To investigate whether H. parasuis can secrete OMVs during in vitro growth, a clinical isolate of H. parasuis H45 was grown in tryptic soy broth (TSB) and observed with electron microscopy. The H45 strain had the typical morphology of HPS, which has various forms, from a single short bacillus to a long rod (Figure 1A). Further observation revealed that some vesicle-like structures were found in and around the bacterium (Figures 1B,C). As displayed in Figure 1C, the spherical vesicles budded from bacterial outer membranes. These results suggested that H. parasuis H45 could secrete OMVs during in vitro growth.

The cell-free OMV fractions isolated from H. parasuis broth cultures were repurified by density gradient ultracentrifugation. The electron microscopy results showed that OMVs' fraction exhibited bilayered spherical vesicles (Figures 2A,B). The vesicle diameters ranged from 20 to 160 nm, with an average 50–70 nm diameter. Dynamic light scattering resulted that the Rh of the vesicles is ~69.8 nm; the size of purified OMVs ranged in diameter from ~50 to 100 nm, which was consistent with other reports (Figure 2C). To further determine whether the vesicles carried bacterial proteins, OMPs, OPs, and CPs were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) along with OMV fraction (Figure 2D). High similarity of protein bands among OMP, OP, CP, and the OMV fraction was observed, suggesting that the vesicles were mainly responsible for carrying the extracellular proteome of Haemophilus parasuis.

To identify proteins associated with H. parasuis-derived OMVs, proteome analysis was performed by liquid chromatography with MS/MS. Firstly, the purified OMVs were digested with trypsin enzyme to obtain the freeze-dried peptide liquid without salt. Then, the dried peptide samples were separated by Shimadzu LC-20AD model nanoliter liquid chromatography. The liquid phase chromatography-separated peptides were detected by electrospray ionization (ESI) MS/MS (Triple TOF 5600). Subsequently, aligning the experimental MS/MS data with theoretical MS/MS data from UniProt and National Center for Biotechnology Information database is used to identify the proteins of OMVs. In all, 195,271 spectra were obtained. After identification by the search engine, 1,614 spectra were matched; 915 peptides and 588 proteins were identified in H. parasuis OMVs (Figure 3A). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the iProX partner repository with the dataset identifier PXD029451. The identified proteins were classified into six groups according to the subcellular location of the proteins in the bacteria: cytoplasmic (n = 356), cytoplasmic membrane (n = 78), periplasmic (n = 27), outer membrane (n = 20), extracellular (n = 3), and unknown (n = 104) (Figure 3B). OMVs are enriched in membrane proteins (n = 98), including CPs and OMPs. In contrast, 5% of the total bacterial proteins are OPs. Those subcellular location results indicated that the OMVs were successfully purified from Haemophilus parasuis. Notably, proteomic results indicated that a large number of CPs were highly enriched in H. parasuis OMVs. Furthermore, we carried out a Gene Ontology (GO) functional annotation analysis for all identified proteins. The molecular function and biological processes genes are shown in Figures 3C,D. Similar to other reports, the H. parasuis OMVs were rich in proteins that have catalytic activity (n = 344) and binding function (n = 290) (Figure 3C). The classification of biological processes showed that H. parasuis-derived OMVs also contain numbers of proteins associated with cellular processes (n = 282) and metabolic processes (n = 257) (Figure 3D). These results suggested that OMVs play important roles in the pathogenesis of H. parasuis during infection.

OMVs are considered to represent a specialized bacterial secretion pathway. Several studies showed that OMVs could deliver bacterial bioactive proteins, toxins, and virulence factors (15). According to the proteome result of OMVs, we found that H. parasuis (H45)-derived OMVs are rich in immune modulators and virulence-associated proteins. According to the protein mass score, the detected proteins related to immune modulators and virulence-associated proteins are listed in Table 1. Lots of immunogenic proteins are identified, such as ompP2 (ACL31860.1), oppA (ACL32119.1), ompD15 (ACL31912.1), palA (ACL31779.1), AfuA (ACL31862.1), LppC (ACL32393.1), espP2 (ACL32961.1), and so on. These results indicated that OMV might induce a degree of protective immunity and could be selected as a new type subunit vaccine to prevent H. parasuis infection. Iron is an essential nutrient for almost all bacteria. To acquire enough free iron from the host, pathogenic bacteria have developed different mechanisms to obtain iron from the host (siderophores, hemophores, or host-molecule-binding proteins), which are involved in the expression of surface-exposed proteins (27). The iron metabolism-related proteins yfeA (ACL32714.1), dsbC (ACL31879.1), hxuA (ACL33616.1), fbpA (ACL32742.1), and HAPS_0679 (ACL32327.1) were found in OMV proteome data. In addition, several virulence-associated proteins also were identified: they are vtaA9 (ADZ54070.1), tbpA (ACL33656.1), pgi (ACL32784.1), manB (ACL32480.1), ompP5 (ACL33726.1), TolC (ACL33604.1), and yfeA (ACL32714.1). Serum resistance is potential pathogenesis of H. parasuis, which is associated with systemic diseases in swine. Several serum-resistance associated proteins of H45-derived OMVs were identified, including ctdA (ACL32011.1), ctdB (ACL32010.1), ctdC (ACL32009.1), lsgB (ACL31747.1), galE (ACL32657.1), HAPS_1019 (ACL32638.1), and oppA (ACL32119.1). Among them, ctdA, ctdB, ctdC are considered to contribute to cell cycle arrest, apoptosis, adherence, and invasion. Haemophilus parasuis (H45)-derived OMVs can carry such numbers of proteins that associated with virulence and/or pathogenic mechanism, which suggested OMVs might have an important role in H. parasuis infection.

Severe serous inflammation in multi-tissues is a typical feature of H. parasuis infection. To evaluate the ability of H45 infection for inducing IL-1β transcription and secretion, mouse mononuclear macrophage cells (J774A.1) were infected with H45 at different MOIs of infection. As shown in Figures 4A,B, the IL-1β messenger RNA (mRNA) expression and secretion were significantly increased in a dose-dependent manner during H45 infection. To further investigate whether OMVs take part in IL-1β secretion during H. parasuis H45 infection, J774A.1 were treated with OMVs at different doses for 24 h. Then, the IL-1β mRNA expression and secretion were detected by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. We found that the IL-1β expression and secretion induced by H45-derived OMVs are also in a dose-dependent manner (Figures 4C,D). This result suggested that OMV is an essential component in the inflammatory response caused by H45 infection. At last, PAMs were used to confirm the results mentioned earlier. As expected, the expressed and secreted IL-1β levels were both significantly increased in PAMs after H45 infection at 10 MOI of infection or H45 OMVs stimulation at 10 μg/ml for 24 and 48 h (Figures 4E,F). Taken together, these results indicate that H. parasuis H45 infection induces IL-1β expression and secretion, and OMVs were required for IL-1β secretion.

Haemophilus parasuis H45 strain was a clinically isolated strain in China. H45 belongs to serovar 5, which is high-level virulent and is the most popular strain in China. Bacteria were cultured in TSB (Difco Laboratories, Detroit, MI, USA) supplemented with 1 mg/ml nicotinamide adenine dinucleotide and 10% bovine serum. After incubation at 37°C overnight with circular agitation (200 rpm), bacteria were harvested at an optical density at 600 nm 0.7–0.9. A volume of each strain was mixed with 20% glycerol and stored at −80°C in cryogenic storage tubes for further use.

Mouse mononuclear macrophage cells (J774A.1) were purchased and cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C with 5% CO₂. Four-week-old piglets were used to isolate PAMs as previously described (36) and maintained in RPMI-1640 medium containing 10% heat-inactivated fetal bovine serum (Hyclone), 100 U/ml penicillin, 100 μg/ml streptomycin, and non-essential amino acid (Gibco) at 37°C in a humidified atmosphere of 5% CO_2.

The OMVs of H. parasuis were collected from the supernatant of bacterial culture (1,000 ml). In brief, H. parasuis were inoculated overnight in 5 ml of TSB. The next day, 1,000 ml of TSB with the 1 mL of starter was cultured at 37°C with shaking until the optical density at 600 nm reached 1.0. Bacterial cells were removed with centrifugation at 5,000 × g for 20 min at 4°C. The supernatants were concentrated with a 100-kDa hollow-fiber membrane, and then, the concentrate was vacuum filtered through a 0.22-μm membrane to remove any remaining bacteria. The OMVs were collected by ultracentrifugation at 150,000 × g for 4.5 h at 4°C (rotor Type 50.2 Ti, Beckman-Coulter). The pelleted OMVs were suspended with sterile phosphate-buffered saline (PBS). The resuspended OMVs were purified by density gradient centrifugation with OptiPrep [60% iodixanol (w/v)]. The OptiPrep solution was diluted with 0.85% (w/v) NaCl containing 10-mM 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid–sodium hydroxide pH 7.4 into 45, 40, 35, 30, 25, and 20% (v/v) OptiPrep. The 45% OptiPrep solution was prepared with suspended OMVs and layered 2 ml at the bottom of an Ultra-Clear centrifuge tube (13.2 ml, Beckman Coulter). The discontinuous gradients were loaded in 2-ml increments in degressive concentrations on top of the prior layer [36]. Prepared tubes were centrifuged at 150,000 × g for 16 h at 4°C (rotor SW 41 Ti, BeckmanCoulter). Each 1-ml fraction was carefully collected by pipet from top to bottom. The OMV content of each fraction was analyzed by 12% SDS-PAGE. The fractions containing OMVs were collected and pooled into an ultracentrifugation tube with 27-ml sterile PBS (at least 10-fold of the sample volume) and then ultracentrifuged at 200,500 × g for 2 h at 4°C (rotor Type 45 Ti, Beckman Coulter) to remove the OptiPrep solution. The purified OMVs were resuspended with sterile PBS, and the aliquots were stored at −80°C for future use.

Transmission electron microscopy was used to investigate OMV morphology. A drop of OMV suspension was placed on Formvar/carbon-coated grids and adsorbed for 5 min. Grids were washed with distilled water and blotted with filter paper. For negative staining, grids were treated with 1.5% (wt/vol) uranyl acetate for 1 min, air-dried, and viewed with a Hitachi HT7700 electron microscope operating at 80 kV.

OMV proteins were quantified by BCA Protein Assay Kit (Thermo fisher scientific, Cat# 23225). Vesicles proteins from H. parasuis H45 (Serum type 5) were analyzed by 12% SDS-PAGE. The gel was stained with Coomassie brilliant blue. Protein molecular weight standard (size range 35–180 KDa, Thermo Fisher Scientific) was used.

The vesicle proteins were separated by SDS-PAGE and digested with the trypsin enzyme. The digested peptide liquid was desalted and freeze-dried. The dried peptide samples were reconstituted with mobile phase A (2% ACN, 0.1% FA), and the supernatant after centrifugation at 20,000 × g for 10 min was taken for high-performance liquid chromatography–ESI-MS/MS analysis. The peptide samples were subjected to Shimadzu LC-20AD model nanoliter liquid chromatography (75 μm × 3 μm × 15 cm, C18 column) at a flow rate of 300 nl/min. The liquid phase chromatography-separated peptides were passed to an ESI-MS/MS, which was carried out using a TripleTOF 5600 (SCIEX, Framingham, MA, USA) mass spectrometer equipped with a Nanospray III source, and coupled with an emitter which drawn from quartz material (New Objectives, Woburn, MA, USA). For data acquisition, the mass spectrometer parameters were set as follows: ion source spray voltage 2,300 V, nitrogen pressure 35 psi, spray gas 15, and spray interface temperature 150°C. Scanning in high sensitivity mode, the MS1 scan cumulative time was 250 ms, and the scan quality range was 350–1,500 Da. Based on the MS1 scanning information, according to the ionic strength in the MS1 spectrum from high to low, the first 30 ions exceeding 150 cp were selected for fragmentation, and the MS2 information was scanned. The scan accumulation time of the MS2 mass spectrum was 50 ms. The collision energy was set to “rolling collision energy.”

The protein identification uses experimental MS/MS data and aligns them with theoretical MS/MS data from the database to obtain results. The process starts from converting raw MS data into a peak list and then searching matches in the Mascot search engine (Mascot 2.3.02). The UniProt protein database and National Center for Biotechnology Information database were selected. We used e-value ≤ 0.05 from Mascot as a filtering condition in this pipeline (the PSM FDR column is the Mascot value). Then, based on the parsimony principle, we performed protein inference on peptides and generated a series of protein groups. Finally, functional annotation analyses such as GO, Clusters of Orthologous Groups of protein/EuKaryotic Orthologous Groups, and Pathway analysis are performed from the final protein identification list.

J774A.1 or PAMs cells were stimulated by H. parasuis or OMVs as indicated. After 24 or 48 h, supernatants from cell culture were harvested and assayed with the production of murine IL-1β (R&D Systems, Cat# MLB00C) and swine IL-1β (RayBio, Cat# ELP-IL1b-1) by enzyme-linked immunosorbent assay, in accordance with the manufacturer's instructions. The absorbance was quantified at 450 nm by a microplate spectrophotometer (Synergy H1, BioTek, USA).