Mycobacterium abscessus VapC5 toxin potentiates evasion of antibiotic killing by ribosome overproduction and activation of multiple resistance pathways.

Mycobacterium abscessus (Mab) infections are inexplicably intractable to clearing after aggressive and lengthy treatment regimens. Here we discovered that acquisition of a single toxin-antitoxin system enables Mab to activate a phenotypic switch that enhances survival upon treatment with current first-line antibiotics. This switch is tripped when the VapC5 toxin inactivates tRNASerCGA by cleavage at only one site within its anticodon, leading to growth arrest. Concomitant tRNASerCGA depletion then reprograms the transcriptome to favor synthesis of proteins naturally low in the cognate Ser UCG codon including the transcription factor WhiB7 and members of its regulon as well as the ribosomal protein family. This programmed stockpiling of ribosomes is predicted to override the efficacy of ribosome-targeting antibiotics while the growth arrest phenotype attenuates antibiotics targeting cell wall synthesis. In agreement, VapC5 increases Mab persister formation upon exposure to amikacin and the next-generation oxazolidinone tedizolid (both target ribosomes) or cefoxitin (inhibits cell wall synthesis). These findings expand the repertoire of genetic adaptations harnessed by Mab to survive assaults intended to eradicate it, as well as provide a much-needed framework for selection of shorter and more efficacious alternate treatment options for Mab infections using currently available antimicrobials whose targets are not confounded by VapC5.

tRNAfMet Inactivating Mycobacterium tuberculosis VapBC Toxin-Antitoxin Systems as Therapeutic Targets.

The Mycobacterium tuberculosis genome contains an abundance of toxin-antitoxin (TA) systems, 50 of which belong to the VapBC family. The activity of VapC toxins is controlled by dynamic association with their cognate antitoxins-the toxin is inactive when complexed with VapB antitoxin but active when freed. Here, we determined the cellular target of two phylogenetically related VapC toxins and demonstrate how their properties can be harnessed for drug development. First, we used a specialized RNA sequencing (RNA-seq) approach, 5' RNA-seq, to accurately identify the in vivo RNA target of M. tuberculosis VapC2 and VapC21 toxins. Both toxins exclusively disable initiator tRNAfMet through cleavage at a single, identical site within their anticodon loop. Consistent with the essential role and global requirement for initiator tRNAfMet in bacteria, expression of each VapC toxin resulted in potent translation inhibition followed by growth arrest and cell death. Guided by previous structural studies, we then mutated two conserved amino acids in the antitoxin (WR→AA) that resided in the toxin-antitoxin interface and were predicted to inhibit toxin activity. Both mutants were markedly less efficient in rescuing growth over time, suggesting that screens for high-affinity small-molecule inhibitors against this or other crucial VapB-VapC interaction sites could drive constitutive inactivation of tRNAfMet by these VapC toxins. Collectively, the properties of the VapBC2 and VapBC21 TA systems provide a framework for development of bactericidal antitubercular agents with high specificity for M. tuberculosis cells.

Mycobacterium tuberculosis VapC4 toxin engages small ORFs to initiate an integrated oxidative and copper stress response.

The Mycobacterium tuberculosis (Mtb) VapBC4 toxin-antitoxin system is essential for the establishment of Mtb infection. Using a multitier, systems-level approach, we uncovered the sequential molecular events triggered by the VapC4 toxin that activate a circumscribed set of critical stress survival pathways which undoubtedly underlie Mtb virulence. VapC4 exclusively inactivated the sole transfer RNACys (tRNACys) through cleavage at a single site within the anticodon sequence. Depletion of the pool of tRNACys led to ribosome stalling at Cys codons within actively translating messenger RNAs. Genome mapping of these Cys-stalled ribosomes unexpectedly uncovered several unannotated Cys-containing open reading frames (ORFs). Four of these are small ORFs (sORFs) encoding Cys-rich proteins of fewer than 50 amino acids that function as Cys-responsive attenuators that engage ribosome stalling at tracts of Cys codons to control translation of downstream genes. Thus, VapC4 mimics a state of Cys starvation, which then activates Cys attenuation at sORFs to globally redirect metabolism toward the synthesis of free Cys. The resulting newly enriched pool of Cys feeds into the synthesis of mycothiol, the glutathione counterpart in this pathogen that is responsible for maintaining cellular redox homeostasis during oxidative stress, as well as into a circumscribed subset of cellular pathways that enable cells to defend against oxidative and copper stresses characteristically endured by Mtb within macrophages. Our ability to pinpoint activation or down-regulation of pathways that collectively align with Mtb virulence-associated stress responses and the nonreplicating persistent state brings to light a direct and vital role for the VapC4 toxin in mediating these critical pathways.

The Suf Iron-Sulfur Cluster Biosynthetic System Is Essential in Staphylococcus aureus, and Decreased Suf Function Results in Global Metabolic Defects and Reduced Survival in Human Neutrophils.

Staphylococcus aureus remains a causative agent for morbidity and mortality worldwide. This is in part a result of antimicrobial resistance, highlighting the need to uncover novel antibiotic targets and to discover new therapeutic agents. In the present study, we explored the possibility that iron-sulfur (Fe-S) cluster synthesis is a viable antimicrobial target. RNA interference studies established that Suf (sulfur mobilization)-dependent Fe-S cluster synthesis is essential in S. aureus We found that sufCDSUB were cotranscribed and that suf transcription was positively influenced by sigma factor B. We characterized an S. aureus strain that contained a transposon inserted in the intergenic space between sufC and sufD (sufD*), resulting in decreased transcription of sufSUB Consistent with the transcriptional data, the sufD* strain had multiple phenotypes associated with impaired Fe-S protein maturation. They included decreased activities of Fe-S cluster-dependent enzymes, decreased growth in media lacking metabolites that require Fe-S proteins for synthesis, and decreased flux through the tricarboxylic acid (TCA) cycle. Decreased Fe-S cluster synthesis resulted in sensitivity to reactive oxygen and reactive nitrogen species, as well as increased DNA damage and impaired DNA repair. The sufD* strain also exhibited perturbed intracellular nonchelated Fe pools. Importantly, the sufD* strain did not exhibit altered exoprotein production or altered biofilm formation, but it was attenuated for survival upon challenge by human polymorphonuclear leukocytes. The results presented are consistent with the hypothesis that Fe-S cluster synthesis is a viable target for antimicrobial development.