Brucella abortus efp gene is required for internalization in HeLa cells

IANNINO F; UGALDE JE; IÑON DE IANNINO N

Capital Federal

Congreso; Brucellosis 2011-International Research Conference; 2011

Asociación Argentina de Microbiología

Numerous chromosomal virulence genes (chv) have been shown to play an important role in the ability of Agrobacterium tumefaciens to transform plants. The A. tumefaciens chvH gene encodes a protein similar in sequence to the Escherichia coli elongation factor P (EF-P). In A. tumefaciens this factor is required for tumor formation and for full expression of the vir genes, exerting its activity at a post-transcriptional level. Crosscomplementation assays suggest that the chvH gene and the efp gene of Escherichia coli are functionally homologous. We have cloned and characterized the efp homologue gene in Brucella abortus which has 44% identity to A. tumefaciens chvH and 35% identity to E. coli efp. The gene complemented detergent sensitivity and virulence in the chvH A. tumefaciens mutant, suggesting that both genes are functionally homologous; the growth rate in complex medium also increased to wild type levels. An efp mutant in B. abortus 2308 grew slower in complex media and showed more sensitivity to detergents. Infection assays in J774 macrophage like cells revealed no significant differences between the wild type and the efp mutant strains. The recovery of this mutant from spleens of inoculated mice was equivalent compared to that of the parental strain suggesting that B. abortus efp is not required for virulence in the animal model. However, the efp mutant revealed significant differences at 4 h post-infection in HeLa infection assays compared to the wild type strain, indicating that cellular internalization was affected in non-professional phagocytes. Double immunofluorescence assays for detecting extracellular and intracellular bacteria, demonstrated that the mutant attaches to HeLa cells as the wild type strain but is deficient in the internalization process, thus indicating that efp is involved in the penetration of Brucella in non-professional phagocytes.