Molecular Genetic Analysis of Mycobacterium Tuberculosis

Project Summary Despite broad availability and extensive use of the vaccine Bacille Calmette Guerin (BCG) and sterilizing chemotherapies, tuberculosis (TB) remains responsible for more than 1.5 million deaths annually. Although COVID-19 caused more deaths than TB in 2020 and 2021, TB is expected to resume the position it held for most of the last 100 years as the leading cause of death associated with an infectious agent. The mechanisms through which Mycobacterium tuberculosis (Mtb) successfully evades vaccines and effective chemotherapies remain unknown. We hypothesize that Mtb persister cells enter a physiological state that prevents them from succumbing to death and sterilization. This proposal will use in vitro and in vivo models to dissect the mechanisms enabling persistence, building on our preliminary data showing that persistence is a multifactorial phenomenon involving amino acid metabolism, other metabolic pathways, interactions with the host immune response, and effects on bacterial chromosome structure. Persisters were first identified as the 0.1% to 1% of streptococci and staphylococci that survive treatment with the bacteriocidal antibiotic penicillin. We have established similar, highly robust, reproducible models for isolating Mtb persisters using either isoniazid (INH) or amikacin. In our INH model, 99% to 99.9% of Mtb cells are killed within 3 days of in vitro exposure. Microarray analysis of surviving cells revealed a defined stress-associated signature. We compared the RNA sequencing results for amikacin and INH persisters to those for Mtb under other persister-inducing conditions, including hypoxia, nutrient starvation, and stationary phase. We found 25 commonly upregulated genes, which we hypothesize are important genes for persister formation. We propose to construct deletion mutants for all 25 genes to define pathways leading to persistence. We further hypothesize that Mtb persisters evolved to prevent sterilization by the adaptive immune response, supported by our studies of a new conditionally immune-sterilized auxotrophic (CIMSAUX) Mtb mutant that contains deletions in methionine, leucine, and pantothenate biosynthesis genes. The CIMSAUX mutant persists indefinitely in immunocompromised mice but is sterilized in immunocompetent mice. The genetic factors contributing to the CIMSAUX mutant phenotype will be analyzed, and additional CIMSAUX mutants will be isolated. Novel fluorescent reporter constructs will be engineered to monitor the growth and persistence of the multiple mutants generated in this study. We present preliminary evidence that deletion of the gene encoding the structural maintenance of the chromosome (SMC) protein in Mtb results in a persister formation–defective mutant (Mtb Δsmc), suggesting that Mtb chromosome structure contributes to persister formation. To test this hypothesis, we will delete genes with the potential to impact chromosome structure in wild-type Mtb and Mtb Δsmc and test their persister capacities in vitro and in mouse models. These studies will (i) provide new insights into the Mtb persister phenotype, (ii) generate valuable models for investigating persistence, and (iii) lead to improved strategies for sterilizing Mtb infections.