Loss of a Cardiolipin Synthase in Helicobacter pylori G27 Blocks Flagellum Assembly

ABSTRACT Helicobacter pylori uses a cluster of polar, sheathed flagella for motility, which it requires for colonization of the gastric epithelium in humans. As part of a study to identify factors that contribute to localization of the flagella to the cell pole, we disrupted a gene encoding a cardiolipin synthase (clsC) in H. pylori strains G27 and B128. Flagellum biosynthesis was abolished in the H. pylori G27 clsC mutant but not in the B128 clsC mutant. Transcriptome sequencing analysis showed that flagellar genes encoding proteins needed early in flagellum assembly were expressed at wild-type levels in the G27 clsC mutant. Examination of the G27 clsC mutant by cryo-electron tomography indicated the mutant assembled nascent flagella that contained the MS ring, C ring, flagellar protein export apparatus, and proximal rod. Motile variants of the G27 clsC mutant were isolated after allelic exchange mutagenesis using genomic DNA from the B128 clsC mutant as the donor. Genome resequencing of seven motile G27 clsC recipients revealed that each isolate contained the flgI (encodes the P-ring protein) allele from B128. Replacing the flgI allele in the G27 clsC mutant with the B128 flgI allele rescued flagellum biosynthesis. We postulate that H. pylori G27 FlgI fails to form the P ring when cardiolipin levels in the cell envelope are low, which blocks flagellum assembly at this point. In contrast, H. pylori B128 FlgI can form the P ring when cardiolipin levels are low and allows for the biosynthesis of mature flagella. IMPORTANCE H. pylori colonizes the epithelial layer of the human stomach, where it can cause a variety of diseases, including chronic gastritis, peptic ulcer disease, and gastric cancer. To colonize the stomach, H. pylori must penetrate the viscous mucous layer lining the stomach, which it accomplishes using its flagella. The significance of our research is identifying factors that affect the biosynthesis and assembly of the H. pylori flagellum, which will contribute to our understanding of motility in H. pylori, as well as other bacterial pathogens that use their flagella for host colonization.

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DC-LAMP+Dendritic Cells Are Recruited to Gastric Lymphoid Follicles in Helicobacter pylori-Infected Individuals

Infection and Immunity ◽

10.1128/iai.00801-13 ◽

2013 ◽

Vol 81 (10) ◽

pp. 3684-3692 ◽

Cited By ~ 6

Author(s):

Malin Hansson ◽

Malin Sundquist ◽

Susanne Hering ◽

B. Samuel Lundin ◽

Michael Hermansson ◽

...

Keyword(s):

Helicobacter Pylori ◽

Dendritic Cells ◽

Gastric Mucosa ◽

Human Gastric Mucosa ◽

Lymphoid Follicles ◽

Content Type ◽

Ulcer Disease ◽

Hla Dr ◽

H Pylori ◽

Antigen Presenting

ABSTRACTInfection withHelicobacter pyloriis associated with development of ulcer disease and gastrointestinal adenocarcinoma. The infection leads to a large infiltration of immune cells and the formation of organized lymphoid follicles in the human gastric mucosa. Still, the immune system fails to eradicate the bacteria, and the substantial regulatory T cell (Treg) response elicited is probably a major factor permitting bacterial persistence. Dendritic cells (DCs) are professional antigen-presenting cells that can activate naive T cells, and maturation of DCs is crucial for the initiation of primary immune responses. The aim of this study was to investigate the presence and localization of mature human DCs inH. pylori-infected gastric mucosa. Gastric antral biopsy specimens were collected from patients withH. pylori-associated gastritis and healthy volunteers, and antrum tissue was collected from patients undergoing gastric resection. Immunohistochemistry and flow cytometry showed that DCs expressing the maturation marker dendritic cell lysosome-associated membrane glycoprotein (DC-LAMP; CD208) are enriched in theH. pylori-infected gastric mucosa and that these DCs are specifically localized within or close to lymphoid follicles. Gastric DC-LAMP-positive (DC-LAMP+) DCs express CD11c and high levels of HLA-DR but little CD80, CD83, and CD86. Furthermore, immunofluorescence analyses demonstrated that DC-LAMP+DCs are in the same location as FoxP3-positive putative Tregs in the follicles. In conclusion, we show that DC-LAMP+DCs with low costimulatory capacity accumulate in the lymphoid follicles in humanH. pylori-infected gastric tissue, and our results suggest that Treg-DC interactions may promote chronic infection by rendering gastric DCs tolerogenic.

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Helicobacter pylori AlpA and AlpB Bind Host Laminin and Influence Gastric Inflammation in Gerbils

Infection and Immunity ◽

10.1128/iai.01275-10 ◽

2011 ◽

Vol 79 (8) ◽

pp. 3106-3116 ◽

Cited By ~ 59

Author(s):

Olga A. Senkovich ◽

Jun Yin ◽

Viktoriya Ekshyyan ◽

Carolyn Conant ◽

James Traylor ◽

...

Keyword(s):

Helicobacter Pylori ◽

Wild Type Strain ◽

Host Tissue ◽

Gastric Epithelium ◽

Gastric Tissue ◽

Wild Type ◽

Binding Ability ◽

Content Type ◽

H Pylori ◽

Laminin Binding

ABSTRACTHelicobacter pyloripersistently colonizes humans, causing gastritis, ulcers, and gastric cancer. Adherence to the gastric epithelium has been shown to enhance inflammation, yet only a fewH. pyloriadhesins have been paired with targets in host tissue. ThealpABlocus has been reported to encode adhesins involved in adherence to human gastric tissue. We report that abrogation ofH. pyloriAlpA and AlpB reduces binding ofH. pylorito laminin while expression of plasmid-bornealpAoralpBconfers laminin-binding ability toEscherichia coli. AnH. pyloristrain lacking only AlpB is also deficient in laminin binding. Thus, we conclude that both AlpA and AlpB contribute toH. pylorilaminin binding. Contrary to expectations, theH. pyloriSS1 mutant deficient in AlpA and AlpB causes more severe inflammation than the isogenic wild-type strain in gerbils. Identification of laminin as the target of AlpA and AlpB will facilitate future investigations of host-pathogen interactions occurring duringH. pyloriinfection.

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Immune responses to Helicobacter pylori colonization: mechanisms and clinical outcomes

Clinical Science ◽

10.1042/cs20050232 ◽

2006 ◽

Vol 110 (3) ◽

pp. 305-314 ◽

Cited By ~ 51

Author(s):

Cynthia Portal-Celhay ◽

Guillermo I. Perez-Perez

Keyword(s):

Immune Response ◽

Helicobacter Pylori ◽

Immune Responses ◽

Gastric Lymphoma ◽

Gastric Epithelium ◽

Immune Effector ◽

Ulcer Disease ◽

Gastroduodenal Disease ◽

Increased Risk ◽

H Pylori

Helicobacter pylori colonizes the stomachs of half of the world's population and usually persists in the gastric mucosa of human hosts for decades or life. Although most H. pylori-positive people are asymptomatic, the presence of H. pylori is associated with increased risk for the development of peptic ulcer disease, gastric adenocarcinoma and gastric lymphoma. The development of a sustained gastric inflammatory and immune response to infection appears to be pivotal for the development of disease. During its long co-existence with humans, H. pylori has evolved complex strategies to maintain a mild inflammation of the gastric epithelium while limiting the extent of immune effector activity. In this review, the nature of the host immune response to H. pylori infection and the mechanism employed by the bacterium to evade them is considered. Understanding the mechanisms of colonization, persistence and virulence factors of the bacterium as well as the innate and adaptive immune responses of the host are critically important for the development of new strategies to prevent the development of H. pylori-induced gastroduodenal disease.

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In VivoExpression of Helicobacter pylori Virulence Genes in Patients with Gastritis, Ulcer, and Gastric Cancer

Infection and Immunity ◽

10.1128/iai.05845-11 ◽

2011 ◽

Vol 80 (2) ◽

pp. 594-601 ◽

Cited By ~ 12

Author(s):

Francisco Avilés-Jiménez ◽

Adriana Reyes-Leon ◽

Erik Nieto-Patlán ◽

Lori M. Hansen ◽

Juan Burgueño ◽

...

Keyword(s):

Gastric Cancer ◽

Helicobacter Pylori ◽

Environmental Conditions ◽

Virulence Genes ◽

Content Type ◽

Ulcer Disease ◽

Type Iv ◽

H Pylori

ABSTRACTThe best-studiedHelicobacter pylorivirulence factor associated with development of peptic ulcer disease or gastric cancer (GC) rather than asymptomatic nonatrophic gastritis (NAG) is thecagpathogenicity island (cagPAI), which encodes a type IV secretion system (T4SS) that injects the CagA oncoprotein into host epithelial cells. Here we used real-time reverse transcription-PCR (RT-PCR) to measure thein vivoexpression of genes on thecagPAI and of other virulence genes in patients with NAG, duodenal ulcer (DU), or GC.In vivoexpression ofH. pylorivirulence genes was greater overall in gastric biopsy specimens of patients with GC than in those of patients with NAG or DU. However, sincein vitroexpression ofcagAwas not greater inH. pyloristrains from patients with GC than in those from patients with NAG or DU, increased expression in GCin vivois likely a result of environmental conditions in the gastric mucosa, though it may in turn cause more severe pathology. Increased expression of virulence genes in GC may represent a stress response to elevated pH or other environmental conditions in the stomach of patients with GC, which may be less hospitable toH. pyloricolonization than the acidic environment in patients with NAG or DU.

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Twin-Arginine Translocation System in Helicobacter pylori: TatC, but Not TatB, Is Essential for Viability

mBio ◽

10.1128/mbio.01016-13 ◽

2014 ◽

Vol 5 (1) ◽

Cited By ~ 16

Author(s):

Stéphane L. Benoit ◽

Robert J. Maier

Keyword(s):

Helicobacter Pylori ◽

Homologous Recombination ◽

Signal Sequence ◽

Cell Envelope ◽

Wild Type ◽

Content Type ◽

Twin Arginine Translocation ◽

Tat System ◽

Gastric Pathogen ◽

H Pylori

ABSTRACT The twin-arginine translocation (Tat) system, needed to transport folded proteins across biological membranes, has not been characterized in the gastric pathogen Helicobacter pylori. Analysis of all H. pylori genome sequences available thus far reveals the presence of single copies of tatA, tatB, and tatC needed for the synthesis of a fully functional Tat system. Based on the presence of the twin-arginine hallmark in their signal sequence, only four H. pylori proteins appear to be Tat dependent: hydrogenase (HydA), catalase-associated protein (KapA), biotin sulfoxide reductase (BisC), and the ubiquinol cytochrome oxidoreductase Rieske protein (FbcF). In the present study, targeted mutations were aimed at tatA, tatB, tatC, or queA (downstream gene control). While double homologous recombination mutations in tatB and queA were easily obtained, attempts at disrupting tatA proved unsuccessful, while deletion of tatC led to partial mutants following single homologous recombination, with cells retaining a chromosomal copy of tatC. Double homologous recombination tatC mutants were obtained only when a plasmid-borne, isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible copy of tatC was introduced prior to transformation. These conditional tatC mutants could grow only in the presence of IPTG, suggesting that tatC is essential in H. pylori. tatB and tatC mutants had lower hydrogenase and catalase activities than the wild-type strain did, and the ability of tatC mutants to colonize mouse stomachs was severely affected compared to the wild type. Chromosomal complementation of tatC mutants restored hydrogenase and catalase activities to wild-type levels, and additional expression of tatC in wild-type cells resulted in elevated Tat-dependent enzyme activities. Unexpectedly, the tat strains had cell envelope defects. IMPORTANCE This work reports the first characterization of the twin-arginine translocation (Tat) system in the gastric pathogen Helicobacter pylori. While tatB mutants were easily obtained, only single-crossover partial tatC mutants or conditional tatC mutants could be generated, indicating that tatC is essential in H. pylori, a surprising finding given the fact that only four proteins are predicted to be translocated by the Tat system in this bacterium. The levels of activity of hydrogenase and catalase, two of the predicted Tat-dependent enzymes, were affected in these mutants. In addition, all tat mutants displayed cell envelope defects, and tatC mutants were deficient in mouse colonization.

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Flagellar Localization of a Helicobacter pylori Autotransporter Protein

mBio ◽

10.1128/mbio.00613-12 ◽

2013 ◽

Vol 4 (2) ◽

Cited By ~ 17

Author(s):

Jana N. Radin ◽

Jennifer A. Gaddy ◽

Christian González-Rivera ◽

John T. Loh ◽

Holly M. Scott Algood ◽

...

Keyword(s):

Helicobacter Pylori ◽

Bacterial Infections ◽

Secreted Proteins ◽

Wild Type ◽

Gram Negative ◽

Content Type ◽

Ulcer Disease ◽

H Pylori ◽

Type V

ABSTRACTHelicobacter pyloricontains four genes that are predicted to encode proteins secreted by the autotransporter (type V) pathway. One of these, the pore-forming toxin VacA, has been studied in great detail, but thus far there has been very little investigation of three VacA-like proteins. We show here that all three VacA-like proteins are >250 kDa in mass and localized on the surface ofH. pylori. The expression of the threevacA-like genes is upregulated duringH. pyloricolonization of the mouse stomach compared toH. pylorigrowthin vitro, and a wild-typeH. pyloristrain outcompeted each of the three corresponding isogenic mutant strains in its ability to colonize the mouse stomach. One of the VacA-like proteins localizes to a sheath that overlies the flagellar filament and bulb, and therefore, we designate it FaaA (flagella-associated autotransporter A). In comparison to a wild-typeH. pyloristrain, an isogenicfaaAmutant strain exhibits decreased motility, decreased flagellar stability, and an increased proportion of flagella in a nonpolar site. The flagellar localization of FaaA differs markedly from the localization of other known autotransporters, and the current results reveal an important role of FaaA in flagellar localization and motility.IMPORTANCEThe pathogenesis of most bacterial infections is dependent on the actions of secreted proteins, and proteins secreted by the autotransporter pathway constitute the largest family of secreted proteins in pathogenic Gram-negative bacteria. In this study, we analyzed three autotransporter proteins (VacA-like proteins) produced byHelicobacter pylori, a Gram-negative bacterium that colonizes the human stomach and contributes to the pathogenesis of gastric cancer and peptic ulcer disease. We demonstrate that these three proteins each enhance the capacity ofH. pylorito colonize the stomach. Unexpectedly, one of these proteins (FaaA) is localized to a sheath that overliesH. pyloriflagella. The absence of FaaA results in decreasedH. pylorimotility as well as a reduction in flagellar stability and a change in flagellar localization. The atypical localization of FaaA reflects a specialized function of this autotransporter designed to optimizeH. pyloricolonization of the gastric niche.

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Helicobacter pylori Biofilm Involves a Multigene Stress-Biased Response, Including a Structural Role for Flagella

mBio ◽

10.1128/mbio.01973-18 ◽

2018 ◽

Vol 9 (5) ◽

Cited By ~ 14

Author(s):

Skander Hathroubi ◽

Julia Zerebinski ◽

Karen M. Ottemann

Keyword(s):

Helicobacter Pylori ◽

Stress Response ◽

Biofilm Formation ◽

Extracellular Polymeric Substances ◽

Cell Envelope ◽

Flagellar Apparatus ◽

Planktonic Cells ◽

Content Type ◽

H Pylori ◽

Biofilm Matrix

ABSTRACT Helicobacter pylori has an impressive ability to persist chronically in the human stomach. Similar characteristics are associated with biofilm formation in other bacteria. The H. pylori biofilm process, however, is poorly understood. To gain insight into this mode of growth, we carried out comparative transcriptomic analysis between H. pylori biofilm and planktonic cells, using the mouse-colonizing strain SS1. Optimal biofilm formation was obtained with a low concentration of serum and 3 days of growth, conditions that caused both biofilm and planktonic cells to be ∼80% coccoid. Transcriptome sequencing (RNA-seq) analysis found that 8.18% of genes were differentially expressed between biofilm and planktonic cell transcriptomes. Biofilm-downregulated genes included those involved in metabolism and translation, suggesting these cells have low metabolic activity. Biofilm-upregulated genes included those whose products were predicted to be at the cell envelope, involved in regulating a stress response, and surprisingly, genes related to formation of the flagellar apparatus. Scanning electron microscopy visualized flagella that appeared to be a component of the biofilm matrix, supported by the observation that an aflagellated mutant displayed a less robust biofilm with no apparent filaments. We observed flagella in the biofilm matrix of additional H. pylori strains, supporting that flagellar use is widespread. Our data thus support a model in which H. pylori biofilm involves a multigene stress-biased response and that flagella play an important role in H. pylori biofilm formation. IMPORTANCE Biofilms, communities of bacteria that are embedded in a hydrated matrix of extracellular polymeric substances, pose a substantial health risk and are key contributors to many chronic and recurrent infections. Chronicity and recalcitrant infections are also common features associated with the ulcer-causing human pathogen H. pylori. However, relatively little is known about the role of biofilms in H. pylori pathogenesis, as well as the biofilm structure itself and the genes associated with this mode of growth. In the present study, we found that H. pylori biofilm cells highly expressed genes related to cell envelope and stress response, as well as those encoding the flagellar apparatus. Flagellar filaments were seen in high abundance in the biofilm. Flagella are known to play a role in initial biofilm formation, but typically are downregulated after that state. H. pylori instead appears to have coopted these structures for nonmotility roles, including a role building a robust biofilm.

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Helicobacter pylori Outer Membrane Vesicles and Extracellular Vesicles from Helicobacter pylori-Infected Cells in Gastric Disease Development

International Journal of Molecular Sciences ◽

10.3390/ijms22094823 ◽

2021 ◽

Vol 22 (9) ◽

pp. 4823

Author(s):

María Fernanda González ◽

Paula Díaz ◽

Alejandra Sandoval-Bórquez ◽

Daniela Herrera ◽

Andrew F. G. Quest

Keyword(s):

Gastric Cancer ◽

Helicobacter Pylori ◽

Outer Membrane ◽

Extracellular Vesicles ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Host Cells ◽

Gastric Epithelium ◽

Infected Cells ◽

H Pylori

Extracellular vesicles (EVs) are cell-derived vesicles important in intercellular communication that play an essential role in host-pathogen interactions, spreading pathogen-derived as well as host-derived molecules during infection. Pathogens can induce changes in the composition of EVs derived from the infected cells and use them to manipulate their microenvironment and, for instance, modulate innate and adaptive inflammatory immune responses, both in a stimulatory or suppressive manner. Gastric cancer is one of the leading causes of cancer-related deaths worldwide and infection with Helicobacter pylori (H. pylori) is considered the main risk factor for developing this disease, which is characterized by a strong inflammatory component. EVs released by host cells infected with H. pylori contribute significantly to inflammation, and in doing so promote the development of disease. Additionally, H. pylori liberates vesicles, called outer membrane vesicles (H. pylori-OMVs), which contribute to atrophia and cell transformation in the gastric epithelium. In this review, the participation of both EVs from cells infected with H. pylori and H. pylori-OMVs associated with the development of gastric cancer will be discussed. By deciphering which functions of these external vesicles during H. pylori infection benefit the host or the pathogen, novel treatment strategies may become available to prevent disease.

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The Hsp60 Protein of Helicobacter Pylori Exhibits Chaperone and ATPase Activities at Elevated Temperatures

BioChem ◽

10.3390/biochem1010002 ◽

2021 ◽

Vol 1 (1) ◽

pp. 19-25

Author(s):

Jose A. Mendoza ◽

Julian L. Ignacio ◽

Christopher M. Buckley

Keyword(s):

Helicobacter Pylori ◽

Heat Stress ◽

Molecular Chaperone ◽

Elevated Temperatures ◽

Citrate Synthase ◽

Gastric Epithelium ◽

Acidic Conditions ◽

H Pylori ◽

Induced Aggregation ◽

Hydrolysis Of

The heat-shock protein, Hsp60, is one of the most abundant proteins in Helicobacter pylori. Given its sequence homology to the Escherichia coli Hsp60 or GroEL, Hsp60 from H. pylori would be expected to function as a molecular chaperone in this organism. H. pylori is a type of bacteria that grows on the gastric epithelium, where the pH can fluctuate between neutral and 4.5, and the intracellular pH can be as low as 5.0. We previously showed that Hsp60 functions as a chaperone under acidic conditions. However, no reports have been made on the ability of Hsp60 to function as a molecular chaperone under other stressful conditions, such as heat stress or elevated temperatures. We report here that Hsp60 could suppress the heat-induced aggregation of the enzymes rhodanese, malate dehydrogenase, citrate synthase, and lactate dehydrogenase. Moreover, Hsp60 was found to have a potassium and magnesium-dependent ATPase activity that was stimulated at elevated temperatures. Although, Hsp60 was found to bind GTP, the hydrolysis of this nucleotide could not be observed. Our results show that Hsp60 from H. pylori can function as a molecular chaperone under conditions of heat stress.

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Nutrient Deficiency Promotes the Entry of Helicobacter pylori Cells into Candida Yeast Cells

Biology ◽

10.3390/biology10050426 ◽

2021 ◽

Vol 10 (5) ◽

pp. 426

Author(s):

Kimberly Sánchez-Alonzo ◽

Fabiola Silva-Mieres ◽

Luciano Arellano-Arriagada ◽

Cristian Parra-Sepúlveda ◽

Humberto Bernasconi ◽

...

Keyword(s):

Helicobacter Pylori ◽

Nutrient Deficiency ◽

Gastric Epithelium ◽

Yeast Cells ◽

Nutrient Concentrations ◽

Fetal Bovine ◽

H Pylori ◽

Pcr Techniques ◽

Strain Dependent

Helicobacter pylori, a Gram-negative bacterium, has as a natural niche the human gastric epithelium. This pathogen has been reported to enter into Candida yeast cells; however, factors triggering this endosymbiotic relationship remain unknown. The aim of this work was to evaluate in vitro if variations in nutrient concentration in the cultured medium trigger the internalization of H. pylori within Candida cells. We used H. pylori–Candida co-cultures in Brucella broth supplemented with 1%, 5% or 20% fetal bovine serum or in saline solution. Intra-yeast bacteria-like bodies (BLBs) were observed using optical microscopy, while intra-yeast BLBs were identified as H. pylori using FISH and PCR techniques. Intra-yeast H. pylori (BLBs) viability was confirmed using the LIVE/DEAD BacLight Bacterial Viability kit. Intra-yeast H. pylori was present in all combinations of bacteria–yeast strains co-cultured. However, the percentages of yeast cells harboring bacteria (Y-BLBs) varied according to nutrient concentrations and also were strain-dependent. In conclusion, reduced nutrients stresses H. pylori, promoting its entry into Candida cells. The starvation of both H. pylori and Candida strains reduced the percentages of Y-BLBs, suggesting that starving yeast cells may be less capable of harboring stressed H. pylori cells. Moreover, the endosymbiotic relationship between H. pylori and Candida is dependent on the strains co-cultured.

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