A snapshot of Bothrops jararaca snake venom gland subcellular proteome.

Snake venom protein synthesis undergoes finely regulated processes in the specialized secretory epithelium within the venom gland. Such processes occur within a defined period in the cell and at specific cellular locations. Thus, the determination of subcellular proteomes allows the characterization of protein groups for which the site may be relevant to their biological roles, thereby allowing the deconvolution of complex biological circuits into functional information. In this regard, we performed subcellular fractionation of proteins from B. jararaca venom gland, focusing on nuclear proteins since this cellular compartment comprises key effectors that shape gene expression. Our results provided a snapshot of B. jararaca's subcellular venom gland proteome and pointed to a 'conserved' proteome core among different life stages (newborn and adult) and between sexes (adult male and female). Overall, the top 15 highly abundant proteins identified in B. jararaca venom glands mirrored the panel of highly expressed genes in human salivary glands. Therefore, the expression profile observed for such a protein set could be considered a conserved core signature of salivary gland secretory epithelium. Moreover, the newborn venom gland displayed a unique expression signature of transcription factors involved in regulating transcription and biosynthetic processes and may mirror biological constraints of the ontogenetic development of B. jararaca, contributing to venom proteome diversity.

Hidden by the name: A new fluorescent pumpkin toadlet from the Brachycephalus ephippium group (Anura: Brachycephalidae).

Species of Brachycephalus has been having taxonomical issues due its morphological similarity and genetic conservatism. Herein, we describe a new species of Brachycephalus from the south Mantiqueira mountain range and semidecidual forests in the municipalities of Mogi das Cruzes, Campinas and Jundiaí, state of São Paulo, Brazil, based on an integrative approach. It can be distinguished from all species of the B. ephippium species group based on morphological characters (especially osteology and head shape), advertisement call and divergence in partial mitochondrial DNA gene sequences (16S). The new species is genetically similar to B. margaritatus and morphologically similar to B. ephippium. It can be differentiated from B. ephippium by the presence of dark faded spots on skull and post-cranial plates, presence of black connective tissue connective tissue scattered over dorsal musculature, parotic plate morphology, smaller snout-vent length (adult SVL: males 13.46-15.92 mm; females 16.04-17.69 mm) and 3% genetic distance. We also present natural history data and discuss the robustness of the integrative approach, geographic distribution, genetic data, behaviour, fluorescence in ontogeny, and conservation status.

The distinct N-terminomes of Bothrops jararaca newborn and adult venoms.

Using approaches of transcriptomics and proteomics we have shown that the phenotype of Bothrops jararaca venom undergoes a significant rearrangement upon neonate to adult transition. Most regulatory processes in biology are intrinsically related to modifications of protein structure, function, and abundance. However, it is unclear to which extent intrinsic proteolysis affects toxins and snake venom phenotypes upon ontogenesis. Here we assessed the natural N-terminome of Bothrops jararaca newborn and adult venoms and explored the degree of N-terminal protein truncation in ontogenetic-based proteome variation. To this end we applied the Terminal Amine Isotopic Labeling of Substrates (TAILS) technology to characterize venom collected in the presence of proteinase inhibitors. We identified natural N-terminal sequences in the newborn (71) and adult (84) venoms, from which only 37 were common to both. However, truncated toxins were found in higher number in the newborn (212) than in the adult (140) venom. Moreover, sequences N-terminally blocked by pyroglutamic acid were identified in the newborn (55) and adult (49) venoms. Most toxin classes identified by their natural N-terminal sequences showed a similar number of unique peptides in the newborn and adult venoms, however, those of serine proteinases and C-type lectins were more abundant in the adult venom. Truncated sequences from at least ten toxin classes were detected, however the catalytic and cysteine-rich domains of metalloproteinases were the most prone to proteolysis, mainly in the newborn venom. Our results underscore the pervasiveness of truncations in most toxin classes and highlight variable post-translational events in newborn and adult venoms.

Factors that can influence the survival rates of coral snakes (Micrurus corallinus) for antivenom production.

Envenoming and deaths resulting from snakebites are a particularly important public health problem in rural tropical areas of Africa, Asia, Latin America, and New Guinea. In 2015, The Lancet highlighted snake-bite envenoming as a neglected tropical disease and urged the world to increase antivenom production. In Brazil, around 20,000 snakebites occur per year affecting mostly agricultural workers and children, of which 1% is caused by coral snakes (Micrurus sp.). Although human envenoming by coral snakes is relatively rare due to their semifossorial habits and nonaggressive behavior, they are always considered severe due to the neurotoxic, myotoxic, hemorrhagic, and cardiovascular actions of their venom, which is highly toxic when compared to the venom of other Brazilian venomous snakes as Bothrops sp. (pit vipers), Crotalus sp. (rattlesnakes), and Lachesis sp. (bushmasters). The production of antivenom serum is an important public health issue worldwide and the maintenance of venomous snakes in captivity essential to obtain high-quality venom. Though more than 30 species of Brazilian coral snakes exist, the specific antivenom serum produced with the venom of two species, Micrurus corallinus and M. frontalis, is able to neutralize the accidents caused by the genus in general. M. corallinus is considered a difficult species to maintain in captivity and concerned about this difficulty the Laboratory of Herpetology (LH) at Instituto Butantan, over the last 10 yr, has given special attention to its maintenance in captivity. In more than 20 yr of maintenance, LH has made some changes to improve Micrurus captive husbandry and welfare. The objective of this study was to verify the factors influencing the survival rates of coral snakes in captivity through data generated from 289 M. corallinus from the LH snake facility in the last 10 yr. We observed that survival rates increased significantly with the improvement of nutritional adequacy that included freezing food items before offering them to coral snakes, as well as the development of a new pasty diet to force-feed anorexic animals. Another important factor responsible for increasing life expectancy was the shift of the cage's substrate from Sphagnum to bark in 2010, aiding in the eradication of Blister Disease, which used to be responsible for the death of several coral snakes in previous years.

Genomic features of a multidrug-resistant Enterobacter cloacae ST279 producing CTX-M-15 and AAC(6')-Ib-cr isolated from fatal infectious stomatitis in a crossed pit viper (Bothrops alternatus).

OBJECTIVES: The widespread dissemination of extended-spectrum ß-lactamase-producing Enterobacteriaceae has become a major issue in veterinary medicine. However, until now, there has been no report of bacteria with such a phenotype in infected snakes. The aim of this study was to report the first draft genome sequence of an Enterobacter cloacae isolate (SERP1) recovered from a snake with infectious stomatitis. METHODS: The whole genome of E. cloacae strain SERP1 was sequenced on an Illumina NextSeq platform and was de novo assembled using CLC NGS Cell v.10. Data analysis was performed using online tools from the Center of Genomic Epidemiology. RESULTS: The genome size was calculated at 4966856bp, containing a total of 4796 protein-coding sequences. The strain was assigned to sequence type 279 (ST279) and, besides the clinically relevant blaCTX-M-15 and aac(6')-Ib-cr genes, it also presented resistance genes to ß-lactams, aminoglycosides, phenicols, sulphonamides, tetracyclines, trimethoprim, quinolones and fosfomycin. CONCLUSION: These data offer novel information regarding multidrug-resistant E. cloacae dissemination in wild animals and might contribute to further comparative genomic analysis.

Dynamic Rearrangement in Snake Venom Gland Proteome: Insights into Bothrops jararaca Intraspecific Venom Variation.

We carried out an analysis of the venom gland proteome of Bothrops jararaca taking into account two distinct phases of its ontogenetic development (i.e., newborn and adult) and the marked sexual dimorphism recently reported on its venom proteome. Proteomic data analysis showed a dynamic rearrangement in the proteome landscape of B. jararaca venom gland upon development and gender-related changes. Differentially expressed proteins covered a number of biological pathways related to protein synthesis, including proteins associated with transcription and translation, which were found to be significantly higher expressed in the newborn venom gland. Our results suggest that the variation in the expression levels of cellular proteins might give rise to an even higher variation in the levels of the expressed toxins. Upon aging, the venom gland proteome repertoire related to the protein synthesis together with ecological traits would have an impact on the toxin repertoire, which, in the case of B. jararaca species, would enable the species to deal with different prey types during its lifespan. Proteomic data are available via ProteomeXchange with identifier PXD004186.

Bothrops jararaca venom proteome rearrangement upon neonate to adult transition.

The pharmacological activities displayed by Bothrops jararaca venom undergo a significant ontogenetic shift. Similarly, the diet of this species changes from ectothermic prey in early life to endothermic prey in adulthood. In this study we used large and representative newborn and adult venom samples consisting of pools from 694 and 110 specimens, respectively, and demonstrate a significant ontogenetic shift in the venom proteome complexity of B. jararaca. 2-DE coupled to MS protein identification showed a clear rearrangement of the toxin arsenal both in terms of the total proteome, as of the glycoproteome. N-glycosylation seems to play a key role in venom protein variability between newborn and adult specimens. Upon the snake development, the subproteome of metalloproteinases undergoes a shift from a P-III-rich to a P-I-rich profile while the serine proteinase profile does not vary significantly. We also used isobaric tag labeling (iTRAQ) of venom tryptic peptides for the first time to examine the quantitative changes in the venom toxins of B. jararaca upon neonate to adult transition. The iTRAQ analysis showed changes in various toxin classes, especially the proteinases. Our study expands the in-depth understanding of venom complexity variation particularly with regard to toxin families that have been associated with envenomation pathogenesis.

Bothrops jararaca venom proteome rearrangement upon neonate to adult transition

The pharmacological activities displayed by Bothrops jararaca venom undergo a significant ontogenetic shift. Similarly, the diet of this species changes from ectothermic prey in early life to endothermic prey in adulthood. In this study we used large and representative newborn and adult venom samples consisting of pools from 694 and 110 specimens, respectively, and demonstrate a significant ontogenetic shift in the venom proteome complexity of B. jararaca. 2-DE coupled to MS protein identification showed a clear rearrangement of the toxin arsenalboth in terms of the total proteome, as of the glycoproteome. N-glycosylation seems to play a key role in venom protein variability between newborn and adult specimens. Upon the snakedevelopment, the subproteome of metalloproteinases undergoes a shift from a P-III-rich to a P-I-rich profile while the serine proteinase profile does not vary significantly. We also usedisobaric tag labeling (iTRAQ) of venom tryptic peptides for the first time to examine the quantitative changes in the venom toxins of B. jararaca upon neonate to adult transition. TheiTRAQ analysis showed changes in various toxin classes, especially the proteinases. Our study expands the in-depth understanding of venom complexity variation particularly withregard to toxin families that have been associated with envenomation pathogenesis.