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Background: The common Eastern bumble bee Bombus impatiens is native to North America and is the main commercially reared pollinator in the Americas. There has been extensive research on this species related to its social biology, applied pollination, and genetics. The genome of this species was previously sequenced using short-read technology, but recent technological advances provide an opportunity for substantial improvements. This species is common in agricultural and urban environments, and heavy metal contaminants produced by industrial processes can negatively impact it. To begin to identify possible mechanisms underlying responses to these toxins, we used RNA-sequencing to examine how exposure to a cocktail of four heavy metals at field-realistic levels from industrial areas affected B. impatiens worker gene expression., Results: PacBio long-read sequencing resulted in 544x coverage of the genome, and HiC technology was used to map chromatin contacts. Using Juicer and manual curation, the genome was scaffolded into 18 main pseudomolecules, representing a high quality, chromosome-level assembly. The sequenced genome size is 266.6 Mb and BRAKER3 annotation produced 13,938 annotated genes. The genome and annotation show high completeness, with ≥ 96% of conserved Eukaryota and Hymenoptera genes present in both the assembly and annotated genes. RNA sequencing of heavy metal exposed workers revealed 603 brain and 34 fat body differentially expressed genes. In the brain, differentially expressed genes had biological functions related to chaperone activity and protein folding., Conclusions: Our data represent a large improvement in genomic resources for this important model species-with 10% more genome coverage than previously available, and a high-quality assembly into 18 chromosomes, the expected karyotype for this species. The new gene annotation added 777 new genes. Altered gene expression in response to heavy metal exposure suggests a possible mechanism for how these urban toxins are negatively impacting bee health, specifically by altering protein folding in the brain. Overall, these data are useful as a general high quality genomic resource for this species, and provide insight into mechanisms underlying tissue-specific toxicological responses of bumble bees to heavy metals., Competing Interests: Declarations Ethics approval and consent to participate No permissions were necessary to collect the specimens in this study. Permission to access the field site (Horticulture Research Station) was provided by Iowa State University Farms. Consent for publication not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Rotavirus A is a leading cause of non-bacterial gastroenteritis in humans and domesticated animals. Despite the vast diversity of bovine Rotavirus A strains documented in South Asian countries, there are very few whole genomes available for phylogenetic study. A cross-sectional study identified a high prevalence of the G6P[11] genotype of bovine Rotavirus A circulating in the commercial cattle population in Bangladesh. Next-generation sequencing and downstream phylogenetic analysis unveiled all 11 complete gene segments of this strain (BD_ROTA_CVASU), classifying it under the genomic constellation G6P[11]-I2-R2-C2-M2-A13-N2-T6-E2-H3, which belongs to a classical DS-1-like genomic backbone. We found strong evidence of intragenic recombination between human and bovine strains in the Non-structural protein 4 (NSP4) gene, which encodes a multifunctional enterotoxin. Our analyses highlight frequent zoonotic transmissions of rotaviruses in diverse human-animal interfaces, which might have contributed to the evolution and pathogenesis of this dominant genotype circulating in the commercial cattle population in Bangladesh., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

One of the actively developing areas of drug development is the creation of chimeric toxins, recombinant bifunctional molecules designed to affect target cells selectively. The prevalent approach involves fusing bacterial and plant toxins with molecules that facilitate targeted delivery. However, the therapeutic use of such toxins often encounters challenges associated with negative side effects. Concurrently, viruses encode proteins possessing toxin-like properties, exerting multiple effects on the vital activity of cells. In contrast to bacterial and plant toxins, the impact of viral proteins is typically milder, presenting a significant advantage by potentially reducing the likelihood of side effects. This review delineates the characteristics of extensively studied viral proteins with toxic and immunomodulatory properties and explores the prospects of incorporating them into chimeric toxins., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

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.

Porcine rotavirus (PoRV) is one of the main pathogens causing diarrhea in piglets, and multiple genotypes coexist. However, an effective vaccine is currently lacking. Here, the potential adjuvant of nonstructural protein 4 (NSP4) and highly immunogenic structural protein VP4 prompted us to construct recombinant NSP4 86-175aa (NSP4*) and VP4 26-476aa (VP4*) proteins, combine them as immunogens to evaluate their efficacy. Results indicated that NSP4* enhanced systemic and local mucosal responses induced by VP4*. The VP4*-IgG, VP4*-IgA in feces and IgA-secreting cells in intestines induced by the co-immunization were significantly higher than those induced by VP4* alone. Co-immunization of NSP4* and VP4* also induced strong cellular immunity with significantly increased IFN-λ than the single VP4*. Summarily, the NSP4* as a synergistical antigen exerted limited effects on the PoRV NAbs elevation, but conferred strong VP4*-specific mucosal and cellular efficacy, which lays the foundation for the development of a more effective porcine rotavirus subunit vaccine., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Earthworms, long utilized in traditional medicine, serve as a source of inspiration for modern therapeutics. Lysenin, a defensive factor in the coelom fluid of the earthworm Eisenia fetida, has multiple bioactivities. However, the inherent toxicity of Lysenin as a pore-forming protein (PFP) restricts its application in therapy. Here, a gene therapy strategy based on Lysenin for cancer treatment is presented. The formulation consists of polymeric nanoparticles complexed with the plasmid encoding Lysenin. After transfection in vitro, melanoma cells can express Lysenin, resulting in necrosis, autophagy, and immunogenic cell death. The secretory signal peptide alters the intracellular distribution of the expressed product of Lysenin, thereby potentiating its anticancer efficacy. The intratumor injection of Lysenin gene formulation can efficiently kill the transfected melanoma cells and activate the antitumor immune response. Notably, no obvious systemic toxicity is observed during the treatment. Non-viral gene therapy based on Lysenin derived from Eisenia foetida exhibits potential in cancer therapy, which can inspire future cancer therapeutics., (© 2024 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

Genomic imprinting-the non-equivalence of maternal and paternal genomes-is a critical process that has evolved independently in many plant and mammalian species 1,2 . According to kinship theory, imprinting is the inevitable consequence of conflictive selective forces acting on differentially expressed parental alleles 3,4 . Yet, how these epigenetic differences evolve in the first place is poorly understood 3,5,6 . Here we report the identification and molecular dissection of a parent-of-origin effect on gene expression that might help to clarify this fundamental question. Toxin-antidote elements (TAs) are selfish elements that spread in populations by poisoning non-carrier individuals 7-9 . In reciprocal crosses between two Caenorhabditis tropicalis wild isolates, we found that the slow-1/grow-1 TA is specifically inactive when paternally inherited. This parent-of-origin effect stems from transcriptional repression of the slow-1 toxin by the PIWI-interacting RNA (piRNA) host defence pathway. The repression requires PIWI Argonaute and SET-32 histone methyltransferase activities and is transgenerationally inherited via small RNAs. Remarkably, when slow-1/grow-1 is maternally inherited, slow-1 repression is halted by a translation-independent role of its maternal mRNA. That is, slow-1 transcripts loaded into eggs-but not SLOW-1 protein-are necessary and sufficient to counteract piRNA-mediated repression. Our findings show that parent-of-origin effects can evolve by co-option of the piRNA pathway and hinder the spread of selfish genes that require sex for their propagation., (© 2024. The Author(s).)

Toxin-antitoxin (TA) modules are prevalent in prokaryotic genomes, often in substantial numbers. For instance, the Mycobacterium tuberculosis genome alone harbors close to 100 TA modules, half of which belong to a singular type. Traditionally ascribed multiple biological roles, recent insights challenge these notions and instead indicate a predominant function in phage defense. TAs are often located within Defense Islands, genomic regions that encode various defense systems. The analysis of genes within Defense Islands has unveiled a wide array of systems, including TAs that serve in anti-phage defense. Prokaryotic cells are equipped with anti-phage Viperins that, analogous to their mammalian counterparts, inhibit viral RNA transcription. Additionally, bacterial Structural Maintenance of Chromosome (SMC) proteins combat plasmid intrusion by recognizing foreign DNA signatures. This study undertakes a comprehensive bioinformatics analysis of genetic elements encoding the HicA double-stranded RNA-binding domain, complemented by protein structure modeling. The HicA toxin domains are found in at least 14 distinct contexts and thus exhibit a remarkable genetic diversity. Traditional bicistronic TA operons represent eight of these contexts, while four are characterized by monocistronic operons encoding fused HicA domains. Two contexts involve hicA adjacent to genes that encode bacterial Viperins. Notably, genes encoding RelE toxins are also adjacent to Viperin genes in some instances. This configuration hints at a synergistic enhancement of Viperin-mediated anti-phage action by HicA and RelE toxins. The discovery of a HicA domain merged with an SMC domain is compelling, prompting further investigation into its potential roles.IMPORTANCEProkaryotic organisms harbor a multitude of toxin-antitoxin (TA) systems, which have long puzzled scientists as "genes in search for a function." Recent scientific advancements have shed light on the primary role of TAs as anti-phage defense mechanisms. To gain an overview of TAs it is important to analyze their genetic contexts that can give hints on function and guide future experimental inquiries. This article describes a thorough bioinformatics examination of genes encoding the HicA toxin domain, revealing its presence in no fewer than 14 unique genetic arrangements. Some configurations notably align with anti-phage activities, underscoring potential roles in microbial immunity. These insights robustly reinforce the hypothesis that HicA toxins are integral components of the prokaryotic anti-phage defense repertoire. The elucidation of these genetic contexts not only advances our understanding of TAs but also contributes to a paradigm shift in how we perceive their functionality within the microbial world., Competing Interests: The author declares no conflict of interest.

While the unique symbiotic relationship between anemonefishes and sea anemones is iconic, it is still not fully understood how anemonefishes can withstand and thrive within the venomous environment of their host sea anemone. In this study, we used a proteotranscriptomics approach to elucidate the proteinaceous toxin repertoire from the most common host sea anemone, Entacmaea quadricolor . Although 1251 different toxin or toxin-like RNA transcripts were expressed in E. quadricolor tentacles (0.05% of gene clusters, 1.8% of expression) and 5375 proteins were detected in milked venom, only 4% of proteins detected in venom were putative toxins (230), and they only represent on average 14% of the normalised protein expression in the milked venom samples. Thus, most proteins in milked venom do not appear to have a toxin function. This work raises the perils of defining a dominant venom phenotype based on transcriptomics data alone in sea anemones, as we found that the dominant venom phenotype differs between the transcriptome and proteome abundance data. E. quadricolor venom contains a mixture of toxin-like proteins of unknown and known function. A newly identified toxin protein family, Z3, rich in conserved cysteines of unknown function, was the most abundant at the RNA transcript and protein levels. The venom was also rich in toxins from the Protease S1, Kunitz-type and PLA2 toxin protein families and contains toxins from eight venom categories. Exploring the intricate venom toxin components in other host sea anemones will be crucial for improving our understanding of how anemonefish adapt to the venomous environment.

Toxin-antitoxin systems (TAs) are generally two-component genetic modules present in almost every prokaryotic genome. The production of the free and active toxin is able to disrupt key cellular processes leading to the growth inhibition or death of its host organism in absence of its cognate antitoxin. The functions attributed to TAs rely on this lethal phenotype ranging from mobile genetic elements stabilization to phage defense. Their abundance in prokaryotic genomes as well as their lethal potential make them attractive targets for new antibacterial strategies. The hijacking of TAs requires a deep understanding of their regulation to be able to design such approach. In this review, we summarize the accumulated knowledge on how bacteria cope with these toxic genes in their genome. The characterized TAs can be grouped based on the way they prevent toxicity. Some systems rely on a tight control of the expression to prevent the production of the toxin while others control the activity of the toxin at the post-translational level., (Copyright © 2023 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Venomous organisms have independently evolved the ability to produce toxins 101 times during their evolutionary history, resulting in over 200 000 venomous species. Collectively, these species produce millions of toxins, making them a valuable resource for bioprospecting and understanding the evolutionary mechanisms underlying genetic diversification. RNA-seq is the preferred method for characterizing toxin repertoires, but the analysis of the resulting data remains challenging. While early approaches relied on similarity-based mapping to known toxin databases, recent studies have highlighted the importance of structural features for toxin detection. The few existing pipelines lack an integration between these complementary approaches, and tend to be difficult to run for non-experienced users. To address these issues, we developed DeTox, a comprehensive and user-friendly tool for toxin research. It combines fast execution, parallelization and customization of parameters. DeTox was tested on published transcriptomes from gastropod mollusks, cnidarians and snakes, retrieving most putative toxins from the original articles and identifying additional peptides as potential toxins to be confirmed through manual annotation and eventually proteomic analysis. By integrating a structure-based search with similarity-based approaches, DeTox allows the comprehensive characterization of toxin repertoire in poorly-known taxa. The effect of the taxonomic bias in existing databases is minimized in DeTox, as mirrored in the detection of unique and divergent toxins that would have been overlooked by similarity-based methods. DeTox streamlines toxin annotation, providing a valuable tool for efficient identification of venom components that will enhance venom research in neglected taxa., (© The Author(s) 2024. Published by Oxford University Press.)

Three-finger toxins (3FTXs) are a functionally diverse family of toxins, apparently unique to venoms of caenophidian snakes. Although the ancestral function of 3FTXs is antagonism of nicotinic acetylcholine receptors, redundancy conferred by the accumulation of duplicate genes has facilitated extensive neofunctionalization, such that derived members of the family interact with a range of targets. 3FTXs are members of the LY6/UPAR family, but their non-toxin ancestor remains unknown. Combining traditional phylogenetic approaches, manual synteny analysis, and machine learning techniques (including AlphaFold2 and ProtT5), we have reconstructed a detailed evolutionary history of 3FTXs. We identify their immediate ancestor as a non-secretory LY6, unique to squamate reptiles, and propose that changes in molecular ecology resulting from loss of a membrane-anchoring domain and changes in gene expression, paved the way for the evolution of one of the most important families of snake toxins., (© 2023. Springer Nature Limited.)

Thermoacidophilic archaea lack sigma factors and the large inventory of heat shock proteins (HSPs) widespread in bacterial genomes, suggesting other strategies for handling thermal stress are involved. Heat shock transcriptomes for the thermoacidophilic archaeon Saccharolobus (f. Sulfolobus) solfataricus 98/2 revealed genes that were highly responsive to thermal stress, including transcriptional regulators YtrA Ss (Ssol_2420) and FadR Ss (Ssol_0314), as well as type II toxin-antitoxin (TA) loci VapBC6 (Ssol_2337, Ssol_2338) and VapBC22 (Ssol_0819, Ssol_0818). The role, if any, of type II TA loci during stress response in microorganisms, such as Escherichia coli, is controversial. But, when genes encoding YtrA Ss , FadR Ss , VapC22, VapB6, and VapC6 were systematically mutated in Sa. solfataricus 98/2, significant up-regulation of the other genes within this set was observed, implicating an interconnected regulatory network during thermal stress response. VapBC6 and VapBC22 have close homologues in other Sulfolobales, as well as in other archaea (e.g. Pyrococcus furiosus and Archaeoglobus fulgidus), and their corresponding genes were also heat shock responsive. The interplay between VapBC TA loci and heat shock regulators in Sa solfataricus 98/2 not only indicates a cellular mechanism for heat shock response that differs from bacteria but one that could have common features within the thermophilic archaea., (© 2023 Applied Microbiology International and John Wiley & Sons Ltd.)

Secreted protein toxins are widely used weapons in conflicts between organisms. Elucidating how organisms genetically adapt to defend themselves against these toxins is fundamental to understanding the coevolutionary dynamics of competing organisms. Within yeast communities, "killer" toxins are secreted to kill nearby sensitive yeast, providing a fitness advantage in competitive growth environments. Natural yeast isolates vary in their sensitivity to these toxins, but to date, no polymorphic genetic factors contributing to defense have been identified. We investigated the variation in resistance to the killer toxin K28 across diverse natural isolates of the Saccharomyces cerevisiae population. Using large-scale linkage mapping, we discovered a novel defense factor, which we named KTD1 . We identified many KTD1 alleles, which provided different levels of K28 resistance. KTD1 is a member of the DUP240 gene family of unknown function, which is rapidly evolving in a region spanning its two encoded transmembrane helices. We found that this domain is critical to KTD1 's protective ability. Our findings implicate KTD1 as a key polymorphic factor in the defense against K28 toxin.

The evolution of venom and the selection pressures that act on toxins have been increasingly researched within toxinology in the last two decades, in part due to the exceptionally high rates of diversifying selection observed in animal toxins. In 2015, Sungar and Moran proposed the 'two-speed' model of toxin evolution linking evolutionary age of a group to the rates of selection acting on toxins but due to a lack of data, mammals were not included as less than 30 species of venomous mammal have been recorded, represented by elusive species which produce small amounts of venom. Due to advances in genomics and transcriptomics, the availability of toxin sequences from venomous mammals has been increasing. Using branch- and site-specific selection models, we present the rates of both episodic and pervasive selection acting upon venomous mammal toxins as a group for the first time. We identified seven toxin groups present within venomous mammals, representing Chiroptera, Eulipotyphla and Monotremata: KLK1, Plasminogen Activator, Desmallipins, PACAP, CRiSP, Kunitz Domain One and Kunitz Domain Two. All but one group (KLK1) was identified by our results to be evolving under both episodic and pervasive diversifying selection with four toxin groups having sites that were implicated in the fitness of the animal by TreeSAAP (Selection on Amino Acid Properties). Our results suggest that venomous mammal ecology, behaviour or genomic evolution are the main drivers of selection, although evolutionary age may still be a factor. Our conclusion from these results indicates that mammalian toxins are following the two-speed model of selection, evolving predominately under diversifying selection, fitting in with other younger venomous taxa like snakes and cone snails-with high amounts of accumulating mutations, leading to more novel adaptions in their toxins.

Biological replicators, from genes within a genome to whole organisms, are locked in conflicts. Comparative genomics has revealed a staggering diversity of molecular armaments and mechanisms regulating their deployment, collectively termed biological conflict systems. These encompass toxins used in inter- and intraspecific interactions, self/nonself discrimination, antiviral immune mechanisms, and counter-host effectors deployed by viruses and intragenomic selfish elements. These systems possess shared syntactical features in their organizational logic and a set of effectors targeting genetic information flow through the Central Dogma, certain membranes, and key molecules like NAD + . These principles can be exploited to discover new conflict systems through sensitive computational analyses. This has led to significant advances in our understanding of the biology of these systems and furnished new biotechnological reagents for genome editing, sequencing, and beyond. We discuss these advances using specific examples of toxins, restriction-modification, apoptosis, CRISPR/second messenger-regulated systems, and other enigmatic nucleic acid-targeting systems.

Nemertea is a phylum of nonsegmented worms (supraphylum: Spiralia), also known as ribbon worms. The members of this phylum contain various toxins, including peptide toxins. Here, we provide a transcriptomic analysis of peptide toxins in 14 nemertean species, including Cephalothrix cf. simula , which was sequenced in the current study. The summarized data show that the number of toxin transcripts in the studied nemerteans varied from 12 to 82. The most represented groups of toxins were enzymes and ion channel inhibitors, which, in total, reached a proportion of 72% in some species, and the least represented were pore-forming toxins and neurotoxins, the total proportion of which did not exceed 18%. The study revealed that nemerteans possess a much greater variety of toxins than previously thought and showed that these animals are a promising object for the investigation of venom diversity and evolution, and in the search for new peptide toxins.

The phyllosphere microbiome plays an important role in plant fitness. Recently, bacteriophages have been shown to play a role in shaping the bacterial community composition of the phyllosphere. However, no studies on the diversity and abundance of phyllosphere bacteriophage communities have been carried out until now. In this study, we extracted, sequenced, and characterized the dsDNA and ssDNA viral community from a phyllosphere for the first time. We sampled leaves from winter wheat ( Triticum aestivum ), where we identified a total of 876 virus operational taxonomic units (vOTUs), mostly predicted to be bacteriophages with a lytic lifestyle. Remarkably, 848 of these vOTUs corresponded to new viral species, and we estimated a minimum of 2.0 × 10 6 viral particles per leaf. These results suggest that the wheat phyllosphere harbors a large and active community of novel bacterial viruses. Phylloviruses have potential applications as biocontrol agents against phytopathogenic bacteria or as microbiome modulators to increase plant growth-promoting bacteria.

Multiple representatives of eulipotyphlan mammals such as shrews have oral venom systems. Venom facilitates shrews to hunt and/or hoard preys. However, little is known about their venom composition, and especially the mechanism to hoard prey in comatose states for meeting their extremely high metabolic rates. A toxin (BQTX) was identified from venomous submaxillary glands of the shrew Blarinella quadraticauda. BQTX is specifically distributed and highly concentrated (~ 1% total protein) in the organs. BQTX shares structural and functional similarities to toxins from snakes, wasps and snails, suggesting an evolutional relevancy of venoms from mammalians and non-mammalians. By potentiating thrombin and factor-XIIa and inhibiting plasmin, BQTX induces acute hypertension, blood coagulation and hypokinesia. It also shows strong analgesic function by inhibiting elastase. Notably, the toxin keeps high plasma stability with a 16-h half-life in-vivo, which likely extends intoxication to paralyze or immobilize prey hoarded fresh for later consumption and maximize foraging profit., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Animal venoms are among the most complex natural secretions known, comprising a mixture of bioactive compounds often referred to as toxins. Venom arsenals are predominately made up of cysteine-rich peptide toxins that manipulate molecular targets, such as ion channels and receptors, making these venom peptides attractive candidates for the development of therapeutics to benefit human health. With the rise of omic strategies that utilize transcriptomic, proteomic, and bioinformatic methods, we are able to identify more venom proteins and peptides than ever before. However, identification and characterization of bioactive venom peptides remains a significant challenge due to the unique chemical structure and enormous number of peptides found in each venom arsenal (upward of 200 per organism). Here, we introduce a rapid and user-friendly in silico bioinformatic pipeline for the de novo identification and characterization of raw RNAseq reads from venom glands to elucidate cysteine-rich peptides from the arsenal of venomous organisms.Implementation: This project develops a user-friendly automated bioinformatics pipeline via a Galaxy workflow to identify novel venom peptides from raw RNAseq reads of terebrid snails. While designed for venomous terebrid snails, with minor adjustments, this pipeline can be made universal to identify secreted disulfide-rich peptide toxins from any venomous organism., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Bacteria compete against related individuals by delivering toxins. In myxobacteria, a key delivery and kin discrimination mechanism is called outer membrane (OM) exchange (OME). Here, cells that display compatible polymorphic cell surface receptors recognize one another and bidirectionally transfer OM content. Included in the cargo is a suite of polymorphic SitA lipoprotein toxins. Consequently, OME between compatible cells that are not clonemates results in intoxication, while exchange between clonemates is harmonious because cells express a cognate repertoire of immunity proteins, which themselves are not transferred. SitA toxins belong to six nonhomologous families classified by sequence conservation within their N-terminal "escort domains" (EDs), while their C termini contain polymorphic nucleases that target the cytoplasmic compartment. To investigate how toxins delivered to the OM by OME translocate to the cytoplasm, we selected transposon mutants resistant to each family. Our screens identified eight genes that conferred resistance in a SitA family-specific manner. Most of these genes are predicted to localize to the cell envelope, and some resemble proteins that colicins exploit to gain cell entry. By constructing functional chimeric SitAs between families, we show that the ED determines the specificity of resistance. Importantly, a mutant that confers resistance to all six SitA families was discovered. This gene was named traC and plays an accessory role with traAB in OME. This work thus provides insight into the mechanism of kin discrimination in myxobacteria and provides working models for how SitA toxins exploit host proteins to gain cytoplasmic entry. IMPORTANCE Many bacterial species use diverse systems to deliver bacteriocins or toxins to neighboring competing cells. These systems are often selective in targeting cells that are related to themselves and therefore compete in the same niches for resources. How these systems specifically identify target cells and deliver toxins to particular cellular compartments is a fundamental question. This study uses the model social bacterium Myxococcus xanthus to unravel how its kin discrimination system, called outer membrane exchange, works. Along with the TraA polymorphic cell surface receptor that identifies related individuals with compatible receptors, this work discovered a new protein, called TraC, that functions in this discrimination system. Additionally, genetic screens identified host factors that are proposed to be involved in the cytoplasmic entry of lipoprotein toxins from the OM. This work complements and broadens our mechanistic understanding of how bacteria use transport systems to discriminate against related foes to build clonal populations.

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., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Rotavirus is known to be responsible for remarkable numbers of severe diarrheal episodes and even death in infants and young children. In this study, we aimed to survey genetic diversity and variation analysis of viroporin, which is encoded by the rotavirus NSP4 segment. Thirty-five rotavirus-positive specimens were obtained, and RNA extraction and polymerase chain reaction amplification were performed. After the sequencing process, four specimens were excluded, and the final 31 samples remained for genetic diversity and variation analysis. The predominant single G/P combination was G1P[8] (~78%), followed by G2P[8] (~13%), and equal percentages (3%) of G2P[4], G3P[8], and G-non-typeable-P[8]. Further analyses revealed that variations could be found in the three regions of NSP4, including VP4 binding site (aa 112-146), double-layered particle binding site (aa 161-175), and finally, in the predicted amphipathic alpha-helix. Phylogenic tree analysis demonstrated that the mentioned samples clustered with genotype E1 and E2 reference sequences. As previously reported in the literature, in this study, it was revealed that no apparent correlation exists in the deduced amino acid sequences corresponding to this region between the rotaviruses collected from patients with and without diarrhea., (© 2021 Wiley Periodicals LLC.)

Viral infections lead to expeditious activation of the host's innate immune responses, most importantly the interferon (IFN) response, which manifests a network of interferon-stimulated genes (ISGs) that constrain escalating virus replication by fashioning an ill-disposed environment. Interestingly, most viruses, including rotavirus, have evolved numerous strategies to evade or subvert host immune responses to establish successful infection. Several studies have documented the induction of ISGs during rotavirus infection. In this study, we evaluated the induction and antiviral potential of viperin, an ISG, during rotavirus infection. We observed that rotavirus infection, in a stain independent manner, resulted in progressive upregulation of viperin at increasing time points post-infection. Knockdown of viperin had no significant consequence on the production of total infectious virus particles. Interestingly, substantial escalation in progeny virus release was observed upon viperin knockdown, suggesting the antagonistic role of viperin in rotavirus release. Subsequent studies unveiled that RV-NSP4 triggered relocalization of viperin from the ER, the normal residence of viperin, to mitochondria during infection. Furthermore, mitochondrial translocation of NSP4 was found to be impeded by viperin, leading to abridged cytosolic release of Cyt c and subsequent inhibition of intrinsic apoptosis. Additionally, co-immunoprecipitation studies revealed that viperin associated with NSP4 through regions including both its radical SAM domain and its C-terminal domain. Collectively, the present study demonstrated the role of viperin in restricting rotavirus egress from infected host cells by modulating NSP4 mediated apoptosis, highlighting a novel mechanism behind viperin's antiviral action in addition to the intricacy of viperin-virus interaction.

The extracellular Contractile Injection System (eCIS) is a toxin-delivery particle that evolved from a bacteriophage tail. Four eCISs have previously been shown to mediate interactions between bacteria and their invertebrate hosts. Here, we identify eCIS loci in 1,249 bacterial and archaeal genomes and reveal an enrichment of these loci in environmental microbes and their apparent absence from mammalian pathogens. We show that 13 eCIS-associated toxin genes from diverse microbes can inhibit the growth of bacteria and/or yeast. We identify immunity genes that protect bacteria from self-intoxication, further supporting an antibacterial role for some eCISs. We also identify previously undescribed eCIS core genes, including a conserved eCIS transcriptional regulator. Finally, we present our data through an extensive eCIS repository, termed eCIStem. Our findings support eCIS as a toxin-delivery system that is widespread among environmental prokaryotes and likely mediates antagonistic interactions with eukaryotes and other prokaryotes.

The venoms of small rear-fanged snakes (RFS) remain largely unexplored, despite increased recognition of their importance in understanding venom evolution more broadly. Sequencing the transcriptome of venom-producing glands has greatly increased the ability of researchers to examine and characterize the toxin repertoire of small taxa with low venom yields. Here, we use RNA-seq to characterize the Duvernoy's gland transcriptome of the Plains Black-headed Snake, Tantilla nigriceps , a small, semi-fossorial colubrid that feeds on a variety of potentially dangerous arthropods including centipedes and spiders. We generated transcriptomes of six individuals from three localities in order to both characterize the toxin expression of this species for the first time, and to look for initial evidence of venom variation in the species. Three toxin families-three-finger neurotoxins (3FTxs), cysteine-rich secretory proteins (CRISPs), and snake venom metalloproteinases (SVMPIIIs)-dominated the transcriptome of T. nigriceps ; 3FTx themselves were the dominant toxin family in most individuals, accounting for as much as 86.4% of an individual's toxin expression. Variation in toxin expression between individuals was also noted, with two specimens exhibiting higher relative expression of c-type lectins than any other sample (8.7-11.9% compared to <1%), and another expressed CRISPs higher than any other toxin. This study provides the first Duvernoy's gland transcriptomes of any species of Tantilla , and one of the few transcriptomic studies of RFS not predicated on a single individual. This initial characterization demonstrates the need for further study of toxin expression variation in this species, as well as the need for further exploration of small RFS venoms.

The bacteriophage ΦX174 causes large pore formation in Escherichia coli and related bacteria. Lysis is mediated by the small membrane-bound toxin ΦX174-E, which is composed of a transmembrane domain and a soluble domain. The toxin requires activation by the bacterial chaperone SlyD and inhibits the cell wall precursor forming enzyme MraY. Bacterial cell wall biosynthesis is an important target for antibiotics; therefore, knowledge of molecular details in the ΦX174-E lysis pathway could help to identify new mechanisms and sites of action. In this study, cell-free expression and nanoparticle technology were combined to avoid toxic effects upon ΦX174-E synthesis, resulting in the efficient production of a functional full-length toxin and engineered derivatives. Pre-assembled nanodiscs were used to study ΦX174-E function in defined lipid environments and to analyze its membrane insertion mechanisms. The conformation of the soluble domain of ΦX174-E was identified as a central trigger for membrane insertion, as well as for the oligomeric assembly of the toxin. Stable complex formation of the soluble domain with SlyD is essential to keep nascent ΦX174-E in a conformation competent for membrane insertion. Once inserted into the membrane, ΦX174-E assembles into high-order complexes via its transmembrane domain and oligomerization depends on the presence of an essential proline residue at position 21. The data presented here support a model where an initial contact of the nascent ΦX174-E transmembrane domain with the peptidyl-prolyl isomerase domain of SlyD is essential to allow a subsequent stable interaction of SlyD with the ΦX174-E soluble domain for the generation of a membrane insertion competent toxin., (© 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Bacteriophage vB&#95;EcoM&#95;fHy-Eco03 (fHy-Eco03 for short) was isolated from a sewage sample based on its ability to infect an Escherichia coli clinical blood culture isolate. Altogether, 32 genes encoding hypothetical proteins of unknown function (HPUFs) were identified from the genomic sequence of fHy-Eco03. The HPUFs were screened for toxic properties (toxHPUFs) with a novel, Next Generation Sequencing (NGS)-based approach. This approach identifies toxHPUF-encoding genes through comparison of gene-specific read coverages in DNA from pooled ligation mixtures before electroporation and pooled transformants after electroporation. The performance and reliability of the NGS screening assay was compared with a plating efficiency-based method, and both methods identified the fHy-Eco03 gene g05 product as toxic. While the outcomes of the two screenings were highly similar, the NGS screening assay outperformed the plating efficiency assay in both reliability and efficiency. The NGS screening assay can be used as a high throughput method in the search for new phage-inspired antimicrobial molecules.

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 IC 50  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., (© 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

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., (© 2020 Wiley-VCH GmbH.)

Toxin-antidote elements (TAs) are selfish genetic dyads that spread in populations by selectively killing non-carriers. TAs are common in prokaryotes, but very few examples are known in animals. Here, we report the discovery of maternal-effect TAs in both C. tropicalis and C. briggsae, two distant relatives of C. elegans. In C. tropicalis, multiple TAs combine to cause a striking degree of intraspecific incompatibility: five elements reduce the fitness of >70% of the F 2 hybrid progeny of two Caribbean isolates. We identified the genes underlying one of the novel TAs, slow-1/grow-1, and found that its toxin, slow-1, is homologous to nuclear hormone receptors. Remarkably, although previously known TAs act during embryonic development, maternal loading of slow-1 in oocytes specifically slows down larval development, delaying the onset of reproduction by several days. Finally, we found that balancing selection acting on linked, conflicting TAs hampers their ability to spread in populations, leading to more stable genetic incompatibilities. Our findings indicate that TAs are widespread in Caenorhabditis species and target a wide range of developmental processes and that antagonism between them may cause lasting incompatibilities in natural populations. We expect that similar phenomena exist in other animal species., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Protein-bound uremic toxins (Indoxyl sulfate [IS] and p-cresyl sulfate [PCS]) are both associated with cardiovascular (CV) and all-cause mortality in subjects with chronic kidney disease (CKD). Possible mechanisms have not been elucidated. In hemodialysis patients, we investigated the relationship between the free form of IS and PCS and 181 CV-related proteins. First, IS or PCS concentrations were checked, and high levels were associated with an increased risk of acute coronary syndrome (ACS) in 333 stable HD patients. CV proteins were further quantified by a proximity extension assay. We examined associations between the free form protein-bound uremic toxins and the quantified proteins with correction for multiple testing in the discovery process. In the second step, the independent association was evaluated by multivariable-adjusted models. We rank the CV proteins related to protein-bound uremic toxins by bootstrapped confidence intervals and ascending p-value. Six proteins (signaling lymphocytic activation molecule family member 5, complement component C1q receptor, C-C motif chemokine 15 [CCL15], bleomycin hydrolase, perlecan, and cluster of differentiation 166 antigen) were negatively associated with IS. Fibroblast growth factor 23 [FGF23] was the only CV protein positively associated with IS. Three proteins (complement component C1q receptor, CCL15, and interleukin-1 receptor-like 2) were negatively associated with PCS. Similar findings were obtained after adjusting for classical CV risk factors. However, only higher levels of FGF23 was related to increased risk of ACS. In conclusion, IS and PCS were associated with several CV-related proteins involved in endothelial barrier function, complement system, cell adhesion, phosphate homeostasis, and inflammation. Multiplex proteomics seems to be a promising way to discover novel pathophysiology of the uremic toxin.

Venomics, the study of biological venoms, could potentially provide a new source of therapeutic compounds, yet information on the venoms from marine organisms, including cnidarians (sea anemones, corals, and jellyfish), is limited. This study identified the putative toxins of two species of jellyfish-edible jellyfish Rhopilema esculentum Kishinouye, 1891, also known as flame jellyfish, and Amuska jellyfish Sanderia malayensis Goette, 1886. Utilizing nano-flow liquid chromatography tandem mass spectrometry (nLC-MS/MS), 3000 proteins were identified from the nematocysts in each of the above two jellyfish species. Forty and fifty-one putative toxins were identified in R. esculentum and S. malayensis , respectively, which were further classified into eight toxin families according to their predicted functions. Amongst the identified putative toxins, hemostasis-impairing toxins and proteases were found to be the most dominant members (>60%). The present study demonstrates the first proteomes of nematocysts from two jellyfish species with economic and environmental importance, and expands the foundation and understanding of cnidarian toxins.

Geographical variation of animal venom is common among venomous animals. This kind of intraspecific variation based on geographical location mainly concerned from envenomation cases and brought new problems in animal venom studies, including venom components regulatory mechanisms, differentiation of venom activities, and clinical treatment methods. At present, food is considered as the most related factor influencing venom development. Related research defined the variational venomous animal species by the comparison of venom components and activities in snakes, jellyfish, scorpions, cone snails, ants, parasitoid wasps, spiders and toads. In snake venom studies, researchers found that antivenom effectiveness was variated to different located venom samples. As described in some snake venom research, developing region-specific antivenom is the development trend. The difficulties of developing region-specific antivenom and theoretical solutions have been discussed. This review summarized biological studies of animal venom geographical variation by species, compared venom components and major biological activities of the vary venom from the same species, and listed the basic methods in comparing venom protein compositions and major toxicity differences to provide a comprehensive reference., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Rotavirus C (RVC) is an enteric pathogen that affects humans and animals around the world., Methods: In this study, we characterized the genetic diversity of RVC strains detected in asymptomatic Brazilian pigs by sequencing the NSP4, NSP5 and VP6 genes., Results: The results of reverse transcription polymerase chain reaction showed that 53 of 579 samples (9.2%) contained RVC. Positive samples were genotyped by sequencing gene segments NSP4, and NSP5. Most of the RCV strains encountered were classified into typically porcine genotypes: E1-H1. In two strains, BP182 and BP208, the NSP4 gene grouped with E2-RVC human strains with 94.2%-96.5% nucleotide identity, although the NSP5 gene was porcine-like (H1). In strain SD67, the NSP5 gene grouped with human H2-RVC with 92.5%-98.7% nucleotide identity and the NSP4 gene grouped with porcine strains (E1). Two strains (BP208 and SD67) were also genotyped by sequencing gene segment VP6. The VP6 gene grouped with porcine strains, I6 (89.3%-90.2% nucleotide identity) and I5 (88.7%-90.5% nucleotide identity), for strains BP208 and SD67, respectively., Conclusions: These results are indicative of genomic reassortment between RVC strains of human and porcine origin. In recent years, the incidence of RVC infection among humans has increased significantly. It is important to measure the frequency of interspecies transmission in order to monitor the evolution of these viruses and to identify rearranged strains that may lead to an epidemic., (© 2020 Blackwell Verlag GmbH.)

In life's constant battle for survival, it takes one to kill but two to conquer. Toxin-antitoxin or toxin-antidote (TA) elements are genetic dyads that cheat the laws of inheritance to guarantee their transmission to the next generation. This seemingly simple genetic arrangement-a toxin linked to its antidote-is capable of quickly spreading and persisting in natural populations. TA elements were first discovered in bacterial plasmids in the 1980s and have recently been characterized in fungi, plants, and animals, where they underlie genetic incompatibilities and sterility in crosses between wild isolates. In this review, we provide a unified view of TA elements in both prokaryotic and eukaryotic organisms and highlight their similarities and differences at the evolutionary, genetic, and molecular levels. Finally, we propose several scenarios that could explain the paradox of the evolutionary origin of TA elements and argue that these elements may be key evolutionary players and that the full scope of their roles is only beginning to be uncovered.

Species A rotaviruses are a major cause of acute gastroenteritis in infants and young children worldwide. Reassortment is a common phenomenon due to the segmented nature of the rotavirus genome. The complete coding sequences of a species A rotavirus strain isolated from the feces of a child with acute gastroenteritis in Japan in 2018 were determined using an unbiased viral metagenomics approach. The genetic analysis revealed that the rotavirus strain had an unusual genomic constellation (G1-P[8]-I1-R1-C1-M1-A1-N1-T1-E2-H1), suggesting reassortment of a genotype 1 with a genotype 2 rotavirus, from which the NSP4-encoding gene was acquired.

Pore-forming proteins/toxins (PFPs/PFTs) are the distinct class of membrane-damaging proteins. They act by forming oligomeric pores in the plasma membranes. PFTs and PFPs from diverse organisms share a common mechanism of action, in which the designated pore-forming motifs of the membrane-bound protein molecules insert into the membrane lipid bilayer to create the water-filled pores. One common characteristic of these pore-forming motifs is that they are amphipathic in nature. In general, the hydrophobic sidechains of the pore-forming motifs face toward the hydrophobic core of the membranes, while the hydrophilic residues create the lining of the water-filled pore lumen. Interestingly, pore-forming motifs of the distinct subclass of PFPs/PFTs share very little sequence similarity with each other. Therefore, the common guiding principle that governs the sequence-to-structure paradigm in the mechanism of action of these PFPs/PFTs still remains an enigma. In this article, we discuss this notion using the examples of diverse groups of membrane-damaging PFPs/PFTs.

Amyloids are fibrillar protein aggregates with an ordered spatial structure called "cross-β". While some amyloids are associated with development of approximately 50 incurable diseases of humans and animals, the others perform various crucial physiological functions. The greatest diversity of amyloids functions is identified within prokaryotic species where they, being the components of the biofilm matrix, function as adhesins, regulate the activity of toxins and virulence factors, and compose extracellular protein layers. Amyloid state is widely used by different pathogenic bacterial species in their interactions with eukaryotic organisms. These amyloids, being functional for bacteria that produce them, are associated with various bacterial infections in humans and animals. Thus, the repertoire of the disease-associated amyloids includes not only dozens of pathological amyloids of mammalian origin but also numerous microbial amyloids. Although the ability of symbiotic microorganisms to produce amyloids has recently been demonstrated, functional roles of prokaryotic amyloids in host-symbiont interactions as well as in the interspecies interactions within the prokaryotic communities remain poorly studied. Here, we summarize the current findings in the field of prokaryotic amyloids, classify different interspecies interactions where these amyloids are involved, and hypothesize about their real occurrence in nature as well as their roles in pathogenesis and symbiosis.

Improved genome engineering methods that enable automation of large and precise edits are essential for systematic investigations of genome function. We adapted peel-1 negative selection to an optimized Dual-Marker Selection (DMS) cassette protocol for CRISPR-Cas9 genome engineering in Caenorhabditis elegans and observed robust increases in multiple measures of efficiency that were consistent across injectors and four genomic loci. The use of Peel-1-DMS selection killed animals harboring transgenes as extrachromosomal arrays and spared genome-edited integrants, often circumventing the need for visual screening to identify genome-edited animals. To demonstrate the applicability of the approach, we created deletion alleles in the putative proteasomal subunit pbs-1 and the uncharacterized gene K04F10.3 and used machine vision to automatically characterize their phenotypic profiles, revealing homozygous essential and heterozygous behavioral phenotypes. These results provide a robust and scalable approach to rapidly generate and phenotype genome-edited animals without the need for screening or scoring by eye., Competing Interests: The authors have declared that no competing interests exist.

Ribosome Inactivating Proteins (RIPs) are RNA N-glycosidases that depurinate a specific adenine residue in the conserved sarcin/ricin loop of the 28S rRNA. The occurrence of RIP genes has been described in a wide range of plant taxa, as well as in several species of bacteria and fungi. A remarkable case is the presence of these genes in metazoans belonging to the Culicinae subfamily. We reported that these genes are derived from a single horizontal gene transfer event, most likely from a bacterial donor species. Moreover, we have shown evidence that mosquito RIP genes are evolving under purifying selection, suggesting that these toxins have acquired a functional role in these organisms. In the present work, we characterized the intra-specific sequence variability of Aedes aegypti RIP genes (RIPAe1, RIPAe2, and RIPAe3) and tested their expression at the mRNA level. Our results show that RIPAe2 and RIPAe3 are transcribed and polyadenylated, and their expression levels are modulated across the developmental stages. Varibility among genes was observed, including the existence of null alleles for RIPAe1 and RIPAe2, with variants showing partial deletions. These results further support the existence of a physiological function for these foreign genes in mosquitoes. The possible nature of this functionality is discussed., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

Animal venoms are recognised as unique biological systems in which to study molecular evolution. Venom use has evolved numerous times among the insects, and insects today use venom to capture prey, defend themselves from predators, or to subdue and modulate host responses during parasitism. However, little is known about most insect venom toxins or the mode and tempo by which they evolve. Here, I review the evolutionary dynamics of insect venom toxins, and argue that insects offer many opportunities to examine novel aspects of toxin evolution. The key questions addressed are: How do venomous animals evolve from non-venomous animals, and how does this path effect the composition and pharmacology of the venom? What genetic processes (gene duplication, co-option, neofunctionalisation) are most important in toxin evolution? What kinds of selection pressures are acting on toxin-encoding genes and their cognate targets in envenomated animals? The emerging evidence highlights that venom composition and pharmacology adapts quickly in response to changing selection pressures resulting from new ecological interactions, and that such evolution occurs through a stunning variety of genetic mechanisms. Insects offer many opportunities to investigate the evolutionary dynamics of venom toxins due to their evolutionary history rich in venom-related adaptations, and their quick generation time and suitability for culture in the laboratory., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Stenodactylin is one of the most potent type 2 ribosome-inactivating proteins (RIPs); its high toxicity has been demonstrated in several models both in vitro and in vivo. Due to its peculiarities, stenodactylin could have several medical and biotechnological applications in neuroscience and cancer treatment. In this work, we report the complete amino acid sequence of stenodactylin and 3D structure prediction. The comparison between the primary sequence of stenodactylin and other RIPs allowed us to identify homologies/differences and the amino acids involved in RIP toxic activity. Stenodactylin RNA was isolated from plant caudex, reverse transcribed through PCR and the cDNA was amplificated and cloned into a plasmid vector and further analyzed by sequencing. Nucleotide sequence analysis showed that stenodactylin A and B chains contain 251 and 258 amino acids, respectively. The key amino acids of the active site described for ricin and most other RIPs are also conserved in the stenodactylin A chain. Stenodactylin amino acid sequence shows a high identity degree with volkensin (81.7% for A chain, 90.3% for B chain), whilst when compared with other type 2 RIPs the identity degree ranges from 27.7 to 33.0% for the A chain and from 42.1 to 47.7% for the B chain.

Slow lorises are enigmatic animal that represent the only venomous primate lineage. Their defensive secretions have received little attention. In this study we determined the full length sequence of the protein secreted by their unique brachial glands. The full length sequences displayed homology to the main allergenic protein present in cat dander. We thus compared the molecular features of the slow loris brachial gland protein and the cat dander allergen protein, showing remarkable similarities between them. Thus we postulate that allergenic proteins play a role in the slow loris defensive arsenal. These results shed light on these neglected, novel animals., Competing Interests: The authors declare no conflict of interest.

Wnt dependency and Lgr5 expression define multiple mammalian epithelial stem cell types. Under defined growth factor conditions, such adult stem cells (ASCs) grow as 3D organoids that recapitulate essential features of the pertinent epithelium. Here, we establish long-term expanding venom gland organoids from several snake species. The newly assembled transcriptome of the Cape coral snake reveals that organoids express high levels of toxin transcripts. Single-cell RNA sequencing of both organoids and primary tissue identifies distinct venom-expressing cell types as well as proliferative cells expressing homologs of known mammalian stem cell markers. A hard-wired regional heterogeneity in the expression of individual venom components is maintained in organoid cultures. Harvested venom peptides reflect crude venom composition and display biological activity. This study extends organoid technology to reptilian tissues and describes an experimentally tractable model system representing the snake venom gland., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Rotavirus A (RVA) has been detected in patients with gastroenteritis even after vaccine introduction in Japan. To investigate circulating RVA strains, RVA-positive stool specimens obtained in Tokyo in 2017 and 2018 were analyzed using next-generation sequencing. A total of 50 and 21 RVA samples were obtained in 2017 and 2018, respectively. In 2017, G2P[4] (40.0%) was the most prevalent strain, followed by G3P[8] (DS-1-like) (28.0%), G8P[8] (10.0%), G3P[8] (Wa-like) (8.0%), G9P[8]-E1 (8.0%), and mixed infection (6.0%). In 2018, G3P[8] (DS-1-like) (28.6%) and G9P[8]-E2 (28.6%) were the most prevalent strains, followed by G9P[8]-E1 (19.0%), G2P[4] (9.5%), G8P[8] (9.5%), and mixed infection (4.8%). Six G9P[8]-E2 strains detected in 2018 showed an atypical genotype constellation (G9P[8]-I1-R1-C1-M1-A1-N1-T1-E2-H1), which had not been reported previously. Phylogenetic analyses suggested that the RVA virus was generated by inter-genogroup reassortment between commonly circulating G9P[8] and G2P[4] strains in Japan. The G9P[8] strain seemed to be reassorted with only the NSP4 gene of the E2 genotype of the G2P[4] strain. Since this newly-emerged G9P[8]-E2 virus was detected in different locations in Tokyo, the virus appears to have already begun to spread to a wider area.

Biomphalaria glabrata is a freshwater Planorbidae snail. In its environment, this mollusk faces numerous microorganisms or pathogens, and has developed sophisticated innate immune mechanisms to survive. The mechanisms of recognition are quite well understood in Biomphalaria glabrata , but immune effectors have been seldom described. In this study, we analyzed a new family of potential immune effectors and characterized five new genes that were named Glabralysins. The five Glabralysin genes showed different genomic structures and the high degree of amino acid identity between the Glabralysins, and the presence of the conserved ETX/MTX2 domain, support the hypothesis that they are pore-forming toxins. In addition, tertiary structure prediction confirms that they are structurally related to a subset of Cry toxins from Bacillus thuringiensis , including Cry23, Cry45, and Cry51. Finally, we investigated their gene expression profiles in snail tissues and demonstrated a mosaic transcription. We highlight the specificity in Glabralysin expression following immune stimulation with bacteria, yeast or trematode parasites. Interestingly, one Glabralysin was found to be expressed in immune-specialized hemocytes, and two others were induced following parasite exposure., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Many viruses that replicate in the cytoplasm dramatically remodel and stimulate the accumulation of host cell membranes for efficient replication by poorly understood mechanisms. For rotavirus, a critical step in virion assembly requires the accumulation of membranes adjacent to virus replication centers called viroplasms. Early electron microscopy studies describe viroplasm-associated membranes as "swollen" endoplasmic reticulum (ER). We previously demonstrated that rotavirus infection initiates cellular autophagy and that membranes containing the autophagy marker protein LC3 and the rotavirus ER-synthesized transmembrane glycoprotein NSP4 traffic to viroplasms, suggesting that NSP4 must exit the ER. This study aimed to address the mechanism of NSP4 exit from the ER and determine whether the viroplasm-associated membranes are ER derived. We report that (i) NSP4 exits the ER in COPII vesicles, resulting in disrupted COPII vesicle transport and ER exit sites; (ii) COPII vesicles are hijacked by LC3 II, which interacts with NSP4; and (iii) NSP4/LC3 II-containing membranes accumulate adjacent to viroplasms. In addition, the ER transmembrane proteins SERCA and calnexin were not detected in viroplasm-associated membranes, providing evidence that the rotavirus maturation process of "budding" occurs through autophagy-hijacked COPII vesicle membranes. These findings reveal a new mechanism for rotavirus maturation dependent on intracellular host protein transport and autophagy for the accumulation of membranes required for virus replication. IMPORTANCE In a morphogenic step that is exceedingly rare for nonenveloped viruses, immature rotavirus particles assemble in replication centers called viroplasms, and bud through cytoplasmic cellular membranes to acquire the outer capsid proteins for infectious particle assembly. Historically, the intracellular membranes used for particle budding were thought to be endoplasmic reticulum (ER) because the rotavirus nonstructural protein NSP4, which interacts with the immature particles to trigger budding, is synthesized as an ER transmembrane protein. This present study shows that NSP4 exits the ER in COPII vesicles and that the NSP4-containing COPII vesicles are hijacked by the cellular autophagy machinery, which mediates the trafficking of NSP4 to viroplasms. Changing the paradigm for rotavirus maturation, we propose that the cellular membranes required for immature rotavirus particle budding are not an extension of the ER but are COPII-derived autophagy isolation membranes., (Copyright © 2019 American Society for Microbiology.)

Gene transfer and homologous recombination in Streptococcus pyogenes has the potential to trigger the emergence of pandemic lineages, as exemplified by lineages of emm 1 and emm 89 that emerged in the 1980s and 2000s, respectively. Although near-identical replacement gene transfer events in the nga (NADase) and slo (streptolysin O) loci conferring high expression of these toxins underpinned the success of these lineages, extension to other emm genotype lineages is unreported. The emergent emm 89 lineage was characterized by five regions of homologous recombination additional to nga-slo , including complete loss of the hyaluronic acid capsule synthesis locus hasABC , a genetic trait replicated in two other leading emm types and recapitulated by other emm types by inactivating mutations. We hypothesized that other leading genotypes may have undergone similar recombination events. We analyzed a longitudinal data set of genomes from 344 clinical invasive disease isolates representative of locations across England, dating from 2001 to 2011, and an international collection of S. pyogenes genomes representing 54 different genotypes and found frequent evidence of recombination events at the nga - slo locus predicted to confer higher toxin genotype. We identified multiple associations between recombination at this locus and inactivating mutations within hasAB , suggesting convergent evolutionary pathways in successful genotypes. This included common genotypes emm 28 and emm 87. The combination of no or low capsule and high expression of nga and slo may underpin the success of many emergent S. pyogenes lineages of different genotypes, triggering new pandemics, and could change the way S. pyogenes causes disease. IMPORTANCE Streptococcus pyogenes is a genetically diverse pathogen, with over 200 different genotypes defined by emm typing, but only a minority of these genotypes are responsible for the majority of human infection in high-income countries. Two prevalent genotypes associated with disease rose to international dominance following recombination of a toxin locus that conferred increased expression. Here, we found that recombination of this locus and promoter has occurred in other diverse genotypes, events that may allow these genotypes to expand in the population. We identified an association between the loss of hyaluronic acid capsule synthesis and high toxin expression, which we propose may be associated with an adaptive advantage. As S. pyogenes pathogenesis depends both on capsule and toxin production, new variants with altered expression may result in abrupt changes in the molecular epidemiology of this pathogen in the human population over time., (Copyright © 2019 Turner et al.)

With advancements in synthetic biology, the cost and the time needed for designing and synthesizing customized gene products have been steadily decreasing. Many research laboratories in academia as well as industry routinely create genetically engineered proteins as a part of their research activities. However, manipulation of protein sequences could result in unintentional production of toxic proteins. Therefore, being able to identify the toxicity of a protein before the synthesis would reduce the risk of potential hazards. Existing methods are too specific, which limits their application. Here, we extended general function prediction methods for predicting the toxicity of proteins. Protein function prediction methods have been actively studied in the bioinformatics community and have shown significant improvement over the last decade. We have previously developed successful function prediction methods, which were shown to be among top-performing methods in the community-wide functional annotation experiment, CAFA. Based on our function prediction method, we developed a neural network model, named NNTox, which uses predicted GO terms for a target protein to further predict the possibility of the protein being toxic. We have also developed a multi-label model, which can predict the specific toxicity type of the query sequence. Together, this work analyses the relationship between GO terms and protein toxicity and builds predictor models of protein toxicity.