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Background: Interference with the L -arginine/nitric oxide pathway may be a virulence strategy for the gastric pathogen Helicobacter pylori . This study evaluates a bioassay for such inhibitory actions on nitric oxide synthase. Methods: Cultured murine macrophages were stimulated by lipopolysaccharide and interferon-gamma. Nitric oxide synthesis and the expression of inducible nitric oxide synthase (iNOS) at increasing concentrations of L -arginine were analysed using chemiluminescence and Western blotting, respectively. Results: The bioassay was evaluated against nitrite accumulation and two established NOS inhibitors. Bacterial extracts or whole cells of one H. pylori strain inhibited nitric oxide production at low L -arginine concentrations (2-20 μM). A higher concentration of L -arginine (200 μM) was not associated with such inhibition. The iNOS expression was not affected by any of the additives compared to stimulated controls. Conclusions: This bioassay is a reliable and simple method for analysing iNOS inhibition, resolving effects on enzyme activity or enzyme expression. H. pylori water extract and whole cells exert an L -arginine-dependent NOS inhibition, not influencing iNOS expression. [ABSTRACT FROM AUTHOR]

Haemophilus ducreyi and Klebsiella (Calymmatobacterium) granulomatis are sexually transmitted bacteria that cause characteristic, persisting ulceration on external genitals called chancroid and granuloma inguinale, respectively. Those ulcers are endemic in developing countries or exist, as does granuloma inguinale, only in some geographic "hot spots."H. ducreyi is placed in the genus Haemophilus (family Pasteurellacae); however, this phylogenetic position is not obvious. The multiple ways in which the bacterium may be adapted to its econiche through specialized nutrient acquisitions; defenses against the immune system; and virulence factors that increase attachment, fitness, and persistence within genital tissue are discussed below. The analysis of K. granulomatis phylogeny demonstrated a high degree of identity with other Klebsiella species, and the name K. granulomatis comb. nov. was proposed. Because of the difficulty in growing this bacterium on artificial media, its characteristics have not been sufficiently defined. More studies are needed to understand bacterial genetics related to the pathogenesis and evolution of K. granulomatis., (© 2011 New York Academy of Sciences.)

Aims: In this study, the main objective was to verify the hypothesis of induction of 'viable but non-culturable' (VBNC) forms of enterotoxigenic Escherichia coli (ETEC) during incubation in water., Methods and Results: Six clinically isolated ETEC strains were studied. Viable counts showed culturable ETEC bacteria for up to 3 months in freshwater but only two out of six strains were culturable in seawater at this time point. Although the bacterial cells remained intact, no production or secretion of heat-labile (LT) or heat-stable (ST) enterotoxins was observed using GM1-ELISA methods. However, genes encoding ETEC toxins (STh and LT), colonization factors (CS7 and CS17), gapA and 16S RNA were expressed during 3 months in both sea water and freshwater microcosms as determined by real-time RT-PCR on cDNA derived from the bacteria., Conclusions: Clinically isolated ETEC strains can survive for long periods in both sea water and freshwater. The bacterial cells remain intact, and the gene expression of virulence genes and genes involved in metabolic pathways are detected after 3 months., Significance and Impact of the Study: These results indicate that ETEC bacteria can enter a VBNC state during stressful conditions and suggest that ETEC has the potential to be infectious after long-term incubation in water.

The presence and persistence of enterotoxigenic Escherichia coli (ETEC) is poorly investigated in marine habitats. Here we compared ETEC with the more studied fecal contaminant, Salmonella enterica serotype Typhimurium (S. enterica) and the marine bacteria Vibrio parahaemolyticus. All three species of bacteria were culturable on agar plates during 8 weeks of incubation in seawater. However, the culturability of ETEC was positively affected by low temperature whereas V. parahaemolyticus was negatively affected. High-nutrient conditions favored the growth of ETEC but not the other bacteria. When the bacteria were fed to blue mussels, V. parahaemolyticus inhibited the filtration activity and the ingestion was lower than that of the enterobacteria. On the other hand, the mussels were less efficient in eliminating V. parahaemolyticus and an in vitro study showed that the hemocytes of three different species of bivalves were not able to kill this strain of V. parahaemolyticus. The bactericidal capacity of bivalves was seemingly an efficient elimination pathway for S. enterica and ETEC. This study showed that ETEC in endemic areas should, to the same degree as S. enterica and V. parahaemolyticus, be taken in consideration when assessing the role of marine environments as a source of enteric infection.

Aims: We aimed to develop an assay for sensitive detection and quantification of enterotoxigenic Escherichia coli (ETEC) in different types of water samples., Methods and Results: Real-time polymerase chain reaction (PCR) assays with primers against ETEC enterotoxin genes estA (STh) estB (STp) and eltB (LT) were designed and the detection levels were determined to be three bacteria per PCR reaction. Gene copy numbers were estimated to be four (LT), two (STh) and one (STp) per bacteria. Twenty-six household and 13 environmental water samples from Bangladesh were filtered through 0.22-microm filters; DNA was extracted from the filters and analysed by real-time PCR. The results were compared with toxin GM1-enzyme-linked immunosorbent assay (ELISA), in which colonies were tested for toxin production after cultivation of the filters. Out of the 39 samples tested, 18 household and 8 environmental samples were positive for ETEC in real-time PCR, but only 6 positive samples were found with GM1-ELISA., Conclusions: The method allows for highly sensitive detection and quantification of ETEC based on detection of toxin DNA in water samples., Significance and Impact of the Study: The method facilitates detection and identification of ETEC in water and allows comparison between water contamination and incidence of ETEC diarrhoea in endemic areas.

To express high quantities of colonization factor antigen I (CFA/I) derived from enterotoxigenic Escherichia coli (ETEC) for use in ETEC vaccines, the entire CFA/I operon consisting of four genes (cfa-A, -B, -C, -E) was cloned into plasmid expression vectors that could be maintained either with or without antibiotic selection. Expression from the powerful tac promoter was under the control of the lacIq repressor present on the plasmids. Fimbriae were expressed on the surface of both a non-toxigenic E. coli K12 strain and a non-toxigenic strain of Vibrio cholerae following induction with isopropyl-beta-D-thiogalactopyranoside (IPTG). It was found that the recombinant E. coli strains expressed up to 16-fold higher levels of CFA/I fimbriae compared to a reference strain which had previously been shown to be among the highest natural producers of the CFA/I fimbriae among tested wild type ETEC strains. Oral immunization with formalin-killed recombinant E. coli bacteria over-expressing CFA/I induced significantly higher serum IgA and IgG+M antibodies responses compared to the reference strain. Oral immunization with formalin-killed recombinant V. cholerae bacteria also induce strong CFA/I-specific serum IgA and IgG+M responses. We conclude that our constructs may be useful as candidate strains in an oral killed CF-ETEC vaccine.

Objective: Peroxynitrite formation, as reflected by nitrotyrosine expression, is low in Helicobacter pylori-infected Mongolian gerbils despite pronounced expression of radical-forming enzymes. The aim of the present study was to investigate in vivo whether H. pylori inhibits either one or both of the nitro- and oxyradical formation pathways., Material and Methods: Male Mongolian gerbils were infected with two different H. pylori strains, TN2GF4 and SS1. Six months after inoculation, direct measurement of NO and H2O2 was performed in vivo using electrochemical microsensors positioned in close proximity to the gastric mucosa., Results: In the TN2GF4-infected animals the level of NO was significantly lower than that in controls. No significant difference in NO levels was detected between the SS1-infected group and the controls. H2O2 was significantly increased in the SS1 animals compared with that in controls after 6 months. The H2O2 level in the TN2GF4 group did not differ from that in controls., Conclusions: The results indicate that H. pylori infection is associated with strain-dependent functional inhibition of both the NO and oxyradical formation pathways in the gastric mucosa.

Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrhea among children in developing countries and in travelers to areas of ETEC endemicity. ETEC strains isolated from humans may produce a heat-labile enterotoxin (LT) and two types of the heat-stable enterotoxin STa, called STh and STp, encoded by the estA gene. Two commonly used assay methods for the detection of STa, the infant mouse assay or different enzyme-linked immunosorbent assays, are unable to distinguish between the two subtypes of ST. Different genotypic methods, such as DNA probes or PCR assays, may, however, allow such discrimination. Using gene probes, it has recently been reported that ETEC strains producing STp as the only enterotoxin are not associated with diarrhea. In this study, we have used highly specific PCR methods, including newly designed primers for STh together with previously described STp primers, to compare the relative distribution of STh and STp in ETEC isolated from children with diarrhea in three different geographically distinct areas, i.e., Bangladesh, Egypt, and Guatemala, and from travelers to Mexico and Guatemala. It was found that ETEC strains producing STp were as commonly isolated from cases of diarrhea as strains producing STh both in Egypt and Guatemala, whereas STp strains were considerably less common in Bangladesh. No difference was found in the relative distribution of STh and STp in ETEC strains isolated from travelers with diarrhea and from asymptomatic carriers. Irrespective of ST genotype, the disease symptoms were also similar in both children and travelers.

Objective: For obscure reasons Helicobacter pylori infection of the gastric mucosa is maintained despite a pronounced host defence response. The present study elucidates possible H. pylori-related interference in the oxy- and nitro-radical formation pathways., Material and Methods: Male Mongolian gerbils were infected with two different H. pylori strains, TN2GF4 and SS1. At 3, 6, 12 or 18 months after inoculation, gastric expressions of myeloperoxidase (MPO), inducible nitric oxide synthase (iNOS) and nitrotyrosine were assessed by Western blotting., Results: Expression of both iNOS and MPO was markedly up-regulated in the H. pylori-infected animals compared with non-infected controls. The TN2GF4-infected animals initially (at 3 and 6 months) demonstrated pronounced expression of both iNOS and MPO. The SSI-infected animals exhibited a slower onset with significantly increased iNOS after 12 and 18 months. Nitrotyrosine expression was slightly elevated in the infected groups at 3 and 6 months compared with that in the controls. Nitrotyrosine levels then decreased and were no longer significantly different from those of controls (TN2GF4-infected animals) or were lower (SS1-infected animals) than in the controls., Conclusions: The results indicate that peroxynitrite formation as reflected by nitrotyrosine expression is low or even inhibited in chronic H. pylori infection despite pronounced expression of enzymes representing both the oxy- and nitro-radical formation pathways. The results support the theory that H. pylori survival is related to functional inhibition of mucosal enzymatic NO and/or oxy-radical formation.

Infection with the human gastric pathogen Helicobacter pylori can give rise to chronic gastritis, peptic ulcer, and gastric cancer. All H. pylori strains express the surface-localized protein HpaA, a promising candidate for a vaccine against H. pylori infection. To study the physiological importance of HpaA, a mutation of the hpaA gene was introduced into a mouse-adapted H. pylori strain. To justify that the interruption of the hpaA gene did not cause any polar effects of downstream genes or was associated with a second site mutation, the protein expression patterns of the mutant and wild-type strains were characterized by two different proteomic approaches. Two-dimensional differential in-gel electrophoresis analysis of whole-cell extracts and subcellular fractionation combined with nano-liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometry for outer membrane protein profiling revealed only minor differences in the protein profile between the mutant and the wild-type strains. Therefore, the mutant strain was tested for its colonizing ability in a well-established mouse model. While inoculation with the wild-type strain resulted in heavily H. pylori-infected mice, the HpaA mutant strain was not able to establish colonization. Thus, by combining proteomic analysis and in vivo studies, we conclude that HpaA is essential for the colonization of H. pylori in mice.

The technique of random amplified polymorphic DNA (RAPD) was adapted and optimized to study Haemophilus ducreyi isolates. A panel of 43 strains isolated from chancroid patients from different countries in Africa, Europe, North America, and Asia were characterized. The strains were also studied with respect to lipooligosaccharide (LOS) migration and immunoblotting patterns and the presence of cytolethal distending toxin genes. The RAPD method with the OPJ20 primer generated nine banding patterns (1 to 9). The majority of the isolates were clustered into two major profiles, 14 and 13 strains into profiles 1 and 2, respectively, and just a few strains revealed patterns 3 and 4. The isolates from Thailand were exceptional in that they showed greater diversity and were represented by six different RAPD patterns, i.e., patterns 3 and 5 to 9. The LOS migration and immunoblotting analyses revealed two different patterns, which indicated long and short forms of LOS; the former was found in 20/23 tested strains. Two strains that expressed the short form of LOS were grouped into RAPD pattern 4. The absence of cdtABC genes was observed in only 4/23 strains, and three of these isolates were assigned to RAPD pattern 4. Our results showed limited genotypic and phenotypic variations among H. ducreyi strains, as supported by the conserved RAPD and LOS profiles shared by the majority of the studied strains. However, the RAPD method identified differences between strains, including those from different geographic areas, which indicate the potential of RAPD as an epidemiological tool for the typing of H. ducreyi isolates in countries where chancroid is endemic.

Objective: The Mongolian gerbil is considered as the model of choice when studying adenocarcinoma related to Helicobacter pylori infection. The purpose of this study was to compare two different H. pylori strains and elucidate whether adenocarcinomas developed in gerbils., Material and Methods: Male gerbils were separated into three groups: one control and two groups infected with two different strains of H. pylori, TN2GF4 and SS1. At 3, 6, 12 or 18 months after inoculation 5 animals from each group were sacrificed. The stomach was used for culture, and for histology., Results: Inflammation was seen after 3 months in all the infected animals. In the controls no pathology was found at any time. Intestinal metaplasia was found in both the infected groups. Glands buried in the submucusal layer, changes that might be misinterpreted as adenocarcinoma, were found in 10% of the SS1 and in 65% of the TN2GF4 animals. Adenocarcinoma was not found in any of the gerbils., Conclusions: All studies claiming to have found H. pylori-induced adenocarcinomas in gerbils describe atypical glands penetrating into the muscularis propria and interpret these as invasive growths due to cancer. An alternative interpretation is that the deranged glandular structures grow in and below the submucosa. It is suggested that atypical glands in the muscularis layer are not enough as a diagnostic criterion for gastric adenocarcinoma. It is concluded that adenocarcinoma has not yet been shown convincingly to develop in Mongolian gerbils infected with H. pylori. Nevertheless, it is a model well suited for studying gastritis, gastric ulcer and premalignant changes such as metaplasia.

Background: Interference with the L-arginine/nitric oxide pathway may be a virulence strategy for the gastric pathogen Helicobacter pylori. This study evaluates a bioassay for such inhibitory actions on nitric oxide synthase., Methods: Cultured murine macrophages were stimulated by lipopolysaccharide and interferon-gamma. Nitric oxide synthesis and the expression of inducible nitric oxide synthase (iNOS) at increasing concentrations of L-arginine were analysed using chemiluminescence and Western blotting, respectively., Results: The bioassay was evaluated against nitrite accumulation and two established NOS inhibitors. Bacterial extracts or whole cells of one H. pylori strain inhibited nitric oxide production at low L-arginine concentrations (2-20 microM). A higher concentration of L-arginine (200 microM) was not associated with such inhibition. The iNOS expression was not affected by any of the additives compared to stimulated controls., Conclusions: This bioassay is a reliable and simple method for analysing iNOS inhibition, resolving effects on enzyme activity or enzyme expression. H. pylori water extract and whole cells exert an L-arginine-dependent NOS inhibition, not influencing iNOS expression.

Helicobacter pylori infection causes active chronic inflammation with a continuous recruitment of neutrophils to the inflamed gastric mucosa. To evaluate the role of endothelial cells in this process, we have examined adhesion molecule expression and chemokine and cytokine production from human umbilical vein endothelial cells stimulated with well-characterized H. pylori strains as well as purified proteins. Our results indicate that endothelial cells actively contribute to neutrophil recruitment, since stimulation with H. pylori bacteria induced upregulation of the adhesion molecules VCAM-1, ICAM-1, and E-selectin as well as the chemokines interleukin 8 (IL-8) and growth-related oncogene alpha (GRO-alpha) and the cytokine IL-6. However, there were large variations in the ability of the different H. pylori strains to stimulate endothelial cells. These interstrain variations were seen irrespective of whether the strains had been isolated from patients with duodenal ulcer disease or asymptomatic carriers and were not solely related to the expression of known virulence factors, such as the cytotoxin-associated gene pathogenicity island, vacuolating toxin A, and Lewis blood group antigens. In addition, one or several unidentified proteins which act via NF-kappaB activation seem to induce endothelial cell activation. In conclusion, human endothelial cells produce neutrophil-recruiting factors and show increased adhesion molecule expression after stimulation with certain H. pylori strains. These effects probably contribute to the continuous recruitment of neutrophils to H. pylori-infected gastric mucosa and may also contribute to tissue damage and ulcer formation.

The expression of the virulence-associated genes ureA, encoding the urease subunit A, and nap, encoding the neutrophil activating protein, in Helicobacter pylori grown both in the stomach of C57/Bl6 mice and in Brucella broth was quantified by quantitative competitive reverse transcriptase-PCR using a homologous RNA standard (competitor) and an external standard (16S rRNA). The results showed that the ureA and nap transcripts were increased up to 15 and 80 times, respectively, in vivo compared to in vitro. The transcription of ureA and nap also differed in that ureA showed highest expression early in infection in mice whereas nap transcription was variable throughout the 18-week infection period.

Due to earlier contradictory results regarding the localization of the putative Helicobacter pylori adhesin A (HpaA), we aimed to compare the gene and protein expression and surface localization of HpaA in different H. pylori strains. Five H. pylori strains were cultivated for 11 days and analysed by Northern blot analysis, flow cytometry (FCM), semi-quantitative dot blot, colony blot, immuno-electron microscopy (IEM), and phase-contrast microscopy. The highest transcriptional activity of the hapA gene as observed after 3-4 days of cultivation and two mRNA transcripts of 1600 and 3100 nucleotides, respectively, were detected in all five strains with the hpaA probe. We also showed by reverse transcription-polymerase chain reaction (RT-PCR) that the hpaA gene is co-transcribed with the downstream omp18 gene. The highest total HpaA protein production in bacteria occurred between day 3 and 7, as determined by semi-quantitative dot blot, and was similar in the different strains. The maximal proportion of cells with HpaA on the bacterial surface, detected by FCM, was for strain SS1, 90%; Hel 344, 60%; CCUG 17875, 61%; CCUG 17874, 86% and for strain AH 244 only 35%. By IEM HpaA was detected in all strains both on the bacterial surface and on the flagellar sheath., (Copyright 2001 Academic Press.)

Previous studies on the localization of several different Helicobacter pylori antigens have been contradictory. We have therefore examined by using both one- and two-color flow cytometry (FCM), immunofluorescence (IF), and immunoelectron microscopy (IEM), the possible surface localization of some H. pylori antigens that may be important virulence factors. All four methods detected the lipopolysaccharide and the N-acetyl-neuroaminyllactose-binding hemagglutinin protein (HpaA) as surface-exposed, while the urease enzyme was not detected at all and the neutrophil activating protein only in low concentration on the surface of the H. pylori bacteria during culture of H. pylori in liquid broth for 11 days. The FCM analysis was found to be quite sensitive and specific and also extremely fast compared with IF and IEM, and therefore the preferred method for detection of surface-localized antigens of H. pylori.

Background: The 26 kDa protein, which is an alkyl hydroperoxide reductase (AhpC) homologue, has earlier been described as specific for Helicobacter pylori. The aims of this study were to analyse whether this protein, or the corresponding gene, could be identified in other Helicobacter species., Materials and Methods: Two different monoclonal antibodies (Mabs), which recognise the 26 kDa protein in H. pylori, were used in immunoblots to determine the presence of the protein in 10 Helicobacter species. PCR was performed in order to analyse whether the gene was detectable and the PCR products were sequenced. Southern and Northern blot analyses were done on chromosomal DNA and total RNA, respectively, isolated from some selected Helicobacter species in order to compare the genes and mRNA transcripts to H. pylori., Results: The 26 kDa protein was identified in H. nemestrinae (primate), H. acinonychis (cheetah), H. bilis (mouse), H. felis (cat) and H. salomonis (dog) but not in H. mustelae (ferret), H. cinaedi (human), H. canis (dog), H. fennelliae (human) or H. pullorum (poultry). By PCR the gene was also recognised in H. mustelae, H. cinaedi and H. pullorum. The PCR products showed high sequence homology (66-98%) compared to H. pylori. The gene was also highly conserved in four H. pylori strains (94-99% homology). Southern blot showed that the H. nemestrinae and H. acinonychis chromosomal DNA contained a single copy of the gene and the Northern blot analyses indicated mono-cistronic transcription of the gene in these two species, as has been found in H. pylori., Conclusions: A gene similar to ahpC was found in eight out of 10 Helicobacter species analysed.

The 26 kDa protein of Helicobacter pylori, with 67% amino acid identity to alkyl hydroperoxide reductase (AhpC) of Campylobacter jejuni, was studied. We wanted to evaluate it the protein has AhpC activity. Therefore, an Escherichia coli mutant defective for alkyl hydroperoxide reductase and a plasmid expressing the 26 kDa protein from H. pylori were used in complementation studies. The complemented E. coli mutant showed a decreased sensitivity to cumene hydroperoxide indicating that the 26 kDa protein of H. pylori has AhpC activity and could be of importance in the defence against oxidative stress. Furthermore, Northern blot analysis detected one mRNA transcript of approximately 700 bp which is in agreement with the gene being transcribed as a single gene with its own promoter. This promoter region was further characterized by primer extension experiments. Additional studies on how environmental factors, such as long term growth and pH, can affect the transcription of the gene were performed on two H. pylori strains. We found that low pH and long term growth repressed transcription of the gene. Attempts to construct a mutant deficient for the gene in H. pylori were unsuccessful., (Copyright 2000 Academic Press.)

Background: We have investigated the possibility that the same patients may be colonized by Helicobacter pylori strains of different genotypes or phenotypes in the antrum as compared to in the duodenum. The strains were typed for DNA fingerprints, different lipopolysaccharides (LPS), and Lewis antigen expression on the O-side chains of LPS., Materials and Methods: Polymerase chain reaction (PCR) amplifications using primer sequences (i.e., the Enterobacterial Repetitive Intergenic Consensus [ERIC]) and randomly amplified polymorphic DNA (RAPD) elements were performed to asses chromosomal DNA diversity between H. pylori strains. The expression of different LPS types and Lewis antigens in the various H. pylori isolates were determined by whole bacterial enzyme-linked immunosorbent assays using monoclonal antibodies., Results: Duodenal ulcer patients had different H. pylori genotypes in the duodenum as compared to in the antrum as shown by ERIC-PCR (44%) and by RAPD-PCR (75%). Different DNA patterns were found among the strains that were isolated from various regions of the duodenum in 4 of 16 patients (25%) as shown by ERIC-PCR and in 8 of 16 patients (50%) as shown by RAPD-PCR. Sixty-three percent of the duodenal ulcer patients had H. pylori strains with a different Lewis antigen phenotype in the duodenum as compared to in the antrum, and 3 of 16 patients (19%) had strains with different Lewis antigens expressed by strains from different duodenal biopsies from the same patient., Conclusion: The results suggest that a mixed population of different H. pylori strains with marked variation, both genotypically and phenotypically, colonize the same patient.

In this study we have determined systemic and local antibody responses against different Helicobacter pylori antigens in H. pylori-infected and noninfected subjects. In addition, we studied whether differences in antibody responses between patients with duodenal ulcers and asymptomatic H. pylori carriers might explain the different outcomes of infection. Sera and in most instances gastric aspirates were collected from 19 duodenal ulcer patients, 15 asymptomatic H. pylori carriers, and 20 noninfected subjects and assayed for specific antibodies against different H. pylori antigens, i.e., whole membrane proteins (MP), lipopolysaccharides, flagellin, urease, the neuraminyllactose binding hemagglutinin HpaA, and a 26-kDa protein, by enzyme-linked immunosorbent assay. The H. pylori-infected subjects had significantly higher antibody titers against MP, flagellin, and urease in both sera and gastric aspirates compared with the noninfected subjects. Furthermore, the antibody titers against HpaA were significantly elevated in sera but not in gastric aspirates from the infected subjects. However, no differences in antibody titers against any of the tested antigens could be detected between the duodenal ulcer patients and the asymptomatic H. pylori carriers, either in sera or in gastric aspirates.

Background & Aims: The pathophysiology behind Helicobacter pylori-induced gastroduodenal dysfunction is incompletely understood. The aim of this study was to investigate if a water extract of H. pylori distorts acid-induced duodenal mucosal alkaline secretion., Methods: Chloralose-anesthetized rats were prepared for duodenal luminal perfusion and in situ pH-stat titration of mucosal alkaline secretion., Results: Mucosal bicarbonate secretion increased approximately 55%-60% after a 5-minute exposure to 10 mmol/L HCl. This response was absent when water extracts of three strains of H. pylori (protein content, 0.2-20 microg/mL) had been added to the perfusate. Presence of 3 mmol/L L-arginine, but not the stereoisomer D-arginine, in the luminal perfusate reversed the H. pylori extract blockade of acid-induced mucosal alkaline secretion. High-performance liquid chromatography-based analyses showed that the endogenous nitric oxide synthase inhibitor asymmetric dimethyl arginine (ADMA) increased fourfold in duodenal perfusate and fivefold in duodenal tissue after H. pylori extract exposure. In vitro proteolysis of H. pylori extract also resulted in a substantial accumulation of ADMA. Exogenously administered ADMA, giving similar tissue concentrations, inhibited the mucosal alkaline response to acid exposure., Conclusions: Water extracts of H. pylori inhibit acid-induced mucosal alkaline secretion via interference with mucosal NO synthase.

The capacity of Helicobacter pylori to induce strain specific immune responses was studied in adult Swedish volunteers. Sera and gastric aspirates from 11 H. pylori-infected subjects were tested for specific antibody levels against, respectively, lipopolysaccharides (LPS) and total membrane preparations (MPs) prepared from the study subjects' own strains, as well as with corresponding antigens from two reference H. pylori strains or heterologous strains collected from other subjects within the study. It was found that sera from five of the 11 subjects had significantly higher IgA antibody titres against LPS from the homologous strain than against LPS from either of the reference strains and in five cases sera reacted with higher IgG titres against the homologous LPS than with LPS preparations from the reference or heterologous patient strains. Analyses of specific titres against MPs revealed that six sera had higher IgA titres and four sera had higher IgG titres against MPs prepared from the subjects' own strains than against MPs from either of the two reference strains. Determination of specific antibodies in gastric aspirates revealed significantly higher IgA titres against LPS from the homologous H. pylori isolate than against LPS from the two reference strains in five cases, and six aspirates reacted in higher IgA titre with the homologous H. pylori MPs. Results from immunoblotting analyses of sera support induction of strain specific immune responses against H. pylori LPS. By means of specific monoclonal antibodies against H. pylori LPS, antigenic heterogeneity between the different LPS preparations tested was confirmed., (Copyright 1997 Academic Press Limited.)

Monoclonal antibodies (MAbs) against membrane preparations of Helicobacter pylori were produced. One MAb was found to be specific for H. pylori, because it did not react with a number of other bacterial species, including Helicobacter felis and Campylobacter jejuni. This MAb reacted with a 30-kDa protein found in outer membrane preparations of H. pylori. The protein was also detected on the cell surface on intact bacteria when analyzed by immunoelectron microscopy. To facilitate the identification of H. pylori isolates after culturing of biopsies, an immunodot blot assay based on the reaction of this MAb was developed. This assay was found to be highly specific for H. pylori. Sixty-six clinical isolates typed as H. pylori by conventional biochemical tests were found to be positive, whereas no other bacterial species tested gave a positive result. By this method, reliable and rapid identification of H. pylori could be accomplished.

Virulent Yersinia species possess a common plasmid that encodes essential virulence determinants (Yops) which are regulated by the extracellular stimuli Ca2+ and temperature. The V antigen operon was recently shown to be involved in the Ca2(+)-regulated negative pathway (A. Forsberg and H. Wolf-Watz, Mol. Microbiol. 2:121-133, 1988). We show here that the V antigen-containing operon of Yersinia pseudotuberculosis is a polycistronic operon having the gene order lcrGVH-yopBD. DNA sequencing analysis of lcrGVH revealed a high homology to the corresponding genes of Yersinia pestis. LcrG was conserved and LcrH showed only one amino acid difference, while LcrV showed only 96.6% identity. The amino acid substitutions of LcrV occurred in the central domain of the protein, while the two ends of the protein were conserved. Northern (RNA) blotting experiments showed that the operon is regulated at the transcriptional level by the extracellular stimuli temperature and calcium. One 4.6-kb transcriptional product of the operon was identified. This mRNA is rapidly processed at its 5' end, resulting in different mRNA species of variable stability. By genetic analysis, the lcrV and lcrH gene products were found to be regulatory proteins having important roles in the Ca2(+)-controlled regulation of Yop expression. The activity of LcrH is modulated by a gene product of the operon that inhibits the negative action of LcrH on yop transcription in the absence of Ca2+.

The adherence of Yersinia pseudotuberculosis to the surface of HeLa cells at 4 degrees C was studied. This temperature allows adhesion of bacteria but prevents engulfment. Adhesion between the bacteria and the cells was not dependent upon the presence of serum, Ca2+ or Mg2+ in the medium. Maximum adhesion was obtained at pH 6.5-7.9 and pretreatment of the cells with formaldehyde or glutaraldehyde inhibited the attachment of the bacteria. The interaction between the bacteria and the cell surface seems to involve cellular processes that are mostly microvilli. An intimate association between the bacteria and the cellular glycocalyx was found. Three virulent bacterial strains adhered more easily to the cell surface than five avirulent strains. Maximum adherence was obtained with bacteria from late logarithmic and early stationary phases of growth. The bacteria gradually lose their adhesive property when cultivated for several generations at 37 degrees C in nutrient broth but not when cultivated at 20 degrees C. Treatment of the bacteria with protease IV from Streptomyces caespitosus markedly reduced the efficiency of attachment.

The structural genes of the outer membrane polypeptides of Yersinia spp. (YOPs) and the V antigen of plasmid pIB1 of Yersinia pseudotuberculosis were recently cloned and mapped (A. Forsberg, I. Bölin, L. Norlander, and H. Wolf-Watz, Microb. Pathogen. 2:123-137, 1987). The corresponding genes were localized on pYV019 and pYV8081 of Yersinia pestis and Yersinia enterocolitica, respectively. No obvious differences were observed on comparison of pIB1 and pYV019, whereas pYV8081 showed intragenic as well as extragenic changes. However, one region of plasmid pYV8081, which coded for the V antigen, YOP3, and YOP4a, was essentially conserved among the three plasmids. Since this region is connected with the Ca2+ region, we suggest that the conserved region of the virulence plasmids of Yersinia spp. be extended to include both of these regions. Low amounts of the YOPs were detected in the membrane fraction at 37 degrees C in the presence of 2.5 mM calcium. Only minor differences were noticed when the individual YOPs of Y. pestis and Y. pseudotuberculosis were compared. Several differences were observed when the YOPs of Y. enterocolitica were included for comparison. All Y. enterocolitica proteins, except YOP1, YOP4b, and the V antigen, exhibited changes in their characteristic molecular sizes. Although these differences were within a range of +/- 2 kilodaltons, the isoelectric point was retained for each protein type.

A number of plasmid-associated proteins (YOPs) of Y. pseudotuberculosis are induced and expressed at high levels when the pathogen is grown at 37 degrees C in absence of Ca2+ ions. These proteins were recovered both from the outer membrane fraction and the culture supernatant. Two hours after a temperature-shift the YOPs were only found in the culture supernatant, amounting to about 5% of the total cell protein. After 4 h of incubation they were also detected in the outer membrane fraction. Separation by 2-D gel electrophoresis revealed that the YOPs could be separated into 6 different polypeptides; YOP2a (45 kDa), YOP2b (45 kDa), YOP3 (41-42 kDa), YOP4a (34 kDa), YOP4b (34 kDa) and YOP5 (26 kDa). The structural genes of all of these YOPs, except the YOP2a gene, were cloned to pBR322 and their respective genetic localization was established. It was found that the genes were not part of a common operon but scattered around plasmid plB1. Only the YOP4b protein was found to map within the Ca2+ region. The hybrid plasmid plB572 coded for a number of plasmid plB1 specific proteins, one of which showed a molecular weight of 38 kDa. This polypeptide could be precipitated by monospecific V-antiserum, showing that this protein is the V-antigen.

Transposon Tn5 insertion derivatives of the virulence plasmid pYV019 of Yersinia pestis were transferred by P1 transduction into a plasmid-free strain of Y. pseudotuberculosis. One of these plasmid derivatives conferred virulence upon the Y. pseudotuberculosis strain. This strain had the ability to express temperature-inducible plasmid-coded outer membrane proteins and was also found to be Ca2+ dependent.

The ingestion into human fibroblasts of different strains (avirulent and virulent) of Yersinia pseudotuberculosis was investigated. The kinetics of ingestion was studied with the addition of 10(7) bacteria to human lung fibroblast cultures. The number of ingested bacteria after 0.5-3 h was measured with viable count technique. All strains were ingested except for one avirulent strain. The other strains were ingested at roughly the same rate. The bacterial uptake seemed to be mediated by a phagocytic-like procedure and the intracellular bacteria resided in vacuoles. The bacteria survived and also multiplied in the cells (for at least 7 days).

Virulence plasmid-containing cells of Yersinia pseudotuberculosis had the ability to inhibit phagocytosis by mouse peritoneal macrophages cultured in vitro, but cells of its plasmid-cured derivative did not. Inhibition was most pronounced when the pathogen was incubated under Ca2+-deficient conditions, which allowed a high level of expression of outer membrane proteins (Yops). The addition of 2.5 mM Ca2+ to the growth medium reduced the degree of inhibition by the pathogen, but it was still significantly higher than that of the plasmid-cured strain. An avirulent mutant strain, from which the entire yopH gene was deleted, was impaired in its phagocytosis inhibition ability. This mutant could be trans-complemented by the yopH+ gene back to the wild-type phenotype with respect to virulence, as well as the ability to inhibit phagocytosis, demonstrating that the ability to inhibit phagocytosis is an important virulence function. The mutant strain was still cytotoxic for HeLa cells, indicating that inhibition of phagocytosis can be genetically separated from the ability to cause a cytotoxic effect.

The basic Yop2b protein, encoded by the virulence plasmid pIBI of Yersinia pseudotuberculosis, is produced under Ca2+-deficient conditions. A mutant deleted for the entire yopH gene, which encodes the Yop2b protein, was found to be avirulent. Virulence could be restored by trans-complementation. The DNA-sequence of yopH predicted a 50 737 D polypeptide lacking a typical signal peptide. Transcription of yopH is regulated by both temperature and Ca2+-concentration. Mutations within the region of the virulence plasmid known to be involved in regulating gene expression in response to Ca2+ abolished transcription of yopH. Other temperature-sensitive mutations in the Ca2+-regulatory locus showed a high level of transcription regardless of Ca2+-concentration. These responses were similar to those of the yopE gene. The promoter region of the yopE and yopH genes were compared and four conserved motifs identified.

The expression of the temperature-inducible plasmid-coded outer membrane proteins (YOPs) of Yersinia pseudotuberculosis was studied. These proteins were not recovered in the outer membrane fraction when the strain was grown in minimal medium at 37 degrees C, but they were expressed under these conditions. A strict correlation was found between Ca2+ dependency in the virulent strain, YPIII(pIB1), and ability to express YOPs. Ca2+-independent plasmid mutants or RNA-polymerase mutants harboring the virulence plasmid were unable to express YOPs, in contrast to the wild-type strain. These strains were also found to be avirulent. Sera recovered from patients or animals undergoing infection with either Y. pseudotuberculosis, Y. pestis, or Y. enterocolitica possessed antibodies directed against YOPs, indicating that they were expressed in all three pathogenic Yersinia species during infection. The YOPs of the three different species showed high immunological relatedness.

The autoagglutination of Yersinia enterocolitica was dependent on the presence of the virulence plasmid and on the active growth of bacteria in tissue culture media at 37 degrees C. Cultures with a high initial concentration of bacteria failed to autoagglutinate , indicating that synthesis of new virulence plasmid-associated surface factors was essential for autoagglutination. The synthesis of a plasmid-encoded polypeptide (molecular weight, 240,000), designated P1, that could be dissociated under strongly reducing conditions into subunits of 52,500 daltons was found to be correlated with autoagglutination. Further, a strain of Yersinia pseudotuberculosis [ YPIII ( PIB102 )], which has Tn5 inserted within the structural gene of P1 that prevents the synthesis of P1, failed to autoagglutinate , in contrast to the wild-type strain, strongly indicating that P1 is involved in this phenomenon. It was also found by immunoblotting that in addition to the common response to temperature, the P1 proteins of Y. enterocolitica and Y. pseudotuberculosis were immunologically related.