Differential composition of culture supernatants from wild-type Brucella abortus and its isogenic virB mutants

DELPINO, M. V.; DIEGO JOSE COMERCI; WAGNER, M. A.; ESCHENBRENNER, M.; MUJER, C. V.; UGALDE, R. A.; FOSSATI, C. A.; BALDI, P. C.; DELVECCHIO, V. G.

ARCHIVES OF MICROBIOLOGY

Springer

Lugar: HEedelberg; Año: 2009 vol. 191 p. 571 - 581

0302-8933

The virB genes coding type IV secretion system are necessary for the intracellular survival and replication of Brucella spp. In this study, extracellular proteins from B. abortus 2308 (wild type, WT) and its isogenic virB10 polar mutant were compared. Culture supernatants harvested in the early stationary phase were concentrated and subjected to 2D electrophoresis. Spots present in the WT strain but absent in the virB10 mutant (diVerential spots) were considered extracellular proteins released in a virB-related manner, and were identiWed by MALDI-TOF analysis and matching with Brucella genomes. Among the 11 diferential proteins identified, DnaK chaperone (Hsp70), choloylglycine hydrolase (CGH) and a peptidyl-prolyl cis–trans isomerase (PPIase) were chosen for further investigation because of their homology with extracellular and/or virulence factors from other bacteria. The three proteins were obtained in recombinant form and specific monoclonal antibodies (mAbs) were prepared. By Western blot with these mAbs, the three proteins were detected in supernatants from the WT but not in those from the virB10 polar mutant or from strains carrying non-polar mutations in virB10 or virB11 genes. These results suggest that the expression of virB genes aVects the extracellular release of DnaK, PPIase and CGH, and possibly other proteins from B. abortusvirB genes coding type IV secretion system are necessary for the intracellular survival and replication of Brucella spp. In this study, extracellular proteins from B. abortus 2308 (wild type, WT) and its isogenic virB10 polar mutant were compared. Culture supernatants harvested in the early stationary phase were concentrated and subjected to 2D electrophoresis. Spots present in the WT strain but absent in the virB10 mutant (diVerential spots) were considered extracellular proteins released in a virB-related manner, and were identiWed by MALDI-TOF analysis and matching with Brucella genomes. Among the 11 diferential proteins identified, DnaK chaperone (Hsp70), choloylglycine hydrolase (CGH) and a peptidyl-prolyl cis–trans isomerase (PPIase) were chosen for further investigation because of their homology with extracellular and/or virulence factors from other bacteria. The three proteins were obtained in recombinant form and specific monoclonal antibodies (mAbs) were prepared. By Western blot with these mAbs, the three proteins were detected in supernatants from the WT but not in those from the virB10 polar mutant or from strains carrying non-polar mutations in virB10 or virB11 genes. These results suggest that the expression of virB genes aVects the extracellular release of DnaK, PPIase and CGH, and possibly other proteins from B. abortus