Phosphorylation of VapB antitoxins affects intermolecular interactions to regulate VapC toxin activity in .

Toxin-antitoxin modules are present in many bacterial pathogens. The VapBC family is particularly abundant in members of the complex, with 50 modules present in the genome. In type IIA modules, the VapB antitoxin protein binds to and inhibits the activity of the co-expressed cognate VapC toxin protein.

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 tRNA by cleavage at only one site within its anticodon, leading to growth arrest.

tRNA 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.

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

The (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 RNA (tRNA) through cleavage at a single site within the anticodon sequence.

The Sole MazF Toxin Targets tRNA to Impart Highly Selective, Codon-Dependent Proteome Reprogramming.

Survival of mycobacteria, both free-living and host-dependent pathogenic species, is dependent on their ability to evade being killed by the stresses they routinely encounter. Toxin-antitoxin (TA) systems are unique to bacteria and archaea and are thought to function as stress survival proteins. Here, we study the activity of the endoribonuclease toxin derived from the MazEF TA system in , designated MazEF-ms.

Accurate target identification for Mycobacterium tuberculosis endoribonuclease toxins requires expression in their native host.

The Mycobacterium tuberculosis genome harbors an unusually high number of toxin-antitoxin (TA) systems. These TA systems have been implicated in establishing the nonreplicating persistent state of this pathogen during latent tuberculosis infection. More than half of the M.

Cloaked dagger: tRNA slicing by an unlikely culprit.

The unusually high number of toxin-antitoxin (TA) systems in Mycobacterium tuberculosis, the etiological agent of tuberculosis, is thought to contribute to the unique ability of this pathogen to evade killing by the immune system and persist as a latent infection. One TA family, designated mazEF (for the MazE antitoxin and MazF toxin), comprises 10 of the >80 TA systems in the M. tuberculosis genome.

tRNA is a new target for cleavage by a MazF toxin.

Toxin-antitoxin (TA) systems play key roles in bacterial persistence, biofilm formation and stress responses. The MazF toxin from the Escherichia coli mazEF TA system is a sequence- and single-strand-specific endoribonuclease, and many studies have led to the proposal that MazF family members exclusively target mRNA. However, recent data indicate some MazF toxins can cleave specific sites within rRNA in concert with mRNA.

tRNAs taking charge.

Most bacterial toxins derived from chromosomally encoded toxin-antitoxin (TA) systems that have been studied to date appear to protect cells from relatively short pulses of stress by triggering a reversible state of growth arrest. In contrast to many bacterial toxins that are produced as defense mechanisms and secreted from their hosts, TA toxins exert their protective effect from within the cell that produces them. TA toxin-mediated growth arrest is most frequently achieved through their ability to selectively cleave RNA species that participate in protein synthesis.

Growth-regulating Mycobacterium tuberculosis VapC-mt4 toxin is an isoacceptor-specific tRNase.

Toxin-antitoxin (TA) systems are implicated in the downregulation of bacterial cell growth associated with stress survival and latent tuberculosis infection, yet the activities and intracellular targets of these TA toxins are largely uncharacterized. Here, we use a specialized RNA-seq approach to identify targets of a Mycobacterium tuberculosis VapC TA toxin, VapC-mt4 (also known as VapC4), which have eluded detection using conventional approaches. Distinct from the one other characterized VapC toxin in M.

An RNA-seq method for defining endoribonuclease cleavage specificity identifies dual rRNA substrates for toxin MazF-mt3.

Toxin-antitoxin (TA) systems are widespread in prokaryotes. Among these, the mazEF TA system encodes an endoribonucleolytic toxin, MazF, that inhibits growth by sequence-specific cleavage of single-stranded RNA. Defining the physiological targets of a MazF toxin first requires the identification of its cleavage specificity, yet the current toolkit is antiquated and limited.

23S rRNA as an a-Maz-ing new bacterial toxin target.

Mycobacterium tuberculosis, the causative agent of tuberculosis in humans, is a bacterium with the unique ability to persist for years or decades as a latent infection. This latent state, during which bacteria have a markedly altered physiology and are thought to be dormant, is crucial for the bacteria to survive the stressful environments it encounters in the human host. Importantly, M.

Doc toxin is a kinase that inactivates elongation factor Tu.

The Doc toxin from bacteriophage P1 (of the phd-doc toxin-antitoxin system) has served as a model for the family of Doc toxins, many of which are harbored in the genomes of pathogens. We have shown previously that the mode of action of this toxin is distinct from the majority derived from toxin-antitoxin systems: it does not cleave RNA; in fact P1 Doc expression leads to mRNA stabilization. However, the molecular triggers that lead to translation arrest are not understood.

Mycobacterial toxin MazF-mt6 inhibits translation through cleavage of 23S rRNA at the ribosomal A site.

The Mycobacterium tuberculosis genome contains an unusually high number of toxin-antitoxin modules, some of which have been suggested to play a role in the establishment and maintenance of latent tuberculosis. Nine of these toxin-antitoxin loci belong to the mazEF family, encoding the intracellular toxin MazF and its antitoxin inhibitor MazE. Nearly every MazF ortholog recognizes a unique three- or five-base RNA sequence and cleaves mRNA.

Clostridium difficile MazF toxin exhibits selective, not global, mRNA cleavage.

Clostridium difficile is an important, emerging nosocomial pathogen. The transition from harmless colonization to disease is typically preceded by antimicrobial therapy, which alters the balance of the intestinal flora, enabling C. difficile to proliferate in the colon.

Growth and translation inhibition through sequence-specific RNA binding by Mycobacterium tuberculosis VapC toxin.

The Mycobacterium tuberculosis genome harbors an unusually large number of toxin-antitoxin (TA) modules. Curiously, over half of these are VapBC (virulence-associated protein) family members. Nonetheless, the cellular target, precise mode of action, and physiological role of the VapC toxins in this important pathogen remain unclear.

Bacterial toxin RelE mediates frequent codon-independent mRNA cleavage from the 5' end of coding regions in vivo.

The enzymatic activity of the RelE bacterial toxin component of the Escherichia coli RelBE toxin-antitoxin system has been extensively studied in vitro and to a lesser extent in vivo. These earlier reports revealed that 1) RelE alone does not exhibit mRNA cleavage activity, 2) RelE mediates mRNA cleavage through its association with the ribosome, 3) RelE-mediated mRNA cleavage occurs at the ribosomal A site and, 4) Cleavage of mRNA by RelE exhibits high codon specificity. More specifically, RelE exhibits a preference for the stop codons UAG and UGA and sense codons CAG and UCG in vitro.

Noncognate Mycobacterium tuberculosis toxin-antitoxins can physically and functionally interact.

The Mycobacterium tuberculosis genome harbors a striking number (>40) of toxin-antitoxin systems. Among them are at least seven MazF orthologs, designated MazF-mt1 through MazF-mt7, four of which have been demonstrated to function as mRNA interferases that selectively target mRNA for cleavage at distinct consensus sequences. As is characteristic of all toxin-antitoxin systems, each of the mazF-mt toxin genes is organized in an operon downstream of putative antitoxin genes.

Crystal structures of Phd-Doc, HigA, and YeeU establish multiple evolutionary links between microbial growth-regulating toxin-antitoxin systems.

Bacterial toxin-antitoxin (TA) systems serve a variety of physiological functions including regulation of cell growth and maintenance of foreign genetic elements. Sequence analyses suggest that TA families are linked by complex evolutionary relationships reflecting likely swapping of functional domains between different TA families. Our crystal structures of Phd-Doc from bacteriophage P1, the HigA antitoxin from Escherichia coli CFT073, and YeeU of the YeeUWV systems from E.

Bacterial toxin HigB associates with ribosomes and mediates translation-dependent mRNA cleavage at A-rich sites.

Most pathogenic Proteus species are primarily associated with urinary tract infections, especially in persons with indwelling catheters or functional/anatomic abnormalities of the urinary tract. Urinary tract infections caused by Proteus vulgaris typically form biofilms and are resistant to commonly used antibiotics. The Rts1 conjugative plasmid from a clinical isolate of P.

Bacterial toxin YafQ is an endoribonuclease that associates with the ribosome and blocks translation elongation through sequence-specific and frame-dependent mRNA cleavage.

Toxin-antitoxin (TA) systems on the chromosomes of free-living bacteria appear to facilitate cell survival during intervals of stress by inducing a state of reversible growth arrest. However, upon prolonged stress, TA toxin action leads to cell death. They have been implicated in several clinically important phenomena--bacterial persistence during antibiotic treatment, biofilm formation and bacterial pathogenesis--and serve as attractive new antibiotic targets for pathogens.

Bacterial addiction module toxin Doc inhibits translation elongation through its association with the 30S ribosomal subunit.

Bacterial toxin-antitoxin (TA) systems (or "addiction modules") typically facilitate cell survival during intervals of stress by inducing a state of reversible growth arrest. However, upon prolonged stress, TA toxin action leads to cell death. TA systems have also been implicated in several clinically important phenomena: biofilm formation, bacterial persistence during antibiotic treatment, and bacterial pathogenesis.

Single protein production in living cells facilitated by an mRNA interferase.

We designed a single-protein production (SPP) system in living E. coli cells that exploits the unique properties of MazF, a bacterial toxin that is an ssRNA- and ACA-specific endoribonuclease. In effect, MazF functions as an "mRNA interferase," because it efficiently and selectively degrades all cellular mRNAs in vivo, resulting in a precipitous drop in total protein synthesis.