Transcription regulation by CarD in mycobacteria is guided by basal promoter kinetics.

Bacterial pathogens like Mycobacterium tuberculosis (Mtb) employ transcription factors to adapt their physiology to the diverse environments within their host. CarD is a conserved bacterial transcription factor that is essential for viability in Mtb. Unlike classical transcription factors that recognize promoters by binding to specific DNA sequence motifs, CarD binds directly to the RNA polymerase to stabilize the open complex intermediate (RPo) during transcription initiation. We previously showed using RNA-sequencing that CarD is capable of both activating and repressing transcription in vivo. However, it is unknown how CarD achieves promoter-specific regulatory outcomes in Mtb despite binding indiscriminate of DNA sequence. We propose a model where CarD's regulatory outcome depends on the promoter's basal RPo stability and test this model using in vitro transcription from a panel of promoters with varying levels of RPo stability. We show that CarD directly activates full-length transcript production from the Mtb ribosomal RNA promoter rrnAP3 (AP3) and that the degree of transcription activation by CarD is negatively correlated with RPo stability. Using targeted mutations in the extended -10 and discriminator region of AP3, we show that CarD directly represses transcription from promoters that form relatively stable RPo. DNA supercoiling also influenced RPo stability and affected the direction of CarD regulation, indicating that the outcome of CarD activity can be regulated by factors beyond promoter sequence. Our results provide experimental evidence for how RNA polymerase-binding transcription factors like CarD can exert specific regulatory outcomes based on the kinetic properties of a promoter.

Transcription regulation by CarD in mycobacteria is guided by basal promoter kinetics.

Bacterial pathogens like Mycobacterium tuberculosis ( Mtb ) employ transcription factors to adapt their physiology to the diverse environments within their host. CarD is a conserved bacterial transcription factor that is essential for viability in Mtb . Unlike classical transcription factors that recognize promoters by binding to specific DNA sequence motifs, CarD binds directly to the RNA polymerase (RNAP) to stabilize the open complex intermediate (RP o ) during transcription initiation. We previously showed using RNA-sequencing that CarD is capable of both activating and repressing transcription in vivo . However, it is unknown how CarD achieves promoter specific regulatory outcomes in Mtb despite binding indiscriminate of DNA sequence. We propose a model where CarD's regulatory outcome depends on the promoter's basal RP o stability and test this model using in vitro transcription from a panel of promoters with varying levels of RP o stability. We show that CarD directly activates full-length transcript production from the Mtb ribosomal RNA promoter rrnA P3 (AP3) and that the degree of transcription activation by CarD is negatively correlated with RP o stability. Using targeted mutations in the extended -10 and discriminator region of AP3, we show that CarD directly represses transcription from promoters that form relatively stable RP o . DNA supercoiling also influenced RP o stability and affected the direction of CarD regulation, indicating that the outcome of CarD activity can be regulated by factors beyond promoter sequence. Our results provide experimental evidence for how RNAP-binding transcription factors like CarD can exert specific regulatory outcomes based on the kinetic properties of a promoter.

Molecular dissection of RbpA-mediated regulation of fidaxomicin sensitivity in mycobacteria.

RNA polymerase (RNAP) binding protein A (RbpA) is essential for mycobacterial viability and regulates transcription initiation by increasing the stability of the RNAP-promoter open complex (RPo). RbpA consists of four domains: an N-terminal tail (NTT), a core domain (CD), a basic linker, and a sigma interaction domain. We have previously shown that truncation of the RbpA NTT and CD increases RPo stabilization by RbpA, implying that these domains inhibit this activity of RbpA. Previously published structural studies showed that the NTT and CD are positioned near multiple RNAP-σA holoenzyme functional domains and predict that the RbpA NTT contributes specific amino acids to the binding site of the antibiotic fidaxomicin (Fdx), which inhibits the formation of the RPo complex. Furthermore, deletion of the NTT results in decreased Mycobacterium smegmatis sensitivity to Fdx, but whether this is caused by a loss in Fdx binding is unknown. We generated a panel of rbpA mutants and found that the RbpA NTT residues predicted to directly interact with Fdx are partially responsible for RbpA-dependent Fdx activity in vitro, while multiple additional RbpA domains contribute to Fdx activity in vivo. Specifically, our results suggest that the RPo-stabilizing activity of RbpA decreases Fdx activity in vivo. In support of the association between RPo stability and Fdx activity, we find that another factor that promotes RPo stability in bacteria, CarD, also impacts to Fdx sensitivity. Our findings highlight how RbpA and other factors may influence RNAP dynamics to affect Fdx sensitivity.

Perspectives and Advances in the Understanding of Tuberculosis.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), remains a leading cause of death due to infection in humans. To more effectively combat this pandemic, many aspects of TB control must be developed, including better point of care diagnostics, shorter and safer drug regimens, and a protective vaccine. To address all these areas of need, better understanding of the pathogen, host responses, and clinical manifestations of the disease is required. Recently, the application of cutting-edge technologies to the study of Mtb pathogenesis has resulted in significant advances in basic biology, vaccine development, and antibiotic discovery. This leaves us in an exciting era of Mtb research in which our understanding of this deadly infection is improving at a faster rate than ever, and renews hope in our fight to end TB. In this review, we reflect on what is known regarding Mtb pathogenesis, highlighting recent breakthroughs that will provide leverage for the next leaps forward in the field.

CarD contributes to diverse gene expression outcomes throughout the genome of Mycobacterium tuberculosis.

The ability to regulate gene expression through transcription initiation underlies the adaptability and survival of all bacteria. Recent work has revealed that the transcription machinery in many bacteria diverges from the paradigm that has been established in Escherichia coliMycobacterium tuberculosis (Mtb) encodes the RNA polymerase (RNAP)-binding protein CarD, which is absent in E. coli but is required to form stable RNAP-promoter open complexes (RPo) and is essential for viability in Mtb The stabilization of RPo by CarD has been proposed to result in activation of gene expression; however, CarD has only been examined on limited promoters that do not represent the typical promoter structure in Mtb In this study, we investigate the outcome of CarD activity on gene expression from Mtb promoters genome-wide by performing RNA sequencing on a panel of mutants that differentially affect CarD's ability to stabilize RPo In all CarD mutants, the majority of Mtb protein encoding transcripts were differentially expressed, demonstrating that CarD had a global effect on gene expression. Contrary to the expected role of CarD as a transcriptional activator, mutation of CarD led to both up- and down-regulation of gene expression, suggesting that CarD can also act as a transcriptional repressor. Furthermore, we present evidence that stabilization of RPo by CarD could lead to transcriptional repression by inhibiting promoter escape, and the outcome of CarD activity is dependent on the intrinsic kinetic properties of a given promoter region. Collectively, our data support CarD's genome-wide role of regulating diverse transcription outcomes.

Chemical disarming of isoniazid resistance in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) killed more people in 2017 than any other single infectious agent. This dangerous pathogen is able to withstand stresses imposed by the immune system and tolerate exposure to antibiotics, resulting in persistent infection. The global tuberculosis (TB) epidemic has been exacerbated by the emergence of mutant strains of Mtb that are resistant to frontline antibiotics. Thus, both phenotypic drug tolerance and genetic drug resistance are major obstacles to successful TB therapy. Using a chemical approach to identify compounds that block stress and drug tolerance, as opposed to traditional screens for compounds that kill Mtb, we identified a small molecule, C10, that blocks tolerance to oxidative stress, acid stress, and the frontline antibiotic isoniazid (INH). In addition, we found that C10 prevents the selection for INH-resistant mutants and restores INH sensitivity in otherwise INH-resistant Mtb strains harboring mutations in the katG gene, which encodes the enzyme that converts the prodrug INH to its active form. Through mechanistic studies, we discovered that C10 inhibits Mtb respiration, revealing a link between respiration homeostasis and INH sensitivity. Therefore, by using C10 to dissect Mtb persistence, we discovered that INH resistance is not absolute and can be reversed.

The stringent response and Mycobacterium tuberculosis pathogenesis.

During infection, the host restrains Mycobacterium tuberculosis (Mtb) from proliferating by imposing an arsenal of stresses. Despite this onslaught of attacks, Mtb is able to persist for the lifetime of the host, indicating that this pathogen has substantial molecular mechanisms to resist host-inflicted damage. The stringent response is a conserved global stress response in bacteria that involves the production of the hyperphosphorylated guanine nucleotides ppGpp and pppGpp (collectively called (p)ppGpp). (p)ppGpp then regulates a number of cellular processes to adjust the physiology of the bacteria to promote survival in different environments. Survival in the presence of host-generated stresses is an essential quality of successful pathogens, and the stringent response is critical for the intracellular survival of a number of pathogenic bacteria. In addition, the stringent response has been linked to virulence gene expression, persistence, latency and drug tolerance. In Mtb, (p)ppGpp synthesis is required for survival in low nutrient conditions, long term culture and during chronic infection in animal models, all indicative of a strict requirement for (p)ppGpp during exposure to stresses associated with infection. In this review we discuss (p)ppGpp metabolism and how this functions as a critical regulator of Mtb virulence.