Multidrug-resistant (MDR) tuberculosis, defined as tuberculosis resistant to the two first-line drugs isoniazid and rifampin, poses a serious problem for global tuberculosis control strategies. Lack of a safe and convenient model organism hampers progress in combating the spread of MDR strains of Mycobacterium tuberculosis. We reasoned that auxotrophic MDR mutants of M. tuberculosis would provide a safe means for studying MDR M. tuberculosis without the need for a biosafety level 3 (BSL3) laboratory. Two different sets of triple auxotrophic mutants of M. tuberculosis were generated, which were auxotrophic for the nutrients leucine, pantothenate, and arginine or for leucine, pantothenate, and methionine. These triple auxotrophic strains retained their acid-fastness, their ability to generate both a drug persistence phenotype and drug-resistant mutants, and their susceptibility to plaque-forming mycobacterial phages. MDR triple auxotrophic mutants were obtained in a two-step fashion, selecting first for solely isoniazid-resistant or rifampin-resistant mutants. Interestingly, selection for isoniazid-resistant mutants of the methionine auxotroph generated isolates with single point mutations in katG, which encodes an isoniazid-activating enzyme, whereas similar selection using the arginine auxotroph yielded isoniazid-resistant mutants with large deletions in the chromosomal region containing katG. These M. tuberculosis MDR strains were readily sterilized by second-line tuberculosis drugs and failed to kill immunocompromised mice. These strains provide attractive candidates for M. tuberculosis biology studies and drug screening outside the BSL3 facility.

Mycobacterium tuberculosis infects ~25% of the world’s population and causes tuberculosis (TB), the second leading cause of death from infectious disease. Heme is an essential metabolite for M. tuberculosis , and targeting the unique heme biosynthetic pathway of M. tuberculosis could serve as an effective therapeutic strategy.

Heme is both an essential cofactor and an abundant source of nutritional iron for the human pathogen Mycobacterium tuberculosis (Mtb). While heme is required for Mtb survival and virulence, it is also potentially cytotoxic. Since Mtb has the ability to both make and uptake heme, the de novo synthesis of heme and its acquisition from the host must be balanced in order to mitigate heme toxicity. However, the mechanisms employed by Mtb to regulate heme uptake, synthesis, and bioavailability are poorly understood. By integrating ratiometric heme sensors with mycobacterial genetics, cell biology, and biochemistry, we determined that the terminal heme biosynthetic enzyme, coproheme decarboxylase (ChdC), plays a role in regulating both heme bioavailability and uptake in Mtb. Moreover, we found that Mtb has a preference for scavenging reduced ferrous heme and exhibits a cell surface heme reductase activity that is regulated by ChdC. In Mtb, ChdC expression is down-regulated when iron is limiting, which in-turn increases both heme import and bioavailability. Such a mechanism may serve to protect cells from heme toxicity while trying to meet the nutritional demand for iron. Our results demonstrate that heme synthesis and uptake are tightly integrated in mycobacteria and represent the first example of a heme synthetic enzyme playing a role in controlling heme uptake.