RESUMEN
Pneumolysin (PLY) is a cholesterol-dependent cytolysin (CDC) from Streptococcus pneumoniae, the main cause for bacterial pneumonia. Liberation of PLY during infection leads to compromised immune system and cytolytic cell death. Here, we report discovery, development, and validation of targeted small molecule inhibitors of PLY (pore-blockers, PB). PB-1 is a virtual screening hit inhibiting PLY-mediated hemolysis. Structural optimization provides PB-2 with improved efficacy. Cryo-electron tomography reveals that PB-2 blocks PLY-binding to cholesterol-containing membranes and subsequent pore formation. Scaffold-hopping delivers PB-3 with superior chemical stability and solubility. PB-3, formed in a protein-templated reaction, binds to Cys428 adjacent to the cholesterol recognition domain of PLY with a KD of 256 nM and a residence time of 2000 s. It acts as anti-virulence factor preventing human lung epithelial cells from PLY-mediated cytolysis and cell death during infection with Streptococcus pneumoniae and is active against the homologous Cys-containing CDC perfringolysin (PFO) as well.
Asunto(s)
Proteínas Bacterianas , Toxinas Bacterianas , Proteínas Hemolisinas , Hemólisis , Streptococcus pneumoniae , Estreptolisinas , Estreptolisinas/metabolismo , Estreptolisinas/química , Humanos , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/antagonistas & inhibidores , Streptococcus pneumoniae/efectos de los fármacos , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/antagonistas & inhibidores , Hemólisis/efectos de los fármacos , Proteínas Hemolisinas/metabolismo , Proteínas Hemolisinas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Bibliotecas de Moléculas Pequeñas/química , Células A549 , Colesterol/metabolismo , Microscopía por Crioelectrón , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Factores de Virulencia/metabolismoRESUMEN
In Southeast Asia, the penaeid shrimp aquaculture production faces a new pandemic bacterial disease called acute hepatopancreatic necrosis disease (AHPND). The highly profitable pacific white shrimp, Litopenaeus vannamei, has become a challenging species due to severe lethal infections. Recent research has identified a critical pathogen, Vibrio parahaemolyticus, which caused significant loss in the shrimp industry. The disease pathway involves a virulence plasmid encoding binary protein toxins (PirA/B) that cause cell death. The protein toxins were inherited and conjugatively transferred to other Vibrio species through a post-segregational killing system. In this study, "in silico" (Glide, 2021) analysis identified four phytocompounds as myricetin (Myr), ( +)-taxifolin (TF), (-)-epigallocatechin gallate (EGCG), and strychnine (STN) which could be most effective against both the toxins concerning its docking score and affinity. The interactions of complexes and the critical amino acids involved in docking were analyzed using the Discovery Studio (version 2016). Molecular dynamic studies showed lower root mean square deviations (RMSD) and improved stabilization of ( +)-taxifolin (TF) and (-)-epigallocatechin-3-gallate (EGCG) against both the protein toxins. The antibacterial potential of all four selected compounds had tested against pathogenic strains of V. parahaemolyticus through minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The best MBC results were observed at concentrations of 1 mg/mL for EGCG and 1.25 mg/mL for TF. Moreover, the complete reduction of viable cell counts in the in vitro bactericidal activity had recorded after 24 h of incubation.
Asunto(s)
Artemia , Toxinas Bacterianas , Fitoquímicos , Vibrio parahaemolyticus , Animales , Acuicultura , Artemia/microbiología , Toxinas Bacterianas/antagonistas & inhibidores , Toxinas Bacterianas/química , Catequina/química , Hepatopáncreas , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Quercetina/química , Vibrio parahaemolyticus/fisiología , Fitoquímicos/química , Fitoquímicos/farmacologíaRESUMEN
Clostridium difficile infection is mediated by two major exotoxins: toxins A (TcdA) and B (TcdB). Inhibiting the biocatalytic activities of these toxins with targeted peptide-based drugs can reduce the risk of C. difficile infection. In this work, we used a computational strategy that integrates a peptide binding design (PepBD) algorithm and explicit-solvent atomistic molecular dynamics simulation to determine promising toxin A-targeting peptides that can recognize and bind to the catalytic site of the TcdA glucosyltransferase domain (GTD). Our simulation results revealed that two out of three in silico discovered peptides, viz. the neutralizing peptides A (NPA) and B (NPB), exhibit lower binding free energies when bound to the TcdA GTD than the phage-display discovered peptide, viz. the reference peptide (RP). These peptides may serve as potential inhibitors against C. difficile infection. The efficacy of the peptides RP, NPA, and NPB to neutralize the cytopathic effects of TcdA was tested in vitro in human jejunum cells. Both phage-display peptide RP and in silico peptide NPA were found to exhibit strong toxin-neutralizing properties, thereby preventing the TcdA toxicity. However, the in silico peptide NPB demonstrates a relatively low efficacy against TcdA.
Asunto(s)
Toxinas Bacterianas/antagonistas & inhibidores , Simulación por Computador , Enterotoxinas/antagonistas & inhibidores , Péptidos/farmacología , Toxinas Bacterianas/química , Diseño de Fármacos , Enterotoxinas/química , Modelos Moleculares , Péptidos/química , Unión Proteica , Conformación Proteica , Dominios Proteicos , Reproducibilidad de los ResultadosRESUMEN
A series of acyclic nucleoside phosphonates (ANPs) was designed as inhibitors of bacterial adenylate cyclases (ACs), where adenine was replaced with 2-amino-4-arylthiazoles. The target compounds were prepared using the halogen dance reaction. Final AC inhibitors were evaluated in cell-based assays (prodrugs) and cell-free assays (phosphono diphosphates). Novel ANPs were potent inhibitors of adenylate cyclase toxin (ACT) from Bordetella pertussis and edema factor (EF) from Bacillus anthracis, with substantial selectivity over mammalian enzymes AC1, AC2, and AC5. Six of the new ANPs were more potent or equipotent ACT inhibitors (IC50 =9-18â nM), and one of them was more potent EF inhibitor (IC50 =12â nM), compared to adefovir diphosphate (PMEApp) with IC50 =18â nM for ACT and IC50 =36â nM for EF. Thus, these compounds represent the most potent ACT/EF inhibitors based on ANPs reported to date. The potency of the phosphonodiamidates to inhibit ACT activity in J774A.1 macrophage cells was somewhat weaker, where the most potent derivative had IC50 =490â nM compared to IC50 =150â nM of the analogous adefovir phosphonodiamidate. The results suggest that more efficient type of phosphonate prodrugs would be desirable to increase concentrations of the ANP-based active species in the cells in order to proceed with the development of ANPs as potential antitoxin therapeutics.
Asunto(s)
Toxina de Adenilato Ciclasa/antagonistas & inhibidores , Inhibidores de Adenilato Ciclasa/farmacología , Toxinas Bacterianas/antagonistas & inhibidores , Halógenos/farmacología , Organofosfonatos/farmacología , Tiazoles/farmacología , Toxina de Adenilato Ciclasa/metabolismo , Inhibidores de Adenilato Ciclasa/síntesis química , Inhibidores de Adenilato Ciclasa/química , Antígenos Bacterianos/metabolismo , Bacillus anthracis/química , Toxinas Bacterianas/metabolismo , Bordetella pertussis/enzimología , Relación Dosis-Respuesta a Droga , Halógenos/química , Estructura Molecular , Organofosfonatos/química , Relación Estructura-Actividad , Tiazoles/síntesis química , Tiazoles/químicaRESUMEN
Clostridium difficile causes life-threatening diarrhea and is the leading cause of healthcare-associated bacterial infections in the United States. TcdA and TcdB bacterial toxins are primary determinants of disease pathogenesis and are attractive therapeutic targets. TcdA and TcdB contain domains that use UDP-glucose to glucosylate and inactivate host Rho GTPases, resulting in cytoskeletal changes causing cell rounding and loss of intestinal integrity. Transition state analysis revealed glucocationic character for the TcdA and TcdB transition states. We identified transition state analogue inhibitors and characterized them by kinetic, thermodynamic and structural analysis. Iminosugars, isofagomine and noeuromycin mimic the transition state and inhibit both TcdA and TcdB by forming ternary complexes with Tcd and UDP, a product of the TcdA- and TcdB-catalyzed reactions. Both iminosugars prevent TcdA- and TcdB-induced cytotoxicity in cultured mammalian cells by preventing glucosylation of Rho GTPases. Iminosugar transition state analogues of the Tcd toxins show potential as therapeutics for C. difficile pathology.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Toxinas Bacterianas/antagonistas & inhibidores , Clostridioides difficile/efectos de los fármacos , Clostridioides difficile/enzimología , Infecciones por Clostridium/microbiología , Enterotoxinas/antagonistas & inhibidores , Antibacterianos/química , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Clostridioides difficile/química , Clostridioides difficile/genética , Enterotoxinas/química , Enterotoxinas/metabolismo , Humanos , CinéticaRESUMEN
Serious infection caused by multi-drug-resistant bacteria is a major threat to human health. Bacteria can invade the host tissue and produce various toxins to damage or kill host cells, which may induce life-threatening sepsis. Here, we aimed to explore whether fructose-coated Ångstrom-scale silver particles (F-AgÅPs), which were prepared by our self-developed evaporation-condensation system and optimized coating approach, could kill bacteria and sequester bacterial toxins to attenuate fatal bacterial infections. Methods: A series of in vitro assays were conducted to test the anti-bacterial efficacy of F-AgÅPs, and to investigate whether F-AgÅPs could protect against multi-drug resistant Staphylococcus aureus (S. aureus)- and Escherichia coli (E. coli)-induced cell death, and suppress their toxins (S. aureus hemolysin and E. coli lipopolysaccharide)-induced cell injury or inflammation. The mouse models of cecal ligation and puncture (CLP)- or E. coli bloodstream infection-induced lethal sepsis were established to assess whether the intravenous administration of F-AgÅPs could decrease bacterial burden, inhibit inflammation, and improve the survival rates of mice. The levels of silver in urine and feces of mice were examined to evaluate the excretion of F-AgÅPs. Results: F-AgÅPs efficiently killed various bacteria that can cause lethal infections and also competed with host cells to bind with S. aureus α-hemolysin, thus blocking its cytotoxic activity. F-AgÅPs inhibited E. coli lipopolysaccharide-induced endothelial injury and macrophage inflammation, but not by directly binding to lipopolysaccharide. F-AgÅPs potently reduced bacterial burden, reversed dysregulated inflammation, and enhanced survival in mice with CLP- or E. coli bloodstream infection-induced sepsis, either alone or combined with antibiotic therapy. After three times injections within 48 h, 79.18% of F-AgÅPs were excreted via feces at the end of the 14-day observation period. Conclusion: This study suggests the prospect of F-AgÅPs as a promising intravenous agent for treating severe bacterial infections.
Asunto(s)
Toxinas Bacterianas/antagonistas & inhibidores , Sepsis/tratamiento farmacológico , Plata/farmacología , Animales , Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Modelos Animales de Enfermedad , Escherichia coli/efectos de los fármacos , Fructosa/farmacología , Proteínas Hemolisinas/antagonistas & inhibidores , Inflamación/tratamiento farmacológico , Lipopolisacáridos/antagonistas & inhibidores , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Ratones , Nanopartículas/uso terapéutico , Sepsis/microbiología , Infecciones Estafilocócicas/tratamiento farmacológico , Staphylococcus aureus/efectos de los fármacosRESUMEN
Toxin-antitoxin modules are widespread in prokaryotes, and the capacity of toxin accumulation to increase the tolerances of bacteria to antibiotics has been well documented. The conventional model for this functionality implies that an overabundance of toxin arrests bacterial growth, which inhibits processes targeted by antibiotics and thereby limits their corruption and the lethal damage that would ensue. Implicit in this model is that toxins exert their influence on antibiotic lethality before and/or during treatment, even though they are also present and functional after treatment concludes. Given recent evidence establishing that the period following antibiotic treatment (recovery) is important for the survival of nongrowing bacterial populations treated with fluoroquinolones (FQs), we assayed to what extent toxins influence bacterial survival during the recovery period. With both LdrD and MazF, toxins of type I and II systems, respectively, controlling accumulation to occur only after FQ treatment of nongrowing cultures resulted in significant increases in persisters. Further genetic investigation revealed important roles for homologous recombination and nucleotide excision repair machinery. Focusing on the wild type, we did not observe any SOS-induced toxin functioning in this manner; however, an analogous phenomenon was observed for wild-type Escherichia coli as well as uropathogenic E. coli (UPEC) when transcription or translation was inhibited during the post-FQ recovery period. Collectively, these data reveal the capacity of toxins to thwart FQ killing even after the treatment has concluded and show that FQ treatment of nongrowing bacteria can be rendered largely ineffective if bacteria cannot readily resume translation and growth at the conclusion of treatment. IMPORTANCE Overabundances of toxins have been shown to increase the antibiotic tolerances of bacteria. Largely, these effects have been attributed to the abilities of toxins to inhibit bacterial growth before and during antibiotic exposure. In this study, we assessed to what extent toxins can influence bacterial survival following antibiotic treatment, rather than before or during. Using two mechanistically distinct toxins, we show that their accumulations after antibiotic exposure have the capacity to increase the abundances of fluoroquinolone persisters from nongrowing populations. Further, we show with wild-type and uropathogenic E. coli that chemical inhibition of growth, not just that induced by toxins, produces analogous results. These observations reveal another dimension of how toxins influence antibiotic tolerance and highlight the importance of postantibiotic physiology on bacterial survival.
Asunto(s)
Antibacterianos/farmacología , Toxinas Bacterianas/antagonistas & inhibidores , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Fluoroquinolonas/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , Sistemas Toxina-Antitoxina/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Toxinas Bacterianas/genética , Escherichia coli Uropatógena/efectos de los fármacos , Escherichia coli Uropatógena/genéticaRESUMEN
Soft rot disease of edible mushrooms leads to rapid degeneration of fungal tissue and thus severely affects farming productivity worldwide. The bacterial mushroom pathogen Burkholderia gladioli pv. agaricicola has been identified as the cause. Yet, little is known about the molecular basis of the infection, the spatial distribution and the biological role of antifungal agents and toxins involved in this infectious disease. We combine genome mining, metabolic profiling, MALDI-Imaging and UV Raman spectroscopy, to detect, identify and visualize a complex of chemical mediators and toxins produced by the pathogen during the infection process, including toxoflavin, caryoynencin, and sinapigladioside. Furthermore, targeted gene knockouts and inâ vitro assays link antifungal agents to prevalent symptoms of soft rot, mushroom browning, and impaired mycelium growth. Comparisons of related pathogenic, mutualistic and environmental Burkholderia spp. indicate that the arsenal of antifungal agents may have paved the way for ancestral bacteria to colonize niches where frequent, antagonistic interactions with fungi occur. Our findings not only demonstrate the power of label-free, inâ vivo detection of polyyne virulence factors by Raman imaging, but may also inspire new approaches to disease control.
Asunto(s)
Agaricales/efectos de los fármacos , Toxinas Bacterianas/análisis , Imagen Molecular , Enfermedades de las Plantas/inducido químicamente , Agaricales/metabolismo , Antifúngicos/farmacología , Toxinas Bacterianas/antagonistas & inhibidores , Toxinas Bacterianas/metabolismo , Burkholderia gladioli/efectos de los fármacos , Burkholderia gladioli/metabolismo , Burkholderia gladioli/patogenicidad , Pruebas de Sensibilidad MicrobianaRESUMEN
α-Hemolysin (Hla) is an extracellular protein secreted by methicillin-resistant Staphylococcus aureus (MRSA) strains that plays a critical role in the pathogenesis of pulmonary, intraperitoneal, intramammary, and corneal infections, rendering Hla a potential therapeutic target. In this study, 10 unreported polycyclic polyprenylated acylphloroglucinol (PPAP) derivatives, garciyunnanins C-L (1-10), with diverse skeletons, were isolated from Garcinia yunnanensis Hu. The structures of these new compounds were determined by HRMS, NMR, electronic circular dichroism (ECD) calculations, single-crystal X-ray diffraction, and biomimetic transformation. Garciyunnanins C and D (1 and 2) were found to be potent Hla inhibitors in the anti-virulence efficacy evaluation against MRSA strain.
Asunto(s)
Antibacterianos/farmacología , Toxinas Bacterianas/antagonistas & inhibidores , Garcinia/química , Proteínas Hemolisinas/antagonistas & inhibidores , Floroglucinol/farmacología , Staphylococcus aureus/efectos de los fármacos , Antibacterianos/química , Antibacterianos/aislamiento & purificación , Toxinas Bacterianas/biosíntesis , Relación Dosis-Respuesta a Droga , Proteínas Hemolisinas/biosíntesis , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Floroglucinol/química , Floroglucinol/aislamiento & purificación , Staphylococcus aureus/metabolismo , Relación Estructura-ActividadRESUMEN
The clinically highly relevant Clostridioides (C.) difficile releases several AB-type toxins that cause diseases such as diarrhea and pseudomembranous colitis. In addition to the main virulence factors Rho/Ras-glycosylating toxins TcdA and TcdB, hypervirulent strains produce the binary AB-type toxin CDT. CDT consists of two separate proteins. The binding/translocation B-component CDTb facilitates uptake and translocation of the enzyme A-component CDTa to the cytosol of cells. Here, CDTa ADP-ribosylates G-actin, resulting in depolymerization of the actin cytoskeleton. We previously showed that CDTb exhibits cytotoxicity in the absence of CDTa, which is most likely due to pore formation in the cytoplasmic membrane. Here, we further investigated this cytotoxic effect and showed that CDTb impairs CaCo-2 cell viability and leads to redistribution of F-actin without affecting tubulin structures. CDTb was detected at the cytoplasmic membrane in addition to its endosomal localization if CDTb was applied alone. Chloroquine and several of its derivatives, which were previously identified as toxin pore blockers, inhibited intoxication of Vero, HCT116, and CaCo-2 cells by CDTb and CDTb pores in vitro. These results further strengthen pore formation by CDTb in the cytoplasmic membrane as the underlying cytotoxic mechanism and identify pharmacological pore blockers as potent inhibitors of cytotoxicity induced by CDTb and CDTa plus CDTb.
Asunto(s)
Toxinas Bacterianas/antagonistas & inhibidores , Clostridioides difficile/patogenicidad , Actinas/metabolismo , Animales , Toxinas Bacterianas/farmacología , Células CACO-2 , Calcio/metabolismo , Supervivencia Celular/efectos de los fármacos , Chlorocebus aethiops , Cloroquina/farmacología , Humanos , Células VeroRESUMEN
Incidence of drug resistance in clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) is attributed to its diverse repertoire of virulence factors. Of these virulence determinants, Panton-Valentine Leukocidin (PVL) has been experimentally validated as a prospective drug target due to its conspicuous and comprehensive role in nosocomial infections. This study encompassed an in silico approach to elucidate the antimicrobial potentiality of human cathelicidin LL-37 against PVL toxin of MRSA. Molecular docking studies of LL-37 and its segments with the PVL toxin subunits LukS and LukF were carried out using PatchDock server and the results were refined using FireDock server. The paramount ligand-receptor combination was selected and analyzed based on diverse parametric attributes and compared with the commercial inhibitors of PVL viz. Andrimid, Beclobrate, Beta-sitosterol, Diathymosulfone, and Probucol to determine the most potent inhibitor among them. Our results elucidated that the interaction of LL-37 with the LukS subunit of PVL toxin (minimum global energy of -61.82 kcal/mol) depicted 34 molecular interactions, while the commercial PVL inhibitors depicted fewer and insubstantial interactions. SWISS-ADME (Absorption, Distribution, Metabolism, and Excretion) and ToxinPred analysis of LL-37 further corroborated its null potency of toxicity in systemic milieu. The results obtained may credit this study as basis for the development of LL-37 as a potential inhibitor against virulent MRSA toxins, thereby exalting the treatment regimes for nosocomial infections in health care facilities worldwide.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/farmacología , Toxinas Bacterianas/antagonistas & inhibidores , Exotoxinas/antagonistas & inhibidores , Leucocidinas/antagonistas & inhibidores , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Péptidos Catiónicos Antimicrobianos/farmacocinética , Humanos , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , CatelicidinasRESUMEN
Guanylate cyclase 2C (GC-C), encoded by the GUCY2C gene, is implicated in hereditary early onset chronic diarrhea. Several families with chronic diarrhea symptoms have been identified with autosomal dominant, gain-of-function mutations in GUCY2C. We have identified a Mennonite patient with a novel GUCY2C variant (c.2381A > T; p.Asp794Val) with chronic diarrhea and an extensive maternal family history of chronic diarrhea and bowel dilatation. Functional studies including co-segregation analysis showed that all family members who were heterozygous for this variant had GI-related symptoms. HEK-293 T cells expressing the Asp794Val GC-C variant showed increased cGMP production when stimulated with Escherichia coli heat-stable enterotoxin STp (HST), which was reversed when 5-(3-Bromophenyl)-5,11-dihydro-1,3-dimethyl-1H-indeno[2',1':5,6]pyrido[2,3-d]pyrimidine-2,4,6(3H)-trione (BPIPP; a GC-C inhibitor) was used. In addition, cystic fibrosis transmembrane conductance regulator (CFTR) activity measured with SPQ fluorescence assay was increased in these cells after treatment with HST, indicating a crucial role for CFTR activity in the pathogenesis of this disorder. These results support pathogenicity of the GC-C Asp794Val variant as a cause of chronic diarrhea in this family. Furthermore, this work identifies potential candidate drug, GC-C inhibitor BPIPP, to treat diarrhea caused by this syndrome.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Diarrea/genética , Predisposición Genética a la Enfermedad , Receptores de Enterotoxina/genética , Adolescente , Toxinas Bacterianas/antagonistas & inhibidores , Toxinas Bacterianas/genética , Niño , Diarrea/tratamiento farmacológico , Diarrea/patología , Enterotoxinas/antagonistas & inhibidores , Enterotoxinas/genética , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas de Escherichia coli/genética , Femenino , Mutación con Ganancia de Función/genética , Células HEK293 , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Humanos , Masculino , Linaje , Adulto JovenRESUMEN
Biofilms are rigid and largely impenetrable three-dimensional matrices constituting virulence determinants of various pathogenic bacteria. Here, we demonstrate that molecular tweezers, unique supramolecular artificial receptors, modulate biofilm formation of Staphylococcus aureus. In particular, the tweezers affect the structural and assembly properties of phenol-soluble modulin α1 (PSMα1), a biofilm-scaffolding functional amyloid peptide secreted by S. aureus. The data reveal that CLR01, a diphosphate tweezer, exhibits significant S. aureus biofilm inhibition and disrupts PSMα1 self-assembly and fibrillation, likely through inclusion of lysine side chains of the peptide. In comparison, different peptide binding occurs in the case of CLR05, a tweezer containing methylenecarboxylate units, which exhibits lower affinity for the lysine residues yet disrupts S. aureus biofilm more strongly than CLR01. Our study points to a possible role for molecular tweezers as potent biofilm inhibitors and antibacterial agents, particularly against untreatable biofilm-forming and PSM-producing bacteria, such as methicillin-resistant S. aureus.
Asunto(s)
Amiloide/antagonistas & inhibidores , Antibacterianos/farmacología , Toxinas Bacterianas/antagonistas & inhibidores , Biopelículas/efectos de los fármacos , Proteínas Hemolisinas/antagonistas & inhibidores , Staphylococcus aureus/efectos de los fármacos , Amiloide/metabolismo , Antibacterianos/química , Toxinas Bacterianas/metabolismo , Proteínas Hemolisinas/metabolismo , Pruebas de Sensibilidad Microbiana , Pinzas Ópticas , Staphylococcus aureus/metabolismoRESUMEN
Staphylococcus aureus (SA), especially the methicillin-resistant variant (MRSA), is becoming a serious threat to human health in hospitals and communities, making the development of an effective vaccine urgent. Alpha-hemolysin (Hla) is a key virulence factor and also a good target for the development of SA vaccines. However, the epitopes in Hla recognized by human immunity are not characterized in detail, which hinders the design of epitope-based human vaccines against SA. In this study, we collected sera from volunteers in a phase 1b clinical trial of a novel recombinant five-antigen SA vaccine (NCT03966040). Using a Luminex-based assay, we characterized the human serologic response against Hla, and identified Hla121-138 as a neutralizing epitope. In addition, we successfully produced ferritin nanoparticles carrying the neutralizing Hla121-138 epitope (EpNP) in E. coli. EpNP presented as homogenous nanoparticles in aqueous solution. Immunization with EpNP elicited potent hemolysis-neutralizing antibodies and conferred significant protection in a mouse model of SA skin infection. Our data suggest that EpNP, carrying the neutralizing epitope Hla121-138, is a good candidate for a vaccine against SA.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Anticuerpos Neutralizantes/inmunología , Toxinas Bacterianas/inmunología , Proteínas Hemolisinas/inmunología , Staphylococcus aureus Resistente a Meticilina/inmunología , Infecciones Estafilocócicas/prevención & control , Animales , Toxinas Bacterianas/antagonistas & inhibidores , Vacunas Bacterianas/inmunología , Epítopos/inmunología , Femenino , Proteínas Hemolisinas/antagonistas & inhibidores , Humanos , Nanopartículas del Metal/química , Staphylococcus aureus Resistente a Meticilina/patogenicidad , Ratones , Ratones Endogámicos BALB C , Infecciones Estafilocócicas/microbiología , Vacunación , Factores de VirulenciaRESUMEN
Novel therapeutics are needed to treat pathologies associated with the Clostridioides difficile binary toxin (CDT), particularly when C. difficile infection (CDI) occurs in the elderly or in hospitalized patients having illnesses, in addition to CDI, such as cancer. While therapies are available to block toxicities associated with the large clostridial toxins (TcdA and TcdB) in this nosocomial disease, nothing is available yet to treat toxicities arising from strains of CDI having the binary toxin. Like other binary toxins, the active CDTa catalytic subunit of CDT is delivered into host cells together with an oligomeric assembly of CDTb subunits via host cell receptor-mediated endocytosis. Once CDT arrives in the host cell's cytoplasm, CDTa catalyzes the ADP-ribosylation of G-actin leading to degradation of the cytoskeleton and rapid cell death. Although a detailed molecular mechanism for CDT entry and host cell toxicity is not yet fully established, structural and functional resemblances to other binary toxins are described. Additionally, unique conformational assemblies of individual CDT components are highlighted herein to refine our mechanistic understanding of this deadly toxin as is needed to develop effective new therapeutic strategies for treating some of the most hypervirulent and lethal strains of CDT-containing strains of CDI.
Asunto(s)
Proteínas Bacterianas/antagonistas & inhibidores , Toxinas Bacterianas/antagonistas & inhibidores , Clostridioides difficile/patogenicidad , Infección Hospitalaria/tratamiento farmacológico , Enterocolitis Seudomembranosa/tratamiento farmacológico , Enterotoxinas/antagonistas & inhibidores , ADP-Ribosilación/efectos de los fármacos , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestructura , Actinas/deficiencia , Actinas/genética , Antibacterianos/uso terapéutico , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Sitios de Unión , Clostridioides difficile/efectos de los fármacos , Clostridioides difficile/genética , Clostridioides difficile/metabolismo , Infección Hospitalaria/metabolismo , Infección Hospitalaria/microbiología , Infección Hospitalaria/patología , Endocitosis/efectos de los fármacos , Enterocolitis Seudomembranosa/metabolismo , Enterocolitis Seudomembranosa/microbiología , Enterocolitis Seudomembranosa/patología , Enterotoxinas/química , Enterotoxinas/genética , Enterotoxinas/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Células Epiteliales/ultraestructura , Humanos , Modelos Moleculares , Unión Proteica , Dominios Proteicos , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de ProteínaRESUMEN
OBJECTIVES: We and others have previously shown that epigallocatechin gallate (EGCg) inhibits the activity of an important virulence factor, leukotoxin (LtxA), produced by the oral bacterium Aggregatibacter actinomycetemcomitans, suggesting the potential use of this molecule as an anti-virulence strategy to treat periodontal infections. Here, we sought to better understand the effects of EGCg on toxin secretion and A. actinomycetemcomitans pathogenicity in a co-culture model. METHODS: We used a quantitative immunoblot assay to determine the concentrations of LtxA in the bacterial supernatant and on the bacterial cell surface. Using a co-culture model, consisting of A. actinomycetemcomitans and THP-1 cells, we studied the impact of EGCg-mediated changes in LtxA secretion on the toxicity of A. actinomycetemcomitans. KEY FINDINGS: EGCg increased production of LtxA and changed the localization of secreted LtxA from the supernatant to the surface of the bacterial cells. In the co-culture model, a single low dose of EGCg did not protect host THP-1 cells from A. actinomycetemcomitans-mediated cytotoxicity, but a multiple dosing strategy had improved effects. CONCLUSIONS: Together, these results demonstrate that EGCg has important, but complicated, effects on toxin secretion and activity; new dosing strategies and comprehensive model systems may be required to properly develop these anti-virulence activities.
Asunto(s)
Aggregatibacter actinomycetemcomitans , Catequina/análogos & derivados , Exotoxinas , Periodontitis , Aggregatibacter actinomycetemcomitans/efectos de los fármacos , Aggregatibacter actinomycetemcomitans/metabolismo , Aggregatibacter actinomycetemcomitans/patogenicidad , Antibacterianos/farmacología , Toxinas Bacterianas/antagonistas & inhibidores , Toxinas Bacterianas/metabolismo , Catequina/farmacología , Técnicas de Cocultivo/métodos , Relación Dosis-Respuesta a Droga , Exotoxinas/antagonistas & inhibidores , Exotoxinas/metabolismo , Humanos , Periodontitis/tratamiento farmacológico , Periodontitis/microbiología , Virulencia/efectos de los fármacosRESUMEN
Pseudomonas aeruginosa (PA) is a leading cause of nosocomial infections and death in cystic fibrosis patients. The study was conducted to evaluate the physicochemical structure, biological activity and serum stability of a recombinant anti-PcrV single chain variable antibody fragment genetically attached to the mCH3cc domain. The stereochemical properties of scFv-mCH3 (YFL001) and scFv (YFL002) proteins as well as molecular interactions towards Pseudomonas aeruginosa PcrV were evaluated computationally. The subcloned fragments encoding YFL001 and YFL002 in pET28a were expressed within the E. coli BL21-DE3 strain. After Ni-NTA affinity chromatography, the biological activity of the proteins in inhibition of PA induced hemolysis as well as cellular cytotoxicity was assessed. In silico analysis revealed the satisfactory stereochemical quality of the models as well as common residues in their interface with PcrV. The structural differences of proteins through circular dichroism spectroscopy were confirmed by NMR analysis. Both proteins indicated inhibition of ExoU positive PA strains in hemolysis of red blood cells compared to ExoU negative strains as well as cytotoxicity effect on lung epithelial cells. The ELISA test showed the longer serum stability of the YFL001 molecule than YFL002. The results were encouraging to further evaluation of these two scFv molecules in animal models.
Asunto(s)
Antibacterianos/farmacología , Toxinas Bacterianas/antagonistas & inhibidores , Infección Hospitalaria/tratamiento farmacológico , Proteínas Citotóxicas Formadoras de Poros/antagonistas & inhibidores , Infecciones por Pseudomonas/tratamiento farmacológico , Anticuerpos de Cadena Única/farmacología , Antibacterianos/aislamiento & purificación , Antibacterianos/uso terapéutico , Antígenos Bacterianos/metabolismo , Toxinas Bacterianas/metabolismo , Línea Celular Tumoral , Clonación Molecular , Simulación por Computador , Infección Hospitalaria/inmunología , Infección Hospitalaria/microbiología , Semivida , Humanos , Simulación del Acoplamiento Molecular , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Infecciones por Pseudomonas/inmunología , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/uso terapéutico , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/aislamiento & purificación , Anticuerpos de Cadena Única/uso terapéuticoRESUMEN
With the rapid growth of antibiotic-resistant bacteria, it is urgent to develop alternative therapeutic strategies. Pore-forming toxins (PFTs) belong to the largest family of virulence factors of many pathogenic bacteria and constitute the most characterized classes of pore-forming proteins (PFPs). Recent studies revealed the structural basis of several PFTs, both as soluble monomers, and transmembrane oligomers. Upon interacting with host cells, the soluble monomer of bacterial PFTs assembles into transmembrane oligomeric complexes that insert into membranes and affect target cell-membrane permeability, leading to diverse cellular responses and outcomes. Herein we have reviewed the structural basis of pore formation and interaction of PFTs with the host cell membrane, which could add valuable contributions in comprehensive understanding of PFTs and searching for novel therapeutic strategies targeting PFTs and interaction with host receptors in the fight of bacterial antibiotic-resistance.
Asunto(s)
Bacterias/metabolismo , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Membrana Celular/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Animales , Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Bacterias/patogenicidad , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/química , Toxinas Bacterianas/antagonistas & inhibidores , Toxinas Bacterianas/química , Membrana Celular/efectos de los fármacos , Membrana Celular/microbiología , Resistencia a Medicamentos/efectos de los fármacos , Interacciones Huésped-Patógeno , Humanos , Terapia Molecular Dirigida , Proteínas Citotóxicas Formadoras de Poros/antagonistas & inhibidores , Proteínas Citotóxicas Formadoras de Poros/química , Conformación Proteica , Relación Estructura-Actividad , VirulenciaRESUMEN
Toxin-antitoxin (TA) systems regulate key cellular functions in bacteria. Here, we report a unique structure of the Streptococcus pneumoniae HigBA system and a novel antimicrobial agent that activates HigB toxin, which results in mRNA degradation as an antibacterial strategy. In this study, protein structure-based peptides were designed and successfully penetrated the S. pneumoniae cell membrane and exerted bactericidal activity. This result represents the time during which inhibitors triggered S. pneumoniae cell death via the TA system. This discovery is a remarkable milestone in the treatment of antibiotic-resistant S. pneumoniae, and the mechanism of bactericidal activity is completely different from those of current antibiotics. Furthermore, we found that the HigBA complex shows a crossed-scissor interface with two intermolecular ß-sheets at both the N and C termini of the HigA antitoxin. Our biochemical and structural studies provided valuable information regarding the transcriptional regulation mechanisms associated with the structural variability of HigAs. Our in vivo study also revealed the potential catalytic residues of HigB and their functional relationships. An inhibition study with peptides additionally proved that peptide binding may allosterically inhibit HigB activity. Overall, our results provide insights into the molecular basis of HigBA TA systems in S. pneumoniae, which can be applied for the development of new antibacterial strategies. DATABASES: Structural data are available in the PDB database under the accession number 6AF4.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/farmacología , Antitoxinas/química , Toxinas Bacterianas/antagonistas & inhibidores , Descubrimiento de Drogas , Streptococcus pneumoniae/efectos de los fármacos , Sistemas Toxina-Antitoxina/efectos de los fármacos , Regulación Alostérica/efectos de los fármacos , Sitio Alostérico , Péptidos Catiónicos Antimicrobianos/síntesis química , Antitoxinas/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Permeabilidad de la Membrana Celular , Clonación Molecular , Cristalografía por Rayos X , Diseño de Fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Ingeniería de Proteínas/métodos , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/crecimiento & desarrollo , Streptococcus pneumoniae/patogenicidad , Relación Estructura-ActividadRESUMEN
Aeromonas hydrophila (A. hydrophila) can cause a number of diseases in both human and animals. A. hydrophila-related infections in aquaculture cause severe economic losses every year throughout the world. The emergence of antibiotic resistance that is due to the abuse of antibiotics has limited the application of antibiotics. Thus, novel approaches are needed to combat with treatment failure of antibiotics caused by resistant bacterial strains. Aerolysin plays a critical role in the pathogenesis of A. hydrophila and has been considered as a novel target for developing drugs based on anti-virulence strategies. Here, we reported that luteolin, a natural product with no anti-A. hydrophila activity, could reduce aerolysin-induced hemolysis by inhibiting aerolysin activity. The binding mode was simulated by molecular docking and dynamics simulation. Then the main binding sites were confirmed by fluorescence quenching assays. We found that luteolin could hindered the formation of functional heptamer of aerolysin according to the results of the oligomerization assay. Moreover, luteolin could protect A549 cells from aerolysin mediated cell death and increase the survival rate of A. hydrophila-infected channel catfish. These findings suggest a novel approach to developing drugs fighting against A. hydrophila, and luteolin can be a promising drug candidate for treatment of A. hydrophila-associated infections.