RESUMO
Resistance to aminoglycoside antibiotics is a serious problem, typically arising from inactivating enzymes, reduced uptake, or increased efflux in the important pathogens for which they are used as treatment. Conjugating aminoglycosides to proline-rich antimicrobial peptides (PrAMPs), which also target ribosomes and have a distinct bacterial uptake mechanism, might mutually benefit their individual activities. To this aim we have developed a strategy for noninvasively modifying tobramycin to link it to a Cys residue and through this covalently link it to a Cys-modified PrAMP by formation of a disulfide bond. Reduction of this bridge in the bacterial cytosol should release the individual antimicrobial moieties. We found that the conjugation of tobramycin to the well-characterized N-terminal PrAMP fragment Bac7(1-35) resulted in a potent antimicrobial capable of inactivating not only tobramycin-resistant bacterial strains but also those less susceptible to the PrAMP. To a certain extent, this activity also extends to the shorter and otherwise poorly active fragment Bac7(1-15). Although the mechanism that allows the conjugate to act when its individual components do not is as yet unclear, results are very promising and suggest this may be a way of resensitizing pathogens that have developed resistance to the antibiotic.
Assuntos
Antibacterianos , Anti-Infecciosos , Antibacterianos/farmacologia , Antibacterianos/química , Aminoglicosídeos/farmacologia , Tobramicina/farmacologia , Peptídeos Antimicrobianos , Prolina , Bactérias , Testes de Sensibilidade MicrobianaRESUMO
The 3D structure and surface characteristics of proteins and peptides are crucial for interactions with receptors or ligands and can be modified to some extent to modulate their biological roles and pharmacological activities. The introduction of halogen atoms on the side-chains of amino acids is a powerful tool for effecting this type of tuning, influencing both the physico-chemical and structural properties of the modified polypeptides, helping to first dissect and then rationally modify features that affect their mode of action. This review provides examples of the influence of different types of halogenation in amino acids that replace native residues in proteins and peptides. Examples of synthetic strategies for obtaining halogenated amino acids are also provided, focusing on some representative compounds and their biological effects. The role of halogenation in native and designed antimicrobial peptides (AMPs) and their mimetics is then discussed. These are in the spotlight for the development of new antimicrobial drugs to counter the rise of antibiotic-resistant pathogens. AMPs represent an interesting model to study the role that natural halogenation has on their mode of action and also to understand how artificially halogenated residues can be used to rationally modify and optimize AMPs for pharmaceutical purposes.
Assuntos
Antibacterianos/química , Peptídeos Antimicrobianos/química , Halogenação , Halogênios/química , Peptidomiméticos/metabolismo , Prolina/análogos & derivados , Antibacterianos/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , Peptidomiméticos/química , Peptoides/química , Prolina/química , Relação Estrutura-AtividadeRESUMO
The human cathelicidin hCAP18/LL-37 has become a paradigm for the pleiotropic roles of peptides in host defence. It has a remarkably wide functional repertoire that includes direct antimicrobial activities against various types of microorganisms, the role of 'alarmin' that helps to orchestrate the immune response to infection, the capacity to locally modulate inflammation both enhancing it to aid in combating infection and limiting it to prevent damage to infected tissues, the promotion of angiogenesis and wound healing, and possibly also the elimination of abnormal cells. LL-37 manages to carry out all its reported activities with a small and simple, amphipathic, helical structure. In this review we consider how different aspects of its primary and secondary structures, as well as its marked tendency to form oligomers under physiological solution conditions and then bind to molecular surfaces as such, explain some of its cytotoxic and immunomodulatory effects. We consider its modes of interaction with bacterial membranes and capacity to act as a pore-forming toxin directed by our organism against bacterial cells, contrasting this with the mode of action of related peptides from other species. We also consider its different membrane-dependent effects on our own cells, which underlie many of its other activities in host defence. This article is part of a Special Issue entitled: Pore-Forming Toxins edited by Mauro Dalla Serra and Franco Gambale.
Assuntos
Peptídeos Catiônicos Antimicrobianos/imunologia , Membrana Celular/imunologia , Imunomodulação , Infecções/imunologia , Neovascularização Fisiológica/imunologia , Cicatrização/imunologia , Humanos , Estrutura Secundária de Proteína , Relação Estrutura-Atividade , CatelicidinasRESUMO
Pseudomonas aeruginosa infections represent a serious threat to worldwide health. Proline-rich antimicrobial peptides (PR-AMPs), a particular group of peptide antibiotics, have demonstrated in vitro activity against P. aeruginosa strains. Here we show that the mammalian PR-AMP Bac7(1-35) is active against some multidrug-resistant cystic fibrosis isolates of P. aeruginosa By confocal microscopy and cytometric analyses, we investigated the mechanism of killing against P. aeruginosa strain PAO1 and three selected isolates, and we observed that the peptide inactivated the target cells by disrupting their cellular membranes. This effect is deeply different from that previously described for PR-AMPs in Escherichia coli and Salmonella enterica serovar Typhimurium, where these peptides act intracellularly after having been internalized by means of the transporter SbmA without membranolytic effects. The heterologous expression of SbmA in PAO1 cells enhanced the internalization of Bac7(1-35) into the cytoplasm, making the bacteria more susceptible to the peptide but at the same time more resistant to the membrane lysis, similarly to what occurs in E. coli The results evidenced a new mechanism of action for PR-AMPs and indicate that Bac7 has multiple and variable modes of action that depend on the characteristics of the different target species and the possibility to be internalized by bacterial transporters. This feature broadens the spectrum of activity of the peptide and makes the development of peptide-resistant bacteria a more difficult process.
Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Membrana Celular/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Pseudomonas aeruginosa/efeitos dos fármacos , Animais , Antibacterianos/síntese química , Peptídeos Catiônicos Antimicrobianos/síntese química , Bovinos , Membrana Celular/ultraestrutura , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/ultraestrutura , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Testes de Sensibilidade Microbiana , Microscopia Confocal , Transporte Proteico , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/ultraestrutura , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Especificidade da Espécie , TransgenesRESUMO
The human cathelicidin LL-37 is a multifunctional host defense peptide with immunomodulatory and antimicrobial roles. It kills bacteria primarily by altering membrane barrier properties, although the exact sequence of events leading to cell lysis has not yet been completely elucidated. Random insertion mutagenesis allowed isolation of Escherichia coli mutants with altered susceptibility to LL-37, pointing to factors potentially relevant to its activity. Among these, inactivation of the waaY gene, encoding a kinase responsible for heptose II phosphorylation in the LPS inner core, leads to a phenotype with decreased susceptibility to LL-37, stemming from a reduced amount of peptide binding to the surface of the cells, and a diminished capacity to lyse membranes. This points to a specific role of the LPS inner core in guiding LL-37 to the surface of Gram-negative bacteria. Although electrostatic interactions are clearly relevant, the susceptibility of the waaY mutant to other cationic helical cathelicidins was unaffected, indicating that particular structural features or LL-37 play a role in this interaction.
Assuntos
Catelicidinas/metabolismo , Membrana Celular/efeitos dos fármacos , Proteínas de Escherichia coli/metabolismo , Escherichia coli/efeitos dos fármacos , Deleção de Genes , Lipopolissacarídeos/metabolismo , Sequência de Aminoácidos , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas Sanguíneas/química , Proteínas Sanguíneas/metabolismo , Catelicidinas/química , Membrana Celular/química , Membrana Celular/metabolismo , Farmacorresistência Bacteriana , Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Expressão Gênica , Heptoses/química , Heptoses/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Lipopolissacarídeos/química , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fosforilação , Ligação Proteica , Proteínas/química , Proteínas/metabolismo , Eletricidade EstáticaRESUMO
Patients with cystic fibrosis require pharmacological treatment against chronic lung infections. The alpha-helical antimicrobial peptides BMAP-27 and BMAP-28 have shown to be highly active in vitro against planktonic and sessile forms of multidrug-resistant Pseudomonas aeruginosa, Staphylococcus aureus, and Stenotrophomonas maltophilia cystic fibrosis strains. To develop small antibacterial peptides for therapeutic use, we tested shortened/modified BMAP fragments, and selected the one with the highest in vitro antibacterial activity and lowest in vivo acute pulmonary toxicity. All the new peptides have shown to roughly maintain their antibacterial activity in vitro. The 1-18 N-terminal fragment of BMAP-27, showing MIC90 of 16 µg/ml against P. aeruginosa isolates and strain-dependent anti-biofilm effects, showed the lowest pulmonary toxicity in mice. However, when tested in a murine model of acute lung infection by P. aeruginosa, BMAP-27(1-18) did not show any curative effect. If exposed to murine broncho-alveolar lavage fluid BMAP-27(1-18) was degraded within 10 min, suggesting it is not stable in pulmonary environment, probably due to murine proteases. Our results indicate that shortened BMAP peptides could represent a starting point for antibacterial drugs, but they also indicate that they need a further optimization for effective in vivo use.
Assuntos
Biofilmes/efeitos dos fármacos , Fibrose Cística/tratamento farmacológico , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Peptídeos , Pneumonia Estafilocócica/tratamento farmacológico , Proteínas , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/fisiologia , Staphylococcus aureus/fisiologia , Animais , Peptídeos Catiônicos Antimicrobianos , Biofilmes/crescimento & desenvolvimento , Modelos Animais de Doenças , Humanos , Camundongos , Peptídeos/química , Peptídeos/farmacologia , Proteínas/química , Proteínas/farmacologiaRESUMO
Cathelicidins, a major family of vertebrate antimicrobial peptides (AMPs), have a recognized role in the first line of defense against infections. They have been identified in several salmonid species, where the putative mature peptides are unusually long and rich in serine and glycine residues, often arranged in short multiple repeats (RLGGGS/RPGGGS) intercalated by hydrophobic motifs. Fragments of 24-40 residues, spanning specific motifs and conserved sequences in grayling or brown, rainbow and brook trout, were chemically synthesized and examined for antimicrobial activity against relevant Gram-positive and Gram-negative salmonid pathogens, as well as laboratory reference strains. They were not active in complete medium, but showed varying potency and activity spectra in diluted media. Bacterial membrane permeabilization also occurred only under these conditions and was indicated by rapid propidium iodide uptake in peptide-treated bacteria. However, circular dichroism analyses indicated that they did not significantly adopt ordered conformations in membrane-like environments. The peptides were not hemolytic or cytotoxic to trout cells, including freshly purified head kidney leukocytes (HKL) and the fibroblastic RTG-2 cell line. Notably, when exposed to them, HKL showed increased metabolic activity, while a growth-promoting effect was observed on RTG-2 cells, suggesting a functional interaction of salmonid cathelicidins with host cells similar to that shown by mammalian ones. The three most active peptides produced a dose-dependent increase in phagocytic uptake by HKL simultaneously stimulated with bacterial particles. The peptide STF(1-37), selected for further analyses, also enhanced phagocytic uptake in the presence of autologous serum, and increased intracellular killing of live E. coli. Furthermore, when tested on HKL in combination with the immunostimulant ß-glucan, it synergistically potentiated both phagocytic uptake and the respiratory burst response, activities that play a key role in fish immunity. Collectively, these data point to a role of salmonid cathelicidins as modulators of fish microbicidal mechanisms beyond a salt-sensitive antimicrobial activity, and encourage further studies also in view of potential applications in aquaculture.
Assuntos
Catelicidinas/genética , Catelicidinas/farmacologia , Salmonidae/imunologia , Sequência de Aminoácidos , Animais , Anti-Infecciosos/química , Anti-Infecciosos/isolamento & purificação , Anti-Infecciosos/farmacologia , Catelicidinas/química , Catelicidinas/isolamento & purificação , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/farmacologia , Domínios Proteicos , Salmonidae/genética , Salmonidae/microbiologia , Alinhamento de Sequência/veterináriaRESUMO
Two new synthetic ionophores in which the hydrophobic portion is represented by a short helical Aib-peptide (Aib=α-amino-isobutyric acid) and the hydrophilic one is a poly-amino (1a) or a polyether (1b) chain have been prepared. The two conjugates show a high ionophoric activity in phospholipid membranes being able to efficiently dissipate a pH gradient and, in the case of 1b, to transport Na(+) across the membrane. Bioactivity evaluation of the two conjugates shows that 1a has a moderate antimicrobial activity against a broad spectrum of microorganisms and it is able to permeabilize the inner and the outer membrane of Escherichia coli cells.
Assuntos
Antibacterianos/farmacologia , Oligopeptídeos/farmacologia , Organofosfatos/farmacologia , Ionóforos de Próton/farmacologia , Ionóforos de Sódio/farmacologia , Antibacterianos/síntese química , Candida albicans/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Cryptococcus neoformans/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Cinética , Oligopeptídeos/síntese química , Organofosfatos/síntese química , Ionóforos de Próton/síntese química , Força Próton-Motriz , Pseudomonas aeruginosa/efeitos dos fármacos , Ionóforos de Sódio/síntese química , Staphylococcus aureus/efeitos dos fármacos , Lipossomas UnilamelaresRESUMO
SbmA is an inner membrane protein of Gram-negative bacteria that is involved in the internalization of glycopeptides and prokaryotic and eukaryotic antimicrobial peptides, as well as of peptide nucleic acid (PNA) oligomers. The SbmA homolog BacA is required for the development of Sinorhizobium meliloti bacteroids within plant cells and favors chronic infections with Brucella abortus and Mycobacterium tuberculosis in mice. Here, we investigated functional features of SbmA/BacA using the proline-rich antimicrobial peptide Bac7(1-35) as a substrate. Circular dichroism and affinity chromatography studies were used to investigate the ability of SbmA to bind the peptide, and a whole-cell transport assay with fluorescently labeled peptide allowed the determination of transport kinetic parameters with a calculated Km value of 6.95 ± 0.89 µM peptide and a Vmax of 53.91 ± 3.17 nmol/min/mg SbmA. Use of a bacterial two-hybrid system coupled to SEC-MALLS (size exclusion chromatography coupled with multiangle laser light scattering) analyses established that SbmA is a homodimer in the membrane, and treatment of the cells with arsenate or ionophores indicated that the peptide transport mediated by SbmA is driven by the electrochemical gradient. Overall, these results shed light on the SbmA-mediated internalization of peptide substrates and suggest that the transport of an unknown substrate(s) represents the function of this protein.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Trifosfato de Adenosina/metabolismo , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/metabolismo , Dicroísmo Circular , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Membrana Transportadoras/genética , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Ligação Proteica , Prótons , Proteínas Recombinantes de FusãoRESUMO
BACKGROUND: Treatment of cystic fibrosis-associated lung infections is hampered by the presence of multi-drug resistant pathogens, many of which are also strong biofilm producers. Antimicrobial peptides, essential components of innate immunity in humans and animals, exhibit relevant in vitro antimicrobial activity although they tend not to select for resistant strains. RESULTS: Three α-helical antimicrobial peptides, BMAP-27 and BMAP-28 of bovine origin, and the artificial P19(9/B) peptide were tested, comparatively to Tobramycin, for their in vitro antibacterial and anti-biofilm activity against 15 Staphylococcus aureus, 25 Pseudomonas aeruginosa, and 27 Stenotrophomonas maltophilia strains from cystic fibrosis patients. All assays were carried out in physical-chemical experimental conditions simulating a cystic fibrosis lung. All peptides showed a potent and rapid bactericidal activity against most P. aeruginosa, S. maltophilia and S. aureus strains tested, at levels generally higher than those exhibited by Tobramycin and significantly reduced biofilm formation of all the bacterial species tested, although less effectively than Tobramycin did. On the contrary, the viability-reducing activity of antimicrobial peptides against preformed P. aeruginosa biofilms was comparable to and, in some cases, higher than that showed by Tobramycin. CONCLUSIONS: The activity shown by α-helical peptides against planktonic and biofilm cells makes them promising "lead compounds" for future development of novel drugs for therapeutic treatment of cystic fibrosis lung disease.
Assuntos
Peptídeos Catiônicos Antimicrobianos/administração & dosagem , Biofilmes/efeitos dos fármacos , Fibrose Cística/complicações , Pneumonia Bacteriana/terapia , Pseudomonas aeruginosa/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Stenotrophomonas maltophilia/efeitos dos fármacos , Animais , Antibacterianos/administração & dosagem , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bovinos , Humanos , Testes de Sensibilidade Microbiana , Pneumonia Bacteriana/prevenção & controle , Pseudomonas aeruginosa/fisiologia , Staphylococcus aureus/fisiologia , Stenotrophomonas maltophilia/fisiologiaRESUMO
Proline-rich antimicrobial peptides are a group of cationic host defense peptides of vertebrates and invertebrates characterized by a high content of proline residues, often associated with arginine residues in repeated motifs. Those isolated from some mammalian and insect species, although not evolutionarily related, use a similar mechanism to selectively kill Gram-negative bacteria, with a low toxicity to animals. Unlike other types of antimicrobial peptides, their mode of action does not involve the lysis of bacterial membranes but entails penetration into susceptible cells, where they then act intracellularly. Some aspects of the transport system and cytoplasmic targets have been elucidated. These features make them attractive both as anti-infective lead compounds and as a new class of potential cell-penetrating peptides capable of internalising membrane-impermeant drugs into both bacterial and eukaryotic cells.
Assuntos
Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Domínios Proteicos Ricos em Prolina , Sequência de Aminoácidos , Animais , Bactérias/efeitos dos fármacos , Insetos/química , Dados de Sequência Molecular , Prolina/química , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Bac7 is a proline-rich peptide with a potent in vitro antimicrobial activity against Gram-negative bacteria. Here we investigated its activity in biological fluids and in vivo using a mouse model of S. typhimurium infection. RESULTS: The efficacy of the active 1-35 fragment of Bac7 was assayed in serum and plasma, and its stability in biological fluids analyzed by Western blot and mass spectrometry. The ability of the peptide to protect mice against Salmonella was assayed in a typhoid fever model of infection by determination of survival rates and bacterial load in liver and spleen of infected animals. In addition, the peptide's biodistribution was evaluated by using time-domain optical imaging. Bac7(1-35) retained a substantial in vivo activity showing a very low toxicity. The peptide increased significantly the number of survivors and the mean survival times of treated mice reducing the bacterial load in their organs despite its rapid clearance. CONCLUSIONS: Our results provide a first indication for a potential development of Bac7-based drugs in the treatment of salmonellosis and, eventually, other Gram-negative infections. The in vivo activity for this peptide might be substantially enhanced by decreasing its excretion rate or modifying the treatment schedule.
Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Salmonelose Animal/tratamento farmacológico , Salmonella typhimurium , Animais , Peptídeos Catiônicos Antimicrobianos/farmacocinética , Fígado/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos CBA , Salmonelose Animal/microbiologia , Baço/microbiologia , Distribuição TecidualRESUMO
The synthesis and screening of neutral and cationic, linear and cyclic peptoids (N-alkylglycine peptidomimetics) is described. Structure-activity relationship studies show that the in vitro activities of the tested peptoids depend on both cyclization and decoration with cationic groups. The most powerful N-lysine cyclopeptoid derivatives showed good antifungal activity against Candida albicans (ATCC90029 and L21) and Candida famata (SA550, Amph B-resistant) and low hemolytic activity. The effects of the cyclic peptoids on membrane permeabilization were evaluated by the propidium iodide exclusion assay.
Assuntos
Antibacterianos/síntese química , Antifúngicos/síntese química , Cátions/química , Peptídeos Cíclicos/síntese química , Antibacterianos/química , Antibacterianos/farmacologia , Antifúngicos/química , Antifúngicos/farmacologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Desenho de Fármacos , Eritrócitos/efeitos dos fármacos , Hemólise , Humanos , Testes de Sensibilidade Microbiana , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Relação Estrutura-AtividadeRESUMO
In this paper, we describe a rapid flow cytometry method to identify antimicrobial peptides that are internalized into bacterial cells and differentiate them from those that are membrane active. The method was applied to fluorescently labeled Bac7(1-35) and polymyxin B, whose mechanisms of action are, respectively, based on cell penetration and on membrane binding and permeabilization. Identification of peptides with the former mechanism is of considerable interest for the intracellular delivery of membrane-impermeant drugs.
Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacocinética , Bactérias Gram-Negativas/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Citometria de Fluxo , Bactérias Gram-Negativas/efeitos dos fármacos , Microscopia Confocal , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacocinética , Polimixina B/química , Polimixina B/farmacocinéticaRESUMO
In this work we study (i) the formation and stabilization of silver nanoparticles in a bioactive chitosan-derived polysaccharide solution, (ii) the antimicrobial properties, either in solution or in 3D hydrogel structures, obtained by mixtures with the polysaccharide alginate, and (iii) the cytotoxicity of the latter nanocomposite materials on different eukaryotic cell lines. Antimicrobial results show that these nanocomposite systems display a very effective bactericidal activity toward both Gram+ and Gram- bacteria. However, the hydrogel does not show any cytotoxic effect toward three different eukaryotic cell lines. This is due to the fact that the nanoparticles, immobilized in the gel matrix, can exert their antimicrobial activity by simple contact with the bacterial membrane, while they can not be uptaken and internalized by eukaryotic cells. This novel finding could advantageously contribute to responding to the growing concerns on the toxicity of nanoparticles and facilitate the use of silver-biopolymer composites in the preparation of biomaterials.
Assuntos
Antibacterianos/farmacologia , Nanopartículas Metálicas , Prata/química , Antibacterianos/química , Escherichia coli/efeitos dos fármacos , Citometria de Fluxo , Hidrogéis , Espectrometria de Massas , Testes de Sensibilidade Microbiana , Microscopia Eletrônica de Transmissão , Pseudomonas aeruginosa/efeitos dos fármacos , Espectrofotometria Ultravioleta , Staphylococcus aureus/efeitos dos fármacosRESUMO
The proline-rich antimicrobial peptides (PRPs) are considered to act by crossing bacterial membranes without altering them and then binding to, and functionally modifying, one or more specific targets. This implies that they can be used as molecular hooks to identify the intracellular or membrane proteins that are involved in their mechanism of action and that may be subsequently used as targets for the design of novel antibiotics with mechanisms different from those now in use. The targets can be identified by using peptide-based affinity columns or via the genetic approach described here. This approach depends on chemical mutagenesis of a PRP-susceptible bacterial strain to select mutants that are either more resistant or more susceptible to the relevant peptide. The genes conferring the mutated phenotype can then be isolated and identified by subcloning and sequencing. In this manner, we have currently identified several genes that are involved in the mechanism of action of these peptides, including peptide-transport systems or potential resistance factors, which can be used or taken into account in drug design efforts.
Assuntos
Antibacterianos , Peptídeos Catiônicos Antimicrobianos , Desenho de Fármacos , Prolina/química , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/genética , Testes de Sensibilidade Microbiana , FenótipoRESUMO
Proline-rich peptides are a unique group of antimicrobial peptides that exert their activity selectively against Gram-negative bacteria through an apparently non-membranolytic mode of action that is not yet well understood. We have investigated the mechanism underlying the antibacterial activity of the proline-rich cathelicidin Bac7 against Salmonella enterica and Escherichia coli. The killing and membrane permeabilization kinetics as well as the cellular localization were assessed for the fully active N-terminal fragment Bac7(1-35), its all-D enantiomer and for differentially active shortened fragments. At sub-micromolar concentrations, Bac7(1-35) rapidly killed bacteria by a non-lytic, energy-dependent mechanism, whereas its D-enantiomer was inactive. Furthermore, while the L-enantiomer was rapidly internalized into bacterial cells, the D-enantiomer was virtually excluded. At higher concentrations (>or=64 microM), both L- and D-Bac7(1-35) were instead able to kill bacteria also via a lytic mechanism. Overall, these results suggest that Bac7 may inactivate bacteria via two different modes of action depending on its concentration: (i) at near-MIC concentrations via a mechanism based on a stereospecificity-dependent uptake that is likely followed by its binding to an intracellular target, and (ii) at concentrations several times the MIC value, via a non-stereoselective, membranolytic mechanism.
Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Antibacterianos/farmacocinética , Peptídeos Catiônicos Antimicrobianos/farmacocinética , Membrana Celular/efeitos dos fármacos , Citometria de Fluxo , Microscopia Imunoeletrônica , Farmacocinética , Prolina/química , Estereoisomerismo , Fatores de TempoRESUMO
BMAP-28, a bovine antimicrobial peptide of the cathelicidin family, induces membrane permeabilization and death in human tumor cell lines and in activated, but not resting, human lymphocytes. In addition, we found that BMAP-28 causes depolarization of the inner mitochondrial membrane in single cells and in isolated mitochondria. The effect of the peptide was synergistic with that of Ca(2+) and inhibited by cyclosporine, suggesting that depolarization depends on opening of the mitochondrial permeability transition pore. The occurrence of a permeability transition was investigated on the basis of mitochondrial permeabilization to calcein and cytochrome c release. We show that BMAP-28 permeabilizes mitochondria to entrapped calcein in a cyclosporine-sensitive manner and that it releases cytochrome c in situ. Our results demonstrate that BMAP-28 is an inducer of the mitochondrial permeability transition pore and that its cytotoxic potential depends on its effects on mitochondrial permeability.
Assuntos
Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Canais Iônicos , Leucócitos Mononucleares/efeitos dos fármacos , Proteínas de Membrana/efeitos dos fármacos , Proteínas/farmacologia , Animais , Cálcio/metabolismo , Cálcio/farmacologia , Bovinos , Morte Celular/efeitos dos fármacos , Células Cultivadas , Ciclosporina/farmacologia , Grupo dos Citocromos c/metabolismo , Relação Dose-Resposta a Droga , Fluoresceínas/metabolismo , Corantes Fluorescentes , Humanos , Imunidade Inata/fisiologia , Células K562 , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial , Poro de Transição de Permeabilidade Mitocondrial , Células U937RESUMO
Nine Chlamydia suis isolates, obtained from pigs with conjunctivitis, were molecularly characterized by ompA sequencing and their in vitro susceptibility to six cathelicidin peptides (SMAP-29, BAC-7, BMAP-27, BMAP-27, BMAP-28, PG-1, LL-37) determined in cell culture. SMAP-29 was the most active peptide, reducing the intracellular inclusion number by > or =50% at a concentration of 10 microg/ml (3 microM) in six of the nine isolates tested. Three molecularly identical isolates were insensitive at a concentration as high as 80 microg/ml (25 microM). Of the remaining cathelicidin peptides tested, BAC-7 and BMAP-27 were active against six C. suis isolates at a concentration of 80 microg/ml (25 and 26 microM, respectively). Cathelicidins LL-37 and PG-1 did not show any anti-chlamydial activity at 80 microg/ml.
Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Chlamydia/classificação , Chlamydia/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/química , Catelicidinas , Bovinos , Linhagem Celular , Chlorocebus aethiops , Humanos , SuínosRESUMO
A distinct group of antimicrobial peptides kills bacteria by interfering with internal cellular functions and without concurrent lytic effects on cell membranes. Here we describe some methods to investigate the mechanisms of action of these antimicrobial peptides. They include assays to detect the possible temporal separation between membrane permeabilization and bacterial killing events, to assess the capacity of antimicrobial peptides to cross the bacterial membranes and reside in the cytoplasm, and later to inhibit vital cell functions such as DNA transcription and protein translation.