Skip to main content
Log in

Omp16, a conserved peptidoglycan-associated lipoprotein, is involved in Brucella virulence in vitro

  • Microbial Pathogenesis and Host-Microbe Interaction
  • Published:
Journal of Microbiology Aims and scope Submit manuscript

Abstract

Brucella, the bacterial agent of common zoonotic brucellosis, primarily infects specific animal species. The Brucella outer membrane proteins (Omps) are particularly attractive for developing vaccine and improving diagnostic tests and are associated with the virulence of smooth Brucella strains. Omp16 is a homologue to peptidoglycan-associated lipoproteins (Pals), and an omp16 mutant has not been generated in any Brucella strain until now. Very little is known about the functions and pathogenic mechanisms of Omp16 in Brucella. Here, we confirmed that Omp16 has a conserved Pal domain and is highly conserved in Brucella. We attempted to delete omp16 in Brucella suis vaccine strain 2 (B. suis S2) without success, which shows that Omp16 is vital for Brucella survival. We acquired a B. suis S2 Omp16 mutant via conditional complementation. Omp16 deficiency impaired Brucella outer membrane integrity and activity in vitro. Moreover, inactivation of Omp16 decreased bacterial intracellular survival in macrophage RAW 264.7 cells. B. suis S2 and its derivatives induced marked expression of IL-1β, IL-6, and TNF-a mRNA in Raw 264.7 cells. Whereas inactivation of Omp16 in Brucella enhanced IL-1β and IL-6 expression in Raw 264.7 cells. Altogether, these findings show that the Brucella Omp16 mutant was obtained via conditional complementation and confirmed that Omp16 can maintain outer membrane integrity and be involved in bacterial virulence in Brucella in vitro and in vivo. These results will be important in uncovering the pathogenic mechanisms of Brucella.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Bowden, R.A., Estein, S.M., Zygmunt, M.S., Dubray, G., and Cloeckaert, A. 2000. Identification of protective outer membrane antigens of Brucella ovis by passive immunization of mice with monoclonal antibodies. Microbes Infect. 2, 481–488.

    PubMed  CAS  Google Scholar 

  • Caro-Hernández, P., Fernández-Lago, L., de Miguel, M.J., Martín-Martín, A.I., Cloeckaert, A., Grilló, M.J., and Vizcaíno, N. 2007. Role of the Omp25/Omp31 family in outer membrane properties and virulence of Brucella ovis. Infect. Immun. 75, 4050–4061.

    PubMed  PubMed Central  Google Scholar 

  • Celli, J. 2015. The changing nature of the Brucella-containing vacuole. Cell. Microbiol. 17, 951–958.

    PubMed  PubMed Central  CAS  Google Scholar 

  • Cloeckaert, A., Tibor, A., and Zygmunt, M.S. 1999. Brucella outer membrane lipoproteins share antigenic determinants with bacteria of the family Rhizobiaceae. Clin. Diagn. Lab. Immunol. 6, 627–629.

    PubMed  PubMed Central  CAS  Google Scholar 

  • de Figueiredo, P., Ficht, T.A., Rice-Ficht, A., Rossetti, C.A., and Adams, L.G. 2015. Pathogenesis and immunobiology of Brucellosis. Review of Brucella-host interactions. Am. J. Pathol. 185, 1505–1517.

    PubMed  PubMed Central  CAS  Google Scholar 

  • Dorneles, E.M.S., Sriranganathan, N., and Lage, A.P. 2015. Recent advances in Brucella abortus vaccines. Vet. Res. 46, 76.

    PubMed  PubMed Central  Google Scholar 

  • Edmonds, M.D., Cloeckaert, A., and Elzer, P.H. 2002. Brucella species lacking the major outer membrane protein Omp25 are attenuated in mice and protect against Brucella melitensis and Brucella ovis. Vet. Microbiol. 88, 205–221.

    PubMed  CAS  Google Scholar 

  • Elfaki, M.G., Alaidan, A.A., and Al-Hokail, A.A. 2015. Host response to Brucella infection: review and future perspective. J. Infect. Dev. Ctries 9, 697–701.

    PubMed  Google Scholar 

  • Feng, Y., Peng, X.W., Jiang, H., Peng, Y., Zhu, L., and Ding, J. 2017. Rough Brucella strain RM57 is attenuated and confers protection against Brucella melitensis. Microb. Pathog. 107, 270–275.

    PubMed  CAS  Google Scholar 

  • Giambartolomei, G.H., Zwerdling, A., Cassataro, J., Bruno, L., Fossati, C.A., and Philipp, M.T. 2004. Lipoproteins, not lipopolysaccharide, are the key mediators of the proinflammatory response elicited by heat-killed Brucella abortus. J. Immunol. 173, 4635–4642.

    PubMed  CAS  Google Scholar 

  • Hielpos, M.S., Fernández, A.G., Falivene, J., Paiva, I.M.A., González, F.M., Ferrero, M.C., Campos, P.C., Vieira, A.T., Oliveira, S.C., and Baldi, P.C. 2018. IL-1R and inflammasomes mediate early pulmonary protective mechanisms in respiratory Brucella abortus infection. Front. Cell. Infect. Microbiol. 8, 391.

    PubMed  PubMed Central  CAS  Google Scholar 

  • Hirakawa, H., Suzue, K., Kurabayashi, K., and Tomita, H. 2019. The Tol-Pal system of uropathogenic Escherichia coli is responsible for optimal internalization into and aggregation within bladder epithelial cells, colonization of the urinary tract of mice, and bacterial motility. Front. Microbiol. 10, 1827.

    PubMed  PubMed Central  Google Scholar 

  • Hop, H.T., Huy, T.X.N., Reyes, A.W.B., Arayan, L.T., Vu, S.H., Min, W.G., Lee, H.J., Kang, C.K., Kim, D.H., Tark, D.S., et al. 2019a. Interleukin 6 promotes Brucella abortus clearance by controlling bactericidal activity of macrophages and CD8+ T cell differentiation. Infect. Immun. 87, e00431–19.

    PubMed  PubMed Central  CAS  Google Scholar 

  • Hop, H.T., Reyes, A.W.B., Arayan, L.T., Huy, T.X.N., Vu, S.H., Min, W., Lee, H.J., Kang, C.K., Rhee, M.H., and Kim, S. 2019b. Interleukin 1 alpha (IL-1α) restricts Brucella abortus 544 survival through promoting lysosomal-mediated killing and NO production in macrophages. Vet. Microbiol. 232, 128–136.

    PubMed  CAS  Google Scholar 

  • Hunter, C.A. and Jones, S.A. 2015. IL-6 as a keystone cytokine in health and disease. Nat. Immunol. 16, 448–457.

    PubMed  CAS  Google Scholar 

  • Im, Y.B., Park, W.B., Jung, M., Kim, S., and Yoo, H.S. 2018. Comparative analysis of immune responses to outer membrane antigens OMP10, OMP19, and OMP28 of Brucella abortus. Jpn. J. Infect. Dis. 71, 197–204.

    PubMed  CAS  Google Scholar 

  • Kaushik, P., Singh, D.K., Kumar, S.V., Tiwari, A.K., Shukla, G., Dayal, S., and Chaudhuri, P. 2010. Protection of mice against Brucella abortus 544 challenge by vaccination with recombinant OMP28 adjuvanted with CpG oligonucleotides. Vet. Res. Commun. 34, 119–132.

    PubMed  Google Scholar 

  • Kovach, M.E., Phillips, R.W., Elzer, P.H., Roop, R.M.2nd, and Peterson, K.M. 1994. pBBR1MCS: a broad-host-range cloning vector. BioTechniques 16, 800–802.

    PubMed  CAS  Google Scholar 

  • Lalsiamthara, J. and Lee, J.H. 2017. Development and trial of vaccines against Brucella. J. Vet. Sci. 18, 281–290.

    PubMed  PubMed Central  Google Scholar 

  • Lloubes, R., Cascales, E., Walburger, A., Bouveret, E., Lazdunski, C., Bernadac, A., and Journet, L. 2001. The Tol-Pal proteins of the Escherichia coli cell envelope: an energized system required for outer membrane integrity? Res. Microbiol. 152, 523–529.

    PubMed  CAS  Google Scholar 

  • Luo, D., Ni, B., Li, P., Shi, W., Zhang, S., Han, Y., Mao, L., He, Y., Wu, Y., and Wang, X. 2006. Protective immunity elicited by a divalent DNA vaccine encoding both the L7/L12 and Omp16 genes of Brucella abortus in BALB/c mice. Infect. Immun. 74, 2734–2741.

    PubMed  PubMed Central  CAS  Google Scholar 

  • Luo, X., Zhang, X., Wu, X., Yang, X., Han, C., Wang, Z., Du, Q., Zhao, X., Liu, S.L., Tong, D., et al. 2017. Brucella downregulates tumor necrosis factor-alpha to promote intracellular survival via Omp25 regulation of different microRNAs in porcine and murine macrophages. Front. Immunol. 8, 2013.

    PubMed  Google Scholar 

  • Moriyón, I. and López-Goni, I. 1998. Structure and properties of the outer membranes of Brucella abortus and Brucella melitensis. Int. Microbiol. 1, 19–26.

    PubMed  Google Scholar 

  • Mustafa, A.A. and Abusowa, M. 1993. Field-oriented trial of the chinese Brucella suis strain 2 vaccine on sheep and goats in Libya. Vet. Res. 24, 422–429.

    PubMed  CAS  Google Scholar 

  • Olsen, S.C. and Palmer, M.V. 2014. Advancement of knowledge of Brucella over the past 50 years. Vet. Pathol. 51, 1076–1089.

    PubMed  CAS  Google Scholar 

  • Pasquevich, K.A., Estein, S.M., García Samartino, C., Zwerdling, A., Coria, L.M., Barrionuevo, P., Fossati, C.A., Giambartolomei, G.H., and Cassataro, J. 2009. 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, 436–445.

    PubMed  CAS  Google Scholar 

  • Pasquevich, K.A., García Samartino, C., Coria, L.M., Estein, S.M., Zwerdling, A., Ibañez, A.E., Barrionuevo, P., Oliveira, F.S., Carvalho, N.B., Borkowski, J., et al. 2010. The protein moiety of Brucella abortus outer membrane protein 16 is a new bacterial pathogen-associated molecular pattern that activates dendritic cells in vivo, induces a Th1 immune response, and is a promising self-adjuvanting vaccine against systemic and oral acquired brucellosis. J. Immunol. 184, 5200–5212.

    PubMed  CAS  Google Scholar 

  • Roop, R.M. 2nd, Gaines, J.M., Anderson, E.S., Caswell, C.C., and Martin, D.W. 2009. Survival of the fittest: how Brucella strains adapt to their intracellular niche in the host. Med. Microbiol. Immunol. 198, 221–238.

    PubMed  Google Scholar 

  • Sadikaliyeva, S.O., Sultankulova, K.T., Shorayeva, K.A., Strochkov, V.M., Chervyakova, O.V., Zaitsev, V.L., Tabynov, K.K., Sandybayev, N.T., Sansyzbay, A.R., and Egorov, A.Y. 2015. [Genetic stability of the HA, NA, and NS genes of the recombinant vector virus Flu-NS1-124-Omp16 (H5N1) expressing the brucellar gene]. Vopr. Virusol. 60, 18–23.

    PubMed  CAS  Google Scholar 

  • Sancho, P., Tejedor, C., Sidhu-Muñoz, R.S.S., Fernández-Lago, L., and Vizcaíno, N. 2014. Evaluation in mice of Brucella ovis attenuated mutants for use as live vaccines against B. ovis infection. Vet. Res. 45, 61.

    PubMed  PubMed Central  Google Scholar 

  • Seleem, M.N., Boyle, S.A., and Sriranganathan, N. 2008. Brucella: a pathogen without classic virulence genes. Vet. Microbiol. 129, 1–14.

    PubMed  CAS  Google Scholar 

  • Sidhu-Muñoz, R.S., Sancho, P., and Vizcaino, N. 2016. 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.

    PubMed  Google Scholar 

  • Sola-Landa, A., Pizarro-Cerdá, J., Grilló, M.J., Moreno, E., Moriyón, I., Blasco, J.M., Gorvel, J.P., and López-Goñi, I. 1998. A two-component regulatory system playing a critical role in plant pathogens and endosymbionts is present in Brucella abortus and controls cell invasion and virulence. Mol. Microbiol. 29, 125–138.

    PubMed  CAS  Google Scholar 

  • Tabynov, K., Sansyzbay, A., Kydyrbayev, Z., Yespembetov, B., Ryskeldinova, S., Zinina, N., Assanzhanova, N., Sultankulova, K., Sandybayev, N., Khairullin, B., et al. 2014. Influenza viral vectors expressing the Brucella OMP16 or L7/L12 proteins as vaccines against B. abortus infection. Virol. J. 11, 69.

    PubMed  PubMed Central  Google Scholar 

  • Tian, M., Qu, J., Bao, Y., Gao, J., Liu, J., Wang, S., Sun, Y., Ding, C., and Yu, S. 2016. Construction of pTM series plasmids for gene expression in Brucella species. J. Microbiol. Methods 123, 18–23.

    PubMed  CAS  Google Scholar 

  • Utaisincharoen, P., Kespichayawattana, W., Anuntagool, N., Chaisuriya, P., Pichyangkul, S., Krieg, A.M., and Sirisinha, S. 2003. CpG ODN enhances uptake of bacteria by mouse macrophages. Clin. Exp. Immunol. 132, 70–75.

    PubMed  PubMed Central  CAS  Google Scholar 

  • Van der Henst, C., de Barsy, M., Zorreguieta, A., Letesson, J.J., and De Bolle, X. 2013. The Brucella pathogens are polarized bacteria. Microbes Infect. 15, 998–1004.

    PubMed  CAS  Google Scholar 

  • Velásquez, L.N., Milillo, M.A., Delpino, M.V., Trotta, A., Fernández, P., Pozner, R.G., Lang, R., Balboa, L., Giambartolomei, G.H., and Barrionuevo, P. 2017. Brucella abortus down-regulates MHC class II by the IL-6-dependent inhibition of CIITA through the downmodulation of IFN regulatory factor-1 (IRF-1). J. Leukoc. Biol. 101, 759–773.

    PubMed  Google Scholar 

  • Verdiguel-Fernández, L., Oropeza-Navarro, R., Basurto-Alcántara, F.J., Castañeda-Ramírez, A., and Verdugo-Rodríguez, A. 2017. Omp31 plays an important role on outer membrane properties and intracellular survival of Brucella melitensis in murine macrophages and HeLa cells. Arch. Microbiol. 199, 971–978.

    PubMed  Google Scholar 

  • von Bargen, K., Gorvel, J.P., and Salcedo, S.P. 2012. Internal affairs: investigating the Brucella intracellular lifestyle. FEMS Microbiol. Rev. 36, 533–562.

    PubMed  CAS  Google Scholar 

  • Walburger, A., Lazdunski, C., and Corda, Y. 2002. The Tol/Pal system function requires an interaction between the C-terminal domain of TolA and the N-terminal domain of TolB. Mol. Microbiol. 44, 695–708.

    PubMed  CAS  Google Scholar 

  • Wang, Y.F., Chen, Z.L., Qiao, F., Zhong, Z.J., Xu, J., Wang, Z.J., Du, X.Y., Qu, Q., Yuan, J., Jia, L.L., et al. 2010. The type IV secretion system affects the expression of Omp25/Omp31 and the outer membrane properties of Brucella melitensis. FEMS Microbiol. Lett. 303, 92–100.

    PubMed  CAS  Google Scholar 

  • Wang, X., Lin, P., Li, Y., Xiang, C., Yin, Y., Chen, Z., Du, Y., Zhou, D., Jin, Y., and Wang, A. 2016. Brucella suis vaccine strain 2 induces endoplasmic reticulum stress that affects intracellular replication in goat trophoblast cells in vitro. Front. Cell. Infect. Microbiol. 6, 19.

    PubMed  PubMed Central  Google Scholar 

  • Zhi, F., Zhou, D., Bai, F., Li, J., Xiang, C., Zhang, G., Jin, Y., and Wang, A. 2019. VceC mediated IRE1 pathway and inhibited CHOP-induced apoptosis to support Brucella replication in goat trophoblast cells. Int. J. Mol. Sci. 20, 4104.

    PubMed Central  CAS  Google Scholar 

Download references

Acknowledgments

This research was funded by the National Key R&D Program of China (2018YFD0500900); the National Natural Science Foundation of China (31672584, 31702310).

Author information

Authors and Affiliations

Authors

Corresponding authors

Correspondence to Yaping Jin or Aihua Wang.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhi, F., Zhou, D., Li, J. et al. Omp16, a conserved peptidoglycan-associated lipoprotein, is involved in Brucella virulence in vitro. J Microbiol. 58, 793–804 (2020). https://doi.org/10.1007/s12275-020-0144-y

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12275-020-0144-y

Keywords

Navigation