Cholera Toxin Encapsulated within Several Vibrio cholerae O1 Serotype Inaba Outer Membrane Vesicles Lacks a Functional B-Subunit

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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Outer Membrane Vesicles of Vibrio cholerae Protect and Deliver Active Cholera Toxin to Host Cells via Porin-Dependent Uptake

mBio ◽

10.1128/mbio.00534-21 ◽

2021 ◽

Vol 12 (3) ◽

Author(s):

Franz G. Zingl ◽

Himadri B. Thapa ◽

Martina Scharf ◽

Paul Kohl ◽

Anna M. Müller ◽

...

Keyword(s):

Outer Membrane ◽

Cholera Toxin ◽

Vibrio Cholerae ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Host Cells ◽

Intestinal Cells ◽

Gram Negative ◽

Content Type ◽

Outer Membrane Porins

ABSTRACT Outer membrane vesicles (OMVs) are an emerging research field due to their multifactorial composition and involvement in interspecies and intraspecies communication. Recent studies indicate that vesicle release by Gram-negative bacterial pathogens is increased during in vivo colonization, as exemplified by the facultative human pathogen Vibrio cholerae upon oral ingestion by the host. In this study, we investigate the fate of OMVs produced by the Gram-negative facultative pathogen V. cholerae. We show that vesicles produced by the clinically relevant El Tor biotype are readily taken up by human intestinal cell lines. We identify outer membrane porins of V. cholerae, i.e., OmpU and OmpT, as the required surface effectors on OMVs for cellular uptake, and we pinpoint the uptake mechanism as caveolin-mediated endocytosis. Furthermore, we show that OMVs derived from V. cholerae grown under virulence-inducing conditions act as potent vehicles for delivery of bioactive cholera toxin to intestinal epithelial cells. In contrast to free cholera toxin secreted via the type II secretion system, OMV-associated cholera toxin is protected from degradation by intestinal proteases. Taken together, these data show that OMV-associated cholera toxin can sustain longer periods in the intestinal tract and preserve toxin effects, as indicated by a prolonged increase of cAMP levels in the intestinal tissue. IMPORTANCE Cholera is still a massive global health burden because it causes large outbreaks with millions of infections and thousands of deaths every year. Several studies have contributed to the knowledge of this pathogen, although key parts are still missing. We aim to broaden our understanding of Vibrio cholerae infections, virulence, and toxicity by drawing attention to the involvement of OMVs in these core processes. Upon host entry, V. cholerae increases secretion of OMVs, which can carry the main virulence factor, cholera toxin, to distant host intestinal cells. We show that specific outer membrane porins on the vesicle surface mediate endocytosis of the vesicles into intestinal cells. With protection by the vesicles, cholera toxin activity endures even in the presence of intestinal proteases. It is tempting to hypothesize that the extended half-life of vesicle-associated cholera toxin allows it to target host cells distant from the primary colonization sites.

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Aggregatibacter actinomycetemcomitans Outer Membrane Vesicles Are Internalized in Human Host Cells and Trigger NOD1- and NOD2-Dependent NF-κB Activation

Infection and Immunity ◽

10.1128/iai.01980-14 ◽

2014 ◽

Vol 82 (10) ◽

pp. 4034-4046 ◽

Cited By ~ 59

Author(s):

Bernard Thay ◽

Anna Damm ◽

Thomas A. Kufer ◽

Sun Nyunt Wai ◽

Jan Oscarsson

Keyword(s):

Outer Membrane ◽

Hela Cells ◽

Inflammatory Responses ◽

Membrane Vesicles ◽

Aggregatibacter Actinomycetemcomitans ◽

Outer Membrane Vesicles ◽

Biologically Active ◽

Host Cells ◽

Content Type ◽

Human Embryonic Kidney Cells

ABSTRACTAggregatibacter actinomycetemcomitansis an oral and systemic pathogen associated with aggressive forms of periodontitis and with endocarditis. We recently demonstrated that outer membrane vesicles (OMVs) disseminated byA. actinomycetemcomitanscould deliver multiple proteins, including biologically active cytolethal distending toxin (CDT), into the cytosol of HeLa cells and human gingival fibroblasts (HGF). In the present work, we have used immunoelectron and confocal microscopy analysis and fluorescently labeled vesicles to further investigate mechanisms forA. actinomycetemcomitansOMV-mediated delivery of bacterial antigens to these host cells. Our results supported that OMVs were internalized into the perinuclear region of HeLa cells and HGF. Colocalization analysis revealed that internalized OMVs colocalized with the endoplasmic reticulum and carried antigens, detected using an antibody specific to wholeA. actinomycetemcomitansserotype a cells. Consistent with OMV internalization mediating intracellular antigen exposure, the vesicles acted as strong inducers of cytoplasmic peptidoglycan sensor NOD1- and NOD2-dependent NF-κB activation in human embryonic kidney cells. Moreover, NOD1 was the main sensor of OMV-delivered peptidoglycan in myeloid THP1 cells, contributing to the overall inflammatory responses induced by the vesicles. This work reveals a role ofA. actinomycetemcomitansOMVs as a trigger of innate immunity via carriage of NOD1- and NOD2-active pathogen-associated molecular patterns (PAMPs).

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

PLoS ONE ◽

10.1371/journal.pone.0106731 ◽

2014 ◽

Vol 9 (9) ◽

pp. e106731 ◽

Cited By ~ 35

Author(s):

Sridhar Elluri ◽

Constance Enow ◽

Svitlana Vdovikova ◽

Pramod K. Rompikuntal ◽

Mitesh Dongre ◽

...

Keyword(s):

Outer Membrane ◽

Vibrio Cholerae ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Biologically Active

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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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Aggregatibacter actinomycetemcomitans Leukotoxin Is Delivered to Host Cells in an LFA-1-Indepdendent Manner When Associated with Outer Membrane Vesicles

Toxins ◽

10.3390/toxins10100414 ◽

2018 ◽

Vol 10 (10) ◽

pp. 414 ◽

Cited By ~ 9

Author(s):

Justin Nice ◽

Nataliya Balashova ◽

Scott Kachlany ◽

Evan Koufos ◽

Eric Krueger ◽

...

Keyword(s):

Outer Membrane ◽

Membrane Vesicles ◽

Aggregatibacter Actinomycetemcomitans ◽

Aggressive Periodontitis ◽

Outer Membrane Vesicles ◽

Host Cells ◽

Type I ◽

Outer Membranes ◽

Independent Mechanism ◽

Bacterial Outer Membrane

The Gram-negative bacterium, Aggregatibacter actinomycetemcomitans, has been associated with localized aggressive periodontitis (LAP). In particular, highly leukotoxic strains of A. actinomycetemcomitans have been more closely associated with this disease, suggesting that LtxA is a key virulence factor for A. actinomycetemcomitans. LtxA is secreted across both the inner and outer membranes via the Type I secretion system, but has also been found to be enriched within outer membrane vesicles (OMVs), derived from the bacterial outer membrane. We have characterized the association of LtxA with OMVs produced by the highly leukotoxic strain, JP2, and investigated the interaction of these OMVs with host cells to understand how LtxA is delivered to host cells in this OMV-associated form. Our results demonstrated that a significant fraction of the secreted LtxA exists in an OMV-associated form. Furthermore, we have discovered that in this OMV-associated form, the toxin is trafficked to host cells by a cholesterol- and receptor-independent mechanism in contrast to the mechanism by which free LtxA is delivered. Because OMV-associated toxin is trafficked to host cells in an entirely different manner than free toxin, this study highlights the importance of studying both free and OMV-associated forms of LtxA to understand A. actinomycetemcomitans virulence.

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Proteomic Characterization of the Whole Secretome of Legionella pneumophila and Functional Analysis of Outer Membrane Vesicles

Infection and Immunity ◽

10.1128/iai.01396-07 ◽

2008 ◽

Vol 76 (5) ◽

pp. 1825-1836 ◽

Cited By ~ 116

Author(s):

Frank Galka ◽

Sun Nyunt Wai ◽

Harald Kusch ◽

Susanne Engelmann ◽

Michael Hecker ◽

...

Keyword(s):

Outer Membrane ◽

Legionella Pneumophila ◽

Protein Identification ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Laser Scanning Microscopy ◽

Host Cells ◽

Confocal Laser ◽

Lipolytic Enzyme ◽

New Findings

ABSTRACT Secretion of effector molecules is one of the major mechanisms by which the intracellular human pathogen Legionella pneumophila interacts with host cells during infection. Specific secretion machineries which are responsible for the subfraction of secreted proteins (soluble supernatant proteins [SSPs]) and the production of bacterial outer membrane vesicles (OMVs) both contribute to the protein composition of the extracellular milieu of this lung pathogen. Here we present comprehensive proteome reference maps for both SSPs and OMVs. Protein identification and assignment analyses revealed a total of 181 supernatant proteins, 107 of which were specific to the SSP fraction and 33 of which were specific to OMVs. A functional classification showed that a large proportion of the identified OMV proteins are involved in the pathogenesis of Legionnaires' disease. Zymography and enzyme assays demonstrated that the SSP and OMV fractions possess proteolytic and lipolytic enzyme activities which may contribute to the destruction of the alveolar lining during infection. Furthermore, it was shown that OMVs do not kill host cells but specifically modulate their cytokine response. Binding of immunofluorescently stained OMVs to alveolar epithelial cells, as visualized by confocal laser scanning microscopy, suggested that there is delivery of a large and complex group of proteins and lipids in the infected tissue in association with OMVs. On the basis of these new findings, we discuss the relevance of protein sorting and compartmentalization of virulence factors, as well as environmental aspects of the vesicle-mediated secretion.

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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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