Whitefly hijacks a plant detoxification gene that neutralizes plant toxins.

Plants protect themselves with a vast array of toxic secondary metabolites, yet most plants serve as food for insects. The evolutionary processes that allow herbivorous insects to resist plant defenses remain largely unknown. The whitefly Bemisia tabaci is a cosmopolitan, highly polyphagous agricultural pest that vectors several serious plant pathogenic viruses and is an excellent model to probe the molecular mechanisms involved in overcoming plant defenses. Here, we show that, through an exceptional horizontal gene transfer event, the whitefly has acquired the plant-derived phenolic glucoside malonyltransferase gene BtPMaT1. This gene enables whiteflies to neutralize phenolic glucosides. This was confirmed by genetically transforming tomato plants to produce small interfering RNAs that silence BtPMaT1, thus impairing the whiteflies' detoxification ability. These findings reveal an evolutionary scenario whereby herbivores harness the genetic toolkit of their host plants to develop resistance to plant defenses and how this can be exploited for crop protection.

Peptide toxins that target vertebrate voltage-gated sodium channels underly the painful stings of harvester ants.

Harvester ants (genus Pogonomyrmex) are renowned for their stings which cause intense, long-lasting pain, and other neurotoxic symptoms in vertebrates. Here, we show that harvester ant venoms are relatively simple and composed largely of peptide toxins. One class of peptides is primarily responsible for the long-lasting local pain of envenomation via activation of peripheral sensory neurons. These hydrophobic, cysteine-free peptides potently modulate mammalian voltage-gated sodium (NaV) channels, reducing the voltage threshold for activation and inhibiting channel inactivation. These toxins appear to have evolved specifically to deter vertebrates.

PAT: a comprehensive database of prokaryotic antimicrobial toxins.

Antimicrobial toxins help prokaryotes win competitive advantages in intraspecific or interspecific conflicts and are also a critical factor affecting the pathogenicity of many pathogens that threaten human health. Although many studies have revealed that antagonism based on antimicrobial toxins plays a central role in prokaryotic life, a database on antimicrobial toxins remains lacking. Here, we present the prokaryotic antimicrobial toxin database (PAT, http://bioinfo.qd.sdu.edu.cn/PAT/), a comprehensive data resource collection on experimentally validated antimicrobial toxins. PAT has organized information, derived from the reported literature, on antimicrobial toxins, as well as the corresponding immunity proteins, delivery mechanisms, toxin activities, structural characteristics, sequences, etc. Moreover, we also predict potential antimicrobial toxins in prokaryotic reference genomes and show the taxonomic information and environmental distribution of typical antimicrobial toxins. These details have been fully incorporated into the PAT database, where users can browse, search, download, analyse and view informative statistics and detailed information. PAT resources have already been used in our prediction and identification of prokaryotic antimicrobial toxins and may contribute to promoting the efficient investigation of antimicrobial toxin functions, the discovery of novel antimicrobial toxins, and an improved understanding of the biological roles and significance of these toxins.

A peptide toxin in ant venom mimics vertebrate EGF-like hormones to cause long-lasting hypersensitivity in mammals.

Venoms are excellent model systems for studying evolutionary processes associated with predator-prey interactions. Here, we present the discovery of a peptide toxin, MIITX2-Mg1a, which is a major component of the venom of the Australian giant red bull ant Myrmecia gulosa and has evolved to mimic, both structurally and functionally, vertebrate epidermal growth factor (EGF) peptide hormones. We show that Mg1a is a potent agonist of the mammalian EGF receptor ErbB1, and that intraplantar injection in mice causes long-lasting hypersensitivity of the injected paw. These data reveal a previously undescribed venom mode of action, highlight a role for ErbB receptors in mammalian pain signaling, and provide an example of molecular mimicry driven by defensive selection pressure.

Identification of a cooperative effect between amino acids 169 and 174 in the rotavirus NSP4 double-layered particle-binding domain.

Segmented RNA viruses are capable of exchanging genome segments via reassortment as a means of immune evasion and to maintain viral fitness. Reassortments of single-genome segments are common among group A rotaviruses. Multiple instances of co-reassortment of two genome segments, GS6(VP6) and GS10(NSP4), have been documented in surveillance. Specifically, a division between NSP4 genotypes has been observed in the NSP4 double-layered particle (DLP)-binding domain. A previously hypothesized mechanism for this co-reassortment has been suggested to be the interaction between VP6 and NSP4 during DLP transport from viroplasms for particle maturation. In this study, we used sequence analysis, RNA secondary structure prediction, molecular dynamics and reverse genetics to form a hypothesis regarding the role of the NSP4 DLP-binding domain. Sequence analysis showed that the polarity of NSP4 DLP-binding domain amino acids 169 and 174 is clearly divided between E1 and E2 NSP4 genotypes. Viruses with E1 NSP4s had 169A/I or 169S/T with 174S. E2 NSP4s had 169R/K and 174A. RNA secondary structure prediction showed that mutation in both 545 (aa169) and 561 (aa174) causes global structure remodelling. Molecular dynamics showed that the NSP4/VP6 interaction stability is increased by mutating both aa positions 169 and 174. Using reverse genetics, we showed that an R169I mutation alone does not prevent rescue. Conversely, 174A to 174S prevented rescue, and rescue could be returned by combining 174S with 169I. When compared to rSA11 NSP4-wt, both rSA11 NSP4-R169I and rSA11 NSP4-R169I/A174S had a negligible but significant reduction in titre at specific time points. This study suggests that amino acid 174 of NSP4 may be essential in maintaining the VP6/NSP4 interaction required for DLP transport. Our results suggest that maintenance of specific polarities of amino acids at positions 169 and 174 may be required for the fitness of rotavirus field strains.

Friends or Foes? Emerging Impacts of Biological Toxins.

Toxins are substances produced from biological sources (e.g., animal, plants, microorganisms) that have deleterious effects on a living organism. Despite the obvious health concerns of being exposed to toxins, they are having substantial positive impacts in a number of industrial sectors. Several toxin-derived products are approved for clinical, veterinary, or agrochemical uses. This review sets out the case for toxins as 'friends' that are providing the basis of novel medicines, insecticides, and even nucleic acid sequencing technologies. We also discuss emerging toxins ('foes') that are becoming increasingly prevalent in a range of contexts through climate change and the globalisation of food supply chains and that ultimately pose a risk to health.

Neurotoxic and cytotoxic peptides underlie the painful stings of the tree nettle Urtica ferox.

The stinging hairs of plants from the family Urticaceae inject compounds that inflict pain to deter herbivores. The sting of the New Zealand tree nettle (Urtica ferox) is among the most painful of these and can cause systemic symptoms that can even be life-threatening; however, the molecular species effecting this response have not been elucidated. Here we reveal that two classes of peptide toxin are responsible for the symptoms of U. ferox stings: Δ-Uf1a is a cytotoxic thionin that causes pain via disruption of cell membranes, while ß/δ-Uf2a defines a new class of neurotoxin that causes pain and systemic symptoms via modulation of voltage-gated sodium (NaV) channels. We demonstrate using whole-cell patch-clamp electrophysiology experiments that ß/δ-Uf2a is a potent modulator of human NaV1.5 (EC50: 55 nM), NaV1.6 (EC50: 0.86 nM), and NaV1.7 (EC50: 208 nM), where it shifts the activation threshold to more negative potentials and slows fast inactivation. We further found that both toxin classes are widespread among members of the Urticeae tribe within Urticaceae, suggesting that they are likely to be pain-causing agents underlying the stings of other Urtica species. Comparative analysis of nettles of Urtica, and the recently described pain-causing peptides from nettles of another genus, Dendrocnide, indicates that members of tribe Urticeae have developed a diverse arsenal of pain-causing peptides.

ToxCodAn: a new toxin annotator and guide to venom gland transcriptomics.

MOTIVATION: Next-generation sequencing has become exceedingly common and has transformed our ability to explore nonmodel systems. In particular, transcriptomics has facilitated the study of venom and evolution of toxins in venomous lineages; however, many challenges remain. Primarily, annotation of toxins in the transcriptome is a laborious and time-consuming task. Current annotation software often fails to predict the correct coding sequence and overestimates the number of toxins present in the transcriptome. Here, we present ToxCodAn, a python script designed to perform precise annotation of snake venom gland transcriptomes. We test ToxCodAn with a set of previously curated transcriptomes and compare the results to other annotators. In addition, we provide a guide for venom gland transcriptomics to facilitate future research and use Bothrops alternatus as a case study for ToxCodAn and our guide. RESULTS: Our analysis reveals that ToxCodAn provides precise annotation of toxins present in the transcriptome of venom glands of snakes. Comparison with other annotators demonstrates that ToxCodAn has better performance with regard to run time ($>20x$ faster), coding sequence prediction ($>3x$ more accurate) and the number of toxins predicted (generating $>4x$ less false positives). In this sense, ToxCodAn is a valuable resource for toxin annotation. The ToxCodAn framework can be expanded in the future to work with other venomous lineages and detect novel toxins.

Dramatic dietary shift maintains sequestered toxins in chemically defended snakes.

Unlike other snakes, most species of Rhabdophis possess glands in their dorsal skin, sometimes limited to the neck, known as nucho-dorsal and nuchal glands, respectively. Those glands contain powerful cardiotonic steroids known as bufadienolides, which can be deployed as a defense against predators. Bufadienolides otherwise occur only in toads (Bufonidae) and some fireflies (Lampyrinae), which are known or believed to synthesize the toxins. The ancestral diet of Rhabdophis consists of anuran amphibians, and we have shown previously that the bufadienolide toxins of frog-eating species are sequestered from toads consumed as prey. However, one derived clade, the Rhabdophis nuchalis Group, has shifted its primary diet from frogs to earthworms. Here we confirm that the worm-eating snakes possess bufadienolides in their nucho-dorsal glands, although the worms themselves lack such toxins. In addition, we show that the bufadienolides of R. nuchalis Group species are obtained primarily from fireflies. Although few snakes feed on insects, we document through feeding experiments, chemosensory preference tests, and gut contents that lampyrine firefly larvae are regularly consumed by these snakes. Furthermore, members of the R. nuchalis Group contain compounds that resemble the distinctive bufadienolides of fireflies, but not those of toads, in stereochemistry, glycosylation, acetylation, and molecular weight. Thus, the evolutionary shift in primary prey among members of the R. nuchalis Group has been accompanied by a dramatic shift in the source of the species' sequestered defensive toxins.

Claudin-9 structures reveal mechanism for toxin-induced gut barrier breakdown.

The human pathogenic bacterium Clostridium perfringens secretes an enterotoxin (CpE) that targets claudins through its C-terminal receptor-binding domain (cCpE). Isoform-specific binding by CpE causes dissociation of claudins and tight junctions (TJs), resulting in cytotoxicity and breakdown of the gut epithelial barrier. Here, we present crystal structures of human claudin-9 (hCLDN-9) in complex with cCpE at 3.2 and 3.3 Å. We show that hCLDN-9 is a high-affinity CpE receptor and that hCLDN-9-expressing cells undergo cell death when treated with CpE but not cCpE, which lacks its cytotoxic domain. Structures reveal cCpE-induced alterations to 2 epitopes known to enable claudin self-assembly and expose high-affinity interactions between hCLDN-9 and cCpE that explain isoform-specific recognition. These findings elucidate the molecular bases for hCLDN-9 selective ion permeability and binding by CpE, and provide mechanisms for how CpE disrupts gut homeostasis by dissociating claudins and TJs to affect epithelial adhesion and intercellular transport.

Pharmacological Screening of Venoms from Five Brazilian Micrurus Species on Different Ion Channels.

Coral snake venoms from the Micrurus genus are a natural library of components with multiple targets, yet are poorly explored. In Brazil, 34 Micrurus species are currently described, and just a few have been investigated for their venom activities. Micrurus venoms are composed mainly of phospholipases A2 and three-finger toxins, which are responsible for neuromuscular blockade-the main envenomation outcome in humans. Beyond these two major toxin families, minor components are also important for the global venom activity, including Kunitz-peptides, serine proteases, 5' nucleotidases, among others. In the present study, we used the two-microelectrode voltage clamp technique to explore the crude venom activities of five different Micrurus species from the south and southeast of Brazil: M. altirostris, M. corallinus, M. frontalis, M. carvalhoi and M. decoratus. All five venoms induced full inhibition of the muscle-type α1ß1δε nAChR with different levels of reversibility. We found M. altirostris and M. frontalis venoms acting as partial inhibitors of the neuronal-type α7 nAChR with an interesting subsequent potentiation after one washout. We discovered that M. altirostris and M. corallinus venoms modulate the α1ß2 GABAAR. Interestingly, the screening on KV1.3 showed that all five Micrurus venoms act as inhibitors, being totally reversible after the washout. Since this activity seems to be conserved among different species, we hypothesized that the Micrurus venoms may rely on potassium channel inhibitory activity as an important feature of their envenomation strategy. Finally, tests on NaV1.2 and NaV1.4 showed that these channels do not seem to be targeted by Micrurus venoms. In summary, the venoms tested are multifunctional, each of them acting on at least two different types of targets.

Cysteine-Rich Peptides: Hyperstable Scaffolds for Protein Engineering.

Cysteine-rich peptides (CRPs) are small proteins of less than 100 amino acids in length characterized by the presence of disulfide bridges and common end-to-end macrocyclization. These properties confer hyperstability against high temperatures, salt concentration, serum presence, and protease degradation to CRPs. Moreover, their intercysteine domains (loops) are susceptible to residue hypervariability. CRPs have been successfully applied as stable scaffolds for molecular grafting, a protein engineering process in which cysteine-rich structures provide higher thermodynamic and metabolic stability to an epitope and acquire new biological function(s). This review describes the successes and limitations of seven cysteine-rich scaffolds, their bioactive epitopes, and the resulting grafted peptides.

Structure Revision of Protoaculeine B, a Post-translationally Modified N-Terminal Residue in the Peptide Toxin Aculeine B.

The structure of protoaculeine B, the N-terminal residue of the marine peptide toxin aculeine B, is revised to the cis-1,3-disubstituted tetrahydro-ß-carboline framework. We prepared two truncated model compounds that lack a long-chain polyamine using the one-step Pictet-Spengler reaction of tryptophan and compared their NMR, mass spectra, and chemical reactivity with those of the natural protoaculeine B. The synthetic models reproduced the profiles of the natural product well, which confirmed the appropriateness of the structure revision.

Phaseocyclopentenones A and B, Phytotoxic Penta- and Tetrasubstituted Cyclopentenones Produced by Macrophomina phaseolina, the Causal Agent of Charcoal Rot of Soybean in Argentina.

Two new penta- and tetrasubstituted cyclopentenones, named phaseocyclopentenones A and B (1 and 2), together with guignardone A (3), were isolated from Macrophomina phaseolina cultures. The phytopathogenic fungus was isolated from infected soybean tissues showing charcoal rot symptoms in Argentina. Charcoal rot is a devastating disease considering that soybean is one of the main legumes cultivated in the world. Phaseocyclopentenones A and B were characterized by 1D and 2D 1H and 13C NMR spectroscopic and HRESIMS spectrometric data and chemical methods as 4-benzoyl-3,4,5-trihydroxy-2-phenylcyclopent-2-enone and 3,5-dihydroxy-2,4-diphenylcyclopent-2-enone, respectively. The relative configuration of phaseocyclopentenones A and B was assigned by 1H and NOESY NMR methods, while their absolute configurations were assigned by electronic circular dichroism methods. When assayed on a nonhost plant (Solanum lycopersicum L.) by the leaf puncture assay, phaseocyclopentenones A and B and guignardone A showed phytotoxic activity, while only 1 and 2 were toxic when tested on cuttings of the same plant. No phytotoxicity or antifungal activity was detected for the three compounds on the host plant soybean (Glycine max L.) and against some of its fungal pathogens, namely, Cercospora nicotianae and Colletotrichum truncatum, also isolated from infected soybean plants in Argentina.

Multifunctional carbonized nanogels to treat lethal acute hepatopancreatic necrosis disease.

BACKGROUND: Shrimp aquaculture has suffered huge economic losses over the past decade due to the outbreak of acute hepatopancreatic necrosis disease (AHPND), which is mainly caused by the bacteria Vibrio parahaemolyticus (V. parahaemolyticus) with the virulence pVA1 plasmid, which encodes a secretory photorhabdus insect-related (Pir) toxin composed of PirA and PirB proteins. The Pir toxin mainly attacks the hepatopancreas, a major metabolic organ in shrimp, thereby causing necrosis and loss of function. The pandemic of antibiotic-resistant strains makes the impact worse. METHODS: Mild pyrolysis of a mixture of polysaccharide dextran 70 and the crosslinker 1,8-diaminooctane at 180 â„ƒ for 3 h to form carbonized nanogels (DAO/DEX-CNGs) through controlled cross-linking and carbonization. The multifunctional therapeutic CNGs inherit nanogel-like structures and functional groups from their precursor molecules. RESULTS: DAO/DEX-CNGs manifest broad-spectrum antibacterial activity against Vibrio parahaemolyticus responsible for AHPND and even multiple drug-resistant strains. The polymer-like structures and functional groups on graphitic-carbon within the CNGs exhibit multiple treatment effects, including disruption of bacterial membranes, elevating bacterial oxidative stress, and neutralization of PirAB toxins. The inhibition of Vibrio in the midgut of infected shrimp, protection of hepatopancreas tissue from Pir toxin, and suppressing overstimulation of the immune system in severe V. parahaemolyticus infection, revealing that CNGs can effectively guard shrimp from Vibrio invasion. Moreover, shrimps fed with DAO/DEX-CNGs were carefully examined, such as the expression of the immune-related genes, hepatopancreas biopsy, and intestinal microbiota. Few adverse effects on shrimps were observed. CONCLUSION: Our work proposes brand-new applications of multifunctional carbon-based nanomaterials as efficient anti-Vibrio agents in the aquatic industry that hold great potential as feed additives to reduce antibiotic overuse in aquaculture.

Computational exploration of vicine - an alkaloid glycoside mediated pathological hallmark of adenosine kinase to promote neurological disorder.

Epilepsy disease is characterized by the neuronal dysfunction or abnormal neuronal activity of the brain which is regulated by astrocytes. These are glial cells and found to be the major regulators of the brain which are guided by the occurrence of adenosine kinase (ADK) enzyme in the central nervous system (CNS). During the normal physiological environment, ADK maintains the level of adenosine in the CNS. Dysfunction of ADK levels results in accumulation of adenosine levels in the CNS that leads to the pathophysiology of the brain such as astrogliosis which is a pathological hallmark of epileptic seizures. Vicine, an alkaloid glycoside in bitter gourd juice (Momordica charantia) is found to be toxic to the human system if the bitter gourd juice is consumed more. This compound inhibits ADK enzyme activity to lead epilepsy and seizure. Here, the toxic effect of vicine targeting ADK using computational predictions was investigated. The 3-dimensional structure of ADK has been constructed using I-Tasser, which has been refined by ModRefiner, GalaxyRefine, and 3D refine and it was endorsed using PROCHECK, ERRAT, and VADAR. 3D structure of the ligand molecule has been obtained from PubChem. Molecular docking has been achieved using AutoDock 4.2 software, from which the outcome showed the effective interaction between vicine and ADK, which attains binding free energy (∆G) of - 4.13 kcal/mol. Vicine molecule interacts with the active region ARG 149 of ADK and inhibits the functions of ADK that may cause imbalance in energy homeostasis. Also, pre-ADMET results robustly propose in which vicine possesses toxicity, and meanwhile, from the Ames test, it was shown as mutagenic. Hence, the results from our study suggest that vicine was shown to be toxic that suppresses the ADK activity to undergo pathological conditions in the neuronal junctions to lead epilepsy.

Role of human Hv1 channels in sperm capacitation and white blood cell respiratory burst established by a designed peptide inhibitor.

Using a de novo peptide inhibitor, Corza6 (C6), we demonstrate that the human voltage-gated proton channel (hHv1) is the main pathway for H+ efflux that allows capacitation in sperm and permits sustained reactive oxygen species (ROS) production in white blood cells (WBCs). C6 was identified by a phage-display strategy whereby ∼1 million novel peptides were fabricated on an inhibitor cysteine knot (ICK) scaffold and sorting on purified hHv1 protein. Two C6 peptides bind to each dimeric channel, one on the S3-S4 loop of each voltage sensor domain (VSD). Binding is cooperative with an equilibrium affinity (Kd) of ∼1 nM at -50 mV. As expected for a VSD-directed toxin, C6 inhibits by shifting hHv1 activation to more positive voltages, slowing opening and speeding closure, effects that diminish with membrane depolarization.

Characterization of Sialic Acid Affinity of the Binding Domain of Mistletoe Lectin Isoform One.

Sialic acid (Sia) is considered as one of the most important biomolecules of life since its derivatives and terminal orientations on cell membranes and macromolecules play a major role in many biological and pathological processes. To date, there is only a limited number of active molecules that can selectively bind to Sia and this limitation has made the study of this glycan challenging. The lectin superfamily is a well-known family of glycan binding proteins, which encompasses many strong glycan binding peptides with diverse glycan affinities. Mistletoe lectin (ML) is considered one of the most active members of lectin family which was initially classified in early studies as a galactose binding lectin; more recent studies have suggested that the peptide can also actively bind to Sia. However, the details with respect to Sia binding of ML and the domain responsible for this binding are left unanswered because no comprehensive studies have been instigated. In this study, we sought to identify the binding domain responsible for the sialic acid affinity of mistletoe lectin isoform I (MLI) in comparison to the binding activity of elderberry lectin isoform I (SNA), which has long been identified as a potent Sia binding lectin. In order to execute this, we performed computational carbohydrate-protein docking for MLB and SNA with Neu5Ac and ß-Galactose. We further analyzed the coding sequence of both lectins and identified their glycan binding domains, which were later cloned upstream and downstream to green fluorescent protein (GFP) and expressed in Escherichia coli (E. coli). Finally, the glycan affinity of the expressed fusion proteins was assessed by using different biochemical and cell-based assays and the Sia binding domains were identified.

Targeting Toxins toward Tumors.

Many cancer diseases, e.g., prostate cancer and lung cancer, develop very slowly. Common chemotherapeutics like vincristine, vinblastine and taxol target cancer cells in their proliferating states. In slowly developing cancer diseases only a minor part of the malignant cells will be in a proliferative state, and consequently these drugs will exert a concomitant damage on rapidly proliferating benign tissue as well. A number of toxins possess an ability to kill cells in all states independently of whether they are benign or malignant. Such toxins can only be used as chemotherapeutics if they can be targeted selectively against the tumors. Examples of such toxins are mertansine, calicheamicins and thapsigargins, which all kill cells at low micromolar or nanomolar concentrations. Advanced prodrug concepts enabling targeting of these toxins to cancer tissue comprise antibody-directed enzyme prodrug therapy (ADEPT), gene-directed enzyme prodrug therapy (GDEPT), lectin-directed enzyme-activated prodrug therapy (LEAPT), and antibody-drug conjugated therapy (ADC), which will be discussed in the present review. The review also includes recent examples of protease-targeting chimera (PROTAC) for knockdown of receptors essential for development of tumors. In addition, targeting of toxins relying on tumor-overexpressed enzymes with unique substrate specificity will be mentioned.

The Sandarazols are Cryptic and Structurally Unique Plasmid-Encoded Toxins from a Rare Myxobacterium*.

Herein, we describe a new plasmid found in Sandaracinus sp. MSr10575 named pSa001 spanning 209.7 kbp that harbors a cryptic secondary metabolite biosynthesis gene cluster (BGC). Activation of this BGC by homologous-recombination-mediated exchange of the native promoter sequence against a vanillate inducible system led to the production and subsequent isolation and structure elucidation of novel secondary metabolites, the sandarazols A-G. The sandarazols contain intriguing structural features and very reactive functional groups such as an α-chlorinated ketone, an epoxyketone, and a (2R)-2-amino-3-(N,N-dimethylamino)-propionic acid building block. In-depth investigation of the underlying biosynthetic machinery led to a concise biosynthetic model for the new compound family, including several uncommon biosynthetic steps. The chlorinated congener sandarazol C shows an IC50  value of 0.5 µm against HCT 116 cells and a MIC of 14 µm against Mycobacterium smegmatis, which points at the sandarazols' potential function as defensive secondary metabolites or toxins.