ABSTRACT
A temperature sensitive mutation in the cell division protein FtsZ was used in combination with transcriptional analysis to identify biomarkers for inhibition of septum formation. Crystallography and modeling revealed that the glycine for aspartate substitution at amino acid 210 was located in helix 8 of the protein, adjacent to the T7 synergy loop. To verify the molecular behavior of FtsZ D210G, the in vitro activity and structural stability were evaluated as a function of temperature. These analyses confirmed that the FtsZ D210G mutant had reduced GTPase and polymerization activity compared to wild-type FtsZ, and CD spectroscopy demonstrated that both FtsZ D210G and wild-type FtsZ had similar structure and stability. Significantly, the FtsZ D210G merodiploid strain of M. tuberculosis had compromised growth at 37 degrees C, substantiating the suitability of FtsZ D210G as a molecular tool for global analysis in response to improper FtsZ polymerization and septum inhibition. Advanced model-based bioinformatics and transcriptional mapping were used to identify high-content multiple features that provide biomarkers for the development of a rational drug screening platform for discovering novel chemotherapeutics that target cell division.
Subject(s)
Bacterial Proteins/biosynthesis , Cytoskeletal Proteins/biosynthesis , GTP Phosphohydrolases/biosynthesis , Mycobacterium tuberculosis/genetics , Bacterial Proteins/chemistry , Cell Cycle , Cell Division , Circular Dichroism/methods , Crystallography, X-Ray/methods , Cytoskeletal Proteins/chemistry , Gene Expression Regulation, Bacterial , Humans , Mutagenesis, Site-Directed , Mycobacterium tuberculosis/physiology , Transcription, GeneticABSTRACT
Screening of 120 taxanes identified a number of compounds that exhibited significant antituberculosis activity. Rational optimization of selected compounds led to the discovery that the C-seco-taxane-multidrug-resistance (MDR) reversal agents (C-seco-TRAs) are noncytotoxic at the upper limit of solubility and detection (>80 microM), while maintaining MIC(99) values of 1.25-2.5 microM against drug-resistant and drug-sensitive strains of Mycobacterium tuberculosis (MTB). Treatment of MTB cells with TRA 3aa and 10a at the MIC caused filamentation and prolongation of the cells, a phenotypic response to FtsZ inactivation.
