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

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

Toxins ◽

10.3390/toxins11040207 ◽

2019 ◽

Vol 11 (4) ◽

pp. 207 ◽

Cited By ~ 2

Author(s):

Elnaz Rasti ◽

Angela Brown

Keyword(s):

Outer Membrane ◽

Cholera Toxin ◽

Vibrio Cholerae ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Biologically Active ◽

Host Cells ◽

Free Form ◽

Type Ii Secretion ◽

Secondary Mechanism

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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Cytotoxic and Inflammatory Responses Induced by Outer Membrane Vesicle-Associated Biologically Active Proteases from Vibrio cholerae

Infection and Immunity ◽

10.1128/iai.01365-15 ◽

2016 ◽

Vol 84 (5) ◽

pp. 1478-1490 ◽

Cited By ~ 29

Author(s):

Ayan Mondal ◽

Rima Tapader ◽

Nabendu Sekhar Chatterjee ◽

Amit Ghosh ◽

Ritam Sinha ◽

...

Keyword(s):

Outer Membrane ◽

Vibrio Cholerae ◽

Serine Protease ◽

Inflammatory Responses ◽

Membrane Vesicle ◽

Active Form ◽

Outer Membrane Vesicles ◽

Biologically Active ◽

Ileal Loop ◽

Content Type

Proteases inVibrio choleraehave been shown to play a role in its pathogenesis.V. choleraesecretes Zn-dependent hemagglutinin protease (HAP) and calcium-dependent trypsin-like serine protease (VesC) by using the type II secretion system (TIISS). Our present studies demonstrated that these proteases are also secreted in association with outer membrane vesicles (OMVs) and transported to human intestinal epithelial cells in an active form. OMV-associated HAP induces dose-dependent apoptosis in Int407 cells and an enterotoxic response in the mouse ileal loop (MIL) assay, whereas OMV-associated VesC showed a hemorrhagic fluid response in the MIL assay, necrosis in Int407 cells, and an increased interleukin-8 (IL-8) response in T84 cells, which were significantly reduced in OMVs from VesC mutant strain. Our results also showed that serine protease VesC plays a role in intestinal colonization ofV. choleraestrains in adult mice. In conclusion, our study shows thatV. choleraeOMVs secrete biologically active proteases which may play a role in cytotoxic and inflammatory responses.

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NOD-Like Receptor Activation by Outer Membrane Vesicles fromVibrio choleraeNon-O1 Non-O139 Strains Is Modulated by the Quorum-Sensing Regulator HapR

Infection and Immunity ◽

10.1128/iai.00754-10 ◽

2011 ◽

Vol 79 (4) ◽

pp. 1418-1427 ◽

Cited By ~ 48

Author(s):

H. Bielig ◽

P. K. Rompikuntal ◽

M. Dongre ◽

B. Zurek ◽

B. Lindmark ◽

...

Keyword(s):

Quorum Sensing ◽

Immune Responses ◽

Outer Membrane ◽

Inflammatory Responses ◽

Membrane Vesicles ◽

Receptor Activation ◽

Outer Membrane Vesicles ◽

Host Cells ◽

Mammalian Host ◽

Host Immune Responses

ABSTRACTVibrio choleraeis an inhabitant of aquatic systems and one of the causative agents of severe dehydrating diarrhea in humans. It has also emerged as an important cause of different kinds of inflammatory responses, and in particular,V. choleraestrains of the non-O1 non-O139 serogroups (NOVC) have been associated with such infections in human. We analyzed the potential of outer membrane vesicles (OMVs) derived from the NOVC strain V:5/04 to induce inflammatory responses in human host cells. V:5/04 OMVs were taken up by human epithelial cells and induced inflammatory responses. Small interfering RNA (siRNA)-mediated gene knockdown revealed that the inflammatory potential of NOVC OMVs was partially mediated by the nucleotide-binding domain-, leucine-rich repeat-containing family member NOD1. Physiochemical analysis of the content of these OMVs, in conjunction with NOD1 and NOD2 reporter assays in HEK293T cells, confirmed the presence of both NOD1 and NOD2 active peptidoglycan in the OMVs. Furthermore, we show that deletion of the quorum-sensing regulator HapR, which mimics an infective life style, specifically reduced the inflammatory potential of the V:5/04 OMVs and their ability to activate NOD1 and NOD2. In conclusion, our study shows that NOVC OMVs elicit immune responses mediated by NOD1 and NOD2 in mammalian host cells. Moreover, we provide evidence that the quorum-sensing machinery plays an important regulatory role in this process by attenuating the inflammatory potential of OMVs under infective conditions. This work thus identifies a new facet of howVibrioaffects host immune responses and defines a role for the quorum-sensing machinery in this process.

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

Infection and Immunity ◽

10.1128/iai.01277-10 ◽

2011 ◽

Vol 79 (6) ◽

pp. 2182-2192 ◽

Cited By ~ 38

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.

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

Infection and Immunity ◽

10.1128/iai.00161-12 ◽

2012 ◽

Vol 80 (12) ◽

pp. 4089-4098 ◽

Cited By ~ 80

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.

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Vesicular Delivery of the Antifungal Antibiotics of Lysobacter enzymogenes C3

Applied and Environmental Microbiology ◽

10.1128/aem.01353-18 ◽

2018 ◽

Vol 84 (20) ◽

Cited By ~ 4

Author(s):

Paul R. Meers ◽

Carol Liu ◽

Rensa Chen ◽

William Bartos ◽

Julianne Davis ◽

...

Keyword(s):

Antifungal Activity ◽

Outer Membrane ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Physical Contact ◽

Host Cells ◽

Yeast Cells ◽

Content Type ◽

Lysobacter Enzymogenes ◽

Antifungal Antibiotics

ABSTRACT Lysobacter enzymogenes C3 is a predatory strain of Gram-negative gliding bacteria that produces antifungal antibiotics by the polyketide synthetic pathway. Outer membrane vesicles (OMV) are formed as a stress response and can deliver virulence factors to host cells. The production of OMV by C3 and their role in antifungal activity are reported here. Vesicles in the range of 130 to 150 nm in diameter were discovered in the cell-free supernatants of C3 cultures. These OMV contain molecules characteristic of bacterial outer membranes, such as lipopolysaccharide and phospholipids. In addition, they contain chitinase activity and essentially all of the heat-stable antifungal activity in cell supernatants. We show here that C3 OMV can directly inhibit growth of the yeast Saccharomyces cerevisiae as well as that of the filamentous fungus Fusarium subglutinans. The activity is dependent on physical contact between OMV and the cells. Furthermore, fluorescent lipid labeling of C3 OMV demonstrated transfer of the membrane-associated probe to yeast cells, suggesting the existence of a mechanism of delivery for membrane-associated molecules. Mass spectrometric analysis of C3 OMV extracts indicates the presence of molecules with molecular weights identical to some of the previously identified antifungal products of C3. These data together suggest that OMV act as an important remote mobile component of predation by Lysobacter. IMPORTANCE The data presented here suggest a newly discovered function of outer membrane vesicles (OMV) that are produced from the outer membrane of the bacterial species Lysobacter enzymogenes strain C3. We show that these OMV can be released from the surface of the cells to deliver antibiotics to target fungal organisms as a mechanism of killing or growth inhibition. Understanding the role of OMV in antibiotic delivery can generally lead to improved strategies for dealing with antibiotic-resistant organisms. These results also add to the evidence that some bacterially produced antibiotics can be discovered and purified using methods designed for isolation of nanoscale vesicles. Information on these systems can lead to better identification of active molecules or design of delivery vehicles for these molecules.

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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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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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