True Bugs (Insecta: Heteroptera) produce venom or saliva with diverse bioactivities depending on their feeding strategies. However, little is known about the molecular evolution of the venom toxins ...underlying these biological activities. We examined venom of the giant fish-killing water bug
Lethocerus distinctifemur
(Insecta: Belostomatidae) using infrared spectroscopy, transcriptomics, and proteomics. We report 132 venom proteins including putative enzymes, cytolytic toxins, and antimicrobial peptides. Over 73% (96 proteins) showed homology to venom proteins from assassin bugs (Reduviidae), including 21% (28 proteins from seven families) not known from other sources. These data suggest that numerous protein families were recruited into venom and diversified rapidly following the switch from phytophagy to predation by ancestral heteropterans, and then were retained over > 200 my of evolution. In contrast, trophic switches to blood-feeding (e.g. in Triatominae and Cimicidae) or reversions to plant-feeding (e.g., in Pentatomomorpha) were accompanied by rapid changes in the composition of venom/saliva, including the loss of many protein families.
Pseudomonas are a common cause of hospital-acquired infections that may be lethal. ADP-ribosyltransferase activities of Pseudomonas exotoxin-S and -T depend on 14-3-3 proteins inside the host cell. ...By binding in the 14-3-3 phosphopeptide binding groove, an amphipathic C-terminal helix of ExoS and ExoT has been thought to be crucial for their activation. However, crystal structures of the 14-3-3β:ExoS and -ExoT complexes presented here reveal an extensive hydrophobic interface that is sufficient for complex formation and toxin activation. We show that C-terminally truncated ExoS ADP-ribosyltransferase domain lacking the amphipathic binding motif is active when co-expressed with 14-3-3. Moreover, swapping the amphipathic C-terminus with a fragment from Vibrio Vis toxin creates a 14-3-3 independent toxin that ADP-ribosylates known ExoS targets. Finally, we show that 14-3-3 stabilizes ExoS against thermal aggregation. Together, this indicates that 14-3-3 proteins activate exotoxin ADP-ribosyltransferase domains by chaperoning their hydrophobic surfaces independently of the amphipathic C-terminal segment.
Clostridium difficile, an emerging nosocomial pathogen of increasing clinical significance, produces two large protein toxins that are responsible for the cellular damage associated with the disease. ...The precise mechanisms by which toxin synthesis is regulated in response to environmental change have yet to be discovered. The toxin genes (tcdA and tcdB) are located in a pathogenicity locus (PaLoc), along with tcdR and tcdC. TcdR is an alternative RNA polymerase σ factor that directly activates toxin gene expression, while the inverse relationship between expression of tcdR, tcdA and tcdB genes on the one hand and tcdC on the other has led to the suggestion that TcdC somehow interferes with toxin gene expression. This idea is further supported by the finding that many recent C. difficile epidemic strains in which toxin production is increased carry a common tcdC deletion mutation. In this report we demonstrate that TcdC negatively regulates toxin synthesis both in vivo and in vitro. TcdC destabilizes the TcdR-containing holoenzyme before open complex formation, apparently by interaction with TcdR or TcdR-containing RNA polymerase holoenzyme or both. In addition, we show that the hypertoxigenicity phenotype of C. difficile epidemic strains is not due to their common 18 bp in-frame deletion in tcdC.
Summary Background Resistance from antagonistic muscle groups might be a crucial factor reducing function in chronic hemiparesis. The resistance due to spastic co-contraction might be reduced by ...botulinum toxin injections. We assessed the effects of abobotulinumtoxinA injection in the upper limb muscles on muscle tone, spasticity, active movement, and function. Methods In this randomised, placebo-controlled, double-blind study, we enrolled adults (aged 18–80 years) at least 6 months after stroke or brain trauma from 34 neurology or rehabilitation clinics in Europe and the USA. Eligible participants were randomly allocated in a 1:1:1 ratio with a computer-generated list to receive a single injection session of abobotulinumtoxinA 500 U or 1000 U or placebo into the most hypertonic muscle group among the elbow, wrist, or finger flexors (primary target muscle group PTMG), and into at least two additional muscle groups from the elbow, wrist, or finger flexors or shoulder extensors. Patients and investigators were masked to treatment allocation. The primary endpoint was the change in muscle tone (Modified Ashworth Scale MAS) in the PTMG from baseline to 4 weeks. Secondary endpoints were Physician Global Assessment (PGA) at week 4 and change from baseline to 4 weeks in the perceived function (Disability Assessment Scale DAS) in the principal target of treatment, selected by the patient together with physician from four functional domains (dressing, hygiene, limb position, and pain). Analysis was by intention to treat. This study is registered with ClinicalTrials.gov , number NCT01313299. Findings 243 patients were randomly allocated to placebo (n=81), abobotulinumtoxinA 500 U (n=81), or abobotulinumtoxinA 1000 U (n=81). Mean change in MAS score from baseline at week 4 in the PTMG was −0·3 (SD 0·6) in the placebo group (n=79), −1·2 (1·0) in the abobotulinumtoxinA 500 U group (n=80; difference −0·9, 95% CI −1·2 to −0·6; p<0·0001 vs placebo), and −1·4 (1·1) in the abobotulinumtoxinA 1000 U group (n=79; −1·1, −1·4 to −0·8; p<0·0001 vs placebo). Mean PGA score at week 4 was 0·6 (SD 1·0) in the placebo group (n=78), 1·4 (1·1) in the abobotulinumtoxinA 500 U group (n=80; p=0·0003 vs placebo), and 1·8 (1·1) in the abobotulinumtoxinA 1000 U group (n=78; p<0·0001 vs placebo). Mean change from baseline at week 4 in DAS score for the principal target of treatment was −0·5 (0·7) in the placebo group (n=79), −0·7 (0·8) in the abobotulinumtoxinA 500 U group (n=80; p=0·2560 vs placebo), and −0·7 (0·7) in the abobotulinumtoxinA 1000 U group (n=78; p=0·0772 vs placebo). Three serious adverse events occurred in each group and none were treatment related; two resulted in death (from pulmonary oedema in the placebo group and a pre-existing unspecified cardiovascular disorder in the abobotulinumtoxinA 500 U group). Adverse events that were thought to be treatment related occurred in two (2%), six (7%), and seven (9%) patients in the placebo, abobotulinumtoxinA 500 U, and abobotulinumtoxinA 1000 U groups, respectively. The most common treatment-related adverse event was mild muscle weakness. All adverse events were mild or moderate. Interpretation AbobotulinumtoxinA at doses of 500 U or 1000 U injected into upper limb muscles provided tone reduction and clinical benefit in hemiparesis. Future research into the treatment of spastic paresis with botulinum toxin should use active movement and function as primary outcome measures. Funding Ipsen.
Under continuous long-term treatment with abo- or onabotulinum toxin type A (BoNT/A), ~10 to 15% of patients with cervical dystonia (CD) will develop neutralizing antibodies and reduced ...responsiveness over an ~10-year treatment period. Among the botulinum neurotoxin type A preparations so far licensed for CD, incobotulinum toxin A (incoBoNT/A; Xeomin
) is the only one without complex proteins. Whether CD patients with treatment failure under abo- or onaBoNT/A may still respond to incoBoNT/A is unknown. In this cross-sectional, retrospective study, 64 CD patients with secondary treatment failure after abo- or onaBoNT/A therapy who were switched to incoBoNT/A were compared to 34 CD patients exclusively treated with incoBoNT/A. The initial clinical severity of CD, best outcome during abo- or onaBoNT/A therapy, severity at the time of switching to incoBoNT/A and severity at recruitment, as well as all corresponding doses, were analyzed. Furthermore, the impact of neutralizing antibodies (NABs) on the long-term outcome of incoBoNT/A therapy was evaluated. Patients significantly improved after the switch to incoBoNT/A (
< 0.001) but did not reach the improvement level obtained before the development of partial secondary treatment failure or that of patients who were exclusively treated with incoBoNT/A. No difference between abo- and onaBoNT/A pretreatments or between the long-term outcomes of NAB-positive and NAB-negative patients was found. The present study demonstrates significant long-term improvement after a switch to incoBoNT/A in patients with preceding secondary treatment failure after abo- or onaBoNT/A therapy and confirms the low antigenicity of incoBoNT/A.
Type I toxin-antitoxin (TA) systems have been identified in a wide range of bacterial genomes. Here, we report the characterization of a new type I TA system present on the chromosome of the major ...human gastric pathogen, Helicobacter pylori. We show that the aapA1 gene encodes a 30 amino acid peptide whose artificial expression in H. pylori induces cell death. The synthesis of this toxin is prevented by the transcription of an antitoxin RNA, named IsoA1, expressed on the opposite strand of the toxin gene. We further reveal additional layers of post-transcriptional regulation that control toxin expression: (i) transcription of the aapA1 gene generates a full-length transcript whose folding impedes translation (ii) a 3΄ end processing of this message generates a shorter transcript that, after a structural rearrangement, becomes translatable (iii) but this rearrangement also leads to the formation of two stem-loop structures allowing formation of an extended duplex with IsoA1 via kissing-loop interactions. This interaction ensures both the translation inhibition of the AapA1 active message and its rapid degradation by RNase III, thus preventing toxin synthesis under normal growth conditions. Finally, a search for homologous mRNA structures identifies similar TA systems in a large number of Helicobacter and Campylobacter genomes.
Toxin-antitoxin (TA) gene pairs have been identified in nearly all bacterial genomes sequenced to date and are thought to facilitate persistence and antibiotic tolerance. TA loci are classified into ...various types based upon the characteristics of their antitoxins, with those in type II expressing proteic antitoxins. Many toxins from type II modules are ribonucleases that maintain a PilT N-terminal (PIN) domain containing conserved amino acids considered essential for activity. The
(
irulence-
ssociated
rotein) TA system is the largest subfamily in this class and has been linked to pathogenesis of nontypeable
(NTHi). In this study, the crystal structure of the VapBC-1 complex from NTHi was determined to 2.20 Å resolution. Based on this structure, aspartate-to-asparagine and glutamate-to-glutamine mutations of four conserved residues in the PIN domain of the VapC-1 toxin were constructed and the effects of the mutations on protein-protein interactions, growth of
, and pathogenesis
were tested. Finally, a novel model system was designed and utilized that consists of an NTHi Δ
strain complemented in
with the TA module containing a mutated or wild-type toxin at an ectopic site on the chromosome. This enabled the analysis of the effect of PIN domain toxin mutants in tandem with their wild-type antitoxin under the control of the
native promoter and in single copy. This is the first report of a system facilitating the study of TA mutant operons in the background of NTHi during infections of primary human tissues
Herein the crystal structure of the VapBC-1 complex from nontypeable
(NTHi) is described. Our results show that some of the mutations in the PIN domain of the VapC-1 toxin were associated with decreased toxicity in
, but the mutants retained the ability to homodimerize and to heterodimerize with the wild-type cognate antitoxin, VapB-1. A new system was designed and constructed to quantify the effects of these mutations on NTHi survival during infections of primary human tissues
Any mutation to a conserved amino acid in the PIN domain significantly decreased the number of survivors compared to that of the in
wild-type toxin under the same conditions.
Summary
Antitoxins encoded by type II toxin – antitoxin (TA) modules neutralize cognate toxins by direct protein – protein contact and in addition, regulate TA operon transcription by binding to ...operators in the promoter regions. On top of the simple negative feed‐back regulation, canonical type II TA operons are regulated by a mechanism called ‘Conditional Cooperativity’(CC). In CC, the cellular toxin:antitoxin (T:A) ratio controls the transcription‐rate such that low T:A ratios favour repression and high T:A ratios favour de‐repression of TA operon transcription. Here a new molecular mechanism that secures selective synthesis of antitoxin in the presence of excess toxin was unravelled. The hicAB locus of E. coli K‐12 encodes HicA mRNase and HicB antitoxin. It was shown that hicAB is transcribed by two promoters, an upstream one that is activated by CRP‐cAMP and competence factor Sxy and a downstream one that is autorepressed solely by HicB. Excess HicA destabilizes the HicB•operator complex in vitro and consistently, activates hicAB transcription in vivo. Remarkably, the hicAB transcript synthesized from the HicB‐controlled promoter produces HicB but not HicA. Thus, the HicA‐mediated derepression of hicAB transcription provides a mechanism that conditionally and selectively stimulates synthesis of HicB antitoxin under conditions of excess HicA toxin.
The hicAB toxin‐antitoxin locus is transcribed by two promoters, an upstream one, activated by CRP‐cAMP and competence factor Sxy and a downstream one that is autorepressed solely by HicB. Excess HicA destabilizes the HicB•operator complex and activates hicAB transcription. The hicAB transcript synthesized from the HicB‐controlled promoter produces HicB but not HicA. Thus, the HicA‐mediated derepression of hicAB transcription provides a mechanism that stimulates synthesis of HicB antitoxin under conditions of excess HicA toxin.
Toxin complex (Tc) toxins are virulence factors of pathogenic bacteria. Tcs are composed of three subunits: TcA, TcB and TcC. TcA facilitates receptor-toxin interaction and membrane permeation, TcB ...and TcC form a toxin-encapsulating cocoon. While the mechanisms of holotoxin assembly and pore formation have been described, little is known about receptor binding of TcAs. Here, we identify heparins/heparan sulfates and Lewis antigens as receptors for different TcAs from insect and human pathogens. Glycan array screening reveals that all tested TcAs bind negatively charged heparins. Cryo-EM structures of Morganella morganii TcdA4 and Xenorhabdus nematophila XptA1 reveal that heparins/heparan sulfates unexpectedly bind to different regions of the shell domain, including receptor-binding domains. In addition, Photorhabdus luminescens TcdA1 binds to Lewis antigens with micromolar affinity. Here, the glycan interacts with the receptor-binding domain D of the toxin. Our results suggest a glycan dependent association mechanism of Tc toxins on the host cell surface.
Eight marine cyanobacteria strains of the genera Cyanobium, Leptolyngbya, Oscillatoria, Phormidium, and Synechococcus were isolated from rocky beaches along the Atlantic Portuguese central coast and ...tested for ecotoxicity. Strains were identified by morphological characteristics and by the amplification and sequentiation of the 16S rDNA. Bioactivity of dichloromethane, methanol and aqueous extracts was assessed by the Artemia salina bioassay. Peptide toxin production was screened by matrix assisted laser desorption/ionization time of flight mass spectrometry. Molecular analysis of the genes involved in the production of known cyanotoxins such as microcystins, nodularins and cylindrospermopsin was also performed. Strains were toxic to the brine shrimp A. salina nauplii with aqueous extracts being more toxic than the organic ones. Although mass spectrometry analysis did not reveal the production of microcystins or other known toxic peptides, a positive result for the presence of mcyE gene was found in one Leptolyngbya strain and one Oscillatoria strain. The extensive brine shrimp mortality points to the involvement of other unknown toxins, and the presence of a fragment of genes involved in the cyanotoxin production highlight the potential risk of cyanobacteria occurrence on the Atlantic coast.
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