RESUMO
Envenomation by the Trinidad thick-tailed scorpion Tityus trinitatis may result in fatal myocarditis and there is a high incidence of acute pancreatitis among survivors. Peptidomic analysis (reversed-phase HPLC followed by MALDI-TOF mass spectrometry and automated Edman degradation) of T. trinitatis venom led to the isolation and characterization of three peptides with antimicrobial activity. Their primary structures were established asTtAP-1 (FLGSLFSIGSKLLPGVFKLFSRKKQ.NH2), TtAP-2 (IFGMIPGLIGGLISAFK.NH2) and TtAP-3 (FFSLIPSLIGGLVSAIK.NH2). In addition, potassium channel and sodium channel toxins, present in the venom in high abundance, were identified by CID-MS/MS sequence analysis. TtAP-1 was the most potent against a range of clinically relevant Gram-positive and Gram-negative aerobes and against the anaerobe Clostridioides difficile (MIC = 3.1-12.5 µg/mL). At a concentration of 1× MIC, TtAP-1 produced rapid cell death (<15 min against Acinetobacter baumannii and Staphylococcus aureus). The therapeutic potential of TtAP-1 as an anti-infective agent is limited by its high hemolytic activity (LC50 = 18 µg/mL against mouse erythrocytes) but the peptide constitutes a template for the design of analogs that maintain the high bactericidal activity against ESKAPE pathogens but are less toxic to human cells. It is suggested that the antimicrobial peptides in the scorpion venom facilitate the action of the neurotoxins by increasing the membrane permeability of cells from either prey or predator.
RESUMO
Skin secretions of certain frog species represent a source of host-defense peptides (HDPs) with therapeutic potential and their primary structures provide insight into taxonomic and phylogenetic relationships. Peptidomic analysis was used to characterize the HDPs in norepinephrine-stimulated skin secretions from the Amazon River frog Lithobates palmipes (Ranidae) collected in Trinidad. A total of ten peptides were purified and identified on the basis of amino acid similarity as belonging to the ranatuerin-2 family (ranatuerin-2PMa, -2PMb, -2PMc, and-2PMd), the brevinin-1 family (brevinin-1PMa, -1PMb, -1PMc and des(8-14)brevinin-1PMa) and the temporin family (temporin-PMa in C-terminally amidated and non-amidated forms). Deletion of the sequence VAAKVLP from brevinin-1PMa (FLPLIAGVAAKVLPKIFCAISKKC) in des[(8-14)brevinin-1PMa resulted in a 10-fold decrease in potency against Staphylococcus aureus (MIC = 31 µM compared with 3 µM) and a > 50-fold decrease in hemolytic activity but potency against Echerichia coli was maintained (MIC = 62.5 µM compared with 50 µM). Temporin-PMa (FLPFLGKLLSGIF.NH2) inhibited growth of S. aureus (MIC = 16 µM) but the non-amidated form of the peptide lacked antimicrobial activity. Cladistic analysis based upon the primary structures of ranaturerin-2 peptides supports the division of New World frogs of the family Ranidae into the genera Lithobates and Rana. A sister-group relationship between L. palmipes and Warszewitsch's frog Lithobates warszewitschii is indicated within a clade that includes the Tarahumara frog Lithobates tarahumarae. The study has provided further evidence that peptidomic analysis of HDPs in frog skin secretions is a valuable approach to elucidation of the evolutionary history of species within a particular genus.
Assuntos
Ranidae , Staphylococcus aureus , Animais , Sequência de Aminoácidos , Filogenia , Staphylococcus aureus/metabolismo , Ranidae/metabolismo , Proteínas de Anfíbios/metabolismo , Pele/metabolismoRESUMO
Ocellatins are peptides produced in the skins of frogs belonging to the genus Leptodactylus that generally display weak antimicrobial activity against Gram-negative bacteria only. Peptidomic analysis of norepinephrine-stimulated skin secretions from Leptodactylus insularum Barbour 1906 and Leptodactylus nesiotus Heyer 1994, collected in the Icacos Peninsula, Trinidad, led to the purification and structural characterization of five ocellatin-related peptides from L. insularum (ocellatin-1I together with its (1-16) fragment, ocellatin-2I and its (1-16) fragment, and ocellatin-3I) and four ocellatins from L. nesiotus (ocellatin-1N, -2N, -3N, and -4N). While ocellatins-1I, -2I, and -1N showed a typically low antimicrobial potency against Gram-negative bacteria, ocellatin-3N (GIFDVLKNLAKGVITSLAS.NH2) was active against an antibiotic-resistant strain of Klebsiella pneumoniae and reference strains of Escherichia coli, K. pneumoniae, Pseudomonas aeruginosa, and Salmonella typhimurium (minimum inhibitory concentrations (MICs) in the range 31.25-62.5 µM), and was the only peptide active against Gram-positive Staphylococcus aureus (MIC = 31.25 µM) and Enterococcus faecium (MIC = 62.5 µM). The therapeutic potential of ocellatin-3N is limited by its moderate hemolytic activity (LC50 = 98 µM) against mouse erythrocytes. The peptide represents a template for the design of long-acting, non-toxic, and broad-spectrum antimicrobial agents for targeting multidrug-resistant pathogens.
RESUMO
Peptidomic analysis of norepinephrine-stimulated skin secretions from the Greek stream frog Rana graeca Boulenger, 1891 led to the identification and structural characterization of a range of host-defense peptides. These comprised brevinin-1GRa, brevinin-1GRb and an N-terminally extended form of brevinin-1GRb, ranatuerin-2GR together with its oxidized form and (11-28) fragment, temporin-GRa, temporin-GRb and its non-amidated form, and a melittin-related peptide, MRP-GR and its (1-18) fragment. The most abundant peptide, MRP-GR significantly (Pâ¯<â¯0.001) stimulated insulin release from BRIN-BD11 clonal ß-cells at concentrations ≥0.1â¯nM. Rana graeca (formerly Rana graeca graeca) and the morphologically similar Italian stream frog Rana italica Dubois, 1987 (formerly Rana graeca italica) were originally regarded as sub-species. However, the primary structures of the host defense peptides from both frogs support the claim based upon comparisons of the nucleotide sequences of S1 satellite DNA that R. graeca and R. italica are separate species. Cladistic analyses based upon the primary structures of the brevinin-1 and ranatuerin-2 peptides from Eurasian frogs indicate a close phylogenetic relationship between R. graeca and Rana latastei whereas R. italica is most closely related to Rana dalmatina.
Assuntos
Proteínas de Anfíbios/metabolismo , Peptídeos/metabolismo , Filogenia , Ranidae/classificação , Pele/metabolismo , Sequência de Aminoácidos , Proteínas de Anfíbios/química , Animais , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Insulina/metabolismo , Peptídeos/química , Ranidae/metabolismo , Ratos , Especificidade da Espécie , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Peptidomic analysis (reversed-phase HPLC combined with MALDI-TOF mass spectrometry and automated Edman degradation) of norepinephrine-stimulated skin secretions from the Trinidadian leaf frog Phyllomedusa trinitatis Mertens 1926 led to the identification and structural characterization of 26 host-defense peptides. On the basis of amino acid sequence similarity, the peptides may be divided into the followings groups: dermaseptins with the conserved N-terminal region GLWSKIK (6 peptides), dermaseptins with the N-terminal region ALWKXXLK (5 peptides), dermaseptins with the conserved N-terminal region GLFKTLIKGAGKMLGHVAK (4 peptides), C-terminally α-amidated and non-amidated forms of the phylloseptins (9 peptides), phyllocaerulein, a peptide (GLVSGLLNSVTGLLGNLAGGGL) with structural similarity to the plasticins, and a putative antioxidant peptide (LTWKIPTRFCGVT). The primary structures of the peptides support the claim based upon morphological criteria that P. trinitatis and Phyllomedusa tarsius are very closely related phylogenetically but are probably not conspecific. Among the phylloseptins, phylloseptin-1.1TR (FLSLIPKIAGGIASLVKNL.NH2) displayed the most potent antimicrobial activity.