Curcumin Oxidation Is Required for Inhibition of Helicobacter pylori Growth, Translocation and Phosphorylation of Cag A

Curcumin is a potential natural remedy for preventing Helicobacter pylori-associated gastric inflammation and cancer. Here, we analyzed the effect of a phospholipid formulation of curcumin on H. pylori growth, translocation and phosphorylation of the virulence factor CagA and host protein kinase Src in vitro and in an in vivo mouse model of H. pylori infection. Growth of H. pylori was inhibited dose-dependently by curcumin in vitro. H. pylori was unable to metabolically reduce curcumin, whereas two enterobacteria, E. coli and Citrobacter rodentium, which efficiently reduced curcumin to the tetra- and hexahydro metabolites, evaded growth inhibition. Oxidative metabolism of curcumin was required for the growth inhibition of H. pylori and the translocation and phosphorylation of CagA and cSrc, since acetal- and diacetal-curcumin that do not undergo oxidative transformation were ineffective. Curcumin attenuated mRNA expression of the H. pylori virulence genes cagE and cagF in a dose-dependent manner and inhibited translocation and phosphorylation of CagA in gastric epithelial cells. H. pylori strains isolated from dietary curcumin-treated mice showed attenuated ability to induce cSrc phosphorylation and the mRNA expression of the gene encoding for IL-8, suggesting long-lasting effects of curcumin on the virulence of H. pylori. Our work provides mechanistic evidence that encourages testing of curcumin as a dietary approach to inhibit the virulence of CagA.

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Helicobacter pylori DnaB helicase can bypass Escherichia coli DnaC function in vivo

Biochemical Journal ◽

10.1042/bj20050062 ◽

2005 ◽

Vol 389 (2) ◽

pp. 541-548 ◽

Cited By ~ 28

Author(s):

Rajesh K. Soni ◽

Parul Mehra ◽

Gauranga Mukhopadhyay ◽

Suman Kumar Dhar

Keyword(s):

Escherichia Coli ◽

Helicobacter Pylori ◽

Temperature Sensitive ◽

Chromosomal Dna ◽

E Coli ◽

Dnab Helicase ◽

H Pylori

In Escherichia coli, DnaC is essential for loading DnaB helicase at oriC (the origin of chromosomal DNA replication). The question arises as to whether this model can be generalized to other species, since many eubacterial species fail to possess dnaC in their genomes. Previously, we have reported the characterization of HpDnaB (Helicobacter pylori DnaB) both in vitro and in vivo. Interestingly, H. pylori does not have a DnaC homologue. Using two different E. coli dnaC (EcdnaC) temperature-sensitive mutant strains, we report here the complementation of EcDnaC function by HpDnaB in vivo. These observations strongly suggest that HpDnaB can bypass EcDnaC activity in vivo.

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Helicobacter pylori rocF Is Required for Arginase Activity and Acid Protection In Vitro but Is Not Essential for Colonization of Mice or for Urease Activity

Journal of Bacteriology ◽

10.1128/jb.181.23.7314-7322.1999 ◽

1999 ◽

Vol 181 (23) ◽

pp. 7314-7322 ◽

Cited By ~ 81

Author(s):

David J. McGee ◽

Fiona J. Radcliff ◽

George L. Mendz ◽

Richard L. Ferrero ◽

Harry L. T. Mobley

Keyword(s):

Helicobacter Pylori ◽

Urease Activity ◽

Geometric Mean ◽

Acid Exposure ◽

Arginase Activity ◽

Gene Encoding ◽

Acid Sensitivity ◽

H Pylori

ABSTRACT Arginase of the Helicobacter pylori urea cycle hydrolyzes l-arginine to l-ornithine and urea.H. pylori urease hydrolyzes urea to carbon dioxide and ammonium, which neutralizes acid. Both enzymes are involved inH. pylori nitrogen metabolism. The roles of arginase in the physiology of H. pylori were investigated in vitro and in vivo, since arginase in H. pylori is metabolically upstream of urease and urease is known to be required for colonization of animal models by the bacterium. The H. pylori genehp1399, which is orthologous to the Bacillus subtilis rocF gene encoding arginase, was cloned, and isogenic allelic exchange mutants of three H. pylori strains were made by using two different constructs: 236-2 androcF::aphA3. In contrast to wild-type (WT) strains, all rocF mutants were devoid of arginase activity and had diminished serine dehydratase activity, an enzyme activity which generates ammonium. Compared with WT strain 26695 of H. pylori, the rocF::aphA3 mutant was ∼1,000-fold more sensitive to acid exposure. The acid sensitivity of the rocF::aphA3 mutant was not reversed by the addition of l-arginine, in contrast to the WT, and yielded a ∼10,000-fold difference in viability. Urease activity was similar in both strains and both survived acid exposure equally well when exogenous urea was added, indicating that rocF is not required for urease activity in vitro. Finally, H. pylorimouse-adapted strain SS1 and the 236-2 rocF isogenic mutant colonized mice equally well: 8 of 9 versus 9 of 11 mice, respectively. However, the rocF::aphA3 mutant of strain SS1 had moderately reduced colonization (4 of 10 mice). The geometric mean levels of H. pylori recovered from these mice (in log10 CFU) were 6.1, 5.5, and 4.1, respectively. Thus,H. pylori rocF is required for arginase activity and is crucial for acid protection in vitro but is not essential for in vivo colonization of mice or for urease activity.

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Helicobacter pylori Uptake and Efflux: Basis for Intrinsic Susceptibility to Antibiotics In Vitro

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.44.2.248-254.2000 ◽

2000 ◽

Vol 44 (2) ◽

pp. 248-254 ◽

Cited By ~ 77

Author(s):

J. E. Bina ◽

R. A. Alm ◽

M. Uria-Nickelsen ◽

S. R. Thomas ◽

T. J. Trust ◽

...

Keyword(s):

Helicobacter Pylori ◽

Outer Membrane ◽

Polymyxin B ◽

Intrinsic Resistance ◽

In Vitro Susceptibility ◽

E Coli ◽

Uptake Pathway ◽

H Pylori

ABSTRACT We previously demonstrated (M. M. Exner, P. Doig, T. J. Trust, and R. E. W. Hancock, Infect. Immun. 63:1567–1572, 1995) that Helicobacter pylori has at least one nonspecific porin, HopE, which has a low abundance in the outer membrane but forms large channels. H. pylori is relatively susceptible to most antimicrobial agents but less susceptible to the polycationic antibiotic polymyxin B. We demonstrate here that H. pylori is able to take up higher basal levels of the hydrophobic fluorescent probe 1-N-phenylnaphthylamine (NPN) thanPseudomonas aeruginosa or Escherichia coli, consistent with its enhanced susceptibility to hydrophobic agents. Addition of polymyxin B led to a further increase in NPN uptake, indicative of a self-promoted uptake pathway, but it required a much higher amount of polymyxin B to yield a 50% increase in NPN uptake inH. pylori (6 to 8 μg/ml) than in P. aeruginosa or E. coli (0.3 to 0.5 μg/ml), suggesting that H. pylori has a less efficient self-promoted uptake pathway. Since intrinsic resistance involves the collaboration of restricted outer membrane permeability and secondary defense mechanisms, such as periplasmic β-lactamase (which H. pylori lacks) or efflux, we examined the possible role of efflux in antibiotic susceptibility. We had previously identified in H. pylori 11637 the presence of portions of three genes with homology to potential restriction-nodulation-division (RND) efflux systems. It was confirmed that H. pylori contained only these three putative RND efflux systems, named here hefABC, hefDEF, andhefGHI, and that the hefGHI system was expressed only in vivo while the two other RND systems were expressed both in vivo and in vitro. In uptake studies, there was no observable energy-dependent tetracycline, chloramphenicol, or NPN efflux activity in H. pylori. Independent mutagenesis of the three putative RND efflux operons in the chromosome of H. pylori had no effect on the in vitro susceptibility of H. pylori to 19 antibiotics. These results, in contrast to what is observed inE. coli, P. aeruginosa, and other clinically important gram-negative bacteria, suggest that active efflux does not play a role in the intrinsic resistance of H. pylori to antibiotics.

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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.

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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.

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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.

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A Novel 3-Deoxy-d-manno-Octulosonic Acid (Kdo) Hydrolase That Removes the Outer Kdo Sugar of Helicobacter pylori Lipopolysaccharide

Journal of Bacteriology ◽

10.1128/jb.187.10.3374-3383.2005 ◽

2005 ◽

Vol 187 (10) ◽

pp. 3374-3383 ◽

Cited By ~ 34

Author(s):

Christopher Stead ◽

An Tran ◽

Donald Ferguson ◽

Sara McGrath ◽

Robert Cotter ◽

...

Keyword(s):

Helicobacter Pylori ◽

Lipid A ◽

In Vitro Assay ◽

Spectrometric Analysis ◽

Vitro Assay ◽

The Core ◽

H Pylori

ABSTRACT The lipid A domain anchors lipopolysaccharide (LPS) to the outer membrane and is typically a disaccharide of glucosamine that is both acylated and phosphorylated. The core and O-antigen carbohydrate domains are linked to the lipid A moiety through the eight-carbon sugar 3-deoxy-d-manno-octulosonic acid known as Kdo. Helicobacter pylori LPS has been characterized as having a single Kdo residue attached to lipid A, predicting in vivo a monofunctional Kdo transferase (WaaA). However, using an in vitro assay system we demonstrate that H. pylori WaaA is a bifunctional enzyme transferring two Kdo sugars to the tetra-acylated lipid A precursor lipid IVA. In the present work we report the discovery of a Kdo hydrolase in membranes of H. pylori capable of removing the outer Kdo sugar from Kdo2-lipid A. Enzymatic removal of the Kdo group was dependent upon prior removal of the 1-phosphate group from the lipid A domain, and mass spectrometric analysis of the reaction product confirmed the enzymatic removal of a single Kdo residue by the Kdo-trimming enzyme. This is the first characterization of a Kdo hydrolase involved in the modification of gram-negative bacterial LPS.

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3-Pentylcatechol, a Non-Allergenic Urushiol Derivative, Displays Anti-Helicobacter pylori Activity In Vivo

Pharmaceuticals ◽

10.3390/ph13110384 ◽

2020 ◽

Vol 13 (11) ◽

pp. 384

Author(s):

Hang Yeon Jeong ◽

Tae Ho Lee ◽

Ju Gyeong Kim ◽

Sueun Lee ◽

Changjong Moon ◽

...

Keyword(s):

Helicobacter Pylori ◽

Triple Therapy ◽

Inflammatory Cells ◽

Control Group ◽

Vitro Assay ◽

Low Concentrations ◽

H Pylori ◽

Stomach Mucous Membrane

We previously reported that 3-pentylcatechol (PC), a synthetic non-allergenic urushiol derivative, inhibited the growth of Helicobacter pylori in an in vitro assay using nutrient agar and broth. In this study, we aimed to investigate the in vivo antimicrobial activity of PC against H. pylori growing in the stomach mucous membrane. Four-week-old male C57BL/6 mice (n = 4) were orally inoculated with H. pylori Sydney Strain-1 (SS-1) for 8 weeks. Thereafter, the mice received PC (1, 5, and 15 mg/kg) and triple therapy (omeprazole, 0.7 mg/kg; metronidazole, 16.7 mg/kg; clarithromycin, 16.7 mg/kg, reference groups) once daily for 10 days. Infiltration of inflammatory cells in gastric tissue was greater in the H. pylori-infected group compared with the control group and lower in both the triple therapy- and PC-treated groups. In addition, upregulation of cytokine mRNA was reversed after infection, upon administration of triple therapy and PC. Interestingly, PC was more effective than triple therapy at all doses, even at 1/15th the dose of triple therapy. In addition, PC demonstrated synergism with triple therapy, even at low concentrations. The results suggest that PC may be more effective against H. pylori than established antibiotics.

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Full Capacity of Recombinant Escherichia coliHeat-Stable Enterotoxin Fusion Proteins for Extracellular Secretion, Antigenicity, Disulfide Bond Formation, and Activity

Infection and Immunity ◽

10.1128/iai.68.7.4064-4074.2000 ◽

2000 ◽

Vol 68 (7) ◽

pp. 4064-4074 ◽

Cited By ~ 16

Author(s):

Isabelle Batisson ◽

Maurice Der Vartanian ◽

Brigitte Gaillard-Martinie ◽

Michel Contrepois

Keyword(s):

Disulfide Bonds ◽

Secretory Pathway ◽

Biological Properties ◽

Leader Peptide ◽

Dependent Manner ◽

Reporter Protein ◽

E Coli ◽

Heat Stable

ABSTRACT We have successfully used the major subunit ClpG ofEscherichia coli CS31A fimbriae as an antigenic and immunogenic exposure-delivery vector for various heterologous peptides varying in nature and length. However, the ability of ClpG as a carrier to maintain in vitro and in vivo the native biological properties of passenger peptide has not yet been reported. To address this possibility, we genetically fused peptides containing all or part of the E. coli human heat-stable enterotoxin (STh) sequence to the amino or carboxyl ends of ClpG. Using antibodies to the ClpG and STh portions for detecting the hybrids; AMS (4-acetamido-4′-maleimidylstilbene-2,2′-disulfonate), a potent free thiol-trapping reagent, for determining the redox state of STh in the fusion; and the suckling mouse assay for enterotoxicity, we demonstrated that all ClpG-STh proteins were secreted in vitro and in vivo outside the E. coli cells in a heat-stable active oxidized (disulfide-bonded) form. Indeed, in contrast to many earlier studies, blocking the natural NH2 or COOH extremities of STh had, in all cases, no drastic effect on cell release and toxin activity. Only antigenicity of STh C-terminally extended with ClpG was strongly affected in a conformation-dependent manner. These results suggest that the STh activity was not altered by the chimeric structure, and therefore that, like the natural toxin, STh in the fusion had a spatial structure flexible enough to be compatible with secretion and enterotoxicity (folding and STh receptor recognition). Our study also indicates that disulfide bonds were essential for enterotoxicity but not for release, that spontaneous oxidation by molecular oxygen occurred in vitro in the medium, and that the E. coli cell-bound toxin activity in vivo resulted from an effective export processing of hybrids and not cell lysis. None of the ClpG-STh subunits formed hybrid CS31A-STh fimbriae at the cell surface ofE. coli, and a strong decrease in the toxin activity was observed in the absence of CS31A helper proteins. In fact, chimeras translocated across the outer membrane as a free folded monomer once they were guided into the periplasm by the ClpG leader peptide through the CS31A-dependent secretory pathway. In summary, ClpG appears highly attractive as a carrier reporter protein for basic and applied research through the engineering of E. coli for culture supernatant delivery of an active cysteine-containing protein, such as the heat-stable enterotoxin.

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VapC-1 of Nontypeable Haemophilus influenzae Is a Ribonuclease

Journal of Bacteriology ◽

10.1128/jb.00290-07 ◽

2007 ◽

Vol 189 (14) ◽

pp. 5041-5048 ◽

Cited By ~ 52

Author(s):

Dayle A. Daines ◽

Mack H. Wu ◽

Sarah Y. Yuan

Keyword(s):

Haemophilus Influenzae ◽

Growth Inhibition ◽

Gene Pair ◽

Gene Families ◽

Upper Respiratory Tract ◽

Nontypeable Haemophilus Influenzae ◽

E Coli ◽

Conserved Gene

ABSTRACT Nontypeable Haemophilus influenzae (NTHi) organisms are obligate parasites of the human upper respiratory tract that can exist as commensals or pathogens. Toxin-antitoxin (TA) loci are highly conserved gene pairs that encode both a toxin and antitoxin moiety. Seven TA gene families have been identified to date, and NTHi carries two alleles of the vapBC family. Here, we have characterized the function of one of the NTHi alleles, vapBC-1. The gene pair is transcribed as an operon in two NTHi clinical isolates, and promoter fusions display an inverse relationship to culture density. The antitoxin VapB-1 forms homomultimers both in vitro and in vivo. The expression of the toxin VapC-1 conferred growth inhibition to an Escherichia coli expression strain and was successfully purified only when cloned in tandem with its cognate antitoxin. Using total RNA isolated from both E. coli and NTHi, we show for the first time that VapC-1 is an RNase that is active on free RNA but does not degrade DNA in vitro. Preincubation of the purified toxin and antitoxin together results in the formation of a protein complex that abrogates the activity of the toxin. We conclude that the NTHi vapBC-1 gene pair functions as a classical TA locus and that the induction of VapC-1 RNase activity leads to growth inhibition via the mechanism of mRNA cleavage.

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