Characterization of the transcriptional regulator WhiB7 establishes a link between metabolism and intrinsic antibiotic resistance in mycobacteria

Mycobacterium tuberculosis (Mtb), the etiologic agent of tuberculosis, continues to be the world’s deadliest human bacterial pathogen. Current treatments are notoriously limited, lengthy, and becoming increasingly ineffective due to drug-resistant mutant strains. WhiB7, a putative transcriptional regulator, is an essential component of intrinsic antibiotic resistance in Mtb. Unique to Actinobacteria, multiple paralogous WhiB-like proteins have diverse roles in physiology, but little is known about their mode of action or regulation. To investigate WhiB7, a combination of in vitro run-off, two-hybrid assays, protein pull-down experiments, and genetic approaches was used. WhiB7 was characterized as an auto-regulatory, redox-sensitive transcriptional activator, providing the first biochemical proof that a WhiB-like protein directly promotes transcription. WhiB7’s antibiotic resistance function was dependent on three regions: an iron-sulfur cluster binding region likely required for stability; a middle region for binding to the vegetative sigma factor SigA; and a C-terminal DNA-binding region. Mutations disrupting any one of the regions led to an inability of WhiB7 to activate resistance. These experimental constraints were combined with protein modelling techniques to visualize the WhiB7:SigA:DNA complex which may serve as a platform for the design of inhibitors. Additionally, a GFP reporter was constructed to monitor whiB7 induction, and was used to screen our custom library of almost 600 bioactive compounds including the majority of clinical antibiotics. Expression was induced by compounds having diverse structures and targets, which did not correlate with drug susceptibility of the whiB7 mutant. Antibiotic-induced transcription was synergistically increased by the reductant dithiothreitol, an effect mirrored by a whiB7-dependent shift to a highly reduced intracellular condition reflected by the reduced:oxidized mycothiol ratio. Amino acid metabolism also contributed to WhiB7-mediated intrinsic resistance. To gain insights into whether other genetic loci contribute to WhiB7-mediated antibiotic resistance, a transposon library of Mycobacterium smegmatis was screened for WhiB7-like drug susceptibility or resistance. These studies revealed a putative aspartate aminotransferase (MSMEG_4060) that contributed to whiB7 repression and a pair of adjacent hypothetical genes (MSMEG_3637/3638) that contributed to whiB7 induction. Continued characterization of WhiB7 may serve as a paradigm for other WhiB-like proteins and lead to novel and desperately needed therapies for tuberculosis.Science, Faculty ofMicrobiology and Immunology, Department ofGraduat