C terminus of the protein. Analysis of EPIYA motifs in CagA has revealed considerable type variation, depending on the peptide sequence surrounding it, namely, EPIYA A (EPIYAKVNKKK), EPIYA B (EPIYAQVAKKV), or EPIYA C (EPIYATIDDLG) in isolates from Western populations or EPIYA D (EPIYATIDFD) in isolates of Asian origin. In addition, considerable variation in number of repeating EPIYA C or D motifs at the carboxyl terminus of the protein (10, 44) has been observed, and biological activity of CagA was suggested to be determined by variation in these motifs (25) in phosphorylation-dependent as well as -independent ways (23). Hence, the number and type of EPIYA phosphorylation motifs may be viewed as putative virulence determinants of CagA activity and therefore become useful clinical markers that may predict the degree of individual H. pylori strain virulence potential. In this context, we proposed a PCR amplification and sequencing-based strategy for accurate characterization of the number and type of EPIYA motifs of CagA in H. pylori clinical isolates (34).A multifactorial role has also been attributed to the secreted VacA virulence factor (16), a protein with multiple cellular activities, as it can disrupt endocytic trafficking of host cells, promote cell death through apoptosis, suppress the local immune system, and possibly potentiate the development of ulcers (6). Although the vacA gene is present in all H. pylori strains, it contains at least three variable parts, the s region, the i region, and the m region, which encode the signal, intermediate, and middle peptides, respectively, which have all been classified as allelic types 1 and 2. The s1-or-s2/i1/m1-or-m2 and s1-or-s2/i1-i2/m1-or-m2 VacA isotypes induce, in general, high and moderate levels of vacuolation, respectively, whereas the s1-or-s2/i2/m1-or-m2 strains induce very little or no vacuolation (39). Consequently, the vacA s/m genotype can also be regarded as a marker of pathogenicity of individual strains (8). Moreover, phylogenetic linkage analysis studies have indicated that there may be a functional basis for the selection of vacA and cagA isotypes (50), although there is substantial distance between vacA loci and cag genes on the bacterial genome. Furthermore, the intermediate region has been associated with development of gastric cancer (39).In the present study, we investigated the potential association of the CagA and VacA virulence factor polymorphisms with clinicopathological manifestations of the disease in symptomatic Greek children. More specifically, H. pylori clinical strains isolated from symptomatic children were characterized with regard to the number and type of repeating EPIYA phosphorylation motifs in CagA protein and the vacA signal, intermediate, and middle region genotypes. Furthermore, these clinical isolates were carefully assessed for their ability to express phosphorylated CagA as well as induce interleukin-8 (IL-8) secretion following infection of gastric epithelial cells. Finally, the potential association of such functional bacterial determinants with H. pylori-associated histopathology in these patients was assessed.     

Helicobacter pylori Virulence Factor Genotypes in Children in the United States: Clues about Genotype and Outcome Relationships

The most common Helicobacter pylori genotype among 37 U.S. children was cagA positive, vacA s1m1, and oipA “on” (n = 17, 45.9%), followed by cagA negative, vacA s2m2, and oipA “off” (n = 8, 21.6%), similar to the pattern in adults. cagA positivity was more common in blacks than in whites (i.e., 100% versus 56.5%, P = 0.032).Infection with Helicobacter pylori is etiologically associated with gastritis, peptic ulcer disease, gastric atrophy, and gastric cancer. H. pylori is thought to be typically acquired in childhood, with infection continuing for decades if not lifelong. This long bacterial host association involves countless generations of bacteria which are thought to continually evolve as the intragastric conditions change, such that those best suited for the local conditions outgrow and replace less suited neighbors. There has been considerable interest in the molecular epidemiology of H. pylori''s putative virulence factors, especially CagA, VacA, and OipA (outer inflammatory protein A) (8, 11, 12). However, there are few studies of children and only one previous study investigating the relationship between H. pylori virulence factors and ethnic groups of children in the United States (4-6, 11). This study reports the patterns of H. pylori virulence factor genotypes in children of different ethnic groups in the United States.The biopsy specimens and cultures were obtained as part of a multicenter study from 5 widely dispersed sites in the United States. The study was designed to validate the [¹³C]urea breath test for the diagnosis of H. pylori infection in children aged between 2 years and 17 years and 11 months (2). Symptomatic children scheduled for endoscopy were enrolled. Gastric biopsy specimens were evaluated by histology, rapid urease testing, and culture of H. pylori using established techniques (2). H. pylori culture isolates were evaluated for cagA and vacA genotypes using established PCR assays as previously described (9). The number of EPIYA (Glu-Pro-Ile-Tyr-Ala) repeat motifs in the 3′ region of the cagA gene was evaluated using PCR as previously described (7). OipA is a member of the large outer membrane protein family whose functional status is regulated by slipped-strand mispairing based on the number of CT dinucleotide repeats in the 5′ region of the gene (a switch status of “on” indicates the gene is functional, and a switch status of “off” indicates it is nonfunctional) (10). The 5′ region of the oipA gene was amplified using previously described primers (10), and the PCR fragments were purified and directly sequenced at Macrogen, Ltd., in Seoul, South Korea.Forty-eight of 176 children enrolled were H. pylori infected, based on two positive tests or a positive H. pylori culture. The mean age was 11.5 years (range, 3.2 to 17.9 years). Thirty-seven were H. pylori culture positive. None had atrophic gastritis. Only one patient had a significant endoscopic abnormality, a duodenal ulcer (cagA positive, vacA s1m2, and oipA on). The most common H. pylori genotype was cagA positive, vacA s1m1, and oipA on (n = 17, 45.9%), followed by cagA negative, vacA s2m2, and oipA off (n = 8, 21.6%), cagA positive, vacA s1m2, and oipA on (n = 5), cagA positive, vacA s1m1, and oipA on (n = 3), cagA negative, vacA s2m2, and oipA on (n = 2), cagA negative, vacA s1m1, and oipA on (n = 1), and cagA positive, vacA s1m2, and oipA off (n = 1) (Table ​(Table1).1). Overall, 70% of strains were cagA positive, which is similar to is the proportion found in U.S. adults (6).

TABLE 1.

Relationship between H. pylori genotype and ethnic group

Antibody-Mediated Protection against Infection with Helicobacter pylori in a Suckling Mouse Model of Passive Immunity

Studies of active immunization against Helicobacter pylori indicate that antibodies play a minor role in immunity. There is also evidence, however, that the translocation of antibodies in the stomach may be insufficient to achieve functional antibody levels in the gastric lumen. We have used a suckling mouse model of passive immunity to determine if perorally delivered antibodies can protect against infection with H. pylori. Female C57BL/6 mice were immunized parenterally with formalin-fixed cells of three clinical isolates of H. pylori (3HP) or the mouse-adapted H. pylori strain SS1 before mating. Their pups were challenged with the SS1 strain at 4 days of age and left to suckle before determination of bacterial loads 14 days later. Compared to age-matched controls, pups suckled by 3HP-vaccinated dams were significantly protected against infection (>95% reduction in median bacterial load; P < 0.0001). Pups suckled by SS1-vaccinated dams were also significantly protected in terms of both median bacterial load (>99.5% reduction; P < 0.0001) and the number of culture-negative pups (28% versus 2% for immune and nonimmune cohorts, respectively; P < 0.0001). Similar results were obtained with pups suckled by dams immunized with a urease-deficient mutant of SS1. Fostering experiments demonstrated that protection was entirely attributable to suckling from an immunized dam, and antibody isotype analysis suggested that protection was mediated by the immunoglobulin G fraction of immune milk. Analysis of the bacterial loads in pups sampled before and after weaning confirmed that infection had been prevented in culture-negative animals. These data indicate that antibodies can prevent colonization by H. pylori and suppress the bacterial loads in animals that are colonized.Helicobacter pylori colonizes the gastric mucosa of humans and commandeers host defenses to establish chronic active gastritis while increasing the host''s susceptibility to gastroduodenal ulceration or certain gastric malignancies (37). Although H. pylori induces profound systemic and mucosal immune responses, clearance of infection is infrequent, and there is no protection against reinfection following eradication by antimicrobial chemotherapy (15). Consequently, there are no obvious parameters of natural immunity on which to base effective vaccination strategies.Vaccination studies of animal models have suggested that antibody development is not necessary for protective immunity to H. pylori (19) and may even enhance colonization (5, 6). Conversely, cellular immunity, possibly in concert with innate immune factors, such as defensins (59), elicits protection or eradication by exaggerating the gastric inflammatory response induced by H. pylori, thus interrupting colonization without a need to interact with the bacteria directly (3). The importance of the inflammatory response for protection against H. pylori is supported by the association of postimmunization gastritis with vaccine efficacy (6, 23). Nevertheless, the failure of antibody to limit H. pylori colonization is yet to be fully explained. One reason for this failure may be the relatively low level of antibodies in the gastric lumen due to the apparent inability of the mucosal immune system to translocate sufficient quantities of antibody across the gastric mucosa.Although well characterized in the intestine, relatively little is known about antibody secretion into the stomach. Some studies of H. pylori infection have reported that levels of immunoglobulin A (IgA) in gastric juice are significantly lower than those found in the saliva or intestinal contents (33, 34). Evidence that these low levels of IgA are due to inadequate antibody secretion in the stomach includes the following: (i) H. pylori-specific antibodies in gastric juice of infected individuals are predominantly nonsecretory IgA (10); (ii) equivalent amounts of IgG and IgA in the stomach suggest that IgA may leak across the mucosa rather than being actively secreted (14, 18); and (iii) much of the secretory IgA (sIgA) in the stomach is derived from swallowed saliva (17, 54). In addition, compared to the small intestine, the normal mammalian stomach has barely detectable secretory component (SC), suggesting a limited capacity for translocation of polymeric IgA across the gastric mucosa (8, 33). Moreover, despite considerable upregulation of SC by gamma interferon following the development of gastritis, there is no corresponding increase in the concentration of sIgA in gastric juice (4). Consequently, the concentration of sIgA in the stomach is unlikely to be sufficient to prevent or eradicate colonization by H. pylori.On the other hand, there is evidence that passive immunization with antibodies delivered perorally may reduce the extent of gastric colonization by Helicobacter species. This therapeutic approach has shown some promise in adult mice given monoclonal IgA or hyperimmune bovine colostrum against Helicobacter felis (14, 41) or urease-specific, chicken-derived IgY against H. pylori (44). In addition, reports of delayed acquisition of H. pylori by Gambian infants that corresponded to their mothers’ levels of breast milk IgA specific for H. pylori (58) and the protection of infant mice against full colonization by H. felis while suckling from immunized dams (13) suggest that orally delivered antibodies may be beneficial in controlling gastric Helicobacter infections. Despite these favorable reports, there are no tightly controlled studies that conclusively show prevention of H. pylori infection by orally delivered immune antibodies in the absence of additional factors, such as famotidine (44). Moreover, no studies have investigated the refinement of vaccine preparations for use in the production of anti-H. pylori polyclonal antibody products.In this study, we used a suckling mouse model of infection to investigate whether H. pylori-specific antibodies delivered during lactation to the gastric lumen of infant mice can protect against H. pylori infection. The route and adjuvant used to immunize the dams were selected to evoke an immune response similar to that required for the production of commercial quantities of polyclonal monomeric antibodies, such as from hyperimmune bovine colostrum. The model allowed us for the first time to quantify the contribution of passively acquired H. pylori-specific antibodies to protection against infection and provided an opportunity to examine different vaccine preparations for their ability to elicit these antibodies.     

homB Status of Helicobacter pylori as a Novel Marker To Distinguish Gastric Cancer from Duodenal Ulcer

The hom family of Helicobacter pylori outer-membrane proteins, especially the homB gene, has been suggested as a novel virulence factor; however, the clinical association and function of this gene are still unclear. We evaluated the presence of the homA, homB, and cagA genes in 286 strains isolated from patients in the U.S. and Colombian populations (126 with gastritis, 96 with duodenal ulcer, and 64 with gastric cancer) by PCR. The results were compared with the clinical presentation and gastric injury. The prevalence of the homB gene was significantly higher in strains isolated from gastric-cancer patients (71.9%) than in those from duodenal ulcer patients (52.1%) (P = 0.012). In a multivariate analysis, the presence of the cagA gene significantly increased the risk for developing gastric cancer and duodenal ulcer, with the presence of the homB gene acting as a factor that could distinguish gastric cancer from duodenal ulcer (adjusted odds ratio, 3.033; 95% confidence interval, ∼1.37 to ∼6.73). cagA status was correlated with homB status (r = 0.323; P < 0.01). A histological analysis showed that cagA status was associated with inflammation and atrophy both in the antrum and in the corpus, while homB status was associated with inflammation and atrophy in the corpus. homB gene status might be susceptible to gastric-cancer development such that the homB gene is used as a factor for discriminating the risk of gastric cancer from that of duodenal ulcer.Helicobacter pylori infection is one of the most common infections of mankind and is etiologically associated with gastritis, peptic ulcer disease (PUD), gastric cancer (GC), and gastric mucosa-associated lymphoid tissue lymphoma (19). Most infected people remain asymptomatic. Factors thought to be associated with clinical gastroduodenal diseases include H. pylori virulence, host genetics, and environmental factors, such as diet (11). Putative H. pylori virulence factors associated with an increased risk of a clinical outcome include the cag pathogenicity island, CagA, VacA, BabA, and OipA (5, 9, 22). However, none have been exclusively linked to a specific H. pylori-related disease (e.g., GC).The hom family is a small paralogous family of proteins that contain the C-terminal alternating hydrophobic motif and signal sequences typical of outer-membrane proteins. The homB and homA genes are 90% identical; the differences are confined to the central domain (1). Recent studies suggested that there was a close association between the presence of the homB gene and interleukin-8 secretion from human gastric epithelial cells and that the number of H. pylori isolates binding to gastric cells was related to the number of homB copies present (15). Moreover, the authors proposed that the presence of the homB gene was significantly associated with PUD in Portuguese children and adults less than 40 years of age and that it may be a new H. pylori virulence factor (15, 16). However, there is no study for the association between the homB gene and H. pylori-related diseases in other countries. This study investigated whether there was an association between the homA and homB genes and clinical gastroduodenal diseases and the severity of gastric inflammation in the U.S. and Colombian populations.     

Uptake of Helicobacter pylori Outer Membrane Vesicles by Gastric Epithelial Cells

Helicobacter pylori bacteria colonize the human stomach where they stimulate a persistent inflammatory response. H. pylori is considered noninvasive; however, lipopolysaccharide (LPS)-enriched outer membrane vesicles (OMV), continuously shed from the surface of this bacterium, are observed within gastric epithelial cells. The mechanism of vesicle uptake is poorly understood, and this study was undertaken to examine the roles of bacterial VacA cytotoxin and LPS in OMV binding and cholesterol and clathrin-mediated endocytosis in vesicle uptake by gastric epithelial cells. OMV association was examined using a fluorescent membrane dye to label OMV, and a comparison was made between the associations of vesicles from a VacA⁺ strain and OMV from a VacA⁻ isogenic mutant strain. Within 20 min, essentially all associated OMV were intracellular, and vesicle binding appeared to be facilitated by the presence of VacA cytotoxin. Uptake of vesicles from the VacA⁺ strain was inhibited by H. pylori LPS (58% inhibition with 50 μg/ml LPS), while uptake of OMV from the VacA⁻ mutant strain was less affected (25% inhibition with 50 μg/ml LPS). Vesicle uptake did not require cholesterol. However, uptake of OMV from the VacA⁻ mutant strain was inhibited by a reduction in clathrin-mediated endocytosis (42% with 15 μg/ml chlorpromazine), while uptake of OMV from the VacA⁺ strain was less affected (25% inhibition with 15 μg/ml chlorpromazine). We conclude that VacA toxin enhances the association of H. pylori OMV with cells and that the presence of the toxin may allow vesicles to exploit more than one pathway of internalization.Infection with the gastric pathogen Helicobacter pylori results in chronic gastritis (13) and is associated with increased risk for the development of peptic ulcer disease (35), gastric carcinoma (41, 57), and gastric lymphoma (5, 60). H. pylori persistence, in an environment where peristalsis and sloughing of cells are continually occurring, is mediated by a variety of adhesins present on the bacterial surface (14, 21, 36, 40). However, despite the ability to adhere to the gastric epithelium, the majority of organisms remain unattached to surface cells (32), leading to speculation that lipopolysaccharide (LPS)-enriched outer membrane vesicles (OMV) shed by these bacteria (15, 19, 26) contribute to H. pylori pathogenesis via the persistent delivery of bacterial virulence factors (including the vacuolating cytotoxin VacA) and antigens to the gastric mucosa (26, 27). Observations that H. pylori OMV modulate gastric epithelial cell proliferation (22), induce apoptosis (3), stimulate secretion of the proinflammatory cytokine interleukin-8 (22), increase micronucleus formation (8), and are at the luminal surface (15, 26) and within cells of the gastric epithelium (15) support this hypothesis.OMV shedding by Gram-negative bacteria is well described in the literature (reviewed by Kuehn and Kesty [33]), yet little is known of the mechanisms of vesicle adherence to and internalization within mammalian host cells. The adherence of enterotoxigenic Escherichia coli (ETEC) OMV to host cells is mediated via a heat-labile enterotoxin (LT) associated with these OMV (31), whereas leukotoxin, associated with OMV shed by Actinobacillus actinomycetemcomitans, is not involved in vesicle binding (12). The internalization of ETEC, Porphyromonas gingivalis, and Pseudomonas aeruginosa OMV has been shown to involve cholesterol-rich lipid rafts (6, 16, 31), and recently, Kaparakis and colleagues (25) reported that uptake of H. pylori OMV is also lipid raft dependent. This is in contrast to the uptake of Shigella flexneri OMV, which occurs via phagocytosis, with the proposed subsequent fusion of OMV with the phagosomal membrane and the release of vesicle contents into the cell cytoplasm (24).In this study, we sought to examine whether VacA cytotoxin associated with H. pylori OMV was involved in vesicle binding. We also examined the rate of OMV internalization and the involvement of LPS, cholesterol, and clathrin-mediated endocytosis in vesicle uptake by AGS gastric epithelial cells. We report that within 20 min essentially all VacA⁺ OMV associated with AGS cells were intracellular and that uptake was enhanced by the presence of vesicle-associated cytotoxin. Excess H. pylori LPS reduced vesicle uptake, having a more significant effect on VacA⁺ OMV than VacA⁻ vesicle uptake. Uptake of both VacA⁺ and VacA⁻ OMV did not require cholesterol. However, a reduction in clathrin-mediated endocytosis inhibited VacA⁻ OMV uptake and to a lesser extent VacA⁺ OMV internalization.     

Helicobacter pylori Stimulates Dendritic Cells To Induce Interleukin-17 Expression from CD4+ T Lymphocytes

Helicobacter pylori is a human gastroduodenal pathogen that leads to active chronic inflammation characterized by T-cell responses biased toward a Th1 phenotype. It has been accepted that H. pylori infection induces a Th17 response. At mucosal sites, dendritic cells (DCs) have the capacity to induce effector T cells. Here, we evaluate the role of DCs in the H. pylori-induced interleukin-17 (IL-17) response. Immunohistochemistry and immunofluorescence were performed on human gastric mucosal biopsy samples and showed that myeloid DCs in H. pylori-infected patients colocalized with IL-23- and that IL-17-producing lymphocytes were present in H. pylori-infected antral biopsy samples. In parallel, human monocyte-derived DCs stimulated in vitro with live H. pylori cells produced significant levels of IL-23 in the absence of IL-12 release. The subsequent incubation of H. pylori-infected DCs with autologous CD4⁺ T cells led to gamma interferon (IFN-γ) and IL-17 expression. The inhibition of IL-1 and, to a lesser extent, IL-23 inhibited IL-17 production by T cells. Finally, isogenic H. pylori mutant strains not expressing major virulence factors were less effective in inducing IL-1 and IL-23 release by DCs and IL-17 release by T cells than parental strains. Altogether, we can conclude that DCs are potent inducers of IL-23/IL-17 expression following H. pylori stimulation. IL-1/IL-23 as well as H. pylori virulence factors seem to play an important role in mediating this response.Gram-negative Helicobacter pylori is a gastroduodenal pathogen identified as being the causative agent of a variety of disease including gastritis, peptic ulcer, gastric adenocarcinoma, and mucosa-associated lymphoma (23, 27, 41, 42). H. pylori infection of gastric mucosa leads to active chronic inflammation characterized by both a lymphocytic and neutrophil infiltrate with the induction of proinflammatory cytokines, mainly interleukin-1β (IL-1β), tumor necrosis factor alpha (TNF-α), IL-8, and IL-6 (13, 29).The H. pylori-specific gastric mucosal T-cell response is predominantly a CD4⁺ T-cell response polarized toward a T-helper 1 (Th1) phenotype with increased levels of gamma interferon (IFN-γ) (4, 38, 55). Although profound, this immune response does not clear the bacteria, and indeed, the cytokines secreted are more associated with pathogenesis (38, 45). Furthermore, neutrophil responses are associated with tissue damage and ulceration (7, 60). The release of the neutrophil chemoattractant IL-8 by gastric epithelial cells was previously shown to depend on the expression of an H. pylori virulence factor: the cytotoxin-associated gene (cag) pathogenicity island (PAI) (14, 62). The cag PAI encodes the immunodominant protein CagA and the type IV secretion system, which serves to transfer the bacterial CagA protein and other soluble factors, such as peptidoglycans, to the cytoplasm of the host cell (9, 52). Strains expressing the cag PAI have been associated with a more severe inflammatory response than that induced by cag PAI-negative strains (12). The cellular recognition of cag PAI-positive strains is mediated via signaling through the host-intracellular pathogen recognition molecule NOD1 (nucleotide-binding oligomerization domain 1), leading to NF-κB activation and the induction of proinflammatory responses (58).It was previously shown that H. pylori infection is also associated with a marked production of Th17 cytokines (2, 39, 44). By using real-time PCR and Western blotting, it was previously demonstrated that IL-17, a proinflammatory cytokine, is upregulated in H. pylori-infected stomach biopsy specimens in comparison to uninfected specimens (39). IL-17 is a cytokine that characterizes a distinct population of T cells, namely, Th17 (1, 28). IL-17 has been associated with chronic inflammatory conditions such as rheumatoid arthritis (10) and multiple sclerosis (37). In addition, IL-17 proinflammatory function leading to IL-8 stimulation raises the possibility that IL-17 may play a role during bacterial infections (39, 57). Major cytokines associated with the differentiation of human Th17 cells were identified to be IL-23, IL-1β, and IL-6 (11, 61). While IL-12 plays a key role in the differentiation of naïve T cells to Th1 cells, IL-23 promotes the expansion of Th17 cells. In contrast, IL-27, another IL-12 family member, has been shown to limit the development of Th17 cells (25). IL-12 and IL-23 are heterodimers with a shared subunit, p40. Both IL-23 and IL-12 are produced by activated antigen-presenting cells (APCs) such as DCs and macrophages (48, 53).DCs, which play a central role in the induction of adaptive immune responses, are widely distributed in tissues, including gastrointestinal mucosa (32, 33), and were previously shown to be capable of migrating through epithelial tight junctions to gain access to the gastrointestinal lumen (33, 49). Furthermore, we and others have shown that H. pylori interactions with DCs trigger maturation and activation events that lead to the production of cytokines, which are important for the induction and regulation of immune responses (5, 18, 34, 43, 46).Previous studies of DC activation by H. pylori have focused on the induction of the Th1-biased response. Much less is known about the mechanism of induction as well as the cells and cytokine stimuli responsible for the expression of IL-17 in Helicobacter infection. Here, we have reevaluated the role of DCs in the induction of immune responses to Helicobacter infection by addressing the interaction of H. pylori-infected DCs with CD4⁺ T lymphocytes in initiating a Th17 response.     

Expression of the BabA Adhesin during Experimental Infection with Helicobacter pylori

The Helicobacter pylori babA gene encodes an outer membrane protein that mediates binding to fucosylated ABH antigens of the ABO blood group. We recently demonstrated that BabA expression is lost during experimental infection of rhesus macaques with H. pylori J166. We sought to test the generality of this observation by comparison of different H. pylori strains and different animal hosts. Challenge of macaques with H. pylori J99 yielded output strains that lost BabA expression, either by selection and then expansion of a subpopulation of J99 that had a single-base-pair mutation that encoded a stop codon or by gene conversion of babA with a duplicate copy of babB, a paralog of unknown function. Challenge of mice with H. pylori J166, which unlike J99, has 5′ CT repeats in babA, resulted in loss of BabA expression due to phase variation. In the gerbil, Leb binding was lost by replacement of the babA gene that encoded Leb binding with a nonbinding allele that differed at six amino acid residues. Complementation experiments confirmed that change in these six amino acids of BabA was sufficient to eliminate binding to Leb and to gastric tissue. These results demonstrate that BabA expression in vivo is highly dynamic, and the findings implicate specific amino acid residues as critical for binding to fucosylated ABH antigens. We hypothesize that modification of BabA expression during H. pylori infection is a mechanism to adapt to changing conditions of inflammation and glycan expression at the epithelial surface.Helicobacter pylori is a gastric bacterial pathogen of humans that chronically infects an estimated 50% of the world''s population (19). The consequence of long-term infection with H. pylori is typically asymptomatic gastritis, but ca. 10 to 15% of those infected will develop peptic ulcer disease or gastric adenocarcinoma (19). Several bacterial virulence factors are found more commonly in H. pylori strains that are associated with disease, the best studied of which are the cytotoxin-associated gene pathogenicity island (cag PAI) and certain alleles of the vacuolating cytotoxin, VacA. Another bacterial factor that is associated with disease, rather than asymptomatic infection, is the capacity for adherence. Approximately 4% of the H. pylori genome encodes a diverse repertoire of outer membrane proteins (OMPs), the largest of which is the 21-gene Hop family (1). BabA is a member of the Hop family that binds Lewis b (Leb) and related terminal fucose residues found on blood group O (H antigen), A, and B antigens that are expressed on the gastric epithelium (3, 6). Some studies suggest that patients infected with strains that express BabA are more likely to present with ulcer or gastric cancer, particularly if they also have the cag PAI and the s1m1 allele of vacA (12, 35).DNA sequence analysis suggests that among the genes encoding Hop family proteins there is considerable potential for both antigenic variation, in which combinatorial DNA shuffling creates antigenically distinct proteins, and phase variation, in which there is reversible on/off switching of gene expression. For example, there is extensive 5′ and 3′ homology between babA and two other H. pylori OMPs of unknown function called babB and babC (1). The greater similarity among bab paralogues (within a genome) than among orthologues (across genomes) suggests that there is frequent recombination and concerted evolution among these genes (27). Regions of dinucleotide CT repeats in the 5′ coding region of babA, babB, and other Hop genes may promote phase variation by slipped-strand mispairing during DNA replication. Finally, polynucleotide (A or T) tracts in babA and other Hop promoters might alter expression by subtle changes in the spacing between −35 and −10 hexamers. BabA proteins are also highly polymorphic in the variable midregion (26, 27), and they differ by more than 1,500-fold in their affinity for Leb (3).We recently showed that H. pylori strains recovered from experimentally challenged rhesus macaques had lost expression of BabA and the capacity to attach to Leb (33). In some cases the strains underwent a gene conversion event in which the babA gene was replaced by a duplicate copy of babB. In other cases the babA gene was present in H. pylori strains recovered from macaques, but it was not expressed due to phase variation that resulted from gain or loss of one CT repeat in the 5′ coding region of babA. These results demonstrate that H. pylori regulates OMP expression in vivo using both antigenic variation and phase variation. Here we sought to extend these observations by comparison of different animal hosts, including primate, mouse, and gerbil, and different H. pylori strains. The results indicate that loss of BabA expression and Leb attachment is a robust phenomenon that occurs via multiple genetic mechanisms in different H. pylori strains and across diverse animal models. Furthermore, analysis of subtle BabA variants that emerge during colonization permitted the identification of six amino acid changes that are sufficient to eliminate binding to Leb.     

Invasion and Multiplication of Helicobacter pylori in Gastric Epithelial Cells and Implications for Antibiotic Resistance

Helicobacter pylori is a Gram-negative, spiral-shaped bacterium that infects more than 50% of the human population and can cause gastritis, peptic ulcer, or gastric malignancies. It is generally viewed as an extracellular microorganism. In a gentamicin protection assay on AGS or MKN45 cells, H. pylori could invade the epithelial cells and multiply within double-layer vesicles either on the plasma membrane or in the cytoplasm. A 5-fold increase in the number of bacteria was recultured from the infected cells at 12 h, compared with the number of invading cells at 2.5 h postinfection. The autophagic vesicles induced by H. pylori are the sites of replication and also of the degradation of the replicating bacteria after fusion with lysosomes. Many H. pylori bacteria in coccoid form associated with the plasma membrane can be released into culture. Only cell-penetrating antibiotics can enhance the intracellular killing of the replicating bacteria. The multiplication of H. pylori within cells provides a niche for its resistance to antibacterial therapy and has a significant impact on its biological life cycle.Helicobacter pylori is a Gram-negative, flagellated, microaerophilic bacterium that selectively colonizes the gastric mucosa. It infects people worldwide and is correlated with socioeconomic conditions (24). The prevalence among middle-aged adults is over 80% in many developing countries. Overt disease, however, occurs in only 10 to 20% of infected individuals. The most common pathology associated with H. pylori infection is chronic active gastritis and peptic ulceration. A long-term chronic infection will increase the risk of gastric adenocarcinoma and mucosa-associated lymphoid-tissue lymphoma (19). Gastric mucosa is well protected against bacterial infections. However, H. pylori adapts and resides in the mucus and achieves attachment to epithelial cells, evasion of the immune responses, and persistent colonization in the stomach. It is not well understood why the immune system fails to clear H. pylori infection. Furthermore, the mechanisms controlling the induction and maintenance of the H. pylori-induced chronic inflammation are only partly understood.Although H. pylori is generally viewed as a noninvasive pathogen, a number of in vivo and in vitro studies have shown that H. pylori is invasive, and it can reside in the vacuole in the cytoplasm or even replicate on the cell membrane to form a microcolony (2, 11, 25). This suggests that H. pylori can be considered a facultative intracellular organism (6, 20). We have reported that H. pylori can multiply in macrophages and bone marrow-derived dendritic cells with autophagy induction (27, 28). In this study, we further extended this line of research to epithelial cells and found that H. pylori could invade and replicate in epithelial cells. Thus, H. pylori can be considered an intracellular microorganism, and this has an impact on its own biological life cycle and its resistance to antibiotics.     

The Coupling Protein Cagβ and Its Interaction Partner CagZ Are Required for Type IV Secretion of the Helicobacter pylori CagA Protein

Bacterial type IV secretion systems are macromolecule transporters with essential functions for horizontal gene transfer and for symbiotic and pathogenic interactions with eukaryotic host cells. Helicobacter pylori, the causative agent of type B gastritis, peptic ulcers, gastric adenocarcinoma, and mucosa-associated lymphoid tissue (MALT) lymphoma, uses the Cag type IV secretion system to inject its effector protein CagA into gastric cells. This protein translocation results in altered host cell gene expression profiles and cytoskeletal rearrangements, and it has been linked to cancer development. Interactions of CagA with host cell proteins have been studied in great detail, but little is known about the molecular details of CagA recognition as a type IV secretion substrate or of the translocation process. Apart from components of the secretion apparatus, we previously identified several CagA translocation factors that are either required for or support CagA translocation. To identify protein-protein interactions between these translocation factors, we used a yeast two-hybrid approach comprising all cag pathogenicity island genes. Among several other interactions involving translocation factors, we found a strong interaction between the coupling protein homologue Cagβ (HP0524) and the Cag-specific translocation factor CagZ (HP0526). We show that CagZ has a stabilizing effect on Cagβ, and we demonstrate protein-protein interactions between the cytoplasmic part of Cagβ and CagA and between CagZ and Cagβ, using immunoprecipitation and pull-down assays. Together, our data suggest that these interactions represent a substrate-translocation factor complex at the bacterial cytoplasmic membrane.The human gastric pathogen Helicobacter pylori is the principal cause of chronic active gastritis and peptic ulcer disease and is also involved in the development of mucosa-associated lymphoid tissue (MALT) lymphoma and gastric cancer (38, 47). The molecular mechanisms that lead to development of ulcers or cancer are not well understood, but it is clear that both bacterial and host factors contribute to disease development (2). Among bacterial factors, the vacuolating cytotoxin VacA and the cytotoxin-associated gene (Cag) type IV secretion system (T4SS) are considered two of the most important virulence determinants. The Cag secretion system is responsible for induction of a pronounced proinflammatory response in vitro and in vivo, mediated by secretion of chemokines such as interleukin-8 (IL-8) from gastric epithelial cells, and for translocation of the effector protein CagA into various host cells. Although the exact function of CagA translocation during infection is not known, H. pylori strains containing the cag pathogenicity island (cagPAI), a 37-kb genomic island which encodes the Cag T4SS, are clearly associated with an enhanced risk of developing peptic ulcers or adenocarcinoma, and translocation of the CagA protein itself significantly increases the risk of gastric cancer in the Mongolian gerbil model (2, 21).For translocation of the CagA protein and induction of an IL-8 response, the Cag T4SS forms a secretion apparatus composed of at least 14 different proteins (22), including more- or less-distant relatives of most or all essential components of prototypical type IV secretion systems, such as the VirB system of Agrobacterium tumefaciens (6, 29). The putative inner membrane components CagU and CagH and the outer membrane-associated proteins CagM and Cagδ (Cag3) are additional essential components (29, 39). Furthermore, the Cag secretion system elaborates pilus-like appendages on the bacterial surface which contain several apparatus proteins, but the exact composition of these structures has not been determined (4, 42, 48). These surface structures are probably required for interaction with β1 integrins as receptors on the host cell surface, and proteins contributing to this interaction include the apparatus proteins CagI, CagL, and CagY as well as the substrate CagA (28, 30).Accessory factors that are not required for the IL-8 response but are required for translocation of the CagA protein (CagA translocation factors) include the proteins Cagβ (also known as HP0524 or Cag5), CagZ (HP0526), CagF (HP0543) (22), and possibly CagD (HP0545) (13). CagF is the strongest interaction partner of CagA in the bacterial cell, and it has been shown to have a secretion chaperone-like function, possibly recruiting CagA to the secretion apparatus (16, 37). A functional role of CagZ is presently unclear, but cagZ mutants have a colonization advantage in mouse infection experiments (12), suggesting an in vivo relevance. Cagβ has significant homology to proteins of the coupling protein family, which are essential components in all DNA-transporting T4SS and in most protein-transporting T4SS (1). In DNA-transporting systems, interactions of coupling proteins with the cognate substrate complexes (relaxosomes) and with secretion apparatus components of the VirB10 family suggest that they act as substrate recognition proteins and mediate recruitment of T4SS substrates to the secretion apparatus (reviewed in reference 1). Relaxases and most DNA-independent T4SS-secreted effector proteins have C-terminal secretion signals that are necessary and sufficient for translocation, although recognition of these signals may be modulated by the presence of other domains (8). For the A. tumefaciens effector protein VirE2, it has been shown that this C-terminal signal sequence region interacts with the coupling protein VirD4 (5). Additionally, coupling proteins from conjugation systems may have DNA-dependent ATPase activity, suggesting that they might also act as DNA-transporting motor proteins (50). However, coupling proteins from different systems display considerable variability and might thus have divergent functions (1).In agreement with previous studies and consistent with its putative role as a type IV substrate recognition protein, we show here that Cagβ acts as a translocation factor for transport of CagA into epithelial cells and that both proteins interact with each other. Using a yeast two-hybrid screen, we identified a novel interaction between Cagβ and the third CagA translocation factor, CagZ. We confirmed this interaction by biochemical interaction assays, and we showed that CagZ stabilizes Cagβ in H. pylori. Together with the interaction between CagA and CagF, our results suggest that a complex of several translocation factors and the substrate CagA is formed at the bacterial membrane prior to type IV translocation.     

Evaluation of a New Test,GenoType HelicoDR,for Molecular Detection of Antibiotic Resistance in Helicobacter pylori

The eradication rate of Helicobacter pylori by standard therapy is decreasing due to antibiotic resistance, mainly to clarithromycin. Our aim was to provide a new molecular test to guide the treatment of new and relapsed cases. We first studied 126 H. pylori strains for phenotypic (MIC) and genotypic resistance to clarithromycin (rrl mutation) and levofloxacin (gyrA mutation) and then developed a DNA strip genotyping test on the basis of the correlation results and literature data. Clinical strains (n = 92) and gastric biopsy specimens containing H. pylori (n = 105) were tested blindly with the new molecular test GenoType HelicoDR. The presence of mutations or the absence of hybridization with wild-type sequences was predictive, in rrl for clarithromycin resistance in 91 cases (mostly the A2147G mutation) and in gyrA for levofloxacin resistance in 58 cases (mutations at codon 87 or 91). Genotyping revealed a mix of genotypes in 33% of the cases, reflecting a coinfection or selection for resistant mutants. The sensitivity and specificity of detecting resistance were 94% and 99% for clarithromycin and 87% and 98.5% for levofloxacin, respectively. The concordance scores were 0.96 for clarithromycin and 0.94 for levofloxacin. With global resistance rates of 46% for clarithromycin and 25% for levofloxacin, which were observed for consecutive positive biopsy specimens from 2007 and 2008, the positive and negative predictive values for detecting resistance were 99% and 94% for clarithromycin and 96% and 96% for fluoroquinolone. GenoType HelicoDR is efficient at detecting mutations predictive of antibiotic resistance in H. pylori when applied to strains or directly to gastric biopsy specimens.Helicobacter pylori infection is a common chronic gastric infection worldwide with one-third prevalence (6). About 1 out of 10 humans infected with H. pylori suffers from various digestive diseases, such as duodenal and gastric ulcer and nonulcer dyspepsia; 1 out of 100 develops gastric adenocarcinoma; and ≤1 out of 1,000 may develop gastric mucosa-associated lymphoid tissue lymphoma. All consensus guidelines recommend eradication of H. pylori (6, 20) in symptomatic patients. Standard therapy combines a proton pump inhibitor, such as omeprazole, and two antibiotics, chosen from among amoxicillin, clarithromycin, and metronidazole (20). This therapy was assessed in studies in the early 1990s and demonstrated an eradication rate of H. pylori of over 80%. However, the eradication rate is decreasing, with as low as 60% success in some countries, and this is related to the increase in clarithromycin and metronidazole resistance reported worldwide (9, 10, 17). Fluoroquinolones, such as levofloxacin and moxifloxacin, are often used for rescue therapy in a third- or fourth-line treatment (20, 31).Antibiotics used for the treatment of H. pylori infection are usually not chosen on the basis of routine susceptibility testing, because H. pylori is a fastidious microorganism requiring 3 to 10 days in a microaerobic atmosphere, and susceptibility results are not reliable for all antibiotics (17, 22). Indeed, susceptibility breakpoints have been difficult to set due to the lack of standard methods for susceptibility testing and difficulties in assessing the correlation between susceptibility results and clinical outcomes. Phenotypic resistance is correlated with clinical and microbiological failure for clarithromycin, but not for metronidazole (21). The eradication rate drops from 88% in the case of a clarithromycin-susceptible strain to less than 20% in the case of clarithromycin resistance (7, 21). Fluoroquinolone resistance was also shown to be correlated with treatment failure (24). Because resistance rates vary according to the country and patient characteristics, the choice of antibiotics on the basis of susceptibility results might be an effective strategy to improve H. pylori eradication. Since susceptibility testing is cumbersome, molecular methods for detection of resistance may be cost-effective.The mutations leading to resistance are now well known for macrolides and fluoroquinolones, although they are still unclear for metronidazole and amoxicillin. Clarithromycin resistance in H. pylori is due to point mutations in the rrl gene encoding the 23S rRNA, with three major mutations described: A2146C, A2146G, and A2147G (the numeration is from genome sequencing of NC000921 and NC000915, positions 2146 and 2147, formerly described as 2142 and 2143 [reviewed in references 21 and 22]). The resistance of H. pylori to quinolones is due to point mutations in the so-called quinolone resistance-determining region of the gyrA gene coding for the A subunit of the DNA gyrase, mainly at codons 87 and 91 (corresponding to 83 and 87 in Escherichia coli numbering) (1, 4, 23, 30).Our objective was to develop and implement a molecular method to easily detect mutations predictive of clarithromycin and fluoroquinolone resistance in H. pylori. We based our test on the DNA strip methodology used with success for other pathogens, such as Mycobacterium tuberculosis (13). We first designed a prototype test using a panel test of 126 H. pylori strains for which the MICs of clarithromycin and fluoroquinolones and the rrl and gyrA genotypes had been determined. Then, the new test was applied blindly to clinical strains and gastric biopsy specimens containing H. pylori, and the results were compared to those of susceptibility testing done routinely. The specificity of the new test for H. pylori was evaluated by testing strains of Helicobacter species other than H. pylori, as well as negative biopsy specimens. The new test was concordant with reference tests for 94 to 98% of the samples, either performed on isolated strains or directly on gastric biopsy specimens containing H. pylori, and was easy to perform.     

Outer Membrane Protein Expression Profile in Helicobacter pylori Clinical Isolates

The gram-negative gastric pathogen Helicobacter pylori is equipped with an extraordinarily large set of outer membrane proteins (OMPs), whose role in the infection process is not well understood. The Hop (Helicobacter outer membrane porins) and Hor (Hop-related proteins) groups constitute a large paralogous family consisting of 33 members. The OMPs AlpA, AlpB, BabA, SabA, and HopZ have been identified as adhesins or adherence-associated proteins. To better understand the relevance of these and other OMPs during infection, we analyzed the expression of eight different omp genes (alpA, alpB, babA, babB, babC, sabA, hopM, and oipA) in a set of 200 patient isolates, mostly from symptomatic children or young adults. Virtually all clinical isolates produced the AlpA and AlpB proteins, supporting their essential function. All other OMPs were produced at extremely variable rates, ranging from 35% to 73%, indicating a function in close adaptation to the individual host or gastric niche. In 11% of the isolates, BabA was produced, and SabA was produced in 5% of the isolates, but the strains failed to bind their cognate substrates. Interleukin-8 (IL-8) expression in gastric cells was strictly dependent on the presence of the cag pathogenicity island, whereas the presence of OipA clearly enhanced IL-8 production. The presence of the translocated effector protein CagA correlated well with BabA and OipA production. In conclusion, we found unexpectedly diverse omp expression profiles in individual H. pylori strains and hypothesize that this reflects the selective pressure for adhesion, which may differ across different hosts as well as within an individual over time.Helicobacter pylori persistently colonizes the stomachs of one-half of the world population and thereby causes gastric disorders and severe diseases, including active chronic gastritis and gastric or duodenal ulcer. Furthermore, H. pylori is considered a risk factor for the development of gastric cancer and mucosa-associated lymphoid tissue lymphoma (45). Ultrastructural studies of infected human gastric tissue revealed that a minor proportion of the bacteria are intimately attached to the epithelium of the gastric pits, whereas the majority of the H. pylori organisms actively move within the mucus overlying the gastric epithelium (22). Theoretical modeling of the dynamics of H. pylori infection identified adherence to the gastric mucosa as an important mechanism contributing to chronic colonization of the human stomach (7, 24).One characteristic feature of H. pylori is the high plasticity of its genome, which is caused by both an elevated mutation rate and an extensive exchange of genetic material, leading to free recombination of H. pylori genes (46, 50). The micro- and macrodiversity among H. pylori strains acts as an important driving force for adaptation to the hostile gastric environment and the variable living conditions during the inflammation process and/or during transmission between host individuals (32, 44). This genetic heterogeneity becomes especially apparent in the class of outer membrane protein (OMP) genes. The H. pylori genome contains more than 30 omp genes, which have been divided into the hop (helicobacter outer membrane porins) and hor (hop-related) groups (1). All H. pylori adhesins identified so far are in the Hop group of proteins (reviewed in references 3, 17, and 30).The blood group antigen binding adhesin (BabA) binds to ABO histo-blood group antigens and corresponding Lewis b antigens (Le^b), which are expressed in the human gastric mucosa of most individuals (23). A further H. pylori OMP, the sialic acid-binding adhesin (SabA), binds to sialylated carbohydrate structures, which are upregulated as part of complex gangliosides in inflamed gastric tissue. SabA was postulated to contribute to the chronic persistence of the infection (4, 29). Two highly homologous OMPs, the adherence-associated lipoproteins A and B (AlpA and AlpB), are also involved in H. pylori adherence to human gastric histo-tissue sections (35, 36), although a corresponding receptor for these proteins is not known. Interestingly, functional and especially intracellular signaling differences in AlpAB proteins between Western and East Asian H. pylori strains have been reported (28). BabB, BabC, and HP0227 are considered members of the bacterial adhesion family, since their genes belong to the hop group of H. pylori omp genes (1), but a function in adherence for these OMPs has not been demonstrated. In addition, H. pylori adherence to extracellular matrix proteins, including laminin and collagen type IV, has been described (48). H. pylori adherence to laminin was recently attributed to the binding of SabA to sialylated moieties on this molecule, whereas fibronectin binding was independent of the SabA and BabA proteins (49). The glycan structures on laminin have not yet been elucidated. It was thus unknown whether sialyl-dimeric Le^x is carried by this protein. However, SabA not only recognizes sialyl-dimeric Le^x but has also been shown to have a broader sialic acid recognition, including binding to α-2,3-linked Neu5NAc (49) that may be present on laminin (25). The intimate contact between H. pylori and the gastric epithelium enables the bacterium to manipulate signal transduction pathways, resulting in the induction of proinflammatory cytokines, such as interleukin-8 (IL-8), in epithelial cells (11). The IL-8 induction is attributable mainly to the action of the cag type IV secretion system, a 40-kb chromosomal locus, also called the cag pathogenicity island (cag-PAI), which is involved in translocation of the bacterial effector protein CagA into the host cells (34). In many H. pylori strains, the CagA protein itself is not directly involved in IL-8 induction (14), but for several strains a direct contribution of translocated CagA to NF-κB activation and IL-8 induction has been demonstrated (9). In addition, the outer inflammatory protein (OipA), which belongs to the large H. pylori OMP family, was shown to be involved in IL-8 induction in epithelial cells upon H. pylori infection (51, 52). In another study, we reported that an oipA mutant strain induced similar IL-8 values to those in the parental wild-type (wt) strain (33, 51). Thus, the role of the OipA protein in this process remains to be elucidated in more detail.Another H. pylori OMP-encoding gene, hopQ, exists as two highly divergent alleles. The type I hopQ allele is mainly found associated with East Asian H. pylori strains, in close association with the cagA gene, whereas the type II hopQ allele is commonly found in Western H. pylori strains lacking cagA (10). Interestingly, certain HopQ OMPs are able to attenuate H. pylori adherence to gastric epithelial cells and thereby affect the efficiency of CagA translocation into epithelial cells (27). Thus, the expression pattern of H. pylori omp genes is expected to have an important influence on H. pylori adaptation and virulence.In order to obtain the first more global overview of the complexity of H. pylori omp gene expression, we concentrated on a set of eight different OMPs and CagA. No data about their expression or possible adhesive or other functions were available, but all selected omp genes belong to the adhesin-encoding hop gene family. Furthermore, we chose our set of 200 clinical H. pylori isolates from a pediatric clientele and tried to correlate the OMP profile and the cag status with adherence properties of the strains and their capacity to induce IL-8 in epithelial cells.