In VitroandIn VivoActivities of HPi1, a Selective Antimicrobial against Helicobacter pylori

ABSTRACTA high-throughput screen (HTS) was performed to identify molecules specifically active againstHelicobacter pylori, the causative agent of peptic ulcer and gastric carcinoma. Currently, treatment ofH. pyloriinfection is suboptimal, with failure rates approaching 25%, despite triple therapy with two broad-spectrum antibiotics and a proton pump inhibitor or quadruple therapy with added bismuth. The HTS was performed in 384-well plates, and reduction of the metabolic indicator resazurin was used as a reporter for cell growth. Diverse molecules from commercial sources were identified as hits, andin vitrovalidations included measurements of MIC and time-dependent killing as well as anaerobic susceptibility testing against a panel of gut microbes.In vivovalidation included testing in the mouse model ofH. pyloriinfection. The small molecule HPi1 (3-hydrazinoquinoxaline-2-thiol) had excellent potency, with an MIC of 0.08 to 0.16 μg/ml and good selectivity forH. pyloricompared to a panel of commensal bacteria. HPi1 was also effective in a mouse model ofH. pyloriinfection, reducing colony counts to below the limit of detection after oral dosing of 25 mg/kg/day for 3 days. HPi1 is a promising lead in the search for more effective and specificH. pyloritherapeutics.

Download Full-text

In Vitro and In Vivo Antibacterial Activities of Patchouli Alcohol, a Naturally Occurring Tricyclic Sesquiterpene, against Helicobacter pylori Infection

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00122-17 ◽

2017 ◽

Vol 61 (6) ◽

Cited By ~ 16

Author(s):

Y. F. Xu ◽

D. W. Lian ◽

Y. Q. Chen ◽

Y. F. Cai ◽

Y. F. Zheng ◽

...

Keyword(s):

Helicobacter Pylori ◽

Bacterial Resistance ◽

Clinical Isolates ◽

Potential Candidate ◽

Necrosis Factor Alpha ◽

Content Type ◽

Patchouli Alcohol ◽

H Pylori

ABSTRACT This study further evaluated the in vitro and in vivo anti-Helicobacter pylori activities and potential underlying mechanism of patchouli alcohol (PA), a tricyclic sesquiterpene. In the in vitro assay, the capacities of PA to inhibit and kill H. pylori were tested on three standard strains at different pH values and on 12 clinical isolates. The effects of PA on H. pylori adhesion (and its alpA, alpB, and babA genes), motility (and its flaA and flaB genes), ultrastructure, and flagellation were investigated. Moreover, the H. pylori resistance to and postantibiotic effect (PAE) of PA were determined. Furthermore, the in vivo effects of PA on H. pylori eradication and gastritis were examined. Results showed that MICs of PA against three standard strains (pH 5.3 to 9) and 12 clinical isolates were 25 to 75 and 12.5 to 50 μg/ml, respectively. The killing kinetics of PA were time and concentration dependent, and its minimal bactericidal concentrations (MBCs) were 25 to 75 μg/ml. In addition, H. pylori adhesion, motility, ultrastructure, and flagellation were significantly suppressed. PA also remarkably inhibited the expression of adhesion genes (alpA and alpB) and motility genes (flaA and flaB). Furthermore, PA treatment caused a longer PAE and less bacterial resistance than clarithromycin and metronidazole. The in vivo study showed that PA can effectively eradicate H. pylori, inhibit gastritis, and suppress the expression of inflammatory mediators (COX-2, interleukin 1β, tumor necrosis factor alpha, and inducible nitric oxide synthase [iNOS]). In conclusion, PA can efficiently kill H. pylori, interfere with its infection process, and attenuate gastritis with less bacterial resistance, making it a potential candidate for new drug development.

Download Full-text

Hydrogen Peroxide-Mediated Oxygen Enrichment Eradicates Helicobacter pylori In Vitro and In Vivo

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02192-19 ◽

2020 ◽

Vol 64 (5) ◽

Author(s):

Jia Di ◽

Jun Zhang ◽

Lei Cao ◽

Ting-ting Huang ◽

Jun-xia Zhang ◽

...

Keyword(s):

Hydrogen Peroxide ◽

Helicobacter Pylori ◽

Bacterial Cell ◽

Cell Membranes ◽

Enriched Environment ◽

Mongolian Gerbils ◽

Content Type ◽

H Pylori

ABSTRACT Helicobacter pylori is an important risk factor for gastric ulcers. However, antibacterial therapies increase the resistance rate and decrease the eradication rate of H. pylori. Inspired by the microaerophilic characteristics of H. pylori, we aimed at effectively establishing an oxygen-enriched environment to eradicate and prevent the recurrence of H. pylori. The effect and the mechanism of an oxygen-enriched environment in eradicating H. pylori and preventing the recurrence were explored in vitro and in vivo. During oral administration and after drug withdrawal, H. pylori counts were evaluated by Giemsa staining in animal cohorts. An oxygen-enriched environment in which H. pylori could not survive was successfully established by adding hydrogen peroxide into several solutions and rabbit gastric juice. Hydrogen peroxide effectively killed H. pylori in Columbia blood agar and special peptone broth. Minimum inhibition concentrations and minimum bactericidal concentrations of hydrogen peroxide were both relatively stable after promotion of resistance for 30 generations, indicating that hydrogen peroxide did not easily promote resistance in H. pylori. In models of Mongolian gerbils and Kunming mice, hydrogen peroxide has been shown to significantly eradicate and effectively prevent the recurrence of H. pylori without toxicity and damage to the gastric mucosa. The mechanism of hydrogen peroxide causing H. pylori death was related to the disruption of bacterial cell membranes. The oxygen-enriched environment achieved by hydrogen peroxide eradicates and prevents the recurrence of H. pylori by damaging bacterial cell membranes. Hydrogen peroxide thus provides an attractive candidate for anti-H. pylori treatment.

Download Full-text

In VivoExpression of Helicobacter pylori Virulence Genes in Patients with Gastritis, Ulcer, and Gastric Cancer

Infection and Immunity ◽

10.1128/iai.05845-11 ◽

2011 ◽

Vol 80 (2) ◽

pp. 594-601 ◽

Cited By ~ 12

Author(s):

Francisco Avilés-Jiménez ◽

Adriana Reyes-Leon ◽

Erik Nieto-Patlán ◽

Lori M. Hansen ◽

Juan Burgueño ◽

...

Keyword(s):

Gastric Cancer ◽

Helicobacter Pylori ◽

Environmental Conditions ◽

Virulence Genes ◽

Content Type ◽

Ulcer Disease ◽

Type Iv ◽

H Pylori

ABSTRACTThe best-studiedHelicobacter pylorivirulence factor associated with development of peptic ulcer disease or gastric cancer (GC) rather than asymptomatic nonatrophic gastritis (NAG) is thecagpathogenicity island (cagPAI), which encodes a type IV secretion system (T4SS) that injects the CagA oncoprotein into host epithelial cells. Here we used real-time reverse transcription-PCR (RT-PCR) to measure thein vivoexpression of genes on thecagPAI and of other virulence genes in patients with NAG, duodenal ulcer (DU), or GC.In vivoexpression ofH. pylorivirulence genes was greater overall in gastric biopsy specimens of patients with GC than in those of patients with NAG or DU. However, sincein vitroexpression ofcagAwas not greater inH. pyloristrains from patients with GC than in those from patients with NAG or DU, increased expression in GCin vivois likely a result of environmental conditions in the gastric mucosa, though it may in turn cause more severe pathology. Increased expression of virulence genes in GC may represent a stress response to elevated pH or other environmental conditions in the stomach of patients with GC, which may be less hospitable toH. pyloricolonization than the acidic environment in patients with NAG or DU.

Download Full-text

The Oligo-Acyl Lysyl Antimicrobial Peptide C12K-2β12Exhibits a Dual Mechanism of Action and Demonstrates StrongIn VivoEfficacy against Helicobacter pylori

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00689-11 ◽

2011 ◽

Vol 56 (1) ◽

pp. 378-390 ◽

Cited By ~ 20

Author(s):

Morris O. Makobongo ◽

Hanan Gancz ◽

Beth M. Carpenter ◽

Dennis P. McDaniel ◽

D. Scott Merrell

Keyword(s):

Helicobacter Pylori ◽

Mechanism Of Action ◽

Mongolian Gerbils ◽

Nucleic Acid Binding ◽

Western Blots ◽

Content Type ◽

Bacterial Membranes ◽

H Pylori

ABSTRACTHelicobacter pylorihas developed antimicrobial resistance to virtually all current antibiotics. Thus, there is a pressing need to develop new anti-H. pyloritherapies. We recently described a novel oligo-acyl-lysyl (OAK) antimicrobial peptidomimetic, C12K-2β12, that shows potentin vitrobactericidal activity againstH. pylori. Herein, we define the mechanism of action and evaluate thein vivoefficacy of C12K-2β12againstH. pyloriafter experimental infection of Mongolian gerbils. We demonstrate using a 1-N-phenylnaphthylamine (fluorescent probe) uptake assay and electron microscopy that C12K-2β12rapidly permeabilizes the bacterial membrane and creates pores that cause bacterial cell lysis. Furthermore, using nucleic acid binding assays, Western blots, and confocal microscopy, we show that C12K-2β12can cross the bacterial membranes into the cytoplasm and tightly bind to bacterial DNA, RNA, and proteins, a property that may result in inhibition of enzymatic activities and macromolecule synthesis. To define thein vivoefficacy of C12K-2β12,H. pylori-infected gerbils were orogastrically treated with increasing doses and concentrations of C12K-2β121 day or 1 week postinfection. The efficacy of C12K-2β12was strongest in animals that received the largest number of doses at the highest concentration, indicating dose-dependent activity of the peptide (P< 0.001 by analysis of variance [ANOVA]) regardless of the timing of the treatment with C12K-2β12. Overall, our results demonstrate a dual mode of action of C12K-2β12against theH. pylorimembrane and cytoplasmic components. Moreover, and consistent with the previously reportedin vitroefficacy, C12K-2β12shows significantin vivoefficacy againstH. pyloriwhen used as monotherapy. Therefore, OAK peptides may be a valuable resource for therapeutic treatment ofH. pyloriinfection.

Download Full-text

Basal Body Structures Differentially Affect Transcription of RpoN- and FliA-Dependent Flagellar Genes in Helicobacter pylori

Journal of Bacteriology ◽

10.1128/jb.02533-14 ◽

2015 ◽

Vol 197 (11) ◽

pp. 1921-1930 ◽

Cited By ~ 5

Author(s):

Jennifer Tsang ◽

Timothy R. Hoover

Keyword(s):

Helicobacter Pylori ◽

Basal Body ◽

Protein Interactions ◽

Transcript Levels ◽

Content Type ◽

Genes Encoding ◽

H Pylori ◽

Flagellar Biogenesis ◽

Flagellar Genes

ABSTRACTFlagellar biogenesis inHelicobacter pyloriis regulated by a transcriptional hierarchy governed by three sigma factors, RpoD (σ80), RpoN (σ54), and FliA (σ28), that temporally coordinates gene expression with the assembly of the flagellum. Previous studies showed that loss of flagellar protein export apparatus components inhibits transcription of flagellar genes. The FlgS/FlgR two-component system activates transcription of RpoN-dependent genes though an unknown mechanism. To understand better the extent to which flagellar gene regulation is coupled to flagellar assembly, we disrupted flagellar biogenesis at various points and determined how these mutations affected transcription of RpoN-dependent (flaBandflgE) and FliA-dependent (flaA) genes. The MS ring (encoded byfliF) is one of the earliest flagellar structures assembled. Deletion offliFresulted in the elimination of RpoN-dependent transcripts and an ∼4-fold decrease inflaAtranscript levels. FliH is a cytoplasmic protein that functions with the C ring protein FliN to shuttle substrates to the export apparatus. Deletions offliHand genes encoding C ring components (fliMandfliY) decreased transcript levels offlaBandflgEbut had little or no effect on transcript levels offlaA. Transcript levels offlaBandflgEwere elevated in mutants where genes encoding rod proteins (fliEandflgBC) were deleted, while transcript levels offlaAwas reduced ∼2-fold in both mutants. We propose that FlgS responds to an assembly checkpoint associated with the export apparatus and that FliH and one or more C ring component assist FlgS in engaging this flagellar structure.IMPORTANCEThe mechanisms used by bacteria to couple transcription of flagellar genes with assembly of the flagellum are poorly understood. The results from this study identified components of theH. pyloriflagellar basal body that either positively or negatively affect expression of RpoN-dependent flagellar genes. Some of these basal body proteins may interact directly with regulatory proteins that control transcription of theH. pyloriRpoN regulon, a hypothesis that can be tested by examining protein-protein interactionsin vitro.

Download Full-text

Systems Modeling of the Role of Interleukin-21 in the Maintenance of Effector CD4+ T Cell Responses during Chronic Helicobacter pylori Infection

mBio ◽

10.1128/mbio.01243-14 ◽

2014 ◽

Vol 5 (4) ◽

Cited By ~ 36

Author(s):

Adria Carbo ◽

Danyvid Olivares-Villagómez ◽

Raquel Hontecillas ◽

Josep Bassaganya-Riera ◽

Rupesh Chaturvedi ◽

...

Keyword(s):

Gastric Cancer ◽

Helicobacter Pylori ◽

T Cell ◽

Wild Type ◽

Content Type ◽

Interleukin 21 ◽

H Pylori ◽

Deficient Mice

ABSTRACTThe development of gastritis duringHelicobacter pyloriinfection is dependent on an activated adaptive immune response orchestrated by T helper (Th) cells. However, the relative contributions of the Th1 and Th17 subsets to gastritis and control of infection are still under investigation. To investigate the role of interleukin-21 (IL-21) in the gastric mucosa duringH. pyloriinfection, we combined mathematical modeling of CD4+T cell differentiation within vivomechanistic studies. We infected IL-21-deficient and wild-type mice withH. pyloristrain SS1 and assessed colonization, gastric inflammation, cellular infiltration, and cytokine profiles. ChronicallyH. pylori-infected IL-21-deficient mice had higherH. pyloricolonization, significantly less gastritis, and reduced expression of proinflammatory cytokines and chemokines compared to these parameters in infected wild-type littermates. Thesein vivodata were used to calibrate anH. pyloriinfection-dependent, CD4+T cell-specific computational model, which then described the mechanism by which IL-21 activates the production of interferon gamma (IFN-γ) and IL-17 during chronicH. pyloriinfection. The model predicted activated expression of T-bet and RORγt and the phosphorylation of STAT3 and STAT1 and suggested a potential role of IL-21 in the modulation of IL-10. Driven by our modeling-derived predictions, we found reduced levels of CD4+splenocyte-specifictbx21androrcexpression, reduced phosphorylation of STAT1 and STAT3, and an increase in CD4+T cell-specific IL-10 expression inH. pylori-infected IL-21-deficient mice. Our results indicate that IL-21 regulates Th1 and Th17 effector responses during chronicH. pyloriinfection in a STAT1- and STAT3-dependent manner, therefore playing a major role controllingH. pyloriinfection and gastritis.IMPORTANCEHelicobacter pyloriis the dominant member of the gastric microbiota in more than 50% of the world’s population.H. pyloricolonization has been implicated in gastritis and gastric cancer, as infection withH. pyloriis the single most common risk factor for gastric cancer. Current data suggest that, in addition to bacterial virulence factors, the magnitude and types of immune responses influence the outcome of colonization and chronic infection. This study uses a combined computational and experimental approach to investigate how IL-21, a proinflammatory T cell-derived cytokine, maintains the chronic proinflammatory T cell immune response driving chronic gastritis duringH. pyloriinfection. This research will also provide insight into a myriad of other infectious and immune disorders in which IL-21 is increasingly recognized to play a central role. The use of IL-21-related therapies may provide treatment options for individuals chronically colonized withH. pylorias an alternative to aggressive antibiotics.

Download Full-text

Enhanced fitness of a Helicobacter pylori babA mutant in a murine model

Infection and Immunity ◽

10.1128/iai.00725-20 ◽

2021 ◽

Author(s):

M. Lorena Harvey ◽

Aung Soe Lin ◽

Lili Sun ◽

Tatsuki Koyama ◽

Jennifer H. B. Shuman ◽

...

Keyword(s):

Helicobacter Pylori ◽

Outer Membrane Proteins ◽

Population Diversity ◽

Fitness Advantage ◽

Neutral Locus ◽

Mutant Strains ◽

Bacterial Fitness ◽

H Pylori

Helicobacter pylori genomes encode >60 predicted outer membrane proteins (OMPs). Several OMPs in the Hop family act as adhesins, but the functions of most Hop proteins are unknown. To identify hop mutant strains that exhibit altered fitness in vivo compared to fitness in vitro , we used a genetic barcoding method that allowed us to track changes in the proportional abundance of H. pylori strains within a mixed population. We generated a library of hop mutant strains, each containing a unique nucleotide barcode, as well as a library of control strains, each containing a nucleotide barcode in an intergenic region predicted to be a neutral locus unrelated to bacterial fitness. We orogastrically inoculated each of the libraries into mice and analyzed compositional changes in the populations over time in vivo compared to changes detected in the populations during library passage in vitro . The control library proliferated as a relatively stable community in vitro, but there was a reduction in the population diversity of this library in vivo and marked variation in the dominant strains recovered from individual animals, consistent with the existence of a non-selective bottleneck in vivo . We did not identify any OMP mutants exhibiting fitness defects exclusively in vivo without corresponding fitness defects in vitro . Conversely, a babA mutant exhibited a strong fitness advantage in vivo but not in vitro . These findings, when taken together with results of other studies, suggest that production of BabA may have differential effects on H. pylori fitness depending on the environmental conditions.

Download Full-text

Lactobacillus johnsonii La1 Attenuates Helicobacter pylori-Associated Gastritis and Reduces Levels of Proinflammatory Chemokines in C57BL/6 Mice

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.12.12.1378-1386.2005 ◽

2005 ◽

Vol 12 (12) ◽

pp. 1378-1386 ◽

Cited By ~ 56

Author(s):

Dionyssios N. Sgouras ◽

Effrosini G. Panayotopoulou ◽

Beatriz Martinez-Gonzalez ◽

Kalliopi Petraki ◽

Spyros Michopoulos ◽

...

Keyword(s):

Helicobacter Pylori ◽

Lamina Propria ◽

Serum Levels ◽

Favorable Effect ◽

Lactobacillus Johnsonii ◽

Ags Cells ◽

Inflammatory Infiltration ◽

H Pylori

ABSTRACT In clinical settings, Lactobacillus johnsonii La1 administration has been reported to have a favorable effect on Helicobacter pylori-associated gastritis, although the mechanism remains unclear. We administered, continuously through the water supply, live La1 to H. pylori-infected C57BL/6 mice and followed colonization, the development of H. pylori-associated gastritis in the lamina propria, and the levels of proinflammatory chemokines macrophage inflammatory protein 2 (MIP-2) and keratinocyte-derived cytokine (KC) in the serum and gastric tissue over a period of 3 months. We documented a significant attenuation in both lymphocytic (P = 0.038) and neutrophilic (P = 0.003) inflammatory infiltration in the lamina propria as well as in the circulating levels of anti-H. pylori immunoglobulin G antibodies (P = 0.003), although we did not observe a suppressive effect of La1 on H. pylori colonizing numbers. Other lactobacilli, such as L. amylovorus DCE 471 and L. acidophilus IBB 801, did not attenuate H. pylori-associated gastritis to the same extent. MIP-2 serum levels were distinctly reduced during the early stages of H. pylori infection in the La1-treated animals, as were gastric mucosal levels of MIP-2 and KC. Finally, we also observed a significant reduction (P = 0.046) in H. pylori-induced interleukin-8 secretion by human adenocarcinoma AGS cells in vitro in the presence of neutralized (pH 6.8) La1 spent culture supernatants, without concomitant loss of H. pylori viability. These observations suggest that during the early infection stages, administration of La1 can attenuate H. pylori-induced gastritis in vivo, possibly by reducing proinflammatory chemotactic signals responsible for the recruitment of lymphocytes and neutrophils in the lamina propria.

Download Full-text

Therapeutic effects of Zuojin Pill on Helicobacter pylori-induced chronic atrophic gastritis through JMJD2B/COX-2/VEGF signaling axis

10.21203/rs.3.rs-20652/v2 ◽

2020 ◽

Author(s):

Shihua Wu ◽

Chunmei Bao ◽

Ruilin Wang ◽

Xiaomei Zhang ◽

Sijia Gao ◽

...

Keyword(s):

Helicobacter Pylori ◽

Cell Viability ◽

Atrophic Gastritis ◽

Chronic Atrophic Gastritis ◽

Gastric Mucosal Damage ◽

Therapeutic Effects ◽

Cox 2 ◽

H Pylori

Abstract Background: Zuojin Pill (ZJP), a famous Chinese medicinal formula, widely accepted for treatment of chronic atrophic gastritis (CAG) in China. This study aimed to explore the therapeutic effects and mechanisms of ZJP in Helicobacter pylori (H. pylori) - induced chronic atrophic gastritis (CAG) in vivo and in vitro. Methods: CAG rat model was induced by H. pylori. ZJP (0.63, 1.26, and 2.52 g/kg, respectively) was administered orally for four weeks. Therapeutic effects of ZJP were identified by H&E staining and serum indices. In addition, cell viability, morphology and proliferation were detected by cell counting kit-8 (CCK8) and high-content screening assay (HCS), respectively. Moreover, relative mRNA expression and protein expression related to JMJD2B/COX-2/VEGF axis was detected to investigate the potential mechanisms of ZJP in CAG. Results: Results showed the symptoms (weight loss and gastric mucosa damage) of CAG were alleviated, and the contents of TNF-α in serum was markedly decreased after treating with ZJP. Moreover, cell viability, proliferation and morphology changes of GES-1 cells were ameliorated by ZJP intervention. In addition, proinflammatory genes and JMJD2B/COX-2/VEGF axis related genes were suppressed by ZJP administration in vitro and in vivo. Meanwhile, immunohistochemistry (IHC) and western blot confirmed down-regulation of these genes by ZJP intervention. Conclusion: ZJP treatment can alleviate gastric mucosal damage induced by H. pylori via JMJD2B/COX-2/VEGF axis.

Download Full-text

Evaluation of Antibiotic Treatment against “Candidatus Helicobacter suis” in a Mouse Model

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.49.11.4530-4535.2005 ◽

2005 ◽

Vol 49 (11) ◽

pp. 4530-4535 ◽

Cited By ~ 11

Author(s):

A. Hellemans ◽

A. Decostere ◽

F. Haesebrouck ◽

R. Ducatelle

Keyword(s):

Mouse Model ◽

Antibiotic Susceptibility ◽

Susceptibility Testing ◽

Bacterial Dna ◽

Urease Test ◽

Slaughter Pigs ◽

Cessation Of Treatment

ABSTRACT “Helicobacter heilmannii” (proposed name) type 1 colonizes the human stomach. It has been shown to be identical to“ Candidatus Helicobacter suis,” a Helicobacter species colonizing the stomachs of >60% of slaughter pigs. This bacterium has not been isolated in vitro until now. Antibiotic susceptibility testing of “Candidatus Helicobacter suis” has not been carried out so far. For the present study, a mouse model was adopted to evaluate the antibiotic susceptibility of this organism. Mice infected with“ Candidatus Helicobacter suis” were treated with amoxicillin and omeprazole, a therapy which is used to treat H. heilmannii infections in humans. Two different isolates of“ Candidatus Helicobacter suis” were tested. The excretion of bacterial DNA was assessed during treatment, using PCR on fecal samples. At the end of the experiment, 8 days after the cessation of treatment, the presence of infection was evaluated using a urease test and a PCR test on stomach samples. A marked decrease in the excretion of bacterial DNA was observed a few days after the onset of treatment, and the level remained low until the end of the experiment. A difference in susceptibility between the two“ Candidatus Helicobacter suis” isolates was pointed out. The in vivo mouse model infected with“ Candidatus Helicobacter suis” will be useful for further screening of potential therapeutic regimens.

Download Full-text