RESUMO
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.
Assuntos
Algoritmos , Biologia Computacional/métodos , Perfilação da Expressão Gênica/métodos , Venenos de Serpentes/genética , Serpentes/genética , Toxinas Biológicas/genética , Animais , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Filogenia , Venenos de Serpentes/química , Venenos de Serpentes/metabolismo , Serpentes/classificação , Serpentes/metabolismo , Especificidade da Espécie , Toxinas Biológicas/química , Toxinas Biológicas/metabolismoRESUMO
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.
Assuntos
Aedes/genética , Inibidores da Síntese de Proteínas/metabolismo , Proteínas Inativadoras de Ribossomos/metabolismo , Ribossomos/metabolismo , Toxinas Biológicas/metabolismo , Aedes/fisiologia , Animais , Sequência de Bases , Proteínas Inativadoras de Ribossomos/genética , Homologia de Sequência , Toxinas Biológicas/genéticaRESUMO
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.
Assuntos
Infecções por Rotavirus/veterinária , Rotavirus/genética , Doenças dos Suínos/virologia , Toxinas Biológicas/genética , Proteínas não Estruturais Virais/genética , Animais , Fezes/virologia , Genótipo , Humanos , Filogenia , Infecções por Rotavirus/virologia , SuínosRESUMO
Background: Phoneutria nigriventer spider venom contains several cysteine-rich peptide toxins that act on different ion channels. Despite extensive studies on its venom and description of cDNA sequences of several of its toxin precursors, the gene structure of these toxins remains unknown. Methods: Genomic regions encoding the precursors of three previously characterized P. nigriventer toxins - PnTx1, PnTx2-5 and PnTx4(5-5) - were amplified by PCR using specific primers. PCR fragments were cloned and sequenced. Obtained sequences were compared with their corresponding cDNA sequences. Results: The size of PCR fragments obtained and sequences corresponding to genomic regions encoding for the toxin precursors matched their cDNA sequences. Conclusions: Despite a few nucleotide substitutions in the genomic regions encoding for the toxin precursors when compared with cDNA sequences, the results of the present work indicate that P. nigriventer toxins do not contain introns in their genes sequences.(AU)
Assuntos
Animais , Venenos de Aranha/genética , Venenos de Aranha/isolamento & purificação , Toxinas Biológicas/isolamento & purificação , Íntrons , Toxinas Biológicas/genética , AranhasRESUMO
Phoneutria nigriventer is one of the largest existing true spiders and one of the few considered medically relevant. Its venom contains several neurotoxic peptides that act on different ion channels and chemical receptors of vertebrates and invertebrates. Some of these venom toxins have been shown as promising models for pharmaceutical or biotechnological use. However, the large diversity and the predominance of low molecular weight toxins in this venom have hampered the identification and deep investigation of the less abundant toxins and the proteins with high molecular weight. Here, we combined conventional and next-generation cDNA sequencing with Multidimensional Protein Identification Technology (MudPIT), to obtain an in-depth panorama of the composition of P. nigriventer spider venom. The results from these three approaches showed that cysteine-rich peptide toxins are the most abundant components in this venom and most of them contain the Inhibitor Cysteine Knot (ICK) structural motif. Ninety-eight sequences corresponding to cysteine-rich peptide toxins were identified by the three methodologies and many of them were considered as putative novel toxins, due to the low similarity to previously described toxins. Furthermore, using next-generation sequencing we identified families of several other classes of toxins, including CAPs (Cysteine Rich Secretory Protein-CRiSP, antigen 5 and Pathogenesis-Related 1-PR-1), serine proteinases, TCTPs (translationally controlled tumor proteins), proteinase inhibitors, metalloproteinases and hyaluronidases, which have been poorly described for this venom. This study provides an overview of the molecular diversity of P. nigriventer venom, revealing several novel components and providing a better basis to understand its toxicity and pharmacological activities.
Assuntos
Proteômica , Venenos de Aranha/metabolismo , Aranhas/metabolismo , Transcriptoma , Sequência de Aminoácidos , Animais , Biomarcadores Tumorais/química , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , DNA Complementar/química , DNA Complementar/genética , DNA Complementar/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Peptídeos/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Aranhas/genética , Toxinas Biológicas/genética , Toxinas Biológicas/metabolismo , Proteína Tumoral 1 Controlada por TraduçãoRESUMO
The recent publication of high-throughput transcriptomic and proteomic analyses of scorpion venom glands has increased our knowledge on the biodiversity of venom components. In this contribution, we report the transcriptome of the venom gland and the proteome of the venom for the scorpion species Paravaejovis schwenkmeyeri, a member of the family Vaejovidae. We report 138 annotated transcripts encoding putative peptides/proteins with sequence identity to known venom components available from different databases. A fingerprint analysis containing the molecular masses of 212 components of the whole soluble venom revealed molecular weights of approximately 700 to 13,800â¯Da, with most detected proteins ranging from 1500 to 3000â¯Da. Amino acid sequencing of venom components by LC-MS/MS allowed the identification of fragments from 27 peptides encoded by transcripts found in the transcriptome analysis. Enzymatic assays conducted with the soluble venom fraction confirmed the presence of enzymes such as hyaluronidases and phospholipases. The database presented here increases our general knowledge on the biodiversity of venom components from neglected non-buthid scorpions.
Assuntos
Proteínas de Artrópodes/metabolismo , Proteoma , Venenos de Escorpião/química , Escorpiões/fisiologia , Transcriptoma , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Biologia Computacional , Canais Iônicos/antagonistas & inibidores , Toxinas Biológicas/química , Toxinas Biológicas/genética , Toxinas Biológicas/metabolismoRESUMO
Nontyphoidal Salmonella (NTS) is a common pathogen in food-producing animals and a public health concern worldwide. Various NTS serovars may be present in apparently healthy animals. This could result in carcass contamination during the slaughter process leading to human exposure. While most genomic research has focused on Salmonella pathogenesis, little is known on the factors associated with subclinical infections and environmental persistence. We report here the widespread distribution of typhoidal toxin genes (i. e. the cdtB islet, hlyE, taiA), among NTS strains from a beef slaughter operation (n = 39) and from epidemiologically unconnected ground beef (n = 20). These genes were present in 76% of the strains, regardless of serovar, isolation source or geographical location. Moreover, strains that predominated in the slaughterhouse carry plasmid-borne type IV secretion systems (T4SS), which have been linked to persistent infections in numerous pathogens. Population genomics supports clonal dissemination of NTS along the food production chain, highlighting its role as reservoir of genetic variability in the environment. Overall, the study provides a thorough characterization of serovar diversity and genomic features of beef-associated NTS in Mexico. Furthermore, it reveals how common genetic factors could partially explain the emergence and persistence of certain NTS serovars in the beef industry.
Assuntos
Plasmídeos/genética , Salmonella/genética , Salmonella/fisiologia , Toxinas Biológicas/genética , Sequenciamento Completo do Genoma , Animais , Bovinos , Variação Genética , Genômica , Interações Hospedeiro-Patógeno , Filogenia , Salmonella/patogenicidade , Estresse Fisiológico , VirulênciaRESUMO
New approaches aimed at neutralizing the primary toxic components present in scorpion venoms, represent a promising alternative to the use of antivenoms of equine origin in humans. New potential therapeutics developed by these approaches correspond to neutralizing antibody fragments obtained by selection and maturation processes from libraries of human origin. The high sequence identity shared among scorpion toxins is associated with an important level of cross reactivity exhibited by these antibody fragments. We have exploited the cross reactivity showed by single chain variable antibody fragments (scFvs) of human origin to re-direct the neutralizing capacity toward various other scorpion toxins. As expected, during these evolving processes several variants derived from a parental scFv exhibited the capacity to simultaneously recognize and neutralize different toxins from Centruroides scorpion venoms. A sequence analyses of the cross reacting scFvs revealed that specific mutations are responsible for broadening their neutralizing capacity. In this work, we generated a set of new scFvs that resulted from the combinatorial insertion of these point mutations. These scFvs are potential candidates to be part of a novel recombinant antivenom of human origin that could confer protection against scorpion stings. A remarkable property of one of these new scFvs (ER-5) is its capacity to neutralize at least three different toxins and its complementary capacity to neutralize the whole venom from Centruroides suffusus in combination with a second scFv (LR), which binds to a different epitope shared by Centruroides scorpion toxins.
Assuntos
Testes de Neutralização , Venenos de Escorpião/química , Toxinas Biológicas/toxicidade , Sequência de Aminoácidos , Animais , Evolução Molecular Direcionada , México , Homologia de Sequência de Aminoácidos , Ressonância de Plasmônio de Superfície , Toxinas Biológicas/genética , Toxinas Biológicas/imunologiaRESUMO
Group A human rotaviruses (HuRVA) are causative agents of acute gastroenteritis. Six viral structural proteins (VPs) and six nonstructural proteins (NSPs) are produced in RV-infected cells. NSP4 is a diarrhoea-inducing viral enterotoxin and NSP4 gene analysis revealed at least 15 (E1-E15) genotypes. This study analysed the NSP4 genetic diversity of HuRVA G2P[4] strains collected in the state of São Paulo (SP) from 1994 and 2006-2010 using reverse transcription-polymerase chain reaction, sequencing and phylogenetic analysis. Forty (97.6%) G2P[4] strains displayed genotype E2; one strain (2.4%) displayed genotype E1. These results are consistent with the proposed linkage between VP4/VP7 (G2P[4]) and the NSP4 (E2) genotype of HuRVA. NSP4 phylogenetic analysis showed distinct clusters, with grouping of most strains by their genotype and collection year, and most strains from SP were clustered together with strains from other Brazilian states. A deduced amino acid sequence alignment for E2 showed many variations in the C-terminal region, including the VP4-binding domain. Considering the ability of NSP4 to generate host immunity, monitoring NSP4 variations, along with those in the VP4 or VP7 protein, is important for evaluating the circulation and pathogenesis of RV. Finally, the presence of one G2P[4]E1 strain reinforces the idea that new genotype combinations emerge through reassortment and independent segregation.
Assuntos
Antígenos Virais/isolamento & purificação , Glicoproteínas/genética , RNA Viral/genética , Rotavirus/genética , Toxinas Biológicas/genética , Proteínas não Estruturais Virais/genética , Adulto , Sequência de Aminoácidos , Sequência de Bases , Brasil , Criança , Fezes/virologia , Ligação Genética/genética , Variação Genética , Genótipo , Humanos , Técnicas Imunoenzimáticas , Dados de Sequência Molecular , Filogenia , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rotavirus/classificação , Rotavirus/imunologia , Alinhamento de SequênciaRESUMO
Group A human rotaviruses (HuRVA) are causative agents of acute gastroenteritis. Six viral structural proteins (VPs) and six nonstructural proteins (NSPs) are produced in RV-infected cells. NSP4 is a diarrhoea-inducing viral enterotoxin and NSP4 gene analysis revealed at least 15 (E1-E15) genotypes. This study analysed the NSP4 genetic diversity of HuRVA G2P[4] strains collected in the state of São Paulo (SP) from 1994 and 2006-2010 using reverse transcription-polymerase chain reaction, sequencing and phylogenetic analysis. Forty (97.6%) G2P[4] strains displayed genotype E2; one strain (2.4%) displayed genotype E1. These results are consistent with the proposed linkage between VP4/VP7 (G2P[4]) and the NSP4 (E2) genotype of HuRVA. NSP4 phylogenetic analysis showed distinct clusters, with grouping of most strains by their genotype and collection year, and most strains from SP were clustered together with strains from other Brazilian states. A deduced amino acid sequence alignment for E2 showed many variations in the C-terminal region, including the VP4-binding domain. Considering the ability of NSP4 to generate host immunity, monitoring NSP4 variations, along with those in the VP4 or VP7 protein, is important for evaluating the circulation and pathogenesis of RV. Finally, the presence of one G2P[4]E1 strain reinforces the idea that new genotype combinations emerge through reassortment and independent segregation.