RESUMO
Superbugs are a public health problem, increasing the need of new drugs and strategies to combat them. Our group has previously identified LyeTxI, an antimicrobial peptide isolated from Lycosa erythrognatha spider venom. From LyeTxI, we synthesized and characterized a derived peptide named LyeTxI-b, which has shown significant in vitro and in vivo activity. In this work, we elucidate the interaction of LyeTxI-b with artificial membranes as well as its effects on resistant strains of bacteria in planktonic conditions or biofilms. Isothermal titration calorimetry revealed that LyeTxI-b interacts more rapidly and with higher intensity with artificial vesicles, showing higher affinity to anionic vesicles, when compared to synthetic LyeTxI. In calcein experiments, LyeTxI-b caused greater levels of vesicle cleavage. Both peptides showed antibacterial activity at concentrations of µmol L-1 against 12 different clinically isolated strains, in planktonic conditions, in a concentration-dependent manner. Furthermore, both peptides elicited a dose-dependent production of reactive oxygen species in methicillin-resistant Staphylococcus aureus. In S. aureus biofilm assay, LyeTxI-b was more potent than LyeTxI. However, none of these peptides reduced Escherichia coli biofilms. Our results show LyeTxI-b as a promising drug against clinically resistant strains, being a template for developing new antibiotics.
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Several research groups have studied the components produced by the venom gland of the scorpion Tityus serrulatus, which has one of the most lethal venoms in the world. Various methodologies have been employed to clarify the complex mechanisms of action of these components, especially neurotoxins and enzymes. Transcriptomes and proteomes have provided important information for pharmacological, biochemical, and immunological research. Next-generation sequencing (NGS) has allowed the description of new transcripts and completion of partial sequence descriptions for peptides, especially those with low expression levels. In the present work, after NGS sequencing, we searched for new putative venom components. We present a total of nine new transcripts with neurotoxic potential (Ts33-41) and describe the sequences of one hyaluronidase (TsHyal_4); three enzymes involved in amidation (peptidyl-glycine alpha-amidating monooxygenase A, peptidyl-alpha-hydroxyglycine alpha-amidating lyase, and peptidylglycine alpha-hydroxylating monooxygenase), which increases the lethal potential of neurotoxins; and also the enzyme Ts_Chitinase1, which may be involved in the venom's digestive action. In addition, we determined the level of transcription of five groups: toxins, metalloproteases, hyaluronidases, chitinases and amidation enzymes, including new components found in this study. Toxins are the predominant group with an expression level of 91.945%, followed by metalloproteases with only 7.790% and other groups representing 0.265%.
Assuntos
Proteoma/química , Venenos de Escorpião/química , Escorpiões , Amidina-Liases , Sequência de Aminoácidos , Animais , Biologia Computacional , Metaloproteases , Oxigenases de Função Mista , Complexos Multienzimáticos , TranscriptomaRESUMO
Cryptic peptides (cryptides) are biologically active peptides formed after proteolysis of native precursors present in animal venoms, for example. Proteolysis is an overlooked post-translational modification that increases venom complexity. The tripeptide KPP (Lys-Pro-Pro) is a peptide encrypted in the C-terminus of Ts14-a 25-mer peptide from the venom of the Tityus serrulatus scorpion that has a positive impact on the cardiovascular system, inducing vasodilation and reducing arterial blood pressure of hypertensive rats among other beneficial effects. A previous study reported that KPP and its native peptide Ts14 act via activation of the bradykinin receptor B2 (B2R). However, the cellular events underlying the activation of B2R by KPP are unknown. To study the cell signaling triggered by the Ts14 cryptide KPP, we incubated cardiac myocytes isolated from C57BL/6 mice with KPP (10-7 mol·L-1) for 0, 5, or 30 min and explored the proteome and phosphoproteome. Our results showed that KPP regulated cardiomyocyte proteins associated with, but not limited to, apoptosis, muscle contraction, protein turnover, and the respiratory chain. We also reported that KPP led to AKT phosphorylation, activating AKT and its downstream target nitric oxide synthase. We also observed that KPP led to dephosphorylation of phospholamban (PLN) at its activation sites (S16 and T17), leading to reduced contractility of treated cardiomyocytes. Some cellular targets reported here for KPP (e.g., AKT, PLN, and ERK) have already been reported to protect the cardiac tissue from hypoxia-induced injury. Hence, this study suggests potential beneficial effects of this scorpion cryptide that needs to be further investigated, for example, as a drug lead for cardiac infarction.
Assuntos
Venenos de Escorpião , Animais , Proteínas de Ligação ao Cálcio , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos , Peptídeos/farmacologia , Proteínas Proto-Oncogênicas c-akt , Ratos , Venenos de Escorpião/farmacologia , Transdução de SinaisRESUMO
Antimicrobial peptides present a broad spectrum of therapeutic applications, including their use as anticancer peptides. These peptides have as target microbial, normal, and cancerous cells. The oncological properties of these peptides may occur by membranolytic mechanisms or non-membranolytics. In this work, we demonstrate for the first time the cytotoxic effects of the cationic alpha-helical antimicrobial peptide LyeTx I-b on glioblastoma lineage U87-MG. The anticancer property of this peptide was associated with a membranolytic mechanism. Loss of membrane integrity occurred after incubation with the peptide for 15 min, as shown by trypan blue uptake, reduction of calcein-AM conversion, and LDH release. Morphological studies using scanning electron microscopy demonstrated disruption of the plasma membrane from cells treated with LyeTx I-b, including the formation of holes or pores. Transmission electron microscopy analyses showed swollen nuclei with mild DNA condensation, cell volume increase with an electron-lucent cytoplasm and organelle vacuolization, but without the rupture of nuclear or plasmatic membranes. Morphometric analyses revealed a high percentage of cells in necroptosis stages, followed by necrosis and apoptosis at lower levels. Necrostatin-1, a known inhibitor of necroptosis, partially protected the cells from the toxicity of the peptide in a concentration-dependent manner. Imaging flow cytometry confirmed that 59% of the cells underwent necroptosis after 3-h incubation with the peptide. It is noteworthy that LyeTx I-b showed only mild cytotoxicity against normal fibroblasts of human and monkey cell lines and low hemolytic activity in human erythrocytes. All data together point out the anticancer potential of this peptide.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Apoptose/efeitos dos fármacos , Fibroblastos/patologia , Glioblastoma/patologia , Neuroblastoma/patologia , Venenos de Aranha/farmacologia , Aranhas/química , Animais , Autofagia , Permeabilidade da Membrana Celular , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Glioblastoma/tratamento farmacológico , Hemólise/efeitos dos fármacos , Humanos , Necrose , Neuroblastoma/tratamento farmacológicoRESUMO
Corynebacterium pseudotuberculosis biovar equi is the etiologic agent of ulcerative lymphangitis. To investigate proteins that could be related to the virulence of this pathogen, we combined an experimental passage process using a murine model and high-throughput proteomics with a mass spectrometry, data-independent acquisition (LC-MSE) approach to identify and quantify the proteins released into the supernatants of strain 258_equi. To our knowledge, this approach allowed characterization of the exoproteome of a C. pseudotuberculosis equi strain for the first time. Interestingly, the recovery of this strain from infected mouse spleens induced a change in its virulence potential, and it became more virulent in a second infection challenge. Proteomic screening performed from culture supernatant of the control and recovered conditions revealed 104 proteins that were differentially expressed between the two conditions. In this context, proteomic analysis of the recovered condition detected the induction of proteins involved in bacterial pathogenesis, mainly related to iron uptake. In addition, KEGG enrichment analysis showed that ABC transporters, bacterial secretion systems and protein export pathways were significantly altered in the recovered condition. These findings show that secretion and secreted proteins are key elements in the virulence and adaptation of C. pseudotuberculosis. Collectively, bacterial pathogenesis-related proteins were identified that contribute to the processes of adherence, intracellular growth and evasion of the immune system. Moreover, this study enhances our understanding of the factors that may influence the pathogenesis of C. pseudotuberculosis.
Assuntos
Proteínas de Bactérias/metabolismo , Infecções por Corynebacterium/microbiologia , Corynebacterium pseudotuberculosis/isolamento & purificação , Meios de Cultura/química , Proteoma/análise , Animais , Cromatografia Líquida , Corynebacterium pseudotuberculosis/crescimento & desenvolvimento , Modelos Animais de Doenças , Ensaios de Triagem em Larga Escala , Espectrometria de Massas , Camundongos , ProteômicaRESUMO
We have previously described a 25mer anti-hypertensive peptide, previously named TsHpt-I (Tityus serrulatus Hypotensin-I), now Ts14, as an agonist of B2 kinin receptor. Bradykinin is known to play physiological roles in angiogenic, inflammatory, and fibrogenic processes, mostly mediated by B2 receptor. Therefore, we investigated whether Ts14 could modulate key events (neovascularization, inflammatory cell recruitment, and extracellular matrix deposition) of the fibrovascular tissue, induced by polyether-polyurethane sponge implants in mice. Sponges were implanted in the dorsum of 7-week-old C57Bl/6 male mice that received daily intrasponge treatment with Ts14 (27.25µg/sponge/day in 10µL PBS) or vehicle (10µL PBS/sponge/day) and were assessed on day 7 after surgery. Hemoglobin content, blood flow (laser Doppler perfusion imaging), and VEGF levels in the implants, used as indices of vascularization, indicated that Ts14 enhanced angiogenesis in implants relative to the PBS-treated group. Interestingly, Ts14 reduced TNF-α levels and neutrophil infiltration, although stimulated macrophage infiltration into implants, as determined by myeloperoxidase (MPO) and N-acetyl-ß-d-glucosaminidase (NAG) enzyme activities, respectively. Regarding the fibrogenic component (soluble collagen content and Sirius-red histological staining), we observed that Ts14 inhibited collagen deposition in the implants. Overall, our results suggest that Ts14 exerts proangiogenic, anti-inflammatory, and anti-fibrogenic activities. These effects may indicate a therapeutical potential of this peptide in conditions where angiogenesis, inflammation, and fibrogenesis contribute to disease progression and chronicity.
Assuntos
Inibidores da Angiogênese/farmacologia , Anti-Inflamatórios não Esteroides/farmacologia , Anti-Hipertensivos/farmacologia , Colágeno/metabolismo , Tecido de Granulação/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Venenos de Escorpião/farmacologia , Inibidores da Angiogênese/química , Animais , Anti-Inflamatórios não Esteroides/química , Anti-Hipertensivos/química , Biomarcadores/análise , Modelos Animais de Doenças , Éteres , Tecido de Granulação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Poliuretanos , Venenos de Escorpião/químicaRESUMO
To date, several families of peptide toxins specifically interacting with ion channels in scorpion venom have been described. One of these families comprise peptide toxins (called KTxs), known to modulate potassium channels. Thus far, 202 KTxs have been reported, belonging to several subfamilies of KTxs (called α, ß, γ, κ, δ, and λ-KTxs). Here we report on a previously described orphan toxin from Tityus serrulatus venom, named Ts11. We carried out an in-depth structure-function analysis combining 3D structure elucidation of Ts11 and electrophysiological characterization of the toxin. The Ts11 structure is highlighted by an Inhibitor Cystine Knot (ICK) type scaffold, completely devoid of the classical secondary structure elements (α-helix and/or ß-strand). This has, to the best of our knowledge, never been described before for scorpion toxins and therefore represents a novel, 6th type of structural fold for these scorpion peptides. On the basis of their preferred interaction with voltage-gated K channels, as compared to all the other targets tested, it can be postulated that Ts11 is the first member of a new subfamily, designated as ε-KTx.
Assuntos
Peptídeos/química , Venenos de Escorpião/química , Toxinas Biológicas/química , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes , Oócitos/metabolismo , Oócitos/fisiologia , Peptídeos/fisiologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/fisiologia , Toxinas Biológicas/fisiologia , Canais de Sódio Disparados por Voltagem/metabolismo , Canais de Sódio Disparados por Voltagem/fisiologia , Xenopus laevisRESUMO
Potassium (K(+)) channels are trans-membrane proteins, which play a key role in cellular excitability and signal transduction pathways. Scorpion toxins blocking the ion-conducting pore from the external side have been invaluable probes to elucidate the structural, functional, and physio-pathological characteristics of these ion channels. This review will focus on the interaction between K(+) channels and their peptide blockers isolated from the venom of the scorpion Tityus serrulatus, which is considered as the most dangerous scorpion in Brazil, in particular in Minas-Gerais State, where many casualties are described each year. The primary mechanisms of action of these K(+) blockers will be discussed in correlation with their structure, very often non-canonical compared to those of other well known K(+) channels blockers purified from other scorpion venoms. Also, special attention will be brought to the most recent data obtained by proteomic and transcriptomic analyses on Tityus serrulatus venoms and venom glands.
Assuntos
Bloqueadores dos Canais de Potássio/isolamento & purificação , Venenos de Escorpião/química , Toxinas Biológicas/isolamento & purificação , Sequência de Aminoácidos , Animais , Modelos Moleculares , Bloqueadores dos Canais de Potássio/química , Bloqueadores dos Canais de Potássio/toxicidade , Espectroscopia de Prótons por Ressonância Magnética , Homologia de Sequência de Aminoácidos , Toxinas Biológicas/química , Toxinas Biológicas/toxicidadeRESUMO
PnTx4(6-1), henceforth renamed δ-Ctenitoxin-Pn1a (δ-CNTX-Pn1a), a peptide from Phoneutria nigriventer spider venom, initially described as an insect toxin, binds to site 3 of sodium channels in nerve cord synaptosomes and slows down sodium current inactivation in isolated axons in cockroaches (Periplaneta americana). δ-CNTX-Pn1a does not cause any apparent toxicity to mice, when intracerebroventricularly injected (30 µg). In this study, we evaluated the antinociceptive effect of δ-CNTX-Pn1a in three animal pain models and investigated its mechanism of action in acute pain. In the inflammatory pain model, induced by carrageenan, δ-CNTX-Pn1a restored the nociceptive threshold of rats, when intraplantarly injected, 2 h and 30 min after carrageenan administration. Concerning the neuropathic pain model, δ-CNTX-Pn1a, when intrathecally administered, reversed the hyperalgesia evoked by sciatic nerve constriction. In the acute pain model, induced by prostaglandin E2, intrathecal administration of δ-CNTX-Pn1a caused a dose-dependent antinociceptive effect. Using antagonists of the receptors, we showed that the antinociceptive effect of δ-CNTX-Pn1a involves both the cannabinoid system, through CB1 receptors, and the opioid system, through µ and δ receptors. Our data show, for the first time, that δ-Ctenitoxin-Pn1a is able to induce antinociception in inflammatory, neuropathic and acute pain models.
Assuntos
Dor Aguda/tratamento farmacológico , Analgésicos/uso terapêutico , Proteínas de Artrópodes/uso terapêutico , Hiperalgesia/tratamento farmacológico , Neuralgia/tratamento farmacológico , Peptídeos/uso terapêutico , Dor Aguda/metabolismo , Analgésicos/farmacologia , Animais , Proteínas de Artrópodes/farmacologia , Antagonistas de Receptores de Canabinoides/farmacologia , Carragenina , Dinoprostona , Hiperalgesia/induzido quimicamente , Hiperalgesia/metabolismo , Masculino , Antagonistas de Entorpecentes/farmacologia , Neuralgia/induzido quimicamente , Neuralgia/metabolismo , Peptídeos/farmacologia , Ratos Wistar , Receptores de Canabinoides/metabolismo , Receptores Opioides/metabolismo , Nervo Isquiático/lesões , Venenos de Aranha/química , AranhasRESUMO
Several studies of the physiological responses of different organisms exposed to extremely low-frequency electromagnetic fields (ELF-EMF) have been described. In this work, we report the minimal effects of in situ exposure to ELF-EMF on the global protein expression of Chromobacterium violaceum using a gel-based proteomic approach. The protein expression profile was only slightly altered, with five differentially expressed proteins detected in the exposed cultures; two of these proteins (DNA-binding stress protein, Dps, and alcohol dehydrogenase) were identified by MS/MS. The enhanced expression of Dps possibly helped to prevent physical damage to DNA. Although small, the changes in protein expression observed here were probably beneficial in helping the bacteria to adapt to the stress generated by the electromagnetic field.
RESUMO
A potent insecticidal toxin, ß/δ-PrIT1, molecular mass of 5598.86 [M+H](+), was characterized from Phoneutria reidyi spider venom. Its partial amino acid sequence showed high similarity with insecticidal spider toxins from the genus Phoneutria. ß/δ-PrIT1 was very toxic (LD50 = 4 nmol/g) to flies (Musca domestica), but not to mice (Mus musculus). Kinetic studies showed that (125)I-ß/δ-PrIT1 binds to two distinct sites in insect sodium channels, with close affinity (Kd1 = 34.7 pM and Kd2 = 35.1 pM). Its association is rather fast (t1/2(1) = 1.4 min, t1/2(2) = 8.5 min) and its dissociation is a slower process (t1/2(1) = 5.4 min, t1/2(2) = 32.8 min). On rat brain synaptosomes ß/δ-PrIT1 partially competed (â¼30%) with the beta-toxin (125)I-CssIV, but did not compete with the alpha-toxin of reference (125)I-AaII, nor with the beta-toxin (125)I-TsVII. On cockroach nerve cord synaptosomes, ß/δ-PrIT1 did not compete with the anti-insect toxin (125)I-LqqIT1, but it competed (IC50 = 80 pM) with the "alpha-like" toxin (125)I-BomIV. In cockroach neurons, ß/δ-PrIT1 inhibited the inactivation of Nav-channels and it shifted the sodium channel activation to hyperpolarizing potentials. These results indicate two different binding sites for ß/δ-PrIT1, leading to two different pharmacological responses. ß/δ-PrIT1 is one of the most toxic spider toxins to insects without apparent toxicity to mammals, and provide new model for the development of insecticides.
Assuntos
Inseticidas/farmacologia , Venenos de Aranha/farmacologia , Aranhas/química , Sinaptossomos/metabolismo , Animais , Sítios de Ligação , Brasil , Baratas/citologia , Baratas/efeitos dos fármacos , Dípteros/efeitos dos fármacos , Feminino , Inseticidas/química , Masculino , Camundongos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ratos , Ratos Wistar , Canais de Sódio/metabolismo , Venenos de Aranha/químicaRESUMO
The scorpion Tityus serrulatus is responsible for the most severe accidents that have been registered in Brazil, mainly in the state of Minas Gerais (MG), being the lung edema (LE), the main cause of death in these accidents. Although an increased in the number of accidents caused to this species in Federal District (Distrito Federal - DF), it seems that this particular species is not responsible for severe scorpionism cases in this region. Given this observation, we tested the toxicity in mice and compared the ability of T. serrulatus venom from DF (Ts-DF) and Minas Gerais State (Ts-MG) to induce LE in rats. The LD50 of Ts-DF venom was 51.6 µg/mouse, almost twice (1.98) higher than that obtained for Ts-MG venom. The ability of venom (0.5 mg/kg) to induce LE in rats was determined by the wet weight differences between treated and untreated lungs, by pulmonary morphological analyses and by pulmonary vascular permeability (PVP) using the Evans blue protocol. Significant differences in the wet weight of lungs and changes in PVP were found in Ts-MG venom treated rats when compared to rats treated with Ts-DF venom or untreated rats (p < 0.001), but no differences occurred when comparing rats treated with Ts-DF venom and untreated rats (p < 0.05). These results were confirmed by evaluation of pulmonary morphology. Comparison of chromatographic profiles obtained from these venoms (Ts-DF and Ts-MG) using the fractal dimension (D) analysis and the molecular mass fingerprint of the chromatographic fractions showed a higher number of components between 35 and 40% acetonitrile in Ts-MG venom than in Ts-DF venom, indicating a higher diversity of sodium channel modulators in that venom.
Assuntos
Discinesia Induzida por Medicamentos/patologia , Variação Genética , Edema Pulmonar/induzido quimicamente , Venenos de Escorpião/toxicidade , Escorpiões/química , Análise de Variância , Animais , Brasil , Permeabilidade Capilar/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Relação Dose-Resposta a Droga , Dispneia/induzido quimicamente , Azul Evans , Fractais , Geografia , Dose Letal Mediana , Contagem de Leucócitos , Masculino , Camundongos , Edema Pulmonar/patologia , Ratos , Ratos Wistar , Venenos de Escorpião/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
This study involves the comparison between the exoproteomes of two different strains of Corynebacterium pseudotuberculosis, the etiologic agent of caseous lymphadenitis in small ruminants. In a previous study, based on a gel-free system (TPP-LC/MS(E)), 70 exoproteins for the strain 1002 and 67 for the strain C231, totaling 93 different extracellular proteins for C. pseudotuberculosis, were identified. In the present work, we have used 2D gel electrophoresis to resolve the extracellular proteins of both strains, which were then digested with trypsin, analyzed by MALDI-TOF/TOF and identified with the software MASCOT(®). A total of 45 extracellular proteins of C. pseudotuberculosis were identified by this approach. The comparative analysis between the strains 1002 and C231 identified 13 and 3 strain-specific proteins, respectively, 11 of which are novel. These newly identified proteins may play an important role in the physiology and virulence of C. pseudotuberculosis.
Assuntos
Proteínas de Bactérias/análise , Corynebacterium pseudotuberculosis/química , Corynebacterium pseudotuberculosis/classificação , Proteoma , Animais , Proteínas de Bactérias/química , Infecções por Corynebacterium/microbiologia , Infecções por Corynebacterium/veterinária , Eletroforese em Gel Bidimensional/métodos , Linfadenite/microbiologia , Linfadenite/veterinária , Proteômica/métodos , Especificidade da Espécie , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
Tarantulas are included in the mygalomorph spider family Theraphosidae. Although the pharmacological diversity of theraphosid toxins (theraphotoxins) is broad, studies dedicated to the characterization of biologically active molecules from the theraphosid genus Acanthoscurria have been restricted to the investigation of antimicrobial peptides and polyamines produced by the hemocytes of Acanthoscurria gomesiana. The present study reports the purification, primary structure determination and electrophysiological effects of an anti-insect toxin, named µ-theraphotoxin-An1a (µ-TRTX-An1a), from the venom of Acanthoscurria natalensis - a tarantula species occurring in the Brazilian biomes caatinga and cerrado. The analysis of the primary structure of µ-TRTX-An1a revealed the similarity of this toxin to theraphosid toxins bearing a huwentoxin-II-like fold. Electrophysiological experiments showed that µ-TRTX-An1a (100 nM) induces membrane depolarization, increases the spontaneous firing frequency and reduces spike amplitude of cockroach dorsal unpaired median (DUM) neurons. In addition, under voltage-clamp conditions, µ-TRTX-An1a (100 nM) only partially blocks voltage-dependent sodium current amplitudes in DUM neurons without any effect on their voltage dependence. This effect correlates well with the reduction of the spontaneous action potential amplitudes. Altogether, these last results suggest that µ-TRTX-An1a affects insect neuronal voltage-dependent sodium channels, which are among possible channels targeted by this promiscuous toxin.
Assuntos
Inseticidas/farmacologia , Venenos de Aranha/farmacologia , Aranhas/química , Potenciais de Ação/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Agentes de Controle Biológico , Brasil , Baratas/efeitos dos fármacos , Baratas/crescimento & desenvolvimento , Feminino , Insetos/efeitos dos fármacos , Insetos/crescimento & desenvolvimento , Dados de Sequência Molecular , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Alinhamento de Sequência , Canais de Sódio/efeitos dos fármacos , Canais de Sódio/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em TandemRESUMO
Corynebacterium pseudotuberculosis is the etiologic agent of caseous lymphadenitis a chronic infectious disease affecting small ruminants. The 2D-DIGE technique was used to compare the exoproteomes of two C. pseudotuberculosis biovar ovis strains isolated from goat (strain 1002) and sheep (strain C231). Seventeen proteins differentially produced were identified here. Nine proteins appeared over-produced in the exoproteome of 1002 goat strain and 8 in that of C231 sheep strain. These proteins were related to various biological functions, such as the cell envelope, respiratory metabolism and proteolysis. This proteomic analysis revealed strain-specific exoproteins although each of the corresponding genes was found in both strain genomes. Such differential expression pattern may reflect inter-strain differences in adaptation to a specific host, in pathogenicity and or in antigenicity of this pathogenic bacterium.
Assuntos
Proteínas de Bactérias/química , Infecções por Corynebacterium/veterinária , Corynebacterium pseudotuberculosis/isolamento & purificação , Corynebacterium pseudotuberculosis/metabolismo , Doenças das Cabras/microbiologia , Proteômica , Doenças dos Ovinos/microbiologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Infecções por Corynebacterium/microbiologia , Corynebacterium pseudotuberculosis/química , Corynebacterium pseudotuberculosis/genética , Cabras , OvinosRESUMO
Sea anemone (Cnidaria, Anthozoa) venom is an important source of bioactive compounds used as tools to study the pharmacology and structure-function of voltage-gated K+ channels (KV). These neurotoxins can be divided into four different types, according to their structure and mode of action. In this work, for the first time, two toxins were purified from the venom of Bunodosoma caissarum population from Saint Peter and Saint Paul Archipelago, Brazil. Sequence alignment and phylogenetic analysis reveals that BcsTx1 and BcsTx2 are the newest members of the sea anemone type 1 potassium channel toxins. Their functional characterization was performed by means of a wide electrophysiological screening on 12 different subtypes of KV channels (KV1.1-KV1.6; KV2.1; KV3.1; KV4.2; KV4.3; hERG and Shaker IR). BcsTx1 shows a high affinity for rKv1.2 over rKv1.6, hKv1.3, Shaker IR and rKv1.1, while Bcstx2 potently blocked rKv1.6 over hKv1.3, rKv1.1, Shaker IR and rKv1.2. Furthermore, we also report for the first time a venom composition and biological activity comparison between two geographically distant populations of sea anemones.
Assuntos
Venenos de Cnidários/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/antagonistas & inibidores , Anêmonas-do-Mar/química , Animais , Brasil , Venenos de Cnidários/química , Fenômenos Eletrofisiológicos , Humanos , Filogenia , Bloqueadores dos Canais de Potássio/química , Bloqueadores dos Canais de Potássio/isolamento & purificação , Ratos , Alinhamento de SequênciaRESUMO
Cyanobacteria produce a high variety of bioactive oligopeptides, which function, ecological, physiological roles and responses to environmental changes are still unclear. The influence of light intensity on the cell quota and the diversity of oligopeptides of two strains of the cyanobacterium Radiocystis fernandoii were experimentally tested. The peptides were quantified by HPLC and identified by a MALDI-TOF-TOF. Microcystins (MC) were generally more abundant in the treatment with low light. A compensatory mechanism was observed for the different variants of microcystin, whereby MC-RR responses were contrary to those observed for the other three variants and showed higher concentration in the treatment with intermediate light. Two microviridins were also produced at higher amounts at intermediate irradiance. For cyanopeptolins and a third microviridin no significant difference among treatments was found. The absence of a similar response for all peptides suggests that these compounds may have unique cellular functions, which better understanding could help explaining changes in toxicity. Finally, we observed that each chemical profile reflected in physiological differences between strains, strengthening the idea that chemotypes may act as distinct ecotypes in nature.
Assuntos
Cianobactérias/metabolismo , Cianobactérias/efeitos da radiação , Oligopeptídeos/biossíntese , Cianobactérias/química , Cianobactérias/genética , Luz , Espectrometria de Massas , Oligopeptídeos/química , Oligopeptídeos/toxicidadeRESUMO
Sea anemones are known to contain a wide diversity of biologically active peptides, mostly unexplored according to recent peptidomic and transcriptomic studies. In the present work, the neurotoxic fractions from the exudates of Stichodactyla helianthus and Bunodosoma granulifera were analyzed by reversed-phase chromatography and mass spectrometry. The first peptide fingerprints of these sea anemones were assessed, revealing the largest number of peptide components (156) so far found in sea anemone species, as well as the richer peptide diversity of B. granulifera in relation to S. helianthus. The transcriptomic analysis of B. granulifera, performed by massive cDNA sequencing with 454 pyrosequencing approach allowed the discovery of five new APETx-like peptides (U-AITX-Bg1a-e - including the full sequences of their precursors for four of them), which together with type 1 sea anemone sodium channel toxins constitute a very distinguishable feature of studied sea anemone species belonging to genus Bunodosoma. The molecular modeling of these new APETx-like peptides showed a distribution of positively charged and aromatic residues in putative contact surfaces as observed in other animal toxins. On the other hand, they also showed variable electrostatic potentials, thus suggesting a docking onto their targeted channels in different spatial orientations. Moreover several crab paralyzing toxins (other than U-AITX-Bg1a-e), which induce a variety of symptoms in crabs, were isolated. Some of them presumably belong to new classes of crab-paralyzing peptide toxins, especially those with molecular masses below 2kDa, which represent the smallest peptide toxins found in sea anemones.
Assuntos
Toxinas Marinhas/metabolismo , Mapeamento de Peptídeos/métodos , Peptídeos/metabolismo , Anêmonas-do-Mar/metabolismo , Sequência de Aminoácidos , Animais , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Biologia Computacional , Toxinas Marinhas/genética , Toxinas Marinhas/isolamento & purificação , Dados de Sequência Molecular , Peptídeos/genética , Peptídeos/isolamento & purificação , Anêmonas-do-Mar/genética , Homologia de Sequência de AminoácidosRESUMO
Coral snakes from Micrurus genus are the main representatives of the Elapidae family in South America. However, biochemical and pharmacological features regarding their venom constituents remain poorly investigated. Here, venomic analyses were carried out aiming at a deeper understanding on the composition of M. frontalis, M. ibiboboca, and M. lemniscatus venoms. In the three venoms investigated, proteins ranging from 6 to 8 kDa (3FTx) and 12 to 14 kDa (PLA(2)) were found to be the most abundant. Also, the N-terminal sequences of four new proteins, purified from the M. lemniscatus venom, similar to 3FTx, PLA(2) and Kunitz-type protease inhibitor from other Micrurus and elapid venoms are reported. Cross-reactivity among different Micrurus venoms and homologous or heterologous antivenoms was carried out by means of 2D-electrophoresis and immunoblotting. As, expected, the heterologous anti-Elapid venom displayed the highest degree of cross-reactivity. Conversely, anti-M. corallinus reacted weakly against the tested venoms. In gel digestions, followed by mass spectrometry sequencing and similarity searching, revealed the most immunogenic protein families as similar to short and long neurotoxins, weak neurotoxins, PLA(2), ß-bungarotoxin, venom protein E2, frontoxin III, LAO and C-type lectin. The implications of our results for the production of Micrurus antivenoms are discussed.
Assuntos
Antivenenos/uso terapêutico , Venenos Elapídicos/análise , Elapidae , Animais , Antivenenos/imunologia , Biologia Computacional/métodos , Reações Cruzadas/imunologia , Proteômica/métodos , América do SulRESUMO
Accidents involving venomous animals have always caught the attention of mankind due to their lethality and other clinical implications. However, since the molecules obtained from animal venoms have been the product of millions of years of evolutionary process, toxins could be used to probe physiological mechanisms and could serve as leads for drug development. The present work reviews the state of the art pertaining to venom molecules from Brazilian medically important arachnid species bearing potential biotechnological applications. Special focus is given to toxins isolated from the scorpion Tityus serrulatus and the spiders Phoneutria nigriventer and Lycosa erythrognatha, whose venoms possess molecules acting as erectile function modulators and as antihypertensive, analgesic, neuroprotective and antimicrobial agents.