Nancy A. Woychik

Mycobacterium abscessus (Mab) is an emerging opportunistic pathogen characterized by extensive intrinsic drug resistance, underscoring the need for precise genetic tools to define mechanisms of pathogenicity and persistence. The ORBIT ( o ligonucleotide-mediated recombineering followed by B xb1 integrase targeting) system developed by the Sassetti group (1) enables precise gene deletions but has primarily been optimized for Mycobacterium tuberculosis and Mycobacterium smegmatis . Although ORBIT has been used in Mab, here we describe an optimized ORBIT workflow for efficient gene deletion in Mab. We tested key parameters, including plasmid selection, oligonucleotide design, and recombineering conditions. We evaluated oligonucleotide length and purification to improve recovery of targeted mutants and established conditions that enabled reproducible deletion of multiple target genes. Our findings establish a robust and adaptable ORBIT protocol tailored to the genetic characteristics of Mab. This optimized workflow provides a practical framework for precise genetic manipulation and can facilitate functional studies of genes that contribute to Mab physiology, antimicrobial resistance, and pathogenesis. IMPORTANCE: Mycobacterium abscessus causes the majority of intractable infections attributable to members of the M. abscessus complex: M. abscessus, M. massiliense and M. bolletii. M. abscessus infections most commonly present as chronic lung disease or as skin and soft tissue infections, and they are among the most difficult bacterial infections to eradicate, often persisting despite aggressive, prolonged treatment underscoring the need for reliable genetic tools to dissect the biology underlying pathogenesis. The ORBIT system enables precise, markerless gene deletion in mycobacteria, but its application to M. abscessus has been limited. Here we present an optimized ORBIT workflow tailored specifically to M. abscessus that will facilitate studies on the genes contributing to physiology and pathogenesis of this important pathogen.

Among clinically important non-tuberculous mycobacteria, Mycobacterium abscessus (Mab) has a large and highly dynamic accessory genome within an open pangenome, contributing traits that enhance drug resistance, bacterial persistence, stress survival, metabolic flexibility, and/or virulence. Defining how accessory loci promote survival during antimicrobial exposure is essential for understanding why Mab frequently evades killing by standard drug regimens and for improving therapeutic outcomes. We recently identified a potent accessory toxin–antitoxin (TA) system, VapB5-VapC5, that substantially enhances the already formidable drug resistance capacity of Mab clinical isolates. Acquisition of this single system reprograms Mab physiology to inhibit growth, activate the WhiB7 regulon, and promote ribosome accumulation. Here, we further define the contribution of the active component of this TA system, the VapC5 toxin, by assessing its impact on a more comprehensively defined WhiB7 regulon. We also studied the effects of VapC5 on growth, persistence, and the Mab transcriptome independently of WhiB7. Finally, we determined that VapC5-mediated resistance is mechanism-dependent. Because our previous study showed that VapC5 promotes survival during exposure to frontline antimicrobials that target cell-wall biosynthesis or protein translation, we reasoned that antibiotics with distinctly different mechanisms of action would be more likely to retain activity. As predicted, DNA gyrase or RNA polymerase inhibitors circumvented VapC5-mediated resistance and retained in vitro efficacy. These findings establish VapC5 as a broad physiological modulator that selectively reshapes Mab antimicrobial susceptibility based on drug mechanism. Screening Mab clinical isolates for acquisition of this accessory VapBC5 toxin-antitoxin system could risk-stratify treatment decisions by directing clinicians toward existing antibiotics targeting processes unaffected by VapC5, thereby improving treatment outcomes.

Mycobacterium tuberculosis (Mtb) remains a major global health threat, sustained by lengthy, multidrug treatment regimens that promote poor compliance and antibiotic resistance. The pathogen’s ability to evade clearance is facilitated in part by a transition to a nonreplicating, persistent state, a process thought to involve the abundant type II toxin-antitoxin (TA) systems. The Mtb chromosome encodes 11 MazEF TA systems whose MazF toxins are single-strand, sequence-specific endoribonucleases yet the precise RNA targets and physiological consequences of most remain unknown. Here we applied a specialized 5’-end RNA-seq approach to pinpoint the Mtb MazF-mt1 toxin recognition sequence and RNA target. MazF-mt1 specifically cleaves at U↓CCUU within the anti-Shine-Dalgarno (aSD) sequence, releasing it from the 3’ end of the 16S rRNA. Notably, the recently described MazF-mt11 toxin also targets the aSD for removal, cleaving at a different sequence 4 nts upstream of the MazF-mt1 site. Consistent with the essential role of the aSD in translation, both toxins rapidly inhibit new protein synthesis. This functional redundancy prompted us to mine Mtb transcriptomic datasets to compare how the two toxins respond to infection-relevant stresses. MazF-mt1 and MazF-mt11 are differentially regulated following exposure to diverse antibiotic and host-imposed stresses, with distinct temporal expression patterns. Thus, Mtb may coordinate transcription of these functionally redundant toxins to more precisely control the establishment and maintenance of nonreplicating persistence, thereby confounding pathogen clearance. Importance: The ability of M. tuberculosis to resist immune-mediated clearance and antibiotic treatment is central to its success as a pathogen. Here, we identify the target of the endoribonuclease toxin MazF-mt1 as the anti-Shine-Dalgarno sequence near the 3’ end of 16S rRNA, cleavage of which causes rapid growth arrest and translation inhibition. Base-pairing between anti-Shine-Dalgarno/Shine-Dalgarno sequences is required for efficient ribosome recruitment and translation of most bacterial mRNAs. Because we previously reported that the MazF-mt11 toxin also cleaves the 16S rRNA 3’ end to remove the anti-Shine-Dalgarno sequence, MazF-mt1 expression phenocopies MazF-mt11. We further show that this toxin pair responds in contrasting ways to several first- and second-line antibiotics and a wide range of environmental stress conditions. Thus, M. tuberculosis encodes functionally redundant, ribosome-targeting toxins that are differentially deployed under stress, illuminating a potential mechanism to rapidly suppress translation and promote entry into a drug-tolerant, nonreplicating persistent state during infection.

Infections caused by the emerging pathogen Mycobacterium abscessus (Mab) require prolonged multidrug treatment regimens that frequently fail because its accessory genome has incorporated a daunting array of poorly defined resistance mechanisms. However, the processes governing acquisition and retention of new genetic material within the Mab accessory genome remain poorly understood. Here, we first identified two Mab clinical strains that harbor the same uncharacterized toxin-antitoxin (TA) system at different genomic positions. Using computational analyses of the surrounding DNA context, we discovered that each TA system resides within identical mobile genetic element (MGE) cassettes whose positions exchange between the two strains via site-specific recombinases. We then applied a specialized 5′ RNA-seq approach to identify initiator tRNA ^fMet as the exclusive RNA target of the VapC2 TA toxin residing within the MGE. VapC2-mediated tRNA ^fMet inactivation by cleavage at its anticodon stem loop leads to growth arrest and global translation inhibition. This activity ensures stable inheritance of the MGE and its resident cargo genes through a mechanism similar to postsegregational killing. Our findings uncover the mechanism for durable expansion of the Mab accessory genome and suggest a selective advantage whereby toxin-mediated entry into a reversible growth-arrested state may reduce antibiotic susceptibility. Together, these results illustrate how retention of horizontally acquired MGEs containing a potent TA system can drive adaptive genome evolution to enhance fitness in this highly drug-resistant pathogen. IMPORTANCE: Mycobacterium abscessus (Mab) causes chronic lung disease and skin and soft tissue infections, posing a major threat to individuals with cystic fibrosis, underlying lung disease, lung transplants, or weakened immune systems. Unlike Mycobacterium tuberculosis , Mab has a large and highly plastic accessory genome generally thought to be predominantly shaped by horizontal gene transfer and recombination. However, the mechanisms that stabilize newly acquired genetic material in this pathogen remain poorly understood. Here, we identify a toxin-antitoxin (TA) system embedded within a mobile genetic element (MGE) predicted to enforce stable retention of the MGE through a mechanism similar to postsegregational killing. The toxin inactivates initiator tRNA ^fMet , inducing reversible growth arrest that likely reduces antibiotic efficacy. By functioning as an addiction module, this TA system also ensures stable inheritance of co-transferred accessory genes within the MGE, providing a mechanism for long-term retention of horizontally acquired genetic material that enhances pathogen fitness.

Mycobacterium tuberculosis (Mtb) must adapt to host-imposed stresses that limit its growth and ability to establish an infection. One important immune-regulated host defense mechanism involves competition between host and pathogen for tryptophan (Trp). Although Mtb is a Trp prototroph capable of synthesizing its own Trp, it normally acquires this amino acid from host macrophages because of the high energetic cost of its synthesis. However, interferon-γ-activated macrophages attempt to restrict Mtb growth by inducing indoleamine 2,3-dioxygenase 1 (IDO1) that degrades Trp. In response, Mtb senses and adapts to limitation of this essential amino acid by inducing its own Trp biosynthetic pathway, although the mechanisms coupling Trp limitation and compensatory Trp biosynthesis remain unknown. Here, we studied the Mtb VapC37 toxin and demonstrate that it arrests growth and is a highly selective tRNase that exclusively depletes tRNA ^Trp . Consequently, VapC37 triggers a state of perceived Trp starvation to upregulate production of Trp biosynthetic enzymes. Therefore, this toxin appears to link intracellular Trp availability—a well-established regulator of Mtb growth—to a codon-dependent adaptive response to increase the Trp concentration. VapC37 also broadly reprograms the Mtb transcriptome and translatome toward synthesis of mRNAs and proteins with low or no Trp codons. However, there is one striking exception: key enzymes in the phthiocerol dimycocerosate (PDIM) biosynthetic pathway were robustly synthesized despite being large and Trp-numerous, resulting in a > 2-fold increase in PDIM lipid production upon VapC37 expression. Thus, Mtb selectively preserves PDIM production under conditions of tRNA ^Trp limitation. Finally, VapBC37 and PDIM transcripts are upregulated in Mtb cells subjected to nitrosative stress and upon Mtb infection of THP-1 macrophage-like cells. This might implicate early macrophage-associated stresses as activators of VapC37, possibly enabling this toxin to reallocate metabolic priorities toward Trp biosynthesis while selectively preserving PDIM production critical for Mtb early immune escape and virulence.

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.

The Mycobacterium tuberculosis (Mtb) VapC4 endoribonuclease toxin exclusively cleaves and inactivates tRNACys, which leads to extensive ribosome stalling at Cys codons. Serendipitously, the precise position of stalled ribosomes is revealed within our 5′ RNA-seq datasets used to identify and validate the tRNA target of the toxin, precluding the need for Ribo-seq. Here we show how mapping of stalled ribosomes can be harnessed as an innovative tool for reliable detection of new Cys-containing Mtb open reading frames (ORFs). Using proteogenomics we unmasked 96 unannotated ORFs; of which 54% are small ORFs ≤50 amino acids. We validated 69% of the 96 ORFs by mass spectrometry, including four whose spectra was matched to synthetic controls Also, 25% of these unannotated ORFs were identified by previously published Ribo-RET. Some of the 96 ORFs are Cys-responsive attenuators or encode stable Cys-containing proteins that map immediately before, or within, genes in the opposite, or same, orientation. These ORF sequences can also reveal functional clues, e.g. zinc-binding motifs or encode novel EsxB-like proteins. Our findings demonstrate that toxin-mediated ribosome stalling can serve as a robust genome annotation tool that is applicable to mycobacteria and other bacteria, with unique advantages that complement existing genome annotation methods.

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.