Recombinant Production and Characterization of a New Toxin from Cryptops iheringi Centipede Venom Revealed by Proteome and Transcriptome Analysis

Among the Chilopoda class of centipede, the Cryptops genus is one of the most associated with envenomation in humans in the metropolitan region of the state of São Paulo. To date, there is no study in the literature about the toxins present in its venom. Thus, in this work, a transcriptomic characterization of the Cryptops iheringi venom gland, as well as a proteomic analysis of its venom, were performed to obtain a toxin profile of this species. These methods indicated that 57.9% of the sequences showed to be putative toxins unknown in public databases; among them, we pointed out a novel putative toxin named Cryptoxin-1. The recombinant form of this new toxin was able to promote edema in mice footpads with massive neutrophils infiltration, linking this toxin to envenomation symptoms observed in accidents with humans. Our findings may elucidate the role of this toxin in the venom, as well as the possibility to explore other proteins found in this work.

Mycotoxin-associated diseases in Yakutia

В странах ЕС любое негативное изменение продуктивности сельскохозяйственных животных (снижение привеса, удоя, репродуктивных показателей, а также повышение индекса заболеваемости) связывают с влиянием микотоксинов, поступающих, как правило, вместе с кормами. Поэтому основной задачей при диагностике микотоксикозов является выявление возможных продуцентов микотоксинов — «плесневых» грибов — в рационах животных. Имея полную картину выявленных в кормах грибов, можно предугадать виды токсинов, характер их воздействия на организм, возможный синергический эффект и порядок превентивного действия для снижения экономического ущерба от микотоксикозов. В статье отражены наиболее важные характеристики микроскопических грибов и их метаболитов; основные лимитирующие условия природно-климатического характера для токсинообразования в растительных кормах; описаны экстремальные условия введения животноводства в Якутии; приведено краткое описание результатов первых микологических исследований, проведённых в ходе научной экспедиции 1925–1926 годов, и первого случая микотоксикоза лошадей, выявленного научным сотрудником Якутского НИИСХ П. И. Николаевым (1983). Кроме того, проведён анализ официальных данных микологического изучения кормов растительного происхождения; установлена связь скармливания поражённых плеснеобразующими грибами кормов с болезнями микотического характера сельскохозяйственных животных. Для снижения количества плесневых грибов при заготовке сена, снижения чувствительности животных к негативному воздействию недоброкачественных кормов наиболее эффективно использование аэробных спорообразующих микроорганизмов Bacillus subtilis, а также природных сорбентов из местных ресурсов. Вопросы нейтрализации и снижения токсигенного эффекта кормов, контаминированных плесневыми грибами и их микотоксинами, остаются актуальными и требуют дальнейших научных исследований. European Union correlates low livestock productivity with mycotoxin poisoning through consumed feed (decreased body weight gain, milk yield, reproductivity and increased susceptibility to various diseases). When analyzing mycotoxicoses it is highly important to identify putative toxin-producing strains of fungi in forage. Accurate identification of fungus species provides valuable information on toxin types, their influence on an organism, possible synergistic effects allowing effective application of preventive measures. This paper provides information on the most important characteristics of fungi and their metabolites. Environmental impact on toxin biosynthesis in plants is presented. Special aspects of Animal Husbandry in Yakutia are described. First mycological investigations of 1925–1926 are reviewed. Forage contaminations with fungi are analyzed. The correlation between fungus-infested forage consumption and disease outbreaks was determined. Treatment of fodder with spore-forming bacteria Bacillus subtilis or use of natural sorbents are able to prevent fungus spread in plant material as well as improve livestock health. Toxin poisoning still remains a serious problem in Agriculture.

A new contact killing toxin permeabilizes cells and belongs to a large protein family

Vibrio cholerae is an aquatic Gram-negative bacterium that causes severe diarrheal cholera disease when ingested by humans. To eliminate competitor cells in both the external environment and inside hosts, V. cholerae uses the Type VI Secretion System (T6SS). The T6SS is a macromolecular weapon employed by many Gram-negative bacteria to deliver cytotoxic proteins into adjacent cells. In addition to canonical T6SS gene clusters encoded by all sequenced V. cholerae isolates, strain BGT49 encodes an additional locus, which we named auxiliary cluster 4 (Aux 4). The Aux 4 cluster is located on a mobile genetic element and can be used by killer cells to eliminate both V. cholerae and Escherichia coli cells in a T6SS-dependent manner. A putative toxin encoded in the cluster, which we name TpeV (Type VI Permeabilizing Effector Vibrio), shares no homology to known proteins and does not contain motifs or domains indicative of function. Ectopic expression of TpeV in the periplasm of E. coli permeabilizes cells and disrupts the membrane potential. Using confocal microscopy, we confirm that susceptible target cells become permeabilized when competed with killer cells harboring the Aux 4 cluster. We also determine that tpiV, the gene located immediately downstream of tpeV, encodes an immunity protein that neutralizes the toxicity of TpeV. Finally, we show that TpeV homologs are broadly distributed across important animal and plant pathogens and are localized in proximity to other T6SS genes. Our results suggest that TpeV is a toxin that belongs to a large family of T6SS proteins.

Bodo saltans (Kinetoplastida) is dependent on a novel Paracaedibacter-like endosymbiont that possesses multiple putative toxin-antitoxin systems

Bacterial endosymbiosis has been instrumental in eukaryotic evolution, and includes both mutualistic, dependent and parasitic associations. Here we characterize an intracellular bacterium inhabiting the flagellated protist Bodo saltans (Kinetoplastida). We present a complete bacterial genome comprising a 1.39 Mb circular chromosome with 40.6% GC content. Fluorescent in situ hybridisation confirms that the endosymbiont is located adjacent to the nuclear membrane, and a detailed model of its intracellular niche is generated using serial block-face scanning electron microscopy. Phylogenomic analysis shows that the endosymbiont belongs to the Holosporales, most closely related to other α-proteobacterial endosymbionts of ciliates and amoebae. Comparative genomics indicates that it has a limited metabolism and is nutritionally host-dependent. However, the endosymbiont genome does encode diverse symbiont-specific secretory proteins, including a type VI secretion system and three separate toxin-antitoxin systems. We show that these systems are actively transcribed and hypothesize they represent a mechanism by which B. saltans becomes addicted to its endosymbiont. Consistent with this idea, attempts to cure Bodo of endosymbionts led to rapid and uniform cell death. This study adds kinetoplastid flagellates to ciliates and amoebae as hosts of Paracaedibacter-like bacteria, suggesting that these antagonistic endosymbioses became established very early in Eukaryotic evolution.

Size Matters: An Evaluation of the Molecular Basis of Ontogenetic Modifications in the Composition of Bothrops jararacussu Snake Venom

Ontogenetic changes in venom composition have been described in Bothrops snakes, but only a few studies have attempted to identify the targeted paralogues or the molecular mechanisms involved in modifications of gene expression during ontogeny. In this study, we decoded B. jararacussu venom gland transcripts from six specimens of varying sizes and analyzed the variability in the composition of independent venom proteomes from 19 individuals. We identified 125 distinct putative toxin transcripts, and of these, 73 were detected in venom proteomes and only 10 were involved in the ontogenetic changes. Ontogenetic variability was linearly related to snake size and did not correspond to the maturation of the reproductive stage. Changes in the transcriptome were highly predictive of changes in the venom proteome. The basic myotoxic phospholipases A2 (PLA2s) were the most abundant components in larger snakes, while in venoms from smaller snakes, PIII-class SVMPs were the major components. The snake venom metalloproteinases (SVMPs) identified corresponded to novel sequences and conferred higher pro-coagulant and hemorrhagic functions to the venom of small snakes. The mechanisms modulating venom variability are predominantly related to transcriptional events and may consist of an advantage of higher hematotoxicity and more efficient predatory function in the venom from small snakes.

Transcriptional Landscape of Waddlia chondrophila Aberrant Bodies Induced by Iron Starvation

Chronic infections caused by obligate intracellular bacteria belonging to the Chlamydiales order are related to the formation of persistent developmental forms called aberrant bodies (ABs), which undergo DNA replication without cell division. These enlarged bacteria develop and persist upon exposure to different stressful conditions such as β-lactam antibiotics, iron deprivation and interferon-γ. However, the mechanisms behind ABs biogenesis remain uncharted. Using an RNA-sequencing approach, we compared the transcriptional profile of ABs induced by iron starvation to untreated bacteria in the Chlamydia-related species Waddliachondrophila, a potential agent of abortion in ruminants and miscarriage in humans. Consistent with the growth arrest observed following iron depletion, our results indicate a significant reduction in the expression of genes related to energy production, carbohydrate and amino acid metabolism and cell wall/envelope biogenesis, compared to untreated, actively replicating bacteria. Conversely, three putative toxin-antitoxin modules were among the most up-regulated genes upon iron starvation, suggesting that their activation might be involved in growth arrest in adverse conditions, an uncommon feature in obligate intracellular bacteria. Our work represents the first complete transcriptomic profile of a Chlamydia-related species in stressful conditions and sets the grounds for further investigations on the mechanisms underlying chlamydial persistence.

Gut Fungi Possess a Conserved Toxin Immunity Gene of Bacterial Origin

Prokaryotes and some unicellular eukaryotes routinely overcome evolutionary pressures with the help of horizontally acquired genes. In contrast, it is unusual for multicellular eukaryotes to adapt through horizontal gene transfer (HGT). Recent studies identified several cases of adaptive acquisition in the gut-dwelling multicellular fungal phylum Neocallimastigomycota. Here, we add to these cases the acquisition of a putative bacterial toxin immunity gene, PoNi, by an ancient common ancestor of four extant Neocallimastigomycota genera through HGT from an extracellular Ruminococcus bacterium. The PoNi homologs in these fungal genera share extraordinarily high (>70%) amino acid sequence identity with their bacterial donor xenolog, providing definitive evidence of HGT as opposed to lineage-specific gene retention. Furthermore, PoNi genes are nested on native sections of chromosomal DNA in multiple fungal genomes and are also found in polyadenylated fungal transcriptomes, confirming that these genes are authentic fungal genomic regions rather than sequencing artifacts from bacterial contamination. The HGT event, which is estimated to have occurred at least 66 (±10) million years ago in the gut of a Cretaceous mammal, gave the fungi a putative toxin immunity protein (PoNi) which likely helps them survive toxin-mediated attacks by bacterial competitors in the mammalian gut microbiome.SignificanceAdaptation via horizontal gene transfer (HGT) is uncommon in multicellular eukaryotes. Here, we report another bona fide case of adaptive evolution involving the horizontal transfer of a bacterial toxin immunity gene from extracellular Ruminococcus bacteria to gut-dwelling multicellular fungi. The acquired gene may help the fungi compete against bacterial neighbors in the gut.

The Paracaedibacter-like endosymbiont of Bodo saltans (Kinetoplastida) uses multiple putative toxin-antitoxin systems to maintain its host association

Bacterial endosymbiosis has been instrumental in eukaryotic evolution, and includes both mutualistic, dependent and parasitic associations. Here we characterize an intracellular bacterium inhabiting the flagellated protist Bodo saltans (Kinetoplastida). We present a complete bacterial genome comprising a 1.39 Mb circular chromosome with 40.6% GC content. Fluorescent in situ hybridisation confirms that the endosymbiont is located adjacent to the nuclear membrane, and a detailed model of its intracellular niche is generated using serial block-face scanning electron microscopy. Phylogenomic analysis shows that the endosymbiont belongs to the Holosporales, most closely related to other α-proteobacterial endosymbionts of ciliates and amoebae. Comparative genomics indicates that it has a limited metabolism and is nutritionally host-dependent. However, the endosymbiont genome does encode diverse symbiont-specific secretory proteins, including a type VI secretion system and three separate toxin-antitoxin systems. We show that these systems are actively transcribed and hypothesize they represent a mechanism by which B. saltans becomes addicted to its endosymbiont. Consistent with this idea, attempts to cure Bodo of endosymbionts led to rapid and uniform cell death. This study adds kinetoplastid flagellates to ciliates and amoebae as hosts of Paracaedibacter-like bacteria, suggesting that these antagonistic endosymbioses became established very early in Eukaryotic evolution.

Beyond Cholera: Characterization of zot-Encoding Filamentous Phages in the Marine Fish Pathogen Vibrio anguillarum

Zonula occludens toxin (Zot) is a conserved protein in filamentous vibriophages and has been reported as a putative toxin in Vibrio cholerae. Recently, widespread distribution of zot-encoding prophages was found among marine Vibrio species, including environmental isolates. However, little is known about the dynamics of these prophages beyond V. cholerae. In this study, we characterized and quantified the zot-encoding filamentous phage VAIϕ, spontaneously induced from the fish pathogen V. anguillarum. VAIϕ contained 6117 bp encoding 11 ORFs, including ORF8pVAI, exhibiting 27%–73% amino acid identity to Inovirus Zot-like proteins. A qPCR method revealed an average of four VAIϕ genomes per host genome during host exponential growth phase, and PCR demonstrated dissemination of induced VAIϕ to other V. anguillarum strains through re-integration in non-lysogens. VAIϕ integrated into both chromosomes of V. anguillarum by recombination, causing changes in a putative ORF in the phage genome. Phylogenetic analysis of the V. anguillarum Inoviridae elements revealed mosaic genome structures related to mainly V. cholerae. Altogether, this study contributes to the understanding of Inovirus infection dynamics and mobilization of zot-like genes beyond human pathogenic vibrios, and discusses their potential role in the evolution of the fish pathogen V. anguillarum.