RESUMO
AIMS: Crotalicidin (Ctn), a cathelicidin-related antimicrobial peptide from the South American rattlesnake venom gland, and its C-terminal Ctn[15-34] fragment, have exhibited important activities against micro-organisms, trypanosomatid protozoa and certain lines of tumour cells. Herein, the activity against clinical strains of fluconazole-resistant Candida albicans and of amphotericin B and fluconazole-resistant Cryptococcus neoformans was investigated. METHODS AND RESULTS: Microdilution and luminescent cell viability tests were used to evaluate and compare the susceptibility of pathogenic yeasts to these peptides. The time-kill curves of the most active Ctn[15-34] alone or in combination with fluconazole against drug-resistant yeasts were determined. Concomitantly, the fungicidal and/or fungistatic effects of Ctn[15-34] were visualized by the spotting test. The peptides were active against all strains, including those resistant to antifungal agents. The association of fluconazole with both Ctn and Ctn[15-34], although not synergic, was additive. In contrast, such pattern was not observed for C. neoformans. CONCLUSIONS: Overall, Ctn and Ctn[15-34] are potential antifungal leads displaying anti-yeast activities against clinical isolates endowed with drug resistance mechanisms. SIGNIFICANCE AND IMPACT OF THE STUDY: The effective peptide activity against resistant strains of pathogenic yeasts demonstrates that crotalicidin-derived peptides are promising templates to develop new antifungal pharmaceuticals.
Assuntos
Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Cryptococcus neoformans/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Anfotericina B/farmacologia , Candida albicans/crescimento & desenvolvimento , Cryptococcus neoformans/crescimento & desenvolvimento , Fluconazol/farmacologia , Testes de Sensibilidade Microbiana , Triazóis/farmacologiaRESUMO
Synthetic peptides mimicking protective B- and T-cell epitopes are good candidates for safer, more effective FMD vaccines. Nevertheless, previous studies of immunization with linear peptides showed that they failed to induce solid protection in cattle. Dendrimeric peptides displaying two or four copies of a peptide corresponding to the B-cell epitope VP1 [136-154] of type O FMDV (O/UKG/11/2001) linked through thioether bonds to a single copy of the T-cell epitope 3A [21-35] (termed B2T and B4T, resp.) afforded protection in vaccinated pigs. In this work, we show that dendrimeric peptides B2T and B4T can elicit specific humoral responses in cattle and confer partial protection against the challenge with a heterologous type O virus (O1/Campos/Bra/58). This protective response correlated with the induction of specific T-cells as well as with an anamnestic antibody response upon virus challenge, as shown by the detection of virus-specific antibody-secreting cells (ASC) in lymphoid tissues distal from the inoculation point.
Assuntos
Linfócitos B/imunologia , Vírus da Febre Aftosa/imunologia , Febre Aftosa/imunologia , Linfócitos T/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/sangue , Bovinos , Dendrímeros/química , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Ativação Linfocitária , Peptídeos/química , Peptídeos/imunologia , Suínos , VacinaçãoRESUMO
The increase in both Mycobacterium tuberculosis human clinical isolates resistant to the essential drugs and cases of disseminated micobacteriosis due to Mycobacterium avium Complex, underlines the need to investigate new antimicobacterial agents. The antimicrobial peptides are a new group of active antibiotics with a particular mechanism of action. Some of them, like cecropin and melittin, isolated from insects, have demonstrated good in vitro activity against Gram-positive and Gram-negative bacteria. Synthetic hybrids of those peptides have been more active than individual peptides. In this study, the in vitro activity of two hybrid synthetic peptides from melittin and cecropin against M. tuberculosis, M. avium Complex, Mycobacterium fortuitum and Mycobacterium smegmatis has been evaluated. The minimal inhibitory concentration was determined by using the broth macrodilution technique. The minimal bactericide concentration in Lowenstein Jensen medium was then obtained. The peptides studied were active, in vitro, against M. smegmatis, but they did not show any activity against the other mycobacteria analyzed.
Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Meliteno/farmacologia , Mycobacterium/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Antibacterianos/síntese química , Peptídeos Catiônicos Antimicrobianos/síntese química , Avaliação Pré-Clínica de Medicamentos , Meliteno/síntese química , Testes de Sensibilidade Microbiana , Mycobacterium avium/efeitos dos fármacos , Mycobacterium fortuitum/efeitos dos fármacos , Mycobacterium smegmatis/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Fragmentos de Peptídeos/síntese químicaRESUMO
The 34-residue peptide CTVAEIYLGNLAGADLILASGLPFWAITIANNFD (TM-34), corresponding to the 64-97 sequence of the rat bradykinin, receptor, was selected as a model of hydrophobic transmembrane peptide segment for systematic study of synthesis and purification strategies. Application of conventional Boc/Bzl chemistry resulted in very low yield of the synthesis (around 4%) when DMF was used as the solvent for coupling reactions. As shorter resin-bound fragments of TM-34 showed improved swelling in 80% NMP/DMSO, the synthesis was repeated in this mixed solvent and the yield increased to 12%. A comparative synthesis using optimized Fmoc chemistry and Fmoc-(FmocHmb) derivatives of Ala and Leu to prevent aggregation did not provide any detectable TM-34. Taken together, these results illustrate the synthetic problems associated with hydrophobic sequences, almost regardless of the chemistry used. As expected, the hydrophobicity of TM-34 and of most of its minor fragments made them scarcely soluble in common solvents. Purification could be achieved by loading the crude materials dissolved in 90% AcOH onto a C4 HPLC column and eluting with a TFA/MeCN linear gradient. CD studies of the TM-34 and of the shorter fragment with the 74-97 sequence (TM-24) showed a higher percentage of alpha-helix structure for the latter. This suggests that the shorter sequence may better represent the correct transmembrane region of the second helix of the rat bradykinin receptor.
Assuntos
Fragmentos de Peptídeos/síntese química , Receptores da Bradicinina/química , Sequência de Aminoácidos , Animais , Membrana Celular/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Dicroísmo Circular , Indicadores e Reagentes , Espectrometria de Massas , Dados de Sequência Molecular , Oligopeptídeos/síntese química , Oligopeptídeos/química , Oligopeptídeos/isolamento & purificação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Conformação Proteica , RatosRESUMO
A large-scale vaccination experiment involving a total of 138 cattle was carried out to evaluate the potential of synthetic peptides as vaccines against foot-and-mouth disease. Four types of peptides representing sequences of foot-and-mouth disease virus (FMDV) C3 Argentina 85 were tested: A, which includes the G-H loop of capsid protein VP1 (site A); AT, in which a T-cell epitope has been added to site A; AC, composed of site A and the carboxy-terminal region of VP1 (site C); and ACT, in which the three previous capsid motifs are colinearly represented. Induction of neutralizing antibodies, lymphoproliferation in response to viral antigens, and protection against challenge with homologous infectious virus were examined. None of the tested peptides, at several doses and vaccination schedules, afforded protection above 40%. Protection showed limited correlation with serum neutralization activity and lymphoproliferation in response to whole virus. In 12 of 29 lesions from vaccinated cattle that were challenged with homologous virus, mutant FMDVs with amino acid substitutions at antigenic site A were identified. This finding suggests the rapid generation and selection of FMDV antigenic variants in vivo. In contrast with previous studies, this large-scale vaccination experiment with an important FMDV host reveals considerable difficulties for vaccines based on synthetic peptides to achieve the required levels of efficacy. Possible modifications of the vaccine formulations to increase protective activity are discussed.
Assuntos
Capsídeo/imunologia , Doenças dos Bovinos/prevenção & controle , Febre Aftosa/prevenção & controle , Peptídeos/imunologia , Vacinas Virais/imunologia , Sequência de Aminoácidos , Animais , Aphthovirus/genética , Aphthovirus/imunologia , Capsídeo/síntese química , Capsídeo/genética , Proteínas do Capsídeo , Bovinos , Doenças dos Bovinos/imunologia , Linhagem Celular , Cricetinae , Febre Aftosa/imunologia , Esquemas de Imunização , Dados de Sequência Molecular , Mutagênese , Peptídeos/síntese química , Relação Estrutura-Atividade , Vacinação , Vacinas Sintéticas/imunologiaRESUMO
One of the major obstacles to the design of effective antiviral vaccines is the frequent generation of antigenic viral variants in the field. The types of variants that will become dominant during disease outbreaks is often unpredictable. However, here we report the genetic and antigenic characterization of emerging foot-and-mouth disease virus (FMDV) variants with antigenically critical amino acid substitutions predicted by model studies using reference viruses and monoclonal antibodies. The new variants belong to serotype C and have caused a number of recent disease outbreaks in Argentina. The variants harbor antigenically drastic amino acid substitutions in each of the antigenic sites identified in FMDV. In particular, a substitution found at a major antigenic site (site A, the G-H loop of VP1) had been repeatedly selected in viruses resistant to neutralization by monoclonal and polyclonal antibodies. The association of critical amino acid replacements at predicted positions with new FMD outbreaks has a number of implications for FMD epidemiology and for the design of vaccines intended to control diseases caused by highly variable RNA viruses.