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Mycobacterium tuberculosis (Mtb) WhiB3 is an iron-sulfur cluster-containing transcription factor belonging to a subclass of the WhiB-Like (Wbl) family that is widely distributed in the phylum Actinobacteria. WhiB3 plays a crucial role in the survival and pathogenesis of Mtb. It binds to the conserved region 4 of the principal sigma factor (σ A ) in the RNA polymerase holoenzyme to regulate gene expression like other known Wbl proteins in Mtb. However, the structural basis of how WhiB3 coordinates with σ 4 to bind DNA and regulate transcription is unclear. Here we determined crystal structures of the WhiB3:σ A complex without and with DNA at 1.5 Å and 2.45 Å, respectively, to elucidate how WhiB3 interacts with DNA to regulate gene expression. These structures reveal that the WhiB3:σ 4 complex shares a molecular interface similar to other structurally characterized Wbl proteins and also possesses a subclass-specific Arg-rich DNA-binding motif. We demonstrate that this newly defined Arg-rich motif is required for WhiB3 binding to DNA in vitro and transcriptional regulation in Mycobacterium smegmatis. Together, our study provides empirical evidence of how WhiB3 regulates gene expression in Mtb by partnering with σ A 4 complex without and with DNA at 1.5 Å and 2.45 Å, respectively, to elucidate how WhiB3 interacts with DNA to regulate gene expression. These structures reveal that the WhiB3:σ A 4 complex shares a molecular interface similar to other structurally characterized Wbl proteins and also possesses a subclass-specific Arg-rich DNA-binding motif. We demonstrate that this newly defined Arg-rich motif is required for WhiB3 binding to DNA in vitro and transcriptional regulation in Mycobacterium smegmatis. Together, our study provides empirical evidence of how WhiB3 regulates gene expression in Mtb by partnering with σ A 4 and engaging with DNA via the subclass-specific structural motif, distinct from the modes of DNA interaction by WhiB1 and WhiB7., Competing Interests: Conflict of interest The authors declare no conflict of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Members of the WhiB-like (Wbl) family of proteins are found in Acintomycetes and are somewhat recalcitrant to overproduction as soluble proteins in the laboratory protein expression workhorse Esherichia coli. The aim of this study was to evaluate the effects of culture conditions and co-expression of the chaperone protein, trigger factor (TF), on the soluble production of recombinant Mycobacterium tuberculosis (Mtb) WhiB3. A pET28a derived expression plasmid coding for His 6 -WhiB3 was created and the effects of varying the concentration of inducer (IPTG), the timing of induction, the nature of the inducer (auto-induction medium) and the temperature of the cultivation on the production of soluble His 6 -WhiB3 were tested. Whilst His 6 -WhiB3 protein was readily detected, the overwhelming majority of the protein was present in the insoluble fraction of cell-free extracts. However, co-expression of the tig from pTf16, coding for TF, increased His 6 -WhiB3 solubility dramatically, facilitating its isolation by affinity chromatography. Purified His 6 -WhiB3 was shown to be monomeric, and UV-visible spectra suggested that ∼10% of the isolated protein possessed a [4Fe-4S] cluster. The secondary structural properties of His 6 -WhiB3 were altered by acquisition of an iron-sulfur cluster. By developing a protocol to readily overproduce and purify WhiB3, this study paves the way for future structure-function experiments., Competing Interests: Declaration of competing interest None., (Crown Copyright © 2022. Published by Elsevier Inc. All rights reserved.)

The physiological state of the human macrophage may impact the metabolism and the persistence of Mycobacterium tuberculosis . This pathogen senses and counters the levels of O 2 , CO, reactive oxygen species (ROS), and pH in macrophages. M. tuberculosis responds to oxidative stress through WhiB3. The goal was to determine the effect of NADPH oxidase (NOX) modulation and oxidative agents on the expression of whiB3 and genes involved in lipid metabolism ( lip-Y , Icl-1 , and tgs-1 ) in intracellular mycobacteria. Human macrophages were first treated with NOX modulators such as DPI (ROS inhibitor) and PMA (ROS activator), or with oxidative agents (H 2 O 2 and generator system O 2 •- ), and then infected with mycobacteria. We determined ROS production, cell viability, and expression of whiB3 , as well as genes involved in lipid metabolism. PMA, H 2 O 2 , and O 2 •- increased ROS production in human macrophages, generating oxidative stress in bacteria and augmented the gene expression of whiB3 , lip-Y , Icl-1 , and tgs-1 . Our results suggest that ROS production in macrophages induces oxidative stress in intracellular bacteria inducing whiB3 expression. This factor may activate the synthesis of reserve lipids produced to survive in the latency state, which allows its persistence for long periods within the host.

WhiB3 is a conserved cytoplasmic redox sensor which is required in the infection and lipid anabolism of Mycobacterium tuberculosis . The response of WhiB3 to environmental nutrient and its regulatory cascades are crucial during the persistent infection, while little is known about the relationship between WhiB3 and emergence of nutrient stress in this process. Here, we found that nitrogen regulator GlnR directly interacted with the WhiB3 promoter region and activated its transcription in response to nitrogen availability. In whiB3 promoter region, the typical GlnR-box was also identified. Moreover, GlnR controlled cell resistance to redox stress and SL-1 lipid anabolism by directly activating whiB3 expression. These results demonstrated that GlnR regulated redox sensor WhiB3 at the transcriptional level and mediated the interplay among nitrogen metabolism, redox sensing, and lipid anabolism.

Mycobacteria propelled modulation of host responses is of considerable interest in the face of emerging drug resistance. Although it is known that Abl tyrosine kinases affect entry and persistence of mycobacteria, mechanisms that couple c-Abl to proximal signaling pathways during immunity are poorly understood. Loss-of-function of c-Abl through Imatinib, in a mouse model of tuberculosis or RNA interference, identified bone morphogenesis protein (BMP) signaling as its cellular target. We demonstrate that c-Abl promotes mycobacterial survival through epigenetic modification brought about by KAT5-TWIST1 at Bmp loci. c-Abl-BMP signaling deregulated iNOS, aggravating the inflammatory balance. Interestingly, BMP signaling was observed to have far-reaching effects on host immunity, as it attenuated TLR3 pathway by engaging miR27a. Significantly, these events were largely mediated via WhiB3 and DosR/S/T but not SecA signaling pathway of mycobacteria. Our findings suggest molecular mechanisms of host pathways hijacked by mycobacteria and expand our understanding of c-Abl inhibitors in potentiating innate immune responses.