In VivoSequence Variation in HopZ, a Phase-Variable Outer Membrane Protein of Helicobacter pylori

ABSTRACTTheHelicobacter pyloriouter membrane protein HopZ is regulated by a phase-variable CT repeat and occurs in two distinct allelic variants. Whole-genome comparisons of isolates from one human volunteer recently provided evidence forin vivoselection for thehopZON status. We explored the frequency of sequence variation inhopZduring acute and chronic human infection and studied the association ofhopZwith the phylogeographic population structure ofH. pylori. hopZON variants were cultured from 24 out of 33 volunteers challenged with thehopZOFF strain BCS 100. Transmission ofH. pyloriwithin families was also frequently associated with a status change ofhopZ. In contrast,hopZsequences obtained from 26 sets of sequential isolates from chronically infected individuals showed no changes of status, suggesting that thehopZstatus selected during early infection is subsequently stable. Mutations leading to amino acid changes in HopZ occurred more frequently in ON than in OFF status isolates during chronic infection, indicating that sequence changes are more likely the result of positive selection in ON isolates than of a loss of negative selection pressure in OFF isolates. Analysis of 63 isolates from chronically infected individuals revealed no significant correlation ofhopZstatus with chronic atrophic gastritis.hopZsequences were obtained from a globally representative collection of 54H. pyloristrains. AllH. pyloripopulations containedhopZ-positive isolates. The data suggest thathopZhas been acquired and split into the two variants before the human migration out of Africa.

Download Full-text

The Helicobacter pylori Autotransporter ImaA (HP0289) Modulates the Immune Response and Contributes to Host Colonization

Infection and Immunity ◽

10.1128/iai.00312-12 ◽

2012 ◽

Vol 80 (7) ◽

pp. 2286-2296 ◽

Cited By ~ 13

Author(s):

William E. Sause ◽

Andrea R. Castillo ◽

Karen M. Ottemann

Keyword(s):

Immune Response ◽

Helicobacter Pylori ◽

Membrane Protein ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Dependent Manner ◽

Wild Type ◽

Content Type ◽

H Pylori ◽

Murine Infections

ABSTRACTThe human pathogenHelicobacter pyloriemploys a diverse collection of outer membrane proteins to colonize, persist, and drive disease within the acidic gastric environment. In this study, we sought to elucidate the function of the host-induced geneHP0289, which encodes an uncharacterized outer membrane protein. We first generated an isogenicH. pylorimutant that lacksHP0289and found that the mutant has a colonization defect in single-strain infections and is greatly outcompeted in mouse coinfection experiments with wild-typeH. pylori. Furthermore, we used protease assays and biochemical fractionation coupled with an HP0289-targeted peptide antibody to verify that the HP0289 protein resides in the outer membrane. Our previous findings showed that theHP0289promoter is upregulated in the mouse stomach, and here we demonstrate thatHP0289expression is induced under acidic conditions in an ArsRS-dependent manner. Finally, we have shown that theHP0289mutant induces greater expression of the chemokine interleukin-8 (IL-8) and the cytokine tumor necrosis factor alpha (TNF-α) in gastric carcinoma cells (AGS). Similarly, transcription of the IL-8 homolog keratinocyte-derived chemokine (KC) is elevated in murine infections with the HP0289 mutant than in murine infections with wild-typeH. pylori. On the basis of this phenotype, we renamed HP0289 ImaA forimmunomodulatoryautotransporter protein. Our work has revealed that genes inducedin vivoplay an important role inH. pyloripathogenesis. Specifically, the outer membrane protein ImaA modulates a component of the host inflammatory response, and thus may allowH. pylorito fine tune the host immune response based on ImaA expression.

Download Full-text

Oral Immunization with Recombinant Mycobacterium smegmatis Expressing the Outer Membrane Protein 26-Kilodalton Antigen Confers Prophylactic Protection against Helicobacter pylori Infection

Clinical and Vaccine Immunology ◽

10.1128/cvi.05306-11 ◽

2011 ◽

Vol 18 (11) ◽

pp. 1957-1961 ◽

Cited By ~ 6

Author(s):

Lin Lü ◽

Han-qing Zeng ◽

Pi-long Wang ◽

Wei Shen ◽

Ting-xiu Xiang ◽

...

Keyword(s):

Helicobacter Pylori ◽

Membrane Protein ◽

Outer Membrane ◽

Chronic Gastritis ◽

Outer Membrane Protein ◽

Mycobacterium Smegmatis ◽

Interleukin 2 ◽

Oral Immunization ◽

Content Type ◽

H Pylori

ABSTRACTHelicobacter pyloriinfection is prevalent worldwide and results in chronic gastritis, which may lead to gastric mucosa-associated lymphoid tissue lymphoma and gastric cancer. We have previously reported that oral immunization with recombinantMycobacterium smegmatisexpressing theH. pyloriouter membrane protein 26-kilodalton (Omp26) antigen affords therapeutic protection againstH. pyloriinfection in mice. In the present study, we investigated the prophylactic effects of this vaccine candidate onH. pylorichallenge in mice. We found that oral immunization with recombinantMycobacteriumOmp26 significantly reducedH. pyloricolonization in the stomach compared to inoculation with wild-typeM. smegmatisin control mice. Six of the recombinantMycobacterium-immunized mice (60%) were completely protected fromH. pyloriinfection. The severity ofH. pylori-associated chronic gastritis assessed histologically was significantly milder in mice vaccinated with recombinantMycobacteriumthan in control animals. Mice immunized with recombinantMycobacteriumshowed enhanced antigen-specific lymphocyte proliferation and antibody responses. Moreover, immunization with recombinantMycobacteriumresulted in an increased expression of interleukin-2 and gamma interferon in the stomach and spleen, as determined by reverse transcription-PCR analysis. Our results collectively suggest that vaccination with recombinantMycobacteriumOmp26 confers prophylactic protection againstH. pyloriinfection. The inhibition ofH. pyloricolonization is associated with the induction of antigen-specific humoral and cell-mediated immune responses.

Download Full-text

Identification of Functional Interactome of Gastric Cancer Cells with Helicobacter pylori Outer Membrane Protein HpaA by HPLC-MS/MS

BioMed Research International ◽

10.1155/2020/1052926 ◽

2020 ◽

Vol 2020 ◽

pp. 1-10

Author(s):

Ruyue Fan ◽

Xiurui Han ◽

Di Xiao ◽

Lihua He ◽

Yanan Gong ◽

...

Keyword(s):

Gastric Cancer ◽

Helicobacter Pylori ◽

Epithelial Cells ◽

Membrane Protein ◽

Outer Membrane ◽

Cell Lines ◽

Outer Membrane Protein ◽

Regulation Of Transcription ◽

Gastric Epithelial Cells ◽

H Pylori

HpaA as an outer membrane protein of Helicobacter pylori (H. pylori) plays a significant role in the adhesion to the human stomach, but the functional relation between HpaA and gastric epithelial cells is still not clear. To screen the interaction between HpaA and cellular proteins in gastric epithelial cells, the HpaA protein from H. pylori 26695 fused with a tag (6× His) was expressed and purified successfully, the secondary structure was estimated by the Circular Dichroism (CD) spectrum, and the purified recombinant protein was used to perform the pull-down assays with gastric cancer cell lines (AGS and SGC-7901) lysates, respectively. The pull-down proteins were identified by high-performance liquid chromatography tandem mass spectrometry system (HPLC-MS/MS). A total of 9 and 13 proteins related were analyzed from AGS and SGC-7901 cell lysates, respectively. ANXA2 was considered as putative HpaA functional partner discovered from lysates of both cell lines with high score and coverage. It is hypothesized that HpaA may be involved in the biological process of regulation of transcription and nucleic acid metabolism during the adhesion of H. pylori to human gastric epithelial cells, and HpaA-binding proteins also be used as targets for the development of antiadhesion drugs against H. pylori.

Download Full-text

Heterogeneity among Helicobacter pylori Strains in Expression of the Outer Membrane Protein BabA

Infection and Immunity ◽

10.1128/iai.72.6.3429-3435.2004 ◽

2004 ◽

Vol 72 (6) ◽

pp. 3429-3435 ◽

Cited By ~ 52

Author(s):

Ewa E. Hennig ◽

Ray Mernaugh ◽

Jennifer Edl ◽

Ping Cao ◽

Timothy L. Cover

Keyword(s):

Helicobacter Pylori ◽

Membrane Protein ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Recombinant Antibody ◽

Wild Type ◽

Single Chain ◽

H Pylori ◽

Different Strains

ABSTRACT The BabA adhesin of Helicobacter pylori is an outer membrane protein that binds to the fucosylated Lewis b histo-blood group antigen on the surface of gastric epithelial cells. We screened a phage-displayed ScFv (single-chain fragment variable) recombinant antibody library for antibodies reactive with a recombinant BabA fragment and identified two such antibodies. Each antibody recognized an ∼75-kDa protein present in wild-type H. pylori strain J99 but absent from an isogenic babA mutant strain. An immunoreactive BabA protein was detected by at least one of the antibodies in 18 (46%) of 39 different wild-type H. pylori strains and was detected more commonly in cagA-positive strains than in cagA-negative strains. Numerous amino acid polymorphisms were detected among BabA proteins expressed by different strains, with the greatest diversity occurring in the middle region of the proteins. Among the 18 strains that expressed a detectable BabA protein, there was considerable variation in the level of binding to Lewis b in vitro. Heterogeneity among H. pylori strains in expression of the BabA protein may be a factor that contributes to differing clinical outcomes among H. pylori-infected humans.

Download Full-text

Helicobacter pylori outer membrane protein and virulence marker differences in expatriate patients

Epidemiology and Infection ◽

10.1017/s095026881600025x ◽

2016 ◽

Vol 144 (10) ◽

pp. 2200-2208 ◽

Cited By ~ 1

Author(s):

J. YAKOOB ◽

Z. ABBAS ◽

W. JAFRI ◽

R. KHAN ◽

S A. SALIM ◽

...

Keyword(s):

Helicobacter Pylori ◽

Membrane Protein ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Virulence Marker ◽

Virulence Markers ◽

Local Residents ◽

H Pylori

SUMMARYWe studied the prevalence of Helicobacter pylori virulence markers, e.g. cytotoxin associated gene (cagA), cagA promoter, vacuolating associated cytotoxin A (vacA) alleles induced by contact with epithelium (iceA type), and outer membrane protein Q (hopQ) in expatriates and compared them with those in local residents. Gastric biopsies were obtained at endoscopy for culture, histology and PCR for virulence marker and hopQ. Of 309 patients, 236 (76%) were males with a mean age of 45 years. A total of 102 patients were expatriates. hopQ type 1 was present in 98 (47%) local residents compared to 88 (86%) expatriates (P < 0·001), while hopQ type 2 was present in 176 (85%) local residents, compared to 60 (59%) expatriates (P < 0·001). H. pylori virulence marker cagA was positive in 97 (47%) local residents compared to 86 (84%) expatriates (P < 0·001) while cagA-P was positive in 72 (35%) local residents compared to 87 (85%) expatriates (P < 0·001). iceA type 1 was positive in 157 (76%) local residents compared to 45 (44%) expatriates (P < 0·001), while iceA type 2 was positive in 81 (39%) local residents compared to 86 (84%) expatriates (P < 0·001). Distribution of H. pylori cagA, cagA promoter, iceA and hopQ type in local residents and expatriates was different. H. pylori virulence markers were associated with severe pathology in expatriates.

Download Full-text

In Vivo Genome and Methylome Adaptation of cag-Negative Helicobacter pylori during Experimental Human Infection

mBio ◽

10.1128/mbio.01803-20 ◽

2020 ◽

Vol 11 (4) ◽

Author(s):

Iratxe Estibariz ◽

Florent Ailloud ◽

Sabrina Woltemate ◽

Boyke Bunk ◽

Cathrin Spröer ◽

...

Keyword(s):

Helicobacter Pylori ◽

Bacterial Genome ◽

Vaccine Trial ◽

Phase Variable ◽

Genetic Changes ◽

Content Type ◽

Challenge Strain ◽

Human Volunteers ◽

H Pylori

ABSTRACT Multiple studies have demonstrated rapid bacterial genome evolution during chronic infection with Helicobacter pylori. In contrast, little was known about genetic changes during the first stages of infection, when selective pressure is likely to be highest. Using single-molecule, real-time (SMRT) and Illumina sequencing technologies, we analyzed genome and methylome evolution during the first 10 weeks of infection by comparing the cag pathogenicity island (cagPAI)-negative H. pylori challenge strain BCS 100 with pairs of H. pylori reisolates from gastric antrum and corpus biopsy specimens of 10 human volunteers who had been infected with this strain as part of a vaccine trial. Most genetic changes detected in the reisolates affected genes with a surface-related role or a predicted function in peptide uptake. Apart from phenotypic changes of the bacterial envelope, a duplication of the catalase gene was observed in one reisolate, which resulted in higher catalase activity and improved survival under oxidative stress conditions. The methylomes also varied in some of the reisolates, mostly by activity switching of phase-variable methyltransferase (MTase) genes. The observed in vivo mutation spectrum was remarkable for a very high proportion of nonsynonymous mutations. Although the data showed substantial within-strain genome diversity in the challenge strain, most antrum and corpus reisolates from the same volunteers were highly similar to each other, indicating that the challenge infection represents a major selective bottleneck shaping the transmitted population. Our findings suggest rapid in vivo selection of H. pylori during early-phase infection providing adaptation to different individuals by common mechanisms of genetic and epigenetic alterations. IMPORTANCE Exceptional genetic diversity and variability are hallmarks of Helicobacter pylori, but the biological role of this plasticity remains incompletely understood. Here, we had the rare opportunity to investigate the molecular evolution during the first weeks of H. pylori infection by comparing the genomes and epigenomes of H. pylori strain BCS 100 used to challenge human volunteers in a vaccine trial with those of bacteria reisolated from the volunteers 10 weeks after the challenge. The data provide molecular insights into the process of establishment of this highly versatile pathogen in 10 different human individual hosts, showing, for example, selection for changes in host-interaction molecules as well as changes in epigenetic methylation patterns. The data provide important clues to the early adaptation of H. pylori to new host niches after transmission, which we believe is vital to understand its success as a chronic pathogen and develop more efficient treatments and vaccines.

Download Full-text

Outer Membrane Targeting of Passenger Proteins by the Vacuolating Cytotoxin Autotransporter of Helicobacter pylori

Infection and Immunity ◽

10.1128/iai.69.11.6769-6775.2001 ◽

2001 ◽

Vol 69 (11) ◽

pp. 6769-6775 ◽

Cited By ~ 52

Author(s):

Wolfgang Fischer ◽

Renate Buhrdorf ◽

Elke Gerland ◽

Rainer Haas

Keyword(s):

Helicobacter Pylori ◽

Membrane Protein ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Shuttle Vector ◽

Expression System ◽

Cholera Toxin B Subunit ◽

Vacuolating Cytotoxin ◽

H Pylori ◽

Passenger Proteins

ABSTRACT Helicobacter pylori produces a number of proteins associated with the outer membrane, including adhesins and the vacuolating cytotoxin. These proteins are supposed to integrate into the outer membrane by β-barrel structures, characteristic of the family of autotransporter proteins. By using the SOMPES (shuttle vector-based outer membrane protein expression) system for outer membrane protein production, we were able to functionally express inH. pylori the cholera toxin B subunit genetically fused to the C-terminal VacA domain. We demonstrate that the fusion protein is translocated to the H. pylori outer membrane and that the CtxB domain is exposed on the H. pylori surface. Thus, we provide the first experimental evidence that the C-terminal β-domain of VacA can transport a foreign passenger protein to theH. pylori surface and hence acts as a functional autotransporter.

Download Full-text

Helicobacter pylori HP1512 Is a Nickel-Responsive NikR-Regulated Outer Membrane Protein

Infection and Immunity ◽

10.1128/iai.01188-06 ◽

2006 ◽

Vol 74 (12) ◽

pp. 6811-6820 ◽

Cited By ~ 36

Author(s):

Gregg S. Davis ◽

Erika L. Flannery ◽

Harry L. T. Mobley

Keyword(s):

Helicobacter Pylori ◽

Membrane Protein ◽

Outer Membrane ◽

Real Time ◽

Outer Membrane Protein ◽

Intergenic Region ◽

Calf Serum ◽

Nickel Concentration ◽

Wild Type ◽

H Pylori

ABSTRACT Helicobacter pylori is dependent upon the production of the highly abundant and active metalloenzyme urease for colonization of the human stomach. Thus, H. pylori has an absolute requirement for the transition metal nickel, a required cofactor for urease. To investigate the contribution of genes that are factors in this process, microarray analysis comparing the transcriptome of wild-type H. pylori 26695 cultured in brucella broth containing fetal calf serum (BBF) alone or supplemented with 100 μM NiCl2 suggested that HP1512 is repressed in the presence of 100 μM supplemental nickel. When measured by comparative real-time quantitative PCR (qPCR), HP1512 transcription was reduced 43-fold relative to the value for the wild type when cultured in BBF supplemented with 10 μM NiCl2. When grown in unsupplemented BBF, urease activity of an HP1512::cat mutant was significantly reduced compared to the wild type, 4.9 ± 0.5 μmol/min/mg of protein (n = 7) and 17.1 ± 4.9 μmol/min/mg of protein (n = 13), respectively (P < 0.0001). In silico analysis of the HP1511-HP1512 (HP1511-1512) intergenic region identified a putative NikR operator upstream of HP1512. Gel shift analysis with purified recombinant NikR verified nickel-dependent binding of H. pylori NikR to the HP1511-1512 intergenic region. Furthermore, comparative real-time qPCR of four nickel-related genes suggests that mutation of HP1512 results in reduced intracellular nickel concentration relative to wild-type H. pylori 26695. Taken together, these data suggest that HP1512 encodes a NikR-nickel-regulated outer membrane protein.

Download Full-text