Unchanged characteristics of Helicobacter pylori during its morphological conversion.

Helicobacter pylori strains RH 54 and NCTC 11637 were grown in brain-heart infusion broth up to 56 days, and the coccoid form was obtained during prolonged incubation. Two morphological types of coccoids were observed, one of which was electron-dense and had an intact cellular membrane and flagella, indicating that it was likely to be viable. The other coccoid form was sphaeroblast-like and weakly stained, showing features of degeneration. Catalase activity was positive for aged cultures even up to 160 days. Sodium dodecyl sulphate polyacrylamide gel electrophoresis showed that most of the protein bands appeared to be similar in both the spiral and coccoid forms. In addition, Lewis blood group antigens were detected in cultures of up to 8 weeks. Furthermore, two sets of primers for the vacA and cagA genes were used in polymerase chain reaction, and these two important genes remained conserved in both the spiral and coccoid forms. The present study shows that the coccoid form of H. pylori retained many important characteristics present in the spiral form despite the morphological conversion, and thus supports the notion that some of the coccoid forms of H. pylori are likely to be viable.     

Characteristics of Helicobacter pylori attachment to human primary antral epithelial cells

Conventional cell lines are commonly used to study infection characteristics of the human gastric pathogen Helicobacter pylori. We sought to investigate bacterial attachment to human antral primary epithelial cells, a cell model that more closely resembles the human stomach than transformed cell lines. Primary cells were infected for 24 and 48 h with H. pylori. Morphological appearance of both the pathogen and the cells as well as features of colonization, attachment and internalization were evaluated by electron microscopy and compared to features observed with cultured AGS cells. H. pylori exhibited various shapes during colonization including the spiral, U-shaped, donut, and coccoid forms. The prevalence of each form seemed to be dependent on the infected donor tissue but, in general, changed with time to the coccoid form. Bacterial cell membranes progressively enlarged and appeared at times to be connected with microvilli. Bacterial attachment occurred to cells that were either unchanged, or had formed cup-like structures. Simultaneously, outer membrane vesicles were increasingly secreted from the bacteria, coinciding with increased cellular damage. We conclude that bacterial shape conversion, adherence and secretion of outer membrane vesicles are features of H. pylori infection. Primary gastric cell cultures closely imitate the antral environment and present an appropriate and useful model to study H. pylori pathogenesis.     

Oxygen tension regulates reactive oxygen generation and mutation of Helicobacter pylori

Although both bacillary and coccoid forms of Helicobacter pylori reside in human stomach, the pathophysiological significance of the two forms remains obscure. The present work describes the effect of oxygen tension on the transformation and reactive oxygen species (ROS) metabolism of this pathogen. Most H. pylori cultured under an optimum O2 concentration (7%) were the bacillary form, whereas about 80% of cells cultured under aerobic or anaerobic conditions were the coccoid form. The colony-forming unit of H. pylori decreased significantly under both aerobic and anaerobic culture conditions. The bacillary form of H. pylori generated predominantly superoxide radical, whereas the coccoid form generated preferentially hydroxyl radical. Specific activities of cellular respiration, urease, and superoxide dismatase decreased markedly after transformation of the bacillary form to the coccoid form, with concomitant generation of protein carbonyls and 8-hydroxyguanine. The frequency of mutation of cells increased significantly during culture under nonoptimum O2 conditions. These results indicate that ROS generated by H. pylori catalyze the oxidative modification of cellular DNA, thereby enhancing the transformation from the bacillary to the coccoid form. The enhanced generation of mutagenic hydroxyl radicals in the coccoid form might accelerate mutation and increase the genetic diversity of H. pylori.     

球形与螺旋形幽门螺杆菌基因表达差异的研究

目的：从基因转录水平了解球形幽门螺杆菌(Helicobacter pylori,Hp)基因表达的变化。方法：幽门螺杆菌标准株NCTC ll637和2株临床分离株,经抗生素—灭滴灵诱导球变。提取等菌量螺旋形和球形Hp的RNA,RT—PCR扩增Hp25种基因,这些基因包括毒力基因(kat、aphA、rdxA、frxA、cysS、ureB glmM、cagA、vacA、fldA、iceAl、hpaA、fliD、napA、oipA、alpAB、babB、hopZ);看家基因(scoB、atpD、g1nA、recA)和功能不明的外膜蛋白基因(hopW、hopX)。扩增产物经分子定量成像系统进行扫描定量。结果和结论：等菌量的球形HpRNA量比螺旋形Hp减少。NCTC ll637、F44和F49菌株的螺旋菌分别有25、20、19个基因RT—PCR阳性,对应的球形菌相应的基因RT—PCR也为阳性。定量分析结果表明：被检测的基因在球形菌中的表达均比螺旋菌降低。提示球形菌致病性的降低与基因的低表达有关。3株球形菌不同基因表达的变化并不完全一致,表达量变化较小且各菌间较一致的是g1mM、oipA、g1nA;表达量变化较大且菌问较一致的基因是：napA、sodB、recA、fliD。     

Oxidative cellular damage associated with transformation of Helicobacter pylori from a bacillary to a coccoid form

Exposure to unfavorable conditions results in the transformation of Helicobacter pylori, a gastric pathogen, from a bacillary form to a coccoid form. The mechanism and pathophysiological significance of this transformation remain unclear. The generation of the superoxide radical by H. pylori has previously been shown to inhibit the bactericidal action of nitric oxide, the concentration of which is relatively high in gastric juice. With the use of chemiluminescence probes, both the quality and quantity of reactive oxygen species generated by H. pylori have now been shown to change markedly during the transformation from the bacillary form to the coccoid form. The transformation of H. pylori was associated with oxidative modification of cellular proteins, including urease, an enzyme required for the survival of this bacterium in acidic gastric juice. Although the cellular abundance of urease protein increased during the transformation, the specific activity of the enzyme decreased and it underwent aggregation. Specific activities of both superoxide dismutase and catalase in H. pylori also decreased markedly during the transformation. The transformation of H. pylori was also associated with oxidative modification of DNA, as revealed by the generation of 8-hydroxyguanine, and subsequent DNA fragment. These observations indicate that oxidative stress elicited by endogenously generated reactive oxygen species might play an important role in the transformation of H. pylori from the bacillary form to the coccoid form.     

Morphological changes of synchronized Campylobacter jejuni populations during growth in single phase liquid culture

Campylobacter jejuni strains demonstrate a variety of growth phase-linked distinct morphological forms when grown in liquid culture. The typical spiral form of the organism, evident during logarithmic phase, undergoes elongation during stationary phase before becoming coccoid via the formation of membrane blebs and budded forms in decline phase. Cellular elongation and coccoid formation occurred despite the inhibition of protein synthesis and without a detectable change in the protein components of the inner and outer cell membranes.     

Colony formation by Helicobacter pylori after long-term incubation under anaerobic conditions

To evaluate the viability of Helicobacter pylori cultured under anaerobic conditions, H. pylori strain TK1029 was grown on blood agar in a microaerophilic environment at 37 degrees C for 4 days, and subsequently cultured under anaerobic conditions for 1 to 35 days. Colony formation by bacteria on blood agar plates cultured under anaerobic conditions was observed only for up to 4 days of microaerophilic incubation. By Gram staining, the morphological form of the bacteria was shown to be predominantly coccoid. However, bacteria cultured under anaerobic conditions for 15 to 35 days formed colonies on blood agar after pre-incubation of bacteria with PBS, but not without pre-incubation. These results suggest that H. pylori survives long-term culture under anaerobic conditions and that both pre-incubation in non-nutrient solution and high density of bacterial concentration might be important for recovery of H. pylori cultured for a prolonged time under anaerobic conditions.     

Alteration in the composition of cholesteryl glucosides and other lipids in Helicobacter pylori undergoing morphological change from spiral to coccoid form

In this analysis of membrane lipid compositions in Helicobacter pylori, the membrane lipid profiles drastically changed during coccoid formation: cholesteryl-6-O-tetradecanoyl-alpha-D-glucopyranoside levels increased, cholesteryl-alpha-D-glucopyranoside and phosphatidyl ethanolamine decreased, and a coordinated increase in cardiolipin and decrease in phosphatidyl glycerol were observed. Cholesteryl-6-O-phosphatidyl-alpha-D-glucopyranoside was hardly detected in the spiral forms in the logarithmic phase, but subsequently increased throughout the coccoid conversion. These results suggest that environmental stresses induce the expression of certain regulatory systems for lipid metabolism in H. pylori, and that the resulting alterations in lipid composition play an important role in inducing the coccoid conversion.     

Gastrointestinal colonisation of BALB/cA mice by Helicobacter pylori monitored by heparin magnetic separation

Abstract Immunocompetent and immunodeficient BALB/cA mice were fed orally with 10⁸ colony forming units of 2-day-old spiral or coccoid (12 days old) Helicobacter pylori strain NCTC 11637. Immunocompetent BALB/cA mice were also fed orally with decreasing numbers of spiral or coccoid forms of H. pylori . The gastrointestinal colonisation process was monitored for 34 days post-infection by heparin magnetic separation and subsequent enzyme immunoassay (EIA) for the detection of the H. pylori cells. Both mice types were colonised with H. pylori . The coccoid form of H. pylori gave higher EIA absorbance values and more efficient colonisation in the mice than the spiral form. Immunocompetent BALB/cA mice fed with the coccoid form of H. pylori exhibited an acute inflammation process in histopathological samples from the stomachs. In conclusion, H. pylori can infect both immunocompetent as well as immunodeficient BALB/cA mice and coccoids (viable but non-culturable) obtained after 12 days of culturing can infect BALB/cA mice.     

The bactericidal and morphological effects of peroxynitrite on Helicobacter pylori

Peroxynitrite (ONOO-) is correlated with the pathogenesis of Helicobacter pylori-induced peptic ulcer diseases. We aimed to investigate the time- and concentration-dependent bactericidal and morphological effects of ONOO- on H. pylori. Authentic ONOO- was synthesized as quenched-flow method. A stock culture of H. pylori NCTC 11637 was exposed to different concentrations of ONOO- (0.1-40 micromol/L) or decomposed ONOO- or fresh medium. Samples were taken at 0, 15, 30, 60, and 120 minutes, for the evaluation of viable bacteria and bacterial morphology with gram strain and transmission electron microscopy. Decomposed ONOO- showed no bactericidal activity against H. pylori. ONOO- application caused a decrease in the number of viable bacteria within the first 15 minutes. The significant conversion of H. pylori from spiral form to coccoid form was determined with 10 micromol/L of ONOO-, and higher concentrations caused lysis of the cells. Separation of cell wall, bleb formation, vacuolization, decrease of secretory granules, and lysis of bacteria were the morphological effects of ONOO- on H. pylori. Because the morphology of the bacteria is one of the important factors in virulence; peroxynitrite-related morphological effects might have an impact in the progress of the H. pylori-induced peptic ulcer diseases.     

Egg Passage of Rodshaped and Coccoid Forms of Helicobacter pylori: Preliminary Studies

Background. We used egg passage of bacteria stored in water to evaluate the culturability of the coccoid form of Helicobacter pylori , as a complement to the results obtained from various animal models. Egg passage was performed, as it is a simple, rapid, and well-characterized old method by which to culture and evaluate culturability of bacteria compared to experiments in animal models. Egg passage has been used in such experiments since 1938 for isolation and growth of, for example, Rickettsiae sp. and Chlamydia sp.
Materials and Methods. The rod-shaped form of H. pylori was produced by plate cultures for 4 and 7 days. The coccoid form of H. pylori was produced by culture on agar plates for 10 days, followed by storage in water. These preparations then were inoculated into the yolk sac of differently aged fertilized eggs.
Results. Positive culture was obtained from 14 of 17 eggs (82%) inoculated with rod-shaped H. pylori compared to 0 of 22 eggs (0%) inoculated with the coccoid form.
Conclusion. Culturability of H. pylori is reduced when it converts into the coccoid form produced by starvation and age followed by storage in water for several weeks at room temperature. Egg passage did not raise the culturability of the coccoid form of H. pylori. Our study demonstrates some clear differences between fresh rods and stored cocci forms of H. pylori in terms of culturability when passed through eggs.     

Lewis epitopes on outer membrane vesicles of relevance to Helicobacter pylori pathogenesis

BACKGROUND: Helicobacter pylori extrudes protein- and lipopolysaccharide-enriched outer membrane vesicles from its cell surface which have been postulated to act to deliver virulence factors to the host. Lewis antigen expression by lipopolysaccharide of H. pylori cells has been implicated in a number of pathogenic roles. The aim of this study was to further characterize the expression of lipopolysaccharide on the surface of these outer membrane vesicles and, in particular, expression of Lewis antigens and their association with antibody production in the host. MATERIALS AND METHODS: H. pylori strains were examined for outer membrane vesicle production using transmission electron microscopy and Lewis antigen expression probed using immunoelectron microscopy. Sera from patients were analyzed for cross-reacting anti-Lewis antibodies and, subsequently, absorbed using outer membrane vesicle preparations to remove the cross-reacting antibodies. RESULTS: The formation of outer membrane vesicles by H. pylori was observed in both in vitro and in vivo samples. Furthermore, vesicles were produced following culture in either liquid or solid medium by all strains examined. Moreover, we observed the presence of Lewis epitopes on outer membrane vesicles using immunoelectron microscopy and immunoblotting. Circulating anti-Lewis antibodies were found in the sera of gastric cancer patients but not in the sera of H. pylori-negative control subjects. Absorption of patient sera with outer membrane vesicles decreased the levels of anti-Lewis autoantibodies. CONCLUSIONS: Our results demonstrate the ability of H. pylori to generate outer membrane vesicles bearing serologically recognizable Lewis antigens on lipopolysaccharide molecules which may contribute to the chronic immune stimulation of the host. The ability of these vesicles to absorb anti-Lewis autoantibodies indicates that they may, in part, play a role in putative autoimmune aspects of H. pylori pathogenesis.     

Role of AmiA in the morphological transition of Helicobacter pylori and in immune escape

The human gastric pathogen Helicobacter pylori is responsible for peptic ulcers and neoplasia. Both in vitro and in the human stomach it can be found in two forms, the bacillary and coccoid forms. The molecular mechanisms of the morphological transition between these two forms and the role of coccoids remain largely unknown. The peptidoglycan (PG) layer is a major determinant of bacterial cell shape, and therefore we studied H. pylori PG structure during the morphological transition. The transition correlated with an accumulation of the N-acetyl-D-glucosaminyl-beta(1,4)-N-acetylmuramyl-L-Ala-D-Glu (GM-dipeptide) motif. We investigated the molecular mechanisms responsible for the GM-dipeptide motif accumulation, and studied the role of various putative PG hydrolases in this process. Interestingly, a mutant strain with a mutation in the amiA gene, encoding a putative PG hydrolase, was impaired in accumulating the GM-dipeptide motif and transforming into coccoids. We investigated the role of the morphological transition and the PG modification in the biology of H. pylori. PG modification and transformation of H. pylori was accompanied by an escape from detection by human Nod1 and the absence of NF-kappaB activation in epithelial cells. Accordingly, coccoids were unable to induce IL-8 secretion by AGS gastric epithelial cells. amiA is, to our knowledge, the first genetic determinant discovered to be required for this morphological transition into the coccoid forms, and therefore contributes to modulation of the host response and participates in the chronicity of H. pylori infection.     

Searching the point of no return in Helicobacter pylori life: necrosis and/or programmed death?

AIMS: Ultrastructural and molecular studies to support the hypothesis of programmed cell death in Helicobacter pylori were conducted. METHODS AND RESULTS: Evidence of programmed death in H. pylori is provided through electron microscopic detection and cytochemical labelling of electrondense bodies (EDB), containing packaged DNA in coccoid cells, resembling micronuclei of apoptotic eukaryotic cells. This morphological evidence is also supported by DNA cleavage in homogeneous fragments of about 100 base pairs. Programmed cell death was observed in H. pylori cultures at 37 degrees C, with a maximum of 37.5% of EDB coccoid cells after 7 days. The non-permissive temperature of 4 degrees C anticipated this process, with 40% of EDB coccoid forms within 3 days, and it remained substantially unaffected during the observation time of 14 days. CONCLUSION: In these experiments, deprivation of nutrients and a non-permissive temperature acted as a powerful trigger for programmed cell death. SIGNIFICANCE AND IMPACT OF THE STUDY: Helicobacter pylori bacterial populations, under stressing stimuli, can respond with programmed cell suicide as a means of species preservation.     

Population dynamics in ageing Helicobacter pylori

The aim of this work was to characterize population changes occurring in aged broth cultures of Helicobacter pylori. Experiments were performed using clinical strains cultured immediately after isolation and after multiple subcultures in solid medium. Morphological changes in the ageing bacteria during a 7-day broth culture were analysed by optical and electron microscopy. The expression of the virulence factor, CagA, together with the presence of the cell cycle regulator, cGMP, were also assessed. The transition from bacillary to coccoid forms was the main morphological change observed in freshly isolated bacteria, together with the increase in cGMP from 1 to 2.25 nmoles/mg of proteins within the first 7 days of broth culture. A similar trend of morphological and physiological changes was observed in cells after multiple subcultures in solid medium with a major presence of large cell clusters. The cagA gene product was always expressed in all experimental conditions evaluated. These data show a significant morphological and physiological diversity in fresh, ageing and aged cultures of H. pylori.     

Characterization of the outer membrane protein profile from disease-related Helicobacter pylori isolates by subcellular fractionation and nano-LC FT-ICR MS analysis

Because of the important role of membrane proteins in adhesion, invasion, and intracellular survival of pathogens in the host, membrane proteins are of potential interest in the search for drug targets or biomarkers. We have established a mass spectrometry-based method that allows characterization of the outer membrane protein (OMP) profile of clinical isolates from of the human gastric pathogen Helicobacter pylori. Subcellular fractionation and one-dimensional gel electrophoresis (1D-GE) analysis was combined with nano-liquid chromatography Fourier transform-ion cyclotron resonance mass spectrometry (nano-LC FT-ICR MS) and tandem mass spectrometry (MS/MS) analysis of fifteen H. pylori strains associated either with duodenal ulcers, gastric cancer, or isolated from asymptomatic H. pylori infected carriers. Over 60 unique membrane or membrane-associated proteins, including 30 of the 33 theoretically predicted OMPs, were identified from the strains. Several membrane proteins, including Omp11 and BabA, were found to be expressed by all strains. In the search for clinical markers we found that Omp26 was expressed by all disease-related strains but was only present in one out of five strains from asymptomatic carriers, which makes Omp26 a potential target for further investigation in the search for proteins unique to disease-related H. pylori strains. In addition, presence of Omp30 and absence of Omp6 seemed to be associated with H. pylori strains causing duodenal ulcer.     

Coccoid forms of Helicobacter pylori can be viable.

Controversy exists as to whether the coccoid form of Helicobacter pylori can exist in a viable form. Conversion of helical to coccoid morphology occurs in culture over several days. In this study, the morphology was correlated with parameters of genetic integrity in the reference NCTC 11637 strain over 21 days of culture. The capacity to regrow colonies of helical form was demonstrated from a culture where the coccoid form constituted up to 95% and negligible urease activity could be detected. Urease enzyme activity and its mRNA decreased between day 0 and 10 while 26 kD mRNA and 16S rRNA were expressed unchanged for up to 14 and 21 days of culture, respectively. Expression of mRNA for the Cag A gene behaved in a similar fashion to that of urease. No evidence of DNA fragmentation was detected. These data suggest that a viable form of non-urease producing H. pylori exists after short to intermediate culture and that some if not all of these viable bacteria have coccoid morphology.