Inhibits Toxin Activity Research Articles

Increasing the Scalability of Toxin-Intein Orthogonal Combinations.

Inteins are proteins embedded into host proteins from which they are excised in an autocatalytic reaction. Specifically, split inteins are separated into two independent fragments that reconstitute the host protein during the catalytic process. We recently developed a novel strategy for the specific killing of pathogenic and antibiotic resistant bacteria based on toxin-intein combinations. Bacterial type II toxin-antitoxin systems are protein modules in which the toxin can provoke cell death whereas the antitoxin inhibits toxin activity. Although our previous system was based on a split intein (iDnaE) and the CcdB toxin, we demonstrated that iDnaE is able to reconstitute four different toxins. To expand the applicability of our system by widening the repertoire of toxin-intein combinations for complex set-ups, we introduced a second intein, iDnaX, which was artificially split. We demonstrate that iDnaX is able to reconstitute the four toxins, and we manage to reduce its scar size to facilitate their use. In addition, we prove the orthogonality of both inteins (iDnaE and iDnaX) through a toxin reconstitution assay, thus opening the possibility for complex set-ups based on these toxin-intein modules. This could be used to develop specific antimicrobial and other biotechnological applications.

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.

All major cholesterol-dependent cytolysins use glycans as cellular receptors.

Cholesterol-dependent cytolysins (CDCs) form pores in cholesterol-rich membranes, but cholesterol alone is insufficient to explain their cell and host tropism. Here, we show that all eight major CDCs have high-affinity lectin activity that identifies glycans as candidate cellular receptors. Streptolysin O, vaginolysin, and perfringolysin O bind multiple glycans, while pneumolysin, lectinolysin, and listeriolysin O recognize a single glycan class. Addition of exogenous carbohydrate receptors for each CDC inhibits toxin activity. We present a structure for suilysin domain 4 in complex with two distinct glycan receptors, P1 antigen and αGal/Galili. We report a wide range of binding affinities for cholesterol and for the cholesterol analog pregnenolone sulfate and show that CDCs bind glycans and cholesterol independently. Intermedilysin binds to the sialyl-TF O-glycan on its erythrocyte receptor, CD59. Removing sialyl-TF from CD59 reduces intermedilysin binding. Glycan-lectin interactions underpin the cellular tropism of CDCs and provide molecular targets to block their cytotoxic activity.

Toward the identification of a type I toxin-antitoxin system in the plasmid DNA of dairy Lactobacillus rhamnosus

Plasmids carry genes that give bacteria beneficial traits and allow them to survive in competitive environments. In many cases, they also harbor toxin-antitoxin (TA) systems necessary for plasmid maintenance. TA systems are generally characterized by a stable “toxin”, a protein or peptide capable of killing the cell upon plasmid loss and by an unstable “antitoxin”, a protein or a non-coding RNA that inhibits toxin activity. Here we report data toward the identification of a RNA-regulated TA system in the plasmid DNA of L. rhamnosus isolated from cheese. The proposed TA system comprises two convergently transcribed RNAs: a toxin RNA encoding a 29 amino acid peptide named Lpt and an antitoxin non-coding RNA. Both toxin and antitoxin RNAs resulted upregulated under conditions mimicking cheese ripening. The toxicity of the Lpt peptide was demonstrated in E. coli by cloning the Lpt ORF under the control of an inducible promoter. Bioinformatics screening of the bacterial nucleotide database, shows that regions homologous to the Lpt TA locus are widely distributed in the Lactobacillus genus, particularly within the L. casei group, suggesting a relevant role of TA systems in plasmid maintenance of cheese microbiota.

Importance of the E. coli DinJ antitoxin carboxy terminus for toxin suppression and regulated proteolysis

Toxin-antitoxin genes play important roles in the regulation of bacterial growth during stress. One response to stress is selective proteolysis of antitoxin proteins which releases their cognate toxin partners causing rapid inhibition of growth. The features of toxin-antitoxin complexes that are important to inhibit toxin activity as well as to release the active toxin remain elusive. Furthermore, it is unclear how antitoxins are selected for proteolysis by cellular proteases. Here, we test the minimal structural requirements of the Escherichia coli DinJ antitoxin to suppress its toxin partner, YafQ. We find that DinJ-YafQ complex formation is critically dependent on the last ten C-terminal residues of DinJ. However, deletion of these 10 DinJ residues has little effect on transcriptional autorepression suggesting that the YafQ toxin is not a critical component of the repression complex in contrast to other toxin-antitoxin systems. We further demonstrate that loop 5 preceding these ten C-terminal residues is important for Lon-mediated proteolysis. These results provide important insights into the critical interactions between toxin-antitoxin pairs necessary to inhibit toxin activity and the regulated proteolysis of antitoxins.

The Antitoxin Protein of a Toxin-Antitoxin System from Xylella fastidiosa Is Secreted via Outer Membrane Vesicles.

The Xylella fastidiosa subsp pauca strain 9a5c is a Gram-negative, xylem-limited bacterium that is able to form a biofilm and affects citrus crops in Brazil. Some genes are considered to be involved in biofilm formation, but the specific mechanisms involved in this process remain unknown. This limited understanding of how some bacteria form biofilms is a major barrier to our comprehension of the progression of diseases caused by biofilm-producing bacteria. Several investigations have shown that the toxin-antitoxin (TA) operon is related to biofilm formation. This operon is composed of a toxin with RNAse activity and its cognate antitoxin. Previous reports have indicated that the antitoxin is able to inhibit toxin activity and modulate the expression of the operon as well as other target genes involved in oxidative stress and mobility. In this study, we characterize a toxin-antitoxin system consisting of XfMqsR and XfYgiT, respectively, from X. fastidiosa subsp. pauca strain 9a5c. These proteins display a high similarity to their homologs in X. fastidiosa strain Temecula and a predicted tridimensional structure that is similar to MqsR-YgiT from Escherichia coli. The characterization was performed using in vitro assays such as analytical ultracentrifugation (AUC), size exclusion chromatography, isothermal titration calorimetry, and Western blotting. Using a fluorometric assay to detect RNAses, we demonstrated that XfMqsR is thermostable and can degrade RNA. XfMqsR is inhibited by XfYgiT, which interacts with its own promoter. XfYgiT is known to be localized in the intracellular compartment; however, we provide strong evidence that X. fastidiosa secretes wild-type XfYgiT into the extracellular environment via outer membrane vesicles, as confirmed by Western blotting and specific immunofluorescence labeling visualized by fluorescence microscopy. Taken together, our results characterize the TA system from X. fastidiosa strain 9a5c, and we also discuss the possible influence of wild-type XfYgiT in the cell.

Plant-Derived Polyphenols Interact with Staphylococcal Enterotoxin A and Inhibit Toxin Activity.

This study was performed to investigate the inhibitory effects of 16 different plant-derived polyphenols on the toxicity of staphylococcal enterotoxin A (SEA). Plant-derived polyphenols were incubated with the cultured Staphylococcus aureus C-29 to investigate the effects of these samples on SEA produced from C-29 using Western blot analysis. Twelve polyphenols (0.1–0.5 mg/mL) inhibited the interaction between the anti-SEA antibody and SEA. We examined whether the polyphenols could directly interact with SEA after incubation of these test samples with SEA. As a result, 8 polyphenols (0.25 mg/mL) significantly decreased SEA protein levels. In addition, the polyphenols that interacted with SEA inactivated the toxin activity of splenocyte proliferation induced by SEA. Polyphenols that exerted inhibitory effects on SEA toxic activity had a tendency to interact with SEA. In particular, polyphenol compounds with 1 or 2 hexahydroxydiphenoyl groups and/or a galloyl group, such as eugeniin, castalagin, punicalagin, pedunculagin, corilagin and geraniin, strongly interacted with SEA and inhibited toxin activity at a low concentration. These polyphenols may be used to prevent S. aureus infection and staphylococcal food poisoning.

Toxin-antitoxin systems in the mobile genome of Acidithiobacillus ferrooxidans.

Toxin-antitoxin (TA) systems are genetic modules composed of a pair of genes encoding a stable toxin and an unstable antitoxin that inhibits toxin activity. They are widespread among plasmids and chromosomes of bacteria and archaea. TA systems are known to be involved in the stabilization of plasmids but there is no consensus about the function of chromosomal TA systems. To shed light on the role of chromosomally encoded TA systems we analyzed the distribution and functionality of type II TA systems in the chromosome of two strains from Acidithiobacillus ferrooxidans (ATCC 23270 and 53993), a Gram-negative, acidophilic, environmental bacterium that participates in the bioleaching of minerals. As in other environmental microorganisms, A. ferrooxidans has a high content of TA systems (28-29) and in twenty of them the toxin is a putative ribonuclease. According to the genetic context, some of these systems are encoded near or within mobile genetic elements. Although most TA systems are shared by both strains, four of them, which are encoded in the active mobile element ICEAfe1, are exclusive to the type strain ATCC 23270. We demostrated that two TA systems from ICEAfe1 are functional in E. coli cells, since the toxins inhibit growth and the antitoxins counteract the effect of their cognate toxins. All the toxins from ICEAfe1, including a novel toxin, are RNases with different ion requirements. The data indicate that some of the chromosomally encoded TA systems are actually part of the A. ferrooxidans mobile genome and we propose that could be involved in the maintenance of these integrated mobile genetic elements.

A regulatory role for Staphylococcus aureus toxin–antitoxin system PemIKSa

Toxin-antitoxin systems were shown to be involved in plasmid maintenance when they were initially discovered, but other roles have been demonstrated since. Here we identify and characterize a novel toxin-antitoxin system (pemIKSa) located on Staphylococcus aureus plasmid pCH91. The toxin (PemKSa) is a sequence-specific endoribonuclease recognizing the tetrad sequence U↓AUU, and the antitoxin (PemISa) inhibits toxin activity by physical interaction. Although the toxin-antitoxin system is responsible for stable plasmid maintenance our data suggest the participation of pemIKSa in global regulation of staphylococcal virulence by alteration of the translation of large pools of genes. We propose a common mechanism of reversible activation of toxin-antitoxin systems based on antitoxin transcript resistance to toxin cleavage. Elucidation of this mechanism is particularly interesting because reversible activation is a prerequisite for the proposed general regulatory role of toxin-antitoxin systems.

The crystal structure of the Rv0301‐Rv0300 VapBC‐3 toxin—antitoxin complex from M. tuberculosis reveals a Mg2+ ion in the active site and a putative RNA‐binding site

VapBC pairs account for 45 out of 88 identified toxin-antitoxin (TA) pairs in the Mycobacterium tuberculosis (Mtb) H37Rv genome. A working model suggests that under times of stress, antitoxin molecules are degraded, releasing the toxins to slow the metabolism of the cell, which in the case of VapC toxins is via their RNase activity. Otherwise the TA pairs remain bound to their promoters, autoinhibiting transcription. The crystal structure of Rv0301-Rv0300, an Mtb VapBC TA complex determined at 1.49 Å resolution, suggests a mechanism for these three functions: RNase activity, its inhibition by antitoxin, and its ability to bind promoter DNA. The Rv0301 toxin consists of a core of five parallel beta strands flanked by alpha helices. Three proximal aspartates coordinate a Mg²⁺ ion forming the putative RNase active site. The Rv0300 antitoxin monomer is extended in structure, consisting of an N-terminal beta strand followed by four helices. The last two helices wrap around the toxin and terminate near the putative RNase active site, but with different conformations. In one conformation, the C-terminal arginine interferes with Mg²⁺ ion coordination, suggesting a mechanism by which the antitoxin can inhibit toxin activity. At the N-terminus of the antitoxin, two pairs of Ribbon-Helix-Helix (RHH) motifs are related by crystallographic twofold symmetry. The resulting hetero-octameric complex is similar to the FitAB system, but the two RHH motifs are about 30 Å closer together in the Rv0301-Rv0300 complex, suggesting either a different span of the DNA recognition sequence or a conformational change.

Staphylococcus aureus leucocidin ED contributes to systemic infection by targeting neutrophils and promoting bacterial growth in vivo

Bloodstream infection with Staphylococcus aureus is common and can be fatal. However, virulence factors that contribute to lethality in S. aureus bloodstream infection are poorly defined. We discovered that LukED, a commonly overlooked leucotoxin, is critical for S. aureus bloodstream infection in mice. We also determined that LukED promotes S. aureus replication in vivo by directly killing phagocytes recruited to sites of haematogenously seeded tissue. Furthermore, we established that murine neutrophils are the primary target of LukED, as the greater virulence of wild-type S. aureus compared with a lukED mutant was abrogated by depleting neutrophils. The in vivo toxicity of LukED towards murine phagocytes is unique among S. aureus leucotoxins, implying its crucial role in pathogenesis. Moreover, the tropism of LukED for murine phagocytes highlights the utility of murine models to study LukED pathobiology, including development and testing of strategies to inhibit toxin activity and control bacterial infection.

Within-host competition selects for plasmid-encoded toxin–antitoxin systems

Toxin-antitoxin (TA) systems are commonly found on bacterial plasmids. The antitoxin inhibits toxin activity unless the system is lost from the cell. Then the shorter lived antitoxin degrades and the cell becomes susceptible to the toxin. Selection for plasmid-encoded TA systems was initially thought to result from their reducing the number of plasmid-free cells arising during growth in monoculture. However, modelling and experiments have shown that this mechanism can only explain the success of plasmid TA systems under a restricted set of conditions. Previously, we have proposed and tested an alternative model explaining the success of plasmid TA systems as a consequence of competition occurring between plasmids during co-infection of bacterial hosts. Here, we test a further prediction of this model, that competition between plasmids will lead to the biased accumulation of TA systems on plasmids relative to chromosomes. Transposon-encoded TA systems were added to populations of plasmid-containing cells, such that TA systems could insert into either plasmids or chromosomes. These populations were enriched for transposon-containing cells and then incubated in environments that did, or did not, allow effective within-host plasmid competition to occur. Changes in the ratio of plasmid- to chromosome-encoded TA systems were monitored. In agreement with our model, we found that plasmid-encoded TA systems had a competitive advantage, but only when host cells were sensitive to the effect of TA systems. This result demonstrates that within-host competition between plasmids can select for TA systems.

An improved method for development of toxoid vaccines and antitoxins

Botulinum neurotoxins are the most potent toxins known and causative agents of human botulism. Treatment comprises of administering purified polyclonal antitoxin or the prophylactic use of a vaccine containing formaldehyde inactivated toxoid. Whilst formaldehyde inhibits toxin activity, it induces so many structural changes in the molecule that immunisation often results in low levels of neutralising antibodies. We describe here for the first time a simple, less time consuming, novel method for producing a non-toxic toxoid that is structurally and antigenically more similar to the native toxin. Toxin is chemically inactivated by alkylation with iodoacetamide in the presence of reversibly denaturing conditions. This reduces neurotoxic activity by at least 7-orders of magnitude to undetectable levels. Following immunisation, in vivo neutralising antibody levels were 600-times higher than those produced with formaldehyde toxoid, despite generating equivalent ELISA antitoxin binding titres. These studies demonstrate that the new toxoid retains more of the native toxins structure and critical epitopes responsible for inducing life-saving neutralising antibody. Toxoid produced by the new method should substantially improve both antitoxin and vaccine production and be applicable to other toxins and immunogens.

Development of a novel non-formaldehyde botulinum toxoid inducing high levels of neutralising antibody

Botulinum neurotoxins are the most potent toxins known and causative agents of human botulism. Treatment comprises of administering purified polyclonal antitoxin or the prophylactic use of a vaccine containing formaldehyde inactivated toxoid. Whilst formaldehyde inhibits toxin activity, it induces so many structural changes in the molecule that immunisation often results in low levels of neutralising antibodies. We describe here for the first time a simple, less time consuming, novel method for producing a non-toxic toxoid that is structurally and antigenically more similar to the native toxin. Toxin is chemically inactivated by alkylating with iodoacetamide in the presence of reversibly denaturing conditions. This reduces neurotoxic activity by at least 7-orders of magnitude, to undetectable levels. Following immunisation, in vivo neutralising antibody levels were 600 times higher than those produced with formaldehyde toxoid, despite producing equivalent ELISA anti-toxin binding titres. These studies demonstrate that the new toxoid retains more of the native toxin's structure and critical epitopes responsible for inducing life-saving neutralising antibody. Toxoid produced by the new method should substantially improve both antitoxin and vaccine production and be applicable to other toxins.

Brevetoxin derivatives that inhibit toxin activity

Background: The brevetoxins are marine neurotoxins that interfere with the normal functions of the voltage-gated Na + channel. We have identified two brevetoxin derivatives that do not exhibit pharmacological properties typical of the brevetoxins and that function as brevetoxin antagonists. Results: PbTx-3 and benzoyl-PbTx-3 elicited Na + channel openings during steady-state depolarizations; however, two PbTx-3 derivatives retained their ability to bind to the receptor, but did not elicit Na + channel openings. α-Naphthoyl-PbTx-3 acted as a PbTx-3 antagonist but did not affect Na + channels that were not exposed to PbTx-3. β-Naphthoyl-PbTx-3 reduced openings of Na + channels that were not exposed to PbTx-3. Conclusions: Some modifications to the brevetoxin molecule do not alter either the binding properties or the activity of these toxins. Larger modifications to the K-ring sidechain do not interfere with binding but have profound effects on their pharmacological properties. This implies a critical function for the K-ring sidechain of the native toxin.

Pathogenesis of shigella diarrhea: evidence for an N-linked glycoprotein shigella toxin receptor and receptor modulation by beta-galactosidase.

Pathogenic mechanisms in infectious diseases often involve specific receptor-ligand interactions of cells and soluble molecules. To further elucidate structure-function relations for shigella toxin receptors, we studied binding of purified 125I-labeled toxin and biologic response under various conditions in an experimental model using HeLa cells. Response to toxin was reversibly inhibited by treatment of cells with trypsin or tunicamycin, an inhibitor of glycoprotein synthesis that also significantly inhibited toxin binding, a result indicating that the receptor is an N-linked glycoprotein. Removal of terminal beta-linked galactose from the HeLa cell surface with beta-galactosidase increased toxin binding and activity, and it also potentiated the effects of lysozyme and wheat-germ agglutinin, which recognize oligomeric beta 1----4-linked N-acetyl-D-glucosamine and inhibit toxin activity as well. Incubation of cells with beta-N-acetylglucosaminidase, which cleaves terminal beta-linked N-acetyl-D-glucosamine, inhibited toxin activity. Effects of beta-galactosidase were reversed by readdition of galactose to cell-surface oligosaccharide acceptors. The data demonstrate that alterations of a single sugar on cell-surface glycoproteins may have a dramatic effect on receptor activity and indicate that shigella toxin is a sugar-binding protein with specificity for beta 1----4-linked N-acetyl-D-glucosamine.