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.

     

The Doc toxin and Phd antidote proteins of the bacteriophage P1 plasmid addiction system form a heterotrimeric complex.

The toxin (Doc) and antidote (Phd) proteins of the plasmid addiction system of bacteriophage P1 were purified as a complex. Cocrystals of the complex contained a 2:1 molar ratio of Phd:Doc as assayed by dye binding following SDS-polyacrylamide gel electrophoresis and as determined by amino acid analysis. Gel filtration and analytical ultracentrifugation revealed that the two addiction proteins interact in solution to form a P2D trimer composed of one Doc and two Phd molecules. These results support a model in which Phd inhibits the toxic activity of Doc by direct binding. Circular dichroism experiments showed that changes in secondary structure accompany formation of the heterotrimeric complex, raising the possibility that Phd may act by an allosteric mechanism. Studies of Phd and Doc molecules labeled with fluorescent energy donor and acceptor groups gave an equilibrium dissociation constant of about 0.8 microM(2) and a very short, sub second half-life of complex dissociation. As a consequence, low concentrations of free Doc toxin are likely to be present both transiently and in the steady state in cells containing the Phd antidote, making mechanisms of single-hit Doc toxicity improbable.     

Doc of prophage P1 is inhibited by its antitoxin partner Phd through fold complementation

Prokaryotic toxin-antitoxin modules are involved in major physiological events set in motion under stress conditions. The toxin Doc (death on curing) from the phd/doc module on phage P1 hosts the C-terminal domain of its antitoxin partner Phd (prevents host death) through fold complementation. This Phd domain is intrinsically disordered in solution and folds into an alpha-helix upon binding to Doc. The details of the interactions reveal the molecular basis for the inhibitory action of the antitoxin. The complex resembles the Fic (filamentation induced by cAMP) proteins and suggests a possible evolutionary origin for the phd/doc operon. Doc induces growth arrest of Escherichia coli cells in a reversible manner, by targeting the protein synthesis machinery. Moreover, Doc activates the endogenous E. coli RelE mRNA interferase but does not require this or any other known chromosomal toxin-antitoxin locus for its action in vivo.     

The chromosomal mazEF locus of Streptococcus mutans encodes a functional type II toxin-antitoxin addiction system

Type II chromosomal toxin-antitoxin (TA) modules consist of a pair of genes that encode two components: a stable toxin and a labile antitoxin interfering with the lethal action of the toxin through protein complex formation. Bioinformatic analysis of Streptococcus mutans UA159 genome identified a pair of linked genes encoding a MazEF-like TA. Our results show that S. mutans mazEF genes form a bicistronic operon that is cotranscribed from a σ70-like promoter. Overproduction of S. mutans MazF toxin had a toxic effect on S. mutans which can be neutralized by coexpression of its cognate antitoxin, S. mutans MazE. Although mazF expression inhibited cell growth, no cell lysis of S. mutans cultures was observed under the conditions tested. The MazEF TA is also functional in E. coli, where S. mutans MazF did not kill the cells but rather caused reversible cell growth arrest. Recombinant S. mutans MazE and MazF proteins were purified and were shown to interact with each other in vivo, confirming the nature of this TA as a type II addiction system. Our data indicate that MazF is a toxic nuclease arresting cell growth through the mechanism of RNA cleavage and that MazE inhibits the RNase activity of MazF by forming a complex. Our results suggest that the MazEF TA module might represent a cell growth modulator facilitating the persistence of S. mutans under the harsh conditions of the oral cavity.     

Adenine aminohydrolase from Leishmania donovani: unique enzyme in parasite purine metabolism

Adenine aminohydrolase (AAH) is an enzyme that is not present in mammalian cells and is found exclusively in Leishmania among the protozoan parasites that infect humans. AAH plays a paramount role in purine metabolism in this genus by steering 6-aminopurines into 6-oxypurines. Leishmania donovani AAH is 38 and 23% identical to Saccharomyces cerevisiae AAH and human adenosine deaminase enzymes, respectively, catalyzes adenine deamination to hypoxanthine with an apparent K(m) of 15.4 μM, and does not recognize adenosine as a substrate. Western blot analysis established that AAH is expressed in both life cycle stages of L. donovani, whereas subcellular fractionation and immunofluorescence studies confirmed that AAH is localized to the parasite cytosol. Deletion of the AAH locus in intact parasites established that AAH is not an essential gene and that Δaah cells are capable of salvaging the same range of purine nucleobases and nucleosides as wild type L. donovani. The Δaah null mutant was able to infect murine macrophages in vitro and in mice, although the parasite loads in both model systems were modestly reduced compared with wild type infections. The Δaah lesion was also introduced into a conditionally lethal Δhgprt/Δxprt mutant in which viability was dependent on pharmacologic ablation of AAH by 2'-deoxycoformycin. The Δaah/Δhgprt/Δxprt triple knock-out no longer required 2'-deoxycoformycin for growth and was avirulent in mice with no persistence after a 4-week infection. These genetic studies underscore the paramount importance of AAH to purine salvage by L. donovani.     

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.     

大肠埃希菌TA系统mazEF的研究进展

大部分细菌的遗传物质中含有毒素-抗毒素系统(TA)的遗传基因.mazEF是大肠埃希菌染色体上的一对毒素抗毒素基因,由毒素基因mazF和抗毒素基因mazE组成.其在细菌的生长调控和细胞程序性死亡中发挥了重要的作用.环境压力激活mazEF后,MazF可以通过对mRNA的剪切作用造成翻译停止.mazEF的存在可以增加细菌对环境压力的耐受性、保持细菌遗传物质的稳定、参与抗生素引起的细胞死亡、也在细菌的耐药性中发挥重要作用.     

The Intrinsically Disordered Domain of the Antitoxin Phd Chaperones the Toxin Doc against Irreversible Inactivation and Misfolding

The toxin Doc from the phd/doc toxin-antitoxin module targets the cellular translation machinery and is inhibited by its antitoxin partner Phd. Here we show that Phd also functions as a chaperone, keeping Doc in an active, correctly folded conformation. In the absence of Phd, Doc exists in a relatively expanded state that is prone to dimerization through domain swapping with its active site loop acting as hinge region. The domain-swapped dimer is not capable of arresting protein synthesis in vitro, whereas the Doc monomer is. Upon binding to Phd, Doc becomes more compact and is secured in its monomeric state with a neutralized active site.     

MazF-induced Growth Inhibition and Persister Generation in Escherichia coli

Toxin-antitoxin systems are ubiquitous in nature and present on the chromosomes of both bacteria and archaea. MazEF is a type II toxin-antitoxin system present on the chromosome of Escherichia coli and other bacteria. Whether MazEF is involved in programmed cell death or reversible growth inhibition and bacterial persistence is a matter of debate. In the present work the role of MazF in bacterial physiology was studied by using an inactive, active-site mutant of MazF, E24A, to activate WT MazF expression from its own promoter. The ectopic expression of E24A MazF in a strain containing WT mazEF resulted in reversible growth arrest. Normal growth resumed on inhibiting the expression of E24A MazF. MazF-mediated growth arrest resulted in an increase in survival of bacterial cells during antibiotic stress. This was studied by activation of mazEF either by overexpression of an inactive, active-site mutant or pre-exposure to a sublethal dose of antibiotic. The MazF-mediated persistence phenotype was found to be independent of RecA and dependent on the presence of the ClpP and Lon proteases. This study confirms the role of MazEF in reversible growth inhibition and persistence.     

Functional RelBE-Family Toxin-Antitoxin Pairs Affect Biofilm Maturation and Intestine Colonization in Vibrio cholerae

Toxin–antitoxin (TA) systems are small genetic elements that typically encode a stable toxin and its labile antitoxin. These cognate pairs are abundant in prokaryotes and have been shown to regulate various cellular functions. Vibrio cholerae, a human pathogen that is the causative agent of cholera, harbors at least thirteen TA loci. While functional HigBA, ParDE have been shown to stabilize plasmids and Phd/Doc to mediate cell death in V. cholerae, the function of seven RelBE-family TA systems is not understood. In this study we investigated the function of the RelBE TA systems in V. cholerae physiology and found that six of the seven relBE loci encoded functional toxins in E. coli. Deletion analyses of each relBE locus indicate that RelBE systems are involved in biofilm formation and reactive oxygen species (ROS) resistance. Interestingly, all seven relBE loci are induced under the standard virulence induction conditions and two of the relBE mutants displayed a colonization defect, which was not due to an effect on virulence gene expression. Although further studies are needed to characterize the mechanism of action, our study reveals that RelBE systems are important for V. cholerae physiology.     

Impact of the lectin chaperone calnexin on the stress response, virulence and proteolytic secretome of the fungal pathogen Aspergillus fumigatus

Calnexin is a membrane-bound lectin chaperone in the endoplasmic reticulum (ER) that is part of a quality control system that promotes the accurate folding of glycoproteins entering the secretory pathway. We have previously shown that ER homeostasis is important for virulence of the human fungal pathogen Aspergillus fumigatus, but the contribution of calnexin has not been explored. Here, we determined the extent to which A. fumigatus relies on calnexin for growth under conditions of environmental stress and for virulence. The calnexin gene, clxA, was deleted from A. fumigatus and complemented by reconstitution with the wild type gene. Loss of clxA altered the proteolytic secretome of the fungus, but had no impact on growth rates in either minimal or complex media at 37°C. However, the ΔclxA mutant was growth impaired at temperatures above 42°C and was hypersensitive to acute ER stress caused by the reducing agent dithiothreitol. In contrast to wild type A. fumigatus, ΔclxA hyphae were unable to grow when transferred to starvation medium. In addition, depleting the medium of cations by chelation prevented ΔclxA from sustaining polarized hyphal growth, resulting in blunted hyphae with irregular morphology. Despite these abnormal stress responses, the ΔclxA mutant remained virulent in two immunologically distinct models of invasive aspergillosis. These findings demonstrate that calnexin functions are needed for growth under conditions of thermal, ER and nutrient stress, but are dispensable for surviving the stresses encountered in the host environment.     

Use of a Gluconobacter frateurii mutant to prevent dihydroxyacetone accumulation during glyceric acid production from glycerol

To prevent dihydroxyacetone (DHA) by-production during glyceric acid (GA) production from glycerol using Gluconobacter frateurii, we used a G. frateurii THD32 mutant, ΔsldA, in which the glycerol dehydrogenase subunit-encoding gene (sldA) was disrupted, but ΔsldA grew much more slowly than the wild type, growth starting after a lag of 3 d under the same culture conditions. The addition of 1% w/v D-sorbitol to the medium improved both the growth and the GA productivity of the mutant, and ΔsldA produced 89.1 g/l GA during 4 d of incubation without DHA accumulation.     

Chromosomal bacterial type II toxin-antitoxin systems

Most prokaryotic chromosomes contain a number of toxin-antitoxin (TA) modules consisting of a pair of genes that encode 2 components, a stable toxin and its cognate labile antitoxin. TA systems are also known as addiction modules, since the cells become "addicted" to the short-lived antitoxin product (the unstable antitoxin is degraded faster than the more stable toxin) because its de novo synthesis is essential for their survival. While toxins are always proteins, antitoxins are either RNAs (type I, type III) or proteins (type II). Type II TA systems are widely distributed throughout the chromosomes of almost all free-living bacteria and archaea. The vast majority of type II toxins are mRNA-specific endonucleases arresting cell growth through the mechanism of RNA cleavage, thus preventing the translation process. The physiological role of chromosomal type II TA systems still remains the subject of debate. This review describes the currently known type II toxins and their characteristics. The different hypotheses that have been proposed to explain their role in bacterial physiology are also discussed.