Analysis of Non-Typeable Haemophilous influenzae VapC1 Mutations Reveals Structural Features Required for Toxicity and Flexibility in the Active Site

Bacteria have evolved mechanisms that allow them to survive in the face of a variety of stresses including nutrient deprivation, antibiotic challenge and engulfment by predator cells. A switch to dormancy represents one strategy that reduces energy utilization and can render cells resistant to compounds that kill growing bacteria. These persister cells pose a problem during treatment of infections with antibiotics, and dormancy mechanisms may contribute to latent infections. Many bacteria encode toxin-antitoxin (TA) gene pairs that play an important role in dormancy and the formation of persisters. VapBC gene pairs comprise the largest of the Type II TA systems in bacteria and they produce a VapC ribonuclease toxin whose activity is inhibited by the VapB antitoxin. Despite the importance of VapBC TA pairs in dormancy and persister formation, little information exists on the structural features of VapC proteins required for their toxic function in vivo. Studies reported here identified 17 single mutations that disrupt the function of VapC1 from non-typeable H. influenzae in vivo. 3-D modeling suggests that side chains affected by many of these mutations sit near the active site of the toxin protein. Phylogenetic comparisons and secondary mutagenesis indicate that VapC1 toxicity requires an alternative active site motif found in many proteobacteria. Expression of the antitoxin VapB1 counteracts the activity of VapC1 mutants partially defective for toxicity, indicating that the antitoxin binds these mutant proteins in vivo. These findings identify critical chemical features required for the biological function of VapC toxins and PIN-domain proteins.     

Structural and functional studies of the Mycobacterium tuberculosis VapBC30 toxin-antitoxin system: implications for the design of novel antimicrobial peptides

Toxin-antitoxin (TA) systems play important roles in bacterial physiology, such as multidrug tolerance, biofilm formation, and arrest of cellular growth under stress conditions. To develop novel antimicrobial agents against tuberculosis, we focused on VapBC systems, which encompass more than half of TA systems in Mycobacterium tuberculosis. Here, we report that theMycobacterium tuberculosis VapC30 toxin regulates cellular growth through both magnesium and manganese ion-dependent ribonuclease activity and is inhibited by the cognate VapB30 antitoxin. We also determined the 2.7-Å resolution crystal structure of the M. tuberculosis VapBC30 complex, which revealed a novel process of inactivation of the VapC30 toxin via swapped blocking by the VapB30 antitoxin. Our study on M. tuberculosis VapBC30 leads us to design two kinds of VapB30 and VapC30-based novel peptides which successfully disrupt the toxin-antitoxin complex and thus activate the ribonuclease activity of the VapC30 toxin. Our discovery herein possibly paves the way to treat tuberculosis for next generation.     

VapC toxins from Mycobacterium tuberculosis are ribonucleases that differentially inhibit growth and are neutralized by cognate VapB antitoxins

The chromosome of Mycobacterium tuberculosis (Mtb) encodes forty seven toxin-antitoxin modules belonging to the VapBC family. The role of these modules in the physiology of Mtb and the function(s) served by their expansion are unknown. We investigated ten vapBC modules from Mtb and the single vapBC from M. smegmatis. Of the Mtb vapCs assessed, only Rv0549c, Rv0595c, Rv2549c and Rv2829c were toxic when expressed from a tetracycline-regulated promoter in M. smegmatis. The same genes displayed toxicity when conditionally expressed in Mtb. Toxicity of Rv2549c in M. smegmatis correlated with the level of protein expressed, suggesting that the VapC level must exceed a threshold for toxicity to be observed. In addition, the level of Rv2456 protein induced in M. smegmatis was markedly lower than Rv2549c, which may account for the lack of toxicity of this and other VapCs scored as 'non-toxic'. The growth inhibitory effects of toxic VapCs were neutralized by expression of the cognate VapB as part of a vapBC operon or from a different chromosomal locus, while that of non-cognate antitoxins did not. These results demonstrated a specificity of interaction between VapCs and their cognate VapBs, a finding corroborated by yeast two-hybrid analyses. Deletion of selected vapC or vapBC genes did not affect mycobacterial growth in vitro, but rendered the organisms more susceptible to growth inhibition following toxic VapC expression. However, toxicity of 'non-toxic' VapCs was not unveiled in deletion mutant strains, even when the mutation eliminated the corresponding cognate VapB, presumably due to insufficient levels of VapC protein. Together with the ribonuclease (RNase) activity demonstrated for Rv0065 and Rv0617--VapC proteins with similarity to Rv0549c and Rv3320c, respectively--these results suggest that the VapBC family potentially provides an abundant source of RNase activity in Mtb, which may profoundly impact the physiology of the organism.     

Determination of ribonuclease sequence-specificity using Pentaprobes and mass spectrometry

The VapBC toxin-antitoxin (TA) family is the largest of nine identified TA families. The toxin, VapC, is a metal-dependent ribonuclease that is inhibited by its cognate antitoxin, VapB. Although the VapBCs are the largest TA family, little is known about their biological roles. Here we describe a new general method for the overexpression and purification of toxic VapC proteins and subsequent determination of their RNase sequence-specificity. Functional VapC was isolated by expression of the nontoxic VapBC complex, followed by removal of the labile antitoxin (VapB) using limited trypsin digestion. We have then developed a sensitive and robust method for determining VapC ribonuclease sequence-specificity. This technique employs the use of Pentaprobes as substrates for VapC. These are RNA sequences encoding every combination of five bases. We combine the RNase reaction with MALDI-TOF MS to detect and analyze the cleavage products and thus determine the RNA cut sites. Successful MALDI-TOF MS analysis of RNA fragments is acutely dependent on sample preparation methods. The sequence-specificity of four VapC proteins from two different organisms (VapC(PAE0151) and VapC(PAE2754) from Pyrobaculum aerophilum, and VapC(Rv0065) and VapC(Rv0617) from Mycobacterium tuberculosis) was successfully determined using the described strategy. This rapid and sensitive method can be applied to determine the sequence-specificity of VapC ribonucleases along with other RNA interferases (such as MazF) from a range of organisms.     

In Silico Derived Peptides for Inhibiting the Toxin–Antitoxin Systems of Mycobacterium tuberculosis: Basis for Developing Peptide-Based Therapeutics

Toxin–antitoxin (TA) systems of Mycobacterium tuberculosis (Mtb) is a prerequisite for the bacterium to survive in extreme conditions. Antimicrobial peptides inhibiting the formation of these complexes provide a novel strategy for TB drug discovery process. Absence of TA genes in human, makes these systems as an attractive target for drug development. In this study using Peptiderive server, we have derived a number of potential inhibitory peptides for nine TA complexes—VapBC3, VapBC5, VapBC11, VapBC15, VapBC26, VapBC30, RelBE2, RelJK, MazEF4 of Mtb. We have studied about the common interacting toxin residues with the antitoxin and with the derived peptide. Further, using Cluspro server, we compared the binding efficacy of the in silico derived peptides with the published potential peptides for the toxins VapC26, VapC30 and MazF. Thus, these in silico derived peptides would serve as basis for developing peptide based therapeutics for TA complexes of Mtb.

     

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. However, only one of the seven putative upstream antitoxins (designated MazE-mt1 through MazE-mt7) has significant sequence similarity to Escherichia coli MazE, the cognate antitoxin for E. coli MazF. Interestingly, the M. tuberculosis genome contains two independent operons encoding E. coli MazE orthologs, but they are not paired with mazF-mt-like genes. Instead, the genes encoding these two MazE orthologs are each paired with proteins containing a PIN domain, indicating that they may be members of the very large VapBC toxin-antitoxin family. We tested a spectrum of pair-wise combinations of cognate and noncognate Mtb toxin-antitoxins using in vivo toxicity and rescue experiments along with in vitro interaction experiments. Surprisingly, we uncovered several examples of noncognate toxin-antitoxin association, even among different families (e.g. MazF toxins and VapB antitoxins). These results challenge the “one toxin for one antitoxin” dogma and suggest that M. tuberculosis may enlist a sophisticated toxin-antitoxin network to alter its physiology in response to environmental cues.     

Characterization of a Chromosomal Type II Toxin–Antitoxin System mazEaFa in the Cyanobacterium Anabaena sp. PCC 7120

Cyanobacteria have evolved to survive stressful environmental changes by regulating growth, however, the underlying mechanism for this is obscure. The ability of chromosomal type II toxin-antitoxin (TA) systems to modulate growth or cell death has been documented in a variety of prokaryotes. A chromosomal mazEaFa locus of Anabaena sp. PCC 7120 has been predicted as a putative mazEF TA system. Here we demonstrate that mazEaFa form a bicistronic operon that is co-transcribed under normal growth conditions. Overproduction of MazFa induced Anabaena growth arrest which could be neutralized by co-expression of MazEa. MazFa also inhibited the growth of Escherichia coli cells, and this effect could be overcome by simultaneous or subsequent expression of MazEa via formation of the MazEa-MazFa complex in vivo, further confirming the nature of the mazEaFa locus as a type II TA system. Interestingly, like most TA systems, deletion of mazEaFa had no effect on the growth of Anabaena during the tested stresses. Our data suggest that mazEaFa, or together with other chromosomal type II TA systems, may promote cells to cope with particular stresses by inducing reversible growth arrest of Anabaena.