Outer Membrane Vesicles Biogenesis Research Articles

Inserting Omp22 into the flagellin protein, replacing its hypervariable region, results in stronger protection against lethal Acinetobacter baumannii infection

Acinetobacter baumannii, a common nosocomial pathogen, is known for its rapid acquisition of antimicrobial resistance, underscoring the urgent need to develop an effective vaccine against this pathogen. Outer membrane protein 22 (Omp22) regulates the biogenesis of outer membrane vesicles to transport virulence-promoting factors into the host cells and facilitates the progression of A. baumannii infection. In this study, we used a mouse model to assess a vaccine’s immunogenicity and protective efficacy using recombinant Omp22 protein within the hypervariable region of flagellin (FliC-Omp22). FliC-Omp22 demonstrated superior protection following challenge with a lethal dose of multidrug-resistant (MDR) A. baumannii strain 58ST compared to Omp22 alone. In addition, it elicited increased IgG1/IgG2a and IL-4/IFN-γ ratios, indicating a predominant Th2 immune response. Furthermore, the FliC-Omp22 vaccination elicited strong specific antibodies that inhibited the adhesion and invasion of A. baumannii 58ST and enhanced the opsonic killing activity against the pathogen. FliC-Omp22 immunization significantly reduced bacterial loads in infected mice’s spleen, lungs, and liver, thereby improving their survival against the lethal infection caused by MDR A. baumannii 58ST. This study suggests that integrating Omp22 into the hypervariable domain of flagellin holds promise for developing an effective vaccine against A. baumannii infections.

Engineering Versatile Bacteria-Derived Outer Membrane Vesicles: An Adaptable Platform for Advancing Cancer Immunotherapy.

In recent years, cancer immunotherapy has undergone a transformative shift toward personalized and targeted therapeutic strategies. Bacteria-derived outer membrane vesicles (OMVs) have emerged as a promising and adaptable platform for cancer immunotherapy due to their unique properties, including natural immunogenicity and the ability to be engineered for specific therapeutic purposes. In this review, a comprehensive overview is provided of state-of-the-art techniques and methodologies employed in the engineering of versatile OMVs for cancer immunotherapy. Beginning by exploring the biogenesis and composition of OMVs, unveiling their intrinsic immunogenic properties for therapeutic appeal. Subsequently, innovative approaches employed to engineer OMVs are delved into, ranging from the genetic engineering of parent bacteria to the incorporation of functional molecules. The importance of rational design strategies is highlighted to enhance the immunogenicity and specificity of OMVs, allowing tailoring for diverse cancer types. Furthermore, insights into clinical studies and potential challenges utilizing OMVs as cancer vaccines or adjuvants are also provided, offering a comprehensive assessment of the current landscape and future prospects. Overall, this review provides valuable insights for researchers involved in the rapidly evolving field of cancer immunotherapy, offering a roadmap for harnessing the full potential of OMVs as a versatile and adaptable platform for cancer treatment.

Inappropriate use of antibiotic enhances antibiotic resistance dissemination in ESBL-EC: Role of ydcz in outer membrane vesicles biogenesis and protein transport

Extended-spectrumβ-lactam producing Escherichia coli (ESBL-EC) readily colonizes live poultry and serves as a major source of contamination in retail chicken meat, posing significant threats to public health. This study aims to investigate the impact of inappropriate antibiotic use on the dissemination and exacerbation of antibiotic resistance in ESBL-EC and explore the underlying molecular mechanisms. Through experimental analysis, we propose a hypothesis that inappropriate antibiotic use may exacerbate resistance by affecting vesicle formation and protein secretion. Experimental results demonstrate that under the influence of amoxicillin, the concentration of proteins secreted in outer membrane vehicles (OMVs) by ESBL-EC significantly increases, along with a significant upregulation in the expression of the CTX-M-55-type Extended-spectrum beta-lactamase (CTX-M-55). Proteomic analysis and differential gene knockout experiments identified the key protein YdcZ, associated with OMVs formation and protein transportation in ESBL-EC under amoxicillin treatment. Further investigations reveal direct interactions between YdcZ and other proteins (YdiH and BssR). Upon ydcz gene knockout, a significant decrease in protein concentration within OMVs is observed, accompanied by a noticeable reduction in protection against sensitive bacteria. These findings suggest a critical role of YdcZ in regulating the process of protein transportation to OMVs in ESBL-EC under the influence of amoxicillin. In summary, our research uncovers the significant role of inappropriate antibiotic use in promoting the secretion of OMVs by ESBL-EC, aiding the survival of antibiotic-sensitive bacteria in the vicinity of infection sites. These findings provide new insights into the mechanisms underlying antibiotic-induced bacterial resistance dissemination and offer novel avenues for exploring prevention and control strategies against bacterial resistance propagation.

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.

Screening for Genes Involved in Outer Membrane Vesicle Biogenesis in Bacteroides thetaiotaomicron.

Bacteroides spp. are prominent gut commensals that are believed to modulate the intestinal environment, in part, by producing outer membrane vesicles (OMVs). Bacteroides OMVs have been ascribed many functions in vitro, but the genetic underpinnings behind OMV biogenesis and regulation are unclear. Understanding the mechanismof OMV biogenesis is required to determine the importance of Bacteroides OMVs in vivo. Here, we describe our methodologyfor screening Bacteroides thetaiotaomicron VPI-5482 to identify genes required for OMV biogenesis and regulation in a high-throughput format. This protocol is easily adaptable and can potentially be employed to further our knowledge of OMV biogenesis in other bacteria.

Outer Membrane Vesicles from Acinetobacter baumannii: Biogenesis, Functions, and Vaccine Application.

This review focuses on Acinetobacter baumannii, a Gram-negative bacterium that causes various infections and whose multidrug resistance has become a significant challenge in clinical practices. There are multiple bacterial mechanisms in A. baumannii that participate in bacterial colonization and immune responses. It is believed that outer membrane vesicles (OMVs) budding from the bacteria play a significant role in mediating bacterial survival and the subsequent attack against the host. Most OMVs originate from the bacterial membranes and molecules are enveloped in them. Elements similar to the pathogen endow OMVs with robust virulence, which provides a new direction for exploring the pathogenicity of A. baumannii and its therapeutic pathways. Although extensive research has been carried out on the feasibility of OMV-based vaccines against pathogens, no study has yet summarized the bioactive elements, biological activity, and vaccine applicability of A. baumannii OMVs. This review summarizes the components, biogenesis, and function of OMVs that contribute to their potential as vaccine candidates and the preparation methods and future directions for their development.

Characterization of outer membrane vesicles released by clinical isolates of Neisseria gonorrhoeae.

The sexually transmitted pathogen Neisseria gonorrhoeae releases membrane vesicles including outer membrane vesicles (OMVs) during infections. OMVs traffic outer membrane molecules, such as the porin PorB and lipo-oligosaccharide (LOS), into host innate immune cells, eliciting programmed cell death pathways, and inflammation. Little is known, however, about the proteome and LOS content of OMVs released by clinical strains isolated from different infection sites, and whether these vesicles similarly activate immune responses. Here, we characterized OMVs from four N. gonorrhoeae isolates and determined their size, abundance, proteome, LOS content, and activation of inflammatory responses in macrophages. The overall proteome of the OMVs was conserved between the four different isolates, which included major outer membrane and periplasm proteins. Despite this, we observed differences in the rate of OMV biogenesis and the relative abundance of membrane proteins and LOS. Consequently, OMVs from clinical isolates induced varying rates of macrophage cell death and the secretion of interleukin-1 family members, such as IL-1αandIL-1β. Overall, these findings demonstrate that clinical isolates of N. gonorrhoeae utilize membrane vesicles to release proteins and lipids, which affects innate immune responses.

PmrAB controls virulence-associated traits and outer membrane vesicle biogenesis in Acinetobacter baumannii

The PmrAB two-component system modulates colistin resistance in Acinetobacter baumannii, but its association with the virulence traits of this bacterium remains uncharacterized. This study explored the role of A. baumannii PmrAB in surface motility, biofilm formation, and outer membrane vesicle (OMV) biogenesis using wild-type (WT) A. baumannii 17978 and ΔpmrA and ΔpmrB mutant strains. The two mutant strains exhibited significantly decreased surface motility compared with that of WT strain by the low expression of abaI, abaR, A1S_0113, A1S_0115, and A1S_0116. Biofilm mass also significantly decreased in the two mutant strains at 12 h of incubation, but restored at 24 h. Under static culture conditions for 12 h, the two mutant strains showed low pgaA expression. However, the other biofilm-associated genes, such as csuC, csuE, ompA, and bap, showed different expression between the two mutant strains. Although the size of OMVs was similar among the three strains, the number of OMVs secreted from the two mutant strains slightly decreased compared with that secreted from the WT strain. Protein concentrations in the OMVs of ΔpmrA mutant significantly decreased compared with those in the OMVs of WT and ΔpmrB strains. Overall, PmrAB modulates virulence traits and OMV biogenesis in A. baumannii.

Bacterial outer membrane vesicles as drug delivery carrier for photodynamic anticancer therapy.

Photodynamic Therapy (PDT) is an effective tumor treatment strategy that not only induces photocytotoxicity to kill tumor cells directly but also activates the immune system in the body to generate tumor-specific immunity, preventing cancer metastasis and recurrence. However, some limitations of PDT limit the therapeutic efficacy in deep tumors. Previous studies have used different types of nanoparticles (NPs) as drug carriers of photosensitizers (PSs) to overcome the shortcomings of PDT and improve therapeutic efficacy. Among them, bacterial outer membrane vesicles (OMVs) have natural advantages as carriers for PS delivery. In addition to the targeted delivery of PSs into tumor cells, their unique immunogenicity helps them to serve as immune adjuvants to enhance the PDT-induced immune effect, providing new ideas for photodynamic anticancer therapy. Therefore, in this review, we will introduce the biogenesis and anticancer functions of OMVs and the research on them as drug delivery carriers in PDT. Finally, we also discuss the challenges and prospects of OMVs as a versatile drug delivery carrier for photodynamic anticancer therapy.

Quorum sensing-activated phenylalanine metabolism drives OMV biogenesis to enhance mosquito commensal colonization resistance to Plasmodium

Gut microbiota and its symbiotic relationship with the host are crucial for preventing pathogen infection. However, little is known about the mechanisms that drive commensal colonization. Serratia bacteria, commonly found in Anopheles mosquitoes, potentially mediate mosquito resistance to Plasmodium. Using S.ureilytica Su_YN1 as a model, we show that a quorum sensing (QS) circuit is crucial for stable colonization. After blood ingestion, the QS synthase SueI generates the signaling molecule N-hexanoyl-L-homoserine lactone (C6-HSL). Once C6-HSL binds to the QS receptor SueR, repression of the phenylalanine-to-acetyl-coenzyme A (CoA) conversion pathway is lifted. This pathway regulates outer membrane vesicle (OMV) biogenesis and promotes Serratia biofilm-like aggregate formation, facilitating gut adaptation and colonization. Notably, exposing Serratia Su_YN1-carrying Anopheles mosquitoes to C6-HSL increases Serratia gut colonization and enhances Plasmodium transmission-blocking efficacy. These findings provide insights into OMV biogenesis and commensal gut colonization and identify a powerful strategy for enhancing commensal resistance to pathogens.

The NQR Complex Regulates the Immunomodulatory Function of Bacteroides thetaiotaomicron.

The gut microbiome and intestinal immune system are engaged in a dynamic interplay that provides myriad benefits to host health. However, the microbiome can also elicit damaging inflammatory responses, and thus establishing harmonious immune-microbiome interactions is essential to maintain homeostasis. Gut microbes actively coordinate the induction of anti-inflammatory responses that establish these mutualistic interactions. Despite this, the microbial pathways that govern this dialogue remain poorly understood. We investigated the mechanisms through which the gut symbiont Bacteroides thetaiotaomicron exerts its immunomodulatory functions on murine- and human-derived cells. Our data reveal that B. thetaiotaomicron stimulates production of the cytokine IL-10 via secreted factors that are packaged into outer membrane vesicles, in a TLR2- and MyD88-dependent manner. Using a transposon mutagenesis-based screen, we identified a key role for the B. thetaiotaomicron-encoded NADH:ubiquinone oxidoreductase (NQR) complex, which regenerates NAD+ during respiration, in this process. Finally, we found that disruption of NQR reduces the capacity of B. thetaiotaomicron to induce IL-10 by impairing biogenesis of outer membrane vesicles. These data identify a microbial pathway with a previously unappreciated role in gut microbe-mediated immunomodulation that may be targeted to manipulate the capacity of the microbiome to shape host immunity.

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.

Evaluation of β-Lactamase Enzyme Activity in Outer Membrane Vesicles (OMVs) Isolated from Extended Spectrum β-Lactamase (ESBL) Salmonella Infantis Strains.

Outer membrane vesicles (OMVs) are nanoparticles released by Gram-negative bacteria, which contain different cargo molecules and mediate several biological processes. Recent studies have shown that OMVs are involved in antibiotic-resistance (AR) mechanisms by including β-lactamase enzymes in their lumen. Since no studies have as yet been conducted on Salmonella enterica subs. enterica serovar Infantis' OMVs, the aim of the work was to collect OMVs from five S. Infantis β-lactam resistant strains isolated from a broiler meat production chain and to investigate whether β-lactamase enzymes are included in OMVs during their biogenesis. OMVs were isolated by means of ultrafiltration and a Nitrocefin assay quantified the presence of β-lactamase enzymes in the OMVs. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) were used to identify the OMVs. The results showed that all strains release spherical OMVs, ranging from 60 to 230 nm. The Nitrocefin assay highlighted the presence of β-lactamase enzymes within the OMVs. This suggests that β-lactamase enzymes also get packaged into OMVs from bacterial periplasm during OMV biogenesis. An investigation into the possible role played by OMVs in AR mechanisms would open the door for an opportunity to develop new, therapeutic strategies.

A Robust Protocol to Isolate Outer Membrane Vesicles from Nontypeable Haemophilus influenzae

Outer membrane vesicles (OMVs) are lipid structures containing various biomolecules in their native environment and are spontaneously shed by gram-negative bacteria. OMVs perform several biological functions critical to both bacterial physiology and pathogenicity. Scientific research on OMV function and biogenesis requires a standardized and robust method of isolating these vesicles from bacterial cultures that reliably provide high-purity OMVs. Herein, we describe an optimized protocol to isolate OMVs from overnight cultures of three different strains of nontypeable Haemophilus influenzae (NTHi) for use in different downstream applications. Involving mainly differential centrifugation of the culture supernatant, the procedure described is relatively simple, efficient, and generates high-quality OMV preparations from each strain tested with sufficient yields, while preserving the native outer membrane composition.

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.

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.

Accurate molecular dynamics modeling of Pseudomonas aeruginosa outer membrane interaction with signaling molecules.

Pseudomonas aeruginosa is an opportunistic pathogen responsible for lungs, urinary tract, and surgical wound infections. This gram-negative bacterium releases outer membrane vesicles (OMV) derived from the cell's outer membrane (OM), serving diverse functions in bacterial survival, including nutrient acquisition, signaling, antibiotic resistance, delivery vehicle, and host-immune response modulation. This work aims to develop an accurate in silico model of P. aeruginosa outer membrane using all-atom molecular dynamics (MD) to understand OMV biogenesis formation based on the prediction of the bilayer-couple model. This model predicts the intercalation of a signaling molecule name Pseudomonas Quinolone Signal (PQS) to the OM as the main driver for the vesicle formation. In this study, the asymmetric model OM is composed of PA14 Lipid A in the outer leaflet and a mixture of POPE and POPG phospholipids in the inner leaflet. Due to the use of periodic boundary conditions in MD simulations, the compositional asymmetry imposes a fixed ratio of lipid numbers between the two leaflets, which needs to be set with caution. It has been shown that many widely used methods of defining this ratio lack control over the stress state of the membrane. As a result, the generated membrane could have a non-zero per leaflet tension. We investigate the role of the initial stress state on the intercalation of PQS to the OM. Free energy calculation shows that the favorable position of PQS within the membrane would shift significantly by variation of per leaflet tension, which could have important implications on the bilayer-couple model of OMV biogenesis. In addition, we model various PQS analogs to probe the role of different functional groups of PQS on the spontaneous intercalation of this signaling molecule to the membrane.

Desulfovibrio fairfieldensis-Derived Outer Membrane Vesicles Damage Epithelial Barrier and Induce Inflammation and Pyroptosis in Macrophages.

Sulfate-reducing bacteria Desulfovibrio fairfieldensis is an opportunistic pathogen that widely exists in the human intestine and can cause severe infectious diseases. However, the mechanisms contributing to its pathogenesis remain of great interest. In this study, we aim to investigate the outer membrane vesicles (OMVs) secreted by D. fairfieldensis and their pathogenic effect. The OMVs separated by ultracentrifugation were spherical and displayed a characteristic bilayer lipid structure observed by transmission electron microscopy, with an average hydrodynamic diameter of 75 nm measurement using the particle size analyzer. We identified 1496 and 916 proteins from D. fairfieldensis and its OMVs using label-free non-target quantitative proteomics, respectively. The 560 co-expressed proteins could participate in bacterial life activities by function prediction. The translocation protein TolB, which participates in OMVs biogenesis and transporting toxins was highly expressed in OMVs. The OMVs inhibited the expression of tight junction proteins OCCLUDIN and ZO-1 in human colonic epithelial cells (Caco-2). The OMVs decreased the cell viability of monocyte macrophages (THP-1-Mφ) and activated various inflammatory factors secretion, including interferon-γ (IFN-γ), tumor necrosis factor (TNF-α), and many interleukins. Further, we found the OMVs induced the expression of cleaved-gasdermin D, caspase-1, and c-IL-1β and caused pyroptosis in THP-1-Mφ cells. Taken together, these data reveal that the D. fairfieldensis OMVs can damage the intestinal epithelial barrier and activate intrinsic inflammation.

Characterization of a Bacterial Kinase That Phosphorylates Dihydrosphingosine to Form dhS1P.

ABSTRACTLike other members of the phylum Bacteroidetes, the oral anaerobe Porphyromonas gingivalis synthesizes a variety of sphingolipids, similar to its human host. Studies have shown that synthesis of these lipids (dihydroceramides [DHCs]) is involved in oxidative stress resistance, the survival of P. gingivalis during stationary phase, and immune modulation. Here, we constructed a deletion mutant of P. gingivalis strain W83 with a deletion of the gene encoding DhSphK1, a protein that shows high similarity to a eukaryotic sphingosine kinase, an enzyme that phosphorylates sphingosine to form sphingosine-1-phosphate. Our data show that deletion of the dhSphK1 gene results in a shift in the sphingolipid composition of P. gingivalis cells; specifically, the mutant synthesizes higher levels of phosphoglycerol DHCs (PG-DHCs) than the parent strain W83. Although PG1348 shows high similarity to the eukaryotic sphingosine kinase, we discovered that the PG1348 enzyme is unique, since it preferentially phosphorylates dihydrosphingosine, not sphingosine. Besides changes in lipid composition, the W83 ΔPG1348 mutant displayed a defect in cell division, the biogenesis of outer membrane vesicles (OMVs), and the amount of K antigen capsule. Taken together, we have identified the first bacterial dihydrosphingosine kinase whose activity regulates the lipid profile of P. gingivalis and underlies a regulatory mechanism of immune modulation.IMPORTANCE Sphingoid base phosphates, such as sphingosine-1-phosphate (S1P) and dihydrosphingosine-1-phosphate (dhS1P), act as ligands for S1P receptors, and this interaction is known to play a central role in mediating angiogenesis, vascular stability and permeability, and immune cell migration to sites of inflammation. Studies suggest that a shift in ratio to higher levels of dhS1P in relation to S1P alters downstream signaling cascades due to differential binding and activation of the various S1P receptor isoforms. Specifically, higher levels of dhS1P are thought to be anti-inflammatory. Here, we report on the characterization of a novel kinase in Porphyromonas gingivalis that phosphorylates dihydrosphingosine to form dhS1P.

Lipidomics Analysis of Outer Membrane Vesicles and Elucidation of the Inositol Phosphoceramide Biosynthetic Pathway in Bacteroides thetaiotaomicron.

ABSTRACTApproximately one-third of the human colonic microbiome is formed by bacteria from the genus Bacteroides. These bacteria produce a large amount of uniformly sized outer membrane vesicles (OMVs), which are equipped with hydrolytic enzymes that play a role in the degradation of diet- and host-derived glycans. In this work, we characterize the lipid composition of membranes and OMVs from Bacteroides thetaiotaomicron VPI-5482. Liquid chromatography-mass spectrometry (LC-MS) analysis indicated that OMVs carry sphingolipids, glycerophospholipids, and serine-dipeptide lipids. Sphingolipid species represent more than 50% of the total lipid content of OMVs. The most abundant sphingolipids in OMVs are ethanolamine phosphoceramide (EPC) and inositol phosphoceramide (IPC). Bioinformatics analysis allowed the identification of the BT1522–1526 operon putatively involved in IPC synthesis. Mutagenesis studies revealed that BT1522–1526 is essential for the synthesis of phosphatidylinositol (PI) and IPC, confirming the role of this operon in the biosynthesis of IPC. BT1522–1526 mutant strains lacking IPC produced OMVs that were indistinguishable from the wild-type strain, indicating that IPC sphingolipid species are not involved in OMV biogenesis. Given the known role of sphingolipids in immunomodulation, we suggest that OMVs may act as long-distance vehicles for the delivery of sphingolipids in the human gut.IMPORTANCE Sphingolipids are essential membrane lipid components found in eukaryotes that are also involved in cell signaling processes. Although rare in bacteria, sphingolipids are produced by members of the phylum Bacteroidetes, human gut commensals. Here, we determined that OMVs carry sphingolipids and other lipids of known signaling function. Our results demonstrate that the BT1522–1526 operon is required for IPC biosynthesis in B. thetaiotaomicron.