Delivery of Virulence Factors by Bacterial Membrane Vesicles to Mammalian Host Cells

Pathogenic bacteria interact with cells of their host via many factors. The surface components, i.e., adhesins, lipoproteins, LPS and glycoconjugates, are particularly important in the initial stages of colonization. They enable adhesion and multiplication, as well as the formation of biofilms. In contrast, virulence factors such as invasins and toxins act quickly to damage host cells, causing tissue destruction and, consequently, organ dysfunction. These proteins must be exported from the bacterium and delivered to the host cell in order to function effectively. Bacteria have developed a number of one- and two-step secretion systems to transport their proteins to target cells. Recently, several authors have postulated the existence of another transport system (sometimes called “secretion system type zero”), which utilizes extracellular structures, namely membrane vesicles (MVs). This review examines the role of MVs as transporters of virulence factors and the interaction of toxin-containing vesicles and other protein effectors with different human cell types. We focus on the unique ability of vesicles to cross the blood–brain barrier and deliver protein effectors from intestinal or oral bacteria to the central nervous system.

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Bacteria- and host-derived extracellular vesicles – two sides of the same coin?

Journal of Cell Science ◽

10.1242/jcs.256628 ◽

2021 ◽

Vol 134 (11) ◽

Author(s):

Jeffrey S. Schorey ◽

Yong Cheng ◽

William R. McManus

Keyword(s):

Extracellular Vesicles ◽

Membrane Vesicles ◽

Bacterial Pathogenesis ◽

Host Immunity ◽

Host Cells ◽

Bacterial Membrane ◽

Cell Responses ◽

Two Sides ◽

Compare And Contrast

ABSTRACT Intracellular bacterial pathogens spend portions of their life cycle both inside and outside host cells. While in these two distinct environments, they release or shed bacterial components, including virulence factors that promote their survival and replication. Some of these components are released through extracellular vesicles, which are either derived from the bacteria themselves or from the host cells. Bacteria- and host-derived vesicles have been studied almost exclusively in isolation from each other, with little discussion of the other type of secreted vesicles, despite the fact that both are generated during an in vivo infection and both are likely play a role in bacterial pathogenesis and host immunity. In this Review, we aim to bridge this gap and discuss what we know of bacterial membrane vesicles in their generation and composition. We will compare and contrast this with the composition of host-derived vesicles with regard to bacterial components. We will also compare host cell responses to the different vesicles, with a focus on how these vesicles modulate the immune response, using Mycobacterium, Listeria and Salmonella as specific examples for these comparisons.

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Virulence and Immunomodulatory Roles of Bacterial Outer Membrane Vesicles

Microbiology and Molecular Biology Reviews ◽

10.1128/mmbr.00031-09 ◽

2010 ◽

Vol 74 (1) ◽

pp. 81-94 ◽

Cited By ~ 474

Author(s):

Terri N. Ellis ◽

Meta J. Kuehn

Keyword(s):

Outer Membrane ◽

Virulence Factors ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Host Cells ◽

Gram Negative ◽

Stress Response Pathway ◽

Degradative Enzymes ◽

Bacterial Virulence Factor ◽

Molecular Patterns

SUMMARY Outer membrane (OM) vesicles are ubiquitously produced by Gram-negative bacteria during all stages of bacterial growth. OM vesicles are naturally secreted by both pathogenic and nonpathogenic bacteria. Strong experimental evidence exists to categorize OM vesicle production as a type of Gram-negative bacterial virulence factor. A growing body of data demonstrates an association of active virulence factors and toxins with vesicles, suggesting that they play a role in pathogenesis. One of the most popular and best-studied pathogenic functions for membrane vesicles is to serve as natural vehicles for the intercellular transport of virulence factors and other materials directly into host cells. The production of OM vesicles has been identified as an independent bacterial stress response pathway that is activated when bacteria encounter environmental stress, such as what might be experienced during the colonization of host tissues. Their detection in infected human tissues reinforces this theory. Various other virulence factors are also associated with OM vesicles, including adhesins and degradative enzymes. As a result, OM vesicles are heavily laden with pathogen-associated molecular patterns (PAMPs), virulence factors, and other OM components that can impact the course of infection by having toxigenic effects or by the activation of the innate immune response. However, infected hosts can also benefit from OM vesicle production by stimulating their ability to mount an effective defense. Vesicles display antigens and can elicit potent inflammatory and immune responses. In sum, OM vesicles are likely to play a significant role in the virulence of Gram-negative bacterial pathogens.

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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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Entry of Porphyromonas gingivalis Outer Membrane Vesicles into Epithelial Cells Causes Cellular Functional Impairment

Infection and Immunity ◽

10.1128/iai.00841-09 ◽

2009 ◽

Vol 77 (11) ◽

pp. 4761-4770 ◽

Cited By ~ 67

Author(s):

Nobumichi Furuta ◽

Hiroki Takeuchi ◽

Atsuo Amano

Keyword(s):

Epithelial Cells ◽

Outer Membrane ◽

Porphyromonas Gingivalis ◽

Virulence Factors ◽

Functional Impairment ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Cellular Migration ◽

Host Cells ◽

Periodontal Pathogen

ABSTRACT Porphyromonas gingivalis, a periodontal pathogen, secretes outer membrane vesicles (MVs) that contain major virulence factors, including proteases termed gingipains (Arg-gingipain [Rgp] and Lys-gingipain [Kgp]). We recently showed that P. gingivalis MVs swiftly enter host epithelial cells via an endocytosis pathway and are finally sorted to lytic compartments. However, it remains unknown whether MV entry impairs cellular function. Herein, we analyzed cellular functional impairment following entry of P. gingivalis into epithelial cells, including HeLa and immortalized human gingival epithelial (IHGE) cells. After being taken up by endocytic vacuoles, MVs degraded the cellular transferrin receptor (TfR) and integrin-related signaling molecules, such as paxillin and focal adhesion kinase (FAK), which resulted in depletion of intracellular transferrin and inhibition of cellular migration. Few Rgp-null MVs entered the cells, and these negligibly degraded TfR, whereas paxillin and FAK degradation was significant. In contrast, Kgp-null MVs clearly entered the cells and degraded TfR, while they scarcely degraded paxillin and FAK. In addition, both wild-type and Kgp-null MVs significantly impaired cellular migration, whereas the effect of Rgp-null MVs was limited. Our findings suggest that, following entry of P. gingivalis MVs into host cells, MV-associated gingipains degrade cellular functional molecules such as TfR and paxillin/FAK, resulting in cellular impairment, indicating that P. gingivalis MVs are potent vehicles for transmission of virulence factors into host cells and are involved in the etiology of periodontitis.

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Detection of Bacterial Membrane Vesicles by NOD-Like Receptors

International Journal of Molecular Sciences ◽

10.3390/ijms22031005 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1005

Author(s):

Ella L. Johnston ◽

Begoña Heras ◽

Thomas A. Kufer ◽

Maria Kaparakis-Liaskos

Keyword(s):

Inflammatory Diseases ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Innate Immune ◽

Host Cells ◽

Bacterial Membrane ◽

Gram Positive ◽

Gram Negative ◽

Gram Positive Bacteria ◽

Key Driver

Bacterial membrane vesicles (BMVs) are nanoparticles produced by both Gram-negative and Gram-positive bacteria that can function to modulate immunity in the host. Both outer membrane vesicles (OMVs) and membrane vesicles (MVs), which are released by Gram-negative and Gram-positive bacteria, respectively, contain cargo derived from their parent bacterium, including immune stimulating molecules such as proteins, lipids and nucleic acids. Of these, peptidoglycan (PG) and lipopolysaccharide (LPS) are able to activate host innate immune pattern recognition receptors (PRRs), known as NOD-like receptors (NLRs), such as nucleotide-binding oligomerisation domain-containing protein (NOD) 1, NOD2 and NLRP3. NLR activation is a key driver of inflammation in the host, and BMVs derived from both pathogenic and commensal bacteria have been shown to package PG and LPS in order to modulate the host immune response using NLR-dependent mechanisms. Here, we discuss the packaging of immunostimulatory cargo within OMVs and MVs, their detection by NLRs and the cytokines produced by host cells in response to their detection. Additionally, commensal derived BMVs are thought to shape immunity and contribute to homeostasis in the gut, therefore we also highlight the interactions of commensal derived BMVs with NLRs and their roles in limiting inflammatory diseases.

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