The Effects of HP0044 and HP1275 Knockout Mutations on the Structure and Function of Lipopolysaccharide in Helicobacter pylori Strain 26695

Helicobacter pylori infection is associated with several gastric diseases, including gastritis, peptic ulcer, gastric adenocarcinoma and mucosa-associated lymphatic tissue (MALT) lymphoma. Due to the prevalence and severeness of H. pylori infection, a thorough understanding of this pathogen is necessary. Lipopolysaccharide, one of the major virulence factors of H. pylori, can exert immunomodulating and immunostimulating functions on the host. In this study, the HP0044 and HP1275 genes were under investigation. These two genes potentially encode GDP-D-mannose dehydratase (GMD) and phosphomannomutase (PMM)/phosphoglucomutase (PGM), respectively, and are involved in the biosynthesis of fucose. HP0044 and HP1275 knockout mutants were generated; both mutants displayed a truncated LPS, suggesting that the encoded enzymes are not only involved in fucose production but are also important for LPS construction. In addition, these two gene knockout mutants exhibited retarded growth, increased surface hydrophobicity and autoaggregation as well as being more sensitive to the detergent SDS and the antibiotic novobiocin. Furthermore, the LPS-defective mutants also had significantly reduced bacterial infection, adhesion and internalization in the in vitro cell line model. Moreover, disruptions of the HP0044 and HP1275 genes in H. pylori altered protein sorting into outer membrane vesicles. The critical roles of HP0044 and HP1275 in LPS biosynthesis, bacterial fitness and pathogenesis make them attractive candidates for drug inventions against H. pylori infection.

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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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Carbonic Anhydrases: New Perspectives on Protein Functional Role and Inhibition in Helicobacter pylori

Frontiers in Microbiology ◽

10.3389/fmicb.2021.629163 ◽

2021 ◽

Vol 12 ◽

Author(s):

Cristina Campestre ◽

Viviana De Luca ◽

Simone Carradori ◽

Rossella Grande ◽

Vincenzo Carginale ◽

...

Keyword(s):

Helicobacter Pylori ◽

Pathogenic Bacteria ◽

Acid Tolerance ◽

Membrane Vesicles ◽

Research Field ◽

Outer Membrane Vesicles ◽

Bacterial Toxin ◽

Carbonic Anhydrases ◽

Ph Homeostasis ◽

H Pylori

Our understanding of the function of bacterial carbonic anhydrases (CAs, EC 4.2.1.1) has increased significantly in the last years. CAs are metalloenzymes able to modulate CO2, HCO3– and H+ concentration through their crucial role in catalysis of reversible CO2 hydration (CO2 + H2O ⇄ HCO3– + H+). In all living organisms, CA activity is linked to physiological processes, such as those related to the transport and supply of CO2 or HCO3–, pH homeostasis, secretion of electrolytes, biosynthetic processes and photosynthesis. These important processes cannot be ensured by the very low rate of the non-catalyzed reaction of CO2 hydration. It has been recently shown that CAs are important biomolecules for many bacteria involved in human infections, such as Vibrio cholerae, Brucella suis, Salmonella enterica, Pseudomonas aeruginosa, and Helicobacter pylori. In these species, CA activity promotes microorganism growth and adaptation in the host, or modulates bacterial toxin production and virulence. In this review, recent literature in this research field and some of the above-mentioned issues are discussed, namely: (i) the implication of CAs from bacterial pathogens in determining the microorganism growth and virulence; (ii) the druggability of these enzymes using classical CA inhibitors (CAIs) of the sulfonamide-type as examples; (iii) the role played by Helicobacter pylori CAs in the acid tolerance/adaptation of the microbe within the human abdomen; (iv) the role of CAs played in the outer membrane vesicles spawned by H. pylori in its planktonic and biofilm phenotypes; (v) the possibility of using H. pylori CAIs in combination with probiotic strains as a novel anti-ulcer treatment approach. The latter approach may represent an innovative and successful strategy to fight gastric infections in the era of increasing resistance of pathogenic bacteria to classical antibiotics.

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Cholesteryl α-D-glucoside 6-acyltransferase enhances the adhesion of Helicobacter pylori to gastric epithelium

Communications Biology ◽

10.1038/s42003-020-0855-y ◽

2020 ◽

Vol 3 (1) ◽

Cited By ~ 2

Author(s):

Hau-Ming Jan ◽

Yi-Chi Chen ◽

Tsai-Chen Yang ◽

Lih-Lih Ong ◽

Chia-Chen Chang ◽

...

Keyword(s):

Helicobacter Pylori ◽

Bacterial Adhesion ◽

Direct Synthesis ◽

Membrane Vesicles ◽

Etiologic Agent ◽

Outer Membrane Vesicles ◽

Host Cells ◽

Gastric Epithelium ◽

Gastric Diseases ◽

Host Cell Membranes

AbstractHelicobacter pylori, the most common etiologic agent of gastric diseases including gastric cancer, is auxotrophic for cholesterol and has to hijack it from gastric epithelia. Upon uptake, the bacteria convert cholesterol to cholesteryl 6′-O-acyl-α-D-glucopyranoside (CAG) to promote lipid raft clustering in the host cell membranes. However, how CAG appears in the host to exert the pathogenesis still remains ambiguous. Herein we identified hp0499 to be the gene of cholesteryl α-D-glucopyranoside acyltransferase (CGAT). Together with cholesteryl glucosyltransferase (catalyzing the prior step), CGAT is secreted via outer membrane vesicles to the host cells for direct synthesis of CAG. This significantly enhances lipid rafts clustering, gathers adhesion molecules (including Lewis antigens and integrins α5, β1), and promotes more bacterial adhesion. Furthermore, the clinically used drug amiodarone was shown as a potent inhibitor of CGAT to effectively reduce the bacterial adhesion, indicating that CGAT is a potential target of therapeutic intervention.

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Functional Analysis of the cag Pathogenicity Island in Helicobacter pylori Isolates from Patients with Gastritis, Peptic Ulcer, and Gastric Cancer

Infection and Immunity ◽

10.1128/iai.72.2.1043-1056.2004 ◽

2004 ◽

Vol 72 (2) ◽

pp. 1043-1056 ◽

Cited By ~ 87

Author(s):

Steffen Backert ◽

Tobias Schwarz ◽

Stephan Miehlke ◽

Christian Kirsch ◽

Christian Sommer ◽

...

Keyword(s):

Gastric Cancer ◽

Helicobacter Pylori ◽

Peptic Ulcer ◽

Pathogenicity Island ◽

Epidemiological Studies ◽

Disease Development ◽

Gastric Diseases ◽

H Pylori ◽

Gastric Cancer Patients

ABSTRACT Helicobacter pylori is the causative agent of a variety of gastric diseases, but the clinical relevance of bacterial virulence factors is still controversial. Virulent strains carrying the cag pathogenicity island (cagPAI) are thought to be key players in disease development. Here, we have compared cagPAI-dependent in vitro responses in H. pylori isolates obtained from 75 patients with gastritis, peptic ulcer, and gastric cancer (n = 25 in each group). AGS gastric epithelial cells were infected with each strain and assayed for (i) CagA expression, (ii) translocation and tyrosine phosphorylation of CagA, (iii) c-Src inactivation, (iv) cortactin dephosphorylation, (v) induction of actin cytoskeletal rearrangements associated with cell elongation, (vi) induction of cellular motility, and (vii) secretion of interleukin-8. Interestingly, we found high but similar prevalences of all of these cagPAI-dependent host cell responses (ranging from 56 to 80%) among the various groups of patients. This study revealed CagA proteins with unique features, CagA subspecies of various sizes, and new functional properties for the phenotypic outcomes. We further showed that induction of AGS cell motility and elongation are two independent processes. Our data corroborate epidemiological studies, which indicate a significant association of cagPAI presence and functionality with histopathological findings in gastritis, peptic ulcer, and gastric cancer patients, thus emphasizing the importance of the cagPAI for the pathogenicity of H. pylori. Nevertheless, we found no significant association of the specific H. pylori-induced responses with any particular patient group. This may indicate that the determination of disease development is highly complex and involves multiple bacterial and/or host factors.

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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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Selective Inhibition of Helicobacter pylori Carbonic Anhydrases by Carvacrol and Thymol Could Impair Biofilm Production and the Release of Outer Membrane Vesicles

International Journal of Molecular Sciences ◽

10.3390/ijms222111583 ◽

2021 ◽

Vol 22 (21) ◽

pp. 11583

Author(s):

Rossella Grande ◽

Simone Carradori ◽

Valentina Puca ◽

Irene Vitale ◽

Andrea Angeli ◽

...

Keyword(s):

Helicobacter Pylori ◽

Outer Membrane ◽

Bacterial Resistance ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Peptic Ulcers ◽

Carbonic Anhydrases ◽

Biofilm Production ◽

Isoform Selectivity ◽

H Pylori

Helicobacter pylori, a Gram-negative neutrophilic pathogen, is the cause of chronic gastritis, peptic ulcers, and gastric cancer in humans. Current therapeutic regimens suffer from an emerging bacterial resistance rate and poor patience compliance. To improve the discovery of compounds targeting bacterial alternative enzymes or essential pathways such as carbonic anhydrases (CAs), we assessed the anti-H. pylori activity of thymol and carvacrol in terms of CA inhibition, isoform selectivity, growth impairment, biofilm production, and release of associated outer membrane vesicles-eDNA. The microbiological results were correlated by the evaluation in vitro of H. pylori CA inhibition, in silico analysis of the structural requirements to display such isoform selectivity, and the assessment of their limited toxicity against three probiotic species with respect to amoxicillin. Carvacrol and thymol could thus be considered as new lead compounds as alternative H. pylori CA inhibitors or to be used in association with current drugs for the management of H. pylori infection and limiting the spread of antibiotic resistance.

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In Silico Screening and In Vitro Assessment of Natural Products with Anti-Virulence Activity against Helicobacter pylori

Molecules ◽

10.3390/molecules27010020 ◽

2021 ◽

Vol 27 (1) ◽

pp. 20

Author(s):

Maciej Spiegel ◽

Paweł Krzyżek ◽

Ewa Dworniczek ◽

Ryszard Adamski ◽

Zbigniew Sroka

Keyword(s):

Helicobacter Pylori ◽

In Silico ◽

Stringent Response ◽

Human Pathogens ◽

Gastric Diseases ◽

Natural Substances ◽

Interesting Candidate ◽

H Pylori ◽

Static Conditions

Helicobacter pylori is one of the most frequent human pathogens and a leading etiological agent of various gastric diseases. As stringent response, coordinated by a SpoT protein, seems to be crucial for the survivability of H. pylori, the main goal of this article was to use in silico computational studies to find phytochemical compounds capable of binding to the active site of SpoT from H. pylori and confirm the ability of the most active candidates to interfere with the virulence of this bacterium through in vitro experiments. From 791 natural substances submitted for the virtual screening procedure, 10 were chosen and followed for further in vitro examinations. Among these, dioscin showed the most interesting parameters (the lowest MIC, the highest anti-biofilm activity in static conditions, and a relatively low stimulation of morphological transition into coccoids). Therefore, in the last part, we extended the research with a number of further experiments and observed the ability of dioscin to significantly reduce the formation of H. pylori biofilm under Bioflux-generated flow conditions and its capacity for additive enhancement of the antibacterial activity of all three commonly used antibiotics (clarithromycin, metronidazole, and levofloxacin). Based on these results, we suggest that dioscin may be an interesting candidate for new therapies targeting H. pylori survivability and virulence.

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The Role of E-cadherin in Helicobacter pylori-Related Gastric Diseases

Current Drug Metabolism ◽

10.2174/1389200219666180625113010 ◽

2019 ◽

Vol 20 (1) ◽

pp. 23-28

Author(s):

Yunzhan Zhang ◽

Danyan Li ◽

Yunkai Dai ◽

Ruliu Li ◽

Yong Gao ◽

...

Keyword(s):

Helicobacter Pylori ◽

Gastric Mucosa ◽

Precancerous Lesions ◽

Research Literature ◽

Gastric Diseases ◽

H Pylori ◽

The Relationship ◽

E Cadherin

Background: Helicobacter pylori (H. pylori)-related gastric diseases are a series of gastric mucosal disorders associated with H. pylori infection. Gastric cancer (GC) is widely believed to evolve from gastritis and gastric ulcer. As an important adhesion molecule of epithelial cells, E-cadherin plays a key role in the development of gastric diseases. In this review, we aim to seek the characteristic of E-cadherin expression at different stages of gastric diseases. Methods: We searched plenty of databases for research literature about E-cadherin expression in H. pylori-related gastric diseases, and reviewed the relationship of E-cadherin and H. pylori, and the role of E-cadherin at different stages of gastric diseases. Results: H. pylori was shown to decrease E-cadherin expression by various ways in vitro, while most of clinical studies have not found the relationship between H. pylori and E-cadherin expression. It is defined that poor outcome of GC is related to loss expression of E-cadherin, but it is still unclear when qualitative change of E-cadherin expression in gastric mucosa emerges. Conclusion: Expression level of E-cadherin in gastric cells may be a consequence of injury factors and body’s selfrepairing ability. More studies on E-cadherin expression in gastric mucosa with precancerous lesions need to be performed, which may be potential and useful for early detection, prevention and treatment of GC.

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The Effects of Sulglycotide on the Adhesion and the Inflammation of Helicobacter Pylori

International Journal of Environmental Research and Public Health ◽

10.3390/ijerph17082918 ◽

2020 ◽

Vol 17 (8) ◽

pp. 2918

Author(s):

Ji Yeong Yang ◽

Pumsoo Kim ◽

Seok-Hoo Jeong ◽

Seong Woong Lee ◽

Yu Sik Myung ◽

...

Keyword(s):

Helicobacter Pylori ◽

Protein A ◽

Enzyme Linked Immunosorbent Assay ◽

Etiologic Factor ◽

Dilution Method ◽

Gastric Diseases ◽

Ags Cells ◽

Human Gastric Adenocarcinoma ◽

H Pylori

Helicobacter pylori (H. pylori) is a primary etiologic factor in gastric diseases. Sulglycotide is a glycopeptide derived from pig duodenal mucin. Esterification of its carbohydrate chains with sulfate groups creates a potent gastroprotective agent used to treat various gastric diseases. We investigated the inhibitory effects of sulglycotide on adhesion and inflammation after H. pylori infection in human gastric adenocarcinoma cells (AGS cells). H. pylori reference strain 60190 (ATCC 49503) was cultured on Brucella agar supplemented with 10% bovine serum. Sulgylcotide-mediated growth inhibition of H. pylori was evaluated using the broth dilution method. Inhibition of H. pylori adhesion to AGS cells by sulglycotide was assessed using a urease assay. Effects of sulglycotide on the translocation of virulence factors was measured using western blot to detect cytotoxin-associated protein A (CagA) and vacuolating cytotoxin A (VacA) proteins. Inhibition of IL-8 secretion was measured using enzyme-linked immunosorbent assay (ELISA) to determine the effects of sulglycotide on inflammation. Sulglycotide did not inhibit the growth of H. pylori, however, after six and 12 hours of infection on AGS cells, H. pylori adhesion was significantly inhibited by approximately 60% by various concentrations of sulglycotide. Sulglycotide decreased H. pylori virulence factor (CagA and VacA) translocation to AGS cells and inhibited IL-8 secretion. Sulglycotide inhibited H. pylori adhesion and inflammation after infection of AGS cells in vitro. These results support the use of sulglycotide to treat H. pylori infections.

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Helicobacter pylori-Derived Outer Membrane Vesicles (OMVs): Role in Bacterial Pathogenesis?

Microorganisms ◽

10.3390/microorganisms8091328 ◽

2020 ◽

Vol 8 (9) ◽

pp. 1328 ◽

Cited By ~ 2

Author(s):

Miroslaw Jarzab ◽

Gernot Posselt ◽

Nicole Meisner-Kober ◽

Silja Wessler

Keyword(s):

Helicobacter Pylori ◽

Outer Membrane ◽

Current Knowledge ◽

Membrane Vesicles ◽

Bacterial Pathogenesis ◽

Outer Membrane Vesicles ◽

Host Cells ◽

Cellular Mechanisms ◽

Wide Range ◽

H Pylori

Persistent infections with the human pathogen Helicobacter pylori (H. pylori) have been closely associated with the induction and progression of a wide range of gastric disorders, including acute and chronic gastritis, ulceration in the stomach and duodenum, mucosa-associated lymphoid tissue (MALT) lymphoma, and gastric adenocarcinoma. The pathogenesis of H. pylori is determined by a complicated network of manifold mechanisms of pathogen–host interactions, which involves a coordinated interplay of H. pylori pathogenicity and virulence factors with host cells. While these molecular and cellular mechanisms have been intensively investigated to date, the knowledge about outer membrane vesicles (OMVs) derived from H. pylori and their implication in bacterial pathogenesis is not well developed. In this review, we summarize the current knowledge on H. pylori-derived OMVs.

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