Outer Membrane Vesicles Biogenesis Research Articles

Molecular dynamics modeling of Pseudomonas aeruginosa outer membranes.

Pseudomonas aeruginosa is a common Gram-negative bacterium and opportunistic human pathogen. The distinctive structure of its outer membrane (OM) and outer membrane vesicles (OMVs) plays a fundamental role in bacterial virulence, colonization ability, and antibiotic resistance. To provide critical insights into OM and OMV functionality, we conducted an all-atom molecular dynamics study of asymmetric membranes that are biologically relevant to P. aeruginosa. We hybridized a GLYCAM06-based lipopolysaccharides force field with the Stockholm lipids force field (Slipids) to model bilayer membranes with Lipid A molecules in one leaflet and physiologically relevant phospholipid molecules in the other, including 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), and 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG). In particular, a membrane with phospholipid composition representing the P. aeruginosa OM was constructed and modeled by mixing the physiologically dominant components. The detailed structure of membranes was characterized by area per lipid, transmembrane mass and charge densities, radial distribution function (RDF), deuterium order parameter (SCD) of acyl chains, and inclination angles of phosphates and disaccharide in Lipid A. The membrane fluidity in equilibrium and the hydration of functional groups were probed and characterized quantitatively. The consistent properties of the Lipid A leaflets in different membranes demonstrate its compatibility with various phospholipids present in the P. aeruginosa OM. The more ordered acyl chains of Lipid A compared to the cytoplasmic cell membrane contribute to the low permeability of bacterial outer membrane. The findings of this computational investigation of P. aeruginosa OM will further the understanding of microbial pathogenesis and enable future study of OMV biogenesis.

Membrane Distribution of the Pseudomonas Quinolone Signal Modulates Outer Membrane Vesicle Production in Pseudomonasaeruginosa.

ABSTRACTThe Pseudomonas quinolone signal (PQS) is an important quorum-sensing molecule in Pseudomonas aeruginosa that also mediates its own packaging and transport by stimulating outer membrane vesicle (OMV) formation. Because OMVs have been implicated in many virulence-associated behaviors, it is critical that we understand how they are formed. Our group proposed the bilayer-couple model for OMV biogenesis, where PQS intercalates into the outer membrane, causing expansion of the outer leaflet and consequently inducing curvature. In accordance with the model, we hypothesized that PQS must be transported from the cytoplasm to the outer membrane before it can initiate OMV formation. We initially examined two laboratory strains of P. aeruginosa and found significant strain-dependent differences. PQS export correlated strongly with OMV production, even though equivalent amounts of total PQS were produced by both strains. Interestingly, we discovered that poor OMV producers sequestered the majority of PQS in the inner membrane, which appeared to be the result of early saturation of the export pathway. Further analysis showed that strain-specific PQS export and OMV biogenesis patterns were stable once established but could be significantly altered by changing the growth medium. Finally, we demonstrated that the associations described for laboratory strains also held for three clinical strains. These results suggest that factors controlling the export of PQS dictate OMV biogenesis. This work provides new insight into PQS-controlled virulence in P. aeruginosa and provides important tools to further study signal export and OMV biogenesis.

Overexpression of MicA induces production of OmpC-enriched outer membrane vesicles that protect against Salmonella challenge

Outer membrane vesicles (OMVs) derived from bacteria are promising candidates for subunit vaccines. Stresses that modulate the composition of outer membrane proteins (OMPs) are important for OMV synthesis. Small RNAs (sRNAs) expressed in response to stress regulate OMPs, although the mechanism underlying sRNA-mediated OMV biogenesis and its utility for developing vaccine platforms remains to be elucidated. Here, we characterized the role of a sRNA, MicA, which regulates OmpA, a major OMP involved in both production of OMVs and reactive immunity against Salmonella challenge. A Salmonella strain overexpressing MicA generated more OMVs than a control strain. In addition, OmpC was the major component of MicA-derived OMV proteins. MicA-derived OMVs induced Th1- and Th17-type immune responses in vitro and reduced Salmonella-mediated lethality in a mouse model. Thus, OmpA-regulatory sRNA-derived OMVs may facilitate production of Salmonella-protective vaccines.

Application of the bacterial outer membrane vesicles in vaccine design

Abstract Outer membrane vesicles (OMVs) are extracellular structures produced by most gram-negative bacteria, including pathogens of humans and animals. OMVs play an important role in the physiology of microorganisms and are an integral part of many biological processes. Following the discovery that they are able to transport many biomolecules, also these which have the ability to interact with the immune system, their potential use as non-replicating vaccines has become an important aspect of immunotherapeutic researches. These nano-sized elements exhibit remarkable potential for immunomodulation of immune response, thanks to the ability to deliver naturally or artificially incorporated antigens within their structure. First vaccine based on outer membrane vesicles was developed almost 30 years ago against Neisseria meningitidis serogroup B. This review presents some basic information on biogenesis and functions of OMVs. It also provides examples of pathogens, whose OMVs (in natural or modified form) have been used in the development of immunogenic vaccines against the organisms from which the vesicles had been obtained. OMVs are proving to be more versatile than first conceived and may become important part of biotechnology research, not limited to medical applications. 1. Introduction. 2. Outer membrane vesicles biogenesis. 3. Biological functions of outer membrane vesicles. 3.1. Role in response to stressors. 3.2. Role in the extracellular transport. 3.3. Role in biofilm formation. 4. OMVs in vaccine construction. 4.1. Neisseria meningitidis. 4.2. Vibrio cholerae. 4.3. Bordetella pertussis. 4.4. Chlamydia trachomatis. 4.5. Burkholderia pseudomallei. 4.6. Acinetobacter baumannii. 4.7. Francisella noatunensis. 4.8. Shigella spp. 4.9. Campylobacter jejuni. 5. Conclusions 1. Wprowadzenie. 2. Biogeneza pęcherzyków zewnątrzbłonowych. 3. Funkcje pęcherzyków zewnątrzkomórkowych. 3.1. Udział w odpowiedzi na czynniki stresogenne. 3.2. Udział w transporcie pozakomórkowym. 3.3. Udział w tworzeniu biofilmu. 4. Pęcherzyki zewnątrzbłonowe w konstrukcji szczepionek. 4.1. Neisseria meningitidis. 4.2. Vibrio cholerae. 4.3. Bordetella pertussis. 4.4. Chlamydia trachomatis. 4.5. Burkholderia pseudomallei. 4.6. Acineto bacter baumannii. 4.7. Francisella noatunensis. 4.8. Shigella spp. 4.9. Campylobacter jejuni. 5. Podsumowanie

Proteomic profiling of Gram-negative bacterial outer membrane vesicles: Current perspectives.

Outer membrane vesicles (OMVs) are extracellular vesicles derived from Gram-negative bacteria. Recent progress in the studies of Gram-negative bacterial extracellular vesicles implies that OMVs may function as intercellular communicasomes in bacteria-bacteria and bacteria-host interactions. Current MS-based high-throughput proteomic analyses of Gram-negative bacterial OMVs have identified thousands of vesicular proteins and provided clues to reveal the biogenesis and pathophysiological functions of Gram-negative bacterial OMVs. The future directions of proteomics of Gram-negative bacterial OMVs may include the isolation strategy of Gram-negative bacterial OMVs to thoroughly exclude nonvesicular contaminants and proteomics of Gram-negative bacterial OMVs derived from diverse conditions as well as body fluids of bacterium-infected hosts. We hope this review will shed light on future research in this emerging field of proteomics of extracellular vesicles derived from Gram-negative bacteria and contribute to the development of OMV-based diagnostic tools and effective vaccines.

Outer Membrane Vesicle Biosynthesis in Salmonella: Is There More to Gram-Negative Bacteria?

ABSTRACTRecent research has focused on the biological role of outer membrane vesicles (OMVs), which are derived from the outer membranes (OMs) of Gram-negative bacteria, and their potential exploitation as therapeutics. OMVs have been characterized in many ways and functions. Until recently, research focused on hypothetical and empirical models that addressed the molecular mechanisms of OMV biogenesis, such as vesicles bulging from the OM in various ways. The recently reported study by Elhenawy et al. (mBio 7:e00940-16, 2016, http://dx.doi.org/10.1128/mBio.00940-16) provided further insights into OMV biogenesis of Salmonella enterica serovar Typhimurium. That study showed that deacylation of lipopolysaccharides (LPS) influences the level of OMV production and, furthermore, determines a sorting of high versus low acylated LPS in OMs and OMVs, respectively. Interestingly, deacylation may inversely correlate with other LPS modifications, suggesting some synergy toward optimized host resistance via best OM compositions for S. Typhimurium.

Bacterial outer membrane vesicle biogenesis: a new mechanism and its implications.

Outer membrane vesicle (OMV) release by Gram-negative bacteria has been observed and studied for decades. First considered as a by-product of cell lysis, it soon became evident that OMVs are actively secreted from the outer membrane (OM) of Gram-negative bacteria. Accordingly, these small particles (~ 10-300 nm in diameter) consist mainly of OM components like phospholipids (PLs), OM proteins, and lipopolysaccharides or lipooligosaccharides. However, OMVs may also comprise periplasmic, inner membrane, or cytoplasmic components. Since the shedding of substantial amounts of OM material represents a significant energy cost to the bacterial cell, OMV production must have some vital biological functions for Gram-negative bacteria. Indeed, intense research on that topic revealed that OMVs play important roles in bacterial physiology and pathogenesis, ranging from secretion and delivery of biomolecules (for example, toxins, DNA, or quorum sensing molecules) over stress response and biofilm formation to immunomodulation and adherence to host cells. Only recently researchers have begun to elucidate the mechanistic aspects of OMV formation, but a general mechanism for the biogenesis of these vesicles is still lacking. Here we review the findings and implications of our recent study published in Nature Communications (Roier S, et al. (2016) Nat. Commun. 7:10515), where we propose a novel and highly conserved bacterial OMV biogenesis mechanism based on PL accumulation in the outer leaflet of the OM. This mechanism might not only have important pathophysiological roles in vivo, but also represents the first general mechanism of OMV formation applicable to all Gram-negative bacteria.

Bacterial Outer Membrane Vesicles as a Delivery System for Virulence Regulation.

Outer membrane vesicles (OMVs) are spherical nanostructures that are ubiquitously shed from gram-negative bacteria both in vitro and in vivo. Recent findings revealed that OMVs, which contain diverse components derived from the parent bacterium, play an important role in communication with neighboring bacteria and the environment. Furthermore, nanoscale proteoliposomes decorated with pathogen-associated molecules attract considerable attention as a non-replicative carrier for vaccines and drug materials. This review introduces recent advances in OMV biogenesis and discusses the roles of OMVs in the context of bacterial communication and virulence regulation. It also describes the remarkable accomplishments in OMV engineering for diverse therapeutic applications.

Release of outer membrane vesicles in Pseudomonas putida as a response to stress caused by cationic surfactants.

Pseudomonas putida A (ATCC 12633), a degrader of cationic surfactants, releases outer membrane vesicles (OMVs) when grown with tetradecyltrimethylammonium bromide (TTAB) as the sole carbon, nitrogen and energy source. The OMVs exhibit a bilayer structure and were found to be composed of lipopolysaccharides, proteins and phospholipids (PLs) such as cardiolipin, phosphatidylcholine, phosphatidic acid and phosphatidylglycerol (PG). The OMVs showed a marked increase in the PG content, approximately 43 % higher than the amount registered in the parent cells from which the vesicles were derived. After growth of P. putida with TTAB, the amount of lipoprotein covalently cross-linked to the peptidoglycan showed a twofold decrease when compared with values found after growth without the surfactant [16 ± 2 and 28 ± 3 μg (mg cell envelope protein)- 1, respectively]. This decrease in the amount of lipoprotein can be related to areas of loss of contact between the outer membrane and the peptidoglycan and, therefore, to OMV production. In addition, due to its amphiphilic nature, TTAB can contribute to OMV biogenesis, through a physical mechanism, by induction of the curvature of the membrane. Taking into account that OVMs were produced when the cells were grown under external stress, caused by the surfactant, and that TTAB was detected in the vesicles [48 nmol TTAB (nmol PL)- 1], we concluded that this system of TTAB elimination is a mechanism that P. putida A (ATCC 12633) would utilize for alleviating stress caused by cationic surfactants.

A novel mechanism for the biogenesis of outer membrane vesicles in Gram-negative bacteria.

Bacterial outer membrane vesicles (OMVs) have important biological roles in pathogenesis and intercellular interactions, but a general mechanism of OMV formation is lacking. Here we show that the VacJ/Yrb ABC (ATP-binding cassette) transport system, a proposed phospholipid transporter, is involved in OMV formation. Deletion or repression of VacJ/Yrb increases OMV production in two distantly related Gram-negative bacteria, Haemophilus influenzae and Vibrio cholerae. Lipidome analyses demonstrate that OMVs from VacJ/Yrb-defective mutants in H. influenzae are enriched in phospholipids and certain fatty acids. Furthermore, we demonstrate that OMV production and regulation of the VacJ/Yrb ABC transport system respond to iron starvation. Our results suggest a new general mechanism of OMV biogenesis based on phospholipid accumulation in the outer leaflet of the outer membrane. This mechanism is highly conserved among Gram-negative bacteria, provides a means for regulation, can account for OMV formation under all growth conditions, and might have important pathophysiological roles in vivo.

Spheres of influence: Porphyromonas gingivalis outer membrane vesicles.

Outer membrane vesicles (OMVs) are asymmetrical single bilayer membranous nanostructures produced by Gram-negative bacteria important for bacterial interaction with the environment. Porphyromonas gingivalis, a keystone pathogen associated with chronic periodontitis, produces OMVs that act as a virulence factor secretion system contributing to its pathogenicity. Despite their biological importance, the mechanisms of OMV biogenesis have not been fully elucidated. The ~14 times more curvature of the OMV membrane than cell outer membrane (OM) indicates that OMV biogenesis requires energy expenditure for significant curvature of the OMV membrane. In P.gingivalis, we propose that this may be achieved by upregulating the production of certain inner or outer leaflet lipids, which causes localized outward curvature of the OM. This results in selection of anionic lipopolysaccharide (A-LPS) and associated C-terminal domain (CTD) -family proteins on the outer surface due to their ability to accommodate the curvature. Deacylation of A-LPS may further enable increased curvature leading to OMV formation. Porphyromonas gingivalis OMVs that are selectively enriched in CTD-family proteins, largely the gingipains, can support bacterial coaggregation, promote biofilm development and act as an intercessor for the transport of non-motile bacteria by motile bacteria. The P.gingivalis OMVs are also believed to contribute to host interaction and colonization, evasion of immune defense mechanisms, and destruction of periodontal tissues. They may be crucial for both micro- and macronutrient capture, especially heme and probably other assimilable compounds for its own benefit and that of the wider biofilm community.

Tracking the Dynamic Relationship between Cellular Systems and Extracellular Subproteomes in Pseudomonas aeruginosa Biofilms.

The transition of the opportunistic pathogen Pseudomonas aeruginosa from free-living bacteria into surface-associated biofilm communities represents a viable target for the prevention and treatment of chronic infectious disease. We have established a proteomics platform that identified 2443 and 1142 high-confidence proteins in P. aeruginosa whole cells and outer-membrane vesicles (OMVs), respectively, at three time points during biofilm development (ProteomeXchange identifier PXD002605). The analysis of cellular systems, specifically the phenazine biosynthetic pathway, demonstrates that whole-cell protein abundance correlates to end product (i.e., pyocyanin) concentrations in biofilm but not in planktonic cultures. Furthermore, increased cellular protein abundance in this pathway results in quantifiable pyocyanin in early biofilm OMVs and OMVs from both growth modes isolated at later time points. Overall, our data indicate that the OMVs being released from the surface of the biofilm whole cells have unique proteomes in comparison to their planktonic counterparts. The relative abundance of OMV proteins from various subcellular sources showed considerable differences between the two growth modes over time, supporting the existence and preferential activation of multiple OMV biogenesis mechanisms under different conditions. The consistent detection of cytoplasmic proteins in all of the OMV subproteomes challenges the notion that OMVs are composed of outer membrane and periplasmic proteins alone. Direct comparisons of outer-membrane protein abundance levels between OMVs and whole cells shows ratios that vary greatly from 1:1 and supports previous studies that advocate the specific inclusion, or "packaging", of proteins into OMVs. The quantitative analysis of packaged protein groups suggests biogenesis mechanisms that involve untethered, rather than absent, peptidoglycan-binding proteins. Collectively, individual protein and biological system analyses of biofilm OMVs show that drug-binding cytoplasmic proteins and porins are potentially shuttled from the whole cell into the OMVs and may contribute to the antibiotic resistance of P. aeruginosa whole cells within biofilms.

Outer membrane vesicles of Tannerella forsythia: biogenesis, composition, and virulence.

Summary Tannerella forsythia is the only ‘red‐complex’ bacterium covered by an S‐layer, which has been shown to affect virulence. Here, outer membrane vesicles (OMVs) enriched with putative glycoproteins are described as a new addition to the virulence repertoire of T. forsythia. Investigations of this bacterium are hampered by its fastidious growth requirements and the recently discovered mismatch of the available genome sequence (92A2 = ATCC BAA‐2717) and the widely used T. forsythia strain (ATCC 43037). T. forsythia was grown anaerobically in serum‐free medium and biogenesis of OMVs was analyzed by electron and atomic force microscopy. This revealed OMVs with a mean diameter of ~100 nm budding off from the outer membrane while retaining the S‐layer. An LC‐ESI‐TOF/TOF proteomic analysis of OMVs from three independent biological replicates identified 175 proteins. Of these, 14 exhibited a C‐terminal outer membrane translocation signal that directs them to the cell/vesicle surface, 61 and 53 were localized to the outer membrane and periplasm, respectively, 22 were predicted to be extracellular, and 39 to originate from the cytoplasm. Eighty proteins contained the Bacteroidales O‐glycosylation motif, 18 of which were confirmed as glycoproteins. Release of pro‐inflammatory mediators from the human monocytic cell line U937 and periodontal ligament fibroblasts upon stimulation with OMVs followed a concentration‐dependent increase that was more pronounced in the presence of soluble CD14 in conditioned media. The inflammatory response was significantly higher than that caused by whole T. forsythia cells. Our study represents the first characterization of T. forsythia OMVs, their proteomic composition and immunogenic potential.

Roles of Outer Membrane Vesicles (OMVs) in Bacterial Virulence

Outer membrane vesicles (OMVs) are ubiquitous membranous structures in all Gram-negative bacteria, including pathogens and non-pathogens. Gram-positive bacteria also release membrane-derived vesicles (MV). Originating from the cell envelope, OMVs are enriched with bacterial antigen molecules that conduct multiple functions as decoys to manipulate the host immune system. Besides, OMVs and their components play diverse roles in nutrient acquisition, biofilm formation, and resistance to antibiotics. Despite the diverse benefits ascribed to OMVs, many questions remain unanswered with regard to OMV biogenesis and cargo selectivity. In this report, we review the advantages of vesiculation in the context of all bacteria and then focus on additional benefits acquired by OMVs in pathogenic bacteria.

A basis for vaccine development: Comparative characterization of Haemophilus influenzae outer membrane vesicles

Outer membrane vesicles (OMVs) are spherical and bilayered particles that are naturally released from the outer membrane (OM) of Gram-negative bacteria. They have been proposed to possess several biological roles in pathogenesis and interbacterial interactions. Additionally, OMVs have been suggested as potential vaccine candidates against infections caused by pathogenic bacteria like Haemophilus influenzae, a human pathogen of the respiratory tract. Unfortunately, there is still a lack of fundamental knowledge regarding OMV biogenesis, protein sorting into OMVs, OMV size and quantity, as well as OMV composition in H. influenzae. Thus, this study comprehensively characterized and compared OMVs and OMs derived from heterologous encapsulated as well as nonencapsulated H. influenzae strains. Semiquantitative immunoblot analysis revealed that certain OM proteins are enriched or excluded in OMVs suggesting the presence of regulated protein sorting mechanisms into OMVs as well as interconnected OMV biogenesis mechanisms in H. influenzae. Nanoparticle tracking analysis, transmission electron microscopy, as well as protein and lipooligosaccharide quantifications demonstrated that heterologous H. influenzae strains differ in their OMV size and quantity. Lipidomic analyses identified palmitic acid as the most abundant fatty acid, while phosphatidylethanolamine was found to be the most dominant phospholipid present in OMVs and the OM of all strains tested. Proteomic analysis confirmed that H. influenzae OMVs contain vaccine candidate proteins as well as important virulence factors. These findings contribute to the understanding of OMV biogenesis as well as biological roles of OMVs and, in addition, may be important for the future development of OMV based vaccines against H. influenzae infections.

Comparative proteomic analysis of outer membrane vesicles from Shigella flexneri under different culture conditions

The production of outer membrane vesicles (OMVs) is a common and regulated process of gram-negative bacteria. Nonetheless, the processes of Shigella flexneri OMV production still remain unclear. S. flexneri is the causative agent of endemic shigellosis in developing countries. The Congo red binding of strains is associated with increased infectivity of S. flexneri. Therefore, understanding the modulation pattern of OMV protein expression induced by Congo red will help to elucidate the bacterial pathogenesis.In the present study, we investigated the proteomic composition of OMVs and the change in OMV protein expression induced by Congo red using mTRAQ-based quantitative comparative proteomics. mTRAQ labelling increased the confidence in protein identification, and 148 total proteins were identified in S. flexneri-derived OMVs. These include a variety of important virulence factors, including Ipa proteins, TolC family, murein hydrolases, and members of the serine protease autotransporters of Enterobacteriaceae (SPATEs) family. Among the identified proteins, 28 and five proteins are significantly up- and down-regulated in the Congo red-induced OMV, respectively. Additionally, by comprehensive comparison with previous studies focused on DH5a-derived OMV, we identified some key node proteins in the protein–protein interaction network that may be involved in OMV biogenesis and are common to all gram-negative bacteria.

Prokaryotic membrane vesicles: new insights on biogenesis and biological roles

Biogenesis and trafficking of membrane vesicles are essential and well-studied processes in eukaryotes. In contrast, vesiculation in bacteria is not well understood. Outer membrane vesicles (OMVs) are produced in Gram-negative bacteria by blebbing of the outer membrane. In addition to the roles in pathogenesis, cell-to-cell communication and stress response, recent work has suggested that OMVs play important roles in immunomodulation and the establishment and balance of the gut microbiota. In this review we discuss the known and novel roles of OMVs and the different biogenesis models proposed, and address the evidence for cargo selection into OMVs. We also discuss the growing evidence for the existence of membrane vesicles in Gram-positive bacteria and Archaea. Due to their biological importance and promising applications in vaccinology, the biogenesis of OMVs is an important topic in microbiology.

Biogenesis and multifaceted roles of outer membrane vesicles from Gram-negative bacteria.

Outer membrane vesicles (OMVs) released from Gram-negative bacteria consist of lipids, proteins, lipopolysaccharides and other molecules. OMVs are associated with several biological functions such as horizontal gene transfer, intracellular and intercellular communication, transfer of contents to host cells, and eliciting an immune response in host cells. Although hypotheses have been made concerning the mechanism of biogenesis of these vesicles, research on OMV formation is far from complete. The roles of outer membrane components, bacterial quorum sensing molecules and some specific proteins in OMV biogenesis have been studied. This review discusses the different models that have been proposed for OMV biogenesis, along with details of the biological functions of OMVs and the likely scope of future research.

Enterohemorrhagic Escherichia coli OmpT regulates outer membrane vesicle biogenesis.

Enterohemorrhagic Escherichia coli (EHEC) infection from food or water often results in severe diarrheal disease and is a leading cause of death globally. Outer membrane vesicles (OMVs) secreted from E. coli induce lethality in mice. The omptin outer membrane protease OmpT from E. coli inactivates antimicrobial peptides and may enhance colonization of the uroepithelium, but its precise function remains unclear. Given OmpT is an outer membrane protease, we hypothesized it may have a role in OMV biogenesis. To further characterize the effect of OmpT on OMV production, a genetic approach using wild type, an ompT deletion mutant and an ompT overexpressing construct in EHEC were employed. ompT gene deletion markedly decreased OMV production and stainable lipid but increased vesicle diameter. Conversely, ompT overexpression profoundly increased OMV biogenesis but decreased stainable lipid, protein content, and vesicle diameter. Alterations in EHEC ompT gene expression have an impact on the biogenesis, composition, and size of OMVs. Changes in ompT gene expression may dynamically alter OMV formation, composition, and diameter in response to different host environments and contribute to cell-free intercellular communication to enhance bacterial growth and survival.

Influence of O Polysaccharides on Biofilm Development and Outer Membrane Vesicle Biogenesis in Pseudomonas aeruginosa PAO1

Pseudomonas aeruginosa is a common opportunistic human pathogen known for its ability to adapt to changes in its environment during the course of infection. These adaptations include changes in the expression of cell surface lipopolysaccharide (LPS), biofilm development, and the production of a protective extracellular exopolysaccharide matrix. Outer membrane vesicles (OMVs) have been identified as an important component of the extracellular matrix of P. aeruginosa biofilms and are thought to contribute to the development and fitness of these bacterial communities. The goal of this study was to examine the relationships between changes in the cell surface expression of LPS O polysaccharides, biofilm development, and OMV biogenesis in P. aeruginosa. We compared wild-type P. aeruginosa PAO1 with three chromosomal knockouts. These knockouts have deletions in the rmd, wbpM, and wbpL genes that produce changes in the expression of common polysaccharide antigen (CPA), O-specific antigen (OSA), or both. Our results demonstrate that changes in O polysaccharide expression do not significantly influence OMV production but do affect the size and protein content of OMVs derived from both CPA(-) and OSA(-) cells; these mutant cells also exhibited different physical properties from wild-type cells. We further examined biofilm growth of the mutants and determined that CPA(-) cells could not develop into robust biofilms and exhibit changes in cell morphology and biofilm matrix production. Together these results demonstrate the importance of O polysaccharide expression on P. aeruginosa OMV composition and highlight the significance of CPA expression in biofilm development.