ALBERT

Description: Background Analysis of the pathogen interactome is a powerful approach for dissecting potential signal transduction and virulence pathways. It also offers opportunities for exploring new drug targets. Results In this study, a protein-protein interaction (PPI) network of Mycobacterium tuberculosis H37Rv was constructed using a homogenous protein mapping method, which has shown molecular chaperones, ribosomal proteins and ABC transporters to be highly interconnected proteins. A further analysis of this network unraveled the function of hypothetical proteins as well as a potential signaling pathway. A hypothetical protein, Rv2752c, which was linked to a metal cation-transporting ATPase, was characterized as a metal-beta-lactamase, through domain analysis in combination with an in vitro activity experiment. A second hypothetical protein, Rv1354c, and an unknown protein kinase, PknK, interacted with a similar group of inner membrane-associated ABC transporters in the PPI network. The interactions of Rv1354 with these proteins were also confirmed by a further bacterial two-hybrid analysis. According to protein domain structures, the unique M. tuberculosis Rv1354c gene was proposed, for the first time, to be responsible for the turnover of cyclic-di-GMP, a second messenger molecule in this bacterium. A further structure-based inhibitors screening for Rv1354c was also performed in silicon. Conclusion We constructed a comprehensive protein-protein interaction network for M. tuberculosis consisting of 738 proteins and 5639 interaction pairs. Our analysis unraveled the function of hypothetical proteins as well as a potential signaling pathway. The group of ABC transporters, PknK, and Rv1354c were proposed to constitute a potential membrane-associated signaling pathway that cooperatively responds to environmental stresses in M. tuberculosis. The study therefore provides valuable clues in exploring new signaling proteins, virulence pathways, and drug targets.

Description: Background The two-component systems of Mycobacterium tuberculosis are apparently required for its growth and resistance in hostile host environments. In such environments, MtrAB has been reported to regulate the expression of the M. tuberculosis replication initiator gene, dnaA. However, the dnaA promoter binding sites and many potential target genes for MtrA have yet to be precisely characterized. Results In this study, a 7 bp sequence motif in the dnaA promoter region was identified for MtrA binding using DNaseI footprinting assays and surface plasmon resonance (SPR) analysis. Approximately 420 target genes potentially regulated by MtrA, including the isoniazid inducible gene iniB, were further characterized from M. tuberculosis and M. smegmatis genomes. When assayed using quantitative real-time PCR (qRT-PCR), many of the target genes demonstrated significant expression changes when the antisense mRNA of the mtrA gene was expressed in M. smegmatis. The recombinant mycobacteria grew in length and were more sensitive to two anti-tuberculosis drugs, isoniazid and streptomycin. Conclusions These findings yield critical information about the regulatory mechanisms of the MtrAB two-component system and its role in the drug resistance of M. smegmatis.