SONG Huimin, RUAN Yilong, LI Ya, YANG Huirong, and ZHANG Weiwei, 2), *

Proteomic and Functional Analyses of Outer Membrane Vesicles Secreted by

SONG Huimin1), RUAN Yilong1), LI Ya1), YANG Huirong1), and ZHANG Weiwei1), 2), *

1),,315832,2),,315211,

is an important opportunistic pathogen ubiquitously present in the marine environment, exhibiting vi- rulence to a variety of cultured animals. The extracellular products secreted byare crucial to bacterial survival and vi- rulence. In this study, the secretion of outer membrane vesicles (OMVs) bywas determined, purified, and morphologi- cally characterized. The protein composition of OMVs was analyzed by proteomic analysis. The results showed that approximately 120 proteins were contained in these OMVs, including outer membrane proteins, flagellins, ABC transporters, protease, and iron re- gulation proteins,., which were involved in bacterial motility, formation of biofilms and the cell membrane components, and cellu- lar localization based on their structural molecule activity, passive transmembrane transporter activity, channel activity, neurotransmitter receptor activity, extracellular ligand-gated ion channel activity, glutamate receptor activity, ligand-gated ion channel activity, and transmembrane signaling receptor activity. To explore the biological functions of OMVs in, the effects of OMVs on the bacterial adaption to iron limitation, antibiotic, and the coelomic fluid of thewere confirmed. This study is the first time to show thatsecretes OMVs, and OMVs carry functional proteins that enhance bacterial survival under vari- ous stresses.

; outer membrane vesicles (OMVs); proteomic analysis; biological function

1 Introduction

is an important member of marine microbial com- munity. Some species are opportunistically pathogenic to both marine vertebrates and invertebrates (Torresi., 2011).is one of the most dominantspecies in the coastal and brackish aquatic environment, sea mud, as well as on the surface or in a variety of marine animals (Zhang and Li, 2021). It can infect a wide range of hosts including(Gao., 2015),(Duperthuy., 2010),L. (Mu?oz-Atienza., 2014), and(Kang., 2022), leading to serious econo- mic losses. In our previous studies, it had been proved thatmainly exerted its virulence as an extracellu- lar bacterium (Dai., 2020), and the virulence factors- include metalloprotease and hemolysin (Liang., 2016; Zhang., 2016), siderophore (Song., 2018), and the effector proteins are released by the secretion system (Zhuang., 2021).

Outer membrane vesicles (OMVs) are considered to be a potential secretion system known as ‘zero-type secretion system’ (Guerrero-Mandujano., 2017). They are nano-particles produced by Gram-negative bacteria along with their growth and metabolism (Avila-Calderón., 2021), and are released into the extracellular niche from the bac- terial cell membrane as double-membrane spherical vesi- cles (Cai., 2018). OMVs produced by different bac- teria vary in size, mainly ranging from 10 nm to 300 nm (Sar- torio., 2021). As bacterial exosomes, OMVs are rich in a variety of biological macromolecules, including pro- teins, lipids, and nucleic acids (Dhital., 2021). Normal- ly, proteins harbored in OMVs contain outer membrane pro- teins, periplasmic proteins, and other proteins that act as ad- hesion and virulence factors (Avila-Calderón., 2021). Lipids are ubiquitous in OMVs, but with species specificity. For example, in addition to phospholipids and lipopolysac- charides (LPS), OMVs secreted by enterotoxigenicmainly contain phosphatidylethanolamine and cardiolipin, which are thought to be related to the curvature of OMVs (Horstman and Kuehn, 2000). Additionally, OMVs also contain the biomacromolecule DNA and RNA (Ha- bier., 2018).

Since OMVs contain a variety of important biomolecules, such as proteases, signaling molecules, virulence factors, ge- netic materials,., they can contribute to the biological processes of environmental adaption, survival, and nutrient acquisition of the bacteria (Furuyama and Sircili, 2021). For example, the scarcity of metal ions leads to competition be- tween both intra-species and inter-species among all the spe- cies in one bacterial community, and OMVs can enrich me- tal ions and act as ‘weapons’ of bacteria in this competitive race (Kulp and Kuehn, 2010). When bacteria are threaten- ed by environmental stresses such as antibiotics or starva- tion, OMVs can also increase bacterial survival‘parcel cargos’, protecting bacteria from slaughter (Klimentová and Stulík, 2015). OMVs can also participate in the gene hori- zontal transfer among bacterial cells. For example, OMVs secreted bycan transfer its plasmids carrying resistance genes to,, and(Dell’Annunziata., 2021). For pathogenic bacteria, OMVs contain biological mole- cules involved in invasion, adhesion, or regulation of the host immunity. In(Wai., 2003),sp. (Liu., 2021), and(Chatterjee and Chaud- huri, 2011), OMVs exported and delivered toxins such as cytolysin A (ClyA) and shiga toxin (STX) to epithelial cells of the stomach and intestine, causing infection and diseases. In, the LPS and outer membrane proteins (OMPs) carried by OMVs activated the immune response of the host and caused over-stimulation in the in- flammatory response (Cahill., 2015).

Considering the important roles of OMVs played in the environmental adaption and virulence of bacteria, the iso- lation, purification, and characterization of OMVs secret- ed bywere performed in this study. Further- more, the protein components and the biological functions of OMVs were also explored.

2 Materials and Methods

2.1 Strains and Culture Conditions

The strain ofAJ01, which is an important pathogen of(Zhang., 2016), is conserved in the China General Microbiological Culture Collection (CGMCC, Beijing, China). It was sequenced by 16S rDNA periodically to verify its status. The 2216E medium consist- ing of tryptone 5.0 g, yeast extract 1.0 g, and FePO40.01 g in 1 L filtered seawater was used to cultureAJ01 (pH 7.6 – 8.0). To prepare solid medium, 1.2 g of agar powder was added to 100 mL of the liquid medium.

2.2 Separation and Purification of OMVs

AJ01 was inoculated into fresh 2216E me- dium at a ratio of 1:100 from overnight culture, and was cul- tured in a shaker at 28℃ with a shaking speed of 180 r min?1. When the cells were grown to the logarithmic growth phase, the bacteria were collected. Cell-free supernatant was obtained by centrifugation at 4500 ×for 15 min at 4℃, fol- lowed by filtration through a 0.22 μm filter (millipore). The OMVs were purified according to the method described by Schild. (2008). The cell-free supernatant was ultracen- trifuged (Beckman, TYPE 45Ti) at 140000 ×for 4 h at 4℃. Then the supernatant was discarded and the pellet was re- suspended in an appropriate volume of PBS. The OMVs suspension was frozen at ?80℃ for further use.

2.3 Morphological Characterization of OMVs

The OMVs associated with bacteria cells or the purified OMVs were observed under transmission electron micro- scope (TEM, Hitachi H-7650) as described by Fulsundar (2015). Briefly, a drop of liquid was taken and dropped on formvar/carbon-coated grids, blotted dry with filter paper, and observed with a TEM. To measure the size of OMVs, 1 mL OMVs suspension was mixed with 3 mL ethanol in 50 mL ddH2O, and the diameter of OMVs in the mixture was then recorded in the nanoparticle tracking analyzer (NTA, Nano ZS).

2.4 Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) Analysis of the OMVs Proteins

12% SDS-PAGE was used to preliminarily analyze the proteins harbored in OMVs. The total proteins ofwere extracted as a control, and the concentrations of total proteins and the OMV proteins were determined by the BCA protein assay (CWBIO, Taizhou, China). Equal amounts of total proteins and OMVs proteins were loaded onto the gel. After electrophoresis, the proteins on the gels were stained with Coomassie brilliant blue and the bands on the gel were imaged in gel imager (Aplegen, Omega Lum C).

2.5 LC-MS/MS Analysis of OMVs Proteins

The proteins in OMVs were analyzed using LC-MS/MS. Briefly, the proteins in OMVs were precipitated by 10% trichloroacetic acid (TCA). After washing with acetone and drying, 100 mmol L?1Tris-HCl solution (pH 8.0) contain- ing 8 mol L?1urea was added to the precipitate to fully dis- solve the proteins. After centrifugation at 12000 ×for 15 min, the supernatant was collected and dithiothreitol (DTT) was added to a final concentration of 10 mmol L?1. The mix- ture was incubated at 37℃ for another 1 h to break the di- sulfide bonds. Subsequently, iodoacetamide was added at a final concentration of 40 mmol L?1, and an alkylation re- action to block sulfhydryl groups was carried out at room temperature in the dark. After these reactions, an appropri- ate volume of 100 mmol L?1Tris-HCl solution (pH 8.0) was added, and the protein concentration was quantified using the Bradford method. Then, the urea concentration was di- luted below 2 mol L?1, trypsin was added at a ratio of 50:1, and the mixture was incubated at 37℃ overnight with shak- ing for enzyme cleavage. Finally, trifluoroacetic acid was added to terminate the digestion, and the pH of the solu- tion was adjusted to about 6.0. After centrifugated at 12000 ×for 15 min, a C18 column was used to desalt the solu- tion. The desalted peptide solution was dried in a centri- fugal concentrator, and then stored at ?20℃ for detection.

Mass spectrometry analysis was performed using a Q Ex- active Plus LC/MS system (Thermo). Samples were sepa- rated by a liquid phase UltiMate 3000 RSLCnano system at nanoliter flow rates. The peptide sample was dissolved in the loading buffer, sucked by the autosampler, bound to a C18 capture column (3 μm, 120 ?, 100 μm × 20 mm), and then eluted to an analytical column (2 μm, 120 ?, 75 μm × 150 mm) for separation. An analytical gradient was estab- lished using two mobile phases (mobile phase A: 3% DMSO, 0.1% formic acid, 97% H2O, and mobile phase B: 3% DMSO, 0.1% formic acid, 97% ACN). The flow rate of the liquid phase was 300 nL min?1. For MS DDA mode analy- sis, each scan cycle consists of one MS full scan (R = 70 K, AGC = 3e6, max IT = 20 ms, scan range = 350 – 1800 m/z), followed by 15 MS/MS scan (R = 17.5 K, AGC = 2e5, max IT = 100 ms). The HCD collision energy was set to 28%. The screening window for the quadrupole was 1.6 Da. The dynamic exclusion time for repeated ion acquisition was 35 s. Mass spectral data generated by Q Exactive Plus were searched by ProteinPilot (V4.5) using the database search algorithm Paragon. The database used for the search wasproteome reference database (Uniprot).

2.6 Protein Bioinformatic Analysis

The localization of proteins in OMVs was acquired by the Uniprot database and predicted by using CELLO2GO (Yu., 2014). To further define the function of proteins in OMVs secreted by, gene ontology (GO) functional annotation analysis was performed. Protein func- tions were obtained by performing functional classification annotations from the Uniprot database. For the GO nodes involved in biological process, cellular component and mole- cular function, the number of all corresponding proteins is listed in a statistical graph.

2.7 Measurement of Protease Activity

Protease activity of OMVs was quantified using azoca- sein as a substrate. 0.1% TritonX-100 was added to 40 μL of OMVs preparation and the same volume of PBS solu- tion, respectively, and the OMVs were lysed at 20℃ for 30 min. Then, 100 μL of azocasein with a stock solution of 5 mg mL?1in 100 mmol L?1Tris-HCl (pH 6.8) was added re- spectively, and the incubation was at 20℃ for 1 h. Totally 100 μL of 10% TCA was added and left on ice for 5 min to precipitate the undecomposed azocasein. After centrifugated at 12000 ×for 5 min, the supernatant was taken to measure its absorbance at 350 nm with a microplate reader (Mole- cular Devices SpectraMax 190).

2.8 Iron Sensitivity Assay

The effect of OMVs on the growth ofun- der iron-limited condition was determined. Briefly, when the OD600of bacterial cell reached approximately 0.1, the OMVs that might be naturally produced during bacterial growth were removed by centrifugation, and the cells were resuspended in 1 mL of 2216E medium. 100 μL of purified OMVs was added to the cell suspension, and 100 μL of PBS was added into another aliquot of the cell suspension as a control sample. Then, 2,2’-bipyridine was added at a final concentration of 320 μmol L?1. The cell suspensions were in- cubated at 28℃ for 1 h, and the suspension was 10-fold se- rially diluted with PBS, 10 μL of each gradient dilution was dropped onto the agar plate. The colony numbers on the plates of both the control group and the experimental group were separately recorded after overnight culture.

2.9 Penicillin Resistance Assay

To test whether the OMVs were associated with antibio- tic resistance of, a penicillin resistance assay was performed. Briefly, after the naturally produced OMVs were removed, 100 μL of purified OMVs was added to the cell suspension, and 100 μL of PBS was added into another aliquot of the cell suspension as a control sample. Then, pe- nicillin at a final concentration of 1 mg mL?1was added to both the experimental and control samples. The cell suspen- sions were then incubated at 28℃ for 1 h, and the suspension was 10-fold serially diluted with PBS, 10 μL of each gradient dilution was dropped onto the agar plate. The co- lony numbers on the plates of the control group and the ex- perimental group were recorded after overnight culture re- spectively.

2.10 Coelomic Fluid Resistance Assay

The effect of OMVs on bacterial survival whenwas challenged with the host immune factors was de- termined. Briefly,was dissected, and the coe- lomic fluid was collected and filtered through 300-mesh sterile gauze. After the naturally produced OMVs were re- moved, 100 μL of purified OMVs was added to the cell sus- pension, and 100 μL of PBS was added into another aliquot of the cell suspension as the control sample. Then, 400 μL coelomic fluid was added into the experimental and control samples respectively. The cell suspensions were incubated at 28℃ for 1 h. Then the suspension was 10-fold serially di- luted with PBS, and 10 μL of each gradient dilution was dropped onto the agar plate. The colony numbers on the plates of both the control group and the experimental group were recorded respectively after overnight culture.

2.11 Strain No. of Bacteria, Accession Number and Statistical Analysis

The isolate ofAJ01, previously designed as Vs1, was deposited into the CGMCC with strain No. 7.242. The mass spectrometry proteomics data have been depo- sited to the ProteomeXchange Consortium (http://proteo- mecentral.proteomexchange.org)the iProX partner re- pository (Ma., 2019) with the dataset identifier PXD- 036928. Statistical analyses were performed by using the two tailed-test. Statistical significance was determined by one-way ANOVA (Mollah., 2015). In all cases, the sig- nificance level was defined as< 0.05.

3 Results

3.1 OMVs Secreted by V. splendidus

Vesicle-like structures were observed in thecul- ture ofwhen its growth reached logarithmic phase, and they were scattered outside the bacteria (Fig.1a). The OMVs secreted bywere isolated, puri- fied, and concentrated from the cell-free supernatant by ul- tra-centrifugation. A large number of isolated nanoscale structures were observed under TEM (Fig.1b). The pheno- type of OMVs was a double-layer spherical structure with a smooth surface and heterogeneous size distribution (Fig. 1c). The particle size of OMVs was mainly distributed be- tween 45 nm and 120 nm, with an average particle size of 77.9 nm (Fig.1d).

Fig.1 (a) TEM image of V. splendidus and the OMVs. OMVs were indicated by arrows. (b) TEM image of purified OMVs. (c) TEM image for the morphology of OMVs. (d) The particle size of OMVs measured by NTA.

3.2 Proteomic Analysis of OMVs Secreted by V. splendidus

The concentration of proteins in OMVs secreted bywas determined to be 1.536 μg μL?1. SDS-PAGE results showed that OMVs contained ample proteins of, and the protein bands were part of the total bacterial proteins, but with distinctly different main bands (Fig.2a)To obtain stable composition of OMVs, three re- plicate samples were collected and identification of OMVs was performed using LC-MS/MS. The information of pro- teins in OMVs could be found in ProteomeXchange Con- sortium. These proteins were classified into five groups ac- cording to the subcellular location of the proteins in the bacteria, which indicated that it contained the proteins from all parts of the bacteria cells. The result showed that the pro- teins from the extracellular, outer membrane, cytoplasmic, periplasmic, and inner membrane were 25.8% (= 55), 23.5% (= 50), 23% (= 49), 20.7% (= 44), and 7% (= 15) of total proteins, respectively (Fig.2b).

Fig.2 (a) SDS-PAGE analysis of total proteins and OMV proteins of V. splendidus. Marker, from the top to the bottom: 180, 135, 100, 75, 63, 48, 35, and 25 kDa; lane 1, total proteins of V. splendidus; lane 2, proteins in OMVs. (b) The sub- cellular localization of proteins in OMVs secreted by V. splendidus.

The GO annotation is classified into three parts: biologi- cal process, cell component, and molecular function, as shown in Fig.3. In terms of cell component, the majority of proteins were encapsulated structures of cells, and 25 proteins were cell projection parts. Other proteins were involved in the formation of the outer cell membrane and flagella. In terms of biological processes, most of the pro- teins were related to the motility of bacteria. In addition, some proteins were involved in the formation of biofilms, cell membrane components, and cellular location. In terms of molecular function, 14 proteins were involved in struc- tural molecule activity, 7 proteins had passive transmem- brane transporter activity, 7 proteins had channel activity, and other proteins were involved in neurotransmitter re- ceptor activity, extracellular ligand-gated ion channel ac- tivity, glutamate receptor activity, ligand-gated ion chan- nel activity, transmembrane signaling receptor activity, and so onThese results indicated that OMVs might be involved in diverse important biological activities of bacterium

Fig.3 GO analysis of proteins in OMVs secreted by V. splendidus.

3.3 OMVs of V. splendidus Exhibited Obvious Protease Activity

OMVs secreted bywere rich in a variety of peptidases up to 41 species, and proteases or the subunits that made up these enzymes included ATP synthase (AtpF, AtpD_2, and AtpA), transferase (AccA), hydrolase (UshA, Ggt, BCU38_19035, and CWN84_07090), metalloprotease (CWO07_25300), and enolase,. These enzymes might be involved in bacterial growth, reproduction, and energy metabolism. After lysis of OMVs by triton X-100, the ac- tivity of protease was determined using azocasein as a sub- strate. The result showed that the proteases in OMVs pos- sessed 2-fold higher activity than those of the PBS control, which suggested obvious protease activity of the proteins in OMVs (Fig.4).

3.4 OMVs of V. splendidus Carried Potential Bacte- rial Survival Factors and Virulence Factors

In the OMVs secreted by, iron regulation related proteins (IrpA, CWO07_11325, CWO07_22930, and A9262_04155), which are involved in the transport, bind- ing, and regulation of iron were identified. Moreover, mul- tiple ABC transporters, including AapJ, YxeM, FliY, and DppA_2, and penicillin-binding proteins LpoA and LpoB were identified, which are supposed to be involved in bac- terial stress responses, especially antibiotic resistance. As an opportunistic pathogen, the OMVs secreted bycontain virulence factors such as aerolysin, which are supposed to be involved in the interaction between the pa- thogen and the host.

Fig.4 Protease activity of OMVs measured using azocasein as a substrate. OMVs preparation or PBS solution were lysed using tritonX-100 and then incubated separately with azocasein. Subsequently, 10% TCA was added to precipi- tate the undecomposed azocasein and the supernatant was taken after centrifugation. The absorbance at 350 nm was measured. Data were presented as mean ± SD (n = 3). Aste- risks indicate significant differences, *P < 0.05.

3.4.1 OMVs enhanced the survival ability under iron-limited condition

OMVs contained iron-regulated proteins, which might be involved in bacterial survival under iron-limited con- dition. Thus, the cells that survived under the iron-limited conditions in with or without OMVs were compared using drop method. Whenwere cultured with OMVs, three colonies appeared in the 105-fold dilution, while co- lonies only appeared in the 104-fold dilution of the control culture. The cell number in the experimental group was 5- fold more than that in the control group without OMVs (Fig. 5a), which indicated that the OMVs could enhance the sur- vival ability ofunder iron-limited condition.

3.4.2 OMVs promoted survival ability under penicillin treatment

OMVs contained penicillin-binding protein activators, which led us to wonder whether OMVs were involved in bacterial resistance to penicillin. The exogenous penicillin was added into the bacterial suspension in the presence or absence of OMVs, respectively. Whenwas cultured with OMVs, two colonies appeared in the 105-fold dilution, while colonies only appeared in the 104-fold dilu- tion of the control culture. The cell number in the treatment group was 5-fold more than that of the control group with- out OMVs (Fig.5b), which indicated that OMVs could en- hance bacterial resistance to penicillin as a defense mecha- nism to improve bacterial survival under antibiotics stress.

3.4.3 OMVs enhanced bacterial resistance to the host immune factors

To demonstrate whether OMVs play roles in its resis- tance to the host immune factors, a coelomic fluid resis- tance assay in the presence of OMVs was performed. Af- ter exposed to the coelomic fluid of, the cul- ture with the supplement of OMVs still showed colonies after a 1011-fold dilution, while the culture without OMVs only showed colonies after a 107-fold dilution (Fig.5c). Thus, OMVs increased bacterial survival up to approximately 104- fold, which suggested that OMVs might helpto survive from the defence when it invaded the host.

4 Discussion

It has been generally accepted that secretion of OMVs has been considered to be an important feature that highly conserves in all Gram-negative bacteria (Deatherage and Cookson, 2012); however, there has been no research on OMVs secreted by. Once the bacterium LGP32 was found to secrete OMVs in 2015, however, the strain was subsequently identified asand then(Vanhove., 2015). Thus, our present study was the first time to show thatsecreted OMVs with a smooth surface and heterogeneous size du- ringgrowth in medium. Similar to the phenotype of OMVs from other Gram-negative bacteria, the shape of the OMVs secreted bywas also regularly sphe- rical. The size of OMVs secreted bywas big- ger than those secreted by bacterium LGP32 (Vanhove., 2015) andUS6-1 (Yun., 2017).

Fig.5 (a) Cell colonies of V. splendidus in the presence and absence of OMVs after treated by 2,2’-bipyridine. (b) The cell colonies of V. splendidus in the presence and absence of OMVs after treated by penicillin. (c) The cell colonies of V. splendidus in the presence and absence of OMVs after treated by coelomic fluid from A. japonicus. After treatment, all the cell suspensions were serially diluted in 10-fold, and 10 μL of each gradient dilution was dropped on the plates. The data were obtained from at least three independent ex- periments and the figures are the representative one.

Usually, OMVs contain different kinds of biological mole- cules such as proteins, lipids, and genetic materials (Kim., 2015), which were involved in critical biological pro- cesses related to bacterial survival, antibiotic resistance, and bacteria-host cell interactions (Schwechheimer and Kuehn, 2015). With advances in MS-based proteomic analysis te- chniques, more than 3500 proteins have been identified in OMVs from different species (Jan, 2017). The OMVs se- creted bycontain an average of 128 proteins. OMVs ofare rich in membrane proteins and periplasmic proteins, which are similar to the proteins in OMVs secreted by(Choi., 2011). This ubiquitous phenomenon confirms the fact that OMVs com- monly originate from the bacterial outer membrane (Gould., 2016). Notably, similar to those secreted by(Zhang., 2021), OMVs secreted bycontain multiple ABC transporters which act as nutrient sensors and transporters, indicating that OMVs can enhance the stress response capacity of. In addition, similar to OMVs ofW3110 that contain flagellar proteins FliC, OMVs ofcontain fla- gellar proteins and flagellar assembly proteins flaB_2, flaD_ 1, FliD, FlgB, FlgC, FlgE FlgF, FlgG, FlgL, and FlaG. Since studies show that it is flagellar proteins that induce the release of OMVs in(Manabe., 2013), whe- ther the flagellar proteins are the inducers of OMVs secre- tion inand whether the OMVs are involved in the bacterial motility need to be studied in the future.

Packaging proteases and peptidases into OMVs plays pro- minent roles in bacterial energy metabolism, nutrient ac- quisition, and virulence (Toyofuku., 2019). Similar to the OMVs ofOMVs ofwere rich in hydrolases. The hydrolases in OMVs ofcan work strongly with some bacterial cells to disin- tegrate them, allowingto feed on the bacte- rial cell lysate (Kadurugamuwa and Beveridge, 1997). It is worth noting that a large number of proteases and pep- tidases in OMVs secreted byand the high protease activities meant thatmight benefit from these hydrolases under natural conditions. Iron is an essential micronutrient in bacterial metabolism, and it is in- volved in the storage and transport of molecular oxygen, carbon and nitrogen assimilation, peroxide decomposition, its electron transfer process, and biological metabolic pro- cess (Kramer., 2020). However, bioavailable iron is low for the normal requirement of bacterial cells (Carpen- ter and Payne, 2014). Therefore, bacteria have developed a variety of iron uptake mechanisms such as synthesis of siderophores, direct utilization of heme and transferrin, and iron reduction in the long-term evolution process (Andrews., 2003). The presence of the iron-regulated proteins that are involved in iron uptake pathways in OMVs leads us to wonder whether OMVs function whenencounters iron-limited condition. The increased bacterial survival in the presence of OMVs under iron-limited con- dition confirmed this speculation. This phenomenon coin- cided with the OMVs ofDS002, which selectively enriched TonB-dependent transporters, allowing the bacterial cells to capture iron more efficient- ly (Dhurve., 2022). In addition to face iron-limited con- dition in the natural and host environment, antibiotics are also a great threat to the normal growth and propagation of bacteria (Richter and Hergenrother, 2019). In, OMVs carry the β-lactamase or efflux pumps that are used to sequester antibiotics from the extracellular environment or reduce antibiotic absorption, allow bacteria to survive transiently (Ciofu., 2000). In our present study, the pro- tective effect of OMVs on the survival ofthat are challenged with penicillin may rely on LpoA and LpoB, which might disclose a new drug resistance path- way depending on OMVs.

As exosomes of pathogenic bacteria, OMVs have also been concerned as a vehicle to deliver survival factors, ad- hesion factors and virulence factors (Cai., 2018). In our present study, the bacterial survival after adding exo- genous OMVs was significantly increased whenwas challenged with the coelomic fluid of, indicating that OMVs might enhance the survival ofwhen it infected. Enolase was also found in OMVs of, and it is an enzyme carried by OMVs ofto catalyze the formation of phosphoenolpyruvate from 2-phosphoglyce- ride to facilitate bacterial colonization in the host of mice and rabbits (Toledo., 2012). Moreover, OMVs are a ‘defense weapon’ for a pathogen because they carry viru- lence factors (Ellis and Kuehn, 2010). For example, deli- very of the hemolysin VvhA by OMVs inresulted in lysis of host red blood cells, providing iron for bacterial growth and pathogenesis (Lee., 2013). In the OMVs of, there existed potential virulence factor aerolysin, which is a hemolysin known to lyse host cell membranes (Méndez., 2012). In addition, GlcNAc- binding protein A (GbpA), an adhesion factor, was also in- cluded in OMVs of. Studies had shown that GbpA promoted the customization ofin the in- testinal epithelium and regulated cholera toxin (CT) produc- tion (Jude., 2009). Thus, it can be concluded that OMVs ofmay play multiple roles in protecting the bacterial cells from the immune killing, helping the bacte- ria adhere to the host, as well as delivering the virulence fac- tors duringinfecting.

In conclusion,can secrete OMVs with he- terogeneous sizes into its extracellular spaces. OMVs con- tain more than 100 proteins, which are from the extracellu- lar, outer membrane, cytoplasmic, periplasmic, and inner membrane of. The proteins in OMVs may have multiple functions during different biological process- es. Correspondingly, OMVs can improve the bacterial sur- vival when the bacteria are under the iron-limited condition, or face the antibiotic stress, or challenge with the host im- mune factors.

Acknowledgements

This work was supported by the Zhejiang Provincial Na- tural Science Foundation for Distinguished Young Scholars (No. LR20C190001), the National Natural Science Foun- dation of China (No. 31972833), the Fundamental Research Funds for the Provincial Universities of Zhejiang (No. SJ LZ2020001), and the K. C. Wong Magna Fund at Ningbo University.

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(June 28, 2022;

September 15, 2022;

December 20, 2022)

? Ocean University of China, Science Press and Springer-Verlag GmbH Germany 2023

. E-mail: zhangweiwei1@nbu.edu.cn

(Edited by Qiu Yantao)