Protein expression using the common lab strains of Escherichia coli and Pichia pastoris is a key pillar of biotechnology, supporting not only basic research but also pharmaceutical development and biomaterial manufacturing^[1-2]. Recently, a fast-growing Vibrio natriegens strain Vmax has been engineered as an additional tool with great potential for producing heterologous proteins^[3]. Due to its fastest growth rate and compatibility with existing expression tools, V. natriegens Vmax has attracted increasing attention focused on genetic engineering and optimizing cytoplasmic expression conditions. These studies include genome engineering^[4], CRISPR-Cas9 editing^[5], expression of natural products and multisubunit membrane proteins^[6-7], as well as the development of a cell-free protein synthesis system^[8]. However, many physiological aspects of V. natriegens are still not fully understood, hindering its application^[9].

Bacterial growth in batch culture exhibits distinct growth phases including lag, exponential, stationary, and long-term stationary phases^[10-13]. Heterologous protein expression is often induced during the exponential phase in which protein expression machinery is expected to function at the highest levels. Although the growth rate of cells remains nearly constant in exponential phase, studies in E. coli have demonstrated that cellular physiological states are not constant but actively changing in response to nutrient depletion and metal availability during growth^[14]. What is the protein expression profiles during the rapid growth of V. natriegens still unknown due to the unexamined proteome.

Outer membrane vesicles (OMVs) are spherical vesicles with a diameter of about 20−250 nm that are ubiquitously secreted by Gram-negative bacteria and enclose outer membrane proteins, periplasmic proteins, lipopolysaccharide, cytoplasmic proteins, outer membrane lipids, DNA, RNA and some virulence factors^[15-20]. OMVs play important roles in many biological processes, including nutrient and iron acquisition, stress response, interbacterial communication, and pathogenesis^[21-25]. In addition, OMVs have been used as vaccine candidates and for therapeutic delivery due to their safety and similar immunogenic properties to whole cells^[26-27]. Heterologous expression of antigens in OMVs is a common strategy for producing engineered OMV vaccines, and target proteins are often fused with vesicle-associated proteins, including ClyA, AIDA and Hbp, for packaging into the OMVs^[28-30]. Proteomic analyses have been used to determine the composition of OMVs in many bacteria, including Fusobacterium nucleatum^[31], Pseudomonas syringae^[32], Helicobacter pylori^[33], Campylobacter jejuni^[34], Xanthomonas campestris pv. campestris^[35], Acinetobacter baumannii^[36], and E. coli^[37]. However, the OMVs in V. natriegens have not been characterized.

In this study, we report the proteomic characterization of whole cell and OMVs at mid-log (OD₆₀₀=0.5) and late-log (OD₆₀₀=1.5) growth phases of V. natriegens strain Vmax. By LC-MS/MS analysis, we identified hundreds of proteins in various physiological pathways in both the whole cell and OMV samples. We also screened some OMV proteins that were present in both mid- and late-log growth phases and demonstrate that an OmpA-domain protein carrier, OmpA24, can deliver a heterologous protein sfGFP (superfolded green fluorescent protein) as a protein-fusion cargo to the OMVs. Collectively, our results provide the first snapshot of protein expression in the whole cell and OMVs during fast growth of V. natriegens and found an efficient protein carrier. The results will facilitate further studies of V. natriegens and its OMVs for both understanding physiological functions and for biotechnological applications.

1 Materials and Methods 1.1 Main reagents and equipment

Mouse monoclonal anti-GFP antibody, Abcam; ECL solution, Bio-Rad; goat anti-mouse IgG HRP, Golden Bridge Biotechnology limited company; RpoB antibody, Biolegend. Filter, Jet Bio-Filtration limited company; Amicon Ultra-4 centrifugal filter, Millipore; ultracentrifuge, Beckman Instruments; transmission electron microscope, FEI company; Zetasizer Nano S, Malvern instruments limited company; Q-Exactive mass spectrometer and Easy nLC HPLC system, Thermo Scientific; inverted microscope, Nikon corporation instruments company.

1.2 Bacterial strains and growth conditions

All the strains and plasmids used in this study are listed in Table 1. The V. natriegens strain Vmax contains a T7 RNA polymerase-mediated expression system^[3]. Bacterial cultures were grown in LB3 medium (Lysogeny Broth with 3% (W/V) final NaCl) for V. natriegens and LB for E. coli strains^[3]. Ampicillin (100 μg/mL) and IPTG (0.001 mmol/L or 0.1 mmol/L) were added when needed. To construct the growth curve, overnight cultures were diluted 1:100 into 50 mL LB3 medium in 250 mL conical flask at 37℃ with agitation at 200 r/min. Growth was monitored by taking OD₆₀₀ readings at specific time points as indicated.

1.3 Construction of V. natriegens derivative strains

Plasmids were constructed by PCR amplifying the target genes and then ligating to the pET-22b-sfGFP vector through restriction enzymes Nde I and Kpn I. The expression plasmid of TolB-sfGFP fusion was constructed by Gibson Assembly. All the constructs were verified by sequencing. Primers are listed in Table 2.

1.4 Preparation of whole cell and OMV samples from V. natriegens

We used a previously established protocol for isolation of OMVs^[38]. Briefly, for wild type bacteria, 5 mL overnight cultures were transferred to 495 mL LB3 broth and grown at 37℃ with 200 r/min shaking. Cultures were grown to mid- and late-exponential phases (OD₆₀₀=0.5 and 1.5) and the supernatants were collected by centrifugation at 4 500×g for 15 min at 4℃. To further remove residual cells and debris, the collected supernatants were filtered through 0.22 μm pore size filter. EDTA-free protease inhibitors were added to prevent from protein degradation. Filtrates were concentrated by Amicon Ultra-4 centrifugal filter unit with 10 kD molecular weight cut-off and then ultracentrifuged for 3 h at 200 000×g, 4℃. The OMV pellets were resuspended in PBS buffer and stored at −80℃. Cultures from mid- and late-exponential phases (OD₆₀₀=0.5 and 1.5) were concentrated or diluted to OD₆₀₀=1.0. One mL cultures of OD₆₀₀=1.0 were collected by centrifugation at 2 500×g for 8 min at room temperature. Then the pellets were resuspended in SDS-loading buffer and used as whole cell samples followed by SDS-PAGE analysis. For inducing the bacteria carrying pET-22b plasmids, overnight cultures were diluted 1:100 in fresh LB3 with appropriate antibiotics and grown at 37℃ with 200 r/min shaking. Cultures were induced at OD₆₀₀=0.5 with 0.1 mmol/L or 0.001 mmol/L IPTG for 2 h or 5 h as indicated prior to OMVs isolation.

1.5 Transmission electron microscopy (TEM) and dynamic light scattering (DLS)

OMV samples were diluted ten-fold with PBS buffer, and 5 μL was placed on 400-mesh copper grids. For negative staining, samples were stained with 2% uranyl acetate for 40 s, blotted with filter paper and air dried. TEM pictures were examined in a transmission electron microscope at 120 kV acceleration voltage. DLS was performed to determine the size of OMVs by using a Zetasizer Nano S with He-Ne laser (633 nm) at 25℃ by angle of 173°.

1.6 SDS-PAGE and in-gel digestion

Proteins of whole cell and OMV samples were analyzed by SDS-PAGE (12% acrylamide gel). The gels were stained with Coomassie Blue R-250 and destained by destaining buffer (25% (V/V) alcohol, 10% (V/V) acetic acid). Gel slices were treated with 200 μL 30% Acetonitrile (ACN) in 100 mmol/L NH₄HCO₃ and then incubated with 10 mmol/L dithiothreitol at 37℃ for 1.5 h. After treatment with 100% ACN for 5 min, 60 mmol/L iodoacetamide was added for the 30 min light-avoidance reaction. Then the samples were incubated with 100 mmol/L NH₄HCO₃ for 15 min at room temperature and treated with 100% ACN for 5 min again. Digestions were performed at 37℃ with 10 ng/μL trypsin for about 20 h. Desalting of digested peptides was performed on C18 Cartridge.

1.7 Liquid Chromatography-Mass Spectrometry (LC-MS/MS)

Mass Spectrometry analysis was performed on the Q-Exactive mass spectrometer coupled with Easy nLC HPLC system. Tryptic peptides were loaded onto the Thermo scientific EASY column (2 cm×100 μm 5 μm-C18), and separated by Thermo scientific EASY column (75 μm×100 mm 3 μm-C18) at 300 nL/min flow rate. Buffer A consisted of 0.1% formic acid in H₂O, and buffer B consisted of 0.1% formic acid in 84% ACN. Peptides were separated and eluted by using the linear gradients of buffer B for 60 min gradient (0%−35% over 50 min; 35%−100% over 50−55 min; 100% over 55−60 min) and for 90 min gradient (0%−55% over 80 min; 55%−100% over 80−85 min; 100% over 85−90 min). The separated peptides were then subjected to the Q-Exactive mass spectrometer for analysis. The detection mode was positive ion mode and the analysis time were 60 min for OMV samples and 90 min for whole cell samples. The parent ion scanning ranged from 300 to 1 800 m/z and the first-order mass spectrometry resolution was 70 000 at m/z 200. The automatic gain control (AGC) target was 3e6, and the maximum IT was 50 ms. MS2 spectra of the 20 debris maps were acquired after each full scan. The MS2 activation type was HCD. In secondary mass spectrometry, the resolution was 17 500 at m/z 200, maximum IT was 60 ms. The microscan was 1, isolation window was 2 m/z, normalized collision energy was 27 eV, dynamic exclusion was 60 s and underfill ratio was 0.1%. Proteome Discoverer 1.4 software was used for data analysis. Proteins that appeared in ≥2 trials among the three independent experiments with ≥ one unique peptide (Σ# unique peptides ≥2) and false discovery rate ≤1% were considered reliable protein identification. LC-MS/MS analysis was performed at Shanghai Applied Protein Technology limited company.

1.8 Bioinformatics analysis

Genome sequences of V. natriegens ATCC 14048 were searched from GenBank under accession numbers CP016345 and CP016346. For protein identification, the spectra were searched using the proteome data of V. natriegens CCUG 16374 from UniProt (www.pir.uniprot.org) database (UP000092577). Subcellular location was analyzed by PSORTb (www.psort.org/psortb/). The functional annotations of identified proteins were defined by the GO and KEGG database. OmpA24 sequence was analyzed with BLASTp to identify homologs and species distribution. In total 20 homologs were manually selected and analyzed by phylogeny together with OmpA24. The phylogenetic tree was constructed using the Maximum-likelihood method in MEGA X software with bootstrap 1 000 iterations, and protein sequences were aligned by MUSCLE algorithms^[39]. Domain analysis was performed using PfamScan tool with default settings (https://www.ebi.ac.uk/Tools/pfa/pfamscan/).

1.9 Fluorescence microscopy

Overnight cultures were diluted 1:100 in fresh LB3 with appropriate antibiotics and grown at 37℃ with 200 r/min shaking. Cells were induced when OD₆₀₀=0.5 with 0.1 mmol/L or 0.001 mmol/L IPTG for 2 h or 5 h. Cultures were then centrifuged for 8 min at 2 500×g, concentrated to OD₆₀₀=10.0 and resuspended in 1×PBS (supplemented to 3% (W/V) NaCl final concentration). One μL bacteria or OMV fractions were spotted onto 1% (W/V) PBS-agarose pad supplemented with 3% (W/V) NaCl. Fluorescence images were acquired with a Nikon Ti-2E inverted microscope using NIS-Elements AR 5.20.00 software and ImageJ was used for image analysis. Exposure time was 500 ms, the minimum and maximum displayed value of brightness and contrast were set at 3 200−3 500 for 2 h-induced-OMV samples and 10 000−15 000 for 5 h-induced-OMV samples.

1.10 Western blot

Protein samples of whole cell and OMVs were fractionated by 12% SDS-PAGE gel and transferred to PVDF membrane by electrophoresis. Membrane was blocked with 5% (W/V) non-fat milk in TBST buffer (50 mmol/L Tris, 150 mmol/L NaCl, 0.5% (V/V) Tween-20, pH 7.6) for 1 h at room temperature. Then the membrane was incubated with primary antibody (mouse monoclonal anti-GFP) in TBST with 1% (W/V) non-fat milk. The membrane was washed three times and incubated with anti-mouse IgG horseradish peroxidase (HRP)-conjugated secondary antibody in TBST with 1% (W/V) non-fat milk for 1 h followed by detection using ECL solution. The beta subunit of RNA polymerase (RpoB) was used as control for cell lysis. The secondary antibody goat anti-mouse IgG HRP and RpoB were used at 1:15 000 dilution, while anti-GFP primary antibody at 1:10 000.

2 Results and Analysis 2.1 Isolation of OMVs in different growth phases of V. natriegens

To determine whole cell and OMVs proteome composition during exponential phase growth, we first monitored bacterial growth in LB3 media and picked OD₆₀₀=0.5 and 1.5 as two sampling points (Figure 1A). The former is when inducers are often added for heterologous protein expression while the latter is when cells are exiting exponential phase growth. Using high-speed centrifugation, we collected OMVs and analyzed them using transmission electron microscopy (TEM) and dynamic light scattering (DLS). Results show that the isolated OMVs were small, spherical particles with an average diameter of 25 nm (Figures 1B and 1C). The size of OMVs between OMV-0.5 and OMV-1.5 samples shows no difference under the observation of TEM and DLS. There were very few membrane debris, pili or fragments of flagella, indicating high quality of OMV preparation.

2.2 Overview of proteomic analysis of whole cell and OMVs from V. natriegens

Using LC-MS/MS, we analyzed protein profiles from whole cell and OMV samples. We identified 1 480 proteins of Cell-0.5, 1 565 proteins of Cell-1.5, 288 proteins of OMV-0.5 and 317 proteins of OMV-1.5 from three independent experiments. These identified proteins appeared in at least two of the three independent experiments, and all of them were predicted with subcellular locations. We identified 1 480 and 1 565 proteins in whole cell samples under mid- and late-exponential phases, of which 226 and 234 proteins were also found in OMV samples, respectively. A total of 185 proteins were present in all four groups, while 1 389 and 254 proteins were found in both mid and late whole cell samples and OMV samples, respectively (Figure 2A). In OMV-0.5 and OMV-1.5 samples, outer membrane, periplasmic, and extracellular proteins were enriched while about 37.15% and 29.97% were cytoplasmic proteins, respectively. In whole cell samples, more than 65% were cytoplasmic proteins (Figure 2B). We also performed prediction of subcellular locations on whole 4 688 proteins of V. natriegens which contained in proteome database of UniProt. The number of proteins in 6 groups, including outer membrane, periplasm, cytoplasm, cytoplasmic membrane, extracellular, and unknown, was taken as the theoretical number. The number of proteins with different locations in four samples (Cell-0.5, Cell-1.5, OMV-0.5, OMV-1.5) was compared with the theoretical number. Comparing with the theoretical number of each category in subcellular location, we identified about 40% of cytoplasmic proteins and a similar portion of periplasmic and outer membrane proteins in both whole cell samples (Figure 2C). OMV proteins are primarily made of outer membrane, periplasmic and extracellular proteins (Figure 2C). In addition, the proteomes of mid- and late-exponential phases share more than 80% identity for both whole cells (Cell-0.5 and Cell-1.5) and OMVs (OMV-0.5 and OMV-1.5), suggesting a relatively stable protein composition during exponential phase growth.

2.3 Functional cluster analysis of whole cell and OMV proteins

We functionally categorized all identified proteins using the GO database and KEGG pathway analyses. We found that proteins in whole cell and OMV samples were abundant in functional groups including cellular process, metabolic process, catalytic activity and binding (Figure 3). As expected, most proteins are involved in growth-related biological functions including metabolic enzymes, translation, and nutrient transport. Figure 4 shows the top 10 KEGG pathways, the most abundant of which are purine metabolism and two-component systems with 55 and 61 proteins in Cell-0.5 and Cell-1.5 samples, respectively. Metabolism of purine and pyrimidine nucleotides is essential for the biochemical processes of DNA and RNA synthesis. The proteins PurF, UPP, and GPT identified from whole cell samples are involved in nucleotide metabolism, for the synthesis of IMP, UMP, GMP and XMP^[40-42]. Two-component systems control many functions including signaling, communication and adaptation, which are critical for bacteria to respond to environmental changes^[43].

Cells growing in exponential phase express a large number of proteins involved in nucleotide and amino acid metabolism, and ribosomal functions to meet the growth demand. The identified top 10 KEGG pathways also contain glycolysis/gluconeogenesis, quorum sensing and biofilm formation. ATP-binding cassette (ABC) transporters are membrane proteins that use ATP hydrolysis to transport various molecules including peptides, ions, amino acids, sugars, polysaccharides, and lipids^[44]. Notably, bacteria mainly use amino acids as nutrient source when growing in LB which contains low concentration of sugars^[14]. Furthermore, more proteins involved in two-component system, quorum sensing and biofilm formation are present in Cell-1.5 sample than in Cell-0.5 sample, suggesting cells might be adjusting to increased density and depleted nutrients.

In OMVs, KEGG pathway analysis shows that several pathways including ABC transporters, flagellar assembly and two-component system are abundant in OMV-0.5 and OMV-1.5 samples (Figure 4). Notably, ribosomal proteins are also present in OMVs. The peptidoglycan-associated protein Pal and periplasmic protein TolB are parts of Tol-Pal system and found in the OMVs. The Tol-Pal complex controls outer membrane integrity and invagination during cell division^[45]. Some cytoplasmic proteins including ribosomal proteins (RplA, RplB, RpsB, RpsC), chaperone proteins (GroL, DnaK, HtpG), DNA-directed RNA polymerase (RpoB, RpoC), elongation factor (FusA), and enzymes (pyruvate dehydrogenase, glutamine synthetase, aconitate hydratase, dihydrolipoyl dehydrogenase) were also identified in OMV samples of V. natriegens.

2.4 OmpA24-sfGFP fusion is enriched in outer membrane vesicles of V. natriegens

A main objective of OMV proteomic profiling is to find candidate carrier proteins in OMVs for delivery of heterologous proteins. The carrier protein should be stable in OMVs, especially in the rapid growth phase. Therefore, two time points OD₆₀₀=0.5 and OD₆₀₀=1.5 were chosen as the representatives of exponential phase to extract OMVs and identify the protein composition. From the proteomic results, we selected outer membrane proteins and periplasmic proteins that appeared in both mid- and late- exponential phases, and avoided enzymes and regulatory factors that may have pleiotropic effects. We selected 8 proteins (OmpW, TolC, OmpA24, Pal, MalE, TolB, Skp and PotD) as OMV carriers and employed sfGFP to make fusion protein with these carriers for the study of cargo protein delivery (Table 3). When these constructs were induced using 0.1 mmol/L IPTG, we found different degrees of growth inhibition (Figure 5). So we changed to a lower dose of IPTG (0.001 mmol/L) and found that there was little effect on growth (Figure 5). We then analyzed expression of target proteins in bacterial cells and OMVs by Western blot and fluorescence microscopy analyses (Figures 6 and 7). All but the OmpW-sfGFP fusion proteins were detected in whole cell samples by Western blot analysis (Figure 6A). In OMV samples, only the OMV sample from pOmpA24 construct was detected by Western blot with an anti-GFP antibody (Figure 6B). Fluorescence microscopy analyses show that all constructs were expressed in cells, with all but TolB-sfGFP highlighting the membrane or periplasm (Figure 7).

Next, we focused on optimizing OMV-expression of the pOmpA24 construct. Because overexpression of the OmpA24-sfGFP fusion protein with 0.1 mmol/L IPTG showed growth inhibition, we lowered the inducer level to 0.001 mmol/L IPTG and extended induction time to 5 h. We found strong fluorescence signals of OmpA24-sfGFP in the OMV samples and confirmed its presence by Western blot and fluorescence microscopy analyses (Figures 8A and 8B). By comparison, plasmid-expressed sfGFP alone was not detected in OMV samples in either fluorescence microscopy or Western blot analyses. These results indicate that sfGFP can be packaged in OMVs of V. natriegens only when it is fused to OmpA24 but not on its own.

2.5 OmpA24 is a conserved membrane protein in many species

OmpA24 contains an OmpA-like domain in its C-terminal and links to sfGFP with a linker. OmpA-like domain-containing proteins can anchor to the peptidoglycan and outer membrane by an N-terminal β-barrel; an N-terminal lipid anchor; or by binding to porins, which could stabilize the cell envelope and outer membrane integrity^[46-50].

We next examined the distribution of OmpA24 (GenBank accession number: ANQ12348.1) using BLASTp, and found OmpA24 homologs are widely distributed in genera of Vibrio, Aliivibrio, and Marinomonas. We searched OmpA24 in non-redundant protein database for its close homologs in Vibrio and in the non-redundant protein database excluding genera Vibrio to include more distant homologs. By ranking the hits by percent identity and excluding redundant species, we selected 20 representative sequences, 10 from each search. The sequence identities of the top 10 homologs in genera Vibrio are more than 88% and those of the 10 homologs from non-Vibrio species are more than 66%. Using phylogenetic tree and conserved domain analyses, we compared these 20 homologs with OmpA24 (Figures 9 and 10). OmpA24 is clustered with homologs from genera Vibrio and divergent from genera Aliivibrio and Marinomonas. Domain analysis shows that OmpA24 and its homologs exhibit conserved N-terminal glycine zipper domains and C-terminal OmpA family domains.

3 Discussion and Conclusion

In this study, we demonstrate that V. natriegens can produce OMVs throughout exponential growth and present the first proteomic snapshot of OMVs and related whole cell samples under different growth phases that are relevant to its application as a promising protein- expression tool. We identified 288, 317, 1 480, 1 565 proteins in OMV-0.5, OMV-1.5, Cell-0.5 and Cell-1.5, respectively. Results show that protein composition remains largely stable during exponential phase growth. By comparing a number of OMV proteins for heterologous sfGFP-fusion cargo enrichment, we show that OmpA24 is the most efficient as a fusion carrier.

Some cytoplasmic proteins and cytoplasmic membrane proteins were identified in OMVs of V. natriegens in this study. Notably, some cytoplasmic proteins identified in our OMVs were also observed in the OMVs of other bacteria including A. baumannii, Francisella novicida and Francisella philomiragia^{[36, 51]}. Besides ribosomal proteins, DNA and RNA have also been identified in OMVs of other bacteria^{[20, 52, 53]}. A previous study examining the membrane vesicles of Pseudomonas aeruginosa has reported a cell-lysis-mediated mechanism to account for the presence of cytosolic components^[54]. However, it remains debatable whether these cytosolic contents play important physiological roles.

Almost all Gram-negative bacteria produce OMVs, V. natriegens grows among the fastest^{[9, 55-57]}. A large amount of OMVs may be harvested in shorter time possibly, which makes V. natriegens a promising tool for OMVs application. OMVs have been used for multiple applications. For example, OMVs isolated from A. baumannii, Bordetella pertussis and Burkholderia pseudomallei, can evoke antibody responses in animal models, and the Meningitis type B OMV vaccine has been approved for clinical application^[58-62]. OMVs have also been used in transdermal nanoplatforms for inducing tumor-targeting drug delivery, in activation of the caspase-11-GSDMD pathway to disperse intravascular coagulation, and in modulating host immune systems^[63-66]. Here, we established a method for extracting OMVs and characterized its proteomic composition. We report that OmpA24-sfGFP can be found in OMVs with the highest efficiency of all fusion proteins tested. Notably, a high induction level of the OmpA24-sfGFP fusion protein showed bacterial growth inhibition. This may be due to the induced levels of OmpA24-sfGFP protein disturbing OmpA24-mediated interaction with peptidoglycan and outer membrane. Although this problem could be mitigated by lowering the induction, optimization using mutagenesis and growth conditions may be needed to further increase yield in future studies.

In summary, our study provides a general understanding of the protein composition of V. natriegens whole cells and OMVs during exponential-phase growth. We demonstrate an OMV-protein OmpA24 is a promising carrier for delivering sfGFP as a protein-fusion cargo to the OMVs. Using similar tools and techniques, we can use the OMVs of V. natriegens to deliver antigens or drugs for a variety of biotechnological applications.