A Helicobacter pylori TolC Efflux Pump Confers Resistance to Metronidazole

ABSTRACT In Helicobacter pylori, the contribution of efflux proteins to antibiotic resistance is not well established. As translocases that act in parallel may have overlapping substrate specificities, the loss of function of one such translocase may be compensated for by that of another translocase with no effect on susceptibilities to antibiotics. The genome of H. pylori 26695 was assessed for the presence of putative translocases and outer membrane efflux or TolC-like proteins which could interact to form efflux systems involved in drug resistance. Twenty-seven translocases were identified, of which HP1184 was the sole representative of the multidrug and toxic compound extrusion family of translocases and which could thus have a unique substrate specificity. In addition, four TolC-like proteins (HP0605, HP0971, HP1327, and HP1489) were identified. Thus, it is feasible that inactivation of a TolC-like protein would affect the functions of multiple translocases. We aimed to determine whether efflux systems contribute to antimicrobial susceptibility by evaluation of the susceptibility profiles of an HP1184-knockout mutant, four mutants in which one of the four TolC homologs was inactivated, as well as a mutant in which both HP0605 and HP0971 were inactivated. The HP1184- and HP1489-knockout mutants both showed increased susceptibilities to ethidium bromide, while the HP0605-knockout mutant exhibited increased susceptibilities to novobiocin and sodium deoxycholate. The HP0605 and HP0971 double-knockout mutant was also more susceptible to metronidazole, in addition to being susceptible to novobiocin and sodium deoxycholate. Thus, active efflux is an eminent means of resistance to antimicrobials in H. pylori and resembles the situation in other bacteria.

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Development of an Interleukin-12-Deficient Mouse Model That Is Permissive for Colonization by a Motile KE26695 Strain of Helicobacter pylori

Infection and Immunity ◽

10.1128/iai.71.5.2534-2541.2003 ◽

2003 ◽

Vol 71 (5) ◽

pp. 2534-2541 ◽

Cited By ~ 34

Author(s):

Paul S. Hoffman ◽

Neeraj Vats ◽

Donna Hutchison ◽

Jared Butler ◽

Kenneth Chisholm ◽

...

Keyword(s):

Helicobacter Pylori ◽

Immune Modulation ◽

Limit Of Detection ◽

Genomic Research ◽

Interleukin 12 ◽

Deficient Mouse ◽

Loss Of Function ◽

Double Knockout ◽

H Pylori ◽

Deficient Mice

ABSTRACT The identification of genes associated with colonization and persistence of Helicobacter pylori in the gastric mucosa has been limited by the lack of robust animal models that support infection by strains whose genomes have been completely sequenced. Here we report that an interleukin-12 (IL-12)-deficient mouse (IL-12−/− p40 subunit knockout in C57BL/6 mouse) is permissive for infection by a motile variant (KE88-3887) of The Institute For Genomic Research-sequenced strain (KE26695) of H. pylori. The IL-12-deficient mouse was also more permissive for colonization by the mouse-colonizing Sydney 1 strain of H. pylori than were wild-type C57BL/6 mice. Differences in colonization efficiency were demonstrated by mouse challenge with SS1 strains containing loss-of-function mutations in two genes (hspR and hrcA), whose products negatively regulate several heat shock genes. At 5 weeks postinfection, double-knockout mutants (SS1 hspR hrcA) efficiently colonized IL-12-deficient mice (5 of 5 animals compared to 4 of 10 for C57BL6 mice) and bacterial counts were higher in stomachs of IL-12-deficient mice (106 versus 105 CFU/g of stomach, respectively). IL-12-deficient mice were efficiently colonized by KE88-3887 (29 of 30), but not by nonmotile KE26695, and bacterial numbers (104 to 105 CFU/g of stomach) were unchanged over an 8-week period postinfection. In contrast, C57BL/6 mice were inefficiently colonized by KE88-3887 (8 of 20 animals with bacterial loads at the limit of detection, ∼103 CFU/g), and infection did not persist much beyond 5 weeks. Cytokine responses (tumor necrosis factor alpha and gamma interferon), pathology, and antral-predominant infection were indistinguishable between IL-12-deficient and C57BL/6 mice. The increased permissiveness of the IL-12-deficient mouse for infection with H. pylori should facilitate whole-genome-based strategies to study genes associated with virulence and immune modulation.

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SHP2-independent tyrosine dephosphorylation of cortactin and vinculin during infection with Helicobacter pylori

European Journal of Microbiology and Immunology ◽

10.1556/1886.2020.00001 ◽

2020 ◽

Vol 10 (1) ◽

pp. 20-27

Author(s):

Jakob Knorr ◽

Steffen Backert ◽

Nicole Tegtmeyer

Keyword(s):

Helicobacter Pylori ◽

Host Cell ◽

Tyrosine Phosphatase ◽

Actin Binding ◽

World Population ◽

Dependent Manner ◽

Knockout Mutant ◽

Ags Cells ◽

Type Iv ◽

H Pylori

The gastric pathogen Helicobacter pylori colonizes approximately half of the human world population. The bacterium injects the effector protein cytotoxin associated gene A (CagA) via a type-IV secretion system into host epithelial cells, where the protein becomes phosphorylated at specific EPIYA-motifs by cellular kinases. Inside the host cell, CagA can interact with over 25 different proteins in both phosphorylation-dependent and phosphorylation-independent manners, resulting in manipulation of host-cell signaling pathways. During the course of an H. pylori infection, certain host-cell proteins undergo tyrosine dephosphorylation in a CagA-dependent manner, including the actin-binding proteins cortactin and vinculin. A predominant response of intracellular CagA is the binding and activation of tyrosine phosphatase, the human Src-homology-region-2-domain-containing-phosphatase-2 (SHP2). Here, we considered the possibility that activated SHP2 might be responsible for the dephosphorylation of cortactin and vinculin. To investigate this, phosphatase inhibitor studies were performed. Additionally, a complete knockout mutant of SHP2 in AGS cells was created by CRISPR/Cas9 technology, and these cells were infected with H. pylori. However, neither the presence of an inhibitor nor the inactivation of SHP2 prevented the dephosphorylation of cortactin and vinculin upon CagA delivery. Tyrosine dephosphorylation of these proteins is therefore independent of SHP2 and instead must be caused by another, as yet unidentified, protein tyrosine phosphatase.

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Enzymes Associated with Reductive Activation and Action of Nitazoxanide, Nitrofurans, and Metronidazole in Helicobacter pylori

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.46.7.2116-2123.2002 ◽

2002 ◽

Vol 46 (7) ◽

pp. 2116-2123 ◽

Cited By ~ 107

Author(s):

Gary Sisson ◽

Avery Goodwin ◽

Ausra Raudonikiene ◽

Nicky J. Hughes ◽

Asish K. Mukhopadhyay ◽

...

Keyword(s):

Helicobacter Pylori ◽

Reductase Activity ◽

Loss Of Function ◽

Reductive Activation ◽

E Coli ◽

Redox Active ◽

Bactericidal Effects ◽

Pyruvate Oxidoreductase ◽

Essential Enzyme ◽

H Pylori

ABSTRACT Nitazoxanide (NTZ) is a redox-active nitrothiazolyl-salicylamide prodrug that kills Helicobacter pylori and also many anaerobic bacterial, protozoan, and helminthic species. Here we describe development and use of a spectrophotometric assay, based on nitroreduction of NTZ at 412 nm, to identify H. pylori enzymes responsible for its activation and mode of action. Three enzymes that reduce NTZ were identified: two related NADPH nitroreductases, which also mediate susceptibility to metronidazole (MTZ) (RdxA and FrxA), and pyruvate oxidoreductase (POR). Recombinant His-tagged RdxA, FrxA, and POR, overexpressed in nitroreductase-deficient Escherichia coli, each rapidly reduced NTZ, whereas only FrxA and to a lesser extent POR reduced nitrofuran substrates (furazolidone, nitrofurantoin, and nitrofurazone). POR exhibited no MTZ reductase activity either in extracts of H. pylori or following overexpression in E. coli; RdxA exhibited no nitrofuran reductase activity, and FrxA exhibited no MTZ reductase activity. Analysis of mutation to rifampin resistance (Rifr) indicated that NTZ was not mutagenic and that nitrofurans were only weakly mutagenic. Alkaline gel DNA electrophoresis indicated that none of these prodrugs caused DNA breakage. In contrast, MTZ caused DNA damage and was strongly mutagenic. We conclude that POR, an essential enzyme, is responsible for most or all of the bactericidal effects of NTZ against H. pylori. While loss-of-function mutations in rdxA and frxA produce a Mtzr phenotype, they do not contribute much to the innate susceptibility of H. pylori to NTZ or nitrofurans.

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Helicobacter pylori and Complex Gangliosides

Infection and Immunity ◽

10.1128/iai.72.3.1519-1529.2004 ◽

2004 ◽

Vol 72 (3) ◽

pp. 1519-1529 ◽

Cited By ~ 37

Author(s):

Niamh Roche ◽

Jonas Ångström ◽

Marina Hurtig ◽

Thomas Larsson ◽

Thomas Borén ◽

...

Keyword(s):

Helicobacter Pylori ◽

Sialic Acid ◽

Mutant Strain ◽

Carbohydrate Chain ◽

Proton Nuclear Magnetic Resonance ◽

Bacterial Cells ◽

Wild Type ◽

Knockout Mutant ◽

Factors Affecting ◽

H Pylori

ABSTRACT Recognition of sialic acid-containing glycoconjugates by the human gastric pathogen Helicobacter pylori has been repeatedly demonstrated. To investigate the structural requirements for H. pylori binding to complex gangliosides, a large number of gangliosides were isolated and characterized by mass spectrometry and proton nuclear magnetic resonance. Ganglioside binding of sialic acid-recognizing H. pylori strains (strains J99 and CCUG 17874) and knockout mutant strains with the sialic acid binding adhesin SabA or the NeuAcα3Galβ4GlcNAcβ3Galβ4GlcNAcβ-binding neutrophil-activating protein HPNAP deleted was investigated using the thin-layer chromatogram binding assay. The wild-type bacteria bound to N-acetyllactosamine-based gangliosides with terminal α3-linked NeuAc, while gangliosides with terminal NeuGcα3, NeuAcα6, or NeuAcα8NeuAcα3 were not recognized. The factors affecting binding affinity were identified as (i) the length of the N-acetyllactosamine carbohydrate chain, (ii) the branches of the carbohydrate chain, and (iii) fucose substitution of the N-acetyllactosamine core chain. While the J99/NAP− mutant strain displayed a ganglioside binding pattern identical to that of the parent J99 wild-type strain, no ganglioside binding was obtained with the J99/SabA− mutant strain, demonstrating that the SabA adhesin is the sole factor responsible for the binding of H. pylori bacterial cells to gangliosides.

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Functional Analysis of the Helicobacter pylori cag Pathogenicity Island Reveals Both VirD4-CagA-Dependent and VirD4-CagA-Independent Mechanisms

Infection and Immunity ◽

10.1128/iai.70.2.665-671.2002 ◽

2002 ◽

Vol 70 (2) ◽

pp. 665-671 ◽

Cited By ~ 119

Author(s):

Matthias Selbach ◽

Stefan Moese ◽

Thomas F. Meyer ◽

Steffen Backert

Keyword(s):

Helicobacter Pylori ◽

Gene Knockout ◽

Shuttle Vector ◽

Pathogenicity Island ◽

Type Iv Secretion ◽

Host Responses ◽

Type Iv ◽

Knockout Mutants ◽

H Pylori ◽

First Time

ABSTRACT The type IV secretion machinery encoded by the cag pathogenicity island (PAI) of Helicobacter pylori has been implicated in a series of host responses during infection. Here, we analyzed the function of 12 cag PAI genes from both cag I and cag II loci, including the complete virB/D complex (virB4, virB7, virB8, virB9, virB10, virB11, and virD4). We monitored interleukin-8 (IL-8) secretion, CagA translocation and tyrosine phosphorylation, and induction of a scattering (“hummingbird”) phenotype upon H. pylori infection of AGS gastric epithelial cells. For the first time, we have complemented individual cag PAI gene knockout mutants with their intact genes expressed from a shuttle vector and showed that complemented CagA and VirD4 restored wild-type function. Our results demonstrate that phenotypic changes and phosphorylation of CagA depended on all virB/D genes and several other genes of the cag PAI. Induction of IL-8 secretion depended largely on the same set of genes but was independent of CagA and VirD4. Thus, CagA translocation and induction of IL-8 secretion are regulated by VirD4-CagA-dependent and VirD4-CagA-independent mechanisms, respectively. The function of VirD4 as a possible adapter protein which guides CagA into the type IV secretion channel is presented in a model.

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Colonization of C57BL/6J and BALB/c Wild-Type and Knockout Mice with Helicobacter pylori: Effect of Vaccination and Implications for Innate and Acquired Immunity

Infection and Immunity ◽

10.1128/iai.71.2.794-800.2003 ◽

2003 ◽

Vol 71 (2) ◽

pp. 794-800 ◽

Cited By ~ 52

Author(s):

Klaus Panthel ◽

Gerhard Faller ◽

Rainer Haas

Keyword(s):

Helicobacter Pylori ◽

Knockout Mice ◽

Tumor Necrosis Factor Receptor ◽

Bacterial Pathogen ◽

Interleukin 4 ◽

Wild Type ◽

Ulcer Disease ◽

Knockout Mutants ◽

Prophylactic Vaccination ◽

H Pylori

ABSTRACT The gram-negative bacterial pathogen Helicobacter pylori is a major cause of peptic ulcer disease and a risk factor for gastric cancer in humans. Adapted H. pylori strains, such as strain SS1, are able to infect mice and are a useful model for gastric colonization and vaccination studies. In this study we used a streptomycin-resistant derivative of H. pylori SS1 to analyze the colonization behavior and the success of vaccination in wild-type (wt) and various knockout mice of the BALB/c and C57BL/6J genetic backgrounds. We here report that BALB/c interleukin-4 knockout (IL-4−/−) mice are weakly overcolonized compared to the wt strain but that the IL-12−/− knockout results in a strong overcolonization (500%). Unexpectedly, in the C57BL/6J background the same knockouts behaved in diametrically opposed manners. The IL-4−/− mutation caused a 50% reduction and the IL-12−/− knockout caused a 95% reduction compared to the wt colonization rate. For C57BL/6J mice we further analyzed the IL-18−/− and Toll-like receptor 2 knockout mutations, which showed reductions to 66 and 57%, respectively, whereas mice with the IL-10−/− phenotype were hardly infected at all (5%). In contrast, the tumor necrosis factor receptor knockout (p55−/− and p55/75−/−) mice showed an overcolonization compared to the C57BL/6J wt strain. With exception of the low-level infected C57BL/6J IL-10−/− and IL-12−/− knockout mice, all knockout mutants were accessible to a prophylactic vaccination and their vaccination behavior was comparable to that of the wt strains.

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Cortactin Promotes Effective AGS Cell Scattering by Helicobacter pylori CagA, but Not Cellular Vacuolization and Apoptosis Induced by the Vacuolating Cytotoxin VacA

Pathogens ◽

10.3390/pathogens11010003 ◽

2021 ◽

Vol 11 (1) ◽

pp. 3

Author(s):

Irshad Sharafutdinov ◽

Jakob Knorr ◽

Delara Soltan Esmaeili ◽

Steffen Backert ◽

Nicole Tegtmeyer

Keyword(s):

Helicobacter Pylori ◽

Cell Movement ◽

Actin Binding ◽

Knockout Mutant ◽

Vacuolating Cytotoxin ◽

Cell Functions ◽

Cell Scattering ◽

Induction Of Apoptosis ◽

H Pylori ◽

Cytoskeletal Rearrangements

Cortactin is an actin-binding protein and actin-nucleation promoting factor regulating cytoskeletal rearrangements in eukaryotes. Helicobacter pylori is a gastric pathogen that exploits cortactin to its own benefit. During infection of gastric epithelial cells, H. pylori hijacks multiple cellular signaling pathways, leading to the disruption of key cell functions. Two bacterial virulence factors play important roles in this scenario, the vacuolating cytotoxin VacA and the translocated effector protein CagA of the cag type IV secretion system (T4SS). Specifically, by overruling the phosphorylation status of cortactin, H. pylori alternates the activity of molecular interaction partners of this important protein, thereby manipulating the performance of cytoskeletal rearrangements, endosomal trafficking and cell movement. Based on shRNA knockdown and other studies, it was previously reported that VacA utilizes cortactin for its cellular uptake, intracellular travel and induction of apoptosis by a mitochondria-dependent mechanism, while CagA induces cell scattering, motility and elongation. To investigate the role of cortactin in these phenotypes in more detail, we produced a complete knockout mutant of cortactin in the gastric adenocarcinoma cell line AGS by CRISPR-Cas9. These cells were infected with H. pylori wild-type or various isogenic mutant strains. Unexpectedly, cortactin deficiency did not prevent the uptake and formation of VacA-dependent vacuoles, nor the induction of apoptosis by internalized VacA, while the induction of T4SS- and CagA-dependent AGS cell movement and elongation were strongly reduced. Thus, we provide evidence that cortactin is required for the function of internalized CagA, but not VacA.

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Bifunctional enzyme SpoT is involved in biofilm formation of Helicobacter pylori with multidrug resistance by upregulating efflux pump Hp1174 (gluP)

10.1101/321026 ◽

2018 ◽

Author(s):

Xiaoran Ge ◽

Yuying Cai ◽

Zhenghong Chen ◽

Sizhe Gao ◽

Xiwen Geng ◽

...

Keyword(s):

Drug Resistance ◽

Helicobacter Pylori ◽

Multidrug Resistance ◽

Biofilm Formation ◽

Efflux Pump ◽

Efflux Pumps ◽

Multi Drug Resistance ◽

Bifunctional Enzyme ◽

Average Expression Level ◽

H Pylori

ABSTRACTThe drug resistance of Helicobacter pylori (H. pylori) is gradually becoming a serious problem. Biofilm formation is an important factor that leads to multidrug resistance in bacteria. The ability of H. pylori to form biofilms on the gastric mucosa has been known. However, there are few studies on the regulation mechanisms of H. pylori biofilm formation and multidrug resistance. Guanosine 3’-diphosphate 5’-triphosphate and guanosine 3’,5’-bispyrophosphate [(p)ppGpp] are global regulatory factors and are synthesized in H. pylori by the bifunctional enzyme SpoT. It has been reported that (p)ppGpp is involved in the biofilm formation and multidrug resistance of various bacteria. In this study, we found that SpoT also plays an important role in H. pylori biofilm formation and multidrug resistance. Therefore, it is necessary to carry out some further studies regarding its regulatory mechanism. Considering that efflux pumps are of great importance in the biofilm formation and multidrug resistance of bacteria, we tried to find if efflux pumps controlled by SpoT participate in these activities. Then, we found that Hp1174 (glucose/galactose transporter, gluP), an efflux pump of the MFS family, is highly expressed in biofilm-forming and multi-drug resistance (MDR) H. pylori and is upregulated by SpoT. Through further research, we determined that gluP involved in H. pylori biofilm formation and multidrug resistance. Furthermore, the average expression level of gluP in clinical MDR strains was considerably higher than that in clinical drug-sensitive strains. Taken together, our results revealed a novel molecular mechanism of H. pylori tolerance to multidrug.

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Exploring the Contribution of the AcrB Homolog MdtF to Drug Resistance and Dye Efflux in a Multidrug Resistant E. coli Isolate

Antibiotics ◽

10.3390/antibiotics10050503 ◽

2021 ◽

Vol 10 (5) ◽

pp. 503

Author(s):

Sabine Schuster ◽

Martina Vavra ◽

Ludwig Greim ◽

Winfried V. Kern

Keyword(s):

Fluorescent Dyes ◽

Efflux Pump ◽

Multidrug Resistant ◽

Coli Strain ◽

Double Knockout ◽

E Coli ◽

Knockout Mutants ◽

Antibiotic Agents ◽

Single Knockout

In Escherichia coli, the role of RND-type drug transporters other than the major efflux pump AcrB has largely remained undeciphered (particularly in multidrug resistant pathogens), because genetic engineering in such isolates is challenging. The present study aimed to explore the capability of the AcrB homolog MdtF to contribute to the extrusion of noxious compounds and to multidrug resistance in an E. coli clinical isolate with demonstrated expression of this efflux pump. An mdtF/acrB double-knockout was engineered, and susceptibility changes with drugs from various classes were determined in comparison to the parental strain and its acrB and tolC single-knockout mutants. The potential of MdtF to participate in the export of agents with different physicochemical properties was additionally assessed using accumulation and real-time efflux assays with several fluorescent dyes. The results show that there was limited impact to the multidrug resistant phenotype in the tested E. coli strain, while the RND-type transporter remarkably contributes to the efflux of all tested dyes. This should be considered when evaluating the efflux phenotype of clinical isolates via dye accumulation assays. Furthermore, the promiscuity of MdtF should be taken into account when developing new antibiotic agents.

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Development of a Markerless Knockout Method for Actinobacillus succinogenes

Applied and Environmental Microbiology ◽

10.1128/aem.00492-14 ◽

2014 ◽

Vol 80 (10) ◽

pp. 3053-3061 ◽

Cited By ~ 11

Author(s):

Rajasi V. Joshi ◽

Bryan D. Schindler ◽

Nikolas R. McPherson ◽

Kanupriya Tiwari ◽

Claire Vieille

Keyword(s):

High Efficiency ◽

Direct Selection ◽

Selection Marker ◽

Actinobacillus Succinogenes ◽

Knockout Mutant ◽

Double Knockout ◽

Pyruvate Formate Lyase ◽

Content Type ◽

Knockout Mutants ◽

Encoding Genes

ABSTRACTActinobacillus succinogenesis one of the best natural succinate-producing organisms, but it still needs engineering to further increase succinate yield and productivity. In this study, we developed a markerless knockout method forA. succinogenesusing natural transformation or electroporation. TheEscherichia coliisocitrate dehydrogenase gene with flanking flippase recognition target sites was used as the positive selection marker, making use ofA. succinogenes's auxotrophy for glutamate to select for growth on isocitrate. TheSaccharomyces cerevisiaeflippase recombinase (Flp) was used to remove the selection marker, allowing its reuse. Finally, the plasmid expressingflpwas cured using acridine orange. We demonstrate that at least two consecutive deletions can be introduced into the same strain using this approach, that no more than a total of 1 kb of DNA is needed on each side of the selection cassette to protect from exonuclease activity during transformation, and that no more than 200 bp of homologous DNA is needed on each side for efficient recombination. We also demonstrate that electroporation can be used as an alternative transformation method to obtain knockout mutants and that an enriched defined medium can be used for direct selection of knockout mutants on agar plates with high efficiency. Single-knockout mutants of the fumarate reductase and of the pyruvate formate lyase-encoding genes were obtained using this knockout strategy. Double-knockout mutants were also obtained by deleting the citrate lyase-, β-galactosidase-, and aconitase-encoding genes in the pyruvate formate lyase knockout mutant strain.

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