Outer Membrane Vesicles Mediate Transport of Biologically Active Vibrio cholerae Cytolysin (VCC) from V. cholerae Strains

Cholera toxin (CT), the major virulence factor of Vibrio cholerae, is an AB5 toxin secreted through the type II secretion system (T2SS). Upon secretion, the toxin initiates endocytosis through the interaction of the B pentamer with the GM1 ganglioside receptor on small intestinal cells. In addition to the release of CT in the free form, the bacteria secrete CT in association with outer membrane vesicles (OMVs). Previously, we demonstrated that strain 569B releases OMVs that encapsulate CT and which interact with host cells in a GM1-independent mechanism. Here, we have demonstrated that OMV-encapsulated CT, while biologically active, does not exist in an AB5 form; rather, the OMVs encapsulate two enzymatic A-subunit (CTA) polypeptides. We further investigated the assembly and secretion of the periplasmic CT and found that a major fraction of periplasmic CTA does not participate in the CT assembly process and instead is continuously encapsulated within the OMVs. Additionally, we found that the encapsulation of CTA fragments in OMVs is conserved among several Inaba O1 strains. We further found that under conditions in which the amount of extracellularly secreted CT increases, the concentration of OMV-encapsulated likewise CTA increases. These results point to a secondary mechanism for the secretion of biologically active CT that does not depend on the CTB-GM1 interaction for endocytosis.

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Naturally Occurring IgG Antibodies Provide Innate Protection against Vibrio cholerae Bacteremia by Recognition of the Outer Membrane Protein U

Journal of Innate Immunity ◽

10.1159/000443646 ◽

2016 ◽

Vol 8 (3) ◽

pp. 269-283 ◽

Cited By ~ 17

Author(s):

Kyaw Min Aung ◽

Annika E. Sjöström ◽

Ulrich von Pawel-Rammingen ◽

Kristian Riesbeck ◽

Bernt Eric Uhlin ◽

...

Keyword(s):

Membrane Protein ◽

Outer Membrane ◽

Vibrio Cholerae ◽

Outer Membrane Protein ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Immune Defense ◽

Dependent Manner ◽

Naturally Occurring ◽

Serum Killing

Cholera epidemics are caused by Vibrio cholerae serogroups O1 and O139, whereas strains collectively known as non-O1/non-O139 V. cholerae are found in cases of extraintestinal infections and bacteremia. The mechanisms and factors influencing the occurrence of bacteremia and survival of V. cholerae in normal human serum have remained unclear. We found that naturally occurring IgG recognizing V. cholerae outer membrane protein U (OmpU) mediates a serum-killing effect in a complement C1q-dependent manner. Moreover, outer membrane vesicles (OMVs) containing OmpU caused enhanced survival of highly serum-sensitive classical V. cholerae in a dose-dependent manner. OMVs from wild-type and ompU mutant V. cholerae thereby provided a novel means to verify by extracellular transcomplementation the involvement of OmpU. Our data conclusively indicate that loss, or reduced expression, of OmpU imparts resistance to V. cholerae towards serum killing. We propose that the difference in OmpU protein levels is a plausible reason for differences in serum resistance and the ability to cause bacteremia observed among V. cholerae biotypes. Our findings provide a new perspective on how naturally occurring antibodies, perhaps induced by members of the microbiome, may play a role in the recognition of pathogens and the provocation of innate immune defense against bacteremia.

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Cytotoxic Necrotizing Factor Type 1 Delivered by Outer Membrane Vesicles of Uropathogenic Escherichia coli Attenuates Polymorphonuclear Leukocyte Antimicrobial Activity and Chemotaxis

Infection and Immunity ◽

10.1128/iai.00637-06 ◽

2006 ◽

Vol 74 (8) ◽

pp. 4401-4408 ◽

Cited By ~ 54

Author(s):

Jon M. Davis ◽

Humberto M. Carvalho ◽

Susan B. Rasmussen ◽

Alison D. O'Brien

Keyword(s):

Escherichia Coli ◽

Outer Membrane ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Biologically Active ◽

Single Amino Acid ◽

Upec Strain ◽

Cytotoxic Necrotizing Factor ◽

Factor Type

ABSTRACT Cytotoxic necrotizing factor type 1 (CNF1), a toxin produced by many strains of uropathogenic Escherichia coli (UPEC), constitutively activates small GTPases of the Rho family by deamidating a single amino acid within these target proteins. Such activated GTPases not only stimulate actin polymerization within affected cells but also, as we previously reported, decrease membrane fluidity on mouse polymorphonuclear leukocytes (PMNs). In that same investigation we found that this diminished membrane movement impedes the clustering of the complement receptor CD11b/CD18 on PMNs and, in turn, decreases PMN phagocytic capacity and microbicidal activity on PMNs in direct contact with CNF1-expressing UPEC as well as on those in proximity to wild-type UPEC. The latter observation suggested to us that CNF1 is released from neighboring bacteria, although at the time of initiation of the study described here, no specific mechanism for export of CNF1 from UPEC had been described. Here we present evidence that CNF1 is released from the CNF1-expressing UPEC strain CP9 (serotype O4/H5/K54) in a complex with outer membrane vesicles (OMVs) and that these CNF1-bearing vesicles transfer biologically active CNF1 to PMNs and attenuate phagocyte function. Furthermore, we show that CNF1-bearing vesicles act in a dose-dependent fashion on PMNs to inhibit their chemotactic response to formyl-Met-Leu-Phe, while purified CNF1 does not. We conclude that OMVs provide a means for delivery of CNF1 from a UPEC strain to PMNs and thus negatively affect the efficacy of the acute inflammatory response to these organisms.

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Immunization of Mice With Vibrio cholerae Outer-Membrane Vesicles Protects Against Hyperinfectious Challenge and Blocks Transmission

The Journal of Infectious Diseases ◽

10.1093/infdis/jir756 ◽

2011 ◽

Vol 205 (3) ◽

pp. 412-421 ◽

Cited By ~ 27

Author(s):

Anne L. Bishop ◽

Abdullah A. Tarique ◽

Bharathi Patimalla ◽

Stephen B. Calderwood ◽

Firdausi Qadri ◽

...

Keyword(s):

Outer Membrane ◽

Vibrio Cholerae ◽

Membrane Vesicles ◽

Outer Membrane Vesicles

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Faculty Opinions recommendation of Vibrio cholerae Outer Membrane Vesicles Inhibit Bacteriophage Infection.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.733061186.793558877 ◽

2019 ◽

Author(s):

Chris Waters

Keyword(s):

Outer Membrane ◽

Vibrio Cholerae ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Bacteriophage Infection

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Immunization with Vibrio cholerae Outer Membrane Vesicles Induces Protective Immunity in Mice

Infection and Immunity ◽

10.1128/iai.00532-08 ◽

2008 ◽

Vol 76 (10) ◽

pp. 4554-4563 ◽

Cited By ~ 104

Author(s):

Stefan Schild ◽

Eric J. Nelson ◽

Andrew Camilli

Keyword(s):

Immune Responses ◽

Outer Membrane ◽

Vibrio Cholerae ◽

High Titer ◽

Immunoglobulin A ◽

Membrane Vesicles ◽

Oral Route ◽

Outer Membrane Vesicles ◽

Intranasal Route ◽

Female Mice

ABSTRACT The gram-negative bacterium Vibrio cholerae releases outer membrane vesicles (OMVs) during growth. In this study, we immunized female mice by the intranasal, intragastric, or intraperitoneal route with purified OMVs derived from V. cholerae. Independent of the route of immunization, mice induced specific, high-titer immune responses of similar levels against a variety of antigens present in the OMVs. After the last immunization, the half-maximum total immunoglobulin titer was stable over a 3-month period, indicating that the immune response was long lasting. The induction of specific isotypes, however, was dependent on the immunization route. Immunoglobulin A, for example, was induced to a significant level only by mucosal immunization, with the intranasal route generating the highest titers. We challenged the offspring of immunized female mice with V. cholerae via the oral route in two consecutive periods, approximately 30 and 95 days after the last immunization. Regardless of the route of immunization, the offspring was protected against colonization with V. cholerae in both challenge periods. Our results show that mucosal immunizations via both routes with OMVs derived from V. cholerae induce long-term protective immune responses against this gastrointestinal pathogen. These findings may contribute to the development of “nonliving,” OMV-based vaccines against V. cholerae and other enteric pathogens, using the oral or intranasal route of immunization.

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Acellular outer membrane vesicles of Brucella abortus strain 19 elicits both humoral and cell mediated immune response in mice

Indian Journal of Animal Research ◽

10.18805/ijar.b-3428 ◽

2018 ◽

Author(s):

Losa Rose ◽

Bablu Kumar ◽

Ankita Jain ◽

M K Singh ◽

Abhishek .

Keyword(s):

Immune Response ◽

Outer Membrane ◽

Brucella Abortus ◽

Outer Membrane Proteins ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Biologically Active ◽

Cell Mediated Immunity ◽

Humoral Immune ◽

Sds Page

Outer membrane vesicles (OMVs) contain biologically active proteins, lipoolysaccharide (LPS), periplasmic and membrane-bound proteins and are known to perform diverse biological functions. OMVs from Brucella abortus S19 were isolated and characterized by transmission electron microscopy (TEM), SDS-PAGE and immunoreactivity was investigated by western blotting. On TEM, bilayered spherical structures of 50-200 nm were observed. SDS-PAGE of OMVs revealed approximate bands size of 82 kDa, 68 kDa, 38 kDa, 32 kDa, 29 kDa and 18 kDa. Western blot analysis of OMVs revealed a dominant immunoreactive band of 38 kDa that correspond to some major outer membrane proteins. Humoral immune response was measured by indirect ELISA which showed that OMV specific antibodies were detected from 7th day post immunization (DPI) onwards and showed a rising trend up to 35th DPI. Cell mediated immune (CMI) response against OMVs as evidenced by the proliferation of splenocytes have also been observed. Thus OMVs were found to possess immunogenic proteins which had potential to induce both humoral as well as cell mediated immunity. After correlating this immune response with protection it has been concluded that OMV can be used as one of the vaccine candidate against brucellosis.

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A combined vaccine approach against Vibrio cholerae and ETEC based on outer membrane vesicles

Frontiers in Microbiology ◽

10.3389/fmicb.2015.00823 ◽

2015 ◽

Vol 6 ◽

Cited By ~ 30

Author(s):

Deborah R. Leitner ◽

Sabine Lichtenegger ◽

Philipp Temel ◽

Franz G. Zingl ◽

Desiree Ratzberger ◽

...

Keyword(s):

Outer Membrane ◽

Vibrio Cholerae ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Vaccine Approach ◽

Combined Vaccine

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Perinuclear Localization of Internalized Outer Membrane Vesicles Carrying Active Cytolethal Distending Toxin from Aggregatibacter actinomycetemcomitans

Infection and Immunity ◽

10.1128/iai.06069-11 ◽

2011 ◽

Vol 80 (1) ◽

pp. 31-42 ◽

Cited By ~ 61

Author(s):

Pramod Kumar Rompikuntal ◽

Bernard Thay ◽

Muhammad Khanzeb Khan ◽

Jonna Alanko ◽

Anna-Maija Penttinen ◽

...

Keyword(s):

Outer Membrane ◽

Membrane Vesicles ◽

Cell Types ◽

Aggregatibacter Actinomycetemcomitans ◽

Outer Membrane Vesicles ◽

Biologically Active ◽

Cytolethal Distending Toxin ◽

Content Type ◽

Serotype A ◽

Tight Association

ABSTRACTAggregatibacter actinomycetemcomitansis implicated in aggressive forms of periodontitis. Similarly to several other Gram-negative species, this organism produces and excretes a cytolethal distending toxin (CDT), a genotoxin associated with cell distention, G2cell cycle arrest, and/or apoptosis in many mammalian cell types. In this study, we have identifiedA. actinomycetemcomitansouter membrane vesicles (OMVs) as a vehicle for simultaneous delivery of multiple proteins, including CDT, into human cells. The OMV proteins were internalized in both HeLa cells and human gingival fibroblasts (HGF) via a mechanism of OMV fusion with lipid rafts in the plasma membrane. The active toxin unit, CdtB, was localized inside the nucleus of the intoxicated cells, whereas OmpA and proteins detected using an antibody specific to wholeA. actinomycetemcomitansserotype a cells had a perinuclear distribution. In accordance with a tight association of CdtB with OMVs, vesicles isolated fromA. actinomycetemcomitansstrain D7SS (serotype a), in contrast to OMVs from a D7SScdtABCmutant, induced a cytolethal distending effect on HeLa and HGF cells, indicating that OMV-associated CDT was biologically active. Association of CDT with OMVs was also observed inA. actinomycetemcomitansisolates belonging to serotypes b and c, indicating that OMV-mediated release of CDT may be conserved inA. actinomycetemcomitans. Although the role ofA. actinomycetemcomitansOMVs in periodontal disease has not yet been elucidated, our present data suggest that OMVs could deliver biologically active CDT and additional virulence factors into susceptible cells of the periodontium.

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