POTRA domains of the TamA insertase interact with the outer membrane and modulate membrane properties.

The outer membrane (OM) of gram-negative bacteria serves as a vital organelle that is densely populated with OM proteins (OMPs) and plays pivotal roles in cellular functions and virulence. The assembly and insertion of these OMPs into the OM represent a fundamental process requiring specialized molecular chaperones. One example is the translocation and assembly module (TAM), which functions as a transenvelope chaperone promoting the folding of specific autotransporters, adhesins, and secretion systems. The catalytic unit of TAM, TamA, comprises a catalytic ß-barrel domain anchored within the OM and three periplasmic polypeptide-transport-associated (POTRA) domains that recruit the TamB subunit. The latter acts as a periplasmic ladder that facilitates the transport of unfolded OMPs across the periplasm. In addition to their role in recruiting the auxiliary protein TamB, our data demonstrate that the POTRA domains mediate interactions with the inner surface of the OM, ultimately modulating the membrane properties. Through the integration of X-ray crystallography, molecular dynamic simulations, and biomolecular interaction methodologies, we located the membrane-binding site on the first and second POTRA domains. Our data highlight a binding preference for phosphatidylglycerol, a minor lipid constituent present in the OM, which has been previously reported to facilitate OMP assembly. In the context of the densely OMP-populated membrane, this association may serve as a mechanism to secure lipid accessibility for nascent OMPs through steric interactions with existing OMPs, in addition to creating favorable conditions for OMP biogenesis.

Engineering bionanoreactor in bacteria for efficient hydrogen production.

Hydrogen production through water splitting is a vital strategy for renewable and sustainable clean energy. In this study, we developed an approach integrating nanomaterial engineering and synthetic biology to establish a bionanoreactor system for efficient hydrogen production. The periplasmic space (20 to 30 nm) of an electroactive bacterium, Shewanella oneidensis MR-1, was engineered to serve as a bionanoreactor to enhance the interaction between electrons and protons, catalyzed by hydrogenases for hydrogen generation. To optimize electron transfer, we used the microbially reduced graphene oxide (rGO) to coat the electrode, which improved the electron transfer from the electrode to the cells. Native MtrCAB protein complex on S. oneidensis and self-assembled iron sulfide (FeS) nanoparticles acted in tandem to facilitate electron transfer from an electrode to the periplasm. To enhance proton transport, S. oneidensis MR-1 was engineered to express Gloeobacter rhodopsin (GR) and the light-harvesting antenna canthaxanthin. This led to efficient proton pumping when exposed to light, resulting in a 35.6% increase in the rate of hydrogen production. The overexpression of native [FeFe]-hydrogenase further improved the hydrogen production rate by 56.8%. The bionanoreactor engineered in S. oneidensis MR-1 achieved a hydrogen yield of 80.4 µmol/mg protein/day with a Faraday efficiency of 80% at a potential of -0.75 V. This periplasmic bionanoreactor combines the strengths of both nanomaterial and biological components, providing an efficient approach for microbial electrosynthesis.

FlhE functions as a chaperone to prevent formation of periplasmic flagella in Gram-negative bacteria.

The bacterial flagellum, which facilitates motility, is composed of ~20 structural proteins organized into a long extracellular filament connected to a cytoplasmic rotor-stator complex via a periplasmic rod. Flagellum assembly is regulated by multiple checkpoints that ensure an ordered gene expression pattern coupled to the assembly of the various building blocks. Here, we use epifluorescence, super-resolution, and transmission electron microscopy to show that the absence of a periplasmic protein (FlhE) prevents proper flagellar morphogenesis and results in the formation of periplasmic flagella in Salmonella enterica. The periplasmic flagella disrupt cell wall synthesis, leading to a loss of normal cell morphology resulting in cell lysis. We propose that FlhE functions as a periplasmic chaperone to control assembly of the periplasmic rod, thus preventing formation of periplasmic flagella.

Periplasmic electron transfer network in Geobacter sulfurreducens revealed by biomolecular interaction studies.

Multiheme cytochromes located in different compartments are crucial for extracellular electron transfer in the bacterium Geobacter sulfurreducens to drive important environmental processes and biotechnological applications. Recent studies have unveiled that for particular sets of electron terminal acceptors, discrete respiratory pathways selectively recruit specific cytochromes from both the inner and outer membranes. However, such specificity was not observed for the abundant periplasmic cytochromes, namely the triheme cytochrome family PpcA-E. In this work, the distinctive NMR spectroscopic signatures of these proteins in different redox states were explored to monitor pairwise interactions and electron transfer reactions between each pair of cytochromes. The results showed that the five proteins interact transiently and can exchange electrons between each other revealing intra-promiscuity within the members of this family. This discovery is discussed in the light of the establishment of an effective electron transfer network by this pool of cytochromes. This network is advantageous to the bacteria as it enables the maintenance of the functional working potential redox range within the cells.

A production platform for disulfide-bonded peptides in the periplasm of Escherichia coli.

BACKGROUND: Recombinant peptide production in Escherichia coli provides a sustainable alternative to environmentally harmful and size-limited chemical synthesis. However, in-vivo production of disulfide-bonded peptides at high yields remains challenging, due to degradation by host proteases/peptidases and the necessity of translocation into the periplasmic space for disulfide bond formation. RESULTS: In this study, we established an expression system for efficient and soluble production of disulfide-bonded peptides in the periplasm of E. coli. We chose model peptides with varying complexity (size, structure, number of disulfide bonds), namely parathyroid hormone 1-84, somatostatin 1-28, plectasin, and bovine pancreatic trypsin inhibitor (aprotinin). All peptides were expressed without and with the N-terminal, low molecular weight CASPON™ tag (4.1 kDa), with the expression cassette being integrated into the host genome. During BioLector™ cultivations at microliter scale, we found that most of our model peptides can only be sufficiently expressed in combination with the CASPON™ tag, otherwise expression was only weak or undetectable on SDS-PAGE. Undesired degradation by host proteases/peptidases was evident even with the CASPON™ tag. Therefore, we investigated whether degradation happened before or after translocation by expressing the peptides in combination with either a co- or post-translational signal sequence. Our results suggest that degradation predominantly happened after the translocation, as degradation fragments appeared to be identical independent of the signal sequence, and expression was not enhanced with the co-translational signal sequence. Lastly, we expressed all CASPON™-tagged peptides in two industry-relevant host strains during C-limited fed-batch cultivations in bioreactors. We found that the process performance was highly dependent on the peptide-host-combination. The titers that were reached varied between 0.6-2.6 g L-1, and exceeded previously published data in E. coli. Moreover, all peptides were shown by mass spectrometry to be expressed to completion, including full formation of disulfide bonds. CONCLUSION: In this work, we demonstrated the potential of the CASPON™ technology as a highly efficient platform for the production of soluble peptides in the periplasm of E. coli. The titers we show here are unprecedented whenever parathyroid hormone, somatostatin, plectasin or bovine pancreatic trypsin inhibitor were produced in E. coli, thus making our proposed upstream platform favorable over previously published approaches and chemical synthesis.

Extending Linker Sequences between Antigen-Recognition Modules Provides More Effective Production of Bispecific Nanoantibodies in the Periplasma of E. coli.

Technology of production of single-domain antibodies (NANOBODY® molecules, also referred to as nanoantibodies, nAb, or molecules based on other stable protein structures) and their derivatives to solve current problems in biomedicine is becoming increasingly popular. Indeed, the format of one small, highly soluble protein with a stable structure, fully functional in terms of specific recognition, is very convenient as a module for creating multivalent, bi-/oligo-specific genetically engineered targeting molecules and structures. Production of nAb in periplasm of E. coli bacterium is a very convenient and fairly universal way to obtain analytical quantities of nAb for the initial study of the properties of these molecules and selection of the most promising nAb variants. The situation is more complicated with production of bi- and multivalent derivatives of the initially selected nAbs under the same conditions. In this work, extended linker sequences (52 and 86 aa) between the antigen-recognition modules in the cloned expression constructs were developed and applied in order to increase efficiency of production of bispecific nanoantibodies (bsNB) in the periplasm of E. coli bacteria. Three variants of model bsNBs described in this study were produced in the periplasm of bacteria and isolated in soluble form with preservation of functionality of all the protein domains. If earlier our attempts to produce bsNB in the periplasm with traditional linkers no longer than 30 aa were unsuccessful, the extended linkers used here provided a significantly more efficient production of bsNB, comparable in efficiency to the traditional production of original monomeric nAbs. The use of sufficiently long linkers could presumably be useful for increasing efficiency of production of other bsNBs and similar molecules in the periplasm of E. coli bacteria.

Control of biofilm formation by an Agrobacterium tumefaciens pterin-binding periplasmic protein conserved among diverse Proteobacteria.

Biofilm formation and surface attachment in multiple Alphaproteobacteria is driven by unipolar polysaccharide (UPP) adhesins. The pathogen Agrobacterium tumefaciens produces a UPP adhesin, which is regulated by the intracellular second messenger cyclic diguanylate monophosphate (c-di-GMP). Prior studies revealed that DcpA, a diguanylate cyclase-phosphodiesterase, is crucial in control of UPP production and surface attachment. DcpA is regulated by PruR, a protein with distant similarity to enzymatic domains known to coordinate the molybdopterin cofactor (MoCo). Pterins are bicyclic nitrogen-rich compounds, several of which are produced via a nonessential branch of the folate biosynthesis pathway, distinct from MoCo. The pterin-binding protein PruR controls DcpA activity, fostering c-di-GMP breakdown and dampening its synthesis. Pterins are excreted, and we report here that PruR associates with these metabolites in the periplasm, promoting interaction with the DcpA periplasmic domain. The pteridine reductase PruA, which reduces specific dihydro-pterin molecules to their tetrahydro forms, imparts control over DcpA activity through PruR. Tetrahydromonapterin preferentially associates with PruR relative to other related pterins, and the PruR-DcpA interaction is decreased in a pruA mutant. PruR and DcpA are encoded in an operon with wide conservation among diverse Proteobacteria including mammalian pathogens. Crystal structures reveal that PruR and several orthologs adopt a conserved fold, with a pterin-specific binding cleft that coordinates the bicyclic pterin ring. These findings define a pterin-responsive regulatory mechanism that controls biofilm formation and related c-di-GMP-dependent phenotypes in A. tumefaciens and potentially acts more widely in multiple proteobacterial lineages.

Lytic transglycosylase Slt of Pseudomonas aeruginosa as a periplasmic hub protein.

Peptidoglycan is a major constituent of the bacterial cell wall. Its integrity as a polymeric edifice is critical for bacterial survival and, as such, it is a preeminent target for antibiotics. The peptidoglycan is a dynamic crosslinked polymer that undergoes constant biosynthesis and turnover. The soluble lytic transglycosylase (Slt) of Pseudomonas aeruginosa is a periplasmic enzyme involved in this dynamic turnover. Using amber-codon-suppression methodology in live bacteria, we incorporated a fluorescent chromophore into the structure of Slt. Fluorescent microscopy shows that Slt populates the length of the periplasmic space and concentrates at the sites of septation in daughter cells. This concentration persists after separation of the cells. Amber-codon-suppression methodology was also used to incorporate a photoaffinity amino acid for the capture of partner proteins. Mass-spectrometry-based proteomics identified 12 partners for Slt in vivo. These proteomics experiments were complemented with in vitro pulldown analyses. Twenty additional partners were identified. We cloned the genes and purified to homogeneity 22 identified partners. Biophysical characterization confirmed all as bona fide Slt binders. The identities of the protein partners of Slt span disparate periplasmic protein families, inclusive of several proteins known to be present in the divisome. Notable periplasmic partners (KD < 0.5 µM) include PBPs (PBP1a, KD = 0.07 µM; PBP5 = 0.4 µM); other lytic transglycosylases (SltB2, KD = 0.09 µM; RlpA, KD = 0.4 µM); a type VI secretion system effector (Tse5, KD = 0.3 µM); and a regulatory protease for alginate biosynthesis (AlgO, KD < 0.4 µM). In light of the functional breadth of its interactome, Slt is conceptualized as a hub protein within the periplasm.

The critical role of a conserved lysine residue in periplasmic nitrate reductase catalyzed reactions.

Periplasmic nitrate reductase NapA from Campylobacter jejuni (C. jejuni) contains a molybdenum cofactor (Moco) and a 4Fe-4S cluster and catalyzes the reduction of nitrate to nitrite. The reducing equivalent required for the catalysis is transferred from NapC → NapB → NapA. The electron transfer from NapB to NapA occurs through the 4Fe-4S cluster in NapA. C. jejuni NapA has a conserved lysine (K79) between the Mo-cofactor and the 4Fe-4S cluster. K79 forms H-bonding interactions with the 4Fe-4S cluster and connects the latter with the Moco via an H-bonding network. Thus, it is conceivable that K79 could play an important role in the intramolecular electron transfer and the catalytic activity of NapA. In the present study, we show that the mutation of K79 to Ala leads to an almost complete loss of activity, suggesting its role in catalytic activity. The inhibition of C. jejuni NapA by cyanide, thiocyanate, and azide has also been investigated. The inhibition studies indicate that cyanide inhibits NapA in a non-competitive manner, while thiocyanate and azide inhibit NapA in an uncompetitive manner. Neither inhibition mechanism involves direct binding of the inhibitor to the Mo-center. These results have been discussed in the context of the loss of catalytic activity of NapA K79A variant and a possible anion binding site in NapA has been proposed.

The patatin-like protein PlpD forms structurally dynamic homodimers in the Pseudomonas aeruginosa outer membrane.

Members of the Omp85 superfamily of outer membrane proteins (OMPs) found in Gram-negative bacteria, mitochondria and chloroplasts are characterized by a distinctive 16-stranded ß-barrel transmembrane domain and at least one periplasmic POTRA domain. All previously studied Omp85 proteins promote critical OMP assembly and/or protein translocation reactions. Pseudomonas aeruginosa PlpD is the prototype of an Omp85 protein family that contains an N-terminal patatin-like (PL) domain that is thought to be translocated across the OM by a C-terminal ß-barrel domain. Challenging the current dogma, we find that the PlpD PL-domain resides exclusively in the periplasm and, unlike previously studied Omp85 proteins, PlpD forms a homodimer. Remarkably, the PL-domain contains a segment that exhibits unprecedented dynamicity by undergoing transient strand-swapping with the neighboring ß-barrel domain. Our results show that the Omp85 superfamily is more structurally diverse than currently believed and suggest that the Omp85 scaffold was utilized during evolution to generate novel functions.

Subdomains of the Helicobacter pylori Cag T4SS outer membrane core complex exhibit structural independence.

The Helicobacter pylori Cag type IV secretion system (Cag T4SS) has an important role in the pathogenesis of gastric cancer. The Cag T4SS outer membrane core complex (OMCC) is organized into three regions: a 14-fold symmetric outer membrane cap (OMC) composed of CagY, CagX, CagT, CagM, and Cag3; a 17-fold symmetric periplasmic ring (PR) composed of CagY and CagX; and a stalk with unknown composition. We investigated how CagT, CagM, and a conserved antenna projection (AP) region of CagY contribute to the structural organization of the OMCC. Single-particle cryo-EM analyses showed that complexes purified from ΔcagT or ΔcagM mutants no longer had organized OMCs, but the PRs remained structured. OMCCs purified from a CagY antenna projection mutant (CagY∆AP) were structurally similar to WT OMCCs, except for the absence of the α-helical antenna projection. These results indicate that CagY and CagX are sufficient for maintaining a stable PR, but the organization of the OMC requires CagY, CagX, CagM, and CagT. Our results highlight an unexpected structural independence of two major subdomains of the Cag T4SS OMCC.

Biosensor that Detects Stress Caused by Periplasmic Proteins.

Escherichia coli is often used as a factory to produce recombinant proteins. In many cases, the recombinant protein needs disulfide bonds to fold and function correctly. These proteins are genetically fused to a signal peptide so that they are secreted to the oxidizing environment of the periplasm (where the enzymes required for disulfide bond formation exist). Currently, it is difficult to determine in vivo whether a recombinant protein is efficiently secreted from the cytoplasm and folded in the periplasm or if there is a bottleneck in one of these steps because cellular capacity has been exceeded. To address this problem, we have developed a biosensor that detects cellular stress caused by (1) inefficient secretion of proteins from the cytoplasm and (2) aggregation of proteins in the periplasm. We demonstrate how the fluorescence fingerprint obtained from the biosensor can be used to identify induction conditions that do not exceed the capacity of the cell and therefore do not cause cellular stress. These induction conditions result in more effective biomass and in some cases higher titers of soluble recombinant proteins.

Escherichia coli Cytoplasmic Expression of Disulfide-Bonded Proteins: Side-by-Side Comparison between Two Competing Strategies.

The production of disulfide bond-containing recombinant proteins in Escherichia coli has traditionally been done by either refolding from inclusion bodies or by targeting the protein to the periplasm. However, both approaches have limitations. Two broad strategies were developed to allow the production of proteins with disulfide bonds in the cytoplasm of E. coli: i) engineered strains with deletions in the disulfide reduction pathways, e.g. SHuffle, and ii) the co-expression of oxidative folding catalysts, e.g. CyDisCo. However, to our knowledge, the relative effectiveness of these strategies has not been properly evaluated. Here, we systematically compare the purified yields of 14 different proteins of interest (POI) that contain disulfide bonds in their native state when expressed in both systems. We also compared the effects of different background strains, commonly used promoters, and two media types: defined and rich autoinduction. In rich autoinduction media, POI which can be produced in a soluble (non-native) state without a system for disulfide bond formation were produced in higher purified yields from SHuffle, whereas all other proteins were produced in higher purified yields using CyDisCo. In chemically defined media, purified yields were at least 10x higher in all cases using CyDisCo. In addition, the quality of the three POI tested was superior when produced using CyDisCo.

Widespread extracellular electron transfer pathways for charging microbial cytochrome OmcS nanowires via periplasmic cytochromes PpcABCDE.

Extracellular electron transfer (EET) via microbial nanowires drives globally-important environmental processes and biotechnological applications for bioenergy, bioremediation, and bioelectronics. Due to highly-redundant and complex EET pathways, it is unclear how microbes wire electrons rapidly (>106 s-1) from the inner-membrane through outer-surface nanowires directly to an external environment despite a crowded periplasm and slow (<105 s-1) electron diffusion among periplasmic cytochromes. Here, we show that Geobacter sulfurreducens periplasmic cytochromes PpcABCDE inject electrons directly into OmcS nanowires by binding transiently with differing efficiencies, with the least-abundant cytochrome (PpcC) showing the highest efficiency. Remarkably, this defined nanowire-charging pathway is evolutionarily conserved in phylogenetically-diverse bacteria capable of EET. OmcS heme reduction potentials are within 200 mV of each other, with a midpoint 82 mV-higher than reported previously. This could explain efficient EET over micrometres at ultrafast (<200 fs) rates with negligible energy loss. Engineering this minimal nanowire-charging pathway may yield microbial chassis with improved performance.

Molecular Crowding Alters the Interactions of Polymyxin Lipopeptides within the Periplasm of E. coli: Insights from Molecular Dynamics.

The cell envelope of Gram-negative bacteria is a crowded tripartite architecture that separates the cell interior from the external environment. Two membranes encapsulate the aqueous periplasm, which contains the cell wall. Little is known about the mechanisms via which antimicrobial peptides move through the periplasm from the outer membrane to their site of action, the inner membrane. We utilize all-atom molecular dynamics to study two antimicrobial peptides, polymyxins B1 and E, within models of the E. coli periplasm crowded to different extents. In a simple chemical environment, both PMB1 and PME bind irreversibly to the cell wall. The presence of specific macromolecules leads to competition with the polymyxins for cell wall interaction sites, resulting in polymyxin dissociation from the cell wall. Chemical complexity also impacts interactions between polymyxins and Braun's lipoprotein; thus, the interaction modes of lipoprotein antibiotics within the periplasm are dependent upon the nature of the other species present.

Rotation of the Fla2 flagella of Cereibacter sphaeroides requires the periplasmic proteins MotK and MotE that interact with the flagellar stator protein MotB2.

The bacterial flagellum is a complex structure formed by more than 25 different proteins, this appendage comprises three conserved structures: the basal body, the hook and filament. The basal body, embedded in the cell envelope, is the most complex structure and houses the export apparatus and the motor. In situ images of the flagellar motor in different species have revealed a huge diversity of structures that surround the well-conserved periplasmic components of the basal body. The identity of the proteins that form these novel structures in many cases has been elucidated genetically and biochemically, but in others they remain to be identified or characterized. In this work, we report that in the alpha proteobacteria Cereibacter sphaeroides the novel protein MotK along with MotE are essential for flagellar rotation. We show evidence that these periplasmic proteins interact with each other and with MotB2. Moreover, these proteins localize to the flagellated pole and MotK localization is dependent on MotB2 and MotA2. These results together suggest that the role of MotK and MotE is to activate or recruit the flagellar stators to the flagellar structure.

Characterization of ß-Barrel Outer Membrane Proteins and Their Interactions with Chaperones by Chemical-Crosslinking Mass Spectrometry.

Chemical crosslinking-mass spectrometry (XL-MS) is an established tool that can be used to study the architecture and dynamics of proteins and protein assemblies. Here the application of XL-MS to study outer membrane proteins (OMPs) and their interactions with periplasmic chaperones is described, to inform on the molecular mechanisms underpinning OMP assembly. XL-MS data are especially powerful when used to complement high-resolution structural data, data from structural prediction or to drive molecular modeling of proteins and protein assemblies. The approach described here could be applied to the study of any protein assembly (including other membrane proteins).

Recent Advances in Modeling Membrane ß-Barrel Proteins Using Molecular Dynamics Simulations: From Their Lipid Environments to Their Assemblies.

Spurred by advances in AI-driven modeling and experimental methods, molecular dynamics simulations are now acting as a platform to integrate these different approaches. This combination of methods is especially useful to understand ß-barrel proteins from the molecular level, e.g., identifying specific interactions with lipids or small molecules, up to assemblies comprised of hundreds of proteins and thousands of lipids. In this minireview, we will discuss recent advances, mainly from the last 5 years, in modeling ß-barrel proteins and their assemblies. These approaches require specific kinds of modeling and potentially different model resolutions that we will first describe in Subheading 1. We will then focus on different aspects of ß-barrel protein modeling: how different types of molecules can diffuse through ß-barrel proteins (Subheading 2); how lipids can interact with these proteins (Subheading 3); how ß-barrel proteins can interact with membrane partners (Subheading 4) or periplasmic extensions and partners (Subheading 5) to form large assemblies.

Monitoring Drug-Protein Interactions in the Bacterial Periplasm by Solution Nuclear Magnetic Resonance Spectroscopy.

The rapid spread of antimicrobial resistance across bacterial pathogens poses a serious risk to the efficacy and sustainability of available treatments. This puts pressure on research concerning the development of new drugs. Here, we present an in-cell NMR-based research strategy to monitor the activity of the enzymes located in the periplasmic space delineated by the inner and outer membranes of Gram-negative bacteria. We demonstrate its unprecedented analytical power in monitoring in situ and in real time (i) the hydrolysis of ß-lactams by ß-lactamases, (ii) the interaction of drugs belonging to the ß-lactam family with their essential targets, and (iii) the binding of inhibitors to these enzymes. We show that in-cell NMR provides a powerful analytical tool for investigating new drugs targeting the molecular components of the bacterial periplasm.

Characterization and immunological effect of outer membrane vesicles from Pasteurella multocida on macrophages.

Pasteurella multocida is an important bacterial pathogen that can cause diseases in both animals and humans. Its elevated morbidity and mortality rates in animals result in substantial economic repercussions within the livestock industry. The prevention of diseases caused by P. multocida through immunization is impeded by the absence of a safe and effective vaccine. Outer membrane vesicles (OMVs) secreted from the outer membrane of Gram-negative bacteria are spherical vesicular structures that encompass an array of periplasmic components in conjunction with a diverse assortment of lipids and proteins. These vesicles can induce antibacterial immune responses within the host. P. multocida has been shown to produce OMVs. Nonetheless, the precise characteristics and immunomodulatory functions of P. multocida OMVs have not been fully elucidated. In this study, OMVs were isolated from P. multocida using an ultrafiltration concentration technique, and their morphology, protein constitution, and immunomodulatory properties in RAW264.7 cells were studied. Transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) revealed that the OMVs exhibited typical spherical and bilayered lipid vesicular architecture, exhibiting an average diameter of approximately 147.5 nm. The yield of OMVs was 2.6 × 1011 particles/mL. Proteomic analysis revealed a high abundance of membrane-associated proteins within P. multocida OMVs, with the capability to instigate the host's immune response. Furthermore, OMVs stimulated the proliferation and cellular uptake of macrophages and triggered the secretion of cytokines, such as TNF-ɑ, IL-1ß, IL-6, IL-10, and TGF-ß1. Consequently, our results indicated that OMVs from P. multocida could directly interact with macrophages and regulate their immune function in vitro. These results supported the prospective applicability of P. multocida OMVs as a platform in the context of vaccine development. KEY POINTS: • Preparation and characterization of P. multocida OMVs. • P. multocida OMVs possess a range of antigens and lipoproteins associated with the activation of the immune system. • P. multocida OMVs can activate the proliferation, internalization, and cytokine secretion of macrophages in vitro.