The aim of this study is to investigate the role of outer membrane protein Omp19 of Brucella in its pathogenesis. In this research, the Brucella2308 ∆Omp19 deletion mutant was constructed and its intracellular survivability was evaluated in murine macrophages RAW264.7 and BALB/c mice. We also analyzed the lysosomal fusion induced by and cytotoxicity of Brucella in murine macrophages RAW264.7. The results showed that the Brucella 2308 ∆Omp19 induced higher levels of immunoprotective cytokines in vitro than Brucella 2308, and the number of intracellular bacteria in RAW264.7 macrophages was lower after Brucella 2308 ∆Omp19 infection than after Brucella 2308 infection. In vivo, Brucella 2308 ∆Omp19 induced protective immune response in mice, and the splenic bacterial load of this deletion mutant was lower than that of Brucella 2308. In addition, the Brucella 2308 ∆Omp19 also promoted the fusion of Brucella-containing vacuoles and lysosomes, in macrophages. Therefore, Brucella 2308 ∆Omp19 reduced virulence compared with Brucella 2308 and activate the immune response of the host, and is a promising candidate for a live attenuated vaccine.
Bu çalışmanın amacı Brucella’nın dış zar proteini Omp19’un bakterinin patogenezindeki rolünü araştırmaktır. Bu araştırmada, Brucella 2308 ∆Omp19 delesyon mutantı oluşturularak faregil makrofajları RAW264.7 ve BALB/c farelerindeki hücre içi canlılığı değerlendirildi. Ayrıca faregil makrofajları RAW264.7’de Brucella’nın neden olduğu lizozomal füzyonu ve sitotoksisite de analiz edildi. Sonuçlar, Brucella 2308 ∆Omp19’un in vitro olarak immünoprotektif sitokin üretimini Brucella 2308’den daha fazla uyardığını ve RAW264.7 makrofajlarındaki hücre içi bakteri sayısının Brucella 2308 ∆Omp19 enfeksiyonunda Brucella 2308 enfeksiyonundan daha düşük olduğunu gösterdi. In vivo olarak, Brucella 2308 ∆Omp19, farelerde koruyucu bağışıklık tepkisine neden oldu ve bu delesyon mutantının dalak bakteri yükü, Brucella 2308’den daha düşüktü. Bunun yanı sıra Brucella 2308 ∆Omp19, makrofajlarda Brucella içeren vakuollerin ve lizozomların füzyonunu da uyardı. Brucella 2308 ∆Omp19, Brucella 2308 ile karşılaştırıldığında düşük virulansla konağın bağışıklık tepkisini aktive etmiştir ve zayıflatılmış canlı aşı için umut verici bir adaydır.
___
1. Głowacka P, Żakowska D, Naylor K, Niemcewicz M, Bielawska-Drózd A: Brucella - Virulence factors, pathogenesis and treatment. Pol J Microbiol, 67 (2): 151-161, 2018. DOI: 10.21307/pjm-2018-029
2. Elzbieta Monika G, Jerzy Z: Brucellosis in humans-etiology, diagnostics, clinical forms. Ann Agric Environ Med, 20 (2): 233-238, 2013.
3. Pascual DW, Yang X, Wang H, Goodwin Z, Hoffman C, Clapp B: Alternative strategies for vaccination to brucellosis. Microbes Infect, 20, 599-605, 2018. DOI: 10.1016/j.micinf.2017.12.006
4. Pasquevich KA, Estein SM, Samartino CG, Zwerdling A, Coria LM, Barrionuevo P, Fossati CA, Giambartolomei GH, Cassataro J: Immunization with recombinant Brucella species outer membrane protein Omp16 or Omp19 in adjuvant induces specific CD4+ and CD8+ T cells as well as systemic and oral protection against Brucella abortus infection. Infect Immun, 77 (1): 436- 445, 2009. DOI: 10.1128/IAI.01151-08
5. Pasquevich KA, Carabajal MV, Guaimas FF, Bruno L, Roset MS, Coria LM, Rey Serantes DAR, Comerci DJ, Cassataro J: Omp19 enables Brucella abortus to evade the antimicrobial activity from host’s proteolytic defense system. Front Immunol, 10:1436, 2019. DOI: 10.3389/fimmu.2019.01436
6. Amils R: Outer Membrane. In, Gargaud M et al. (Eds) Encyclopedia of Astrobiology. Springer Berlin Heidelberg, 2015. DOI: 10.1007/978-3-662-44185- 5_1131
7. Tibor A, Decelle B, Letesson JJ: Outer membrane proteins Omp10, Omp16, and Omp19 of Brucella spp. are lipoproteins. Infect Immun, 67 (9): 4960-4962, 1999.
8. He Z, Luo P, Hu F, Weng Y, Wang W, Li C: Expression and identification of eukaryotic expression vectors of Brucella melitensis lipoprotein OMP19. Chinese J Cell Mol Immunol, 32 (4): 470-473, 2016.
9. Sidhu-Muñoz RS, Sancho P, Vizcaíno N: Brucella ovis PA mutants for outer membrane proteins Omp10, Omp19, SP41, and BepC are not altered in their virulence and outer membrane properties. Vet Microbiol, 186, 59-66, 2016. DOI: 10.1016/j.vetmic.2016.02.010
10. Cloeckaert A, Salih-Alj Debbarh H, Zygmunt MS, Dubray G: Production and characterisation of monoclonal antibodies to Brucella melitensis cytosoluble proteins that are able to differentiate antibody responses of infected sheep from Rev. 1 vaccinated sheep. J Med Microbiol, 45 (3): 206-213, 1996. DOI: 10.1099/00222615-45-3-206
11. Lacerda TLS, Salcedo SP, Gorvel JP: Brucella T4SS: the VIP pass inside host cells. Curr Opin Microbiol, 16 (1): 45-51, 2013. DOI: 10.1016/j.mib.2012.11.005
12.Boschiroli ML, Ouahrani-Bettache S, Foulongne V, Michaux-Charachon S, Bourg G, Allardet-Servent A, Cazevieille C, Lavigne JP, Liautard JP, Ramuz M, O’Callaghan D: Type IV secretion and Brucella virulence. Vet Microbiol, 90 (1-4): 341-348, 2002. DOI: 10.1016/s0378-1135(02)00219-5
13. Marchesini MI, Seijo SMM, Guaimas FF, Comerci DJ: A T4SS effector targets host cell alpha-enolase contributing to Brucella abortus intracellular lifestyle. Front Cell Infect Microbiol, 6:153, 2016. DOI: 10.3389/fcimb.2016.00153
14. Vemulapalli R, Cravero S, Calvert CL, Toth TE, Sriranganathan N, Boyle SM, Rossetti OL, Schurig GG: Characterization of specific immune responses of mice inoculated with recombinant vaccinia virus expressing an 18-kilodalton outer membrane protein of Brucella abortus. Clin Diagn Lab Immunol, 7 (1): 114-118, 2000. DOI: 10.1128/cdli.7.1.114-118.2000
15. Cloeckaert A, Tibor A, Zygmunt MS: Brucella outer membrane lipoproteins share antigenic determinants with bacteria of the family Rhizobiaceae. Clin Diagn Lab Immunol, 6 (4): 627-629, 1999.
16. Caro-Hernandez P, Fernandez-Lago L, de Miguel MJ, Martin-Martin AI, Cloeckaert A, Grillo MJ, Vizcaino N: Role of the Omp25/Omp31 family in outer membrane properties and virulence of Brucella ovis. Infect Immun, 75 (8): 4050-4061, 2007. DOI: 10.1128/IAI.00486-07
17. Pizarro-Cerdá J, Moreno E, Sanguedolce V, Mege JL, Gorvel JP: Virulent Brucella abortus prevents lysosome fusion and is distributed within autophagosome-like compartments. Infect Immun, 66 (5): 2387-2392, 1998.
18. Watarai M, Makino Sİ, Fujii Y, Okamoto K, Shirahata T: Modulation of Brucella-induced macropinocytosis by lipid rafts mediates intracellular replication. Cell Microbiol, 4 (6): 341-355, 2002. DOI: 10.1046/j.1462-5822.2002.00195.x
19. Ma QL, Liu AC, Ma XJ, Wang YB, Hou YT, Wang ZH: Brucella outer membrane protein OMP25 induces microglial cells in vitro to secrete inflammatory cytokines and inhibit apoptosis. Int J Clin Exp Med, 8 (10): 17530-17535, 2015.
20. Vrioni G, Pappas G, Priavali E, Gartzonika C, Levidiotou S: An eternal microbe: Brucella DNA Load persists for years after clinical cure. Clin Infect Dis, 46 (12): e131-e136, 2008. DOI: 10.1086/588482
21. Ottones F, Liautard J, Gross A, Rabenoelina F, Liautard JP, Favero J: Activation of human Vγ9Vδ2 T cells by a Brucella suis non-peptidic fraction impairs bacterial intracellular multiplication in monocytic infected cells. Immunology, 100 (2): 252-258, 2000. DOI: 10.1046/j.1365-2567. 2000.00024.x
22. Colonne PM, Winchell CG, Voth DE: Hijacking host cell highways: Manipulation of the host actin cytoskeleton by obligate intracellular bacterial pathogens. Front Cell Infect Microbiol, 6:107, 2016. DOI: 10.3389/fcimb. 2016.00107
23. Machelart A, Khadrawi A, Demars A, Willemart K, De Trez C, Letesson JJ, Muraille E: Chronic Brucella infection induces selective and persistent Interferon-gamma-dependent alterations of marginal zone macrophages in the spleen. Infect Immun, 85 (11): e00115-17, 2017. DOI: 10.1128/IAI.00115-17
24. Liu Z, Lee J, Krummey S, Lu W, Cai H, Lenardo MJ: The kinase LRRK2 is a regulator of the transcription factor NFAT that modulates the severity of inflammatory bowel disease. Nat Immunol, 12, 1063-1070, 2011. DOI: 10.1038/ni.2113