scholarly journals Proteins in the periplasmic space and outer membrane vesicles ofRhizobium etliCE3 grown in minimal medium are largely distinct and change with growth phase

2018 ◽  
Author(s):  
Hermenegildo Taboada ◽  
Niurka Meneses ◽  
Michael F. Dunn ◽  
Carmen Vargas-Lagunas ◽  
Natasha Buchs ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Minimal Medium ◽  
Proteome Analysis ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Periplasmic Space ◽  
Rhizobium Etli ◽  
Multidrug Efflux ◽  
Secretion Systems ◽  
Shock Proteins

ABSTRACTRhizobium etliCE3 grown in succinate-ammonium minimal medium (MM) excreted outer membrane vesicles (OMVs) with diameters of 40 to 100 nm. Proteins from the OMVs and the periplasmic space were isolated from 6 and 24 h cultures and identified by proteome analysis. A total 770 proteins were identified: 73.8 and 21.3 % of these proteins occurred only in the periplasm and OMVs, respectively, and only 4.9 % were found in both locations. The majority of proteins found in either location were present only at 6 or 24 h: in the periplasm and OMVs, only 24 and 9 % of proteins, respectively, were present at both sampling times, indicating a time-dependent differential sorting of proteins into the two compartments. The OMVs contained proteins with physiologically varied roles, includingRhizobiumadhering proteins (Rap), polysaccharidases, polysaccharide export proteins, autoaggregation and adherence proteins, glycosyl transferases, peptidoglycan binding and cross-linking enzymes, potential cell wall modifying enzymes, porins, multidrug efflux RND family proteins, ABC transporter proteins, and heat shock proteins. As expected, proteins with known periplasmic localizations (phosphatases, phosphodiesterases, pyrophosphatases) were found only in the periplasm, along with numerous proteins involved in amino acid and carbohydrate metabolism and transport. Nearly one-quarter of the proteins present in the OMVs were also found in our previous analysis of theR. etlitotal exproteome of MM-grown cells, indicating that these nanoparticles are an important mechanism for protein excretion in this species.IMPORTANCEThe reduction of atmospheric nitrogen to ammonia by rhizobia symbiotically associated with legumes is of major importance in sustainable agricultural. Rhizobia excrete a variety of symbiotically important proteins using canonical secretion systems. In this work, we show thatRhizobium etligrown in culture also excretes proteins in membrane-enclosed structures called outer membrane vesicles (OMVs). This study reports OMV production by rhizobia. Proteins identified in the OMVs included Rhizobium adhering (Rap) and autoaggregation proteins, polysaccharidases, RTX toxins, porins and multidrug efflux proteins. Some of these proteins have important roles in theR. etli-common bean symbiosis, and their packaging into OMVs could deliver them to the environment in a concentrated yet diffusible form protected from degradation. The work described here provides a basis for future studies on the function of rhizobial OMVs in free life and symbiosis.

scholarly journals Proteins in the periplasmic space and outer membrane vesicles of Rhizobium etli CE3 grown in minimal medium are largely distinct and change with growth phase

Microbiology ◽  
2019 ◽  
Vol 165 (6) ◽  
pp. 638-650 ◽  
Author(s):  
Hermenegildo Taboada ◽  
Niurka Meneses ◽  
Michael F. Dunn ◽  
Carmen Vargas-Lagunas ◽  
Natasha Buchs ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Growth Phase ◽  
Minimal Medium ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Periplasmic Space ◽  
Rhizobium Etli

scholarly journals Proteome analysis of outer membrane vesicles from a clinicalAcinetobacter baumanniiisolate

2009 ◽  
Vol 297 (2) ◽  
pp. 150-156 ◽  
Author(s):  
Sang-Oh Kwon ◽  
Yong Song Gho ◽  
Je Chul Lee ◽  
Seung Il Kim
Keyword(s):  
Outer Membrane ◽  
Proteome Analysis ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles

scholarly journals Proteome Analysis of Outer Membrane Vesicles From a Highly Virulent Strain of Haemophilus parasuis

2021 ◽  
Vol 8 ◽  
Author(s):  
Kunli Zhang ◽  
Pinpin Chu ◽  
Shuai Song ◽  
Dongxia Yang ◽  
Zhibiao Bian ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Virulent Strain ◽  
Bacterial Pathogen ◽  
Proteome Analysis ◽  
Membrane Vesicles ◽  
Subcellular Location ◽  
Outer Membrane Vesicles ◽  
Haemophilus Parasuis ◽  
Extracellular Milieu ◽  
Virulence Potential

Haemophilus parasuis has emerged as an important bacterial pathogen in pig husbandry, as H. parasuis can coinfect pigs with a variety of pathogenic microorganisms and further cause an aggravation of the disease. It is crucial to investigate its pathogenetic mechanism. Gram-negative bacteria naturally secrete outer membrane vesicles (OMVs), and their potent virulence factors play prominent roles that affect the interaction between bacteria and host. Still, the pathogenesis that is associated with the bacterial OMVs has not been well-elucidated. In this study, we investigated the secretion of OMVs from a clinical H. parasuis isolate strain (H45). In addition, we further analyzed the characterization, the comprehensive proteome, and the virulence potential of OMVs. Our data demonstrated that H. parasuis could secrete OMVs into the extracellular milieu during infection. Using liquid chromatography with tandem mass spectrometry (MS/MS) identification and bio-information analysis, we identified 588 different proteins associated with OMVs. Also, we also analyzed the subcellular location and biological function of those proteins. These proteins are mainly involved in immune and iron metabolism. Moreover, we confirmed the pathogenicity of H. parasuis OMVs by observing a strong inflammatory response in J774A.1 and porcine alveolar macrophages. Taken together, our findings suggested that OMVs from H. parasuis were involved in the pathogenesis of this bacterium during infection.

scholarly journals Discovery of Salmonella Virulence Factors Translocated via Outer Membrane Vesicles to Murine Macrophages

2011 ◽  
Vol 79 (6) ◽  
pp. 2182-2192 ◽  
Author(s):  
Hyunjin Yoon ◽  
Charles Ansong ◽  
Joshua N. Adkins ◽  
Fred Heffron
Keyword(s):  
Outer Membrane ◽  
Virulence Factors ◽  
Outer Membrane Proteins ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Host Cells ◽  
Secretion Systems ◽  
Homologous Proteins ◽  
Content Type ◽  
Small Proteins

ABSTRACTSalmonella entericaserovar Typhimurium, an intracellular pathogen and leading cause of food-borne illness, encodes a plethora of virulence effectors.Salmonellavirulence factors are translocated into host cells and manipulate host cellular activities, providing a more hospitable environment for bacterial proliferation. In this study, we report a new set of virulence factors that is translocated into the host cytoplasm via bacterial outer membrane vesicles (OMV). PagK (or PagK1), PagJ, and STM2585A (or PagK2) are small proteins composed of ∼70 amino acids and have high sequence homology to each other (>85% identity).Salmonellalacking all three homologues was attenuated for virulence in a mouse infection model, suggesting at least partial functional redundancy among the homologues. While each homologue was translocated into the macrophage cytoplasm, their translocation was independent of all threeSalmonellagene-encoded type III secretion systems (T3SSs)–Salmonellapathogenicity island 1 (SPI-1) T3SS, SPI-2 T3SS, and the flagellar system. Selected methods, including direct microscopy, demonstrated that the PagK-homologous proteins were secreted through OMV, which were enriched with lipopolysaccharide (LPS) and outer membrane proteins. Vesicles produced by intracellular bacteria also contained lysosome-associated membrane protein 1 (LAMP1), suggesting the possibility of OMV convergence with host cellular components during intracellular trafficking. This study identified novelSalmonellavirulence factors secreted via OMV and demonstrated that OMV can function as a vehicle to transfer virulence determinants to the cytoplasm of the infected host cell.

Qualitative changes in proteins contained in outer membrane vesicles produced by Rhizobium etli grown in the presence of the nod gene inducer naringenin

2019 ◽  
Vol 201 (9) ◽  
pp. 1173-1194
Author(s):  
Hermenegildo Taboada ◽  
Michael F. Dunn ◽  
Niurka Meneses ◽  
Carmen Vargas-Lagunas ◽  
Natasha Buchs ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Rhizobium Etli ◽  
Qualitative Changes

scholarly journals Campylobacter jejuni Outer Membrane Vesicles Play an Important Role in Bacterial Interactions with Human Intestinal Epithelial Cells

2012 ◽  
Vol 80 (12) ◽  
pp. 4089-4098 ◽  
Author(s):  
Abdi Elmi ◽  
Eleanor Watson ◽  
Pamela Sandu ◽  
Ozan Gundogdu ◽  
Dominic C. Mills ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Campylobacter Jejuni ◽  
Virulence Factors ◽  
Intestinal Epithelial Cells ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Host Cells ◽  
Intestinal Epithelial ◽  
Secretion Systems ◽  
Content Type

ABSTRACTCampylobacter jejuniis the most prevalent cause of food-borne gastroenteritis in the developed world; however, the molecular basis of pathogenesis is unclear. Secretion of virulence factors is a key mechanism by which enteric bacterial pathogens interact with host cells to enhance survival and/or damage the host. However,C. jejunilacks the virulence-associated secretion systems possessed by other enteric pathogens. Many bacterial pathogens utilize outer membrane vesicles (OMVs) for delivery of virulence factors into host cells. In the absence of prototypical virulence-associated secretion systems, OMVs could be an important alternative for the coordinated delivery ofC. jejuniproteins into host cells. Proteomic analysis ofC. jejuni11168H OMVs identified 151 proteins, including periplasmic and outer membrane-associated proteins, but also many determinants known to be important in survival and pathogenesis, including the cytolethal distending toxin (CDT).C. jejuniOMVs contained 16N-linked glycoproteins, indicating a delivery mechanism by which these periplasm-located yet immunogenic glycoproteins can interact with host cells.C. jejuniOMVs possess cytotoxic activity and induce a host immune response from T84 intestinal epithelial cells (IECs), which was not reduced by OMV pretreatment with proteinase K or polymyxin B prior to coincubation with IECs. Pretreatment of IECs with methyl-beta-cyclodextrin partially blocks OMV-induced host immune responses, indicating a role for lipid rafts in host cell plasma membranes during interactions withC. jejuniOMVs. OMVs isolated from aC. jejuni11168HcdtAmutant induced interleukin-8 (IL-8) to the same extent as did wild-type OMVs, suggesting OMV induction of IL-8 is independent of CDT.

scholarly journals Bacterial outer membrane vesicles provide an alternative pathway for trafficking of type III secreted effectors into epithelial cells

2018 ◽  
Author(s):  
Natalie Sirisaengtaksin ◽  
Eloise J. O’Donoghue ◽  
Sara Jabbari ◽  
Andrew J. Roe ◽  
Anne Marie Krachler
Keyword(s):  
Outer Membrane ◽  
Disease Severity ◽  
Type Iii Secretion ◽  
Diarrheal Disease ◽  
Alternative Pathway ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Host Cells ◽  
Secretion Systems ◽  
Type Iii

ABSTRACTOuter membrane vesicles (OMVs) are proteo-liposomes universally shed by Gram-negative bacteria. Their secretion is significantly enhanced by the transition into the intra-host milieu and OMVs have been shown to play critical roles during pathogenesis. EnterohemorrhagicEscherichia coliO157 (EHEC), causes diarrheal disease in humans, and soluble toxins including Shiga-like toxins that contribute to disease severity and clinical complications including hemolytic uremic syndrome, have been shown to be OMV associated. In addition to Shiga-like toxins, EHEC produces a type III secretion system (T3SS), and T3SS effectors are associated with colonization and disease severityin vivo. Here, we show that type III secreted substrates including translocators and effectors are incorporated into OMVs independent of type III secretion activity. EHEC strains with non-functional type III secretion systems shed more OMVs and vesicles enter host cells with accelerated kinetics compared to vesicles shed from wild type EHEC. The T3SS effector translocated intimin receptor (Tir) is trafficked from OMVs into host cells and localizes to the membrane. However, its clustering on the host membrane and co-localization with bacterial pedestals is intimin-dependent. We further show that OMV-delivered Tir can cross-complement an effector-deficient EHEC strain, demonstrating that OMV-associated effectors reach the host cell in a biologically intact form. Finally, we observe that the non-LEE encoded E3 ubiquitin ligase effector NleL is also trafficked to host cells via OMVs, where it ubiquitinylates its target kinase JNK. Together, these data demonstrate that trafficking of OMV-associated effectors is a novel and T3SS-independent pathway for the delivery of active effectors to host cells.

Electrophysiological Characterization of Transport Across Outer Membrane Channels from Gram-Negative Bacteria in Their Native Environment

2019 ◽  
Author(s):  
Jiajun Wang ◽  
Rémi Terrasse ◽  
Jayesh Arun Bafna ◽  
Lorraine Benier ◽  
Mathias Winterhalter
Keyword(s):  
Outer Membrane ◽  
Lipid Membrane ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Multi Drug Resistance ◽  
Gram Negative Bacteria ◽  
Membrane Channels ◽  
Gram Negative ◽  
Electrophysiological Characterization

Multi-drug resistance in Gram-negative bacteria is often associated with low permeability of the outer membrane. To investigate the role of membrane channels in the uptake of antibiotics, we extract, purify and reconstitute them into artificial planar membranes. To avoid this time-consuming procedure, here we show a robust approach using fusion of native outer membrane vesicles (OMV) into planar lipid bilayer which moreover allows also to some extend the characterization of membrane protein channels in their native environment. Two major membrane channels from <i>Escherichia coli</i>, OmpF and OmpC, were overexpressed from the host and the corresponding OMVs were collected. Each OMV fusion revealed surprisingly single or only few channel activities. The asymmetry of the OMV´s translates after fusion into the lipid membrane with the LPS dominantly present at the side of OMV addition. Compared to conventional reconstitution methods, the channels fused from OMVs containing LPS have similar conductance but a much broader distribution. The addition of Enrofloxacin on the LPS side yields somewhat higher association (<i>k<sub>on</sub></i>) and lower dissociation (<i>k<sub>off</sub></i>) rates compared to LPS-free reconstitution. We conclude that using outer membrane vesicles is a fast and easy approach for functional and structural studies of membrane channels in the native membrane.

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