RESUMO
Diverse computational approaches have been widely used to assist in designing antimicrobial peptides with enhanced activities. This tactic has also been used to address the need for new treatment alternatives to combat resistant bacterial infections. Herein, we have designed eight variants from a natural peptide, pro-adrenomedullin N-terminal 20 peptide (PAMP), using an in silico pattern insertion approach, the Joker algorithm. All the variants show an α-helical conformation, but with differences in the helix percentages according to circular dichroism (CD) results. We found that the C-terminal portion of PAMP may be relevant for its antimicrobial activities, as revealed by the molecular dynamics, CD, and antibacterial results. The analogs showed variable antibacterial potential, but most were not cytotoxic. Nevertheless, PAMP2 exhibited the most potent activities against human and animal-isolated bacteria, showing cytotoxicity only at a substantially higher concentration than its minimal inhibitory concentration (MIC). Our results suggest that the enhanced activity in the profile of PAMP2 may be related to their particular physicochemical properties, along with the adoption of an amphipathic α-helical arrangement with the conserved C-terminus portion. Finally, the peptides designed in this study can constitute scaffolds for the design of improved sequences.
Assuntos
Adrenomedulina , Dicroísmo Circular , Testes de Sensibilidade Microbiana , Simulação de Dinâmica Molecular , Humanos , Adrenomedulina/química , Adrenomedulina/farmacologia , Sequência de Aminoácidos , Antibacterianos/farmacologia , Antibacterianos/química , Antibacterianos/síntese química , Animais , Simulação por Computador , Precursores de Proteínas/química , Precursores de Proteínas/farmacologia , Precursores de Proteínas/metabolismo , Peptídeos Antimicrobianos/química , Peptídeos Antimicrobianos/farmacologia , Estrutura Secundária de ProteínaRESUMO
BACKGROUND: The synthetic antimicrobial peptide, PaDBS1R1, has been reported as a powerful anti-Klebsiella pneumoniae antimicrobial. However, there is only scarce knowledge about whether K. pneumoniae could develop resistance against PaDBS1R1 and which resistance mechanisms could be involved. OBJECTIVES: Identify via label-free shotgun proteomics the K. pneumoniae resistance mechanisms developed against PaDBS1R1. METHODS: An adaptive laboratory evolution experiment was performed to obtain a PaDBS1R1-resistant K. pneumoniae lineage. Antimicrobial susceptibility was determined through microdilution assay. Modifications in protein abundances between the resistant and sensitive lineages were measured via label-free quantitative shotgun proteomics. Enriched Gene Ontology terms and KEGG pathways were identified through over-representation analysis. Data are available via ProteomeXchange with identifier PXD033020. RESULTS: K. pneumoniae ATCC 13883 parental strain challenged with increased subinhibitory PaDBS1R1 concentrations allowed the PaDBS1R1-resistant K. pneumoniae lineage to emerge. Proteome comparisons between PaDBS1R1-resistant K. pneumoniae and PaDBS1R1-sensitive K. pneumoniae under PaDBS1R1-induced stress conditions enabled the identification and quantification of 1702 proteins, out of which 201 were differentially abundant proteins (DAPs). The profiled DAPs comprised 103 up-regulated proteins (adjusted P value < 0.05, fold change ≥ 2) and 98 down-regulated proteins (adjusted P value < 0.05, fold change ≤ 0.5). The enrichment analysis suggests that PhoPQ-guided LPS modifications and CpxRA-dependent folding machinery could be relevant resistance mechanisms against PaDBS1R1. CONCLUSIONS: Based on experimental evolution and a label-free quantitative shotgun proteomic approach, we showed that K. pneumoniae developed resistance against PaDBS1R1, whereas PhoPQ-guided LPS modifications and CpxRA-dependent folding machinery appear to be relevant resistance mechanisms against PaDBS1R1.
Assuntos
Anti-Infecciosos , Infecções por Klebsiella , Humanos , Antibacterianos/farmacologia , Klebsiella pneumoniae/genética , Peptídeos Antimicrobianos , Proteômica , Lipopolissacarídeos , Anti-Infecciosos/farmacologia , Testes de Sensibilidade MicrobianaRESUMO
Klebsiella pneumoniae has been implicated in wide-ranging nosocomial outbreaks, causing severe infections without effective treatments due to antibiotic resistance. Here, we performed genome sequencing of 70 extensively drug resistant clinical isolates, collected from Brasília's hospitals (Brazil) between 2010 and 2014. The majority of strains (60 out of 70) belonged to a single clonal complex (CC), CC258, which has become distributed worldwide in the last two decades. Of these CC258 strains, 44 strains were classified as sequence type 11 (ST11) and fell into two distinct clades, but no ST258 strains were found. These 70 strains had a pan-genome size of 10â366 genes, with a core-genome size of ~4476 genes found in 95â% of isolates. Analysis of sequences revealed diverse mechanisms of resistance, including production of multidrug efflux pumps, enzymes with the same target function but with reduced or no affinity to the drug, and proteins that protected the drug target or inactivated the drug. ß-Lactamase production provided the most notable mechanism associated with K. pneumoniae. Each strain presented two or three different ß-lactamase enzymes, including class A (SHV, CTX-M and KPC), class B and class C AmpC enzymes, although no class D ß-lactamase was identified. Strains carrying the NDM enzyme involved three different ST types, suggesting that there was no common genetic origin.
Assuntos
Farmacorresistência Bacteriana Múltipla/genética , Genômica , Klebsiella pneumoniae/genética , Fatores de Virulência/genética , Brasil , DNA Bacteriano/genética , Humanos , Infecções por Klebsiella/epidemiologia , Klebsiella pneumoniae/isolamento & purificação , Filogenia , Virulência/genética , beta-Lactamases/genéticaRESUMO
BACKGROUND: Amylin is a 37-amino-acid peptide hormone co-secreted with insulin, which participates in glucose homeostasis. This hormone is able to aggregate in a ß-sheet conformation and deposit in islet amyloids, a hallmark in type II diabetes. Since amylin is a gene-encoded hormone, this peptide has variants caused by point mutations that can impact its functions. METHODS: Here, we analyzed the structural effects caused by S20G and G33R point mutations which, according to the 1000 Genomes Project, have frequency in East Asian and European populations, respectively. The analyses were performed by means of aggrescan server, SNP functional effect predictors, and molecular dynamics. RESULTS: We found that both mutations have aggregation potential and cause changes in the monomeric forms when compared with wild-type amylin. Furthermore, comparative analyses with pramlintide, an amylin drug analogue, allowed us to infer that second α-helix maintenance may be related to the aggregation potential. CONCLUSIONS: The S20G mutation has been described as pathologically related, which is in agreement with our findings. In addition, our data suggest that the G33R mutation might have a deleterious effect. The data presented here also provide new therapy opportunities, whether for creating more effective drugs for diabetes or implementing specific treatment for patients with these mutations. GENERAL SIGNIFICANCE: Our data could help to better understand the impact of mutations on the wild-type amylin sequence, as a starting point for the evaluation and characterization of other variations. Moreover, these findings could improve the health of patients with type II diabetes.
Assuntos
Diabetes Mellitus Tipo 2/genética , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/genética , Simulação de Dinâmica Molecular , Mutação Puntual , Diabetes Mellitus Tipo 2/patologia , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismoRESUMO
BACKGROUND: Physical exercise is an essential factor in preventing and treating metabolic diseases by promoting systemic benefits throughout the body. The molecular factors involved in this process are poorly understood. Micro RNAs (miRNAs) are small non-coding RNAs that inhibit mRNA transcription. MiRNAs, which can participate in the benefits of exercise to health, circulate in plasma in extracellular particles (EP). Horses that undergo endurance racing are an excellent model to study the impact of long-duration/low intensity exercise in plasma EP miRNAs. OBJECTIVES: To evaluate the effects of 160 km endurance racing on horse plasma extracellular particles and their miRNA population. STUDY DESIGN: Cohort study. METHODS: We collected plasma from five Arabian horses during five time-points of an endurance ride. Extracellular particles were purified from plasma and characterised by electron microscopy, resistive pulse sensing (qNano) and western blotting. Small RNAs were purified from horse plasma EP, and sequencing was performed. RESULTS: Endurance racing increased EP concentration and average diameter compared to before the race. Western blotting showed a high concentration of extracellular vesicles proteins 2 hours after the race, which returned to baseline 15 hours after the race. MicroRNA differential expression analysis revealed increasing levels of eca-miR-486-5p during and after the race, and decreasing levels of eca-miR-9083 after the end. CONCLUSIONS: This study adds new data about the variation in plasma EP concentrations after long-distance exercise and brings new insights about the roles of exercise-derived EP miRNAs during low-intensity endurance exercise.
Assuntos
MicroRNAs , Condicionamento Físico Animal , Animais , Estudos de Coortes , Cavalos , MicroRNAs/genética , Resistência Física , PlasmaRESUMO
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
BACKGROUND: Bacterial infections represent a major worldwide health problem the antimicrobial peptides (AMPs) have been considered as potential alternative agents for treating these infections. Here we demonstrated the antimicrobial activity of EcDBS1R6, a peptide derived from a signal peptide sequence of Escherichia coli that we previously turned into an AMP by making changes through the Joker algorithm. METHODS: Antimicrobial activity was measured by broth microdilution method. Membrane integrity was measured using fluorescent probes and through scanning electron microscopy imaging. A sliding window of truncated peptides was used to determine the EcDBS1R6 active core. Molecular dynamics in TFE/water environment was used to assess the EcDBS1R6 structure. RESULTS: Signal peptides are known to naturally interact with membranes; however, the modifications introduced by Joker transformed this peptide into a membrane-active agent capable of killing bacteria. The C-terminus was unable to fold into an α-helix whereas its fragments showed poor or no antimicrobial activity, suggesting that the EcDBS1R6 antibacterial core was located at the helical N-terminus, corresponding to the signal peptide portion of the parent peptide. CONCLUSION: The strategy of transforming signal peptides into AMPs appears to be promising and could be used to produce novel antimicrobial agents. GENERAL SIGNIFICANCE: The process of transforming an inactive signal peptide into an antimicrobial peptide could open a new venue for creating new AMPs derived from signal peptides.
Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Escherichia coli/química , Sinais Direcionadores de Proteínas , Sequência de Aminoácidos , Bactérias Gram-Negativas/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Simulação de Dinâmica Molecular , Conformação ProteicaRESUMO
Diverse peptides have been evaluated for their activity against pathogenic microorganisms. Here, five mastoparan variants were designed based on mastoparan-L, among which two (R1 and R4) were selected for in-depth analysis. Mastoparan-L (parent/control), R1, and R4 inhibited susceptible/resistant bacteria at concentrations ranging from 2 to 32 µM, whereas only R1 and R4 eradicated Pseudomonas aeruginosa biofilms at 16 µM. Moreover, the toxic effects of mastoparan-L toward mammalian cells were drastically reduced in both variants. In skin infections, R1 at 64 µM was the most effective variant, reducing P. aeruginosa bacterial counts 1000 times on day 4 post-infection. Structurally, all of the peptides showed varying levels of helicity and structural stability in aqueous and membrane-like conditions, which may affect the different bioactivities observed here. By computationally modifying the physicochemical properties of R1 and R4, we reduced the cytotoxicity and optimized the therapeutic potential of these mastoparan-like peptides both in vitro and in vivo.
Assuntos
Antibacterianos/farmacologia , Desenho Assistido por Computador , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Venenos de Vespas/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , Relação Dose-Resposta a Droga , Peptídeos e Proteínas de Sinalização Intercelular/síntese química , Peptídeos e Proteínas de Sinalização Intercelular/química , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , Venenos de Vespas/síntese química , Venenos de Vespas/químicaRESUMO
Bacterial biofilms and associated infections represent one of the biggest challenges in the clinic, and as an alternative to counter bacterial infections, antimicrobial peptides have attracted great attention in the past decade. Here, ten short cationic antimicrobial peptides were generated through a sliding-window strategy on the basis of the 19-amino acid residue peptide, derived from a Pyrobaculum aerophilum ribosomal protein. PaDBS1R6F10 exhibited anti-infective potential as it decreased the bacterial burden in murine Pseudomonas aeruginosa cutaneous infections by more than 1000-fold. Adverse cytotoxic and hemolytic effects were not detected against mammalian cells. The peptide demonstrated structural plasticity in terms of its secondary structure in the different environments tested. PaDBS1R6F10 represents a promising antimicrobial agent against bacteria infections, without harming human cells.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/efeitos dos fármacos , Pyrobaculum/metabolismo , Proteínas Ribossômicas/química , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/química , Proteínas Arqueais/química , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Modelos Animais de Doenças , Humanos , Camundongos , Testes de Sensibilidade Microbiana , Estrutura Secundária de Proteína , Pseudomonas aeruginosa/fisiologiaRESUMO
Antimicrobial peptides (AMPs) are small molecules present in all living beings. Despite their huge sequence variability, AMPs present great structural conservation, mainly in cysteine-stabilized families. Moreover, in non-model plants, it is possible to detect cysteine-stabilized AMPs (cs-AMPs) with different sequences not covered by conventional searches. Here, we described a threading application for cs-AMP identification in the non-model arum lily (Zantedeschia aethiopica) plant, exploring the spathe transcriptome. By using the predicted proteins from the Z. aethiopica transcriptome as our primary source of sequences, we have filtered by using structural alignments of 12 putative cs-AMP sequences. The two unreported sequences were submitted to PCR validation, and ZaLTP7 gene was confirmed. By using the structure alignments, we classified ZaLTP7 as an LTP type 2-like. The successful threading application for cs-AMP identification is an important advance in transcriptomic and proteomic data mining. Besides, the same approach could be applied to the use of NGS public data to discover molecules to combat multidrug-resistant bacteria.