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.
Romero
Ibeth C
IC
Centro Internacional de Entrenamiento e Investigaciones Medicas (CIDEIM), Centro Colombiano de Investigacion en Tuberculosis CCITB, Kra. 125 No. 19-225 Pance, Cali, Colombia. ibicris@gmail.com <ibicris@gmail.com>
Mehaffy
Carolina
C
Burchmore
Richard J
RJ
Dobos-Elder
Karen
K
Brennan
Patrick
P
Walker
John
J
D000217:Acyltransferases / Q000235:genetics*; D000942:Antigens, Bacterial / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D002473:Cell Wall / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005784:Gene Amplification / Q000235:genetics*; D006801:Humans; D013058:Mass Spectrometry; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000302:isolation & purification; D011401:Promoter Regions, Genetic; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014376:Tuberculosis / Q000235:genetics* / Q000378:metabolism; D015854:Up-Regulation
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C567945:MarR-family transcriptional regulatory protein, Mycobacterium; D026901:Membrane Transport Proteins; D014157:Transcription Factors; D000217:Acyltransferases; C112580:antigen 85A, Mycobacterium tuberculosis