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Omp31 plays an important role on outer membrane properties and intracellular survival of Brucella melitensis in murine macrophages and HeLa cells

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Abstract

Brucellosis is an infectious disease that affects practically all species of mammals, including human, and is a major zoonosis worldwide. Brucella spp. are facultative intracellular pathogens that have the ability to survive and multiply in phagocytic and nonphagocytic cells such as trophoblast and epithelial cells. Among the six recognized species of the genus Brucella, Brucella melitensis is the main etiological agent involved in goat brucellosis and is also the most pathogenic for human. It causes significant losses in livestock production as a result of abortions, metritis, infertility, and birth of weak animals. Outer membrane proteins (OMPs) are exposed on the bacterial surface and are in contact with cells and effectors of the host immune response, whereby they could be important virulence factors of Brucella species. To evaluate this hypothesis, the gene encoding for the major outer membrane protein Omp31 was amplified, cloned into pUC18 plasmid, and inactivated by inserting a kanamycin cassette, rendering pLVM31 plasmid which was transformed into B. melitensis wild-type strain to obtain LVM31 mutant strain. The Outer membrane (OM) properties of the mutant strain were compared with B. melitensis Bm133 wild-type and B. melitensis Rev1 vaccine strains, in assessing its susceptibility to polymyxin B, sodium deoxycholate, and nonimmune serum. The mutant strain was assessed in vitro with survival assays in murine macrophages J774.A1 and HeLa cells. Our results demonstrate that LVM31 mutant is more susceptible to polymyxin B, sodium deoxycholate, and nonimmune serum than control strains; moreover, Omp31 mutation caused a decrease in the internalization and a significant decrease in the intracellular survival compared with the reference strains in both cell lines. These results allow us to conclude that Omp31 is important for maintaining OM integrity, but also it is necessary for bacterial internalization, establishment and development of an optimal replication niche, and essential for survival and intracellular multiplication.

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Acknowledgements

This work was supported by PAPIIT IN212610, PAPIIT IN-221513, and PAPIIT IN-222516, UNAM. The authors acknowledge Beatriz Arellano for her technical support and Mrs. Francisca Muñoz for her administrative support.

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Authors and Affiliations

  1. Laboratorio de Microbiología Molecular, Departamento de Microbiología e Inmunología, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de México, Avenida Universidad 3000, colonia UNAM CU, Coyoacán, C.P 04510, CdMx, Mexico

    L. Verdiguel-Fernández & Antonio Verdugo-Rodríguez

  2. Departamento de Microbiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernvaca, Morelos, Mexico

    R. Oropeza-Navarro

  3. Laboratorio de Vacunología, Departamento de Microbiología e Inmunología, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de México, Coyoacán, CdMx, Mexico

    Francisco J. Basurto-Alcántara

  4. Departamento de Zootecnia, Universidad Autónoma de Chapingo, Texcoco, Mexico

    A. Castañeda-Ramírez

Authors
  1. L. Verdiguel-Fernández
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  2. R. Oropeza-Navarro
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  3. Francisco J. Basurto-Alcántara
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  4. A. Castañeda-Ramírez
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  5. Antonio Verdugo-Rodríguez
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Correspondence to Antonio Verdugo-Rodríguez.

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Communicated by Erko Stackebrandt.

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Verdiguel-Fernández, L., Oropeza-Navarro, R., Basurto-Alcántara, F.J. et al. 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 (2017). https://doi.org/10.1007/s00203-017-1360-7

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  • DOI: https://doi.org/10.1007/s00203-017-1360-7

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