Beyond a single species: Mapping virulence traits across the redefined Fusobacterium nucleatum complex

Outer membrane vesicles (OMVs) are small membrane enclosed sacs released from bacteria which serve as carriers of biomolecules that shape interactions with the surrounding environment. The periodontal pathogen, Porphyromonas gingivalis, is a prolific OMV producer. Here, we investigated how the structure of lipid A, a core outer membrane molecule, influences P. gingivalis OMV production, OMV-dependent TLR4 activation, and biofilm formation. We examined mutant strains of P. gingivalis 33277 deficient for enzymes that alter lipid A phosphorylation and acylation status. The lipid A C4'-phosphatase (lpxF)-deficient strain and strains bearing inactivating point mutations in the LpxF active site displayed markedly reduced OMV production relative to WT. In contrast, strains deficient for either the lipid A C1-phosphatase (lpxE) or the lipid A deacylase (PGN_1123; lpxZ) genes did not display alterations in OMV abundance compared to WT. These data indicate that lipid A C4'-phosphate removal is required for typical OMV formation. In addition, OMVs produced by ΔlpxF and ΔlpxZ strains, possessing only penta-acylated lipid A, stimulated robust TLR4 activation, whereas OMVs obtained from WT and ΔlpxE strains, containing predominantly tetra-acylated lipid A, did not. Hence, lipid A remodeling modulates the capacity of OMVs to engage host TLR4-dependent immunity. Finally, we demonstrate an inverse relationship between OMV abundance and biofilm density, with the ∆lpxF mutants forming denser biofilms than either WT, ΔlpxE, or ΔlpxZ strains. Therefore, OMVs may also contribute to pathogenesis by regulating biofilm formation and dispersal.IMPORTANCEPorphyromonas gingivalis is a bacterium strongly associated with periodontitis. P. gingivalis exports lipids, proteins, and other biomolecules that contribute to the bacterium's ability to persist in inflammatory conditions encountered during disease. These biomolecules are exported through several mechanisms, including via outer membrane vesicles (OMVs). Despite their ubiquity, the mechanisms that drive outer membrane vesicle production vary among bacteria and are not fully understood. In this study, we report that C4' dephosphorylation of lipid A, a major outer membrane molecule, is required for robust outer membrane vesicle production and biological function in P. gingivalis. This finding adds to the growing body of evidence that lipid A structure is an important factor in outer membrane vesicle biogenesis in diverse bacterial species.

Outer membrane vesicles (OMVs), released from Gram-negative bacteria, have been attributed to intra- and interspecies communication and pathogenicity in diverse bacteria. OMVs carry various components including genetic material, toxins, signaling molecules, or proteins. Although the molecular mechanism(s) of cargo delivery is not fully understood, recent studies showed that transfer of the OMV content to surrounding cells is mediated by selective interactions. Here, we show that the phytopathogen Agrobacterium tumefaciens, the causative agent of crown gall disease, releases OMVs, which attach to the cell surface of various Gram-negative bacteria. The OMVs contain the conserved small lipoprotein Atu8019. An atu8019-deletion mutant produced wildtype-like amounts of OMVs with a subtle but reproducible reduction in cell-attachment. Otherwise, loss of atu8019 did not alter growth, susceptibility against cations or antibiotics, attachment to plant cells, virulence, motility, or biofilm formation. In contrast, overproduction of Atu8019 in A. tumefaciens triggered cell aggregation and biofilm formation. Localization studies revealed that Atu8019 is surface exposed in Agrobacterium cells and in OMVs supporting a role in cell adhesion. Purified Atu8019 protein reconstituted into liposomes interacted with model membranes and with the surface of several Gram-negative bacteria. Collectively, our data suggest that the small lipoprotein Atu8019 is involved in OMV docking to specific bacteria.

<p>Gram-negative bacteria produce outer-membrane vesicles (OMVs) that have biological roles ranging from biofilm formation, modulation of host-cell interactions & delivery of virulence factors. Several studies have shown a role for OMVs to act as intracellular signals to co-ordinate the behaviour of bacteria. This study showed OMVs generated at sub-lethal ciprofloxacin concentrations were capable of programming naïve P. aeruginosa cultures resulting in premature entry into stationary-phase and a significantly lower final culture density reached after 14 hrs. Pyoverdine production was also initiated after 6 hrs in cultures treated with OMVs. Heat-inactivation of OMVs failed to impede OMV-mediated growth inhibition & pyoverdine production. Chloroform-disruption of OMVs prevented OMV-mediated growth inhibition but did not inhibit OMV-induced pyoverdine production. It is likely that these effects are mediated by multiple signals as opposed to a single mechanism. This suggests that a protein is not responsible for OMV-mediated growth inhibition and an intact OMV lipid bilayer is required. Induction of pyoverdine production is likely due to a lipid (such as a homo-serine lactone) or small molecule present within OMVs. Preincubation with OMVs for 2-4 hrs resulted in a substantial decrease in the final culture density from cultures that were exposed to OMVs during the course of growth. This suggests that OMV fusion is capable of programming naïve bacteria to set a predetermined division limit on subsequent daughter cells. We coin this as the ‘Dayflick’ limit due to the similarities of the Hayflick limit in eukaryotic cells. This shows that OMVs act as intercellular messaging vehicles between bacteria that communicate and program naïve bacteria to adapt to the environment under which they were generated in, aiding survival in harsh environments. Further study is needed to determine what OMV components are responsible for initiating these responses and to determine how long the programming is stable.</p>

ABSTRACTAlthough the immunomodulatory potency of bacterial membrane vesicles (MVs) is widely acknowledged, their interactions with host cells and the underlying signaling pathways have not been well studied. Herein, we provide a comparative analysis of the proinflammatory cytokine profile secreted by human intestinal epithelial cells exposed to MVs derived from 32 gut bacteria. In general, outer membrane vesicles (OMVs) from Gram-negative bacteria induced a stronger proinflammatory response than MVs from Gram-positive bacteria. However, the quality and quantity of cytokine induction varied between MVs from different species, highlighting their unique immunomodulatory properties. OMVs from enterotoxigenic Escherichia coli (ETEC) were among those showing the strongest proinflammatory potency. In depth analyses revealed that the immunomodulatory activity of ETEC OMVs relies on a so far unprecedented two-step mechanism, including their internalization into host cells followed by intracellular recognition. First, OMVs are efficiently taken up by intestinal epithelial cells, which mainly depends on caveolin-mediated endocytosis as well as the presence of the outer membrane porins OmpA and OmpF on the MVs. Second, lipopolysaccharide (LPS) delivered by OMVs is intracellularly recognized by novel caspase- and RIPK2-dependent pathways. This recognition likely occurs via detection of the lipid A moiety as ETEC OMVs with underacylated LPS exhibited reduced proinflammatory potency but similar uptake dynamics compared to OMVs derived from wild-type (WT) ETEC. Intracellular recognition of ETEC OMVs in intestinal epithelial cells is pivotal for the proinflammatory response as inhibition of OMV uptake also abolished cytokine induction. The study signifies the importance of OMV internalization by host cells to exercise their immunomodulatory activities.IMPORTANCE The release of membrane vesicles from the bacterial cell surface is highly conserved among most bacterial species, including outer membrane vesicles (OMVs) from Gram-negative bacteria as well as vesicles liberated from the cytoplasmic membrane of Gram-positive bacteria. It is becoming increasingly evident that these multifactorial spheres, carrying membranous, periplasmic, and even cytosolic content, contribute to intra- and interspecies communication. In particular, gut microbiota and the host engage in a myriad of immunogenic and metabolic interactions. This study highlights the individual immunomodulatory activities of bacterial membrane vesicles from different enteric species and provides new mechanistic insights into the recognition of ETEC OMVs by human intestinal epithelial cells.

Gram-negative pathogens ubiquitously shed outer membrane vesicles (OMVs) that play a central role in initiating and regulating pathogenesis in the host. Due to their highly inflammatory nature, OMVs are extensively being examined for their role in mediating disease in addition to their applications in innovative vaccines. A key mechanism whereby OMVs mediate inflammation and disease progression is dependent on their ability to enter host cells. Currently, the role of OMV size on determining their mechanism of cellular entry and their protein composition remains unknown. In this study, we examined the mechanisms whereby OMV size regulates their mode of entry into epithelial cells, in addition to their protein cargo and composition. We identified that a heterogeneous sized population of Helicobacter pylori OMVs entered epithelial cells via macropinocytosis, clathrin, and caveolin-dependent endocytosis. However, smaller OMVs ranging from 20 to 100 nm in size preferentially entered host cells via caveolin-mediated endocytosis. Whereas larger OMVs ranging between 90 and 450 nm in size entered host epithelial cells via macropinocytosis and endocytosis. Most importantly, we identified the previously unknown contribution that OMV size has on determining their protein content, as fewer and less diverse bacterial proteins were contained within small OMVs compared to larger OMVs. Collectively, these findings identify the importance of OMV size in determining the mechanisms of OMV entry into host cells, in addition to regulating their protein cargo, composition, and subsequent immunogenicity. These findings have significant implications in broadening our understanding of the bacterial regulation of virulence determinants and immunogenic proteins associated with OMVs, their role in mediating pathogenesis and in refining the design and development of OMV-based vaccines.

Neisseria gonorrhoeae is a strict human pathogen that causes the sexually transmitted disease gonorrhoea, which affects approximately 106 million people worldwide. It has evolved a diverse range of strategies to establish long-term colonisation of the human mucosal surfaces. Importantly, N. gonorrhoeae resists clearance by innate immune cells, such as macrophages, which can result in sustained inflammation, tissue damage, and bacterial dissemination. The underlying molecular mechanism of innate immune evasion remains largely unknown, given that there is little evidence that N. gonorrhoeae secrete effector proteins to modulate host cell responses. The major focus of the work described in this thesis was to determine how N. gonorrhoeae controls macrophage immune responses. In particular, I focused on the role of outer membrane vesicles (OMVs) that are shed by an increasing number of bacteria, enabling potential communication with host cells. In chapter 3 of this thesis, I was able to establish a method that allowed me to isolate highly purified OMVs from N. gonorrhoeae. Using quantitative proteomic approaches, I determined that the protein content of purified N. gonorrhoeae OMVs is enriched for outer-membrane proteins, whereby the porin PorB is the most abundant protein, constituting 35 % of the total OMV proteome. Purified OMVs also contained proteins known to be important for N. gonorrhoeae virulence, such as opacity proteins, but also proteins that may affect vesicle formation (NlpD, AmiC, and RmpM) as well as several uncharacterised proteins. In contrast, traditional purification of OMVs resulted in crude preparations that contained numerous cytosolic proteins. PorB has previously been shown to damage the mitochondria of host cells during infection, but the trafficking route from the outer-membrane of N. gonorrhoeae has never been characterised. In Chapter 3, I have now described the cellular route of PorB, which includes the uptake of OMVs by bone marrow-derived macrophages (BMDMs) and trafficking of PorB to mitochondria. In confocal and single molecule localisation microscopy, PorB co-localised to the mitochondrial outer-membrane marker, Tom20. Furthermore, N. gonorrhoeae secreted OMVs induce apoptotic cell death in macrophages, as evidenced by the release of mitochondrial cytochrome c and caspase activation, which followed localisation of PorB to mitochondria. In addition to PorB, host factors also regulate macrophage apoptosis. In this context, I have now shown for the first time that OMVs can induce rapid extrinsic apoptosis dependent on caspase-8 (Chapter 3). In addition, the pro-survival protein BCL-XL prevented OMV-induced intrinsic apoptosis (Chapter 3). Finally, I have uncovered a novel of the pro-apoptotic MCL-1S as an essential host factor that regulates macrophage apoptosis in response to N. gonorrhoeae OMVs (Chapter 3). Little is known about how Gram-negative bacteria regulate OMV biogenesis. The proteome of N. gonorrhoeae OMVs contains several proteins that may directly, or indirectly, affect OMV biogenesis. In Chapter 4, I have focused on the proteins TsaP, NlpD, FtsN, AmiC. HldA and GmhB, which are thought to function in peptidoglycan (PG) binding, cell division, and lipid chain biosynthesis, and may thus regulate OMV abundance. To directly test this, defined genetic mutants of N. gonorrhoeae were generated and OMV biogenesis was determined. By electron microscopy, I identified that loss of AmiC and NlpD induced hyper-blebbing cellular morphology, resulting in increased levels of OMVs compared to wild-type. Given that there is great interest in developing a vaccine against N. gonorrhoeae based on naturally produced OMVs, these genetically-engineered hyper-blebbing mutants may enable more effective vaccine approaches. Thus, the molecular characterisation of OMVs coupled with the identification of their roles in N. gonorrhoeae infection may open up new strategies to improve immune clearance of Neisseria, as well as dampen inflammatory responses, both of which are of interest in the treatment of gonococcal-induced sexually transmitted infections.

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

Tenacibaculum maritimum, the etiological agent of tenacibaculosis in marine fish, constitutively secretes extracellular products (ECPs) in which protein content has not been yet comprehensively studied. In this work, the prevalence of extracellular proteolytic and lipolytic activities related to virulence was analyzed in 64 T. maritimum strains belonging to the O1–O4 serotypes. The results showed the existence of a great intra-specific heterogeneity in the enzymatic capacity, particularly within serotype O4. Thus, the secretome of a strain belonging to this serotype was characterized by analyzing the protein content of ECPs and the possible production of outer membrane vesicles (OMVs). Notably, the ECPs of T. maritimum SP9.1 contain a large amount of OMVs that were characterized by electron microscopy and purified. Thus, ECPs were divided into soluble (S-ECPs) and insoluble fractions (OMVs), and their protein content was analyzed by a high-throughput proteomic approach. A total of 641 proteins were identified in ECPs including some virulence-related factors, which were mainly found in one of the fractions, either OMVs or S-ECPs. Outer membrane proteins such as TonB-dependent siderophore transporters and the type IX secretion system (T9SS)-related proteins PorP, PorT, and SprA appeared to be mainly associated with OMVs. By contrast, putative virulence factors such as sialidase SiaA, chondroitinase CslA, sphingomyelinase Sph, ceramidase Cer, and collagenase Col were found only in the S-ECPs. These findings clearly demonstrate that T. maritimum releases, through surface blebbing, OMVs specifically enriched in TonB-dependent transporters and T9SS proteins. Interestingly, in vitro and in vivo assays also showed that OMVs could play a key role in virulence by promoting surface adhesion and biofilm formation and maximizing the cytotoxic effects of the ECPs. The characterization of T. maritimum secretome provides insights into ECP function and can constitute the basis for future studies aimed to elucidate the full role of OMVs in the pathogenesis of fish tenacibaculosis.

Outer membrane vesicles (OMVs) released by some Gram-negative bacteria have been shown to exert immunomodulatory effects that favor the establishment of the infection. The aim of the present study was to assess the interaction of OMVs from Brucella abortus with human epithelial cells (HeLa) and monocytes (THP-1), and the potential immunomodulatory effects they may exert. Using confocal microscopy and flow cytometry, FITC-labeled OMVs were shown to be internalized by both cell types. Internalization was shown to be partially mediated by clathrin-mediated endocytosis. Pretreatment of THP-1 cells with Brucella OMVs inhibited some cytokine responses (TNF-α and IL-8) to E. coli LPS, Pam3Cys or flagellin (TLR4, TLR2 and TLR5 agonists, respectively). Similarly, pretreatment with Brucella OMVs inhibited the cytokine response of THP-1 cells to B. abortus infection. Treatment of THP-1 cells with OMVs during IFN-γ stimulation reduced significantly the inducing effect of this cytokine on MHC-II expression. OMVs induced a dose-dependent increase of ICAM-1 expression on THP-1 cells and an increased adhesion of these cells to human endothelial cells. The addition of OMVs to THP-1 cultures before the incubation with live B. abortus resulted in increased numbers of adhered and internalized bacteria as compared to cells not treated with OMVs. Overall, these results suggest that OMVs from B. abortus exert cellular effects that promote the internalization of these bacteria by human monocytes, but also downregulate the innate immune response of these cells to Brucella infection. These effects may favor the persistence of Brucella within host cells.

In recent years, bacterial outer membrane vesicles (OMVs)—nanoscale, bilayered membrane structures secreted by Gram-negative bacteria—have attracted considerable attention for their involvement in antibiotic resistance and potential in clinical anti-infective strategies. OMVs encapsulate diverse biomolecules, including proteins, lipids, toxins, and nucleic acids, thereby serving as critical mediators of communication between bacteria and host cells. They contribute to horizontal gene transfer, signal transduction, and biofilm formation, ultimately enhancing bacterial adaptability and resistance. Clinically, OMVs are regarded as promising therapeutic platforms owing to their excellent biocompatibility and intrinsic immunogenicity, with ongoing investigations exploring their roles in vaccine development, targeted drug delivery, and immune modulation. This review highlights the participation of OMVs in resistance mechanisms across common pathogenic bacteria and discusses their emerging applications in infection control. By elucidating the biogenesis and functional mechanisms of OMVs, novel antibacterial strategies may be developed, offering new avenues to address the escalating global challenge of antibiotic resistance.

A double-blind cross-over trial between pheneturide and phenytoin in ninety-four outpatients with epilepsy is described. There was no significant difference between the frequency of seizures in the two groups. The difficulties in comparing two anticonvulsants of similar efficacy are discussed particularly in relation to ethical problems, the selection of patients and trial design.

Outer membrane vesicles (OMVs), released by variety of bacteria, are membrane-enclosed entities enriched in microbial components, toxins, and virulent factors. OMVs could deliver lipopolysaccharide (LPS) into the cytosol of host cells and subsequently activate caspase-11, which critically orchestrates immune responses and mediates septic shock. Although it is known that caspase-11 is activated by intracellular LPS, how OMVs deliver LPS into the cytosol remains largely unknown. Here we show that the activation of toll-like receptor 4 (TLR4), a LPS receptor on the cytoplasmic membrane, licenses macrophages to transport LPS from OMVs into the cytosol through TIR domain-containing adaptor-inducing interferon-β (TRIF). TRIF-mediated cytosolic delivery of LPS from OMVs depends on the production of type 1 interferon and the expression of guanylate-binding proteins (GBPs). Deletion of TRIF or GBPs prevents pyroptosis and lethality induced by OMVs or OMVs-releasing Escherichia coli. Together, these findings provide novel insight into how host coordinates extracellular and intracellular LPS sensing to orchestrate immune responses during gram-negative bacterial infection.

Outer membrane vesicles from pathogenic bacteria initiate an inflammatory response in human endothelial cells

Outer membrane vesicles of Vibrio vulnificus deliver cytolysin–hemolysin VvhA into epithelial cells to induce cytotoxicity

Bacterial outer membrane vesicles (OMVs) are extracellular sacs containing biologically active products, such as proteins, cell wall components and toxins. OMVs are reported to contain DNA, however, little is known about the nature of this DNA, nor whether it can be transported into host cells. Our work demonstrates that chromosomal DNA is packaged into OMVs shed by bacteria during exponential phase. Most of this DNA was present on the external surfaces of OMVs, with smaller amounts located internally. The DNA within the internal compartments of Pseudomonas aeruginosa OMVs were consistently enriched in specific regions of the bacterial chromosome, encoding proteins involved in virulence, stress response, antibiotic resistance and metabolism. Furthermore, we demonstrated that OMVs carry DNA into eukaryotic cells, and this DNA was detectable by PCR in the nuclear fraction of cells. These findings suggest a role for OMV-associated DNA in bacterial-host cell interactions and have implications for OMV-based vaccines.