scholarly journals Isolation Method and Characterization of Outer Membranes Vesicles of Helicobacter pylori Grown in a Chemically Defined Medium

2021 ◽  
Vol 12 ◽  
Author(s):  
Joana Melo ◽  
Vanessa Pinto ◽  
Tânia Fernandes ◽  
Ana R. Malheiro ◽  
Hugo Osório ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Bacterial Growth ◽  
Membrane Vesicles ◽  
Defined Medium ◽  
Outer Membrane Vesicles ◽  
Bona Fide ◽  
Cost Efficient ◽  
H Pylori ◽  
Defined Culture ◽  
Protocol Standardization

Outer membrane vesicles (OMVs) are small vesicles constitutively shed by all Gram-negative bacterium, which have been proposed to play a role in Helicobacter pylori persistence and pathogenesis. The methods currently available for the isolation of H. pylori OMVs are diverse and time-consuming, raising the need for a protocol standardization, which was the main aim of this study. Here, we showed that the chemically defined F12 medium, supplemented with cholesterol, nutritionally supports bacterial growth and maintains H. pylori viability for at least 72 h. Additionally, we developed an abridged protocol for isolation of OMVs from these bacterial cultures, which comprises a low-speed centrifugation, supernatant filtration through a 0.45 μm pore, and two ultracentrifugations for OMVs’ recovery and washing. Using this approach, a good yield of highly pure bona fide OMVs was recovered from cultures of different H. pylori strains and in different periods of bacterial growth, as assessed by nanoparticle tracking analysis, transmission electron microscopy (TEM), and proteomic analyses, confirming the reliability of the protocol. Analysis of the proteome of OMVs isolated from H. pylori F12-cholesterol cultures at different time points of bacterial growth revealed differentially expressed proteins, including the vacuolating cytotoxin VacA. In conclusion, this work proposes a time- and cost-efficient protocol for the isolation of H. pylori OMVs from a chemically defined culture medium that is suitable for implementation in research and in the biopharmaceutical field.

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

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.

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

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.

scholarly journals Induction of iron regulated proteins during normal growth of Neisseria meningitidis in a chemically defined medium

1994 ◽  
Vol 36 (4) ◽  
pp. 301-310 ◽  
Author(s):  
Maria Cristina de Cunto Brandileone ◽  
Rosemeire Cobo Zanella ◽  
Vera Simonsen Dias Vieira ◽  
Claudio Tavares Sacciii ◽  
Lucimar Gonçalves Milagres ◽  
...  
Keyword(s):  
Bacterial Growth ◽  
Culture Supernatant ◽  
Culture Media ◽  
Membrane Vesicles ◽  
Normal Growth ◽  
Growth Conditions ◽  
Defined Medium ◽  
Outer Membrane Vesicles ◽  
Iron Deficient

The expression of iron regulated proteins (IRPs) in vitro has been obtained in the past by adding iron chelators to the culture after bacterial growth, in the presence of an organic iron source. We have investigated aspects concerning full expression of the meningococcal IRPs during normal growth, in defined conditions using Catlin medium, Mueller Hinton and Tryptic Soy Broth (TSB). The expression of IRPs varied between different strains with respect to Ethylenediamine Di-ortho-Hidroxy-phenyl-acetic acid (EDDA) concentrations, and according to culture medium, and also between different lots of TSB. For each strain, a specific set of IRPs were expressed and higher EDDA concentrations, or addition of glucose, or use of different culture media did not resulted in a differential expression of IRPs. We were not able to grow N. meningitidis under normal growth conditions using Desferal. We looked for a good yield of outer membrane vesicles (OMVs) expressing IRPs in iron-deficient Catlin medium containing EDDA and Hemin. Culture for 32 h at 30ºC after growing for 16 h at 37ºC supported good bacterial growth. Bacterial lysis was noted after additional 24 h at 30ºC. Approximately 4 times more OMVs was recoverable from a culture supernatant after 24 h at 30ºC than from the cells after 16 h at 37ºC. The IRP were as well expressed in OMVs from culture supernatant obtained after 24 h at 30ºC as from the cells after 16 h at 37ºC.

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

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.

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

2020 ◽  
Vol 8 (9) ◽  
pp. 1328 ◽  
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.

scholarly journals Helicobacter pylori Virulence Factors Exploiting Gastric Colonization and its Pathogenicity

Toxins ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 677 ◽  
Author(s):  
Shamshul Ansari ◽  
Yoshio Yamaoka
Keyword(s):  
Helicobacter Pylori ◽  
Virulence Factors ◽  
Bacterial Colonization ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Bacterial Virulence ◽  
Effector Proteins ◽  
Host Genetic ◽  
Colonization Factors ◽  
H Pylori

Helicobacter pylori colonizes the gastric epithelial cells of at least half of the world’s population, and it is the strongest risk factor for developing gastric complications like chronic gastritis, ulcer diseases, and gastric cancer. To successfully colonize and establish a persistent infection, the bacteria must overcome harsh gastric conditions. H. pylori has a well-developed mechanism by which it can survive in a very acidic niche. Despite bacterial factors, gastric environmental factors and host genetic constituents together play a co-operative role for gastric pathogenicity. The virulence factors include bacterial colonization factors BabA, SabA, OipA, and HopQ, and the virulence factors necessary for gastric pathogenicity include the effector proteins like CagA, VacA, HtrA, and the outer membrane vesicles. Bacterial factors are considered more important. Here, we summarize the recent information to better understand several bacterial virulence factors and their role in the pathogenic mechanism.

scholarly journals Crude Preparations of Helicobacter pylori Outer Membrane Vesicles Induce Upregulation of Heme Oxygenase-1 via Activating Akt-Nrf2 and mTOR–IκB Kinase–NF-κB Pathways in Dendritic Cells

2016 ◽  
Vol 84 (8) ◽  
pp. 2162-2174 ◽  
Author(s):  
Su Hyuk Ko ◽  
Da Jeong Rho ◽  
Jong Ik Jeon ◽  
Young-Jeon Kim ◽  
Hyun Ae Woo ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Dendritic Cells ◽  
Outer Membrane ◽  
Heme Oxygenase ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Heme Oxygenase 1 ◽  
Content Type ◽  
Iκb Kinase ◽  
H Pylori

Helicobacter pylorisheds outer membrane vesicles (OMVs) that contain many surface elements of bacteria. Dendritic cells (DCs) play a major role in directing the nature of adaptive immune responses againstH. pylori, and heme oxygenase-1 (HO-1) has been implicated in regulating function of DCs. In addition, HO-1 is important for adaptive immunity and the stress response. AlthoughH. pylori-derived OMVs may contribute to the pathogenesis ofH. pyloriinfection, responses of DCs to OMVs have not been elucidated. In the present study, we investigated the role ofH. pylori-derived crude OMVs in modulating the expression of HO-1 in DCs. Exposure of DCs to crudeH. pyloriOMVs upregulated HO-1 expression. Crude OMVs obtained from acagA-negative isogenic mutant strain induced less HO-1 expression than OMVs obtained from a wild-type strain. CrudeH. pyloriOMVs activated signals of transcription factors such as NF-κB, AP-1, and Nrf2. Suppression of NF-κB or Nrf2 resulted in significant attenuation of crude OMV-induced HO-1 expression. Crude OMVs increased the phosphorylation of Akt and downstream target molecules of mammalian target of rapamycin (mTOR), such as S6 kinase 1 (S6K1). Suppression of Akt resulted in inhibition of crude OMV-induced Nrf2-dependent HO-1 expression. Furthermore, suppression of mTOR was associated with inhibition of IκB kinase (IKK), NF-κB, and HO-1 expression in crude OMV-exposed DCs. These results suggest thatH. pylori-derived OMVs regulate HO-1 expression through two different pathways in DCs, Akt-Nrf2 and mTOR–IKK–NF-κB signaling. Following this induction, increased HO-1 expression in DCs may modulate inflammatory responses inH. pyloriinfection.

scholarly journals Helicobacter pylori Outer Membrane Vesicles Modulate Proliferation and Interleukin-8 Production by Gastric Epithelial Cells

2003 ◽  
Vol 71 (10) ◽  
pp. 5670-5675 ◽  
Author(s):  
Salim Ismail ◽  
Mark B. Hampton ◽  
Jacqueline I. Keenan
Keyword(s):  
Helicobacter Pylori ◽  
Epithelial Cells ◽  
Outer Membrane ◽  
Strain Differences ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Interleukin 8 ◽  
Low Grade ◽  
Gastric Epithelial Cells ◽  
H Pylori

ABSTRACT Helicobacter pylori infection, which is always associated with gastritis, can progress to ulceration or malignancy. The diversity in clinical outcomes is partly attributed to the expression of virulence factors and adhesins by H. pylori. However, H. pylori may not have to adhere to the epithelium to cause gastritis. We hypothesize that outer membrane vesicles (OMV), which are constantly shed from the surface of H. pylori, play a role as independent activators of host cell responses. In this study, we found that low doses of OMV from cag PAI+ toxigenic and cag PAI− nontoxigenic strains increased proliferation of AGS gastric epithelial cells. At higher doses, we detected growth arrest, increased toxicity, and interleukin-8 (IL-8) production. The only strain differences detected were vacuolation with the toxigenic strain and higher levels of IL-8 production with OMV from the cag PAI− nontoxigenic strain. In summary, we suggest that constitutively shed OMV play a role in promoting the low-grade gastritis associated with H. pylori infection.

scholarly journals Uptake of Helicobacter pylori Outer Membrane Vesicles by Gastric Epithelial Cells

2010 ◽  
Vol 78 (12) ◽  
pp. 5054-5061 ◽  
Author(s):  
Heather Parker ◽  
Kenny Chitcholtan ◽  
Mark B. Hampton ◽  
Jacqueline I. Keenan
Keyword(s):  
Helicobacter Pylori ◽  
Epithelial Cells ◽  
Outer Membrane ◽  
Mutant Strain ◽  
Membrane Vesicles ◽  
Human Stomach ◽  
Outer Membrane Vesicles ◽  
Gastric Epithelial Cells ◽  
Isogenic Mutant Strain ◽  
H Pylori

ABSTRACT Helicobacter pylori bacteria colonize the human stomach where they stimulate a persistent inflammatory response. H. pylori is considered noninvasive; however, lipopolysaccharide (LPS)-enriched outer membrane vesicles (OMV), continuously shed from the surface of this bacterium, are observed within gastric epithelial cells. The mechanism of vesicle uptake is poorly understood, and this study was undertaken to examine the roles of bacterial VacA cytotoxin and LPS in OMV binding and cholesterol and clathrin-mediated endocytosis in vesicle uptake by gastric epithelial cells. OMV association was examined using a fluorescent membrane dye to label OMV, and a comparison was made between the associations of vesicles from a VacA+ strain and OMV from a VacA− isogenic mutant strain. Within 20 min, essentially all associated OMV were intracellular, and vesicle binding appeared to be facilitated by the presence of VacA cytotoxin. Uptake of vesicles from the VacA+ strain was inhibited by H. pylori LPS (58% inhibition with 50 μg/ml LPS), while uptake of OMV from the VacA− mutant strain was less affected (25% inhibition with 50 μg/ml LPS). Vesicle uptake did not require cholesterol. However, uptake of OMV from the VacA− mutant strain was inhibited by a reduction in clathrin-mediated endocytosis (42% with 15 μg/ml chlorpromazine), while uptake of OMV from the VacA+ strain was less affected (25% inhibition with 15 μg/ml chlorpromazine). We conclude that VacA toxin enhances the association of H. pylori OMV with cells and that the presence of the toxin may allow vesicles to exploit more than one pathway of internalization.

scholarly journals Protective effects of Helicobacter pylori membrane vesicles against stress and antimicrobial agents

Microbiology ◽  
2020 ◽  
Vol 166 (8) ◽  
pp. 751-758 ◽  
Author(s):  
Benjamin Oliver Murray ◽  
Robin Andrew Dawson ◽  
Lolwah Mohammad Alsharaf ◽  
Jody Anne Winter
Keyword(s):  
Helicobacter Pylori ◽  
Type Species ◽  
Pathogenic Bacteria ◽  
Bacterial Species ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Host Cells ◽  
Content Type ◽  
Link Type ◽  
H Pylori

Outer-membrane vesicles (OMVs) produced by Helicobacter pylori deliver bacterial components to host cells, provide a mechanism for stabilization of secreted components and may allow the bacteria to exert ‘long-range’ effects in the gastric niche, promoting persistence. In addition to their well-characterized host cell interactions, membrane vesicles improve stress survival in other bacterial species, and are constitutively produced by both pathogenic and non-pathogenic bacteria. We aimed to determine whether OMVs could improve H. pylori survival of a range of stressors. The effects of purified OMVs on the resistance of H. pylori to a range of environmental and antimicrobial stresses were determined using growth curves and survival assays. Addition of purified OMVs to H. pylori cultures provided dose-dependent protection against hydrogen peroxide-mediated killing. Supplementation with OMVs also partially protected H. pylori against the bactericidal effects of the antibiotics clarithromycin and levofloxacin, but not against amoxicillin nor metronidazole. Addition of purified OMVs allowed H. pylori to grow in the presence of inhibitory concentrations of the antimicrobial peptide LL-37. In the presence of 50 µg OMVs ml−1, significantly enhanced H. pylori growth was observed at higher LL-37 concentrations compared with lower LL-37 concentrations, suggesting that OMV–LL-37 interactions might facilitate release of growth-promoting nutrients. Taken together, these data indicate that production of membrane vesicles could help H. pylori to survive exposure to antibiotics and host antimicrobial defences during infection.

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