Mycobacterium tuberculosis (Mtb) latently infects one third of the world's population and remains a leading cause of death from a single bacterial infection worldwide. Latent infection is thought to be sustained by a slowly- or non-replicating dormant population of Mtb, which is refractory to treatment with most of the currently available antibiotics and arises through adaptation of Mtb to the microenvironment within its host via transcriptional remodeling. The precise molecular events that lead to non-replication, sustain it, attain the dormant state and allow Mtb to survive for decades within its host are largely unknown. Present application aims to define one of the molecular pathways that regulates the non-replicating dormant state of Mtb and to elucidate the role of dihydrolipoamide acyltransferase (DlaT), a mycobacterial component of pyruvate dehydrogenase (PDH) and peroxynitrite reductase, in this pathway for the following reasons: DlaT is required for Mtb's full virulence in mice and survival in guinea pigs;dlaT's chromosomal location outside the PDH operon suggests a function unrelated to coordinate expression of other PDH components;expression of DlaT increases in stationary phase without increased PDH activity;Mtb DlaT binds RNA polymerase;DlaT in Bacillus is a transcriptional regulator for sporulation and DlaT in Pseudomonas controls transcription of type III secretion system regulatory operon;DlaT inhibitors only kill Mtb when Mtb is non-replicating. The hypothesis whether DlaT functions as a transcriptional regulator of Mtb's non-replication will be tested by using Illumina's newest technology in parallel sequencing of multiple DNA fragments. Transcriptional response of Mtb to non- replication and its dependence on DlaT will be studied by mRNA-Seq to compare the transcriptomes of the WT, ?dlaT, and ?dlaT complemented Mtb under replicating and non-replicating conditions. DlaT binding sites on Mtb's DNA will be located by ChIP-Seq and confirmed through secondary assays including in vitro transcription. Information from ChIP-Seq will be used to deduce the putative DlaT DNA-binding consensus sequence. Current application aims at elucidating the mechanism behind DlaT's function as a transcriptional regulator by studying DlaT's expression under different conditions in vitro, in mouse bone marrow macrophages and in mice, by identifying proteins associating with DlaT under those conditions, by producing deletion and enzymatically inactive mutants of DlaT and testing their ability to bind DNA and induce transcription, by probing the interaction of DlaT with RNA polymerase and testing whether competitive with sigma factors and DlaT inhibitors. Finally, the expression of DlaT-dependent genes under a variety of non- replicating conditions in vitro, in mouse macrophages and in mice will be studied. This will generate data on the repertoire of macromolecules and pathways expressed during Mtb non-replication in vivo and provide invaluable information on new targets specifically expressed during the non-replicating state of Mtb.

Public Health Relevance

Mycobacterium tuberculosis (Mtb) represents a leading cause of death from a single bacterial infection and latently infects one third of the world's population. Mtb's latency is believed to result from its ability to adapt to the microenvironmental changes within its host and attain dormancy, however, the exact molecular pathways that lead to and sustain Mtb dormancy are largely unknown. Present application aims to explore the transcriptional response of Mtb to non-replication and suggests that DlaT, an Mtb protein known to fulfill other functions, acts as a transcriptional regulator of Mtb dormant state. Products of genes under the regulation of DlaT will provide new insights into the repertoire of macromolecules and pathways important for non- replication and generate information to find new targets specific for the dormant state of Mtb.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Exploratory/Developmental Grants (R21)
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Prokaryotic Cell and Molecular Biology Study Section (PCMB)
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Lacourciere, Karen A
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Weill Medical College of Cornell University
Schools of Medicine
New York
United States
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