RESUMEN
There is an urgent and imminent need to develop new antimicrobials to fight against antibiotic-resistant bacterial and fungal strains. In this study, a checkerboard method was used to evaluate the synergistic effects of the antimicrobial peptide P-113 and its bulky non-nature amino acid substituted derivatives with vancomycin against vancomycin-resistant Enterococcus faecium, Staphylococcus aureus, and wild-type Escherichia coli. Boron-dipyrro-methene (BODIPY) labeled vancomycin was used to characterize the interactions between the peptides, vancomycin, and bacterial strains. Moreover, neutralization of antibiotic-induced releasing of lipopolysaccharide (LPS) from E. coli by the peptides was obtained. Among these peptides, Bip-P-113 demonstrated the best minimal inhibitory concentrations (MICs), antibiotics synergism, bacterial membrane permeabilization, and supernatant LPS neutralizing activities against the bacteria studied. These results could help in developing antimicrobial peptides that have synergistic activity with large size glycopeptides such as vancomycin in therapeutic applications.
Asunto(s)
Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Sinergismo Farmacológico , Enterococcus faecium/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Proteínas Citotóxicas Formadoras de Poros/farmacología , Staphylococcus aureus/efectos de los fármacos , Vancomicina/farmacología , Antibacterianos/farmacología , Pruebas de Sensibilidad MicrobianaRESUMEN
A strategy was described to design antimicrobial peptides (AMPs) with enhanced salt resistance and antiendotoxin activities by linking two helical AMPs with the Ala-Gly-Pro (AGP) hinge. Among the designed peptides, KR12AGPWR6 demonstrated the best antimicrobial activities even in high salt conditions (NaCl ~300 mM) and possessed the strongest antiendotoxin activities. These activities may be related to hydrophobicity, membrane-permeability, and α-helical content of the peptide. Amino acids of the C-terminal helices were found to affect the peptide-induced permeabilization of LUVs, the α-helicity of the designed peptides under various LUVs, and the LPS aggregation and size alternation. A possible model was proposed to explain the mechanism of LPS neutralization by the designed peptides. These findings could provide a new approach for designing AMPs with enhanced salt resistance and antiendotoxin activities for potential therapeutic applications.
Asunto(s)
Endotoxemia/tratamiento farmacológico , Lipopolisacáridos/antagonistas & inhibidores , Proteínas Citotóxicas Formadoras de Poros/farmacología , Tolerancia a la Sal/efectos de los fármacos , Cloruro de Sodio/farmacología , Secuencia de Aminoácidos , Animales , Recuento de Colonia Microbiana , Evaluación Preclínica de Medicamentos , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Prueba de Limulus , Lipopolisacáridos/toxicidad , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Citotóxicas Formadoras de Poros/síntesis química , Proteínas Citotóxicas Formadoras de Poros/uso terapéutico , Conformación Proteica en Hélice alfa , Relación Estructura-Actividad , Factor de Necrosis Tumoral alfa/sangre , Liposomas UnilamelaresRESUMEN
In the absence of proper immunity, such as in the case of acquired immune deficiency syndrome (AIDS) patients, Candida albicans, the most common human fungal pathogen, may cause mucosal and even life-threatening systemic infections. P-113 (AKRHHGYKRKFH), an antimicrobial peptide (AMP) derived from the human salivary protein histatin 5, shows good safety and efficacy profiles in gingivitis and human immunodeficiency virus (HIV) patients with oral candidiasis. However, little is known about how P-113 interacts with Candida albicans or its degradation by Candida-secreted proteases that contribute to the fungi's resistance. Here, we use solution nuclear magnetic resonance (NMR) methods to elucidate the molecular mechanism of interactions between P-113 and living Candida albicans cells. Furthermore, we found that proteolytic cleavage of the C-terminus prevents the entry of P-113 into cells and that increasing the hydrophobicity of the peptide can significantly increase its antifungal activity. These results could help in the design of novel antimicrobial peptides that have enhanced stability in vivo and that can have potential therapeutic applications.
Asunto(s)
Antifúngicos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Candida albicans/efectos de los fármacos , Farmacorresistencia Fúngica/efectos de los fármacos , Secuencia de Aminoácidos , Antifúngicos/química , Péptidos Catiónicos Antimicrobianos/química , Candida albicans/ultraestructura , Relación Dosis-Respuesta a Droga , Histatinas/química , Humanos , Espectroscopía de Resonancia Magnética , Pruebas de Sensibilidad Microbiana , Proteolisis , Factores de TiempoRESUMEN
P-113, which was originally derived from the human saliva protein histatin 5, is a histidine-rich antimicrobial peptide with the sequence AKRHHGYKRKFH. P-113 is currently undergoing phase II clinical trial as a pharmaceutical agent to fight against fungal infections in HIV patients with oral candidiasis. Previously, we developed a new procedure for the high-yield expression and purification of hG31P, an analogue and antagonist of human CXCL8. Moreover, we have successfully removed lipopolysaccharide (LPS, endotoxin) associated with hG31P in the expression with Escherichia coli. In this paper, we have used hG31P as a novel fusion protein for the expression and purification of P-113. The purity of the expressed P-113 is more than 95% and the yield is 4 mg P-113 per liter of E. coli cell culture in Luria-Bertani (LB) medium. The antimicrobial activity of the purified P-113 was tested. Furthermore, we used circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy to study the structural properties of P-113. Our results indicate that using hG31P as a fusion protein to obtain large quantities of P-113 is feasible and is easy to scale up for commercial production. An effective way of producing enough P-113 for future clinical studies is evident in this study.
Asunto(s)
Péptidos Catiónicos Antimicrobianos , Escherichia coli , Expresión Génica , Histatinas , Péptidos Catiónicos Antimicrobianos/biosíntesis , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/genética , Péptidos Catiónicos Antimicrobianos/aislamiento & purificación , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Histatinas/biosíntesis , Histatinas/genética , Histatinas/aislamiento & purificación , Humanos , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificaciónRESUMEN
Lipopolysaccharide (LPS, endotoxin) is the major component of Gram-negative bacterial outer surface membrane. LPS released from bacteria into bloodstream during infection may cause serious unwanted stimulation of host's immune system and lead to septic shock of the patient. Recently, we have developed a strategy to increase salt resistance and LPS neutralization of short antimicrobial peptides by adding ß-naphthylalanine end-tags to their termini. Herein, correlations between membrane immersion depth, orientation, and antiendotoxin activities of the antimicrobial peptides S1 and S1-Nal-Nal have been investigated via solution structure, paramagnetic resonance enhancement, and saturation transfer difference NMR studies. Unlike the parent peptide S1, S1-Nal-Nal rotated its two terminal ß-naphthylalanine residues into the hydrophobic lipid A motif of LPS micelles. The LPS-induced inflammation may then be prohibited by the blocked lipid A motif.
Asunto(s)
Antiinflamatorios no Esteroideos/síntesis química , Antídotos/síntesis química , Péptidos Catiónicos Antimicrobianos/síntesis química , Lipopolisacáridos/antagonistas & inhibidores , Macrófagos/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Antiinflamatorios no Esteroideos/farmacología , Antídotos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Línea Celular , Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Lipopolisacáridos/farmacología , Macrófagos/citología , Macrófagos/inmunología , Ratones , Modelos Moleculares , Relación Estructura-Actividad , Termodinámica , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Release of lipopolysaccharide (LPS) (endotoxin) from bacteria into the bloodstream may cause serious unwanted stimulation of the host immune system. Some but not all antimicrobial peptides can neutralize LPS-stimulated proinflammatory responses. Salt resistance and serum stability of short antimicrobial peptides can be boosted by adding ß-naphthylalanine to their termini. Herein, significant antiendotoxin effects were observed in vitro and in vivo with the ß-naphthylalanine end-tagged variants of the short antimicrobial peptides S1 and KWWK.
Asunto(s)
Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Bacterias Gramnegativas/efectos de los fármacos , Lipopolisacáridos/antagonistas & inhibidores , Péptidos Catiónicos Antimicrobianos/química , Interacciones Hidrofóbicas e Hidrofílicas , Pruebas de Sensibilidad Microbiana , beta-Alanina/análogos & derivados , beta-Alanina/químicaRESUMEN
The efficacies of many antimicrobial peptides are greatly reduced in the presence of high salt concentrations therefore limiting their development as pharmaceutical compounds. PEM-2-W5K/A9W, a short Trp-rich antimicrobial peptide developed based on the structural studies of PEM-2, has been shown to be highly active against various bacterial strains with less hemolytic activity. Here, correlations between membrane immersion depth, orientation, and salt-resistance of PEM-2 and PEM-2-W5K/A9W have been investigated via solution structure and paramagnetic resonance enhancement studies. The antimicrobial activities of PEM-2-W5K/A9W and PEM-2 against various bacterial and fungal strains including multidrug-resistant and clinical isolates under high salt conditions were tested. The activities of the salt-sensitive peptide PEM-2 were reduced and diminished at high salt concentrations, whereas the activities of PEM-2-W5K/A9W were less affected. The results indicated that the strong salt-resistance of PEM-2-W5K/A9W may arise from the peptide positioning itself deeply into microbial cell membranes and thus able to disrupt the membranes more efficiently.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/farmacología , Cloruro de Sodio/química , Triptófano/química , Péptidos Catiónicos Antimicrobianos/química , Espectroscopía de Resonancia por Spin del Electrón , Espectroscopía de Resonancia Magnética , Micelas , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Conformación ProteicaRESUMEN
The efficacies of many antimicrobial peptides are greatly reduced under high salt concentrations, therefore limiting their use as pharmaceutical agents. Here, we describe a strategy to boost salt resistance and serum stability of short antimicrobial peptides by adding the nonnatural bulky amino acid ß-naphthylalanine to their termini. The activities of the short salt-sensitive tryptophan-rich peptide S1 were diminished at high salt concentrations, whereas the activities of its ß-naphthylalanine end-tagged variants were less affected.
Asunto(s)
Ampicilina/farmacología , Antiinfecciosos/farmacología , Péptidos/farmacología , Cloruro de Sodio/química , beta-Alanina/análogos & derivados , Secuencia de Aminoácidos , Ampicilina/química , Antiinfecciosos/química , Estabilidad de Medicamentos , Eritrocitos/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Hemólisis , Humanos , Pruebas de Sensibilidad Microbiana , Péptidos/química , Suero/química , Triptófano/química , beta-Alanina/químicaRESUMEN
Cecropins are a family of antimicrobial peptides (AMPs) that are widely found in the innate immune system of Cecropia moths. Cecropins exhibit a broad spectrum of antimicrobial and anticancer activities. The structures of Cecropins are composed of 34-39 amino acids with an N-terminal amphipathic α-helix, an AGP hinge and a hydrophobic C-terminal α-helix. KR12AGPWR6 was designed based on the Cecropin-like structural feature. In addition to its antimicrobial activities, KR12AGPWR6 also possesses enhanced salt resistance, antiendotoxin and anticancer properties. Herein, we have developed a strategy to produce recombinant KR12AGPWR6 through a salt-sensitive, pH and temperature dependent intein self-cleavage system. The His6-Intein-KR12AGPWR6 was expressed by E. coli and KR12AGPWR6 was released by the self-cleavage of intein under optimized ionic strength, pH and temperature conditions. The molecular weight and structural feature of the recombinant KR12AGPWR6 was determined by MALDI-TOF mass, CD, and NMR spectroscopy. The recombinant KR12AGPWR6 exhibited similar antimicrobial activities compared to the chemically synthesized KR12AGPWR6. Our results provide a potential strategy to obtain large quantities of AMPs and this method is feasible and easy to scale up for commercial production.
RESUMEN
The global spread of antibiotic-resistant infections has meant that there is an urgent need to develop new antimicrobial alternatives. In this study, we developed a strategy to boost and/or synergize the activity of conventional antibiotics by combination with antimicrobial peptides tagged with the bulky non-natural amino acid ß-naphthylalanine (Nal) to their N- or C-terminus. A checkerboard method was used to evaluate synergistic effects of the parent peptide and the Nal-tagged peptides. Moreover, boron-dipyrro-methene labeled vancomycin was used to characterize the synergistic mechanism of action between the peptides and vancomycin on the bacterial strains. These Nal-tagged antimicrobial peptides also reduced the antibiotic-induced release of lipopolysaccharide from Gram-negative bacteria by more than 99.95%. Our results demonstrate that Nal-tagged peptides could help in developing antimicrobial peptides that not only have enhanced antibacterial activities but also increase the synergistic effects with conventional antibiotics against antibiotic-resistant bacteria.
RESUMEN
HYPOTHESIS: Release of lipopolysaccharides (LPS) from bacteria into bloodstream may cause serious unwanted stimulation of the host immune system. P-113 is a clinically active histidine-rich antimicrobial peptide. Nal-P-113, a ß-naphthylalanine-substituted P-113, is salt-resistant but has limited LPS neutralizing activity. We suspected the size and shape of the non-natural bulky amino acid may affect its LPS neutralizing activity. Herein, antimicrobial, LPS neutralizing, and antiproteolytic effects of phenylalanine- (Phe-P-113), ß-naphthylalanine- (Nal-P-113), ß-diphenylalanine- (Dip-P-113), and ß-(4,4'-biphenyl)alanine- (Bip-P-113) substituted P-113 were studied. EXPERIMENTS: Structure-activity relationships of P-113, Phe-P-113, Nal-P-113, Dip-P-113, and Bip-P-113 were evaluated using antimicrobial activity assays, serum proteolytic assays, peptide-induced permeabilization of large unilamellar vesicles, zeta potential measurements, dynamic light scattering measurement of LPS aggregation, and Limulus amebocyte lysate assays for measuring LPS neutralization. In vitro and in vivo LPS neutralizing activities were further confirmed by LPS-induced inflammation inhibition in an endotoxemia mouse model. FINDINGS: Bip-P-113 and Dip-P-113 had the longest and widest non-nature amino acids, respectively. Bip-P-113 enhanced salt resistance, serum proteolytic stability, peptide-induced permeabilization, zeta potential measurements, LPS aggregation, and in vitro and in vivo LPS neutralizing activities. These results could help design novel antimicrobial peptides that have enhanced stability in vivo and that can have potential therapeutic applications.
Asunto(s)
Aminoácidos/química , Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Endotoxemia/tratamiento farmacológico , Inflamación/tratamiento farmacológico , Lipopolisacáridos/antagonistas & inhibidores , Animales , Antibacterianos/sangre , Antibacterianos/química , Péptidos Catiónicos Antimicrobianos/sangre , Péptidos Catiónicos Antimicrobianos/química , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Dispersión Dinámica de Luz , Endotoxemia/inducido químicamente , Endotoxinas , Escherichia coli/efectos de los fármacos , Fibroblastos , Técnica de Placa Hemolítica , Humanos , Inflamación/inducido químicamente , Lipopolisacáridos/química , Lipopolisacáridos/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Pruebas de Sensibilidad Microbiana , Tamaño de la Partícula , Pseudomonas aeruginosa/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacos , Relación Estructura-Actividad , Propiedades de SuperficieRESUMEN
We describe a strategy to boost anticancer activity and reduce normal cell toxicity of short antimicrobial peptides by adding positive charge amino acids and non-nature bulky amino acid ß-naphthylalanine residues to their termini. Among the designed peptides, K4R2-Nal2-S1 displayed better salt resistance and less toxicity to hRBCs and human fibroblast than Nal2-S1 and K6-Nal2-S1. Fluorescence microscopic studies indicated that the FITC-labeled K4R2-Nal2-S1 preferentially binds cancer cells and causes apoptotic cell death. Moreover, a significant inhibition in human lung tumor growth was observed in the xenograft mice treated with K4R2-Nal2-S1. Our strategy provides new opportunities in the development of highly effective and selective antimicrobial and anticancer peptide-based therapeutics.
Asunto(s)
Antibacterianos/farmacología , Antibióticos Antineoplásicos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Animales , Apoptosis , Línea Celular Tumoral , Escherichia coli/efectos de los fármacos , Humanos , Masculino , Ratones Endogámicos BALB C , Ratones Desnudos , Pruebas de Sensibilidad Microbiana , Pseudomonas aeruginosa/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Streptococcus gordonii, Fusobacterium nucleatum and Porphyromonas gingivalis represent the early, middle and late colonizers of the bacterial accretion in dental plaque biofilms. These sessile communities constitute a protected mode of growth that promotes survival in a hostile environment. This study describes a novel and unrecognized role for a synthetic cationic antimicrobial peptide, Nal-P-113, which inhibits and kills periodontal bacteria in planktonic state, inhibits the formation of biofilms and eradicates polymicrobial biofilms. Nal-P-113 is also stable in saliva, serum and saline solution. At a concentration less than 320 µg/mL which is harmless to normal oral cells, Nal-P-113 can kill bacteria in planktonic state. At a concentration of antimicrobial peptide Nal-P-113 (1280 µg/mL) which only causes slight damages to normal oral cells is needed to kill bacteria in biofilm state. It is worth mentioning that this concentration of Nal-P-113 is harmless to rat oral mucosa compared to chlorhexidine. The mechanism of Nal-P-113 inhibiting and killing periodontal bacteria might rely on the abilities to permeabilize and/or to form pores within the cytoplasmic membranes, thus causes the death of bacteria. Here, we provided a novel and stable antimicrobial peptide with very low mammalian cytotoxicity, which can inhibit and kill periodontal bacteria in both planktonic and polymicrobial biofilm states. STATEMENT OF SIGNIFICANCE: Nal-P-113 is a potent antimicrobial peptide with strong antimicrobial ability, improved deficiency compared with other antibacterial peptides, and remains stable in phosphate buffered saline, saliva, brain-heart infusion medium and bovine calf serum. Nal-P-113 exhibits a broad spectrum of bacteriocidal activity with excellent eradicating capability on oral pathogens and the respective biofilms. In this study, we used propidium iodide staining, scanning electron microscopy and transmission electron microscopy to confirm that Nal-P-113 can perforate plasmalemma thereby resulting in the death of oral pathogens and disintegrate the respective biofilms. Nal-P-113 also showed effective anti-plaque biofilms and cytotoxicity in the rat periodontitis model. No adverse effects can be observed on the gingivomucosa tissue. In short, the antimicrobial peptide Nal-P-113 presented to be an effective yet have low mammalian cytotoxicity agent with potential application in the clinic. This study provides a proof of concept in applying antimicrobial peptides in the clinical perspective.