Mechanism of regulation and neutralization of the AtaR-AtaT toxin-antitoxin system.

GCN5-related N-acetyl-transferase (GNAT)-like enzymes from toxin-antitoxin modules are strong inhibitors of protein synthesis. Here, we present the bases of the regulatory mechanisms of ataRT, a model GNAT-toxin-antitoxin module, from toxin synthesis to its action as a transcriptional de-repressor. We show the antitoxin (AtaR) traps the toxin (AtaT) in a pre-catalytic monomeric state and precludes the effective binding of ac-CoA and its target Met-transfer RNAfMet. In the repressor complex, AtaR intrinsically disordered region interacts with AtaT at two different sites, folding into different structures, that are involved in two separate functional roles, toxin neutralization and placing the DNA-binding domains of AtaR in a binding-compatible orientation. Our data suggests AtaR neutralizes AtaT as a monomer, right after its synthesis and only the toxin-antitoxin complex formed in this way is an active repressor. Once activated by dimerization, later neutralization of the toxin results in a toxin-antitoxin complex that is not able to repress transcription.

The Streptococcus pneumoniaeyefM-yoeB and relBE Toxin-Antitoxin Operons Participate in Oxidative Stress and Biofilm Formation.

Type II (proteic) toxin-antitoxin systems (TAs) are widely distributed among bacteria and archaea. They are generally organized as operons integrated by two genes, the first encoding the antitoxin that binds to its cognate toxin to generate a harmless protein⁻protein complex. Under stress conditions, the unstable antitoxin is degraded by host proteases, releasing the toxin to achieve its toxic effect. In the Gram-positive pathogen Streptococcus pneumoniae we have characterized four TAs: pezAT, relBE, yefM-yoeB, and phD-doc, although the latter is missing in strain R6. We have assessed the role of the two yefM-yoeB and relBE systems encoded by S. pneumoniae R6 by construction of isogenic strains lacking one or two of the operons, and by complementation assays. We have analyzed the phenotypes of the wild type and mutants in terms of cell growth, response to environmental stress, and ability to generate biofilms. Compared to the wild-type, the mutants exhibited lower resistance to oxidative stress. Further, strains deleted in yefM-yoeB and the double mutant lacking yefM-yoeB and relBE exhibited a significant reduction in their ability for biofilm formation. Complementation assays showed that defective phenotypes were restored to wild type levels. We conclude that these two loci may play a relevant role in these aspects of the S. pneumoniae lifestyle and contribute to the bacterial colonization of new niches.

The Mycobacterium tuberculosis relBE toxin:antitoxin genes are stress-responsive modules that regulate growth through translation inhibition.

Toxin-antitoxin (TA) genes are ubiquitous among bacteria and are associated with persistence and dormancy. Following exposure to unfavorable environmental stimuli, several species (Escherichia coli, Staphylococcus aureus, Myxococcus xanthus) employ toxin proteins such as RelE and MazF to downregulate growth or initiate cell death. Mycobacterium tuberculosis possesses three Rel TA modules (Rel Mtb ): RelBE Mtb , RelFG Mtb and RelJK Mtb (Rv1246c-Rv1247c, Rv2865-Rv2866, and Rv3357-Rv3358, respectively), which inhibit mycobacterial growth when the toxin gene (relE, relG, relK) is expressed independently of the antitoxin gene (relB, relF, relJ). In the present study, we examined the in vivo mechanism of the RelE Mtb toxin protein, the impact of RelE Mtb on M. tuberculosis physiology and the environmental conditions that regulate all three rel Mtb modules. RelE Mtb negatively impacts growth and the structural integrity of the mycobacterial envelope, generating cells with aberrant forms that are prone to extensive aggregation. At a time coincident with growth defects, RelE Mtb mediates mRNA degradation in vivo resulting in significant changes to the proteome. We establish that rel Mtb modules are stress responsive, as all three operons are transcriptionally activated following mycobacterial exposure to oxidative stress or nitrogen-limiting growth environments. Here we present evidence that the rel Mtb toxin:antitoxin family is stress-responsive and, through the degradation of mRNA, the RelE Mtb toxin influences the growth, proteome and morphology of mycobacterial cells.

Cut to the chase--Regulating translation through RNA cleavage.

Activation of toxin-antitoxin (TA) systems provides an important mechanism for bacteria to adapt to challenging and ever changing environmental conditions. Known TA systems are classified into five families based on the mechanisms of antitoxin inhibition and toxin activity. For type II TA systems, the toxin is inactivated in exponentially growing cells by tightly binding its antitoxin partner protein, which also serves to regulate cellular levels of the complex through transcriptional auto-repression. During cellular stress, however, the antitoxin is degraded thus freeing the toxin, which is then able to regulate central cellular processes, primarily protein translation to adjust cell growth to the new conditions. In this review, we focus on the type II TA pairs that regulate protein translation through cleavage of ribosomal, transfer, or messenger RNA.

Toxin-Antitoxin systems: their role in persistence, biofilm formation, and pathogenicity.

One of the most pertinent recent outcomes of molecular microbiology efforts to understand bacterial behavior is the discovery of a wide range of toxin-antitoxin (TA) systems that are tightly controlling bacterial persistence. While TA systems were originally linked to control over the genetic material, for example plasmid maintenance, it is now clear that they are involved in essential cellular processes like replication, gene expression, and cell wall synthesis. Toxin activity is induced stochastically or after environmental stimuli, resulting in silencing of the above-mentioned biological processes and entry in a dormant state. In this minireview, we highlight the recent developments in research on these intriguing systems with a focus on their role in biofilms and in bacterial virulence. We discuss their potential as targets in antimicrobial drug discovery.

Molecular mechanisms of multiple toxin-antitoxin systems are coordinated to govern the persister phenotype.

Toxin-antitoxin systems are ubiquitous and have been implicated in persistence, the multidrug tolerance of bacteria, biofilms, and, by extension, most chronic infections. However, their purpose, apparent redundancy, and coordination remain topics of debate. Our model relates molecular mechanisms to population dynamics for a large class of toxin-antitoxin systems and suggests answers to several of the open questions. The generic architecture of toxin-antitoxin systems provides the potential for bistability, and even when the systems do not exhibit bistability alone, they can be coupled to create a strongly bistable, hysteretic switch between normal and toxic states. Stochastic fluctuations can spontaneously switch the system to the toxic state, creating a heterogeneous population of growing and nongrowing cells, or persisters, that exist under normal conditions, rather than as an induced response. Multiple toxin-antitoxin systems can be cooperatively marshaled for greater effect, with the dilution determined by growth rate serving as the coordinating signal. The model predicts and elucidates experimental results that show a characteristic correlation between persister frequency and the number of toxin-antitoxin systems.

Lytic activity of the Vibrio cholerae type VI secretion toxin VgrG-3 is inhibited by the antitoxin TsaB.

The type VI secretion system (T6SS) of Gram-negative bacteria has been implicated in microbial competition; however, which components serve purely structural roles, and which serve as toxic effectors remains unresolved. Here, we present evidence that VgrG-3 of the Vibrio cholerae T6SS has both structural and toxin activity. Specifically, we demonstrate that the C-terminal extension of VgrG-3 acts to degrade peptidoglycan and hypothesize that this assists in the delivery of accessory T6SS toxins of V. cholerae. To avoid self-intoxication, V. cholerae expresses an anti-toxin encoded immediately downstream of vgrG-3 that inhibits VgrG-3-mediated lysis through direct interaction.

ε/ζ systems: their role in resistance, virulence, and their potential for antibiotic development.

Cell death in bacteria can be triggered by activation of self-inflicted molecular mechanisms. Pathogenic bacteria often make use of suicide mechanisms in which the death of individual cells benefits survival of the population. Important elements for programmed cell death in bacteria are proteinaceous toxin-antitoxin systems. While the toxin generally resides dormant in the bacterial cytosol in complex with its antitoxin, conditions such as impaired de novo synthesis of the antitoxin or nutritional stress lead to antitoxin degradation and toxin activation. A widespread toxin-antitoxin family consists of the ε/ζ systems, which are distributed over plasmids and chromosomes of various pathogenic bacteria. In its inactive state, the bacteriotoxic ζ toxin protein is inhibited by its cognate antitoxin ε. Upon degradation of ε, the ζ toxin is released allowing this enzyme to poison bacterial cell wall synthesis, which eventually triggers autolysis. ε/ζ systems ensure stable plasmid inheritance by inducing death in plasmid-deprived offspring cells. In contrast, chromosomally encoded ε/ζ systems were reported to contribute to virulence of pathogenic bacteria, possibly by inducing autolysis in individual cells under stressful conditions. The capability of toxin-antitoxin systems to kill bacteria has made them potential targets for new therapeutic compounds. Toxin activation could be hijacked to induce suicide of bacteria. Likewise, the unique mechanism of ζ toxins could serve as template for new drugs. Contrarily, inhibition of virulence-associated ζ toxins might attenuate infections. Here we provide an overview of ε/ζ toxin-antitoxin family and its potential role in the development of new therapeutic approaches in microbial defense.

Antitoxin MqsA helps mediate the bacterial general stress response.

Although it is well recognized that bacteria respond to environmental stress through global networks, the mechanism by which stress is relayed to the interior of the cell is poorly understood. Here we show that enigmatic toxin-antitoxin systems are vital in mediating the environmental stress response. Specifically, the antitoxin MqsA represses rpoS, which encodes the master regulator of stress. Repression of rpoS by MqsA reduces the concentration of the internal messenger 3,5-cyclic diguanylic acid, leading to increased motility and decreased biofilm formation. Furthermore, the repression of rpoS by MqsA decreases oxidative stress resistance via catalase activity. Upon oxidative stress, MqsA is rapidly degraded by Lon protease, resulting in induction of rpoS. Hence, we show that external stress alters gene regulation controlled by toxin-antitoxin systems, such that the degradation of antitoxins during stress leads to a switch from the planktonic state (high motility) to the biofilm state (low motility).

pSM19035-encoded zeta toxin induces stasis followed by death in a subpopulation of cells.

The toxin-antitoxin operon of pSM19035 encodes three proteins: the omega global regulator, the epsilon labile antitoxin and the stable zeta toxin. Accumulation of zeta toxin free of epsilon antitoxin induced loss of cell proliferation in both Bacillus subtilis and Escherichia coli cells. Induction of a zeta variant (zetaY83C) triggered stasis, in which B. subtilis cells were viable but unable to proliferate, without selectively affecting protein translation. In E. coli cells, accumulation of free zeta toxin induced stasis, but this was fully reversed by expression of the epsilon antitoxin within a defined time window. The time window for reversion of zeta toxicity by expression of epsilon antitoxin was dependent on the initial cellular level of zeta. After 240 min of constitutive expression, or inducible expression of high levels of zeta toxin for 30 min, expression of epsilon failed to reverse the toxic effect exerted by zeta in cells growing in minimal medium. Under the latter conditions, zeta inhibited replication, transcription and translation and finally induced death in a fraction (approximately 50 %) of the cell population. These results support the view that zeta interacts with its specific target and reversibly inhibits cell proliferation, but accumulation of zeta might lead to cell death due to pleiotropic effects.

The PIN-domain toxin-antitoxin array in mycobacteria.

PIN-domains (homologues of the pilT N-terminal domain) are small protein domains of approximately 140 amino acids. They are found in a diverse range of organisms and recent evidence from bioinformatics, biochemistry, structural biology and microbiology suggest that the majority of the prokaryotic PIN-domain proteins are the toxic components of toxin-antitoxin (TA) operons. Several microorganisms have a large cohort of these operons. For example, the genome of Mycobacterium tuberculosis encodes 48 PIN-domain proteins, of which 38 are thought to be involved in TA interactions. This large array of PIN-domain TA operons raises questions as to their evolutionary origin and contemporary functional significance. We suggest that the evolutionary origin of genes encoding mycobacterial PIN-domain TA operons is linked to the mobile gene pool, but that TA operons can become resident within the chromosome of host cells from where they might be recruited to fulfil a variety of roles associated with retardation of cell growth and persistence in stressful environments.

Mycelial culture of Phellinus linteus protects primary cultured rat hepatocytes against hepatotoxins.

Hepatoprotective activity of Phellinus linteus was studied using H(2)O(2)- or galactosamine-injured primary cultures of rat hepatocytes as screening systems. The methanolic extract of the mycelial culture of Phellinus linteus significantly protected against hepatotoxins-induced toxicity in primary cultured rat hepatocytes as seen from the decreased level of glutamic pyruvic transaminase released from the injured hepatocytes. The methanolic extract of the mycelial culture of Phellinus linteus was subsequently fractionated with n-hexane, ethyl acetate, n-butanol and water. Among these fractions, 100 microg/mL of the ethyl acetate fraction was the most active one. The relative protections were 68.9 +/- 5.3% in H(2)O(2)-injured hepatocytes and 46.8 +/- 3.9% in galactosamine-injured hepatocytes, respectively. The ethyl acetate fraction appeared to maintain the glutathione level which was decreased by the treatment of H(2)O(2) or galactosamine and restored the level of RNA synthesis more than two times compared to galactosamine-injured hepatocytes. These results suggest that the ethyl acetate fraction of the mycelial culture of Phellinus linteus protects hepatocytes from H(2)O(2)- or galactosamine-induced injury by maintaining hepatic glutathione level and RNA synthesis as well.

Recombinant antitoxic and antiinflammatory factor from the nonvenomous snake Python reticulatus: phospholipase A2 inhibition and venom neutralizing potential.

From the serum of the nonvenomous snake Python reticulatus, a new phospholipase A(2) (PLA(2)) inhibitor termed phospholipase inhibitor from python (PIP) was purified by sequential chromatography and cloned to elucidate its primary structure and fundamental biochemical characteristics. A cDNA clone encoding PIP was isolated from the liver total RNA by reverse transcriptase-polymerase chain reaction (RT-PCR). It contained a 603 bp open reading frame that encoded a 19-residue signal sequence and a 182-residue protein. PIP showed about 60% sequence homology with those PLA(2) inhibitors having a urokinase-type plasminogen activator receptor-like domain structure. PIP was also functionally expressed as a fusion protein in Escherichia coli to explore its potential therapeutic significance. The recombinant PIP was shown to be identical to the native form in chromatographic behavior and biochemical characteristics. Both the native and recombinant PIP appear to exist as a hexamer of 23-kDa subunits having an apparent molecular mass of approximately 140 kDa. PIP showed ability to bind to the major PLA(2) toxin (daboiatoxin, DbTx) of Daboia russelli siamensis at 1-2-fold molar excess of inhibitor to toxin. It exhibited broad spectra in neutralizing the toxicity of various snake venoms and toxins and inhibited the formation of edema in mice. Our data demonstrate the venom neutralizing potential of the recombinant PIP and suggest that the proline-rich hydrophobic core region may play a role in binding to PLA(2).

Vitronectin and its fragments purified as serum inhibitors of Staphylococcus aureus gamma-hemolysin and leukocidin, and their specific binding to the hlg2 and the LukS components of the toxins.

Staphylococcal gamma-hemolysin and leukocidin are bi-component cytolysins, consisting of LukF (or Hlg1)/Hlg2 and LukF/LukS, respectively. Here, we purified serum inhibitors of gamma-hemolysin and leukocidin from human plasma. Protein sequencing showed that the purified inhibitors of 62, 57, 50 and 38 kDa were the vitronectin fragments with truncation(s) of the C-terminal or both N- and C-terminal regions. The purified vitronectin fragments specifically bound to the Hlg2 component of gamma-hemolysin and the LukS component of leukocidin to form high-molecular-weight complexes with them, leading to inhibition of the toxin-induced lysis of human erythrocytes and human polymorphonuclear leukocytes, respectively. Intact vitronectin also showed inhibitory activity to the toxins. The ability of gamma-hemolysin and leukocidin to bind vitronectin and its fragments is a novel function of the pore-forming cytolysins.

Synthetic peptides of Shiga toxin B subunit induce antibodies which neutralize its biological activity.

Shiga toxin B chain, the binding subunit of Shiga toxin, was recently purified; and the amino acid sequence of this 7,716-dalton polypeptide was determined (N.G. Seidah, A. Donohue-Rolfe, C. Lazure, F. Auclair, G. T. Keusch, and M. Chretien, J. Biol. Chem. 261:13928-13931, 1986). In the present study, synthetic peptides corresponding to three overlapping sequences from the N-terminal region of this subunit were prepared. The peptides synthesized consisted of residues 5 to 18, 13 to 26, and 7 to 26. This region coincides with the major peak of hydrophilicity and surface area residues predicted from a computer analysis. For the purpose of immunization, the peptides either were conjugated with a protein or synthetic carrier or were polymerized with glutaraldehyde. Antisera against these peptide derivatives raised in rabbits reacted not only with the respective homologous peptide but also to a comparable extent with the intact Shiga toxin. The anti-peptide antisera effectively neutralized the various biological activities of the Shiga toxin, namely, cytotoxicity to HeLa cells, enterotoxic activity (the fluid secretion into ligated ileal loops in rats), and neurotoxicity in mice. Furthermore, active immunization with the peptide conjugates was found to protect mice against the lethal effect of Shiga toxin.