Genomic, biochemical, and structural investigation of proteins involved in mycobacterial virulence

Tuberculosis, the deadly disease caused by Mycobacterium tuberculosis (Mtb), infects 2 billion people world-wide, accounting for approximately 1.4 million deaths in 2011. Like all organisms, iron is essential to its growth, and this pathogen relies on host-acquired iron for its survival. Because of iron's poor solubility and toxicity, the host carefully guards this important element. Until recently, only non-heme iron uptake utilizing host transferrin iron was characterized in mycobacteria. In collaboration with the Horwitz lab at UCLA, we have characterized a mycobacterial heme uptake system (PNAS, 2011). Using a genomic approach in the model organism Mycobacterium smegmatis (Msmeg), we have identified the genomic region responsible for heme-iron uptake, msmeg0241-msmeg0250. A deletion mutant of this region in Msmeg resulted in severe attenuation in heme-supplemented media. Complementation with the homologous region in Mtb, Rv0201c-Rv0207c resulted in rescue of growth to wild-type levels in heme-supplemented media, indicating that these genes likely play a role in heme uptake in Mtb also.;Encoded within this region are a putative hemophore, Rv0203, and two proposed transmembrane heme transporters, MmpL11 and MmpL3. We have shown that deletion of either mmpL11 or mmpL3 in Msmeg results in attenuation of the strain in heme-supplemented media. Complementation of MsmegΔmmpL11 with mmpL11 from Mtb restored growth to wild-type levels, again suggesting a conserved role in heme uptake for this protein between the two species. Using a fluorescent heme analog, zinc mesoporphyrin, we have also identified the cytosolic fate of exogenous metalloporhyrin.;Additionally, we have characterized two proteins proposed to be involved in disulfide bond formation in Mtb. Disulfide bond formation is necessary for the proper folding of secreted proteins. Rv2969c (Mtb DsbG) and Rv2968c (Mtb VKOR) are necessary for optimal growth of Mtb, and are found adjacent to each other on the genome. The structure of Mtb DsbG was solved by X-ray crystallography, and shows a thioredoxin fold with a CXXC motif, found in all Dsb proteins. Interestingly, Mtb DsbG was shown to share structural homology with gram-positive DsbA homologs, with a similar inserted helical domain to the Staphylococcus aureus DsbA. Mtb DsbG was shown to have disulfide bond isomerase activity in vitro, and is functionally distinct from previously described mycobacterial disulfide bond forming proteins, Mtb DsbE and Mtb DsbF. Mtb VKOR is a predicted transmembrane protein and the proposed redox partner for Mtb DsbG. In an effort to characterize this interaction, we have expressed, purified and crystallized this membrane protein. The ability of this highly successful pathogen to cause disease even after years of quiet persistence in the host is poorly understood. This work has provided needed insight into proteins involved in mycobacterial virulence and has implicated new roles for proteins in heme-uptake and disulfide bond formation.