Abstract
Vibrio splendidus is an important opportunistic pathogen ubiquitously present in the marine environment, exhibiting virulence to a variety of cultured animals. The extracellular products secreted by V. splendidus are crucial to bacterial survival and virulence. In this study, the secretion of outer membrane vesicles (OMVs) by V. splendidus was determined, purified, and morphologically 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 regulation proteins, etc., which were involved in bacterial motility, formation of biofilms and the cell membrane components, and cellular localization based on their structural molecule activity, passive transmembrane transporter activity, channel activity, neurotrans- mitter 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 V. splendidus, the effects of OMVs on the bacterial adaption to iron limitation, antibiotic, and the coelomic fluid of the Apostichopus japonicus were confirmed. This study is the first time to show that V. splendidus secretes OMVs, and OMVs carry functional proteins that enhance bacterial survival under various stresses.
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Andrews, S. C., Robinson, A. K., and Rodríguez-Quiñones, F., 2003. Bacterial iron homeostasis. FEMS Microbiology Letters, 27 (2–3): 215–237, https://doi.org/10.1016/S0168-6445(03)00055-X.
Avila-Calderón, E. D., Ruiz-Palma, M. D. S., Aguilera-Arreola, M., Velázquez-Guadarrama, N., Ruiz, E. A., Gomez-Lunar, Z., et al., 2021. Outer membrane vesicles of gram-negative bacteria: An outlook on biogenesis. Frontiers in Microbiology, 12: 557902, https://doi.org/10.3389/fmicb.2021.557902.
Cahill, B. K., Seeley, K. W., Gutel, D., and Ellis, T. N., 2015. Klebsiella pneumoniae O antigen loss alters the outer membrane protein composition and the selective packaging of proteins into secreted outer membrane vesicles. Microbiological Research, 180: 1–10, https://doi.org/10.1016/j.micres.2015.06.012.
Cai, W., Kesavan, D. K., Wan, J., Abdelaziz, M. H., Su, Z., and Xu, H., 2018. Bacterial outer membrane vesicles, a potential vaccine candidate in interactions with host cells based. Diagnostic Pathology, 13 (1): 1–12, https://doi.org/10.1186/s13000-018-0768-y.
Carpenter, C., and Payne, S. M., 2014. Regulation of iron transport systems in Enterobacteriaceae in response to oxygen and iron availability. Journal of Inorganic Biochemistry, 133: 110–117, https://doi.org/10.1016/j.**orgbio.2014.01.007.
Chatterjee, D., and Chaudhuri, K., 2011. Association of cholera toxin with Vibrio cholerae outer membrane vesicles which are internalized by human intestinal epithelial cells. FEBS Letters, 585 (9): 1357–1362, https://doi.org/10.1016/j.febslet.2011.04.017.
Choi, D. S., Kim, D. K., Choi, S. J, Lee, J., Choi, J. P., Rho, S., et al., 2011. Proteomic analysis of outer membrane vesicles derived from Pseudomonas aeruginosa. Proteomics, 11 (16): 3424–3429, https://doi.org/10.1002/pmic.201000212.
Ciofu, O., Beveridge, T. J., Kadurugamuwa, J., Walther-Rasmussen, J., and Høiby, N., 2000. Chromosomal β-lactamase is packaged into membrane vesicles and secreted from Pseudomonas aeruginosa. Journal of Antimicrobial Chemotherapy, 45 (1): 9–13, https://doi.org/10.1093/jac/45.1.9.
Dai, F., Zhuang, Q., Zhao, X., Shao, Y., Guo, M., Lv, Z., et al., 2020. Green fluorescent protein-tagged Vibrio splendidus for monitoring bacterial infection in the sea cucumber Apostichopus japonicus. Aquaculture, 523: 735169, https://doi.org/10.1016/j.aquaculture.2020.735169.
Deatherage, B. L., and Cookson, B. T., 2012. Membrane vesicle release in bacteria, eukaryotes, and archaea: A conserved yet underappreciated aspect of microbial life. Infection and Immunity, 80 (6): 1948–1957, https://doi.org/10.1128/IAI.06014-11.
Dell’Annunziata, F., Dell’Aversana, C., Doti, N., Donadio, G., Dal Piaz, F., Izzo, V., et al., 2021. Outer membrane vesicles derived from Klebsiella pneumoniae are a driving force for horizontal gene transfer. International Journal of Molecular Sciences, 22 (16): 8732, https://doi.org/10.3390/ijms22168732.
Dhital, S., Deo, P., Stuart, I., and Naderer, T., 2021. Bacterial outer membrane vesicles and host cell death signaling. Trends in Microbiology, 29 (12): 1106–1116, https://doi.org/10.1016/j.tim.2021.04.003.
Dhurve, G., Madikonda, A. K., Jagannadham, M. V., and Siddavattam, D., 2022. Outer membrane vesicles of Acinetobacter baumannii DS002 are selectively enriched with TonB-dependent transporters and play a key role in iron acquisition. Microbiology Spectrum, 10 (2): e00293–22, https://doi.org/10.1128/spectrum.00293-22.
Duperthuy, M., Binesse, J., Le Roux, F., Romestand, B., Caro, A., Got, P., et al., 2010. The major outer membrane protein OmpU of Vibrio splendidus contributes to host antimicrobial peptide resistance and is required for virulence in the oyster Crassostrea gigas. Environmental Microbiology, 12 (4): 951–963, https://doi.org/10.1111/j.1462-2920.2009.02138.x.
Ellis, T. N., and Kuehn, M. J., 2010. Virulence and immunomodulatory roles of bacterial outer membrane vesicles. Microbiology and Molecular Biology Reviews, 74 (1): 81–94, https://doi.org/10.1128/MMBR.00031-09.
Fulsundar, S., Kulkarni, H. M., Jagannadham, M. V., Nair, R., Keerthi, S., Sant, P., et al., 2015. Molecular characterization of outer membrane vesicles released from Acinetobacter radioresistens and their potential roles in pathogenesis. Microbial Pathogenesis, 83: 12–22, https://doi.org/10.1016/j.micpath.2015.04.005.
Furuyama, N., and Sircili, M. P., 2021. Outer membrane vesicles (OMVs) produced by gram-negative bacteria: Structure, functions, biogenesis, and vaccine application. BioMed Research International, 2021: 1490732, https://doi.org/10.1155/2021/1490732.
Gao, Q., Liao, M., Wang, Y., Li, B., Zhang, Z., Rong, X., et al., 2015. Transcriptome analysis and discovery of genes involved in immune pathways from coelomocytes of sea cucumber (Apostichopus japonicus) after Vibrio splendidus challenge. International Journal of Molecular Sciences, 16 (7): 16347–16377, https://doi.org/10.3390/ijms160716347.
Gould, S. B., Garg, S. G., and Martin, W. F., 2016. Bacterial vesicle secretion and the evolutionary origin of the eukaryotic endomembrane system. Trends in Microbiology, 24 (7): 525–534, https://doi.org/10.1016/j.tim.2016.03.005.
Guerrero-Mandujano, A., Hernández-Cortez, C., Ibarra, J. A., and Castro-Escarpulli, G., 2017. The outer membrane vesicles: Secretion system type zero. Traffic, 18 (7): 425–432, https://doi.org/10.1111/tra.12488.
Habier, J., May, P., Heintz-Buschart, A., Ghosal, A., Wienecke-Baldacchino, A. K., Wilmes, P., et al., 2018. Extraction and analysis of RNA isolated from pure bacteria-derived outer membrane vesicles. Methods in Molecular Biology, 1737: 213–230, https://doi.org/10.1007/978-1-4939-7634-8_13.
Horstman, A. L., and Kuehn, M. J., 2000. Enterotoxigenic Escherichia coli secretes active heat-labile enterotoxin via outer membrane vesicles. Journal of Biological Chemistry, 275 (17): 12489–12496, https://doi.org/10.1074/jbc.275.17.12489.
Jan, A. T., 2017. Outer membrane vesicles (OMVs) of gram-negative bacteria: A perspective update. Frontiers in Microbiology, 8: 1053, https://doi.org/10.3389/fmicb.2017.01053.
Jude, B. A., Martinez, R. M., Skorupski, K., and Taylor, R. K., 2009. Levels of the secreted Vibrio cholerae attachment factor GbpA are modulated by quorum-sensing-induced proteolysis. Journal of Bacteriology, 191 (22): 6911, https://doi.org/10.1128/JB.00747-09.
Kadurugamuwa, J. L., and Beveridge, T. J., 1997. Natural release of virulence factors in membrane vesicles by Pseudomonas aeruginosa and the effect of aminoglycoside antibiotics on their release. Journal of Antimicrobial Chemotherapy, 40 (5): 615–621, https://doi.org/10.1093/jac/40.5.615.
Kang, G., Choi, K. M., Joo, M. S., Woo, W. S., and Park, C. I., 2022. A case report of secondary infection by Vibrio splendidus associated with gas bubble disease in syngnathid fishes (Syngnathus schlegeli and Hippocampus haema). Fisheries and Aquatic Sciences, 25 (1): 40–48, https://doi.org/10.47853/FAS.2022.e5.
Kim, J. H., Lee, J., Park, J., and Gho, Y. S., 2015. Gram-negative and Gram-positive bacterial extracellular vesicles. Seminars in Cell & Developmental Biology, 40: 97–104, https://doi.org/10.1016/j.semcdb.2015.02.006.
Klimentová, J., and Stulík, J., 2015. Methods of isolation and purification of outer membrane vesicles from gram-negative bacteria. Microbiological Research, 170: 1–9, https://doi.org/10.1016/j.micres.2014.09.006.
Kramer, J., Özkaya, Ö., and Kümmerli, R., 2020. Bacterial side-rophores in community and host interactions. Nature Reviews Microbiology, 18 (3): 152–163, https://doi.org/10.1038/s41579-019-0284-4.
Kulp, A., and Kuehn, M. J., 2010. Biological functions and biogenesis of secreted bacterial outer membrane vesicles. Annual Review of Microbiology, 64: 163–184, https://doi.org/10.1146/annurev.micro.091208.073413.
Lee, H. J., Kim, J. A., Lee, M. A., Park, S. J., and Lee, K. H., 2013. Regulation of haemolysin (VvhA) production by ferric uptake regulator (Fur) in Vibrio vulnificus: Repression of vvhA transcription by Fur and proteolysis of VvhA by Fur-repressive exoproteases. Molecular Microbiology, 88 (4): 813–826, https://doi.org/10.1111/mmi.12224.
Liang, W. K., Zhang, C., Liu, N. N., Zhang, W. W., Han, Q. X., and Li, C. H., 2016. Cloning and characterization of Vshppd, a gene inducing haemolysis and immune response of Apostichopus japonicus. Aquaculture, 464 (1): 246–252, https://doi.org/10.1016/j.aquaculture.2016.06.039.
Liu, Q., Tian, H., Li, B., Chen, Y., and Huang, X., 2021. Outer membrane vesicles derived from Salmonella typhimurium can deliver shigella flexneri 2a o-polysaccharide antigen to prevent lethal pulmonary infection in mice. Applied and Environmental Microbiology, 87 (19): AEM0096821, https://doi.org/10.1101/2021.02.01.429291.
Ma, J., Chen, T., Wu, S., Yang, C., Bai, M., Shu, K., et al., 2019. iProX: An integrated proteome resource. Nucleic Acids Research, 47: D1211–D1217, https://doi.org/10.1093/nar/gky869.
Manabe, T., Kato, M., Ueno, T., and Kawasaki, K., 2013. Flagella proteins contribute to the production of outer membrane vesicles from Escherichia coli W3110. Biochemical and Biophysical Research Communications, 441 (1): 151–156, https://doi.org/10.1016/j.bbrc.2013.10.022.
Méndez, J., Reimundo, P., Pérez-Pascual, D., Navais, R., Gómez, E., Cascales, D., et al., 2012. An overview of virulence-associated factors of gram-negative fish pathogenic bacteria. Health and Environment in Aquaculture, 5: 133–156.
Mollah, M. M., Jamal, R., Mokhtar, N. M., Harun, R., and Mollah, M. N., 2015. A hybrid one-way ANOVA approach for the robust and efficient estimation of differential gene expression with multiple patterns. PLoS One, 10 (9): e0138810, https://doi.org/10.1371/journal.pone.0138810.
Muñoz-Atienza, E., Araújo, C., Magadán, S., Hernández, P. E., Herranz, C., Santos, Y., et al., 2014. In vitro and in vivo evaluation of lactic acid bacteria of aquatic origin as probiotics for turbot (Scophthalmus maximus L.) farming. Fish and Shellfish Immunogy, 41 (2): 570–580, https://doi.org/10.1016/j.fsi.2014.10.007.
Richter, M. F., and Hergenrother, P. J., 2019. The challenge of converting Gram-positive-only compounds into broad-spectrum antibiotics. Annals of the New York Academy of Sciences, 1435 (1): 18–38, https://doi.org/10.1111/nyas.13598.
Sartorio, M. G., Pardue, E. J., Feldman, M. F., and Haurat, M. F., 2021. Bacterial outer membrane vesicles: From discovery to applications. Annual Review of Microbiology, 75 (1): 609–630, https://doi.org/10.1146/annurev-micro-052821-031444.
Schild, S., Nelson, E. J., and Camilli, A., 2008. Immunization with Vibrio cholerae outer membrane vesicles induces protective immunity in mice. Infection and Immunity, 76 (10): 4554–4563, https://doi.org/10.1128/IAI.00532-08.
Schwechheimer, C., and Kuehn, M. J., 2015. Outer-membrane vesicles from Gram-negative bacteria: Biogenesis and functions. Nature Reviews Microbiology, 13 (10): 605–619, https://doi.org/10.1038/nrmicro3525.
Song, T., Liu, H., Lv, T., Zhao, X., Shao, Y., Han, Q., et al., 2018. Characteristics of the iron uptake-related process of a pathogenic Vibrio splendidus strain associated with massive mortalities of the sea cucumber Apostichopus japonicus. Journal of Invertebrate Pathology, 155: 25–31, https://doi.org/10.1016/j.jip.2018.05.001.
Toledo, A., Coleman, J. L., Kuhlow, C. J., Crowley, J. T., and Benach, J. L., 2012. The enolase of Borrelia burgdorferi is a plasminogen receptor released in outer membrane vesicles. Infection and Immunity, 80 (1): 359–368, https://doi.org/10.1128/IAI.05836-11.
Torresi, M., Acciari, V. A., Piano, A., Serratore, P., and Migliorati, G., 2011. Detection of Vibrio splendidus and related species in Chamelea gallina sampled in the adriatic along the abruzzi coastline. Eterinaria Italiana, 47 (3): 371–378, 363–370, https://doi.org/10.1017/S1751731111000085.
Toyofuku, M., Nomuram, N., and Eberl, L., 2019. Types and origins of bacterial membrane vesicles. Nature Reviews in Microbiology, 17 (1): 13–24, https://doi.org/10.1038/s41579-018-0112-2.
Vanhove, A. S., Duperthuy, M., Charrière, G. M., Le Roux, F., Goudenège, D., Gourbal, B., et al., 2015. Outer membrane vesicles are vehicles for the delivery of Vibrio tasmaniensis virulence factors to oyster immune cells. Environmental Microbiology, 17 (4): 1152–1165, https://doi.org/10.1111/1462-2920.12535Citations.
Wai, S. N., Lindmark, B., Söderblom, T., Takade, A., Westermark, M., Oscarsson, J., et al., 2003. Vesicle-mediated export and assembly of pore-forming oligomers of the enterobacterial ClyA cytotoxin. Cell, 115 (1): 25–35, https://doi.org/10.1016/S0092-8674(03)00754-2.
Yu, C. S., Cheng, C. W., Su, W. C., Chang, K. C., Huang, S. W., Hwang, J. K., et al., 2014. CELLO2GO: A web server for protein subcellular localization prediction with functional gene ontology annotation. PLoS One, 9 (6): e99368, https://doi.org/10.1371/journal.pone.0099368.
Yun, S. H., Lee, S. Y., Choi, C. W., Lee, H., Ro, H. J., Jun, S., et al., 2017. Proteomic characterization of the outer membrane vesicle of the halophilic marine bacterium Novosphingobium pentaromativorans US6-1. Journal of Microbiology, 55 (1): 56–62, https://doi.org/10.1007/s12275-017-6581-6.
Zhang, C., Liang, W., Zhang, W., and Li, C., 2016. Characterization of a metalloprotease involved in Vibrio splendidus infection in the sea cucumber, Apostichopus japonicus. Microbial Pathogenesis, 101: 96–103, https://doi.org/10.1016/j.micpath.2016.11.005.
Zhang, K., Chu, P., Song, S., Yang, D., Bian, Z., Li, Y., et al., 2021. Proteome analysis of outer membrane vesicles from a highly virulent strain of Haemophilus parasuis. Frontiers in Veterinary Science, 8: 756764, https://doi.org/10.3389/fvets.2021.756764.
Zhang, W., and Li, C., 2021. Virulence mechanisms of Vibrios belonging to the splendidus clade as aquaculture pathogens, from case studies and genome data. Reviews in Aquaculture, 13 (4): 2004–2026, https://doi.org/10.1111/raq.12555.
Zhang, W., Liang, W., and Li, C., 2016. Inhibition of marine Vibrio sp. by pyoverdine from Pseudomonas aeruginosa PA1. Journal of Hazardous Materials, 302: 217–224, https://doi.org/10.1016/j.jhazmat.2015.10.003.
Zhuang, Q., Dai, F., Shao, Y., Li, C., and Zhang, W., 2021. vscN encodes a type III secretion system ATPase in Vibrio splendidus AJ01 that contributes to pathogenicity and Hop secretion. Aquaculture, 533: 736228, https://doi.org/10.1016/j.aquaculture.2020.736228.
Acknowledgements
This work was supported by the Zhejiang Provincial Natural Science Foundation for Distinguished Young Scholars (No. LR20C190001), the National Natural Science Foundation 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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Song, H., Ruan, Y., Li, Y. et al. Proteomic and Functional Analyses of Outer Membrane Vesicles Secreted by Vibrio splendidus. J. Ocean Univ. China 22, 1361–1369 (2023). https://doi.org/10.1007/s11802-023-5481-0
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DOI: https://doi.org/10.1007/s11802-023-5481-0