The effect of altered pH growth conditions on the production, composition, and proteomes of Helicobacter pylori outer membrane vesicles.

Gram-negative bacteria release outer membrane vesicles (OMVs) that contain cargo derived from their parent bacteria. Helicobacter pylori is a Gram-negative human pathogen that produces urease to increase the pH of the surrounding environment to facilitate colonization of the gastric mucosa. However, the effect of acidic growth conditions on the production and composition of H. pylori OMVs is unknown. In this study, we examined the production, composition, and proteome of H. pylori OMVs produced during acidic and neutral pH growth conditions. H. pylori growth in acidic conditions reduced the quantity and size of OMVs produced. Additionally, OMVs produced during acidic growth conditions had increased protein, DNA, and RNA cargo compared to OMVs produced during neutral conditions. Proteomic analysis comparing the proteomes of OMVs to their parent bacteria demonstrated significant differences in the enrichment of beta-lactamases and outer membrane proteins between bacteria and OMVs, supporting that differing growth conditions impacts OMV composition. We also identified differences in the enrichment of proteins between OMVs produced during different pH growth conditions. Overall, our findings reveal that growth of H. pylori at different pH levels is a factor that alters OMV proteomes, which may affect their subsequent functions.

Isolation and Characterization of Cow-, Buffalo-, Sheep- and Goat-Milk-Derived Extracellular Vesicles.

Milk is a complex biological fluid that has high-quality proteins including growth factors and also contains extracellular vesicles (EVs). EVs are a lipid bilayer containing vesicles that contain proteins, metabolites and nucleic acids. Several studies have proposed that EVs in cow milk can survive the gut and can illicit cross-species communication in the consuming host organism. In this study, we isolated and characterized extracellular vesicles from the raw milk of the four species of the Bovidae family, namely cow, sheep, goat and buffalo, that contribute 99% of the total milk consumed globally. A comparative proteomic analysis of these vesicles was performed to pinpoint their potential functional role in health and disease. Vesicles sourced from buffalo and cow milk were particularly enriched with proteins implicated in modulating the immune system. Furthermore, functional studies were performed to determine the anti-cancer effects of these vesicles. The data obtained revealed that buffalo-milk-derived EVs induced significantly higher cell death in colon cancer cells. Overall, the results from this study highlight the potent immunoregulatory and anti-cancer nature of EVs derived from the milk of Bovidae family members.

Planktonic and Biofilm-Derived Pseudomonas aeruginosa Outer Membrane Vesicles Facilitate Horizontal Gene Transfer of Plasmid DNA.

Outer membrane vesicles (OMVs) produced by Gram-negative bacteria package various cargo, including DNA that can be transferred to other bacteria or to host cells. OMV-associated DNA has been implicated in mediating horizontal gene transfer (HGT) between bacteria, which includes the dissemination of antibiotic resistance genes within and between bacterial species. Despite the known ability of OMVs to mediate HGT, the mechanisms of DNA packaging into OMVs remain poorly characterized, as does the effect of bacterial growth conditions on the DNA cargo composition of OMVs and their subsequent abilities to mediate HGT. In this study, we examined the DNA content of OMVs produced by the opportunistic pathogen Pseudomonas aeruginosa grown in either planktonic or biofilm conditions. Analysis of planktonic growth-derived OMVs revealed their ability to package and protect plasmid DNA from DNase degradation and to transfer plasmid-encoded antibiotic resistance genes to recipient, antibiotic-sensitive P. aeruginosa bacteria at a greater efficiency than transformation with plasmid alone. Comparisons of planktonic and biofilm-derived P. aeruginosa OMVs demonstrated that biofilm-derived OMVs were smaller but were associated with more plasmid DNA than planktonic-derived OMVs. Additionally, biofilm-derived P. aeruginosa OMVs were more efficient in the transformation of competent P. aeruginosa bacteria, compared to transformations with an equivalent number of planktonic-derived OMVs. The findings of this study highlight the importance of bacterial growth conditions for the packaging of DNA within P. aeruginosa OMVs and their ability to facilitate HGT, thus contributing to the spread of antibiotic resistance genes between P. aeruginosa bacteria. IMPORTANCE Bacterial membrane vesicles (BMVs) mediate interbacterial communication, and their ability to package DNA specifically contributes to biofilm formation, antibiotic resistance, and HGT between bacteria. However, the ability of P. aeruginosa OMVs to mediate HGT has not yet been demonstrated. Here, we reveal that P. aeruginosa planktonic and biofilm-derived OMVs can deliver plasmid-encoded antibiotic resistance to recipient P. aeruginosa. Additionally, we demonstrated that P. aeruginosa biofilm-derived OMVs were associated with more plasmid DNA compared to planktonic-derived OMVs and were more efficient in the transfer of plasmid DNA to recipient bacteria. Overall, this demonstrated the ability of P. aeruginosa OMVs to facilitate the dissemination of antibiotic resistance genes, thereby enabling the survival of susceptible bacteria during antibiotic treatment. Investigating the roles of biofilm-derived BMVs may contribute to furthering our understanding of the role of BMVs in HGT and the spread of antibiotic resistance in the environment.

The mechanism of Pseudomonas aeruginosa outer membrane vesicle biogenesis determines their protein composition.

Gram-negative bacteria produce outer membrane vesicles (OMVs) and contain bacterial cargo including nucleic acids and proteins. The proteome of OMVs can be altered by various factors including bacterial growth stage, growth conditions, and environmental factors. However, it is currently unknown if the mechanism of OMV biogenesis can determine their proteome. In this study, we examined whether the mechanisms of OMV biogenesis influenced the production and protein composition of Pseudomonas aeruginosa OMVs. OMVs were isolated from three P. aeruginosa strains that produced OMVs either by budding alone, by explosive cell lysis, or by both budding and explosive cell lysis. We identified that the mechanism of OMV biogenesis dictated OMV quantity. Furthermore, a global proteomic analysis comparing the proteome of OMVs to their parent bacteria showed significant differences in the identification of proteins in bacteria and OMVs. Finally, we determined that the mechanism of OMV biogenesis influenced the protein composition of OMVs, as OMVs released by distinct mechanisms of biogenesis differed significantly from one another in their proteome and functional enrichment analysis. Overall, our findings reveal that the mechanism of OMV biogenesis is a main factor that determines the OMV proteome which may affect their subsequent biological functions.

Bacteroides fragilis outer membrane vesicles preferentially activate innate immune receptors compared to their parent bacteria.

The release of bacterial membrane vesicles (BMVs) has become recognized as a key mechanism used by both pathogenic and commensal bacteria to activate innate immune responses in the host and mediate immunity. Outer membrane vesicles (OMVs) produced by Gram-negative bacteria can harbor various immunogenic cargo that includes proteins, nucleic acids and peptidoglycan, and the composition of OMVs strongly influences their ability to activate host innate immune receptors. Although various Gram-negative pathogens can produce OMVs that are enriched in immunogenic cargo compared to their parent bacteria, the ability of OMVs produced by commensal organisms to be enriched with immunostimulatory contents is only recently becoming known. In this study, we investigated the cargo associated with OMVs produced by the intestinal commensal Bacteroides fragilis and determined their ability to activate host innate immune receptors. Analysis of B. fragilis OMVs revealed that they packaged various biological cargo including proteins, DNA, RNA, lipopolysaccharides (LPS) and peptidoglycan, and that this cargo could be enriched in OMVs compared to their parent bacteria. We visualized the entry of B. fragilis OMVs into intestinal epithelial cells, in addition to the ability of B. fragilis OMVs to transport bacterial RNA and peptidoglycan cargo into Caco-2 epithelial cells. Using HEK-Blue reporter cell lines, we identified that B. fragilis OMVs could activate host Toll-like receptors (TLR)-2, TLR4, TLR7 and nucleotide-binding oligomerization domain-containing protein 1 (NOD1), whereas B. fragilis bacteria could only induce the activation of TLR2. Overall, our data demonstrates that B. fragilis OMVs activate a broader range of host innate immune receptors compared to their parent bacteria due to their enrichment of biological cargo and their ability to transport this cargo directly into host epithelial cells. These findings indicate that the secretion of OMVs by B. fragilis may facilitate immune crosstalk with host epithelial cells at the gastrointestinal surface and suggests that OMVs produced by commensal bacteria may preferentially activate host innate immune receptors at the mucosal gastrointestinal tract.

Bacterial subversion of NLR-mediated immune responses.

Members of the mammalian Nod-like receptor (NLR) protein family are important intracellular sensors for bacteria. Bacteria have evolved under the pressure of detection by host immune sensing systems, leading to adaptive subversion strategies to dampen immune responses for their benefits. These include modification of microbe-associated molecular patterns (MAMPs), interception of innate immune pathways by secreted effector proteins and sophisticated instruction of anti-inflammatory adaptive immune responses. Here, we summarise our current understanding of subversion strategies used by bacterial pathogens to manipulate NLR-mediated responses, focusing on the well-studied members NOD1/2, and the inflammasome forming NLRs NLRC4, and NLRP3. We discuss how bacterial pathogens and their products activate these NLRs to promote inflammation and disease and the range of mechanisms used by bacterial pathogens to evade detection by NLRs and to block or dampen NLR activation to ultimately interfere with the generation of host immunity. Moreover, we discuss how bacteria utilise NLRs to facilitate immunotolerance and persistence in the host and outline how various mechanisms used to attenuate innate immune responses towards bacterial pathogens can also aid the host by reducing immunopathologies. Finally, we describe the therapeutic potential of harnessing immune subversion strategies used by bacteria to treat chronic inflammatory conditions.

Considerations for the Analysis of Bacterial Membrane Vesicles: Methods of Vesicle Production and Quantification Can Influence Biological and Experimental Outcomes.

Bacterial membrane vesicles (BMVs) are produced by all bacteria and facilitate a range of functions in host-microbe interactions and pathogenesis. Quantification of BMVs is a critical first step in the analysis of their biological and immunological functions. Historically, BMVs have been quantified by protein assay, which remains the preferred method of BMV quantification. However, recent studies have shown that BMV protein content can vary significantly between bacterial strains, growth conditions, and stages of bacterial growth, suggesting that protein concentration may not correlate directly with BMV quantity. Here, we show that the method used to quantify BMVs can alter experimental outcomes. We compared the enumeration of BMVs using different protein assays and nanoparticle tracking analysis (NTA). We show that different protein assays vary significantly in their quantification of BMVs and that their sensitivity varies when quantifying BMVs produced by different species. Moreover, stimulation of epithelial cells with an equivalent amount of BMV protein quantified using different protein assays resulted in significant differences in interleukin 8 (IL-8) responses. Quantification of Helicobacter pylori, Pseudomonas aeruginosa, and Staphylococcus aureus BMVs by NTA and normalization of BMV cargo to particle number revealed that BMV protein, DNA, and RNA contents were variable between strains and species and throughout bacterial growth. Differences in BMV-mediated activation of Toll-like receptors, NF-κB, and IL-8 responses were observed when stimulations were performed with equivalent BMV particle number but not equivalent protein amount. These findings reveal that the method of BMV quantification can significantly affect experimental outcomes, thereby potentially altering the observed biological functions of BMVs. IMPORTANCE Recent years have seen a surge in interest in the roles of BMVs in host-microbe interactions and interbacterial communication. As a result of such rapid growth in the field, there is a lack of uniformity in BMV enumeration. Here, we reveal that the method used to enumerate BMVs can significantly alter experimental outcomes. Specifically, standardization of BMVs by protein amount reduced the ability to distinguish strain differences in the immunological functions of BMVs. In contrast, species-, strain-, and growth stage-dependent differences in BMV cargo content were evident when BMVs were enumerated by particle number, and this was reflected in differences in their ability to induce immune responses. These findings indicate that parameters critical to BMV function, including bacterial species, strain, growth conditions, and sample purity, should form the basis of standard reporting in BMV studies. This will ultimately bring uniformity to the field to advance our understanding of BMV functions.

Staphylococcus aureus membrane vesicles contain immunostimulatory DNA, RNA and peptidoglycan that activate innate immune receptors and induce autophagy.

Gram-positive bacteria ubiquitously produce membrane vesicles (MVs), and although they contribute to biological functions, our knowledge regarding their composition and immunogenicity remains limited. Here we examine the morphology, contents and immunostimulatory functions of MVs produced by three Staphylococcus aureus strains; a methicillin resistant clinical isolate, a methicillin sensitive clinical isolate and a laboratory-adapted strain. We observed differences in the number and morphology of MVs produced by each strain and showed that they contain microbe-associated molecular patterns (MAMPs) including protein, nucleic acids and peptidoglycan. Analysis of MV-derived RNA indicated the presence of small RNA (sRNA). Furthermore, we detected variability in the amount and composition of protein, nucleic acid and peptidoglycan cargo carried by MVs from each S. aureus strain. S. aureus MVs activated Toll-like receptor (TLR) 2, 7, 8, 9 and nucleotide-binding oligomerization domain containing protein 2 (NOD2) signalling and promoted cytokine and chemokine release by epithelial cells, thus identifying that MV-associated MAMPs including DNA, RNA and peptidoglycan are detected by pattern recognition receptors (PRRs). Moreover, S. aureus MVs induced the formation of and colocalized with autophagosomes in epithelial cells, while inhibition of lysosomal acidification using bafilomycin A1 resulted in accumulation of autophagosomal puncta that colocalized with MVs, revealing the ability of the host to degrade MVs via autophagy. This study reveals the ability of DNA, RNA and peptidoglycan associated with MVs to activate PRRs in host epithelial cells, and their intracellular degradation via autophagy. These findings advance our understanding of the immunostimulatory roles of Gram-positive bacterial MVs in mediating pathogenesis, and their intracellular fate within the host.

Immunomodulatory roles and novel applications of bacterial membrane vesicles.

Bacteria release extracellular vesicles (EVs) known as bacterial membrane vesicles (BMVs) during their normal growth. Gram-negative bacteria produce BMVs termed outer membrane vesicles (OMVs) that are composed of a range of biological cargo and facilitate numerous bacterial functions, including promoting pathogenesis and mediating disease in the host. By contrast, less is understood about BMVs produced by Gram-positive bacteria, which are referred to as membrane vesicles (MVs), however their contribution to mediating bacterial pathogenesis has recently become evident. In this review, we summarise the mechanisms whereby BMVs released by Gram-negative and Gram-positive bacteria are produced, in addition to discussing their key functions in promoting bacterial survival, mediating pathogenesis and modulating host immune responses. Furthermore, we discuss the mechanisms whereby BMVs produced by both commensal and pathogenic organisms can enter host cells and interact with innate immune receptors, in addition to how they modulate host innate and adaptive immunity to promote immunotolerance or drive the onset and progression of disease. Finally, we highlight current and emerging applications of BMVs in vaccine design, biotechnology and cancer therapeutics.

Detection of Bacterial Membrane Vesicles by NOD-Like Receptors.

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.

Helicobacter pylori Growth Stage Determines the Size, Protein Composition, and Preferential Cargo Packaging of Outer Membrane Vesicles.

Gram-negative bacteria release outer membrane vesicles (OMVs) as part of their normal growth that contain a range of cargo from their parent bacterium, including DNA, RNA, and proteins. The protein content of OMVs is suggested to be similar in composition to various sub-cellular locations of their parent bacterium. However, very little is known regarding the effect of bacterial growth stage on the size, content, and selective packaging of proteins into OMVs. In this study, the global proteome of Helicobacter pylori and their OMVs throughout bacterial growth are examined to determine if bacterial growth stage affected OMV cargo composition. Analysis of OMVs produced by H. pylori reveals that bacterial growth stage affects the size, composition, and selection of protein cargo into OMVs. Proteomic analysis identifies that the proteome of H. pylori OMVs is vastly different throughout bacterial growth and that OMVs contain a range of proteins compared to their parent bacteria. In addition, bacterial growth stage affects the ability of OMVs to induce the production of IL-8 by human epithelial cells. Therefore, the findings identify that the size, proteome, and immunogenicity of OMVs produced during various stages of bacterial growth is not comparable. Collectively, these findings highlight the importance of considering the bacterial growth stage from which OMVs are isolated, as this will impact their size, protein composition, and ultimately their biological functions.