Generation of Isogenic Mutant Strains of Helicobacter pylori

ABSTRACTHelicobacter pylorimust be able to rapidly respond to fluctuating conditions within the stomach. Despite this need for constant adaptation,H. pyloriencodes few regulatory proteins. Of the identified regulators, the ferric uptake regulator (Fur), the nickel response regulator (NikR), and the two-component acid response system (ArsRS) are each paramount to the success of this pathogen. While numerous studies have individually examined these regulatory proteins, little is known about their combined effect. Therefore, we constructed a series of isogenic mutant strains that contained all possible single, double, and triple regulatory mutations in Fur, NikR, and ArsS. A growth curve analysis revealed minor variation in growth kinetics across the strains; these were most pronounced in the triple mutant and in strains lacking ArsS. Visual analysis showed that strains lacking ArsS formed large aggregates and a biofilm-like matrix at the air-liquid interface. Biofilm quantification using crystal violet assays and visualization via scanning electron microscopy (SEM) showed that all strains lacking ArsS or containing a nonphosphorylatable form of ArsR (ArsR-D52N mutant) formed significantly more biofilm than the wild-type strain. Molecular characterization of biofilm formation showed that strains containing mutations in the ArsRS pathway displayed increased levels of cell aggregation and adherence, both of which are key to biofilm development. Furthermore, SEM analysis revealed prevalent coccoid cells and extracellular matrix formation in the ArsR-D52N, ΔnikRΔarsS, and ΔfurΔnikRΔarsSmutant strains, suggesting that these strains may have an exacerbated stress response that further contributes to biofilm formation. Thus,H. pyloriArsRS has a previously unrecognized role in biofilm formation.IMPORTANCEDespite a paucity of regulatory proteins, adaptation is key to the survival ofH. pyloriwithin the stomach. While prior studies have focused on individual regulatory proteins, such as Fur, NikR, and ArsRS, few studies have examined the combined effect of these factors. Analysis of isogenic mutant strains that contained all possible single, double, and triple regulatory mutations in Fur, NikR, and ArsS revealed a previously unrecognized role for the acid-responsive two-component system ArsRS in biofilm formation.

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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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Molecular Characterization of a Flagellar Export Locus of Helicobacter pylori

Infection and Immunity ◽

10.1128/iai.67.5.2060-2070.1999 ◽

1999 ◽

Vol 67 (5) ◽

pp. 2060-2070 ◽

Cited By ~ 31

Author(s):

Steffen Porwollik ◽

Brian Noonan ◽

Paul W. O’Toole

Keyword(s):

Helicobacter Pylori ◽

Virulence Factor ◽

Housekeeping Genes ◽

Reading Frame ◽

Reverse Transcription Pcr ◽

Mutant Strains ◽

H Pylori ◽

Export Protein ◽

Successful Colonization

ABSTRACT Motility of Helicobacter species has been shown to be essential for successful colonization of the host. We have investigated the organization of a flagellar export locus in Helicobacter pylori. A 7-kb fragment of the H. pylori CCUG 17874 genome was cloned and sequenced, revealing an operon comprising an open reading frame of unknown function (ORF03), essential housekeeping genes (ileS and murB), flagellar export genes (fliI and fliQ), and a homolog to a gene implicated in virulence factor transport in other pathogens (virB11). A promoter for this operon, showing similarity to the Escherichia coli ς70 consensus, was identified by primer extension. Cotranscription of the genes in the operon was demonstrated by reverse transcription-PCR, and transcription of virB11, fliI, fliQ, andmurB was detected in human or mouse biopsies obtained from infected hosts. The genetic organization of this locus was conserved in a panel of H. pylori clinical isolates. EngineeredfliI and fliQ mutant strains were completely aflagellate and nonmotile, whereas a virB11 mutant still produced flagella. The fliI and fliQ mutant strains produced reduced levels of flagellin and the hook protein FlgE. Production of OMP4, a member of the outer membrane protein family identified in H. pylori 26695, was reduced in both thevirB11 mutant and the fliI mutant, suggesting related functions of the virulence factor export protein (VirB11) and the flagellar export component (FliI).

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Mouse-Colonizing Helicobacter pylori SS1 Is Unusually Susceptible to Metronidazole Due to Two Complementary Reductase Activities

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.44.11.3127-3132.2000 ◽

2000 ◽

Vol 44 (11) ◽

pp. 3127-3132 ◽

Cited By ~ 18

Author(s):

Jin-Yong Jeong ◽

Douglas E. Berg

Keyword(s):

Helicobacter Pylori ◽

Kanamycin Resistance ◽

Phenotypic Traits ◽

Insertion Mutant ◽

Low Concentrations ◽

Mutant Strains ◽

Resistance Markers ◽

Clinically Significant ◽

H Pylori ◽

Derivatives Of

ABSTRACT In most strains of Helicobacter pylori, mutational inactivation of the rdxA (HP0954) gene, which encodes a nitroreductase that converts metronidazole (MTZ) from a harmless prodrug to a mutagenic and bacteriocidal product, is sufficient to make this pathogen resistant to clinically significant levels of MTZ. Here we report that SS1, a strain with the special ability to colonize mice, is unusual in being susceptible to very low concentrations of MTZ (0.5 μg/ml) and in being especially difficult to mutate to MTZ resistance (Mtzr). These phenotypic traits were traced to expression in this strain of the normally quiescent H. pylori frxAgene (HP0642, an rdxA paralog) along with rdxA. Transformation tests using rdxA::camand frxA::kan insertion mutant DNAs, with selection solely for the chloramphenicol and kanamycin resistance markers, and sequence analyses of frxA in spontaneous Mtzr derivatives of rdxA null mutant strains each showed that the development of Mtzr in SS1 required inactivation of both rdxA and frxA. Inactivation of either gene alone left SS1 susceptible to MTZ, although it was readily mutable from an MTZ-susceptible to an Mtzrphenotype. Reverse transcriptase PCR tests showed that frxAmRNA was at least 10-fold more abundant in SS1 than in reference strain 26695. It is proposed that these reductases play primarily nutritional roles during bacterial growth.

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The Entner-Doudoroff Pathway Has Little Effect on Helicobacter pylori Colonization of Mice

Infection and Immunity ◽

10.1128/iai.71.5.2920-2923.2003 ◽

2003 ◽

Vol 71 (5) ◽

pp. 2920-2923 ◽

Cited By ~ 10

Author(s):

Amy E. Wanken ◽

Tyrrell Conway ◽

Kathryn A. Eaton

Keyword(s):

Helicobacter Pylori ◽

Wild Type ◽

Mutant Strains ◽

H Pylori ◽

Type Strains

ABSTRACT Helicobacter pylori mutants deficient in 6-phosphogluconate dehydratase (6PGD) were constructed. Colonization densities were lower and minimum infectious doses were higher for mutant strains than for wild-type strains. In spite of better colonization, however, wild-type strains did not displace the mutant in cocolonization experiments. Loss of 6PGD diminishes the fitness of H. pylori in vivo, but the pathway is nonessential for colonization.

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Novel Functions for Glycosyltransferases Jhp0562 and GalT in Lewis Antigen Synthesis and Variation in Helicobacter pylori

Infection and Immunity ◽

10.1128/iai.00032-12 ◽

2012 ◽

Vol 80 (4) ◽

pp. 1593-1605 ◽

Cited By ~ 9

Author(s):

Mary Ann Pohl ◽

Sabine Kienesberger ◽

Martin J. Blaser

Keyword(s):

Helicobacter Pylori ◽

Wild Type ◽

Sequence Analyses ◽

Upstream Gene ◽

Content Type ◽

Lewis Antigen ◽

Mutant Strains ◽

H Pylori

ABSTRACTLewis (Le) antigens are fucosylated oligosaccharides present in theHelicobacter pylorilipopolysaccharide. Expression of these antigens is believed to be important forH. pyloricolonization, since Le antigens also are expressed on the gastric epithelia in humans. A galactosyltransferase encoded by β-(1,3)galTis essential for production of type 1 (Leaand Leb) antigens. The upstream genejhp0562, which is present in many but not allH. pyloristrains, is homologous to β-(1,3)galTbut is of unknown function. BecauseH. pyloridemonstrates extensive intragenomic recombination, we hypothesized that these two genes could undergo DNA rearrangement. A PCR screen and subsequent sequence analyses revealed that the two genes can recombine at both the 5′ and 3′ ends. Chimeric β-(1,3)galT-like alleles can restore function in a β-(1,3)galTnull mutant, but neither native nor recombinantjhp0562can. Mutagenesis ofjhp0562revealed that it is essential for synthesis of both type 1 and type 2 Le antigens. Transcriptional analyses of both loci showed β-(1,3)galTexpression in all wild-type (WT) and mutant strains tested, whereasjhp0562was not expressed injhp0562null mutants, as expected. Sincejhp0562unexpectedly displayed functions in both type 1 and type 2 Le synthesis, we asked whethergalT, part of the type 2 synthesis pathway, had analogous functions in type 1 synthesis. Mutagenesis and complementation analysis confirmed thatgalTis essential for Lebproduction. In total, these results demonstrate thatgalTandjhp0562have functions that cross the expected Le synthesis pathways and thatjhp0562provides a substrate for intragenomic recombination to generate diverse Le synthesis enzymes.

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RAPID CHARACTERIZATION AND SEPARATION OF ISOGENIC MUTANTS OF H. PYLORI BY DIELECTROPHORESIS

Biomedical Engineering Applications Basis and Communications ◽

10.4015/s101623720900157x ◽

2009 ◽

Vol 21 (06) ◽

pp. 433-436

Author(s):

Chi-Chang Lin ◽

Sheng-Kai Li ◽

Bor-Shyang Sheu ◽

Hsien-Chang Chang

Keyword(s):

Helicobacter Pylori ◽

Individual Cell ◽

Outer Membrane Proteins ◽

Cell Types ◽

Wild Type ◽

Nondestructive Analysis ◽

Isogenic Mutant ◽

Rapid Characterization ◽

H Pylori ◽

Isogenic Mutants

A simple, fast, real-time, and nondestructive analysis of protein expression in biological samples, such as membranes, based on dielectrophoresis is described. On the basis of the distinct differences in the dielectrophoretic properties of individual cell types, the wild-type BabA-positive Helicobacter pylori isolates and its BabA-negative isogenic mutant can be identified and separated. The herein-presented approach of using microelectrodes should be an easy-to-use, cheap, and rapid alternative to separate and distinguish the presence or absence of important outer-membrane proteins.

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Pneumococcal Behavior and Host Responses during Bronchopneumonia Are Affected Differently by the Cytolytic and Complement-Activating Activities of Pneumolysin

Infection and Immunity ◽

10.1128/iai.71.4.1813-1819.2003 ◽

2003 ◽

Vol 71 (4) ◽

pp. 1813-1819 ◽

Cited By ~ 51

Author(s):

Rania Jounblat ◽

Aras Kadioglu ◽

Tim J. Mitchell ◽

Peter W. Andrew

Keyword(s):

Lung Tissue ◽

Inflammatory Cell ◽

Cytolytic Activity ◽

Host Responses ◽

Wild Type ◽

Histological Changes ◽

Cell Recruitment ◽

Isogenic Mutant ◽

Mutant Strains ◽

Inflammatory Cell Recruitment

ABSTRACT Pneumolysin, a multifunctional toxin produced by all clinical isolates of Streptococcus pneumoniae, is strongly implicated in the pathogenesis of pneumococcal bronchopneumonia and septicemia. Using isogenic mutant strains, we examined the effect of deletion of the cytotoxic activity or complement-activating activity of pneumolysin on bacterial growth in lungs and blood, histological changes in infected lung tissue, and the pattern of inflammatory cell recruitment. Both of the activities of pneumolysin contributed to the pathology in the lungs, as well as the timing of the onset of bacteremia. Histological changes in the lungs were delayed after infection with either mutant compared to the changes seen after infection with the wild-type pneumococcus. The complement-activating activity of pneumolysin affected the accumulation of T cells, whereas the toxin's cytolytic activity influenced neutrophil recruitment into lung tissue.

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Mo1929 - Enrichment of Helicobacter Pylori Mutant Strains after Eradication Therapy Analyzed by Gastric Wash Based Pyrosequencing and NGS

Gastroenterology ◽

10.1016/s0016-5085(18)32893-2 ◽

2018 ◽

Vol 154 (6) ◽

pp. S-854

Author(s):

Yoshiyuki Watanabe ◽

Ritsuko Oikawa ◽

Hiroyuki Yamamoto ◽

Fumio Itoh

Keyword(s):

Helicobacter Pylori ◽

Eradication Therapy ◽

Mutant Strains

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Effect of Glycine on Helicobacter pylori In Vitro

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.48.10.3782-3788.2004 ◽

2004 ◽

Vol 48 (10) ◽

pp. 3782-3788 ◽

Cited By ~ 15

Author(s):

Masaaki Minami ◽

Takafumi Ando ◽

Shin-nosuke Hashikawa ◽

Keizo Torii ◽

Tadao Hasegawa ◽

...

Keyword(s):

Helicobacter Pylori ◽

Eradication Therapy ◽

Resistant Mutant ◽

Synergistic Activity ◽

Wild Type ◽

Mutant Strains ◽

The Difference ◽

H Pylori ◽

Isogenic Mutants

ABSTRACT Glycine is the simplest amino acid and is used as a metabolic product in some bacteria. However, an excess of glycine inhibits the growth of many bacteria, and it is used as a nonspecific antiseptic agent due to its low level of toxicity in animals. The effect of glycine on Helicobacter pylori is not precisely known. The present study was conducted to investigate (i) the effect of glycine on clarithromycin (CLR)-resistant and -susceptible strains of H. pylori, (ii) the effect of glycine in combination with amoxicillin (AMX), and (iii) the postantibiotic effect (PAE). The MIC at which 90% of strains are inhibited for glycine was almost 2.5 mg/ml for 31 strains of H. pylori, including CLR-resistant strains. We constructed isogenic CLR-resistant mutant strains by natural transformation and investigated the difference between clinical wild-type strains and isogenic mutants. There were no differences in the MICs between CLR-resistant and -susceptible strains or between clinical wild-type and mutant strains. The combination of AMX and glycine showed synergistic activity, with the minimum bactericidal concentration of AMX with glycine decreasing to 1/10 that of AMX alone. Glycine showed no PAE against H. pylori. These results suggest that glycine may be a useful antimicrobial agent against H. pylori not only alone but also in combination with antibacterial drugs for the treatment of H. pylori-associated diseases. Glycine may represent a component of a new type of eradication therapy for CLR-resistant H. pylori.

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