Evaluation of an immunofluorescence assay for specific detection of immunoglobulin G antibodies directed against Helicobacter pylori, and antigenic cross-reactivity between H. pylori and Campylobacter jejuni.

SUMMARYAn enzyme-linked immunosorbent assay has been used to diagnose serologically the prevalence ofHelicobacter pyloriinfection in Asian life-long vegans. There was no difference in the seropositivity between these individuals and a group of age-and sex-matched Asian meat-eaters, indicating the meat consumption is not a risk factor forH. pyloriinfection. However, both Asian groups had a higher prevalence of infection than age- and sex-matched Caucasian meat-eaters. Additionally, the Asian individuals had a wider range of specific IgG antibody concentrations than the Caucasians. This did not appear to be due to antigenic cross-reactivity betweenH. pyloriandCampylobacter jejuni. The significance of these observations to the establishment of cut-off levels for the serodiagnosis of certain ethnic groups is discussed.

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Tailor-Made Detection of Individual Phosphorylated and Non-Phosphorylated EPIYA-Motifs of Helicobacter pylori Oncoprotein CagA

Cancers ◽

10.3390/cancers11081163 ◽

2019 ◽

Vol 11 (8) ◽

pp. 1163 ◽

Cited By ~ 1

Author(s):

Pachathundikandi ◽

Gutiérrez-Escobar ◽

Tegtmeyer

Keyword(s):

Helicobacter Pylori ◽

Cross Reactivity ◽

Host Cells ◽

Gastric Disease ◽

Effector Protein ◽

Sequence Motifs ◽

Type Iv ◽

Efficient Detection ◽

H Pylori ◽

Epiya Motifs

The gastric pathogen and carcinogen Helicobacter pylori (H. pylori) encodes a type IV secretion system for translocation of the effector protein CagA into host cells. Injected CagA becomes tyrosine-phosphorylated at the five amino acid residue Glutamate-Proline- Isoleucine-Tyrosine-Alanine (EPIYA)-sequence motifs. These phosphorylated EPIYA-sites represent recognition motifs for binding of multiple host factors, which then manipulate signaling pathways to trigger gastric disease. Thus, efficient detection of single phosphorylated EPIYA-motifs in CagA is required. Detection of phospho-CagA is primarily performed using commercial pan-phosphotyrosine antibodies. However, those antibodies were originally generated to recognize many phosphotyrosines in various mammalian proteins and are not optimized for use in bacteria. To address this important limitation, we synthesized 11-mer phospho- and non-phospho-peptides from EPIYA-motifs A, B, and C, and produced three phospho-specific and three non-phospho-specific rabbit polyclonal CagA antibodies. These antibodies specifically recognized the corresponding phosphorylated and non-phosphorylated EPIYA-motifs, while the EPIYA-C antibodies also recognized the related East-Asian EPIYA-D motif. Otherwise, no cross-reactivity of the antibodies among EPIYAs was observed. Western blotting demonstrated that each EPIYA-motif can be predominantly phosphorylated during H. pylori infection. This represents the first complete set of phospho-specific antibodies for an effector protein in bacteria, providing useful tools to gather information for the categorization of CagA phosphorylation, cancer signaling, and gastric disease progression.

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Identification of immunodominant Helicobacter pylori proteins with reactivity to H. pylori-specific egg-yolk immunoglobulin

Journal of Medical Microbiology ◽

10.1099/jmm.0.04978-0 ◽

2003 ◽

Vol 52 (3) ◽

pp. 217-222 ◽

Cited By ~ 28

Author(s):

Ji-Hyun Shin ◽

Seung-Woo Nam ◽

Jung-Taik Kim ◽

Jong-Bok Yoon ◽

Won-Gi Bang ◽

...

Keyword(s):

Helicobacter Pylori ◽

Egg Yolk ◽

Cross Reactivity ◽

Heat Shock Protein 60 ◽

Α Subunit ◽

Whole Cell ◽

Whole Cell Lysate ◽

Cell Lysate ◽

Normal Human ◽

H Pylori

The importance of hens eggs as a source of specific antibodies (IgY) is well recognized. The protective effect of IgY obtained from hens immunized with Helicobacter pylori whole-cell lysate has been reported for the control of H. pylori infection. However, IgY produced by whole-cell lysates presents the possibility of cross-reactivity with other bacteria, including the normal human flora, and this could decrease the efficiency of IgY. In the present study, the immunodominant proteins of H. pylori with reactivity to H. pylori-specific IgY (IgY-Hp) were identified. IgY obtained from hens immunized with various fractions of H. pylori proteins was isolated and purified, titres of IgY-Hp against H. pylori were determined and cross-reactivity between IgY-Hp and normal human bacteria was examined by Western blot analysis. Finally, immunodominant H. pylori proteins were identified by LC/MS analysis. IgY obtained 2 months after immunization with H. pylori whole-cell lysate showed the highest antibody titre. Five immunodominant proteins were identified that were strongly reactive to IgY-Hp: urease β-subunit (62 kDa), heat-shock protein 60 (60 kDa), urease α-subunit (26 kDa), probable peroxiredoxin (22 kDa) and probable thiol peroxidase (18 kDa). Immunization of hens with the immunodominant proteins identified would produce a more specific IgY against H. pylori.

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Antiparasitic Drug Nitazoxanide Inhibits the Pyruvate Oxidoreductases of Helicobacter pylori, Selected Anaerobic Bacteria and Parasites, and Campylobacter jejuni

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01159-06 ◽

2006 ◽

Vol 51 (3) ◽

pp. 868-876 ◽

Cited By ~ 133

Author(s):

Paul S. Hoffman ◽

Gary Sisson ◽

Matthew A. Croxen ◽

Kevin Welch ◽

W. Dean Harman ◽

...

Keyword(s):

Helicobacter Pylori ◽

Campylobacter Jejuni ◽

Trichomonas Vaginalis ◽

Enzymatic Reaction ◽

Anaerobic Bacteria ◽

Spectral Shift ◽

Thiamine Pyrophosphate ◽

Escape Mutation ◽

Thiazole Ring ◽

H Pylori

ABSTRACT Nitazoxanide (NTZ) exhibits broad-spectrum activity against anaerobic bacteria and parasites and the ulcer-causing pathogen Helicobacter pylori. Here we show that NTZ is a noncompetitive inhibitor (Ki , 2 to 10 μM) of the pyruvate:ferredoxin/flavodoxin oxidoreductases (PFORs) of Trichomonas vaginalis, Entamoeba histolytica, Giardia intestinalis, Clostridium difficile, Clostridium perfringens, H. pylori, and Campylobacter jejuni and is weakly active against the pyruvate dehydrogenase of Escherichia coli. To further mechanistic studies, the PFOR operon of H. pylori was cloned and overexpressed in E. coli, and the multisubunit complex was purified by ion-exchange chromatography. Pyruvate-dependent PFOR activity with NTZ, as measured by a decrease in absorbance at 418 nm (spectral shift from 418 to 351 nm), unlike the reduction of viologen dyes, did not result in the accumulation of products (acetyl coenzyme A and CO2) and pyruvate was not consumed in the reaction. NTZ did not displace the thiamine pyrophosphate (TPP) cofactor of PFOR, and the 351-nm absorbing form of NTZ was inactive. Optical scans and 1H nuclear magnetic resonance analyses determined that the spectral shift (A 418 to A 351) of NTZ was due to protonation of the anion (NTZ−) of the 2-amino group of the thiazole ring which could be generated with the pure compound under acidic solutions (pKa = 6.18). We propose that NTZ− intercepts PFOR at an early step in the formation of the lactyl-TPP transition intermediate, resulting in the reversal of pyruvate binding prior to decarboxylation and in coordination with proton transfer to NTZ. Thus, NTZ might be the first example of an antimicrobial that targets the “activated cofactor” of an enzymatic reaction rather than its substrate or catalytic sites, a novel mechanism that may escape mutation-based drug resistance.

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Immunoglobulin G Antibody Response to Infection with Coccoid Forms of Helicobacter pylori

Clinical and Vaccine Immunology ◽

10.1128/cdli.9.5.1067-1071.2002 ◽

2002 ◽

Vol 9 (5) ◽

pp. 1067-1071 ◽

Cited By ~ 4

Author(s):

G. Figueroa ◽

G. Faúndez ◽

M. Troncoso ◽

P. Navarrete ◽

M. S. Toledo

Keyword(s):

Helicobacter Pylori ◽

Immunoglobulin G ◽

Gel Electrophoresis ◽

Potential Role ◽

Enzyme Linked Immunosorbent Assay ◽

Nonulcer Dyspepsia ◽

Duodenal Ulcers ◽

One Dimensional ◽

H Pylori ◽

Asymptomatic Individuals

ABSTRACT An increasing number of studies support a potential role for coccoid forms in Helicobacter pylori infection. Evidence for this was obtained through scanning microscopy, genetic analysis for virulence traits, examination of the presence and activity of key enzymes, and other methods. We studied the serum immunoglobulin G responses to coccoid H. pylori forms by enzyme-linked immunosorbent assay (ELISA) and immunoblotting and compared them with those of bacillary cells. Sera from a total of 295 infected individuals were studied; these included sera from 100 patients with duodenal ulcers, 98 patients with nonulcer dyspepsia, 11 patients with gastroduodenal cancer, and 86 asymptomatic individuals. Initially, we characterized and selected coccoid and bacillary antigenic preparations by one-dimensional (1-D) and 2-D gel electrophoresis and immunoblotting. Data showed that coccoid and bacillary preparations with comparable protein contents have similar patterns in 1-D and 2-D electrophoresis gels and antigenic recognition at blotting. These results revealed that coccoid and spiral antigens in ELISA can equally recognize specific antibodies to H. pylori in sera from infected individuals. The analysis of the spiral and coccoid preparations by Western blotting showed no major differences in antigen recognition. No specific bands or profiles associated with a single gastric condition were identified.

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Dynamics of Helicobacter pylori-Specific Immunoglobulin G for 2 Years after Successful Eradication of Helicobacter pylori Infection in an American Indian and Alaska Native Population

Clinical and Vaccine Immunology ◽

10.1128/cvi.00253-06 ◽

2006 ◽

Vol 14 (1) ◽

pp. 85-86 ◽

Cited By ~ 10

Author(s):

Karen M. Miernyk ◽

Dana L. Bruden ◽

Michael G. Bruce ◽

Brian J. McMahon ◽

Thomas W. Hennessy ◽

...

Keyword(s):

Helicobacter Pylori ◽

American Indian ◽

Immunoglobulin G ◽

Alaska Native ◽

Helicobacter Pylori Infection ◽

Native Population ◽

Specific Immunoglobulin ◽

H Pylori

ABSTRACT Helicobacter pylori antibodies were measured over 24 months in American Indian and Alaska Native persons who cleared their infections. Two months after treatment, 82% of H. pylori-negative persons remained seropositive. While there were declines in H. pylori antibodies for 12 months, after 24 months 71% of persons remained seropositive.

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Sero-Prevalence and Risk Factors for Helicobacter pylori Infection Among Individuals with and those Without Dyspepsia in Zaria, Northwestern Nigeria

Annals of Clinical and Experimental Medicine ◽

10.47838/acem.26011977.11162020.asmeda.12.0 ◽

2020 ◽

Vol 1 (1) ◽

pp. 73-76

Author(s):

Okonkwo Lilian Okwubenata ◽

Musa Bolanle Olufunke Priscilla ◽

Hali Bello ◽

Mustapha Shettima Kagu

Keyword(s):

Risk Factors ◽

Helicobacter Pylori ◽

Low Socioeconomic Status ◽

Immunoglobulin G ◽

Rural Areas ◽

Urban Areas ◽

Helicobacter Pylori Infection ◽

Cross Sectional ◽

H Pylori ◽

Study Participants

Background Helicobacter pylori infection is prevalent in African region especially in areas with low socioeconomic status. This study aimed to determine the prevalence and risk factors for Helicobacter pylori Infection among individuals with and those without dyspepsia Materials and Methods The study was cross sectional in which individuals with and those without dyspepsia were enrolled. The prevalence for Helicobacter pylori infection was determined by the screening of Helicobacter pylori immunoglobulin G and this was compared across variables of interest. IBM SPSS was used for the data analysis. Results Overall prevalence of Helicobacter pylori immunoglobulin G was 44 (37.9 %). There was no association between H pylori infection and dyspepsia (0.894). There was statistically significant association between residing in rural areas and acquiring of Helicobacter pylori infection (P= 0.011). Study participants from rural areas had significantly higher and lower pit latrines and water closet toilets respectively than the study participants who reside in urban areas (P= 0.0001). Conclusion Modest prevalence of Helicobacter pylori infection was observed and no association between H pylori infection and dyspepsia was observed. There is a need to take strategic measures towards improving level of socio-economic status of the rural areas so as to reduce the risk of contracting Helicobacter pylori infection in people living in rural areas

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Responses of Endoscopy Patients in Ladakh, India, to Helicobacter pylori Whole-Cell and CagA Antigens

Clinical and Vaccine Immunology ◽

10.1128/cdli.9.6.1313-1317.2002 ◽

2002 ◽

Vol 9 (6) ◽

pp. 1313-1317 ◽

Cited By ~ 7

Author(s):

Judith Romero-Gallo ◽

Guillermo I. Pérez-Pérez ◽

Richard P. Novick ◽

Patrick Kamath ◽

Tsering Norbu ◽

...

Keyword(s):

Helicobacter Pylori ◽

Immunoglobulin G ◽

Human Stomach ◽

Remote Area ◽

Serum Immunoglobulin ◽

Specific Enzyme ◽

Whole Cell ◽

Cell Antigen ◽

The World ◽

H Pylori

ABSTRACT Although Helicobacter pylori is a cosmopolitan colonizer of the human stomach, the responses among persons in remote populations from whom H. pylori was cultured have not been studied. We report on studies of 189 persons in the Ladakh region of India in whom serum immunoglobulin G responses to H. pylori whole-cell and CagA antigens were measured. H. pylori was isolated from 68 of these patients. An H. pylori whole-cell antigen derived from Ladakhi strains outperformed a similar antigen from U.S. strains, as determined by antigen-specific enzyme-linked immunosorbent assays. In total, 95% of the population was seropositive, including individuals responding only to the CagA antigen. Correlation with culture results showed that these were true positives and, therefore, that the H. pylori whole-cell serology was falsely negative in some cases. In addition to establishing a collection of H. pylori isolates from a remote area in the world, we show that use of H. pylori whole-cell and CagA serology together increases the sensitivity for the detection of colonization.

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Evaluation of the AnaeroPack Campylo System for Growth of Microaerophilic Bacteria

Journal of Clinical Microbiology ◽

10.1128/jcm.37.7.2376-2377.1999 ◽

1999 ◽

Vol 37 (7) ◽

pp. 2376-2377 ◽

Cited By ~ 3

Author(s):

Kenneth Van Horn ◽

Clara Tóth

Keyword(s):

New York ◽

Helicobacter Pylori ◽

Campylobacter Jejuni ◽

Growth Control ◽

Colony Morphology ◽

Generation System ◽

Becton Dickinson ◽

H Pylori ◽

Better Than

Growth of microaerophilic bacteria in the AnaeroPack Campylo (Mitsubishi Gas Chemical America, Inc., New York, N.Y.) atmosphere generation system was compared to growth in the CampyPak Plus jar and CampyPak pouch (Becton-Dickinson Microbiology Systems [BDMS], Cockeysville, Md.). Growth in the AnaeroPack Campylo system was considered equivalent to or better than growth obtained in the CampyPak Plus and CampyPak pouch systems for 48 of the 50 Helicobacter pylori strains and for all 28 Campylobacter species tested. All of the 78 organisms tested were recovered in each system in equivalent colony counts. Two strains of H. pylori grown in the AnaeroPack Campylo system were observed to have colony morphology growth discrepancies when compared to growth in the two BDMS systems. Atmosphere failure with the AnaeroPack Campylo was not detected withCampylobacter jejuni ATCC 33291 used as a growth control. The AnaeroPack Campylo system is easy to use and supports the growth of campylobacters and H. pylori.

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ChePep Controls Helicobacter pylori Infection of the Gastric Glands and Chemotaxis in the Epsilonproteobacteria

mBio ◽

10.1128/mbio.00098-11 ◽

2011 ◽

Vol 2 (4) ◽

Cited By ~ 61

Author(s):

Michael R. Howitt ◽

Josephine Y. Lee ◽

Paphavee Lertsethtakarn ◽

Roger Vogelmann ◽

Lydia-Marie Joubert ◽

...

Keyword(s):

Helicobacter Pylori ◽

Campylobacter Jejuni ◽

Deep Sea ◽

Hydrothermal Vent ◽

Bacterial Chemotaxis ◽

Peptic Ulcers ◽

Gastric Glands ◽

Content Type ◽

H Pylori ◽

Chemotaxis System

ABSTRACTMicrobes use directed motility to colonize harsh and dynamic environments. We discovered thatHelicobacter pyloristrains establish bacterial colonies deep in the gastric glands and identified a novel protein, ChePep, necessary to colonize this niche. ChePep is preferentially localized to the flagellar pole. Although mutants lacking ChePep have normal flagellar ultrastructure and are motile, they have a slight defect in swarming ability. By tracking the movement of single bacteria, we found that ∆ChePep mutants cannot control the rotation of their flagella and swim with abnormally frequent reversals. These mutants even sustain bursts of movement backwards with the flagella pulling the bacteria. Genetic analysis of the chemotaxis signaling pathway shows that ChePep regulates flagellar rotation through the chemotaxis system. By examiningH. pyloriwithin a microscopic pH gradient, we determined that ChePep is critical for regulating chemotactic behavior. ThechePepgene is unique to theEpsilonproteobacteriabut is found throughout this diverse group. We expressed ChePep from other members of theEpsilonproteobacteria, including the zoonotic pathogenCampylobacter jejuniand the deep sea hydrothermal vent inhabitantCaminibacter mediatlanticus, inH. pyloriand found that ChePep is functionally conserved across this class. ChePep represents a new family of chemotaxis regulators unique to theEpsilonproteobacteriaand illustrates the different strategies that microbes have evolved to control motility.IMPORTANCEHelicobacter pyloristrains infect half of all humans worldwide and contribute to the development of peptic ulcers and gastric cancer.H. pyloricannot survive within the acidic lumen of the stomach and uses flagella to actively swim to and colonize the protective mucus and epithelium. The chemotaxis system allowsH. pylorito navigate by regulating the rotation of its flagella. We identified a new protein, ChePep, which controls chemotaxis inH. pylori. ChePep mutants fail to colonize the gastric glands of mice and are completely outcompeted by normalH. pylori. Genes encoding ChePep are found only in the classEpsilonproteobacteria, which includes the human pathogenCampylobacter jejuniand environmental microbes like the deep-sea hydrothermal vent colonizerCaminibacter mediatlanticus, and we show that ChePep function is conserved in this class. Our study identifies a new colonization factor inH. pyloriand also provides insight into the control and evolution of bacterial chemotaxis.

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