RESUMEN
Leishmaniasis, a category I neglected tropical disease, is a group of diseases caused by the protozoan parasite Leishmania species with a wide range of clinical manifestations. Current treatment options can be highly toxic and expensive, with drug relapse and the emergence of resistance. Bacteriocins, antimicrobial peptides ribosomally produced by bacteria, are a relatively new avenue for potential antiprotozoal drugs. Particular interest has been focused on enterocin AS-48, with previously proven efficacy against protozoan species, including Leishmania spp. Sequential characterization of enterocin AS-48 has illustrated that antibacterial bioactivity is preserved in linearized, truncated forms; however, minimal domains of AS-48 bacteriocins have not yet been explored against protozoans. Using rational design techniques to improve membrane penetration activity, we designed peptide libraries using the minimal bioactive domain of AS-48 homologs. Stepwise changes to the charge (z), hydrophobicity (H), and hydrophobic dipole moment (µH) were achieved through lysine and tryptophan substitutions and the inversion of residues within the helical wheel, respectively. A total of 480 synthetic peptide variants were assessed for antileishmanial activity against Leishmania donovani. One hundred seventy-two peptide variants exhibited 50% inhibitory concentration (IC50) values below 20 µM against axenic amastigotes, with 60 peptide variants in the nanomolar range. Nine peptide variants exhibited potent activity against intracellular amastigotes with observed IC50 values of <4 µM and limited in vitro host cell toxicity, making them worthy of further drug development. Our work demonstrates that minimal bioactive domains of naturally existing bacteriocins can be synthetically engineered to increase membrane penetration against Leishmania spp. with minimal host cytotoxicity, holding the promise of novel, potent antileishmanial therapies. IMPORTANCE Leishmaniasis is a neglected tropical disease caused by protozoan parasites of the genus Leishmania. There are three primary clinical forms, cutaneous, mucocutaneous, and visceral, with visceral leishmaniasis being fatal if left untreated. Current drug treatments are less than ideal, especially in resource-limited areas, due to the difficult administration and treatment regimens as well as the high cost and the emergence of drug resistance. Identifying potent antileishmanial agents is of the utmost importance. We utilized rational design techniques to synthesize enterocin AS-48 and AS-48-like bacteriocin-based peptides and screened these peptides against L. donovani using a fluorescence-based phenotypic assay. Our results suggest that bacteriocins, specifically these rationally designed AS-48-like peptides, are promising leads for further development as antileishmanial drugs.
Asunto(s)
Antiprotozoarios , Bacteriocinas , Leishmania donovani , Leishmaniasis , Humanos , Bacteriocinas/farmacología , Bacteriocinas/uso terapéutico , Leishmaniasis/tratamiento farmacológico , Hidrocarburos Aromáticos con Puentes/uso terapéutico , Antiprotozoarios/farmacologíaRESUMEN
The circularized bacteriocin enterocin AS-48 produced by Enterococcus sp. exhibits antibacterial activity through membrane disruption. The membrane-penetrating activity of enterocin AS-48 has been attributed to a specific alpha-helical region on the circular peptide. Truncated, linearized forms containing these domains have been shown to preserve limited bactericidal activity. We utilized the amino acid sequence of the active helical domain of enterocin AS-48 to perform a homology-based search of similar sequences in other bacterial genomes. We identified similar domains in three previously uncharacterized AS-48-like bacteriocin genes in Clostridium sordellii, Paenibacillus larvae, and Bacillus xiamenensis. Enterocin AS-48 and homologs from these bacterial species were used as scaffolds for the design of a minimal peptide library based on the active helical domain of each bacteriocin sequence. 95 synthetic peptide variants of each scaffold peptide, designated Syn-enterocin, Syn-sordellicin, Syn-larvacin, and Syn-xiamensin, were designed and synthesized from each scaffold sequence based on defined biophysical parameters. A total of 384 total peptides were assessed for antibacterial activity against Gram-negative and Gram-positive bacteria. Minimal Inhibitory Concentrations (MICs) as low as 15.6 nM could be observed for the most potent peptide candidate tested, with no significant cytotoxicity to eukaryotic cells. Our work demonstrates for the first time a general workflow of using minimal domains of natural bacteriocin sequences as scaffolds to design and rapidly synthesize a library of bacteriocin-based antimicrobial peptide variants for evaluation.
RESUMEN
The ribosomally produced antimicrobial peptides of bacteria (bacteriocins) represent an unexplored source of membrane-active antibiotics. We designed a library of linear peptides from a circular bacteriocin and show that pore-formation dynamics in bacterial membranes are tunable via selective amino acid substitution. We observed antibacterial interpeptide synergy indicating that fundamentally altering interactions with the membrane enables synergy. Our findings suggest an approach for engineering pore-formation through rational peptide design and increasing the utility of novel antimicrobial peptides by exploiting synergy.
RESUMEN
Bacteriocins, the ribosomally produced antimicrobial peptides of bacteria, represent an untapped source of promising antibiotic alternatives. However, bacteriocins display diverse mechanisms of action, a narrow spectrum of activity, and inherent challenges in natural product isolation making in vitro verification of putative bacteriocins difficult. A subset of bacteriocins exert their antimicrobial effects through favorable biophysical interactions with the bacterial membrane mediated by the charge, hydrophobicity, and conformation of the peptide. We have developed a pipeline for bacteriocin-derived compound design and testing that combines sequence-free prediction of bacteriocins using machine learning and a simple biophysical trait filter to generate 20 amino acid peptides that can be synthesized and evaluated for activity. We generated 28,895 total 20-mer candidate peptides and scored them for charge, α-helicity, and hydrophobic moment. Of those, we selected 16 sequences for synthesis and evaluated their antimicrobial, cytotoxicity, and hemolytic activities. Peptides with the overall highest scores for our biophysical parameters exhibited significant antimicrobial activity against Escherichia coli and Pseudomonas aeruginosa. Our combined method incorporates machine learning and biophysical-based minimal region determination to create an original approach to swiftly discover bacteriocin candidates amenable to rapid synthesis and evaluation for therapeutic use.
Asunto(s)
Antibacterianos/síntesis química , Péptidos Catiónicos Antimicrobianos/síntesis química , Bacteriocinas/química , Biología Computacional/métodos , Antibacterianos/química , Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/farmacología , Diseño de Fármacos , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Interacciones Hidrofóbicas e Hidrofílicas , Aprendizaje Automático , Pruebas de Sensibilidad Microbiana , Dominios Proteicos , Estructura Secundaria de Proteína , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/crecimiento & desarrollo , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/crecimiento & desarrollo , Relación Estructura-ActividadRESUMEN
The bacterial pathogen Group A Streptococcus (GAS) has been shown to induce a variety of human diseases ranging in severity from pharyngitis to toxic shock syndrome and necrotizing fasciitis. GAS produces a powerful peptide toxin known as Streptolysin S (SLS). Though long recognized as a potent cytolysin, recent evidence from our lab has shown that SLS-dependent cytotoxicity is mediated through activation of the pro-inflammatory mediators p38 MAPK and NFκB. These findings led us to hypothesize that activation of p38 MAPK and NFκB signaling drive the production of pro-inflammatory cytokines which, in turn, serve as positive feedback signals to initiate cytotoxicity in infected host cells. To address this hypothesis, we utilized a cytokine array to characterize the SLS-dependent pro-inflammatory cytokine response to GAS infection in human keratinocytes. From these studies, IL-1ß was found to be markedly upregulated in the presence of SLS, and further investigation revealed that this cytokine contributes to cytotoxicity in human keratinocytes during infection. Subcutaneous infection studies were performed in mice to address the physiological impact of increased IL-1ß production. These studies demonstrated that IL-1ß is produced during GAS skin infection in an SLS-dependent manner. Furthermore, inhibition of this cytokine and the upstream kinases and other signaling mediators that drive its production reduced SLS-mediated lesion formation early in the infection process. Together, our findings indicate that pharmacological inhibition of this inflammatory axis holds promise as a therapeutic strategy to reduce tissue destruction during severe invasive Group A Streptococcal infections.
Asunto(s)
Proteínas Bacterianas/metabolismo , Inflamación/patología , Interleucina-1beta/antagonistas & inhibidores , Enfermedades Cutáneas Bacterianas/patología , Piel/patología , Infecciones Estreptocócicas/patología , Streptococcus pyogenes/crecimiento & desarrollo , Estreptolisinas/metabolismo , Línea Celular , Humanos , Factores Inmunológicos/metabolismo , Queratinocitos/efectos de los fármacos , Queratinocitos/patología , Transducción de SeñalRESUMEN
BACKGROUND & AIMS: A 6-food elimination diet induces remission in most children and adults with eosinophilic esophagitis (EoE). The effectiveness of empiric elimination of only 4 foods has not been studied in children. We performed a prospective observational outcome study in children with EoE treated with dietary exclusion of cow's milk, wheat, egg, and soy. The objective was to assess the clinical, endoscopic, and histologic efficacy of this treatment in EoE. METHODS: We recruited children (1-18 years old, diagnosed per consensus guidelines) from 4 medical centers. Study participants (n = 78) were given a proton pump inhibitor twice daily and underwent a baseline esophagogastroduodenoscopy. Subjects were instructed on dietary exclusion of cow's milk, wheat, egg, and soy. Clinical, endoscopic, and histologic assessments were made after 8 weeks. Responders had single foods reintroduced for 8 weeks, with repeat endoscopy to assess for recurrence of active disease. The primary endpoint was histologic remission (fewer than 15 eosinophils per high-powered field). Secondary endpoints included symptom and endoscopic improvements and identification of foods associated with active histologic disease. RESULTS: After 8 weeks on 4-food elimination diet, 50 subjects were in histologic remission (64%). The subjects' mean baseline clinical symptoms score was 4.5, which decreased to 2.3 after 8 weeks of 4-food elimination diet (P < .001). The mean endoscopic baseline score was 2.1, which decreased to 1.3 (P < .001). After food reintroduction, the most common food triggers that induced histologic inflammation were cow's milk (85%), egg (35%), wheat (33%), and soy (19%). One food trigger that induced recurrence of esophageal inflammation was identified in 62% of patients and cow's milk-induced EoE was present in 88% of these patients. CONCLUSIONS: In a prospective study of children with EoE, 8 weeks of 4-food elimination diet induced clinical, endoscopic, and histologic remission in more than 60% of children with EoE. Although less restrictive than 6-food elimination diet, 4-food elimination diet was nearly as effective, and can be recommended as a treatment for children with EoE.