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Helicobacter pylori is associated with chronic gastritis, gastric or duodenal ulcers, and gastric cancer. Since the oral cavity is the entry port and the first component of the gastrointestinal system, the oral cavity has been discussed as a potential reservoir of H. pylori . Accordingly, a potential oral-oral transmission route of H. pylori raises the question concerning whether close contact such as kissing or sharing a meal can cause the transmission of H. pylori . Therefore, this topic has been investigated in many studies, applying different techniques for detection of H. pylori from oral samples, i.e. molecular techniques, immunological or biochemical methods and traditional culture techniques. While molecular, immunological or biochemical methods usually yield high detection rates, there is no definitive evidence that H. pylori has ever been isolated from the oral cavity. The specificity of those methods may be limited due to potential cross-reactivity, especially with H. pylori -like microorganisms such as Campylobacter spp. Furthermore, the influence of gastroesophageal reflux has not been investigated so far. This review aims to summarize and critically discuss previous studies investigating the potential colonization of H. pylori in the oral cavity and suggest novel research directions for targeting this critical research question.

Gastric Helicobacter pylori infection is diagnosed based on histopathological evaluation of gastric mucosal biopsies, urease test, urea breath test, H. pylori culturing, or direct detection using polymerase chain reaction (PCR). This study aimed to evaluate the efficacy of immunohistochemical (IHC) staining in detecting H. pylori in gastric biopsies from patients with chronic gastritis and minimal or atypical infection. Gastric biopsies from 50 patients with chronic gastritis were subjected to routine haematoxylin and eosin (H-E), modified Giemsa, and IHC staining. The results of staining were compared with those of quantitative real-time PCR (qRT-PCR). The qRT-PCR analysis identified 32 (64%) H. pylori-positive cases, whereas IHC, H-E, and modified Giemsa staining identified 29 (58%), 27 (54%), and 21 (42%) positive cases. The sensitivity of IHC staining (87.50%) was higher than that of H-E (59.38%) and modified Giemsa (43.75%) staining. The specificity of H-E, modified Giemsa, and IHC staining was 55.56%, 61.11%, and 94.44%, respectively. IHC staining exhibited the highest diagnostic accuracy (90%), followed by H-E (58%) and modified Giemsa (50%) staining. Active gastritis, intestinal metaplasia, and lymphoid follicles were detected in 32 (64%), 4 (8%), and 22 (44%) cases, respectively, and all of these cases were H. pylori positive. In contrast to routine H-E and modified Giemsa staining, IHC allows for the accurate H. pylori detection in cases with minimal or atypical infection. Moreover, IHC can be an alternative diagnostic method to qRT-PCR for detection of H. pylori in such cases.

Toxin-antitoxin systems are found in many bacterial chromosomes and plasmids with roles ranging from plasmid stabilization to biofilm formation and persistence. In these systems, the expression/activity of the toxin is counteracted by an antitoxin, which, in type I systems, is an antisense RNA. While the regulatory mechanisms of these systems are mostly well defined, the toxins' biological activity and expression conditions are less understood. Here, these questions were investigated for a type I toxin-antitoxin system (AapA1-IsoA1) expressed from the chromosome of the human pathogen Helicobacter pylori We show that expression of the AapA1 toxin in H. pylori causes growth arrest associated with rapid morphological transformation from spiral-shaped bacteria to round coccoid cells. Coccoids are observed in patients and during in vitro growth as a response to different stress conditions. The AapA1 toxin, first molecular effector of coccoids to be identified, targets H. pylori inner membrane without disrupting it, as visualized by cryoelectron microscopy. The peptidoglycan composition of coccoids is modified with respect to spiral bacteria. No major changes in membrane potential or adenosine 5'-triphosphate (ATP) concentration result from AapA1 expression, suggesting coccoid viability. Single-cell live microscopy tracking the shape conversion suggests a possible association of this process with cell elongation/division interference. Oxidative stress induces coccoid formation and is associated with repression of the antitoxin promoter and enhanced processing of its transcript, leading to an imbalance in favor of AapA1 toxin expression. Our data support the hypothesis of viable coccoids with characteristics of dormant bacteria that might be important in H. pylori infections refractory to treatment., Competing Interests: The authors declare no competing interest.

A urease inhibitor with good in vivo profile is considered as an alternative agent for treating infections caused by urease-producing bacteria such as Helicobacter pylori . Here, we report a series of N -monosubstituted thioureas, which act as effective urease inhibitors with very low cytotoxicity. One compound ( b19 ) was evaluated in detail and shows promising features for further development as an agent to treat H. pylori caused diseases. Excellent values for the inhibition of b19 against both extracted urease and urease in intact cell were observed, which shows IC 50 values of 0.16 ± 0.05 and 3.86 ± 0.10 µM, being 170- and 44-fold more potent than the clinically used drug AHA, respectively. Docking simulations suggested that the monosubstituted thiourea moiety penetrates urea binding site. In addition, b19 is a rapid and reversible urease inhibitor, and displays nM affinity to urease with very slow dissociation ( k off =1.60 × 10 -3  s -1 ) from the catalytic domain.

Here we present an easy flow cytometry protocol to study the viability of Helicobacter pylori which also enables the detection of even low live bacteria densities. This protocol has potential utility for a fast and accurate assessment of experimental eradication methods against H. pylori., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Identification of novel Ure inhibitors with high potency has received considerable attention. Methodology & results: Ure inhibition was determined using the indophenol method, the affinities to Ure were estimated via surface plasmon resonance. Seventeen new plus ten known N -monosubstituted thiosemicarbazides were synthesized and identified as novel Ure inhibitors. Out of these compounds, compound b5 shows excellent activity against both crude Ure from Helicobacter pylori (IC 50  = 0.04 μM) and Ure in living cell (IC 50  = 0.27 μM), with the potency being over 600-fold higher than clinical used drug acetohyroxamic acid, respectively. Surface plasmon resonance demonstrated the high affinity ( K d .#x00A0;= 6.32 nM) of b5 to Ure. Conclusion: This work provides a class of novel and promising Ure inhibitors.

Background: In this study, one Helicobacter pylori isolate, from gastric biopsy of a dyspeptic patient that turned into mucoid-coccoid (MC) form upon consecutive subcultures, was identified. The culturability, antibiotic resistance, and lipid contents of MC were compared with those of non-mucoid (NM) spiral H pylori., Materials and Methods: Mucoid-coccoid and NM H pylori were subcultured on Brucella blood agar (BBA) and incubated under aerobic and microaerobic atmospheres at 37°C. Cultures were examined for colony characteristics and bacterial morphology after 1-3 days. The isolates were identified by biochemical tests and detection of H pylori-16S rDNA. Antibiogram was performed with currently used antibiotics for H pylori eradication. Cellular lipid contents were extracted and analyzed by gas chromatography., Results: Compared with pin-pointed and glistening colonies of NM H pylori that appeared under microaerobic conditions, MC H pylori grew well in consecutive subcultures under aerobic and microaerobic atmospheres and produced white patches of mucoid colonies. MC exhibited coccoid and NM spiral morphology. Both isolates were catalase, oxidase, and urease positive and contained 16S rDNA. Compared with NM that was susceptible to almost all the antibiotics, MC was resistant to all the antibiotics. Lipid analyses showed high frequency of unsaturated fatty acids and cholesterol in MC., Conclusions: Coccoid forms with high fatty acid and cholesterol contents that show resistance to antibiotics might resist against other stressful conditions such as gastric acidity and immune response. Moreover, mucoid property may enhance resistance of coccoids to stresses. With mucoid-coccoid lifestyle, H pylori may establish a chronic infection refractory to antimicrobial therapy., (© 2019 John Wiley & Sons Ltd.)

A genetic screen for colonization factors of the human stomach pathogen Helicobacter pylori took a surprising turn with the discovery that some colonization mutants had lost helical cell morphology. Further pursuit of direct morphology screens revealed a large H. pylori 'shapesome' complex consisting of peptidoglycan modification and precursor synthesis enzymes, a cytoskeletal element and putative scaffold or regulatory proteins that promote enhanced asymmetric cell wall growth. Functional characterization of H. pylori shape mutants indicates multiple roles for cell shape during colonization of mucosal surfaces. Conservation of both the molecular constituents of the H. pylori cell shape program and a newly appreciated enrichment of this morphotype at mucosal surface suggests that helical organisms may be particularly well poised to exploit host perturbations to become pathogens., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Helical cell shape is necessary for efficient stomach colonization by Helicobacter pylori , but the molecular mechanisms for generating helical shape remain unclear. The helical centerline pitch and radius of wild-type H. pylori cells dictate surface curvatures of considerably higher positive and negative Gaussian curvatures than those present in straight- or curved-rod H. pylori . Quantitative 3D microscopy analysis of short pulses with either N -acetylmuramic acid or D-alanine metabolic probes showed that cell wall growth is enhanced at both sidewall curvature extremes. Immunofluorescence revealed MreB is most abundant at negative Gaussian curvature, while the bactofilin CcmA is most abundant at positive Gaussian curvature. Strains expressing CcmA variants with altered polymerization properties lose helical shape and associated positive Gaussian curvatures. We thus propose a model where CcmA and MreB promote PG synthesis at positive and negative Gaussian curvatures, respectively, and that this patterning is one mechanism necessary for maintaining helical shape., Competing Interests: JT, BB, SS, KB, HJ, KD, EK, JG, JB, MV, WV, CG, JS, NS No competing interests declared, (© 2020, Taylor et al.)

Background: Proton pump inhibitors (PPIs) with lipophilic nature may interact with lipid components of H. pylori cell membrane, disrupting cell structure and viability. In this study, the effect of PPIs on fatty acid and cholesterol components of H. pylori cell membrane was assessed., Methods: One H. pylori isolate was treated with 1X and 2X MICs (μg/mL) of lansoprazole (LPZ: 8 and 16) and pantoprazole (PAN: 128 and 256) in brain heart infusion broth plus serum. Treated H. pylori was cultured on brucella blood agar (BBA) and tetrazolium egg yolk agar (TEYA). Bacterial cells stained with Live/Dead kit were examined by fluorescent microscopy. Fatty acid and cholesterol contents of treated H. pylori were measured by gas chromatography., Results: PPI-treated H. pylori did not grow on BBA but grew on TEYA. Fluorescent microscopy showed H. pylori stained red. Analyses showed high frequency of saturated fatty acids, C14:0, C16:0 and C18:0. Among unsaturated fatty acids, C18:1 and C18:2c were increased, while five were eliminated and five were synthesized de novo. Cholesteryl-6-O-tetradecanoyl-α-D- glucopyranoside was detected as the only glycosylated cholesterol in treated H. pylori. Growth of PPI-treated H. pylori on cholesterol-rich TEYA showed that occurrence of cholesterol can reverse the growth inhibition by PPIs. Red- bacilli form of H. pylori showed dye entry through damaged cell membrane without lysis., Conclusion: Incorporation of lipophilic PPI into H. pylori cell membrane disrupted lipids and inhibited growth. However, H. pylori adjusted the defected membrane by replacing the lipid components and resisted lysis., (© 2020 The Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.)

Evident in its name, the gastric pathogen Helicobacter pylori has a helical cell morphology which facilitates efficient colonization of the human stomach. An improved light-focusing strategy allowed us to robustly distinguish even subtle perturbations of H. pylori cell morphology by deviations in light-scattering properties measured by flow cytometry. Profiling of an arrayed genome-wide deletion library identified 28 genes that influence different aspects of cell shape, including properties of the helix, cell length or width, cell filament formation, cell shape heterogeneity, and cell branching. Included in this mutant collection were two that failed to form any helical cells, a soluble lytic transglycosylase and a previously uncharacterized putative multipass inner membrane protein HPG27_0728, renamed Csd7. A combination of cell fractionation, mutational, and immunoprecipitation experiments show that Csd7 and Csd2 collaborate to stabilize the Csd1 peptidoglycan (PG) endopeptidase. Thus, both csd2 and csd7 mutants show the same enhancement of PG tetra-pentapeptide cross-linking as csd1 mutants. Csd7 also links Csd1 with the bactofilin CcmA via protein-protein interactions. Although Csd1 is stable in ccmA mutants, these mutants show altered PG tetra-pentapeptide cross-linking, suggesting that Csd7 may directly or indirectly activate as well as stabilize Csd1. These data begin to illuminate a highly orchestrated program to regulate PG modifications that promote helical shape, which includes nine nonessential nonredundant genes required for helical shape and 26 additional genes that further modify H. pylori 's cell morphology. IMPORTANCE The stomach ulcer and cancer-causing pathogen Helicobacter pylori has a helical cell shape which facilitates stomach infection. Using light scattering to measure perturbations of cell morphology, we identified 28 genes that influence different aspects of cell shape. A mutant in a previously uncharacterized protein renamed Csd7 failed to form any helical cells. Biochemical analyses showed that Csd7 collaborates with other proteins to stabilize the cell wall-degrading enzyme Csd1. Csd7 also links Csd1 with a putative filament-forming protein via protein-protein interactions. These data suggest that helical cell shape arises from a highly orchestrated program to regulate cell wall modifications. Targeting of this helical cell shape-promoting program could offer new ways to block infectivity of this important human pathogen., (Copyright © 2019 American Society for Microbiology.)

Half of all humans harbor Helicobacter pylori in their stomachs. Helical cell shape is thought to facilitate H. pylori 's ability to bore into the protective mucus layer in a corkscrew-like motion, thereby enhancing colonization of the stomach. H. pylori cell shape mutants show impaired colonization of the mouse stomach, highlighting the importance of cell shape in infection. To gain a deeper understanding of how helical cell morphology promotes host colonization by H. pylori , we used three-dimensional confocal microscopy to visualize the clinical isolate PMSS1 and an isogenic straight-rod mutant (Δ csd6 ) within thick longitudinal mouse stomach sections. We also performed volumetric image analysis to quantify the number of bacteria residing within corpus and antral glands in addition to measuring total CFU. We found that straight rods show attenuation during acute colonization of the stomach (1 day or 1 week postinfection) as measured by total CFU. Our quantitative imaging revealed that wild-type bacteria extensively colonized antral glands at 1 week postinfection, while csd6 mutants showed variable colonization of the antrum at this time point. During chronic infection (1 or 3 months postinfection), total CFU were highly variable but similar for wild-type and straight rods. Both wild-type and straight rods persisted and expanded in corpus glands during chronic infection. However, the straight rods showed reduced inflammation and disease progression. Thus, helical cell shape contributes to tissue interactions that promote inflammation during chronic infection, in addition to facilitating niche acquisition during acute infection., (Copyright © 2019 American Society for Microbiology.)

Helicobacter pylori (Hp) infection is related to the pathogenesis of chronic gastric disorders and extragastric diseases. Here, we examined the anorexigenic and anxiogenic effects of Hp vacuolating cytotoxin A (VacA) through activation of hypothalamic urocortin1 (Ucn1). VacA was detected in the hypothalamus after peripheral administration and increased Ucn1 mRNA expression and c-Fos-positive cells in the hypothalamus but not in the nucleus tractus solitarius. c-Fos and Ucn1-double positive cells were detected. CRF1 and CRF2 receptor antagonists suppressed VacA-induced anxiety and anorexia, respectively. VacA activated single paraventricular nucleus neurons and A7r5 cells; this activation was inhibited by phospholipase C (PLC) and protein kinase C (PKC) inhibitors. VacA causes anorexia and anxiety through the intracellular PLC-PKC pathway, migrates across the blood-brain barrier, and activates the Ucn1-CRF receptor axis.

Infection of the human stomach caused by Helicobacter pylori is very common, as the pathogen colonizes more than half of the world's population. It is associated with varied outcomes of infection, such as peptic ulcer disease, gastric ulcers, and mucosa-associated lymphoid tissue lymphoma, and is generally considered a risk factor for the development of gastric adenocarcinoma. Cholesteryl glucosides (CGs) constitute a vital component of the cell wall of H. pylori and contribute to its pathogenicity and virulence. The hp0421 gene, which encodes cholesteryl-α-glucoside transferase (CGT), appears critical for the enzymatic function of integrating unique CGs into the cell wall of H. pylori , and deletion of this gene leads to depletion of CGs and their variants. Herein, we report that the deletion of hp0421 and consequent deficiency of cholesterol alter the morphology, shape, and cell wall composition of H. pylori cells, as demonstrated by high-resolution confocal microscopy and flow cytometry analyses of two different type strains of H. pylori , their isogenic knockouts as well as a reconstituted strain. Moreover, measurement of ethidium bromide (EtBr) influx by flow cytometry showed that lack of CGs increased cell wall permeability. Antimicrobial susceptibility testing revealed that the hp0421 isogenic knockout strains ( Hp26695Δ421 and Hp76Δ421 ) were sensitive to antibiotics, such as fosfomycin, polymyxin B, colistin, tetracycline, and ciprofloxacin, in contrast to the wild-type strains that were resistant to the above antibiotics and tended to form denser biofilms. Lipid profile analysis of both Hp76 and Hp76Δ421 strains showed an aberrant profile of lipopolysaccharides (LPS) in the Hp76Δ421 strain. Taken together, we herein provide a set of mechanistic evidences to demonstrate that CGs play critical roles in the maintenance of the typical spiral morphology of H. pylori and its cell wall integrity, and any alteration in CG content affects the characteristic morphological features and renders the H. pylori susceptible to various antibiotics. IMPORTANCE Helicobacter pylori is an important cause of chronic gastritis leading to peptic ulcer and is a major risk factor for gastric malignancies. Failure in the eradication of H. pylori infection and increasing antibiotic resistance are two major problems in preventing H. pylori colonization. Hence, a deeper understanding of the bacterial survival strategies is needed to tackle the increasing burden of H. pylori infection by an appropriate intervention. Our study demonstrated that the lack of cholesteryl glucosides (CGs) remarkably altered the morphology of H. pylori and increased permeability of the bacterial cell wall. Further, this study highlighted the substantial role of CGs in maintaining the typical H. pylori morphology that is essential for retaining its pathogenic potential. We also demonstrated that the loss of CGs in H. pylori renders the bacterium susceptible to different antibiotics., (Copyright © 2018 Qaria et al.)

There is an ongoing need to develop ultrasensitive nanomechanical instrumentation that has high spatial and force resolution, as well as an ability to operate in various biological environments. Here, we present a compact nanofiber optic force transducer (NOFT) with sub-piconewton force sensitivity and a nanoscale footprint that paves the way to the probing of complex mechanical phenomena inside biomolecular systems. The NOFT platform comprises a SnO 2 nanofiber optic equipped with a thin, compressible polymer cladding layer studded with plasmonic nanoparticles (NPs). This combination allows angstrom-level movements of the NPs to be quantified by tracking the optical scattering of the NPs as they interact with the near-field of the fiber. The distance-dependent optical signals can be converted to force once the mechanical properties of the compressible cladding are fully characterized. In this protocol, the details of the synthesis, characterization, and calibration of the NOFT system are described. The overall protocol, from the synthesis of the nanofiber optic devices to acquisition of nanomechanical data, takes ~72 h.

Chronic infection with Helicobacter pylori can lead to the development of gastric ulcers and stomach cancers. The helical cell shape of H. pylori promotes stomach colonization. Screens for loss of helical shape have identified several periplasmic peptidoglycan (PG) hydrolases and non-enzymatic putative scaffolding proteins, including Csd5. Both over and under expression of the PG hydrolases perturb helical shape, but the mechanism used to coordinate and localize their enzymatic activities is not known. Using immunoprecipitation and mass spectrometry we identified Csd5 interactions with cytosolic proteins CcmA, a bactofilin required for helical shape, and MurF, a PG precursor synthase, as well as the inner membrane spanning ATP synthase. A combination of Csd5 domain deletions, point mutations, and transmembrane domain chimeras revealed that the N-terminal transmembrane domain promotes MurF, CcmA, and ATP synthase interactions, while the C-terminal SH3 domain mediates PG binding. We conclude that Csd5 promotes helical shape as part of a membrane associated, multi-protein shape complex that includes interactions with the periplasmic cell wall, a PG precursor synthesis enzyme, the bacterial cytoskeleton, and ATP synthase., (© 2018 John Wiley & Sons Ltd.)

Two series of ω-phenoxy contained acylhydroxamic acids as novel urease inhibitors were designed and synthesized. Biological activity evaluations revealed that ω-phenoxypropinoylhydroxamic acids were more active than phenoxyacetohydroxamic acids. Out of these compounds, 3-(3,4-dichlorophenoxy)propionylhydroxamic acid c24 showed significant potency against urease in both cell free extract (IC 50  = 0.061 ± 0.003 μM) and intact cell (IC 50  = 0.89 ± 0.05 μM), being over 450- and 120-fold more potent than the clinically prescribed urease inhibitor AHA, repectively. Non-linear fitting of experimental data (V-[S]) suggested a mixed-type inhibition mechanism and a dual site binding mode of these compounds., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

DNA replication and cell division are two fundamental processes in the life cycle of a cell. The majority of prokaryotic cells undergo division by means of binary fission in coordination with replication of the genome. Both processes, but especially their coordination, are poorly understood in Helicobacter pylori. Here, we studied the cell divisome assembly and the subsequent processes of membrane and peptidoglycan synthesis in the bacterium. To our surprise, we found the cell divisome assembly to be polar, which was well-corroborated by the asymmetric membrane and peptidoglycan synthesis at the poles. The divisome components showed its assembly to be synchronous with that of the replisome and the two remained associated throughout the cell cycle, demonstrating a tight coordination among chromosome replication, segregation and cell division in H. pylori. To our knowledge, this is the first report where both DNA replication and cell division along with their possible association have been demonstrated for this pathogenic bacterium., (© 2018 Federation of European Biochemical Societies.)

Helicobacter pylori (H. pylori), a gram-negative microaerophilic bacterial pathogen that colonizes the stomachs of more than half of all humans, is linked to chronic gastritis, peptic ulcers and gastric cancer. Spiral-shaped H. pylori undergo morphologic conversion to a viable but not culturable coccoid form when they transit from the microaerobic stomach into the anaerobic intestinal tract. However, little is known about the morphological and pathogenic characteristics of H. pylori under prolonged anaerobic conditions. In this study, scanning electron microscopy was used to document anaerobiosis-induced morphological changes of H. pylori, from helical to coccoid to a newly defined fragmented form. Western blot analysis indicated that all three forms express certain pathogenic proteins, including the bacterial cytotoxin-associated gene A (CagA), components of the cag-Type IV secretion system (TFSS), the blood group antigen-binding adhesin BabA, and UreA (an apoenzyme of urease), almost equally. Similar urease activities were also detected in all three forms of H. pylori. However, in contrast to the helical form, bacterial motility and TFSS activity were found to have been abrogated in the anaerobiosis-induced coccoid and fragmented forms of H. pylori. Notably, it was demonstrated that some of the anaerobiosis-induced fragmented state cells could be converted to proliferation-competent helical bacteria in vitro. These results indicate that prolonged exposure to the anaerobic intestine may not eliminate the potential for H. pylori to revert to the helical pathogenic state., (© 2018 The Societies and John Wiley & Sons Australia, Ltd.)

Poorly water-soluble and unstable compounds are difficult to develop as drug products using conventional formulation techniques. The aim of the present study was to develop and evaluate a nanoformulation prepared by a hot high-pressure homogenization method, which was a scalable and solvent-free process. We successfully prepared stable nanodispersions to protect a labile antibiotic, erythromycin. The mean diameter of the dispersed droplets was approximately 150 nm, and size distribution was unimodal. Dispersion was physically stable at room temperature for over six months. Using erythromycin as a model compound, we studied its antimicrobial activity in vitro on Helicobacter pylori. Results showed that drug encapsulation improves API stability in an acidic environment and is conducive to a synergistic effect between the drug and the formulation., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Aim: This study aims to investigate the morphological transition of Helicobacter pylori during adaptation to water., Materials & Methods: Different strains were adapted to water. Changes regarding cultivability and cellular morphology were recorded. Expression of 11 genes involved in H. pylori morphological changes was evaluated by real-time PCR., Results: H. pylori presented increased cultivability in water after adaptation. The permanent loss of the spiral shape was observed, but no transition into coccoid form has occurred. Expression levels of genes involved in peptidoglycan assembly of H. pylori 26695 have shown significant changes between adapted and nonadapted strains., Conclusion: Adaption to water favors the culturable phenotype and the morphological transition to the rod shape, into a process that implicates the peptidoglycan turnover.

Evidence indicates that Helicobacter pylori is the causative agent of chronic gastritis and perhaps gastric malignancy. Extracellular vesicles (EVs) play an important role in the evolutional process of malignancy due to their genetic material cargo. We aimed to evaluate the clinical significance and biological mechanism of H. pylori EVs on the pathogenesis of gastric malignancy. We performed 16S rDNA-based metagenomic analysis of gastric juices either from endoscopic or surgical patients. From each sample of gastric juices, the bacteria and EVs were isolated. We evaluated the role of H. pylori EVs on the development of gastric inflammation in vitro and in vivo. IVIS spectrum and confocal microscopy were used to examine the distribution of EVs. The metagenomic analyses of the bacteria and EVs showed that Helicobacter and Streptococcus are the two major bacterial genera, and they were significantly increased in abundance in gastric cancer (GC) patients. H. pylori EVs are spherical and contain CagA and VacA. They can induce the production of tumor necrosis factor-α, interleukin (IL)-6 and IL-1β by macrophages, and IL-8 by gastric epithelial cells. Also, EVs induce the expression of interferon gamma, IL-17 and EV-specific immunoglobulin Gs in vivo in mice. EVs were shown to infiltrate and remain in the mouse stomach for an extended time. H. pylori EVs, which are abundant in the gastric juices of GC patients, can induce inflammation and possibly cancer in the stomach, mainly via the production of inflammatory mediators from gastric epithelial cells after selective uptake by the cells.

It has frequently been hypothesized that the helical body shapes of flagellated bacteria may yield some advantage in swimming ability. In particular, the helical-shaped pathogen Helicobacter pylori is often claimed to swim like a corkscrew through its harsh gastric habitat, but there has been no direct confirmation or quantification of such claims. Using fast time-resolution and high-magnification two-dimensional (2D) phase-contrast microscopy to simultaneously image and track individual bacteria in bacterial broth as well as mucin solutions, we show that both helical and rod-shaped H. pylori rotated as they swam, producing a helical trajectory. Cell shape analysis enabled us to determine shape as well as the rotational and translational speed for both forward and reverse motions, thereby inferring flagellar kinematics. Using the method of regularized Stokeslets, we directly compare observed speeds and trajectories to numerical calculations for both helical and rod-shaped bacteria in mucin and broth to validate the numerical model. Although experimental observations are limited to select cases, the model allows quantification of the effects of body helicity, length, and diameter. We find that due to relatively slow body rotation rates, the helical shape makes at most a 15% contribution to propulsive thrust. The effect of body shape on swimming speeds is instead dominated by variations in translational drag required to move the cell body. Because helical cells are one of the strongest candidates for propulsion arising from the cell body, our results imply that quite generally, swimming speeds of flagellated bacteria can only be increased a little by body propulsion.

Background: Proton-pump inhibitor (PPI) consumption does lead to false-negative results of Helicobacter pylori diagnostic tests such as biopsy culture and rapid urease test (RUT)., Materials and Methods: Helicobacter pylori isolates from 112 dyspeptic patients with (56.5%) or without (43.5%) PPI consumption were recruited for examining the negative effects of omeprazole (OMP), lansoprazole (LPZ), and pantoprazole (PAN) on H. pylori viability, morphology, and urease, in vitro. The effect of a sublethal concentration of OMP on bacterial features and their recovery after removal of OMP was also assessed., Results: Of 112 culture-positive gastric biopsies, 87.5% were RUT positive and 12.5% RUT negative. There was a significant correlation between negative RUT results and PPI consumption (p < .05). OMP (minimum inhibitory concentration, MIC 32 μg/mL) and LPZ (MIC 8 μg/mL) inhibited the growth of 78.6% of H. pylori isolates. OMP and LPZ inhibited urease of 90.3% of isolates between 0 and 40 minutes and 54.4% between 20 and 40 minutes, respectively. PAN did not inhibit H. pylori growth and urease. Three 3-day (9 days) consecutive subcultures of H. pylori on brucella blood agar (BBA) supplemented with OMP resulted in reduced bacterial viability (1+), compared with control (4+), change of spiral morphology to coccoid, and reduction in pink color intensity in urea agar. Bacterial growth (1+), morphology, and urease test did not improve after the first 3-day and second 3-day (6 days) subcultures on BBA. However, relative recovery occurred after the third 3-day (9 days) subculture and complete recovery was observed after the fourth 3-day (12 days) subculture, as confluent growth (4+), 100% spiral cells, and strong urease test., Conclusion: Proton-pump Inhibitors exert transient negative effects on H. pylori viability, morphology, and urease test. Accordingly, cessation of PPI consumption at least 12 days before endoscopy could help avoiding false-negative results of H. pylori diagnostic tests., (© 2015 John Wiley & Sons Ltd.)

Purpose: Helicobacter pylori infection and a high dietary salt intake are each risk factors for the development of gastric cancer. We hypothesize that changes in environmental salt concentrations lead to alterations in the H. pylori membrane proteome., Experimental Design: Label-free and iTRAQ methods were used to identify H. pylori proteins that change in abundance in response to alterations in environmental salt concentrations. In addition, we biotinylated intact bacteria that were grown under high- or low-salt conditions, and thereby analyzed salt-induced changes in the abundance of surface-exposed proteins., Results: Proteins with increased abundance in response to high salt conditions included CagA, the outer membrane protein HopQ, and fibronectin domain-containing protein HP0746. Proteins with increased abundance in response to low salt conditions included VacA, two VacA-like proteins (ImaA and FaaA), outer-membrane iron transporter FecA3, and several proteins involved in flagellar activity. Consistent with the proteomic data, bacteria grown in high salt conditions exhibited decreased motility compared to bacteria grown in lower salt conditions., Conclusion and Clinical Relevance: Alterations in the H. pylori membrane proteome in response to high salt conditions may contribute to the increased risk of gastric cancer associated with a high salt diet., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Helicobacter pylori causes gastrointestinal diseases, including gastric cancer. Its high motility in the viscous gastric mucosa facilitates colonization of the human stomach and depends on the helical cell shape and the flagella. In H. pylori, Csd6 is one of the cell shape-determining proteins that play key roles in alteration of cross-linking or by trimming of peptidoglycan muropeptides. Csd6 is also involved in deglycosylation of the flagellar protein FlaA. To better understand its function, biochemical, biophysical, and structural characterizations were carried out. We show that Csd6 has a three-domain architecture and exists as a dimer in solution. The N-terminal domain plays a key role in dimerization. The middle catalytic domain resembles those of l,d-transpeptidases, but its pocket-shaped active site is uniquely defined by the four loops I to IV, among which loops I and III show the most distinct variations from the known l,d-transpeptidases. Mass analyses confirm that Csd6 functions only as an l,d-carboxypeptidase and not as an l,d-transpeptidase. The d-Ala-complexed structure suggests possible binding modes of both the substrate and product to the catalytic domain. The C-terminal nuclear transport factor 2-like domain possesses a deep pocket for possible binding of pseudaminic acid, and in silico docking supports its role in deglycosylation of flagellin. On the basis of these findings, it is proposed that H. pylori Csd6 and its homologs constitute a new family of l,d-carboxypeptidase. This work provides insights into the function of Csd6 in regulating the helical cell shape and motility of H. pylori., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: Helicobacter pylori infection has been consistently associated with lack of access to clean water and proper sanitation, but no studies have demonstrated that the transmission of H. pylori can occur from drinking contaminated water. In this study, we used a laboratory mouse model to test whether waterborne H. pylori could cause gastric infection., Materials and Methods: Groups of immunocompetent C57/BL6 Helicobacter-free mice were exposed to static concentrations (1.29 × 10(5), 10(6), 10(7), 10(8), and 10(9) CFU/L) of H. pylori in their drinking water for 4 weeks. One group of Helicobacter-free mice was exposed to uncontaminated water as a negative control. H. pylori morphology changes in water were examined using microscopy Live/Dead staining. Following exposure, H. pylori infection and inflammation status in the stomach were evaluated using quantitative culture, PCR, the rapid urease test, and histology., Results: None of the mice in the negative control or 10(5) groups were infected. One of 20 cages (one of 40 mice) of the 10(6) group, three of 19 cages (four of 38 mice) of the 10(7) CFU/L group, 19 of 20 cages (33 of 40 mice) of the 10(8) group, and 20 of 20 cages (39 of 40 mice) of the 10(9) CFU/L group were infected. Infected mice had significantly higher gastric inflammation than uninfected mice (27.86% higher inflammation, p < .0001)., Conclusions: We offer proof that H. pylori in water is infectious in mice, suggesting that humans drinking contaminated water may be at risk of contracting H. pylori infection. Much work needs to be performed to better understand the risk of infection from drinking H. pylori-contaminated water., (© 2015 John Wiley & Sons Ltd.)

Chemotaxis is important for Helicobacter pylori to colonize the stomach. Like other bacteria, H. pylori uses chemoreceptors and conserved chemotaxis proteins to phosphorylate the flagellar rotational response regulator, CheY, and modulate the flagellar rotational direction. Phosphorylated CheY is returned to its non-phosphorylated state by phosphatases such as CheZ. In previously studied cases, chemotaxis phosphatases localize to the cellular poles by interactions with either the CheA chemotaxis kinase or flagellar motor proteins. We report here that the H. pylori CheZ, CheZ(HP), localizes to the poles independently of the flagellar motor, CheA, and all typical chemotaxis proteins. Instead, CheZ(HP) localization depends on the chemotaxis regulatory protein ChePep, and reciprocally, ChePep requires CheZ(HP) for its polar localization. We furthermore show that these proteins interact directly. Functional domain mapping of CheZ(HP) determined the polar localization motif lies within the central domain of the protein and that the protein has regions outside of the active site that participate in chemotaxis. Our results suggest that CheZ(HP) and ChePep form a distinct complex. These results therefore suggest the intriguing idea that some phosphatases localize independently of the other chemotaxis and motility proteins, possibly to confer unique regulation on these proteins' activities., (© 2015 John Wiley & Sons Ltd.)

Helicobacter pylori (H. pylori) infection is highly prevalent, and has a strong association with various gastric diseases, including gastritis, digestive ulcers, and cancer. H. pylori strains with resistance to existing antibiotics have emerged in the past two decades. Currently, treatment of H. pylori infection (involving the use of proton pump inhibitors, followed by triple therapy with broad-spectrum antibiotics) is suboptimal, with high failure rates. As such, there is a clear need for new approaches against H. pylori. Here, we report that Epinecidin-1 (Epi-1) shows effective bactericidal activity against H. Pylori in vitro, and modulates H. Pylori-induced host immune responses in a mouse model. Epi-1 exhibited a low minimum inhibitory concentration (MIC) against antibiotic-sensitive and clinical antibiotic-resistant strains. Moreover, Epi-1 treatment caused 1-N-phenylnaphthylamine (NPN)-fluorescent probe uptake, suggesting it induced membrane lysis; transmission electron micrographs revealed that membranes were destabilized by the generation of saddle-splay membrane curvature. Oral administration of Epi-1 (quaque die dose) in a mouse infection model had strong efficacy (p < 0.00152) against H. pylori, as compared with conventional proton pump inhibitor (PPI)-triple therapeutic antibiotics. Epi-1 inhibited infection through in vivo depletion of CD4+-FOXP3+ T Regulatory and Th17 subset populations, and aided in clearance of persistent H. pylori colonization. Flow cytometry and gene expression analysis of mouse splenic and gastric tissue indicated that Epi-1 inhibits IL-10, and thereby affects FOXP3 expression levels and reduces pro-inflammatory cytokine responses. Crucially, high doses of Epi-1 did not exert toxic effects in oral, dermal, and eye irritation models. Collectively, our results suggest that Epi-1 may be a promising, effective, and safe monotherapeutic agent for the treatment of multi-drug resistant H. pylori infection., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Helicobacter pylori strains that harbor the oncoprotein CagA increase gastric cancer risk, and this risk is augmented under iron-deficient conditions. We demonstrate here that iron depletion induces coccoid morphology in strains lacking cagA. To evaluate the stability of augmented H. pylori virulence phenotypes stimulated by low-iron conditions, H. pylori isolated from iron-depleted conditions in vivo were serially passaged in vitro. Long-term passage decreased the ability of hypervirulent strains to translocate CagA or induce interleukin 8, indicating that hypervirulent phenotypes stimulated by low-level iron conditions are reversible. Therefore, rectifying iron deficiency may attenuate disease among H. pylori-infected persons with no response to antibiotics., (© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Dual-modal fluorescent magnetic glyconanoparticles have been prepared and shown to be powerful in probing lectins displayed on pathogenic and mammalian cell surfaces. Blood group H1- and Le(b)-conjugated nanoparticles were found to bind to BabA displaying Helicobacter pylori, and Le(a)- and Le(b)-modified nanoparticles are both recognized by and internalized into DC-SIGN and SIGN-R1 expressing mammalian cells via lectin-mediated endocytosis. In addition, glyconanoparticles block adhesion of H. pylori to mammalian cells, suggesting that they can serve as inhibitors of infection of host cells by this pathogen. It has been also shown that owing to their magnetic properties, glyconanoparticles are useful tools to enrich lectin expressing cells. The combined results indicate that dual-modal glyconanoparticles are biocompatible and that they can be employed in lectin-associated biological studies and biomedical applications.

Peptidoglycan modifying carboxypeptidases (CPs) are important determinants of bacterial cell shape. Here, we report crystal structures of Csd4, a three-domain protein from the human gastric pathogen Helicobacter pylori. The catalytic zinc in Csd4 is coordinated by a rare His-Glu-Gln configuration that is conserved among most Csd4 homologs, which form a distinct subfamily of CPs. Substitution of the glutamine to histidine, the residue found in prototypical zinc carboxypeptidases, resulted in decreased enzyme activity and inhibition by phosphate. Expression of the histidine variant at the native locus in a H. pylori csd4 deletion strain did not restore the wild-type helical morphology. Biochemical assays show that Csd4 can cleave a tripeptide peptidoglycan substrate analog to release m-DAP. Structures of Csd4 with this substrate analog or product bound at the active site reveal determinants of peptidoglycan specificity and the mechanism to cleave an isopeptide bond to release m-DAP. Our data suggest that Csd4 is the archetype of a new CP subfamily with a domain scheme that differs from this large family of peptide-cleaving enzymes., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

More than 50% of the world's population is infected with Helicobacter pylori (H. pylori) - a Gram negative bacterium, which persists in the human stomach and duodenum, causing gastric or duodenal ulcers and gastric cancer. The majority of H. pylori cells demonstrate rod-shape morphology occurring in two subtypes: spiral and S-shaped. Both are isolated from mucus layer biopsy specimens of the stomach or from short-term cultures. However, results obtained from electron microscopy revealed that H. pylori long-term cultures not only consist of classic, spiral shaped bacteria, but also contain alternative forms of coccoid cells. Further investigations showed that coccoid forms of H. pylori may be divided into two types: viable but non-culturable coccus (VBnC) and a degenerative form, coccoid stage which is probably the effect of bacterial cell death. Transformation from spiral to coccoid form is induced under stress conditions, such as the presence of antibiotics. But still there is no evidence for reversion from the coccoid state to the viable and infectious spiral form. Besides the planktonic form, H. pylori also forms homo-, and heterogenic biofilms, which may constitute a potential environmental reservoir of this bacterium. The antigenic repertoire and the immunomodulatory and infectious properties of different H. pylori forms differ greatly. The variation in those properties suggests that morphological forms of H. pylori are potentially involved in the transmission of the infection. This review presents recent findings on the variability, antigenicity and infectious properties of H. pylori morphological forms and their potential role in the transmission of the infection.

We followed the position of the replication complex in the pathogenic bacterium Helicobacter pylori using antibodies raised against the single-stranded DNA binding protein (HpSSB) and the replicative helicase (HpDnaB). The position of the replication origin, oriC, was also localized in growing cells by fluorescence in situ hybridization (FISH) with fluorescence-labeled DNA sequences adjacent to the origin. The replisome assembled at oriC near one of the cell poles, and the two forks moved together toward the cell center as replication progressed in the growing cell. Termination and resolution of the forks occurred near midcell, on one side of the septal membrane. The duplicated copies of oriC did not separate until late in elongation, when the daughter chromosomes segregated into bilobed nucleoids, suggesting sister chromatid cohesion at or near the oriC region. Components of the replication machinery, viz., HpDnaB and HpDnaG (DNA primase), were found associated with the cell membrane. A model for the assembly and location of the H. pylori replication machinery during chromosomal duplication is presented.

A multifunctional compound that can prevent early gastric cancer is produced by intercalating 3.20% and 1.64% of 5-FU into the interlayer of montmorillonite (MMT) and attapulgite (At), respectively. A low molecular weight cationic polymer, polyethylenimine (PEI1200), is incorporated into the surface of the 5-FU-MMT and 5-FU-At to form the multifunctional layered silicate (NLS). The chemical structure and surface morphology of the NLS are characterized and the model drug of 5-FU is intercalated into the MMT and At. The cell viability determined by the MTT assay on the BGC-823 cell lines show that over 80% of the cells are live under the experimental conditions. The PEI-5-FU-MMT and PEI-5-FU-At can carry the report gene to the BGC-823 and COS-7 cell lines efficiently. Western blotting assay shows that the pTrail protein of the BGC-823 cell lines treated with PEI-5-FU-MMT/pTrail and PEI-5-FU-At/pTrail is up-regulated, whereas the cFLIP protein is down-regulated at 48 h compared to free 5-FU, PEI1200, MMT, and At, providing evidence that the NLS can increase the sensitivity of pTrail gene and improve the effects of pTrail gene therapy. Moreover, the Helicobacter pylori (HP) bacteria are adsorbed and immobilized efficiently on the surface of the NLS according to the LIVE/DEAD(®) BacLight™ Bacterial Viability Kit in the confocal fluorescence analysis. The histochemical analyses provide evidence that NLS/pTrail can prevent early gastric mucosa effectively., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Helicobacter pylori is an adapted gastric pathogen that colonizes the human stomach, causing severe gastritis and gastric cancer. A hallmark of infection is the ability of this organism to evade detection by the human immune system. H. pylori has evolved a number of features to achieve this, many of which involve glyco-conjugates including the lipopolysaccharide, peptidoglycan layer, glycoproteins, and glucosylated cholesterol. These major bacterial components possess unique features from those of other gram-negative organisms, including differences in structure, assembly, and modification. These defining characteristics of H. pylori glycobiology help the pathogen establish a long-lived infection by providing camouflage, modulating the host immune response, and promoting virulence mechanisms. In this way, glyco-conjugates are essential for H. pylori pathogenicity and survival, allowing it to carve out a niche in the formidable environment of the human stomach.

The helical cell shape of Helicobacter pylori is highly conserved and contributes to its ability to swim through and colonize the viscous gastric mucus layer. A multi-faceted peptidoglycan (PG) modification programme involving four recently characterized peptidases and two accessory proteins is essential for maintaining H. pylori's helicity. To expedite identification of additional shape-determining genes, we employed flow cytometry with fluorescence-activated cell sorting (FACS) to enrich a transposon library for bacterial cells with altered light scattering profiles that correlate with perturbed cell morphology. After a single round of sorting, 15% of our clones exhibited a stable cell shape defect, reflecting 37-fold enrichment. Sorted clones with straight rod morphology contained insertions in known PG peptidases, as well as an insertion in csd6, which we demonstrated has ld-carboxypeptidase activity and cleaves monomeric tetrapeptides in the PG sacculus, yielding tripeptides. Other mutants had only slight changes in helicity due to insertions in genes encoding MviN/MurJ, a protein possibly involved in initiating PG synthesis, and the hypothetical protein HPG27&#95;782. Our findings demonstrate FACS robustly detects perturbations of bacterial cell shape and identify additional PG peptide modifications associated with helical cell shape in H. pylori., (© 2013 John Wiley & Sons Ltd.)

Helicobacter pylori colonizes the gastric mucosa of half of the worlds population and persistent infection is related with an increase in the risk of gastric cancer. Adhesion of H. pylori to the gastric epithelium, which is essential for infection, is mediated by bacterial adhesin proteins that recognize specific glycan structures (Gly-R) expressed in the gastric mucosa. The blood group antigen binding adhesin (BabA) recognizes difucosylated antigens such as Lewis B (Leb), while the sialic acid binding adhesin (SabA) recognizes sialylated glycoproteins and glycolipids, such as sialyl-Lewis x (sLex). This work aimed to investigate whether these Gly-Rs (Leb and sLex) can attract and specifically bind H. pylori after immobilization on synthetic surfaces (self-assembled monolayers (SAMs) of alkanethiols on gold). Functional bacterial adhesion assays for (Gly-R)-SAMs were performed using H. pylori strains with different adhesin protein profiles. The results demonstrate that H. pylori binding to surfaces occurs via interaction between its adhesins and cognate (Gly-R)-SAMs and bound H. pylori maintains its characteristic rod-shaped morphology only during conditions of specific adhesin-glycan binding. These results offer new insights into innovative strategies against H. pylori infection based on the scavenging of bacteria from the stomach using specific H. pylori chelating biomaterials., (Copyright © 2013 Acta Materialia Inc. All rights reserved.)

We report a novel pH-responsive gold nanoparticle-stabilized liposome system for gastric antimicrobial delivery. By adsorbing small chitosan-modified gold nanoparticles (diameter ~10 nm) onto the outer surface of negatively charged phospholipid liposomes (diameter ~75 nm), we show that at gastric pH the liposomes have excellent stability with limited fusion ability and negligible cargo releases. However, when the stabilized liposomes are present in an environment with neutral pH, the gold stabilizers detach from the liposomes, resulting in free liposomes that can actively fuse with bacterial membranes. Using Helicobacter pylori as a model bacterium and doxycycline as a model antibiotic, we demonstrate such pH-responsive fusion activity and drug release profile of the nanoparticle-stabilized liposomes. Particularly, at neutral pH the gold nanoparticles detach, and thus the doxycycline-loaded liposomes rapidly fuse with bacteria and cause superior bactericidal efficacy as compared to the free doxycycline counterpart. Our results suggest that the reported liposome system holds a substantial potential for gastric drug delivery; it remains inactive (stable) in the stomach lumen but actively interacts with bacteria once it reaches the mucus layer of the stomach where the bacteria may reside.

In view of the world wide prevalence of Helicobacter pylori infection, its potentially serious consequences, and the increasing emergence of antibiotic resistant H. pylori strains there is an urgent need for the development of alternative strategies to combat the infection. In this study it has been demonstrated that polyethyleneimine (PEI) functionalized zinc oxide (ZnO) nanoparticles (NPs) inhibit the growth of a metronidazole-resistant strain of H. pylori and the molecular basis of the anti-bacterial activity of ZnO-PEI NP has been investigated. The ZnO-PEI NP was synthesized using a wet chemical method with a core size of approximately 3-7 nm. Internalization and distribution of ZnO-PEI NP without agglomeration was observed in H. pylori cytosol by electron microscopy. Several lines of evidence including scanning electron microscopy, propidium iodide uptake and ATP assay indicate severe membrane damage in ZnO-PEI NP treated H. pylori. Intracellular ROS generation increased rapidly following the treatment of H. pylori with ZnO-PEI NP and extensive degradation of 16S and 23S rRNA was observed by quantitative reverse-transcriptase PCR. Finally, considerable synergy between ZnO-PEI NP and antibiotics was observed and it has been demonstrated that the concentration of ZnO-PEI NP (20 µg/ml) that is non-toxic to human cells could be used in combination with sub-inhibitory concentrations of antibiotics for the inhibition of H. pylori growth.

In the model organism Escherichia coli, Min proteins are involved in regulating the division of septa formation. The computational genome analysis of Helicobacter pylori, a gram-negative microaerophilic bacterium causing gastritis and peptic ulceration, also identified MinC, MinD, and MinE. However, MinC (HP1053) shares a low identity with those of other bacteria and its function in H. pylori remains unclear. In this study, we used morphological and genetic approaches to examine the molecular role of MinC. The results were shown that an H. pylori mutant lacking MinC forms filamentous cells, while the wild-type strain retains the shape of short rods. In addition, a minC mutant regains the short rods when complemented with an intact minCHp gene. The overexpression of MinCHp in E. coli did not affect the growth and cell morphology. Immunofluorescence microscopy revealed that MinCHp forms helix-form structures in H. pylori, whereas MinCHp localizes at cell poles and pole of new daughter cell in E. coli. In addition, co-immunoprecipitation showed MinC can interact with MinD but not with FtsZ during mid-exponential stage of H. pylori. Altogether, our results show that MinCHp plays a key role in maintaining proper cell morphology and its function differs from those of MinCEc.

A. Pylori is a very undemanding microorganism needing the in support of complex of conditions including particular atmosphere, temperature of culturing and composition of growth medium. The two-phase growth medium is recommended to sub-culturing in Petri dishes with diameter of 90 mm. The growth medium consists of chocolate agar with addition of Schedler broth and enriched with 10% serum of cattle.

Of the various kinds of cell division, the most common mode is binary fission, the division of a cell into two morphologically identical daughter cells. However, in the case of asymmetric cell division, Caulobacter crescentus produces two morphologically and functionally distinct cell types. Here, we have studied cell cycle progression of the human pathogen Helicobacter pylori using a functional green fluorescent protein (GFP) fusion of FtsZ protein and membrane staining. In small cells, representing newly divided cells, FtsZ localizes to a single cell pole. During the cell cycle, spiral intermediates are formed until an FtsZ ring is positioned with very little precision, such that central as well as acentral rings can be observed. Daughter cells showed considerably different sizes, suggesting that H. pylori divides asymmetrically. Fluorescence recovery after photobleaching (FRAP) analyses demonstrate that the H. pylori FtsZ ring is about as dynamic as that of Escherichia coli but that polar assemblies show less turnover. Strikingly, our results demonstrate that H. pylori cell division follows a different route from that in E. coli and Bacillus subtilis. It is also different from that in C. crescentus, where cytokinesis regulation proteins like MipZ play a role. Therefore, this report provides the first cell-biological analysis of FtsZ dynamics in the human pathogen H. pylori and even in epsilonproteobacteria to our knowledge. In addition, analysis of the filament architecture of H. pylori and E. coli FtsZ filaments in the heterologous system of Drosophila melanogaster S2 Schneider cells revealed that both have different filamentation properties in vivo, suggesting a unique intrinsic characteristic of each protein.

Aims: To identify active phenolic constituents in muscadine grape skin (MGS) extracts and determine interactions among compounds while further exploring their anti-Helicobacter pylori potential in vitro., Methods and Results: The inhibitory effects of quercetin and resveratrol, active polyphenols identified in MGS extracts, against H. pylori were investigated. Quercetin and resveratrol significantly (P < 0.05) reduced H. pylori counts regardless of pH with minimal bactericidal concentrations of 256 and 128 μg ml(-1), respectively. MGS extracts displayed the highest efficacy, suggesting additional unidentified compounds not determined in this study. Time-course viability experiments showed a dose-dependent anti-H. pylori response to quercetin and resveratrol. Interestingly, neither quercetin nor resveratrol affected H. pylori outer membrane (OM) integrity as determined by 1-N-phenylnaphthylamine (NPN) uptake assays. However, treatment with MGS extract did increase NPN uptake, indicating OM destabilization possibly by additional unknown components. Furthermore, quercetin was found to enter H. pylori as measured by HPLC supporting intracellular drug accumulation., Conclusions: Quercetin and resveratrol possess strong anti-H. pylori activity in vitro and are independent of pH. Our results also suggest that these compounds do not affect H. pylori OM integrity as previously hypothesized and that the primary antimicrobial activity of quercetin may be linked to interactions with intracellular components., Significance and Impact of the Study: The anti-H. pylori effects of quercetin and resveratrol suggest that these compounds may be useful in the dietary prevention and/or treatment of H. pylori infection., (© 2013 The Society for Applied Microbiology.)

Helicobacter pylori (H. Pylori) is an actively dividing spiral bacterium that changes to coccoid morphology under stressful environments. The infectivity of the coccoids is still controversial. The aim of this study was to determine the viability and expression of two important virulence genes (babA and cagE), in antibiotic-induced coccoid forms. Three strains of H. pylori, the standard 26695 and two clinical isolates (p1, p2) were converted to coccoid form by amoxicillin. Coccoids were identified according to Gram-staining and microscopic morphology. The viability of the cells was analyzed by flow cytometry. The expression of cagE and babA in coccoid forms were evaluated and compared to the spirals by quantitative PCR assay. The coccoid forms were developed after 72 h exposure of H. pylori to ½ MIC of amoxicillin, and the conversion form was completed (100 %) at 144 h in all of three isolates. Flow cytometry analyses showed that the majority of the induced coccoids (90-99.9 %) were viable. Expression of cagE and babA was seen in coccoids; however, in lower rate (cagE, ~3-fold and babA, ~10-fold) than these in spiral forms. Coccoid forms of two clinical isolates significantly expressed higher rate of cagE and babA than standard 26695 strain (P = 0.01). These results suggest that the induced coccoid form of H. pylori is not a passive entity but can actively infect the human by expression of the virulence genes for long time in stomach and probably play a role in chronic and severe disease.

H. pylori colonizes half of the world's population leading to gastritis, ulcers and gastric cancer. H. pylori strains resistant to antibiotics are increasing which raises the need for alternative therapeutic approaches. Docosahexaenoic acid (DHA) has been shown to decrease H. pylori growth and its associated-inflammation through mechanisms poorly characterized. We aimed to explore DHA action on H. pylori-mediated inflammation and adhesion to gastric epithelial cells (AGS) and also to identify bacterial structures affected by DHA. H. pylori growth and metabolism was assessed in liquid cultures. Bacterial adhesion to AGS cells was visualized by transmission electron microscopy and quantified by an Enzyme Linked Immunosorbent Assay. Inflammatory proteins were assessed by immunoblotting in infected AGS cells, previously treated with DHA. Bacterial total and outer membrane protein composition was analyzed by 2-dimensional gel electrophoresis. Concentrations of 100 µM of DHA decreased H. pylori growth, whereas concentrations higher than 250 µM irreversibly inhibited bacteria survival. DHA reduced ATP production and adhesion to AGS cells. AGS cells infected with DHA pre-treated H. pylori showed a 3-fold reduction in Interleukin-8 (IL-8) production and a decrease of COX2 and iNOS. 2D electrophoresis analysis revealed that DHA changed the expression of H. pylori outer membrane proteins associated with stress response and metabolism and modified bacterial lipopolysaccharide phenotype. As conclusions our results show that DHA anti-H. pylori effects are associated with changes of bacteria morphology and metabolism, and with alteration of outer membrane proteins composition, that ultimately reduce the adhesion of bacteria and the burden of H. pylori-related inflammation.

Helicobacter pylori might be readily affected with photodynamic therapy (PDT) by weak wavelengths, because it has few repair genes. Recently, gastrointestinal endoscopy emitting specific wavelengths (narrowband imaging, NBI) has been developed for the early detection of tumors. Coincidentally, its wavelength (415 nm) is very similar to the wavelength (410 nm) that activates protoporphyrin IX (PpIX) as a photosensitizer (PS). Therefore, we studied in vitro PDT against H. pylori using NBI and conventional white light (WL) according to low or high concentration of PpIX along with exposure time. The bactericidal effects, the degree of oxidative DNA damage and membrane integrity of H. pylori after PDT were evaluated. In the control, the numbers of viable cells remained constant during the experiment. Viable cells after PDT using both endoscopic light irradiation, were decreased approximately 10(3) - 10(5) fold at low concentration of PpIX and below 0.80 × 10 at high concentration of PpIX. Only membrane damage after PDT was observed microscopically in H. pylori without DNA injury. Conclusively, either the bactericidal effect in high concentration or the decrease of bacterial loading in low concentration of PpIX, would be expected with PDT using endoscopic light (NBI or WL)., (Copyright © 2012 Elsevier B.V. All rights reserved.)

We propose two antigenic types of Helicobacter pylori lipopolysaccharides (LPS): highly antigenic epitope-carrying LPS (HA-LPS) and weakly antigenic epitope-carrying LPS (WA-LPS) based on human serum reactivity. Strains carrying WA-LPS are highly prevalent in isolates from gastric cancer patients. WA-LPS exhibits more potent biological activities compared to HA-LPS, namely, upregulation of Toll-like receptor 4 (TLR4) expression and induction of enhanced epithelial cell proliferation. The results of competitive binding assays using monosaccharides and methylglycosides, as well as binding assays using glycosidase-treated LPS, suggested that β-linked N-acetyl-D-glucosamine and β-linked D-galactose residues largely contributed to the highly antigenic epitope and the weakly antigenic epitope, respectively. WA-LPS exhibited greater binding activity to surfactant protein D (SP-D) in a Ca(2+)-dependent manner, and this interaction was inhibited by methyl-β-D-galactoside. The biological activities of WA-LPS were markedly enhanced by the addition of SP-D. Lines of evidence suggested that removal of β-N-acetyl-D-glucosamine residue, which comprises the highly antigenic epitope, results in exposure of the weakly antigenic epitope. The weakly antigenic epitope interacted preferentially with SP-D, and SP-D enhanced the biological activity of WA-LPS.