Production Of Outer Membrane Vesicles Research Articles

Influence of Environmental and Genetic Factors on Proteomic Profiling of Outer Membrane Vesicles from Campylobacter jejuni.

The proteomes of outer membrane vesicles (OMVs) secreted by C. jejuni 81–176 strain, which was exposed to oxygen or antibiotic stress (polymyxin B), were characterized. We also assessed the OMVs production and their content in two mutated strains – ∆dsbI and ∆htrA. OMVs production was significantly increased under the polymyxin B stress and remained unaltered in all other variants. Interestingly, the qualitative load of OMVs was constant regardless of the stress conditions or genetic background. However, certain proteins exhibited notable quantitative changes, ranging from 4-fold decrease to 10-fold increase. Up- or downregulated proteins (e.g. major outer membrane protein porA, iron ABC transporter, serine protease- htrA, 60 kDa chaperonin-groL, enolase) represented various cell compartments (cytoplasm, periplasm, and membrane) and exhibited various functions; nevertheless, one common group was noted that consisted of components of flagellar apparatus, i.e., FlaA/B, FlgC/E, which were mostly upregulated. Some of these proteins are the putative substrates of DsbI protein. Further investigation of the regulation of C. jejuni OMVs composition and their role in virulence will allow a better understanding of the infectious process of C. jejuni.

Studies on the mechanism of multidrug resistance of Acinetobacter baumannii by proteomic analysis of the outer membrane vesicles of the bacterium

Acinetobacter baumannii, an opportunistic pathogen is responsible for pneumonia, meningitis and a wide range of other infections. It is typically involved in nosocomial infections. Outer membrane vesicles (OMVs) released from the bacteria play an important role in the bacterial physiology including pathogenesis. In this study, OMVs were isolated and characterized form sensitive and multidrug-resistant clinical strains of A. baumannii. A comparative proteomics analysis of the OMVs from the A. baumannii sensitive and MDR strain revealed the increased content of carbapenemase and a host of other related antibiotic resistant enzymes in the OMVs of the A. baumannii MDR strain. The OMVs of antibiotic-sensitive strain contained only 8 antibiotic resistance-conferring proteins, whereas the OMVs of multidrug resistance contained 24 proteins. Growth studies on the sensitive and MDR strain in the presence of polymyxin B and OMVs prepared from the respective strains revealed that the OMVs protected the bacteria against the antibiotic. Bacteria were grown in the presence of antibiotic and an indole derivative has shown that the minimum inhibitory concentration was reduced in the presence of derivative indicating that the derivative regulated the production of OMVs. Interestingly, it was also found that the degradosome complex involved in RNA processing was associated with the OMVs. These studies reveal that OMVs may play an important role in antibiotic resistance/persistence.

Towards a subunit vaccine from a Shigella flexneri ΔtolR mutant

Disruption of one or more components of the Tol-Pal system, involved in maintaining the integrity of the outer membrane of Gram-negative bacteria, has been proposed as a method to increase the yield obtained from natural production of outer membrane vesicles (OMV). We present a new OMV-based product, obtained from genetically modified Shigella flexneri 2a with a non-polar deletion in tolR and heat-inactivated (HT-ΔtolR). The S. flexneri ΔtolR strain lead to a higher release of vesicles, more than 8-times when compared to the yield obtained from chemically inactivated wild type strain. S. flexneri mutant strain appeared to be more sensitive to different chemical compounds, including antibiotics, bile salts or human complement and it was also less virulent in both in vitro and in vivo assays. The mutation produced some changes in the LPS O-chain and protein expression. S. flexneri ΔtolR was enriched in long and very long LPS O-chain and expressed a different pattern of surface proteins or lipoproteins. In vitro toxicity and activation properties were determined in Raw 267.4 macrophage cell line. HT-ΔtolR antigenic complex was non-cytotoxic and activation markers, such as MHC-II or CD40, were highly expressed during incubation with this product. Finally, preliminary studies on the antibody response elicited by HT-ΔtolR demonstrated a robust and diverse response in mice. Considering these promising results, HT-ΔtolR antigenic extract appears as a new potential vaccine candidate to face shigellosis.

Vesicular Delivery of the Antifungal Antibiotics of Lysobacter enzymogenes C3.

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 LysobacterIMPORTANCE 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.

Lipidomic Analysis of the Outer Membrane Vesicles from Paired Polymyxin-Susceptible and -Resistant Klebsiella pneumoniae Clinical Isolates.

Gram-negative bacteria produce outer membrane vesicles (OMVs) as delivery vehicles for nefarious bacterial cargo such as virulence factors, which are antibiotic resistance determinants. This study aimed to investigate the impact of polymyxin B treatment on the OMV lipidome from paired polymyxin-susceptible and -resistant Klebsiella pneumoniae isolates. K. pneumoniae ATCC 700721 was employed as a reference strain in addition to two clinical strains, K. pneumoniae FADDI-KP069 and K. pneumoniae BM3. Polymyxin B treatment of the polymyxin-susceptible strains resulted in a marked reduction in the glycerophospholipid, fatty acid, lysoglycerophosphate and sphingolipid content of their OMVs. Conversely, the polymyxin-resistant strains expressed OMVs richer in all of these lipid species, both intrinsically and increasingly under polymyxin treatment. The average diameter of the OMVs derived from the K. pneumoniae ATCC 700721 polymyxin-susceptible isolate, measured by dynamic light scattering measurements, was ~90.6 nm, whereas the average diameter of the OMVs isolated from the paired polymyxin-resistant isolate was ~141 nm. Polymyxin B treatment (2 mg/L) of the K. pneumoniae ATCC 700721 cells resulted in the production of OMVs with a larger average particle size in both the susceptible (average diameter ~124 nm) and resistant (average diameter ~154 nm) strains. In light of the above, we hypothesize that outer membrane remodelling associated with polymyxin resistance in K. pneumoniae may involve fortifying the membrane structure with increased glycerophospholipids, fatty acids, lysoglycerophosphates and sphingolipids. Putatively, these changes serve to make the outer membrane and OMVs more impervious to polymyxin attack.

Vibrio cholerae Outer Membrane Vesicles Inhibit Bacteriophage Infection.

Novel preventatives could help in efforts to limit Vibrio cholerae infection and the spread of cholera. Bacteriophage (phage) treatment has been proposed as an alternative intervention, given the rapid replication of virulent phages, prey specificity, and relative ease of finding new virulent phages. Phage tropism is dictated in part by the presence of phage receptors on the bacterial surface. While many phages that can kill V. cholerae have been isolated, whether this pathogen is able to defend itself by neutralizing phage binding is unknown. Here, we show that secreted outer membrane vesicles (OMVs) act as a defense mechanism that confers protection to V. cholerae against phage predation and that this OMV-mediated inhibition is phage receptor dependent. Our results suggest that phage therapy or prophylaxis should take into consideration the production of OMVs as a bacterial decoy mechanism that could influence the outcome of phage treatment.IMPORTANCE Phages have been increasingly recognized for the significance of their interactions with bacterial cells in multiple environments. Bacteria use myriad strategies to defend against phage infection, including restriction modification, abortive infection, phase variation of cell surface receptors, phage-inducible chromosomal islands, and clustered regularly interspaced short palindromic repeat(s) (CRISPR)-Cas systems. The data presented here suggest that the apparently passive process of OMV release can also contribute to phage defense. By considering the effect of OMVs on V. cholerae infection by three unique virulent phages, ICP1, ICP2, and ICP3, we show that, in vitro, a reproducible reduction in bacterial killing is both dose and phage receptor dependent. This work supports a role for OMVs as natural decoys to defend bacteria from phage predation.

Reciprocal cross-species induction of outer membrane vesicle biogenesis via secreted factors

Delivery of cargo to target cells is fundamental to bacterial competitiveness. One important but poorly understood system, ubiquitous among Gram-negative organisms, involves packaging cargo into outer membrane vesicles (OMVs). These biological nanoparticles are involved in processes ranging from toxin delivery to cell-cell communication. Despite this, we know comparatively little about how OMVs are formed. Building upon the discovery that the Pseudomonas Quinolone Signal (PQS) stimulates OMV biogenesis in Pseudomonas aeruginosa, we proposed a model where PQS interacts with the outer membrane to induce curvature and ultimately OMV formation. Though this model is well supported in P. aeruginosa, it remained unclear whether other organisms produce similar compounds. Here we describe the development of a tightly controlled experimental system to test the interaction of bacterially-produced factors with target cells. Using this system, we show that multiple species respond to PQS by increasing OMV formation, that PQS accumulates in the induced vesicles, and that other bacteria secrete OMV-promoting factors. Analysis of induced vesicles indicates that recipient-mediated mechanisms exist to control vesicle size and that relatedness to the producer organism can dictate susceptibility to OMV-inducing compounds. This work provides evidence that small molecule induced OMV biogenesis is a widely conserved process and that cross-talk between systems may influence OMV production in neighboring bacteria.

A Novel PhoP/PhoQ Regulation Pathway Modulates the Survival of Extraintestinal Pathogenic Escherichia coli in Macrophages.

The extraintestinal pathogenic Escherichia coli (ExPEC) is a typical facultative intracellular bacterial pathogen. Sensing the environmental stimuli and undertaking adaptive change are crucial for ExPEC to successfully colonize in specific extraintestinal niches. The previous studies show that pathogens exploit two-component systems (TCSs) in response to the host environments during its infection. The PhoP/PhoQ is a typical TCS which is ubiquitous in Gram-negative bacteria. However, there is an incompletely understanding about critical regulatory roles of PhoP/PhoQ in ExPEC pathogenesis. Conjugative ColV-related plasmids are responsible for ExPEC virulence, which is associated with ExPEC zoonotic risk. In this study, the molecular characteristics of HlyF, Mig-14 ortholog (Mig-14p), and OmpT variant (OmpTp) encoded by ColV plasmids were identified. Mig-14p and OmpTp played important roles in conferring ExPEC resistance to cationic antimicrobial peptides (CAMPs) during the infection. Moreover, HlyF and Mig-14p acted as intracellular survival factors to promote ExPEC resistance to macrophages killing. The hlyF and Mig-14p formed an operon in ExPEC ColV plasmid, and PhoP acted as a transcriptional activator of hlyF operon by directly binding to the PhlyF promoter. The acidic pH and CAMPs could additively stimulate ExPEC PhoQ/PhoP activities to upregulate the expression of HlyF and Mig-14p. Our studies revealed that the novel PhoP/PhoQ-HlyF signaling pathway directly upregulates the production of ExPEC outer membrane vesicles. Furthermore, our study first clarified that this PhoP/PhoQ-HlyF pathway was essential for ExPEC intracellular survival in macrophages. It was required to prevent the fusion of ExPEC-containing phagosomes with lysosomes. Moreover, PhoP/PhoQ-HlyF pathway facilitated the inhibition of the phagolysosomal acidification and disruption of the phagolysosomal membranes. In addition, this pathway might promote the formation of ExPEC-containing autophagosome during ExPEC replication in macrophages. Collectively, our studies suggested that PhoP/PhoQ system and CloV plasmids could facilitate ExPEC survival and replication within macrophages.

Physicochemical Analysis of Outer Membrane Vesicles Isolated from Escherichia coli Harboring the Pks Genes

Almost all gram‐negative bacteria secrete sphere‐like proteoliposomes named outer membrane vesicles (OMVs). These vesicles provide the bacteria a vehicle for the secretion of outer membrane molecules and periplasmic content. The pks island is a gene cluster present in some strains of bacteria in the human gut microbiota, which has been correlated with the development of colorectal cancer due to the promotion of tumor growth, DNA damage and toxicity in transiently infected eukaryotic cells. The pks island genes encode a sequence of enzymes for the biosynthesis of a compound that we hypothesize is transported from the bacteria to cells by OMVs. The main objective of this investigation is to analyze physically OMVs isolated from E. coli strains harboring and lacking the pks island genes. To achieve this objective, OMVs were analyzed by scanning electron microscopy (SEM), atomic force microscopy (AFM) and dynamic light scattering (DLS) to assess any morphological differences between them. Results show that pks+ E. coli strains produce more vesicles than the pks− strain. As expected, vesicle diameters are around 50 – 280 nm. The OMVs from pks+ and pks− strains have similar diameter distribution between them, but markedly different compared against the lab strain DH10B. We conclude that the presence of pks island is associated with events in the production of OMVs but not with the structure and length of them.Support or Funding InformationRISE Program Grant #5R25GM061151‐16This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Vaccination with outer membrane vesicles and the fimbrial protein FlfA offers improved protection against lesions following challenge with Gallibacterium anatis

Gallibacterium anatis is an opportunistic poultry pathogen belonging to the Pasteurellaceae family. It has been shown to cause oophoritis, salpingitis and peritonitis in hens, as well as being associated with reduced semen quality in cockerels. Widespread multidrug resistance and substantial antigenic variation among strains of Gallibacterium anatis is a major constraint to treatment with antimicrobials and prevention of infection by vaccination. Novel vaccine strategies targeting G. anatis are therefore necessary. Outer membrane vesicles (OMVs) are nanosized vesicles formed from the outer membrane of Gram-negative bacteria. These vesicles have shown promising potential as both adjuvants and as vaccine candidates against numerous bacterial species. A high vesiculating mutant of G. anatis (G. anatis ΔtolR) has previously been made, enabling production of OMVs in large scale. In this study, we elucidated the potential of G. anatis ΔtolR OMVs as adjuvant for the conserved antigens GtxA-N (the N-terminal part of the RTX like toxin Gallibacterium toxin A) and FlfA (F17-like fimbria), as well as evaluated if combinations of OMVs together with antigens could facilitate cross-protective immunity against three different strains of G. anatis. We showed that ΔtolR OMVs function as an adjuvant for GtxA-N by inducing antigen specific antibody production. However, OMVs in combination with GtxA-N failed to induce protection against lesions after challenge infection. In contrast, vaccination with OMVs in combination with FlfA protected against lesions, especially in the salpinx, caused by two diverse strains of G. anatis, thereby indicating a cross-protective potential. No protection against the third G. anatis strain 7990 could be obtained in any of the experimental settings. In conclusion, ΔtolR OMVs and FlfA could serve as potential future vaccine components againt G. anatis.

Amino acid deprivation and central carbon metabolism regulate the production of outer membrane vesicles and tubes by Francisella.

Francisella tularensis is a highly virulent Gram-negative bacterial pathogen that causes the zoonotic disease tularemia. F. novicida, a model tularemia strain, produces spherical outer membrane vesicles (OMV), as well as novel tubular vesicles and extensions of the cell surface. These OMV and tubes (OMV/T) are produced in a regulated manner and contain known virulence factors. Mechanisms by which bacterial vesicles are produced and regulated are not well understood. We performed a genetic screen in F. novicida to decipher the molecular basis for regulated OMV/T formation, and identified both hypo- and hyper-vesiculating mutants. Mutations in fumA and tktA, involved in central carbon metabolism, and in FTN_0908 and FTN_1037, of unknown function, resulted in severe defects in OMV/T production. Cysteine deprivation was identified as the signal that triggers OMV/T formation in F. novicida during growth in rich medium. We also found that fully virulent F. tularensis produces OMV/T in a similarly regulated manner. Further analysis revealed that OMV/T production is responsive to deprivation of essential amino acids in addition to cysteine, and that the hypo-vesiculating mutants are defective in responding to this signal. Thus, amino acid starvation, such as encountered by Francisella during host cell invasion, regulates the production of membrane-derived structures.

The bile salt sodium taurocholate induces Campylobacter jejuni outer membrane vesicle production and increases OMV-associated proteolytic activity.

Campylobacter jejuni, the leading cause of bacterial acute gastroenteritis worldwide, secretes an arsenal of virulence-associated proteins within outer membrane vesicles (OMVs). C.jejuni OMVs contain three serine proteases (HtrA, Cj0511, and Cj1365c) that cleave the intestinal epithelial cell (IEC) tight and adherens junction proteins occludin and E-cadherin, promoting enhanced C.jejuni adhesion to and invasion of IECs. C.jejuni OMVs also induce IECs innate immune responses. The bile salt sodium taurocholate (ST) is sensed as a host signal to coordinate the activation of virulence-associated genes in the enteric pathogen Vibrio cholerae. In this study, the effect of ST on C.jejuni OMVs was investigated. Physiological concentrations of ST do not have an inhibitory effect on C.jejuni growth until the early stationary phase. Coculture of C.jejuni with 0.1% or 0.2% (w/v) ST stimulates OMV production, increasing both lipid and protein concentrations. C.jejuni ST-OMVs possess increased proteolytic activity and exhibit a different protein profile compared to OMVs isolated in the absence of ST. ST-OMVs exhibit enhanced cytotoxicity and immunogenicity to T84 IECs and enhanced killing of Galleria mellonella larvae. ST increases the level of mRNA transcripts of the OMVs-associated serine protease genes and the cdtABC operon that encodes the cytolethal distending toxin. Coculture with ST significantly enhances the OMVs-induced cleavage of E-cadherin and occludin. C.jejuni OMVs also cleave the major endoplasmic reticulum chaperone protein BiP/GRP78 and this activity is associated with the Cj1365c protease. These data suggest that C.jejuni responds to the presence of physiological concentrations of the bile salt ST that increases OMV production and the synthesis of virulence-associated factors that are secreted within the OMVs. We propose that these events contribute to pathogenesis.

High throughput nanoparticle tracking analysis for monitoring outer membrane vesicle production

ABSTRACTOuter membrane vesicles (OMVs) are spherical membrane nanoparticles released by Gram-negative bacteria. OMVs can be quantified in complex matrices by nanoparticle tracking analysis (NTA). NTA can be performed in static mode or with continuous sample flow that results in analysis of more particles in a smaller time-frame. Flow measurements must be performed manually despite the availability of a sample changer on the NanoSight system. Here we present a method for automated measurements in flow mode. OMV quantification in flow mode results in lower variance in particle quantification (coefficient of variation (CV) of 6%, CV static measurements of 14%). Sizing of OMVs was expected to be less favorable in flow mode due to the increased movement of the particles. However, we observed a CV of 3% in flow mode and a CV of 8% in static measurements. Flow rates of up to 5 µL/min displayed correct size and particle measurements, however, particle concentration was slightly lower than in static measurements. The automated method was used to assess OMV release of batch cultures of Neisseria meningitidis. The bacteria released more OMVs in stationary growth phase, while the size of the vesicles remained constant throughout the culture. Taken together, this study shows that automated measurements in flow mode can be established with advanced scripting to reduce the workload for the user.

The Opportunistic Pathogen Vibrio vulnificus Produces Outer Membrane Vesicles in a Spatially Distinct Manner Related to Capsular Polysaccharide.

Vibrio vulnificus, a bacterial species that inhabits brackish waters, is an opportunistic pathogen of humans. V. vulnificus infections can cause acute gastroenteritis, invasive septicemia, tissue necrosis, and potentially death. Virulence factors associated with V. vulnificus include the capsular polysaccharide (CPS), lipopolysaccharide, flagellum, pili, and outer membrane vesicles (OMVs). The aims of this study were to characterize the morphology of V. vulnificus cells and the formation and arrangement of OMVs using cryo-electron microscopy (cryo-EM). cryo-EM and cryo-electron tomography imaging of V. vulnificus strains grown in liquid cultures revealed the presence of OMVs (diameters of ∼45 nm for wild-type, ∼30 nm for the unencapsulated mutant, and ∼50 nm for the non-motile mutant) in log-phase growth. Production of OMVs in the stationary growth phase was limited and irregular. The spacing of the OMVs around the wild-type cells was in regular, concentric rings. In wild-type cells and a non-motile mutant, the spacing between the cell envelope and the first ring of OMVs was ∼200 nm; this spacing was maintained between subsequent OMV layers. The size, arrangement, and spacing of OMVs in an unencapsulated mutant was irregular and indicated that the polysaccharide chains of the capsule regulate aspects of OMV production and order. Together, our results revealed the distinctive organization of V. vulnificus OMVs that is affected by expression of the CPS.

Mannheimia haemolytica A2 secretes different proteases into the culture medium and in outer membrane vesicles

Respiratory diseases in ruminants have a significantly negative impact on the worldwide economy. The bacterium Mannheimia haemolytica is involved in pneumonic infections in bovine and ovine. In gram-negative bacteria, six secretion systems related to the colonization process and host tissue damage have been reported. In addition, in the last two decades, the production of outer membrane vesicles has been studied as a different bacterial strategy to release virulence factors, such as exotoxins, lipopolysaccharides, and proteases. However, in M. haemolytica serotype A2, protease secretion and release in vesicles have not been reported as virulence mechanisms. The aim of this work was to identify proteases released into the culture supernatant and in vesicles of M. haemolytica A2. Our results showed evident differences in the molecular mass and activity of proteases present in culture supernatants and outer membrane vesicles based on zymography assays. The biochemical characterization of M. haemolytica proteases revealed that the main types were cysteine and metalloproteases. A specific metalloprotease of 100 kDa was active in the culture supernatants, but it was not active and was found in low quantities in vesicles. Proteases could be an important virulence factor during the infectious pneumonic process led by M. haemolytica.

Antibiotic-Mediated Modulations of Outer Membrane Vesicles in Enterohemorrhagic Escherichia coli O104:H4 and O157:H7.

Ciprofloxacin, meropenem, fosfomycin, and polymyxin B strongly increase production of outer membrane vesicles (OMVs) in Escherichia coli O104:H4 and O157:H7. Ciprofloxacin also upregulates OMV-associated Shiga toxin 2a, the major virulence factor of these pathogens, whereas the other antibiotics increase OMV production without the toxin. These two effects might worsen the clinical outcome of infections caused by Shiga toxin-producing E. coli Our data support the existing recommendations to avoid antibiotics for treatment of these infections.

Quantitative Evaluation of Recombinant Protein Packaged into Outer Membrane Vesicles of Escherichia coli Cells

Outer membrane vesicles (OMVs) are spherical bilayered proteolipids released from the cell surfaces of bacteria, which have gained traction in the biotechnology fields. Bacterial cellular machinery can be genetically engineered to produce and package heterologous enzymes into OMVs, producing nanocarriers and nanoparticle catalysts. However, the productivity or efficiency of packaging the target protein into OMVs has not been quantitatively evaluated. In this study, we packaged green fluorescence protein (GFP) into the OMVs of Escherichia coli through N-terminal fused expression to outer membrane protein W (OmpW). The OMV productivity and amount of OmpW-GFP packaged in the OMVs were quantitatively compared between two hypervesiculating mutant strains ΔnlpI and ΔdegP. Both strains increased the OMV production, but the ΔnlpI strain additionally enhanced the packaging of OmpW-GFP into OMVs. It was further confirmed that Spr, a peptidoglycan endopeptidase, plays an important role in the enhanced packaging of OmpW-GFP into OMVs through the increased OmpW-GFP expression on the ΔnlpI cells. Finally, the amount of OmpW-GFP released in the OMV fraction of both mutants was determined in terms of the OMV productivity and the packaging efficiency of OmpW-GFP into OMVs. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:51-57, 2018.

Membrane Distribution of the Pseudomonas Quinolone Signal Modulates Outer Membrane Vesicle Production in Pseudomonasaeruginosa.

ABSTRACTThe Pseudomonas quinolone signal (PQS) is an important quorum-sensing molecule in Pseudomonas aeruginosa that also mediates its own packaging and transport by stimulating outer membrane vesicle (OMV) formation. Because OMVs have been implicated in many virulence-associated behaviors, it is critical that we understand how they are formed. Our group proposed the bilayer-couple model for OMV biogenesis, where PQS intercalates into the outer membrane, causing expansion of the outer leaflet and consequently inducing curvature. In accordance with the model, we hypothesized that PQS must be transported from the cytoplasm to the outer membrane before it can initiate OMV formation. We initially examined two laboratory strains of P. aeruginosa and found significant strain-dependent differences. PQS export correlated strongly with OMV production, even though equivalent amounts of total PQS were produced by both strains. Interestingly, we discovered that poor OMV producers sequestered the majority of PQS in the inner membrane, which appeared to be the result of early saturation of the export pathway. Further analysis showed that strain-specific PQS export and OMV biogenesis patterns were stable once established but could be significantly altered by changing the growth medium. Finally, we demonstrated that the associations described for laboratory strains also held for three clinical strains. These results suggest that factors controlling the export of PQS dictate OMV biogenesis. This work provides new insight into PQS-controlled virulence in P. aeruginosa and provides important tools to further study signal export and OMV biogenesis.

Intrahost milieu modulates production of outer membrane vesicles, vesicle-associated Shiga toxin 2a and cytotoxicity in Escherichia coli O157:H7 and O104:H4.

Outer membrane vesicles (OMVs) are important virulence tools of enterohaemorrhagic Escherichia coli (EHEC), but other biological functions of these nanostructures are unknown. We tested the hypothesis that modulation of OMV production enables EHEC to resist the intrahost environment during infection by investigating if simulated human gastrointestinal conditions affect OMV production in EHEC O157:H7 and O104:H4. All the conditions tested including a low pH, simulated ileal and colonic media, presence of mucin, intestinal epithelial cell lysate or antimicrobial peptides, as well as iron limitation, significantly increased OMV production by these pathogens. Accordingly, a maximum vesiculation in EHEC O104:H4 was observed immediately after its isolation from a patient's intestine, and rapidly decreased during passages in vitro. Most of the simulated intrahost conditions also upregulated the OMV-associated Shiga toxin 2a (Stx2a), the major EHEC virulence factor, and, as a result, OMV cytotoxicity. The data indicates that upregulation of OMV production by the human gastrointestinal milieu contributes to EHEC survival and adaptation within the host during infection. Moreover, the intrahost increase of vesiculation and OMV-associated Stx2a may augment EHEC virulence.

Outer membrane vesicles blebbing contributes to B. vulgatus mpk-mediated immune response silencing

ABSTRACTThe Gram negative intestinal symbiont Bacteroides vulgatus mpk is able to prevent from induction of colonic inflammation in Rag1−/− mice and promotes immune balance in Il2−/− mice. These inflammation-silencing effects are associated with B. vulgatus mpk-mediated induction of semi-mature dendritic cells, especially in the colonic lamina propria (cLP). However the beneficial interaction of bacteria with host immune cells is limited due to the existence of a large mucus layer covering the intestinal epithelium. How can intestinal bacteria overcome this physical barrier and contact the host immune system?One mechanism is the production of outer membrane vesicles (OMVs) via ubiquitous blebbing of the outer membrane. These proteoliposomes have the ability to traverse the mucus layer. Hence, OMVs play an important role in immunomodulation and the maintenance of a balanced gut microbiota. Here we demonstrate that the stimulation of bone marrow derived dendritic cells (BMDCs) with isolated OMVs originated from B. vulgatus mpk leads to the induction of a tolerant semi-mature phenotype. Thereby, microbe- associated molecular patterns (MAMPs) delivered by OMVs are crucial for the interaction and the resulting maturation of immune cells. Additional to the binding to host TLR4, a yet unknown ligand to TLR2 is indispensable for the conversion of immature BMDCs into a semi-mature state. Thus, crossing the epithelial mucus layer and directly contact host cells, OMV mediate cross-tolerance via the transport of various Toll-like receptor antigens. These features make OMVs to a key attribute of B. vulgatus mpk for a vigorous acellular prevention and treatment of systemic diseases.