Production Of Outer Membrane Vesicles Research Articles

Outer membrane vesicles generated by an exogenous bacteriophage lysin and protection against Acinetobacter baumannii infection

BackgroundThe outer membrane vesicles (OMVs) produced by Gram-negative bacteria can modulate the immune system and have great potentials for bacterial vaccine development.ResultsA highly active Acinetobacter baumannii phage lysin, LysP53, can stimulate the production of OMVs after interacting with A. baumannii, Escherichia coli, and Salmonella. The OMVs prepared by the lysin (LOMVs) from A. baumannii showed better homogeneity, higher protein yield, lower endotoxin content, and lower cytotoxicity compared to the naturally produced OMVs (nOMVs). The LOMVs contain a significantly higher number of cytoplasmic and cytoplasmic membrane proteins but a smaller number of periplasmic and extracellular proteins compared to nOMVs. Intramuscular immunization with either LOMVs or nOMVs three times provided robust protection against A. baumannii infections in both pneumonia and bacteremia mouse models. Intranasal immunization offered good protection in the pneumonia model but weaker protection (20–40%) in the bacteremia model. However, with a single immunization, LOMVs demonstrated better protection than the nOMVs in the pneumonia mouse model.ConclusionsThe novel lysin approach provides a superior choice compared to current methods for OMV production, especially for vaccine development.Graphical

Stress response of Salmonella Newport with various sequence types toward plasma-activated water: Viable but nonculturable state formation and outer membrane vesicle production

This study aims to investigate the response of Salmonella Newport to plasma-activated water (PAW), a novel disinfectant that attracts attention due to its broad-spectrum antimicrobial efficacy and eco-friendliness. In this work, we demonstrated that S. Newport of different sequence types (STs) could be induced into the viable but nonculturable (VBNC) state by PAW treatment. Notably, a remarkable 99.96% of S. Newport ST45 strain entered the VBNC state after a 12-min PAW treatment, which was the fastest observed among the five S. Newport STs (ST31, ST45, ST46, ST166, ST2364). Secretion of outer membrane vesicles was observed in ST45, suggesting a potential strategy against PAW treatment. Genes related to oxidative stress (sodA, katE, trxA), outer membrane proteins (ompA, ompC, ompD, ompF) and virulence (pagC, sipC, sopE2) were upregulated in the PAW-treated S. Newport, especially in ST45. A reduction of 38–65% in intracellular ATP level after PAW treatment was observed, indicating a contributor to the formation of the VBNC state. In addition, a rapid method for detecting the proportion of VBNC cells in food products based on pagC was established. This study contributes to understanding the formation mechanism of the VBNC state in S. Newport under PAW stress and offers insights for controlling microbial risks in the food industry.

Evaluation of immunogenicity and protective efficacy of outer membrane vesicles from Salmonella Typhimurium and Salmonella Choleraesuis

Outer membrane vesicles (OMVs) are membranous structures frequently observed in Gram-negative bacteria that contain bioactive substances. These vesicles are rich in bacterial antigens that can activate the host’s immune system, making them a promising candidate vaccine to prevent and manage bacterial infections. The aim of this study was to assess the immunogenicity and protective efficacy of OMVs derived from Salmonella enterica serovar Typhimurium and S. Choleraesuis, while also focusing on enhancing OMV production. Initial experiments showed that OMVs from wild-type strains did not provide complete protection against homologous Salmonella challenge, possible due to the presence of flagella in the purified OMVs samples, which may elicit an unnecessary immune response. To address this, flagellin-deficient mutants of S. Typhimurium and S. Choleraesuis were constructed, designated rSC0196 and rSC0199, respectively. These mutants exhibited reduced cell motility and their OMVs were found to be flagellin-free. Immunization with non-flagellin OMVs derived from rSC0196 induced robust antibody responses and improved survival rates in mice, as compared to the OMVs derived from the wild-type UK-1. In order to enhance OMV production, deletions of ompA or tolR were introduced into rSC0196. The deletion of tolR not only increase the yield of OMVs, but also conferred complete protection against homologous S. Typhimurium challenge in mice. Collectively, these findings indicate that the flagellin-deficient OMVs with a tolR mutation have the potential to serve as a versatile vaccine platform, capable of inducing broad-spectrum protection against significant pathogens.

Construction and characterization of a hypervesiculation strain of Escherichia coli Nissle 1917.

Outer membrane vesicles (OMVs) are produced by Gram-negative bacteria and deliver microbial molecules to distant target cells in a host. OMVs secreted by probiotic probiotic strain Escherichia coli Nissle 1917 (EcN) have been reported to induce an immune response. In this study, we aimed to increase the OMV production of EcN. The double gene knockout of mlaE and nlpI was conducted in EcN because the ΔmlaEΔnlpI of experimental strain E. coli K12 showed the highest OMV production in our previous report. The ΔmlaEΔnlpI of EcN showed approximately 8 times higher OMV production compared with the parental (wild-type) strain. Quick-freeze, deep-etch replica electron microscopy revealed that plasmolysis occurred in the elongated ΔmlaEΔnlpI cells and the peptidoglycan (PG) had numerous holes. While these phenomena are similar to the findings for the ΔmlaEΔnlpI of K12, there were more PG holes in the ΔmlaEΔnlpI of EcN than the K12 strain, which were observed not only at the tip of the long axis but also in the whole PG structure. Further analysis clarified that the viability of ΔmlaEΔnlpI of EcN decreased compared with that of the wild-type. Although the amount of PG in ΔmlaEΔnlpI cells was about half of that in wild-type, the components of amino acids in PG did not change in ΔmlaEΔnlpI. Although the viability decreased compared to the wild-type, the ΔmlaEΔnlpI grew in normal culture conditions. The hypervesiculation strain constructed here is expected to be used as an enhanced probiotic strain.

β-lactam-induced OMV release promotes polymyxin tolerance in Salmonella enterica sv. Typhi.

The rise of multidrug-resistant bacteria is a global concern, leading to a renewed reliance on older antibiotics like polymyxins as a last resort. Polymyxins, cationic cyclic peptides synthesized nonribosomally, feature a hydrophobic acyl tail and positively charged residues. Their antimicrobial mechanism involves initial interaction with Gram-negative bacterial outer-membrane components through polar and hydrophobic interactions. Outer membrane vesicles (OMVs), nano-sized proteoliposomes secreted from the outer membrane of Gram-negative bacteria, play a crucial role in tolerating harmful molecules, including cationic peptides such as polymyxins. Existing literature has documented environmental changes' impact on modulating OMV properties in Salmonella Typhimurium. However, less information exists regarding OMV production and characteristics in Salmonella Typhi. A previous study in our laboratory showed that S. Typhi ΔmrcB, a mutant associated with penicillin-binding protein (PBP, a β-lactam antibiotic target), exhibited hypervesiculation. Consequently, this study investigated the potential impact of β-lactam antibiotics on promoting polymyxin tolerance via OMVs in S. Typhi. Our results demonstrated that sub-lethal doses of β-lactams increased bacterial survival against polymyxin B in S. Typhi. This phenomenon stems from β-lactam antibiotics inducing hypervesiculation of OMVs with higher affinity for polymyxin B, capturing and diminishing its biologically effective concentration. These findings suggest that β-lactam antibiotic use may inadvertently contribute to decreased polymyxin effectivity against S. Typhi or other Gram-negative bacteria, complicating the effective treatment of infections caused by these pathogens. This study emphasizes the importance of evaluating the influence of β-lactam antibiotics on the interaction between OMVs and other antimicrobial agents.

Dual membrane-spanning anti-sigma factors regulate vesiculation in Bacteroides thetaiotaomicron

Bacteroidota are abundant members of the human gut microbiota that shape the enteric landscape by modulating host immunity and degrading dietary- and host-derived glycans. These processes are mediated in part by Outer Membrane Vesicles (OMVs). Here, we developed a high-throughput screen to identify genes required for OMV biogenesis and its regulation in Bacteroides thetaiotaomicron (Bt). We identified a family of Dual membrane-spanning anti-sigma factors (Dma) that control OMV biogenesis. We conducted molecular and multiomic analyses to demonstrate that deletion of Dma1, the founding member of the Dma family, modulates OMV production by controlling the activity of the ECF21 family sigma factor, Das1, and its downstream regulon. Dma1 has a previously uncharacterized domain organization that enables Dma1 to span both the inner and outer membrane of Bt. Phylogenetic analyses reveal that this common feature of the Dma family is restricted to the phylum Bacteroidota. This study provides mechanistic insights into the regulation of OMV biogenesis in human gut bacteria.

Synthetic Outer Membrane Vesicles Bearing Tn Antigen

AbstractN‐Alkyl‐thio glycolipids (TGL) are proposed as fundamental components in the production of synthetic outer membrane vesicles (S‐OMVs). A small but representative library of glycolipids was prepared by thiol‐ene coupling (TEC), using unprotected thiols in acceptable to good yields (63‐96 %). Multilamellar giant vesicles (GVs) of varying sizes were assembled from mixtures of TGL and 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphate (POPC). Our results mark an important advancement for the development of synthetic anticancer vaccines, supramolecular adjuvants and drug delivery systems based on glycolipid/phospholipid formulations (10 % mol/mol in 0.1 mm solution).

Calcium phosphate nanoparticles conjugated with outer membrane vesicle of Riemerella anatipestifer for vaccine development in ducklings

Biodegradable calcium phosphate nanoparticles offer a viable substitute for traditional adjuvants such as aluminum in vaccine production. Calcium phosphate nanoparticle adjuvanted with outer membrane vesicle (OMV) of gram negative bacteria may induce efficient immune response in the host. The present study was carried out to evaluate the potential of a mucosal vaccine formulation of calcium phosphate (CAP) nanoparticle using OMV of Riemerella anatipestifer (RA) as antigen against New Duck disease in ducks. The work was initiated with isolation, identification of RA, followed by OMV production and extraction. The CAP-OMV nanoparticle was prepared and characterized. The efficacy of the vaccine formulation and toxicity were studied in ducks. The average OMV yield in terms of protein concentration was found to be 122.33 ± 3.48 mg per liter of BHI broth. In SDS-PAGE, isolated OMVs exhibited presence of 16 distinct protein bands with molecular weight ranging from 142.1 to 12.1 kDa. Seven protein bands of 74.1, 69.3, 55.5, 50.6, 45.6, 25.1 and 13.1 kDa were detected relatively more distinct. The major bands detected in our findings were 42 kDa, 37 kDa and 16 kDa that corresponds to OmpA, OmpH, P6 respectively. The mean zeta size (±SD) and potential of the nanoparticle were 246.20 ± 0.53 nm and −25.60 ± 5.97 respectively. In transmission electron microscopy (TEM), the nanoparticles exhibited an average diameter of 129.80 ± 11.10 nm and displayed spherical morphology. The median protective dose (PD50) of CAP-OMV nanoparticle was 1881.10 μg of protein. Group I ducks received 3762 μg of protein (entrapped protein in CAP-OMV nanoparticle) via intra nasal route and it showed the highest serum IgG and secretory IgA level than other immunized groups. All experimental ducks were challenged with 10 × LD50 on 35 days of post primary immunization. Group I showed 100 % survivability in the challenge study. No gross and biochemical indication of acute or chronic toxicity were recorded. In conclusion, our results suggest that CAP-OMV nanoparticle can be a safe and efficient mucosal vaccine delivery system for RA, eliciting strong immune response in the host.

Molecular imaging of bacterial outer membrane vesicles based on bacterial surface display

The important roles of bacterial outer membrane vesicles (OMVs) in various diseases and their emergence as a promising platform for vaccine development and targeted drug delivery necessitates the development of imaging techniques suitable for quantifying their biodistribution with high precision. To address this requirement, we aimed to develop an OMV specific radiolabeling technique for positron emission tomography (PET). A novel bacterial strain (E. coli BL21(DE3) ΔnlpI, ΔlpxM) was created for efficient OMV production, and OMVs were characterized using various methods. SpyCatcher was anchored to the OMV outer membrane using autotransporter-based surface display systems. Synthetic SpyTag-NODAGA conjugates were tested for OMV surface binding and 64Cu labeling efficiency. The final labeling protocol shows a radiochemical purity of 100% with a ~ 29% radiolabeling efficiency and excellent serum stability. The in vivo biodistribution of OMVs labeled with 64Cu was determined in mice using PET/MRI imaging which revealed that the biodistribution of radiolabeled OMVs in mice is characteristic of previously reported data with the highest organ uptakes corresponding to the liver and spleen 3, 6, and 12 h following intravenous administration. This novel method can serve as a basis for a general OMV radiolabeling scheme and could be used in vaccine- and drug-carrier development based on bioengineered OMVs.

The Legionella autoinducer LAI-1 is delivered by outer membrane vesicles to promote interbacterial and interkingdom signaling

Legionella pneumophila is an environmental bacterium, which replicates in amoeba but also in macrophages, and causes a life-threatening pneumonia called Legionnaires' disease. The opportunistic pathogen employs the α-hydroxy-ketone compound Legionella autoinducer-1 (LAI-1) for intraspecies and interkingdom signaling. LAI-1 is produced by the autoinducer synthase Legionella quorum sensing A (LqsA), but it is not known, how LAI-1 is released by the pathogen. Here, we use a Vibrio cholerae luminescence reporter strain and liquid chromatography-tandem mass spectrometry to detect bacteria-produced and synthetic LAI-1. Ectopic production of LqsA in Escherichia coli generated LAI-1, which partitions to outer membrane vesicles (OMVs) and increases OMV size. These E.coli OMVs trigger luminescence of the V.cholerae reporter strain and inhibit the migration of Dictyostelium discoideum amoeba. Overexpression of lqsA in L.pneumophila under the control of strong stationary phase promoters (PflaA or P6SRNA), but not under control of its endogenous promoter (PlqsA), produces LAI-1, which is detected in purified OMVs. These L. pneumophila OMVs trigger luminescence of the Vibrio reporter strain and inhibit D.discoideum migration. L.pneumophila OMVs are smaller upon overexpression of lqsA or upon addition of LAI-1 to growing bacteria, and therefore, LqsA affects OMV production. The overexpression of lqsA but not a catalytically inactive mutant promotes intracellular replication of L.pneumophila in macrophages, indicating that intracellularly produced LA1-1 modulates the interaction in favor of the pathogen. Taken together, we provide evidence that L.pneumophila LAI-1 is secreted through OMVs and promotes interbacterial communication and interactions with eukaryotic host cells.

Bacterial Outer Membrane Vesicles as a Platform for the Development of a Broadly Protective Human Papillomavirus Vaccine Based on the Minor Capsid Protein L2.

Human papillomaviruses (HPVs) are a large family of viruses with a capsid composed of the L1 and L2 proteins, which bind to receptors of the basal epithelial cells and promote virus entry. The majority of sexually active people become exposed to HPV and the virus is the most common cause of cervical cancer. Vaccines are available based on the L1 protein, which self-assembles and forms virus-like particles (VLPs) when expressed in yeast and insect cells. Although very effective, these vaccines are HPV type-restricted and their costs limit broad vaccination campaigns. Recently, vaccine candidates based on the conserved L2 epitope from serotypes 16, 18, 31, 33, 35, 6, 51, and 59 were shown to elicit broadly neutralizing anti-HPV antibodies. In this study, we tested whether E. coli outer membrane vesicles (OMVs) could be successfully decorated with L2 polytopes and whether the engineered OMVs could induce neutralizing antibodies. OMVs represent an attractive vaccine platform owing to their intrinsic adjuvanticity and their low production costs. We show that strings of L2 epitopes could be efficiently expressed on the surface of the OMVs and a polypeptide composed of the L2 epitopes from serotypes 18, 33, 35, and 59 provided a broad cross-protective activity against a large panel of HPV serotypes as determined using pseudovirus neutralization assay. Considering the simplicity of the OMV production process, our work provides a highly effective and inexpensive solution to produce universal anti-HPV vaccines.

AamA-mediated epigenetic control of genome-wide gene expression and phenotypic traits in Acinetobacter baumannii ATCC 17978.

Individual deletions of three genes encoding orphan DNA methyltransferases resulted in the occurrence of growth defect only in the aamA (encoding AcinetobacterAdenine Methylase A) mutant of A. baumannii strain ATCC 17978. Our single-molecule real-time sequencing-based methylome analysis revealed multiple AamA-mediated DNA methylation sites and proposed a potent census target motif (TTTRAATTYAAA). Loss of Dam led to modulation of genome-wide gene expression, and several Dam-target sites including the promoter region of the trmD operon (rpsP, rimM, trmD, and rplS) were identified through our methylome and transcriptome analyses. AamA methylation also appeared to control the expression of many genes linked to membrane functions (lolAB, lpxO), replication (dnaA) and protein synthesis (trmD operon) in the strain ATCC 17978. Interestingly, cellular resistance against several antibiotics and ethidium bromide through functions of efflux pumps diminished in the absence of the aamA gene, and the complementation of aamA gene restored the wild-type phenotypes. Other tested phenotypic traits such as outer-membrane vesicle production, biofilm formation and virulence were also affected in the aamA mutant. Collectively, our data indicated that epigenetic regulation through AamA-mediated DNA methylation of novel target sites mostly in the regulatory regions could contribute significantly to changes in multiple phenotypic traits in A. baumannii ATCC 17978.

Exploring engineered vesiculation by Pseudomonas putida KT2440 for natural product biosynthesis.

Pseudomonas species have become promising cell factories for the production of natural products due to their inherent robustness. Although these bacteria have naturally evolved strategies to cope with different kinds of stress, many biotechnological applications benefit from engineering of optimised chassis strains with specially adapted tolerance traits. Here, we explored the formation of outer membrane vesicles (OMV) of Pseudomonas putida KT2440. We found OMV production to correlate with the recombinant production of a natural compound with versatile beneficial properties, the tripyrrole prodigiosin. Further, several P. putida genes were identified, whose up- or down-regulated expression allowed controlling OMV formation. Finally, genetically triggering vesiculation in production strains of the different alkaloids prodigiosin, violacein, and phenazine-1-carboxylic acid, as well as the carotenoid zeaxanthin, resulted in up to three-fold increased product yields. Consequently, our findings suggest that the construction of robust strains by genetic manipulation of OMV formation might be developed into a useful tool which may contribute to improving limited biotechnological applications.

Human gut bacteria tailor extracellular vesicle cargo for the breakdown of diet- and host-derived glycans

Extracellular vesicles are produced in all three domains of life, and their biogenesis has common ancient origins in eukaryotes and archaea. Although bacterial vesicles were discovered several decades ago and multiple roles have been attributed to them, no mechanism has been established for vesicles biogenesis in bacteria. For this reason, there is a significant level of skepticism about the biological relevance of bacterial vesicles. Bacteroides thetaiotaomicron (Bt), a prominent member of the human intestinal microbiota, produces significant amounts of outer membrane vesicles (OMVs) which have been proposed to play key physiological roles. Here, we employed a dual marker system, consisting of outer membrane- and OMV-specific markers fused to fluorescent proteins to visualize OMV biogenesis by time-lapse microscopy. Furthermore, we performed comparative proteomic analyses to show that, in Bt, the OMV cargo is adapted for the optimal utilization of different polysaccharides. We also show that a negatively charged N-terminal motif acts as a signal for protein sorting into OMVs irrespective of the nutrient availability. Our results demonstrate that OMV production is the result of a highly regulated process in Bt.

The secretome of the fish pathogen Tenacibaculum maritimum includes soluble virulence-related proteins and outer membrane vesicles

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.

Features of Biogenesis of Vesicles of External Membranes of Microorganisms, theirImmunogenic, Protective and Adjuvant Ability

Relevance. The process of production of outer membrane vesicles by bacteria is the main mechanism in intercellular communication and an intermediary in relationships of a very different nature (symbiosis, commensalism and parasitism), therefore, the study of the role of vesicles in the pathogenesis and immunogenesis of bacteria is an important and timely task.Aims. The purpose of this research was the analysis of scientific publications Russian and foreign journals for the period from 2002 to 2021 from the bibliographic databases of eLibrary.Ru, PubMed®, MEDLINE, dedicated to vesicles of outer membranes formed in various types of pathogenic and non-pathogenic bacteria.Conclusion. The study of the structure, factors of formation, functional significance of the mechanisms of action of bacterial vesicles, as well as the role of these structures in the pathogenesis and immunogenesis of various diseases, including especially dangerous ones, makes it possible to create new preventive drugs based on them. The use of vesicles as means of delivery of biological drugs and various antigens opens up new opportunities for improving the therapy and prevention of infections.

Engineering Outer Membrane Vesicles to Increase Extracellular Electron Transfer of Shewanella oneidensis.

Outer membrane vesicles (OMVs) of Gram-negative bacteria play an essential role in cellular physiology. The underlying regulatory mechanism of OMV formation and its impact on extracellular electron transfer (EET) in the model exoelectrogenShewanella oneidensis MR-1 remain unclear and have not been reported. To explore the regulatory mechanism of OMV formation, we used the CRISPR-dCas9 gene repression technology to reduce the crosslink between the peptidoglycan (PG) layer and the outer membrane, thus promoting the OMV formation. We screened the target genes that were potentially beneficial to the outer membrane bulge, which were classified into two modules: PG integrity module (Module 1) and outer membrane component module (Module 2). We found that downregulation of the penicillin-binding protein-encoding gene pbpC for peptidoglycan integrity (Module 1) and the N-acetyl-d-mannosamine dehydrogenase-encoding gene wbpP involved in lipopolysaccharide synthesis (Module 2) exhibited the highest production of OMVs and enabled the highest output power density of 331.3 ± 1.2 and 363.8 ± 9.9 mW m-2, 6.33- and 6.96-fold higher than that of the wild-typeS. oneidensis MR-1 (52.3 ± 0.6 mW m-2), respectively. To elucidate the specific impacts of OMV formation on EET, OMVs were isolated and quantified for UV-visible spectroscopy and heme staining characterization. Our study showed that abundant outer membrane c-type cytochromes (c-Cyts) including MtrC and OmcA and periplasmic c-Cyts were exposed on the surface or inside of OMVs, which were the vital constituents responsible for EET. Meanwhile, we found that the overproduction of OMVs could facilitate biofilm formation and increase biofilm conductivity. To the best of our knowledge, this study is the first to explore the mechanism of OMV formation and its correlation with EET of S. oneidensis, which paves the way for further study of OMV-mediated EET.

Outer membrane vesicles induce the mussel plantigrade settlement via regulation of c-di-GMP

Despite the importance of outer membrane vesicles (OMVs) in benthic animal settlement, the underlying molecular mechanism remains elusive. Here, the impact of OMVs and OMVs synthesis-related tolB gene in Mytilus coruscus plantigrade settlement was tested. The OMVs were extracted from Pseudoalteromonas marina through density gradient centrifugation, and a tolB knockout strain, achieved by homologous recombination, was utilized for the investigation. Our results demonstrated that OMVs could significantly enhance M. coruscus plantigrades settlement. Deleting the tolB resulted in downregulation of c-di-GMP, accompanied by a reduction of OMV production, a decline in bacterial motility and increasing biofilm-forming ability. Enzyme treatment resulted in a 61.11% reduction in OMV-inducing activity and a 94.87% reduction in LPS content. Thus, OMVs regulate mussel settlement via LPS, and c-di-GMP is responsible for the OMV-inducing capacity. These findings provide new insights into the interactions between bacteria and mussels.

Murine Norovirus Interaction with Enterobacter cloacae Leads to Changes in Membrane Stability and Packaging of Lipid and Metabolite Vesicle Content.

Outer membrane vesicles (OMVs) are a primary means of communication for Gram-negative bacteria. The specific role of vesicle components in cellular communication and how components are packaged are still under investigation, but a correlation exists between OMV biogenesis and content. The two primary mechanisms of OMV biogenesis are membrane blebbing and explosive cell lysis, and vesicle content is based on the biogenesis mechanism. Hypervesiculation, which can be induced by stress conditions, also influences OMV content. Norovirus interaction with Enterobacter cloacae induces stress responses leading to increased OMV production and changes in DNA content, protein content, and vesicle size. The presence of genomic DNA and cytoplasmic proteins in these OMVs suggests some of the vesicles are formed by explosive cell lysis, so reduction or loss of these components indicates a shift away from this mechanism of biogenesis. Based on this, further investigation into bacterial stability and OMV content was conducted. Results showed that norovirus induced a dramatic shift in OMV lipid content. Specifically, the increased accumulation of phospholipids is associated with increased blebbing, thereby supporting previous observations that noroviruses shift the mechanism of OMV biogenesis. Slight differences in OMV metabolite content were also observed. While norovirus induced changes in OMV content, it did not change the lipid content of the bacterial outer membrane or the metabolite content of the bacterial cell. Overall, these results indicate that norovirus induces significant changes to OMV lipid architecture and cargo, which may be linked to a change in the mechanism of vesicle biogenesis. IMPORTANCE Extracellular vesicles from commensal bacteria are recognized for their importance in modulating host immune responses, and vesicle content is related to their impact on the host. Therefore, understanding how vesicles are formed and how their content shifts in response to stress conditions is necessary for elucidating their downstream functions. Our recent work has demonstrated that interactions between noroviruses and Enterobacter cloacae induce bacterial stress responses leading to hypervesiculation. In this article, we characterize and compare the lipid and metabolomic cargo of E. cloacae vesicles generated in the presence and absence of norovirus and show that viral interactions induce significant changes in vesicle content. Furthermore, we probe how these changes and changes to the bacterial cell may be indicative of a shift in the mechanism of vesicle biogenesis. Importantly, we find that noroviruses induce significant changes in vesicle lipid architecture and cargo that may be responsible for the immunogenic activity of these vesicles.

Genomic and physiological characterization of Novosphingobium terrae sp. nov., an alphaproteobacterium isolated from Cerrado soil containing a mega-sized chromid.

A novel bacterial strain, designated GeG2T, was isolated from soils of the native Cerrado, a highly biodiverse savanna-like Brazilian biome. 16S rRNA gene analysis of GeG2T revealed high sequence identity (100%) to the alphaproteobacterium Novosphingobium rosa; however, comparisons with N. rosa DSM 7285T showed several distinctive features, prompting a full characterization of the new strain in terms of physiology, morphology, and, ultimately, its genome. GeG2T cells were Gram-stain-negative bacilli, facultatively anaerobic, motile, positive for catalase and oxidase activities, and starch hydrolysis. Strain GeG2T presented planktonic-sessile dimorphism and cell aggregates surrounded by extracellular matrix and nanometric spherical structures were observed, suggesting the production of exopolysaccharides (EPS) and outer membrane vesicles (OMVs). Despite high 16S rDNA identity, strain GeG2T showed 90.38% average nucleotide identity and 42.60% digital DNA-DNA hybridization identity with N. rosa, below species threshold. Whole-genome assembly revealed four circular replicons: a 4.1Mb chromosome, a 2.7Mb extrachromosomal megareplicon, and two plasmids (212.7 and 68.6kb). The megareplicon contains a few core genes and plasmid-type replication/maintenance systems, consistent with its classification as a chromid. Genome annotation shows a vast repertoire of carbohydrate-active enzymes and genes involved in the degradation of aromatic compounds, highlighting the biotechnological potential of the new isolate. Chemotaxonomic features, including polar lipid and fatty acid profiles, as well as physiological, molecular, and whole-genome comparisons showed significant differences between strain GeG2T and N. rosa, indicating that it represents a novel species, for which the name Novosphingobium terrae is proposed. The type strain is GeG2T (= CBMAI 2313T = CBAS 753T).