Chemotherapeutic treatment of tuberculosis is a prolonged process due to the innate resistance of the bacterium to the drugs available, their relatively low tissue concentration and the limited number of targets. These parameters lead with alarming frequency to the development of single and multi-drug resistant strains. Initiatives towards identification of new targets are needed badly and this current proposal describes an integrated program to facilitate a more global analysis of metabolic pathways active under conditions relevant for infection. The experiments detail microarray analysis of bacterial RNA, coupled with a novel mutant screen to identify regulator elements responsible for mobilizing metabolic networks.
The specific aims are: 1) Cataloging bacterial metabolism under specific growth conditions: Bacteria will be cultivated in a fermentor to generate a homogeneous population grown under defined, tightly-regulated, physiological conditions relevant to the intracellular environment (varying 02 tension, pH, carbon source & element concentration). Patterns of gene expression will be documented by microarray analysis to identify genes up-regulated under relevant experimental conditions. 2) The intracellular validation of potential targets: The role of """"""""target"""""""" genes will be probed through analysis of (i) intracellular expression profiles using green fluorescent protein (GFP) reporter constructs and real-time PCR and (ii) deletion of the gene and analysis of the growth characteristics in culture, and in macrophages. 3) Screening for sensor/regulator cascades operational under specific growth conditions: Once """"""""target"""""""" genes have been identified we will identify the sensor/regulator pathways active in regulation of gene function using the GFP reporter constructs transfected into transposon-mutagenized bacteria and select for aberrant expressors using a FACScaliber sorter.
