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Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells
Published on: August 5, 2016
Brucella abortus efp gene is required for an efficient internalization in HeLa cells
Florencia Iannino1, Juan E Ugalde, Nora Iñón de Iannino
1Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de Chascomús (IIB-INTECH), Consejo de Investigaciones Científicas y Técnicas, Universidad Nacional de San Martín (CONICET-UNSAM), Av. Gral. Paz 5445, Buenos Aires, Argentina.
Microbial Pathogenesis
|October 11, 2011
Summary
The Brucella abortus efp gene, homologous to Agrobacterium tumefaciens chvH, is crucial for bacterial internalization into non-professional phagocytes like HeLa cells, but not for virulence in mice.
Area of Science:
- Microbiology
- Bacterial Pathogenesis
- Molecular Biology
Background:
- Chromosomal virulence (chv) genes are vital for Agrobacterium tumefaciens plant transformation.
- The A. tumefaciens chvH gene encodes a protein similar to E. coli elongation factor P (EF-P), essential for tumor formation and vir gene expression.
- Functional homology between chvH and E. coli efp suggests conserved roles.
Purpose of the Study:
- To clone and characterize the efp homolog gene in Brucella abortus.
- To investigate the functional role of the B. abortus efp gene in bacterial processes.
- To determine the involvement of B. abortus efp in virulence and host cell interaction.
Main Methods:
- Cloning and characterization of the B. abortus efp gene.
- Complementation assays using A. tumefaciens chvH mutants.
- Growth rate and detergent sensitivity assays for B. abortus strains.
- Infection assays in J774 macrophage-like cells and HeLa cells.
- Double immunofluorescence assays to assess bacterial internalization.
Main Results:
- The B. abortus efp gene shares homology with A. tumefaciens chvH and E. coli efp.
- B. abortus efp complemented virulence and detergent sensitivity in A. tumefaciens chvH mutants.
- A B. abortus efp mutant exhibited slower growth and increased detergent sensitivity.
- The B. abortus efp mutant showed no significant difference in mouse spleen recovery or J774 cell infection.
- HeLa cell infection assays revealed that the B. abortus efp mutant is deficient in internalization but not attachment.
Conclusions:
- The B. abortus efp gene is functionally homologous to A. tumefaciens chvH.
- B. abortus efp is not essential for virulence in a murine model or for infection of professional phagocytes.
- The B. abortus efp gene plays a critical role in the internalization of Brucella into non-professional phagocytes.
