Robert N. Husson

In response to host-generated stresses, Mycobacterium tuberculosis (Mtb) reprograms its physiology in myriad ways to establish and maintain an infection, yet the signals that underlie this transformation are not well defined. The abundant toxin-antitoxin (TA) systems harbored in the Mtb genome, including 11 in the mazEF family, are thought to act as stress sensors, yet their roles are largely unknown. Although TA systems from other bacteria are generally thought to impart reversible growth arrest in response to stress, the exquisite specificity of Mtb tRNase toxins instead portends a more nuanced role. Here, we used a proteomics approach to track de novo protein synthesis to uncover molecular events initiated by the Mtb MazF-mt9 toxin (MazF7, Rv2063A). First, we documented striking enrichment of enzymes and transporters derived from the contiguous 36-gene region for phthiocerol dimycocerosate (PDIM) synthesis without an accompanying increase in PDIM lipid production. This paradox was reconciled by concomitant downregulation of proteins comprising the Mce1 transporter (imports host fatty acids), cholesterol breakdown, and β-oxidation enzymes (limiting the PDIM precursor methylmalonyl-CoA). Thus, increased catalytic efficiency of the PDIM pathway appears to offset substrate starvation to ensure adequate production of PDIMs essential for Mtb early immune escape and virulence. Finally, isocitrate lyase 1 levels also increased, which in this context are expected to primarily catalyze the glyoxylate shunt to sustain central carbon metabolism while minimizing carbon loss. These exacting proteomic signatures are paralleled within the bedaquiline-treated Mtb transcriptome, highlighting a critical role for MazF-mt9 in orchestrating Mtb stress survival. IMPORTANCE: The bacterial pathogen that causes tuberculosis, Mycobacterium tuberculosis (Mtb), must survive a gauntlet of immune assaults to establish an infection. Here, we determined that in response to host-imposed stresses, this pathogen enlists the action of a tRNase, the MazF-mt9 toxin, to reprogram the translatome and orchestrate metabolic remodeling to ensure adequate production of specialized phthiocerol dimycocerosate (PDIM) lipids on the cell surface, which contribute to early immune evasion. This toxin also upregulates isocitrate lyase 1 as a complementary survival-oriented adaptation that conserves carbon and sustains central metabolism for essential cellular functions. Thus, this toxin-mediated cooperative reprogramming toward preservation of PDIMs and central metabolism under lipid precursor-limiting conditions likely enables Mtb to successfully infect and survive in the host lung. Overall, the MazF-mt9-mediated protein expression signatures align with the transcriptome signatures of Mtb cells during bedaquiline treatment, suggesting a precise and essential role for this toxin in Mtb stress survival.

Toxin–antitoxin modules are present in many bacterial pathogens. The VapBC family is particularly abundant in members of the Mycobacterium tuberculosis complex, with 50 modules present in the M. tuberculosis genome. In type IIA modules, the VapB antitoxin protein binds to and inhibits the activity of the co-expressed cognate VapC toxin protein. VapB proteins may also bind to promoter region sequences and repress the expression of the vapB-vapC operon. Though VapB–VapC interactions can control the amount of free VapC toxin in the bacterial cell, the mechanisms that affect this interaction are poorly understood. Based on our recent finding of Ser/Thr phosphorylation of VapB proteins in M. tuberculosis , we substituted phosphomimetic or phosphoablative amino acids at the phosphorylation sites of two VapB proteins. We found that phosphomimetic substitution of VapB27 and VapB46 resulted in decreased interaction with their respective cognate VapC proteins, whereas phosphoablative substitution did not alter binding. Similarly, we determined that phosphomimetic substitution interfered with VapB binding to promoter region DNA sequences. Both decreased VapB–VapC interaction and decreased VapB repression of vapB-vapC operon transcription would result in increased free VapC in the M. tuberculosis cell. In growth inhibition experiments, M. tuberculosis strains expressing vapB46-vapC46 constructs containing a phosphoablative vapB mutation resulted in lower toxicity compared to a strain expressing native vapB46 , whereas similar or greater toxicity was observed in the strain expressing the phosphomimetic vapB mutation. These results identify a novel mechanism by which VapC toxicity activity can be regulated by VapB phosphorylation. IMPORTANCE: Intracellular bacterial toxins are present in many bacterial pathogens and have been linked to bacterial survival in response to stresses encountered during infection. The activity of many toxins is regulated by a co-expressed antitoxin protein that binds to and sequesters the toxin protein. The mechanisms by which an antitoxin may respond to stresses to alter toxin activity are poorly understood. Here, we show that antitoxin interactions with its cognate toxin and with promoter DNA required for antitoxin and toxin expression can be altered by Ser/Thr phosphorylation of the antitoxin and, thus, affect toxin activity. This reversible modification may play an important role in regulating toxin activity within the bacterial cell in response to signals generated during infection.

Toxin-antitoxin modules are present in many bacterial pathogens. The VapBC family is particularly abundant in members of the Mycobacterium tuberculosis complex, with 50 modules present in the M. tuberculosis genome. In type IIA modules the VapB antitoxin protein binds to and inhibits the activity of the co-expressed cognate VapC toxin protein. VapB proteins also bind to promoter region sequences and repress expression of the vapB-vapC operon. Though VapB-VapC interactions can control the amount of free VapC toxin in the bacterial cell, the mechanisms that affect this interaction are poorly understood. Based on our recent finding of Ser/Thr phosphorylation of VapB proteins in M. tuberculosis , we substituted phosphomimetic or phosphoablative amino acids at the phosphorylation sites of two VapB proteins. We found that phosphomimetic substitution of VapB27 and VapB46 resulted in decreased interaction with their respective cognate VapC proteins, whereas phosphoablative substitution did not alter binding. Similarly, we determined that phosphomimetic substitution interfered with VapB binding to promoter region DNA sequences. Both decreased VapB-VapC interaction and decreased VapB repression of vapB - vapC operon transcription would result in increased free VapC in the M. tuberculosis cell. M. tuberculosis strains expressing vapB46-vapC46 constructs containing a phosphoablative vapB mutation resulted in lower toxicity compared to a strain expressing native vapB46 , whereas similar or greater toxicity was observed in the strain expressing the phosphomimetic vapB mutation. These results identify a novel mechanism by which VapC toxicity activity can be regulated by VapB phosphorylation, potentially in response to extracytoplasmic as well as intracellular signals. Importance: Intracellular bacterial toxins are present in many bacterial pathogens and have been linked to bacterial survival in response to stresses encountered during infection. The activity of many toxins is regulated by a co-expressed antitoxin protein that binds to and sequesters the toxin protein. The mechanisms by which an antitoxin may respond to stresses to alter toxin activity are poorly understood. Here we show that antitoxin interactions with its cognate toxin, and with promoter DNA required for antitoxin and toxin expression, can be altered by Ser/Thr phosphorylation of the antitoxin, and thus affect toxin activity. This reversible modification may play an important role in regulating toxin activity within the bacterial cell in response to signals generated during infection.

As the world focuses on the COVID-19 pandemic, it is important not to lose sight of other infectious and noninfectious diseases that continue to exact a major toll on the health of children. Childhood tuberculosis (TB) stands among the most serious of these, with an estimated 1.1 million cases of TB disease and more than 200,000 TB deaths in children less than 15 years of age in 2020 [ 1]. The COVID-19 pandemic has markedly decreased the global capacity to diagnose and treat TB in children and adults, resulting in decreased numbers of persons treated for TB in 2020, and the first increase in TB mortality in more than a decade [ 1]. Increased transmission from adults with untreated infectious TB is anticipated to reverse the previous slow but steady decline in TB in both children and adults. As global efforts to manage the COVID-19 pandemic continue, it is important to take stock of the state of childhood TB and evaluate what needs to be done, not just to reach the rate of progress that existed prior to the pandemic, but to advance efforts on multiple fronts to reduce morbidity and mortality from TB in children.

Significance: Toxin–antitoxin (TA) systems are proposed to protect bacteria from deadly stresses encountered in their environmental niches. Yet clear examples supporting this hypothesis are lacking. The abundant Mycobacterium tuberculosis (Mtb) TA systems are implicated in enabling this pathogen to evade killing by our immune system and persist inside macrophages as a latent tuberculosis infection. We used a multiomics approach to track the activity of one TA system toxin, VapC4, and the series of molecular events it triggers. VapC4 first depletes tRNA ^Cys to mimic a state of Cys starvation. This reprograms Mtb metabolism to specifically defend against oxidative and copper stresses. Thus, VapC4 plays a direct and vital role in mediating these critical stress pathways essential for Mtb viability during infection.

The ability of Mycobacterium tuberculosis (Mtb) to adapt to diverse stresses in its host environment is crucial for pathogenesis. Two essential Mtb serine/threonine protein kinases, PknA and PknB, regulate cell growth in response to environmental stimuli, but little is known about their downstream effects. By combining RNA-Seq data, following treatment with either an inhibitor of both PknA and PknB or an inactive control, with publicly available ChIP-Seq and protein–protein interaction data for transcription factors, we show that the Mtb transcription factor (TF) regulatory network propagates the effects of kinase inhibition and leads to widespread changes in regulatory programs involved in cell wall integrity, stress response, and energy production, among others. We also observe that changes in TF regulatory activity correlate with kinase-specific phosphorylation of those TFs. In addition to characterizing the downstream regulatory effects of PknA/PknB inhibition, this demonstrates the need for regulatory network approaches that can incorporate signal-driven transcription factor modifications.

The Mycobacterium tuberculosis Ser/Thr protein kinases PknA and PknB are essential for growth and have been proposed as possible drug targets. We used a titratable conditional depletion system to investigate the functions of these kinases. Depletion of PknA or PknB or both kinases resulted in growth arrest, shortening of cells, and time-dependent loss of acid-fast staining with a concomitant decrease in mycolate synthesis and accumulation of trehalose monomycolate. Depletion of PknA and/or PknB resulted in markedly increased susceptibility to β-lactam antibiotics, and to the key tuberculosis drug rifampin. Phosphoproteomic analysis showed extensive changes in protein phosphorylation in response to PknA depletion and comparatively fewer changes with PknB depletion. These results identify candidate substrates of each kinase and suggest specific and coordinate roles for PknA and PknB in regulating multiple essential physiologies. These findings support these kinases as targets for new antituberculosis drugs and provide a valuable resource for targeted investigation of mechanisms by which protein phosphorylation regulates pathways required for growth and virulence in M. tuberculosis. Tuberculosis is a major global health threat, with approximately 10 million new cases per year and 1.5 million deaths. Although tuberculosis is treatable, cure requires prolonged antibiotic courses and strains that are resistant to current treatments are increasingly common. Understanding how Mycobacterium tuberculosis adapts to the human host during infection can lead to new approaches to treat tuberculosis. A major adaptive mechanism is reversible protein phosphorylation mediated by protein kinases, which detect signals in the extracellular environment and transmit this information to regulate M. tuberculosis physiology. In this study we analyzed two M. tuberculosis kinases, PknA and PknB, by comparing strains in which these kinases are present to strains in which one or both of these kinases has been depleted. We identified features that differ between the kinase replete and kinase depleted strains including bacterial growth and viability, cell shape, the bacterial cell envelope and gene regulation. We also identified proteins that show decreased phosphorylation in the kinase depleted strains, which can result from direct or indirect effects of kinase depletion. These findings provide insight into how these kinases are important for M. tuberculosis pathogenesis and indicate that these kinases may be valuable targets for development of new TB drugs.

Mycobacterium tuberculosis readily adapts to survive a wide range of assaults by modifying its physiology and establishing a latent tuberculosis (TB) infection. Here we report a sophisticated mode of regulation by a tRNA-cleaving toxin that enlists highly selective ribosome stalling to recalibrate the transcriptome and remodel the proteome. This toxin, MazF-mt9, exclusively inactivates one isoacceptor tRNA, tRNA^Lys43-UUU, through cleavage at a single site within its anticodon (UU↓U). Because wobble rules preclude compensation for loss of tRNA^Lys43-UUU by the second M. tuberculosis lysine tRNA, tRNA^Lys19-CUU, ribosome stalling occurs at in-frame cognate AAA Lys codons. Consequently, the transcripts harboring these stalled ribosomes are selectively cleaved by specific RNases, leading to their preferential deletion. This surgically altered transcriptome generates concomitant changes to the proteome, skewing synthesis of newly synthesized proteins away from those rich in AAA Lys codons toward those harboring few or no AAA codons. This toxin-mediated proteome reprogramming may work in tandem with other pathways to facilitate M. tuberculosis stress survival.