Identification of promoter-binding proteins of the fbp A and C genes in Mycobacterium tuberculosis

The antigen 85 (Ag85) complex of Mycobacterium tuberculosis represents a promising candidate as a novel drug target and pathogenesis factor. Ag85 comprises three proteins Ag85A, B and C, (encoded by the genes fbpA, B, and C), which participate in cell wall biosynthesis, and interact with the host macrophage as fibronectin-binding proteins (fbps). Ag85 is also involved in the response to isoniazid (INH) treatment. The objective of this study was to identify potential fbp gene activators involved in the over-expression of fbp genes in response to INH.

The biotinylated upstream promoter regions of fbpA and fbpC were used together with streptavidin-coated magnetic beads in DNA-binding assays, to isolate proteins with high-binding affinities from cytosolic extracts of INH-treated M. tuberculosis. Resolution of the DNA-binding proteins by 1D SDS-PAGE revealed 6 proteins with high-affinity for the fbpA promoter, and 7 with specificity the fbpC promoter. Mass spectrometric analyses [LC-ES(MS/MS)] identified proteins associated with drug resistance and stress/treatment responses, intermediary metabolism and cellular division, hypothetical proteins including a member of the MarR family of bacterial transcriptional regulators. The DNA-binding MarR protein shows potential as an authentic activator of fbp genes and functional validation of this factor is warranted.