Transcription factors that regulate onset of the persister differentially detectable phenotype in Mycobacterium tuberculosis
Differentially detectable Mycobacterium tuberculosis (DD Mtb) are subpopulations of persister Mtb cells that are recoverable by use of liquid limiting dilution (LD) assays but not by CFU on solid agar. DD Mtb cells show profound phenotypic tolerance to anti-TB drugs and may therefore play a role in disease persistence. A better understanding of the underlying biology of DD Mtb cells would aid in the development of better diagnostics for detecting them and better drugs for killing them. Recently, an in vitro model of DD Mtb was developed in which nutrient deprivation in PBS followed by exposure to rifampin induces a DD state in Mtb (the PBS-RIF model). Notably, the same protocol does not induce a DD state in the closely related M. bovis (Mb), which is associated with lethal levels of oxidative damage in Mb, but sublethal levels of oxidative damage in Mtb. A key genetic difference between Mtb and Mb is inactivation of the PhoPR two-component system, which is causally related to the reduced virulence of Mb in comparison to Mtb. Our RNAseq analysis confirms that nearly the entire PhoPR regulon is severely downregulated in Mb versus Mtb during the PBS-RIF protocol. Whether PhoPR is involved in regulation of the DD Mtb phenotype is a focus of this proposal. Our transcriptional profiling studies have identified a second transcription factor, CarD, that may also be involved in regulation of the DD Mtb phenotype. carD is one of the most downregulated genes in the PBS-RIF model, and its downstream target, icl1, is the most downregulated gene. These associations hold up when examining patient sputa: carD and icl1 show the strongest negative correlation between gene expression and relative abundance of DD Mtb in sputa. Notably, Icl1 was recently demonstrated to play a functional role in restraining DD Mtb formation in the PBS-RIF model, suggesting that its upstream regulator, CarD, may also play a similar role. In this proposal, I plan to rigorously evaluate the role of CarD and PhoPR in the establishment and maintenance of the DD Mtb phenotype in the PBS-RIF model. I will subject existing loss-of-function and gain- of-function Mtb mutants of each gene to the PBS-RIF model and measure quantitative effects on DD Mtb generation. Of note, our recent work demonstrates that this in vitro model faithfully recapitulates aspects of DD Mtb biology found in patients, affirming its use for our planned studies. The identification of transcription factor(s) involved in entry of Mtb into a drug-tolerant, persister DD state would be a fundamental contribution to our understanding of Mtb persistence and its regulation thereof.