Multi-drug resistance pattern and genome-wide SNP detection in levofloxacin-resistant uropathogenic Escherichia coli strains.

OBJECTIVES: Antibiotic treatment is extremely stressful for bacteria and has profound effects on their viability. Such administration induces physiological changes in bacterial cells, with considerable impact on their genome structure that induces mutations throughout the entire genome. This study investigated drug resistance profiles and structural changes in the entire genome of uropathogenic Escherichia coli (UPEC) strains isolated from six adapted clones that had evolved under laboratory conditions. METHODS: Eight UPEC strains, including two parental strains and six adapted clones, with different fluoroquinolone resistance levels originally isolated from two patients were used. The minimum inhibitory concentration (MIC) of 28 different antibiotics including levofloxacin was determined for each of the eight strains. In addition, the effects of mutations acquired with increased drug resistance in the levofloxacin-resistant strains on expression of genes implicated to be involved in drug resistance were examined. RESULTS: Of the eight UPEC strains used to test the MIC of 28 different antibiotics, two highly fluoroquinolone-resistant strains showed increased MIC in association with many of the antibiotics. As drug resistance increased, some genes acquired mutations, including the transcriptional regulator acrR and DNA-binding transcriptional repressor marR. Two strain groups with genetically different backgrounds (GUC9 and GFCS1) commonly acquired mutations in acrR and marR. Notably, acquired mutations related to efflux pump upregulation also contributed to increases in MIC for various antibiotics other than fluoroquinolone. CONCLUSIONS: The present results obtained using strains with artificially acquired drug resistance clarify the underlying mechanism of resistance to fluoroquinolones and other types of antibiotics.

Rapid Serotyping of Salmonella Isolates Based on Single Nucleotide Polymorphism-Like Sequence Profiles of a Salmonella-Specific Gene.

Although serotyping is the most important method of identification of taxonomy in Salmonella, conventional serotype determination with a complete set of antisera is time consuming and laborious. Recently, rapid serotyping procedures with polymerase chain reaction (PCR) have been developed. In this study, we established a novel PCR-based rapid serotyping method that employs a unique target gene. Alignment study of Salmonella-specific gene (Salmonella enterotoxin [stn]) revealed a correlation between the stn gene sequence and the serotype of the organism. In 750 bp of stn gene, 55 nucleotides indicated single nucleotide polymorphism (SNP)-like polymorphism, and the correlation between the SNP-like polymorphism and the serotype of the organism suggests that SNP-like sequences in stn gene can serve as an index for serotyping. To develop a rapid serotyping method based on the SNP-like polymorphism, we selected serotype-associated 12 SNP-like sites in the stn gene and established a method based on high-resolution melting (HRM) and PCR, which identifies nucleotides at SNP-like sites within 1.5 h. This newly established rapid serotyping procedure (stn-HRM) could identify nine serotypes, including the frequently isolated serovar Enteritidis. These nine serotypes cover 64.3% of cases of Salmonella, as reported by the World Health Organization/Global Foodborne Infection Network (WHO/GFN) Country Databank from 2001 to 2010. In this study, we employed a unique target gene, stn, which is completely independent of the genes that were targeted in previously reported rapid serotyping procedures. Therefore, the results obtained by our newly developed stn-HRM procedure are independent of the results obtained by other procedures. Besides, stn-HRM can ensure accurate identification of the bacterial species as stn is a Salmonella-specific gene. It is expected that the combination of newly constructed stn-HRM and previously reported procedures could further improve the credibility of Salmonella isolate serotyping.

Genomic Sequences of Uropathogenic Escherichia coli Strains with Various Fluoroquinolone Resistance Profiles.

The emergence of drug-resistant uropathogenic Escherichia coli (UPEC) has hampered antibiotic therapy for urinary tract infections. To elucidate the resistance mechanisms of UPEC, we performed whole-genome sequencing of eight UPEC strains with different fluoroquinolone resistance levels. Here, we report our sequencing data, providing a valuable resource for understanding such mechanisms.

Detection of Cholera Toxin by an Immunochromatographic Test Strip.

As cholera toxin (CT) is responsible for most of the symptoms induced by Vibrio cholerae O1 or O139 infection, detection of CT is an important biomarker for diagnosis of the disease. The procedure for pathogenicity analysis of V. cholerae isolates must be carefully developed for the reason that the amount of CT produced by V. cholerae varies according to the medium used and culture conditions (i.e. temperature and aeration status) applied. Here we describe a reproducible rapid method for analysis of CT production by toxigenic V. cholerae with an immunochromatographic test strip that can detect as low as 10 ng/mL of purified recombinant CT.

In vitro safety assessments and antimicrobial activities of Lactobacillus rhamnosus strains isolated from a fermented mare's milk.

Safety and probiotic characteristics such as antimicrobial activities of three Lactobacillus rhamnosus strains, FSMM15, FSMM22 and FSMM26, previously isolated as potential probiotics from fermented mare's milk were investigated. The three FSMM strains were susceptible to ampicillin, gentamycin, kanamycin, streptomycin, tetracycline and chloramphenicol, whereas they were resistant to erythromycin (minimal inhibitory concentration (MIC) = 4-8 µg/mL) and clindamycin (MIC = 4 µg/mL); bioconversion of bile salts, hemolytic activity and mucin degradation activity were negative; enzymatic activities of α-chymotrypsin and ß-glucosidase were detected, but those of α-galactosidase, ß-glucuronidase and N-acetyl-ß-glucosaminidase, were undetectable. Among the strains, strain FSMM15 was chosen as a safer probiotic candidate due mainly to the lack of plasminogen binding ability. Despite lower acid production of strain FSMM15 than others, its cell-free culture supernatant inhibited growths of Salmonella Typhimurium LT-2, Shigella sonnei, Listeria monocytogenes, and Escherichia coli O157 with comparable levels of ampicillin, suggesting a favorable aspect of strain FSMM15 as a probiotic strain.

Helicobacter pylori VacA, acting through receptor protein tyrosine phosphatase α, is crucial for CagA phosphorylation in human duodenum carcinoma cell line AZ-521.

Helicobacter pylori, a major cause of gastroduodenal diseases, produces vacuolating cytotoxin (VacA) and cytotoxin-associated gene A (CagA), which seem to be involved in virulence. VacA exhibits pleiotropic actions in gastroduodenal disorders via its specific receptors. Recently, we found that VacA induced the phosphorylation of cellular Src kinase (Src) at Tyr418 in AZ-521 cells. Silencing of receptor protein tyrosine phosphatase (RPTP)α, a VacA receptor, reduced VacA-induced Src phosphorylation. Src is responsible for tyrosine phosphorylation of CagA at its Glu-Pro-Ile-Tyr-Ala (EPIYA) variant C (EPIYA-C) motif in Helicobacter pylori-infected gastric epithelial cells, resulting in binding of CagA to SHP-2 phosphatase. Challenging AZ-521 cells with wild-type H. pylori induced phosphorylation of CagA, but this did not occur when challenged with a vacA gene-disrupted mutant strain. CagA phosphorylation was observed in cells infected with a vacA gene-disrupted mutant strain after addition of purified VacA, suggesting that VacA is required for H. pylori-induced CagA phosphorylation. Following siRNA-mediated RPTPα knockdown in AZ-521 cells, infection with wild-type H. pylori and treatment with VacA did not induce CagA phosphorylation. Taken together, these results support our conclusion that VacA mediates CagA phosphorylation through RPTPα in AZ-521 cells. These data indicate the possibility that Src phosphorylation induced by VacA is mediated through RPTPα, resulting in activation of Src, leading to CagA phosphorylation at Tyr972 in AZ-521 cells.

Ex vivo proteomics of Campylobacter jejuni 81-176 reveal that FabG affects fatty acid composition to alter bacterial growth fitness in the chicken gut.

Campylobacter jejuni is one of the leading causes of foodborne gastrointestinal illness worldwide. Here we performed ex vivo proteomic analysis of C. jejuni 81-176 in chicken, a main reservoir for human infection. At 0, 1 and 4 weeks post-infection (p.i.) with the GFP-expressing 81-176 strain, inocula were recovered from chicken ceca by cell sorting using flow cytometry. iTRAQ-coupled 2D-LC-MS/MS analyses that detected 55 C. jejuni proteins, among which either 3 (FabG, HydB, CJJ81176_0876) or 7 (MscS, CetB, FlhF, PurH, PglJ, LpxC, Icd) proteins exhibited >1.4-fold-increased expression at 1 or 4 week(s) p.i. compared with those at 0 weeks p.i., respectively. Deletion of the fabG gene clearly decreased the proportion of bacterial unsaturated fatty acids (UFAs) and chicken colonization. The UFA proportion of the parental strain was not altered when grown at 42 °C. These findings suggest that FabG might play a pivotal role in UFA production, linked to bacterial adaptation in the poultry host. To our knowledge, this is the first example of ex vivo C. jejuni proteomics, in which fatty acid metabolism might affect bacterial adaptation to the chicken host.

Quantitative Detection of Shiga Toxins Directly from Stool Specimens of Patients Associated with an Outbreak of Enterohemorrhagic Escherichia coli in Japan--Quantitative Shiga toxin detection from stool during EHEC outbreak.

Detection of Shiga toxins (Stx) is important for accurate diagnosis of Enterohemorrhagic Escherichia coli infection. In this study, we quantitatively analyzed Stx protein in nine patients' stool during an outbreak that occurred in Japan. Highly sensitive immunoassay (bead enzyme-linked immunosorbent assay (bead-ELISA)) revealed that the concentrations of toxins in stool of patients ranged from 0.71 to 10.44 ng/mL for Stx1 and 2.75 to 51.61 ng/mL for Stx2. To our knowledge, this is the first report that reveals the range of Stx protein concentrations in human stools.

Identification of Helicobacter pylori VacA in human lung and its effects on lung cells.

OBJECTIVE: Prior reports suggested that infection with Helicobacter pylori was associated with respiratory diseases; pathogenetic mechanisms however, were not defined. We tested the hypothesis that VacA, an exotoxin of H. pylori, a gastric pathogen, was aspirated into the lung and could stimulate secretion of inflammatory cytokines by lung epithelial cells. METHODS: The presence of VacA was determined by immunohistochemistry in surgical lung biopsy tissue samples from 72 patients with interstitial pneumonia. The effects of VacA on A549 human alveolar epithelial adenocarcinoma cells and normal human bronchial epithelial cells were determined. After incubation with VacA, the secretions of cytokines were measured by Multiplex Luminex(®) Assays. RESULTS: VacA was detected with anti-VacA antibodies in bronchial epithelial cells and alveolar epithelial cells from 10 of 72 patients with interstitial pneumonia. VacA was more prevalent in lungs of patients with collagen vascular disease-associated interstitial pneumonia than in those of patients with idiopathic pulmonary fibrosis, nonspecific interstitial pneumonia and cryptogenic organizing pneumonia. Incubation of A549 cells and normal human bronchial epithelial cells with VacA for 24 h was cytotoxic, and resulted in vacuolation. VacA induced interleukin-8 production by A549 cells and normal human bronchial epithelial cells and interleukin-6 production by A549 cells. Based on multiplex screening, interleukin-8 and interleukin-6 were the primary secretory products induced by VacA. CONCLUSIONS: H. pylori VacA is present in human lung and can induce interleukin-8 and interleukin-6 production by human lung cells. VacA could have a role in the pathogenesis of respiratory diseases by its cytotoxic effects and by inducing the secretion of interleukin-8 and interleukin-6 by targeted airway epithelial cells.

Expression of marA is remarkably increased from the early stage of development of fluoroquinolone-resistance in uropathogenic Escherichia coli.

BACKGROUND: Analyses of efflux pumps overexpression and mutations in quinolone resistance determining region (QRDR) in early stage of development of resistance to fluoroquinolones (FQs) are valuable to discuss countermeasures against them. We induced levofloxacin (LVFX)-resistant strains from susceptible uropathogenic Escherichia coli in vitro to analyze the mechanisms of development of FQs-resistance. METHODS: 89 strains were exposed to discontinuous elevation of LVFX dose, and mRNA level of efflux pumps and their regulators as well as mutations developed in QRDR of LVFX-resistant strains were analyzed. RESULTS: In 5 strains, a stepwise increase in MIC to LVFX (up to >128 µg/ml)was observed. Compared to the parent strains, additional mutations in QRDR were observed in the strains developing high MIC. Remarkable increase of marA expression was observed even in the early stage of LVFX-resistance development, and it lasted until high-level resistance was developed. On the other hand, moderate increase in acrB expression but only low increase in yhiU, yhiV, mdfA, tolC and sdiA were observed. CONCLUSIONS: These results suggested that marA expression is a sensitive marker for early detection of development of LVFX-resistance.

Study of the Stn protein in Salmonella; a regulator of membrane composition and integrity.

Our studies were undertaken to develop new insights into the function of the Salmonella Stn protein. An analysis of total cell membrane protein fraction suggested the possibility that Stn associates with OmpA. This possibility was confirmed by immunogold labeling using anti-OmpA antibody and far-western blotting. From these results, we conclude that Stn regulates membrane composition and integrity in Salmonella.

Effects of oral administration of heat-killed Enterococcus faecium strain NHRD IHARA in post-weaning piglets.

Probiotic bacteria such as lactic acid bacteria (LAB) have recently received attention as candidates for alternative anti-microbial feed additives. We previously isolated Enterococcus faecium strain NHRD IHARA (FERM BP-11090, NHRD IHARA strain) and reported its probiotic efficacy. However, we have not determined the effect of oral administration of heat-killed cells of this strain. Here, we performed two experiments to investigate the effect of oral administration of the heat-killed NHRD IHARA strain on post-weaning piglets. In Experiment 1, there was a significant improvement in growth performance (P = 0.04) and increase in serum immunoglobulin A (IgA) production (P = 0.03) in the group fed heat-killed cells. These results were similar to previous results we obtained with live cells. We also found changes in serum and fecal IgA production that were unrelated to the patterns of microbiotal change. In Experiment 2, we detected a significant improvement in villus growth in the jejunum (P = 0.0002). In conclusion, oral administration of the heat-killed NHRD IHARA strain in post-weaning piglets had the same efficacy as administration of the live strain. The heat-killed NHRD IHARA strain can be used as feed additives to improve pig growth and health on commercial farms.

A new protocol to detect multiple foodborne pathogens with PCR dipstick DNA chromatography after a six-hour enrichment culture in a broad-range food pathogen enrichment broth.

A quick foodborne pathogen screening method after six-hour enrichment culture with a broad-range food pathogen enrichment broth is described. Pathogenic factors of Salmonella enterica, Shigella spp., enteroinvasive Escherichia coli, and enterohemorrhagic E. coli are amplified with a cocktail primer and rapid polymerase chain reaction (PCR), which finishes amplification in 30 min. The PCR amplicon was differentiated with a dipstick DNA chromatography assay in 5-10 min. Starting from a four- to six-hour enrichment culture, this assay was finished within 45 min. Detection sensitivity of this protocol was less than 2.5 CFU/25 g for S. enterica and 3.3 CFU/25 g for enterohemorrhagic E. coli in spiked ground meat experiments.

Development of an immunochromatographic test strip for detection of cholera toxin.

Because cholera toxin (CT) is responsible for most of the symptoms induced by Vibrio cholerae infection, detection of CT is critical for diagnosis of the disease. In this study, we constructed an immunochromatographic test strip for detection of CT (CT-IC) with polyclonal antibodies developed against purified recombinant whole CT protein. The detection limit of the CT-IC was 10 ng/mL of purified recombinant CT, and it could detect the CT in culture supernatant of all 15 toxigenic V. cholerae isolates examined, whereas no false-positive signal was detected in all 5 nontoxigenic V. cholerae isolates examined. The specificity of the CT-IC was examined with recombinant heat-labile toxin (LT), which shares high homology with CT, and it was revealed that the minimum detection limit for LT was 100 times higher than that for CT. In addition, lt gene-positive enterotoxigenic Escherichia coli (ETEC) was examined by CT-IC. The false-positive signals were observed in 3 out of 12 ETEC isolates, but these signals were considerably faint. The CT-IC did not develop false-positive signals with all 7 V. parahaemolyticus isolates. These results showed the high specificity of CT-IC and the feasible use of it for the detection and surveillance of toxigenic V. cholerae.

Uropathogenic specific protein gene, highly distributed in extraintestinal uropathogenic Escherichia coli, encodes a new member of H-N-H nuclease superfamily.

BACKGROUND: The uropathogenic specific protein (Usp) and three OrfU proteins (OrfU1, OrfU2 and OrfU3) are encoded in the putative small pathogenicity island which is closely associated with Uropathogenic Escherichia coli. Although homology search revealed that Usp and OrfUs have a homology with nuclease-type bacteriocins, which possess H-N-H nuclease motif, and immunity proteins respectively, the molecular activity of these proteins was never investigated. In this study, we try to over-express Usp in E. coli, purify Usp and characterize its molecular activity. METHOD: Recombinant Usp protein was expressed in E. coli BL21(DE3) cells together with 6× Histidine tagged OrfU1 (OrfU1-His) protein, and purified with affinity chromatography using Ni2+ chelating agarose. The nuclease activity of the purified Usp was examined in vitro by using plasmid DNA as a substrate. The importance of H-N-H motif in nuclease activity of Usp was examined by site-directed mutagenesis study. RESULTS: We revealed that pET expression vector encoding Usp alone could not be maintained in E. coli BL21(DE3), and insertion of the orfUs as well as usp in the constructed plasmid diminished the toxic effect, suggesting that co-expressed OrfUs masked the activity of Usp. To purify Usp protein, we employed the expression vector encoding untagged Usp together with OrfU1-His. A tight complex formation could be observed between Usp and OrfU1-His, which allowed the purification of Usp in a single chromatographic step: binding of Usp/OrfU1-His complex to Ni2+ chelating agarose followed by elution of Usp from the complex with denaturing reagent. The purified free Usp was found to have the nuclease activity, and the activity was constitutively higher than Usp/OrfU1-His complex. H-N-H motif, which is found in various types of nucleases including a subfamily of nuclease-type bacteriocin, had been identified in the C-terminal region of Usp. Site-directed mutagenesis study showed that the H-N-H motif in Usp is indispensable for its nuclease activity. CONCLUSION: This is the first evidence of the molecular activity of the new member of H-N-H superfamily and lays the foundation for the biological characterization of Usp and its inhibitor protein, OrfUs.

Salmonella enterotoxin (Stn) regulates membrane composition and integrity.

The mechanism of action of Salmonella enterotoxin (Stn) as a virulence factor in disease is controversial. Studies of Stn have indicated both positive and negative effects on Salmonella virulence. In this study, we attempted to evaluate Stn function and its effects on Salmonella virulence. To investigate Stn function, we first performed in vitro and in vivo analysis using mammalian cells and a murine ileal loop model. In these systems, we did not observe differences in virulence phenotypes between wild-type Salmonella and an stn gene-deleted mutant. We next characterized the phenotypes and molecular properties of the mutant strain under various in vitro conditions. The proteomic profiles of the total cell membrane protein fraction differed between wild type and mutant in that there was an absence of a protein in the mutant strain, which was identified as OmpA. By far-western blotting, OmpA was found to interact directly with Stn. To verify this result, the morphology of Salmonella was examined by transmission electron microscopy, with OmpA localization being analyzed by immunogold labeling. Compared with wild-type Salmonella, the mutant strain had a different pole structure and a thin periplasmic space; OmpA was not seen in the mutant. These results indicate that Stn, via regulation of OmpA membrane localization, functions in the maintenance of membrane composition and integrity.

Toxic marine puffer fish in Thailand seas and tetrodotoxin they contained.

A total of 155 puffers caught from two of Thailand's seas, the Gulf of Siam and the Andaman seas, during April to July 2010 were included in this study. Among 125 puffers from the Gulf of Siam, 18 were Lagocephalus lunaris and 107 were L. spadiceus which were the same two species found previously in 2000-2001. Thirty puffers were collected from the Andaman seas, 28 Tetraodon nigroviridis and two juvenile Arothron reticularis; the two new species totally replaced the nine species found previously in 1992-1993. Conventional mouse bioassay was used to determine the toxicity in all fish tissue extracts, i.e., liver, reproductive tissue, digestive tissue and muscle. One of each of the species L. lunaris and L. spadiceus (5.56 and 0.93%, respectively) were toxic. All 28 T. nigroviridis and 2 A. reticularis (100%) from the Andaman seas were toxic. The toxicity scores in T. nigroviridis tissues were much higher than in the respective tissues of the other three fish species. Liquid chromatography/tandem mass spectrometry (LC-MS/MS) revealed that the main toxic principle was tetrodotoxin (TTX). This study is the first to report TTX in L. spadiceus. Our findings raised a concern for people, not only Thais but also inhabitants of other countries situated on the Andaman coast; consuming puffers of the Andaman seas is risky due to potential TTX intoxication.

Pathogenesis of experimental amyloid protein A amyloidosis in sore hocks-affected rabbits.

Although the experimental transmission of amyloid protein A (AA) amyloidosis with amyloid-enhancing factor has been studied intensively, its pathogenesis remains obscure. We previously found that rabbits affected with 'sore hocks' (SH) uniquely developed AA amyloidosis in response to primary inflammatory stimulation followed by the administration of bovine AA fibrils. However, it is unknown why only the rabbits with preexisting SH developed experimental AA amyloidosis. There may be hidden factors in the SH status that stimulate the mechanism of cross-species transmission of AA amyloidosis. To examine the essential factors in the development of experimental AA amyloidosis in SH-affected rabbits, we studied the etiology of SH in rabbits pathologically and bacteriologically. In addition, we developed artificial SH symptoms in normal rabbits by use of an adjuvant prepared from Staphylococcus aureus (StA) isolated from a spontaneous SH-affected rabbit, and we evaluated the incidence of AA amyloidosis in rabbits with or without experimental SH symptoms. We found that StA administration was extremely efficient at stimulating the induction of experimental AA amyloidosis, and the influence of SH was required. We found that the persistent S. aureus infection in SH facilitates the development of experimental AA amyloidosis in rabbits and that the inflammatory stimulation provided by SH acts as an additional accelerator in experimental AA amyloidosis.

Helicobacter pylori VacA reduces the cellular expression of STAT3 and pro-survival Bcl-2 family proteins, Bcl-2 and Bcl-XL, leading to apoptosis in gastric epithelial cells.

BACKGROUND: Helicobacter pylori vacuolating cytotoxin, VacA, stimulates apoptosis via a mitochondria-dependent pathway. VacA induces apoptosis via activation of the pro-apoptotic B-cell lymphoma (Bcl)-2 family proteins, Bcl-2-associated X protein (Bax) and Bcl-2 homologous antagonist/killer (Bak), while the implication of such pro-survival Bcl-2 family members as Bcl-2 and Bcl-XL in the VacA-induced apoptosis remains unknown. Signal transduction and activator of transcription 3 (STAT3) is a pivotal transcription factor that upregulates Bcl-2 and Bcl-XL. AIMS: This study was conducted to elicit the implication of STAT3 and pro-survival Bcl-2 and Bcl-XL in the intrinsic apoptosis. METHODS: Immunoblot and reverse transcriptase real-time polymerase chain reaction (RT-PCR) were employed to assess the cellular expression of STAT3, Bcl-2, and Bcl-XL in response to purified VacA in gastric adenocarcinoma cell lines. VacA-induced apoptosis was quantitated morphologically following knockdown by each specific small interfering RNA (siRNA) or in the presence of pharmacological inhibitors. RESULTS: VacA reduced STAT3, Bcl-2, and Bcl-XL expression in a dose-dependent manner. Knockdown of STAT3, Bcl-2, and Bcl-XL by siRNA induced apoptosis to a similar extent in the case of sufficient VacA inoculation. The VacA-mediated reduction of STAT3 expression was independent of cellular vacuolization, since a vacuolar-type ATPase inhibitor, bafilomycin A1, did not inhibit VacA-induced reduction of STAT3, Bcl-2, and Bcl-XL expression. Instead, a c-JUN NH2-terminal kinase (JNK) inhibitor, SP600125, restored the VacA-induced reduction of STAT3 expression to the basal level. CONCLUSIONS: VacA-induced apoptosis may be, in part, implicated in the reduction of STAT3 linking to the downregulation of Bcl-2 and Bcl-XL, in association with JNK activity.

Helicobacter pylori CagA inhibits endocytosis of cytotoxin VacA in host cells.

Helicobacter pylori, a common pathogen that causes chronic gastritis and cancer, has evolved to establish persistent infections in the human stomach. Epidemiological evidence suggests that H. pylori with both highly active vacuolating cytotoxin A (VacA) and cytotoxin-associated gene A (CagA), the major virulence factors, has an advantage in adapting to the host environment. However, the mechanistic relationship between VacA and CagA remains obscure. Here, we report that CagA interferes with eukaryotic endocytosis, as revealed by genome-wide screening in yeast. Moreover, CagA suppresses pinocytic endocytosis and the cytotoxicity of VacA in gastric epithelial cells without affecting clathrin-dependent endocytosis. Our data suggest that H. pylori secretes VacA to attack distant host cells while injecting CagA into the gastric epithelial cells to which the bacteria are directly attached, thereby protecting these attached host cells from the cytotoxicity of VacA and creating a local ecological niche. This mechanism might allow H. pylori to balance damage to one population of host cells with the preservation of another, allowing for persistent infection.