RESUMO
Novel antibiotics are urgently needed to combat multidrug-resistant pathogens. Venoms represent previously untapped sources of novel drugs. Here we repurposed mastoparan-L, the toxic active principle derived from the venom of the wasp Vespula lewisii, into synthetic antimicrobials. We engineered within its N terminus a motif conserved among natural peptides with potent immunomodulatory and antimicrobial activities. The resulting peptide, mast-MO, adopted an α-helical structure as determined by NMR, exhibited increased antibacterial properties comparable to standard-of-care antibiotics both in vitro and in vivo, and potentiated the activity of different classes of antibiotics. Mechanism-of-action studies revealed that mast-MO targets bacteria by rapidly permeabilizing their outer membrane. In animal models, the peptide displayed direct antimicrobial activity, led to enhanced ability to attract leukocytes to the infection site, and was able to control inflammation. Permutation studies depleted the remaining toxicity of mast-MO toward human cells, yielding derivatives with antiinfective activity in animals. We demonstrate a rational design strategy for repurposing venoms into promising antimicrobials.
Assuntos
Bacteriemia/tratamento farmacológico , Proteínas Citotóxicas Formadoras de Poros/química , Venenos de Vespas/química , Animais , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Células HEK293 , Humanos , Camundongos , Testes de Sensibilidade Microbiana , Proteínas Citotóxicas Formadoras de Poros/uso terapêutico , Proteínas Citotóxicas Formadoras de Poros/toxicidade , Venenos de Vespas/uso terapêutico , Venenos de Vespas/toxicidadeRESUMO
Reducing hurdles to clinical trials without compromising the therapeutic promises of peptide candidates becomes an essential step in peptide-based drug design. Machine-learning models are cost-effective and time-saving strategies used to predict biological activities from primary sequences. Their limitations lie in the diversity of peptide sequences and biological information within these models. Additional outlier detection methods are needed to set the boundaries for reliable predictions; the applicability domain. Antimicrobial peptides (AMPs) constitute an extensive library of peptides offering promising avenues against antibiotic-resistant infections. Most AMPs present in clinical trials are administrated topically due to their hemolytic toxicity. Here we developed machine learning models and outlier detection methods that ensure robust predictions for the discovery of AMPs and the design of novel peptides with reduced hemolytic activity. Our best models, gradient boosting classifiers, predicted the hemolytic nature from any peptide sequence with 95-97% accuracy. Nearly 70% of AMPs were predicted as hemolytic peptides. Applying multivariate outlier detection models, we found that 273 AMPs (~ 9%) could not be predicted reliably. Our combined approach led to the discovery of 34 high-confidence non-hemolytic natural AMPs, the de novo design of 507 non-hemolytic peptides, and the guidelines for non-hemolytic peptide design.
Assuntos
Desenho de Fármacos , Aprendizado de Máquina , Proteínas Citotóxicas Formadoras de Poros/química , Sequência de Aminoácidos , Análise Custo-Benefício , Hemólise/efeitos dos fármacos , Aprendizado de Máquina/economia , Proteínas Citotóxicas Formadoras de Poros/toxicidadeRESUMO
Bacterial protein toxins are important virulence factors. A particular class of toxins, the pore-form toxins (PFTs), shares the toxigenic mechanism of forming pores in the membrane of target cells. The relationship between autophagy and bacterial PFTs has been described for several toxin-secreting pathogens and in this review we have recapitulated the more recent findings on this issue. A common outcome is that the target cell, by a yet non-completely defined mechanism, senses the toxin attack and builds up complex responses as a protective mechanism for host survival. However, in some cases, this cellular response is beneficial to the microorganism by supplying an intracellular niche or by promoting host-cell death, which facilitates pathogen spreading.
Assuntos
Autofagia/genética , Toxinas Bacterianas/toxicidade , Bactérias Gram-Negativas/metabolismo , Bactérias Gram-Positivas/metabolismo , Proteínas Citotóxicas Formadoras de Poros/toxicidade , Fatores de Virulência/toxicidade , Toxinas Bacterianas/metabolismo , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Sobrevivência Celular/genética , Bactérias Gram-Negativas/patogenicidade , Bactérias Gram-Positivas/patogenicidade , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Fagossomos/efeitos dos fármacos , Fagossomos/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Virulência/metabolismoRESUMO
Experimental evidence shows that the mechanism of pore formation by actinoporins is a multistep process, involving binding of the water-soluble monomer to the membrane and subsequent oligomerization on the membrane surface, leading to the formation of a functional pore. However, as for other eukaryotic pore-forming toxins, the molecular details of the mechanism of membrane insertion and oligomerization are not clear. In order to obtain further insight with regard to the structure-function relationship in sticholysins, we designed and produced three cysteine mutants of recombinant sticholysin I (rStI) in relevant functional regions for membrane interaction: StI E2C and StI F15C (in the N-terminal region) and StI R52C (in the membrane binding site). The conformational characterization derived from fluorescence and CD spectroscopic studies of StI E2C, StI F15C and StI R52C suggests that replacement of these residues by Cys in rStI did not noticeably change the conformation of the protein. The substitution by Cys of Arg5² in the phosphocholine-binding site, provoked noticeable changes in rStI permeabilizing activity; however, the substitutions in the N-terminal region (Glu², Phe¹5) did not modify the toxin's permeabilizing ability. The presence of a dimerized population stabilized by a disulfide bond in the StI E2C mutant showed higher pore-forming activity than when the protein is in the monomeric state, suggesting that sticholysins pre-ensembled at the N-terminal region could facilitate pore formation.
Assuntos
Proteínas Citotóxicas Formadoras de Poros/química , Animais , Arginina/química , Arginina/genética , Sítios de Ligação , Membrana Celular/química , Membrana Celular/metabolismo , Clonagem Molecular , Cisteína/química , Cisteína/genética , Mutagênese Sítio-Dirigida , Mutação , Compostos Orgânicos/química , Compostos Orgânicos/toxicidade , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/toxicidade , Estrutura Terciária de Proteína , Anêmonas-do-Mar/metabolismo , Relação Estrutura-AtividadeRESUMO
Bacillus thuringiensis (Bt) bacteria are insect pathogens that rely on insecticidal pore forming proteins known as Cry and Cyt toxins to kill their insect larval hosts. At least four different non-structurally related families of proteins form the Cry toxin group of toxins. The expression of certain Cry toxins in transgenic crops has contributed to an efficient control of insect pests resulting in a significant reduction in chemical insecticide use. The mode of action of the three domain Cry toxin family involves sequential interaction of these toxins with several insect midgut proteins facilitating the formation of a pre-pore oligomer structure and subsequent membrane insertion that leads to the killing of midgut insect cells by osmotic shock. In this manuscript we review recent progress in understanding the mode of action of this family of proteins in lepidopteran, dipteran and coleopteran insects. Interestingly, similar Cry-binding proteins have been identified in the three insect orders, as cadherin, aminopeptidase-N and alkaline phosphatase suggesting a conserved mode of action. Also, recent data on insect responses to Cry toxin attack is discussed. Finally, we review the different Bt based products, including transgenic crops, that are currently used in agriculture.
Assuntos
Bacillus thuringiensis , Proteínas de Bactérias , Proteínas de Insetos/metabolismo , Insetos/efeitos dos fármacos , Inseticidas , Controle Biológico de Vetores/métodos , Proteínas Citotóxicas Formadoras de Poros , Fosfatase Alcalina/metabolismo , Animais , Bacillus thuringiensis/química , Bacillus thuringiensis/metabolismo , Bacillus thuringiensis/patogenicidade , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/toxicidade , Antígenos CD13/metabolismo , Caderinas/metabolismo , Insetos/genética , Insetos/metabolismo , Modelos Moleculares , Plantas Geneticamente Modificadas , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Proteínas Citotóxicas Formadoras de Poros/toxicidade , Ligação ProteicaRESUMO
Bacillus thuringiensis produces insecticidal Cry proteins that are active against different insect species. The primary action of Cry toxins is to lyse midgut epithelial cells in the target insect by forming lytic pores on the apical membrane. After interaction with cadherin receptor, Cry proteins undergo conformational changes from a monomeric structure to a pre-pore-oligomeric form that is able to interact with a second GPI-anchored aminopeptidase-N receptor and then insert into lipid membranes. Here, we review the recent advances in the understanding of the structural changes presented by Cry1Ab toxin upon membrane insertion. Based on analysis of the Trp fluorescence of pure monomeric and oligomeric Cry1Ab structures in solution and in membrane-bound state we reported that oligomerization caused 27% reduction of Trp exposed to the solvent. After membrane insertion there is another conformational change that allows an additional rearrangement of the Trp residues resulting in a total protection of these residues from exposure to the solvent. The oligomeric structure is membrane insertion competent since more than 96% of the Cry1Ab oligomer inserts into the membrane as a function of lipid:protein ratio, in contrast to the monomer of which only 5-10%, inserts into the membrane. Finally, analysis of the stability of monomeric, pre-pore and pore structures of Cry1Ab toxin after urea and thermal denaturation suggested that a more flexible conformation could be necessary for membrane insertion and this flexible structure is obtained by toxin oligomerization and by alkaline pH. Domain I is involved in the intermolecular interaction within the oligomeric Cry1Ab and this domain is inserted into the membrane in the membrane-inserted state.