Aeromonas hydrophila ML09-119 is an important fish pathogen that severely affects channel catfish aquaculture. To better understand this strain’s virulence factors, outer membrane vesicles (OMVs) were isolated, and their proteome was assessed. Using transmission electron microscopy and dynamic light scattering, OMVs were shown to be monodispersed particles with an average diameter of 120.33 nm. OMV proteins were identified using mass spectrometry, and analysis of the resulting proteome of 74 proteins revealed that many originated from the cytoplasm, but there was an enrichment of outer membrane, periplasmic, and extracellular proteins compared to the total proteome. The majority of the functional classifications were associated with bacterial metabolism. Of the predicted virulence factors, several had a putative function in adherence, and there were type III secretions system proteins as well as three secreted exotoxins. Overall, our data reveal new insights into A. hydrophila OMVs and their potential roles in physiology and virulence.

Bacteroides fragilis is an important, anaerobic commensal of the human gastro-intestinal tract. As a Gram-negative bacterium, B. fragilis produces a large number of outer membrane vesicles (OMV), spherical globules consisting of outer membrane and periplasmic material, which have a range of potential functions and which are known to be able to deliver their cargo to host dendritic cells (DCs). One of the proteins believed to be packaged into the OMV of B. fragilis is BfUbb (encoded by the ubb gene) which shares 63% homology with human ubiquitin. Ubiquitin is a small, common, eukaryotic protein modifier, which is conjugated to target proteins via a series of activating, conjugating and ligating enzymes, and which has known roles in a wide range of eukaryotic cell processes. Due to key differences between the two proteins, BfUbb has the potential to act as a suicide substrate mimic of ubiquitin. BfUbb was therefore assayed for its ability to interact with ubiquitin E2 conjugating enzymes of the ubiquitylation cascade in vitro, and was found to covalently bind the majority of available enzymes in a DTT-sensitive manner. BfUbb showed a preference for three specific E2 enzymes, all of which are involved in the degradation of mitotic check point proteins, suggesting a role for BfUbb in the inhibition of cell cycle progression and, consequently, tumorigenesis. No binding partners of BfUbb were identified outside of the ubiquitylation cascade, however BfUbb was found to form spontaneous multimers in vitro, the biological function of which is unknown. This study also describes the construction of two sets of plasmids. The first set will allow the expression of untagged and fluorescently tagged forms of BfUbb for purification and use in biochemical assays. The second set will allow the expression of his-tagged and fluorescently tagged forms of BfUbb in mammalian cells, so that the effects of BfUbb on the host epithelial cells may be studied. The proteome of the OMV of B. fragilis was solved using LTQ-Orbitrap mass spectrometry. The identified proteins indicated several putative roles for B. fragilis OMV, including nutrient acquisition and protease inhibition. The suitability of techniques used during the isolation and proteomic analysis of OMV in different studies is discussed. BfUbb-carrying B. fragilis OMV were able to inhibit growth of Salmonella enterica Typhimurium, thus indicating a role for BfUbb in the inhibition of competing, pathogenic bacteria in the gastro-intestinal tract. The conclusions of this study are that the putative roles of both BfUbb and the OMV of B. fragilis may promote both survival of the bacterium and the gastro-intestinal health of the host.

archives@tulane.edu / Gram-negative bacteria secrete outer membrane vesicles (OMVs) that play critical roles in intraspecies, interspecies, and bacteria-environment interactions. Some OMVs, such as those produced by Pseudomonas aeruginosa, have previously been shown to possess antimicrobial activity against competitor species. In the current work, we demonstrate that OMVs from Burkholderia thailandensis inhibit the growth of drug-sensitive and drug-resistant bacteria and fungi and exhibit antibiofilm activity against methicillin-resistant S. aureus (MRSA) and Streptococcus mutans. We show that a number of compounds, including peptidoglycan hydrolases, 4-hydroxy-3-methyl-2-(2-non-enyl)-quinoline (HMNQ) and long-chain rhamnolipid present in B. thailandensis OMVs exert antimicrobial activity. Furthermore, we demonstrate that HMNQ and rhamnolipid possess antimicrobial and antibiofilm properties against various microbes. Rhamnolipid is superior at reducing the integrity of biofilms while HMNQ displays greater bactericidal activity. We attempted to use HMNQ and rhamnolipid to combat MRSA and promote wound healing in a murine full-thickness wound model. However, further optimization of the model and characterization of the molecules in antimicrobial efficacy, wound healing, and host immune responses are required. Overall, this work indicates that B. thailandensis secretes antimicrobial OMVs that may impart a survival advantage by eliminating competition. In addition, bacterial OMVs may represent an untapped resource of novel therapeutics effective against biofilm-forming and multidrug-resistant organisms. / 1 / Yihui Wang

<p>Outer membrane vesicles are an important constitutive product of all Gram-negative bacteria. Bacteria have evolved many responses to alleviate all different types of stress. The primary objective of this dissertation is to investigate the role of outer membrane vesicles (OMVs) as a method by which Gram-negative bacteria can quickly act to protect themselves against particular threats. Generally, we find that stressors whose primary effect is on the outer membrane can be protected against by OMVs. Throughout this study, a variety of different microbiological and biochemical methods are used to answer key questions in the innate ability of OMVs to protect against particular antimicrobials. Using Escherichia coli as well as Pseudomonas aeruginosa as model organisms we tested the ability of purified vesicles from each species to protect themselves and other hosts. Using bacteriophage T4, we investigated the ability of OMVs purified from E. coli to adsorb phage as well as how this interaction affected the efficiency of infection. We found that OMVs are protective against antimicrobial peptides, as well as bacteriophage. In the course of understanding this protection we also observed and characterized the cross species effects of both OMV protection as well as phage infection. Where typically a phage infects a specific species, we found that T4 associated OMVs treating a non-native host P. aeruginosa resulted in the production of a novel prophage. Upon further examination, we determined that this induction was occurring via a novel pathway that we attempted to further characterize by performing a genetic screen to identify genes important to this induction. The work within this dissertation fully supports the hypothesis of a regulated response to outer membrane acting stimuli, resulting in the induction of vesiculation and the adsorption of stressor in the extra-cellular milieu. This model of protection agrees with the idea of a bacterial innate defense system, which acts in the short term before the adaptive response can fully occur, resulting in a bridge between the untreated to the treated and resistant culture.</p> / Dissertation

<p>Enterotoxigenic Escherichia coli (ETEC), the leading cause of traveler's diarrhea and childhood mortality due to diarrhea in the developing world, has been shown to secrete heat-labile enterotoxin (LT) in association with outer membrane vesicles. However, studies on the effect of LT have been performed using soluble LT, which is not its physiologically relevant presentation context. The effect of LT associated with vesicles and its trafficking within human intestinal epithelial cells were compared with soluble LT. Cytokine responses and trafficking of standardized samples of soluble LT and vesicle-associated LT were evaluated in polarized intestinal epithelial cells. Using real-time PCR, immunoblotting, and ELISAs, we found that compared to soluble LT, vesicle-bound LT showed delayed kinetics in the activation of LT. Vesicles containing LT or not also produced cytokines through different signaling pathways than soluble LT. We found that this difference in signaling was due to different trafficking within the cell. Interestingly, not all LT associated with vesicles is active within cells. Vesicle-associated LT must bind to the host receptor GM1 in lipid rafts to be active within cells. This suggests that although vesicles can deliver large amounts of LT to a cell, much of the LT would be inactive and not produce a physiological response. To test this hypothesis, we attempted to develop animal models for ETEC-induced diarrhea. Although the models were largely unsuccessful, the mouse model appears promising for determining the physiological response of a host to LT as fluid accumulation was observed in response to vesicles containing LT. The results in this thesis provide further understanding of the mechanism of LT-induced diarrhea and emphasize the importance of study toxins in their natural context.</p> / Dissertation

<p>Bacterial outer membrane vesicles (OMVs) are spherical buds of the outer membrane (OM) containing periplasmic lumenal components. OMVs have been demonstrated to play a critical part in the transmission of virulence factors, immunologically active compounds, and bacterial survival, however vesiculation also appears to be a ubiquitous physiological process for Gram-negative bacteria. Despite their characterized biological roles, especially for pathogens, very little is known about their importance for the originating organism as well as regulation and mechanism of production. Only when we have established their biogenesis can we fully uncover their roles in pathogenesis and bacterial physiology. The overall goal of this research was to characterize bacterial mutants which display altered vesiculation phenotypes using genetic and biochemical techniques, and thereby begin to elucidate the mechanism of vesicle production and regulation. One part of this work elucidated a synthetic genetic growth defect for a strain with reduced OMV production (&#916;nlpA, inner membrane lipoprotein with a minor role in methionine transport) and envelope stress (&#916;degP, dual function periplasmic chaperone/ protease responsible for managing proteinaceous waste). This research showed that the growth defect of &#916;nlpA&#916;degP correlated with reduced OMV production with respect to the hyprevesiculator &#916;degP and the accumulation of protein in the periplasm and DegP substrates in the lumen of OMVs. We further demonstrated that OMVs do not solely act as a stress response pathway to rid the periplasm of otherwise damaging misfolded protein but also of accumulated peptidoglycan (PG) fragments and lipopolysaccharide (LPS), elucidating OMVs as a general stress response pathway critical for bacterial well-being. The second part of this work, focused on the role of PG structure, turnover and covalent crosslinks to the OM in vesiculation. We established a direct link between PG degradation and vesiculation: Mutations in the OM lipoprotein nlpI had been previously established as a very strong hypervesiculation phenotype. In the literature NlpI had been associated with another OM lipoprotein, Spr that was recently identified as a PG hydrolase. The data presented here suggest that NlpI acts as a negative regulator of Spr and that the &#916;nlpI hypervesiculation phenotype is a result of rampantly degraded PG by Spr. Additionally, we found that changes in PG structure and turnover correlate with altered vesiculation levels, as well as non-canonical D-amino acids, which are secreted by numerous bacteria on the onset of stationary phase, being a natural factor to increase OMV production. Furthermore, we discovered an inverse relationship between the concentration of Lpp-mediated, covalent crosslinks and the level of OMV production under conditions of modulated PG metabolism and structure. In contrast, situations that lead to periplasmic accumulation (protein, PG fragments, and LPS) and consequent hypervesiculation the overall OM-PG crosslink concentration appears to be unchanged. Form this work, we conclude that multiple pathways lead to OMV production: Lpp concentration-dependent and bulk driven, Lpp concentration-independent.</p> / Dissertation

The Gram-negative, motile bacterium Campylobacter jejuni is a causative agent of food-borne gastroenteritis. Cytolethal distending toxin (CDT) is one of the important virulence factors for C. jejuni pathogenesis. It was not previously known how CDT is released from C. jejuni into the surrounding environment. In our study, CDT proteins were observed in the periplasmic fraction and all CDT subunits from C. jejuni were released from the bacterial cells in association with OMVs. The OMV-associated toxin caused cytolethal distending effects on tissue culture cells. Our results strongly suggest that the release of OMV-associated CDT is a route by which C. jejuni delivers all CDT toxin subunits (CdtA, CdtB, and CdtC) to the surrounding environment, including infected host tissue.The Gram-negative, motile bacterium Vibrio cholerae is primarily known as the causal organism of the severe dehydrating diarrheal disease cholera. OMVs released from non-O1 non-O139 V. cholerae (NOVC) strain V:5/04 induced an inflammatory response in human host cells. The inflammatory potential is mediated by the nucleotide-binding domain, leucine-rich repeat containing family members NOD1 and NOD2. Physiochemical analysis in conjunction with NOD1/2 reporter assays in HEK293T cells confirmed the presence of the NOD1/2 active peptidoglycan (PGN) in OMVs. Deletion of the quorum sensing master regulator HapR specifically reduced the inflammatory potential of the V:5/04 OMVs and their ability to activate NOD1 and NOD2. These findings suggest that OMVs from a NOVC strain delivered PGN to the host cells, where they elicited an immune response mediated by NOD1 and NOD2.The Gram-negative, non-motile coccobacillus Aggregatibacter actinomycetemcomitans is a natural inhabitant of the oral cavity, but the bacterium can translocate from the oral cavity into the bloodstream and thereby be transported to other regions of the body. A. actinomycetemcomitans is implicated in aggressive forms of periodontitis. The mechanism behind this aggressive periodontitis was not fully known. In addition to several virulence factors, this organism also produces CDT. We have demonstrated that OMVs released by A. actinomycetemcomitans contain several virulence factors, including CDT. We showed that OMVs delivered CDT to the host cells and that CDT was localized inside the nucleus, which led to a cytolethal distending effect on two different cell lines tested: HeLa cells and human gingival fibroblasts (HGF). These results suggest that A. actinomycetemcomitans OMVs could deliver biologically active CDT toxin into the periodontal tissue and may contribute to periodontitis.In our earlier studies, we discovered that an M6 family metalloprotease PrtV was an essential factor for V. cholerae survival from predator grazing. Pure PrtV protein effectively degraded human blood plasma components. In addition, it also showed a dose-dependent cytotoxic effect in the human intestinal HCT8 cell line. V. cholerae produces a large amount of outer membrane vesicles (OMVs) during the normal course of cell growth. OMVs are composed of periplasmic proteins, membrane lipids, lipopolysaccharides and outer membrane proteins. We showed that OMVs can transport several biologically active toxins and enzymes to the surrounding environment and ultimately into the host cells. We have initiated analysis of OMV-associated secretion of virulence factors in V. cholerae. It was observed that PrtV is secreted from V. cholerae wild type strain C6706 into the culture supernatant in association with OMVs and OMV-associated PrtV protein is biologically active and more stable than the free, soluble PrtV protease.

Vibrio cholerae, the causal agent of cholera typically encodes two critical virulence factors: cholera toxin (CT), which is primarily responsible for the diarrhoeal purge, and toxin-co-regulated pilus (TCP), an essential colonisation factor. Nontoxigenic strains expressing TCP can efficiently acquire the CT gene through lysogenic conversion with CTXΦ, a filamentous phage that encodes CT and uses TCP as a receptor.  V. cholerae is a Gram-negative bacterium and a natural inhabitant of estuarine and coastal waters throughout both temperate and tropical regions of the world. In the aquatic environment, V. cholerae encounters several environmental stresses, such as change in salinity, UV stress, nutrient limitation, temperature fluctuations, viral infections and protozoan predation. To fully understand the pathogenic and virulence potential of V. cholerae, knowledge is required of its interactions with, not only human, but also environmental factors. By using the nematode Caenorhabditis elegans as host model, we were able to identify a previously uncharacterised protein, the extracellular protease PrtV. PrtV was shown to be required for the killing of. elegans and also necessary for survival from grazing by the ciliate Tetrahymena pyriformis and the flagellate Cafeteria roenbergensis. The PrtV protein, which belongs to a M6 family of metallopeptidases was cloned and purified for further characterisations. The purified PrtV was cytotoxic against the human intestinal cell line HCT8. By using human blood plasma, fibrinogen, fibronectin and plasminogen were identified as candidate substrates for the PrtV protease. Outer membrane vesicles (OMVs) are released to the surroundings by most Gram-negative bacteria through “bulging and pinching” of the outer membrane.  OMVs have been shown to contain many virulence factors important in pathogenesis. Therefore, we investigated the association of PrtV with OMVs. PrtV was not associated with OMVs from the wild type O1 strain. In contrast, in an LPS mutant lacking two sugar chains in the core oligosaccharide PrtV was found to be associated with the OMVs. The OMV-associated PrtV was shown to be proteolytically and cytotoxically active. V. cholerae strains are grouped into &gt;200 serogroups. Only the O1 and O139 serogroups have been associated with pandemic cholera, a severe diarrhoeal disease.  All other serogroups are collectively referred to as non-O1 non-O139 V. cholerae. Non-O1 non-O139 V. cholerae can cause gastroenteritis and extraintestinal infections, but unlike O1 and O139 strains of V. cholerae, little is known about the virulence gene content and their potential to become human pathogens. We analysed clinical and environmental non-O1 non-O139 isolates for their putative virulence traits. None of them carry the genes encoding CT or the TCP, but other putative virulence factors were present in these isolates. The incidence of serum resistance was found to vary considerably and was independent of encapsulation. Three strains were strongly serum-resistant, and these same strains could also kill C. elegans.

The antimicrobial properties of dextrose encapsulated gold nanoparticles (dGNPs) with average diameters of 25 nm, 60 nm, and 120 nm (± 5 nm) synthesized by green chemistry principles were investigated against both Gram-negative and Gram-positive bacteria. Studies were performed involving the effect of the dGNPs on the growth, morphology and the ultrastructural properties of bacteria. dGNPs were found to have significant dose dependent antibacterial activity which was directly proportional to their size and also their concentration. The microbial assays revealed the dGNPs to be bacteriostatic as well as bactericidal. The dGNPs exhibited their bactericidal action through the disruption of the bacterial cell membrane causing leakage of cytoplasmic content. The overall outcomes of this study suggest that dGNPs hold promise as a potent antimicrobial agent against a wide range of disease causing bacteria and can control and prevent possible infections or diseases.

Untreatable gonorrhea, caused by fully antimicrobial resistant Neisseria gonorrhoeae (GC), is a major global health threat. While a vaccine would greatly help address this crisis, development of a GC vaccine is complicated by the lack of lab models of symptomatic gonorrhea. We hypothesized that overt disease in animal models of gonorrhea is limited by the human-restriction of gonococcal virulence factors, and the impact of the reproductive hormone cycle (estrus and diestrus phases). We tested these hypotheses by examining the host response to infection in transgenic mice expressing targets of bacterial adhesion, human carcinoembryonic antigen-related cell adhesion molecules (hCEACAMs), in uterine versus vaginal infections, and in different phases of the reproductive cycle (estrus and diestrus phases). hCEACAM expression most impacted estrus phase infections, prolonging colonization in vaginal infection and inducing greater inflammation in uterine. Reproductive phase greatly influenced host response to uterine infection as diestrus infection was more inflammatory than estrus. Phase differences in uterine infection were driven by greater activation of a chemokine-centric common anti gonococcal response and unique induction of type 1 interferons in diestrus. These findings suggest that symptomatic uterine and vaginal GC infection can be modeled by transcervically infected wild-type diestrus mice and transgenic, vaginally-infected estrus mice, respectively. A novel approach to GC vaccine development is also needed. Mono-antigenic vaccines have failed to produce immunity suggesting a poly-antigenic antigen, like natural outer membrane vesicles (nOMVs) may be necessary. It has been shown that any GC vaccine must lack the bacterioprotective antigen, reduction modifiable protein (RMP), and no such nOMV has been previously described. Here we report successful isolation of RMP-deficient nOMVs through sequential size and weight restrictive filtration. Vesicle morphology, proteomics, and bioactivity was characterized via various methods. nOMVs were found to be consistent in size, shape and antigenic load. As antigens, nOMVs induced high serum titers and measurable vaginal levels of antigen and GC specific IgG that recognized several nOMV immunogens supporting the vaccine potential of GC nOMVs. These findings lay the groundwork for protective studies of nOMV vaccines in novel models of active gonorrhea moving the field closer to discovering the mechanism of protective anti-gonococcal immunity.