Tuberculosis (TB) is a major global health problem causing about two million deaths per year. The length of TB chemotherapy, the increasing spread of multi-drug resistant strains and the current failure to treat persistent infections with Mycobacterium tuberculosis affecting approximately one-third of the human population, have intensified worldwide efforts to find new antitubercular drugs. The search for new drugs is focused on proteins, which are required for survival of M. tuberculosis in vivo and/or in vitro. One goal is to identify new lead compounds using high-throughput screening technologies. However, the major difficulty in TB chemotherapy is not finding new drug targets, but rather the extremely low permeability of the unusual mycobacterial outer membrane (OM) rendering mycobacteria intrinsically resistant against many antibiotics. Three out of four first-line TB drugs are assumed to cross the OM of M. tuberculosis by water-filled channel proteins. These porins are the key proteins for uptake of hydrophilic solutes in mycobacteria. We recently discovered two porins of M. tuberculosis that complement the permeability defects of a porin mutant of M. smegmatis. Both purified recombinant proteins showed channel activity in lipid bilayer experiments. We want to analyze the physiological role of the porin-mediated OM permeability for M. tuberculosis in vitro and in vivo and to understand the requirements for efficient drug transport into M. tuberculosis. We will identify further porin genes by screening a transposon library of M. bovis BCG and an expression library of M. tuberculosis in a porin mutant of M. smegmatis. We will construct porin mutants of M. tuberculosis and examine the function of the corresponding porins for OM permeability by transport experiments and for growth in vitro and in mice. The role of porins for susceptibility of M. tuberculosis to antibiotics and current TB drugs will be analyzed both by sensitivity and by transport experiments with M. tuberculosis porin mutants. The main porin will be purified from M. tuberculosis to analyze its channel properties in lipid bilayer and liposome swelling experiments. The protein will be over expressed in E. coli, folded and purified to determine its crystal structure. These studies will not only be essential for the design of new TB drugs, but will also be of fundamental interest for our understanding of nutrient uptake by M. tuberculosis.
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