The long term objectives of this proposal are: (i) To investigate the regulatory systems and processes that govern Mycobacterium tuberculosis (MTB) interactions with the host and control responses of this organism to environmental stimuli and stress conditions relevant to in vivo situations; (ii) to uncover new targets for improved control of MTB based on such regulatory systems; and (iii) to develop alternative or improved means for antimycobacterial drug testing and resistance detection. Adaptation of bacterial pathogens to environmental conditions in the host is based on signal recognition resulting in concerted expression of virulence determinants. Regulation of such processes in itself is a virulence determinant; moreover, uncovering of the regulatory elements and genes that control such systems can help identify and characterize important effectors of virulence. This strategy is applied to MTB with the following specific aims: (i) To complete the characterization of, a newly described MTB gene that belongs to the superfamily of two-component signal transduction systems. Additional signal transduction systems will be investigated. Protein phosphorylation processes and their inhibition in vitro will be examined (ii) To isolate MTB promoters (and, subsequently, structural genes controlled by such promoters) that are responsive to oxidative stress, exposure to reactive nitrogen intermediates, nutrient limitation, and low or high oxygen pressure. (iii) To investigate regulation of genes responsive to oxidative stress and a possible connection with the catalase related resistance to isoniazid. The proposed experiments are based on: (i) Our recent identification and partial characterization of signal transduction elements in MTB; and (ii) construction of novel mycobacterial transcriptional fusion vectors. This analysis will provide new information regarding gene regulation in MTB and communications of this organism with its environment. Such systems in other pathogenic bacteria control important virulence determinants and global physiological functions and are currently being tested as potential targets for novel antimicrobial drugs. The analysis of regulatory genes will be complemented by examining promoters regulated by the studied signal transduction elements in MTB, and by random isolation of MTB promoters responsive to environmental conditions encountered in the host. This will permit identification of the corresponding genes which are likely to encode factors contributing to MTB virulence.
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