RESUMEN
Antimicrobial peptides (AMPs) contribute to an effective protection against infections. The antibacterial function of AMPs depends on their interactions with microbial membranes and lipids, such as lipopolysaccharide (LPS; endotoxin). Hyperinflammation induced by endotoxin is a key factor in bacterial sepsis and many other human diseases. Here, we provide a comprehensive profile of peptide-mediated LPS neutralization by systematic analysis of the effects of a set of AMPs and the peptide antibiotic polymyxin B (PMB) on the physicochemistry of endotoxin, macrophage activation, and lethality in mice. Mechanistic studies revealed that the host defense peptide LL-32 and PMB each reduce LPS-mediated activation also via a direct interaction of the peptides with the host cell. As a biophysical basis, we demonstrate modifications of the structure of cholesterol-rich membrane domains and the association of glycosylphosphatidylinositol (GPI)-anchored proteins. Our discovery of a host cell-directed mechanism of immune control contributes an important aspect in the development and therapeutic use of AMPs.
Asunto(s)
Catelicidinas/farmacología , Membrana Celular/metabolismo , Interacciones Huésped-Patógeno , Lipopolisacáridos/farmacología , Pruebas de Neutralización , Polimixina B/farmacología , Animales , Péptidos Catiónicos Antimicrobianos/farmacología , Transporte Biológico/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Colesterol/metabolismo , Femenino , Células HEK293 , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Inflamación/patología , Ratones Endogámicos C57BL , Transducción de Señal/efectos de los fármacosRESUMEN
Microbial cells show a strong natural tendency to adhere to surfaces and to colonize them by forming complex communities called biofilms. In this growth mode, biofilm-forming cells encase themselves inside a dense matrix which efficiently protects them against antimicrobial agents and effectors of the immune system. Moreover, at the physiological level, biofilms contain a very heterogeneous cell population including metabolically inactive organisms and persisters, which are highly tolerant to antibiotics. The majority of human infectious diseases are caused by biofilm-forming microorganisms which are responsible for pathologies such as cystic fibrosis, infective endocarditis, pneumonia, wound infections, dental caries, infections of indwelling devices, etc. AMPs are well suited to combat biofilms because of their potent bactericidal activity of broad spectrum (including resting cells and persisters) and their ability to first penetrate and then to disorganize these structures. In addition, AMPs frequently synergize with antimicrobial compounds and were recently reported to repress the molecular pathways leading to biofilm formation. Finally, there is a very active research to develop AMP-containing coatings that can prevent biofilm formation by killing microbial cells on contact or by locally releasing their active principle. In this chapter we will describe these strategies and discuss the perspectives of the use of AMPs as anti-biofilm agents for human therapy and prophylaxis.
Asunto(s)
Antiinfecciosos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Biopelículas , HumanosRESUMEN
BACKGROUND: Infections by Pseudomonas aeruginosa constitute a serious health threat because this pathogen -particularly when it forms biofilms - can acquire resistance to the majority of conventional antibiotics. This study evaluated the antimicrobial activity of synthetic peptides based on LF11, an 11-mer peptide derived from human lactoferricin against P. aeruginosa planktonic and biofilm-forming cells. We included in this analysis selected N-acylated derivatives of the peptides to analyze the effect of acylation in antimicrobial activity. To assess the efficacy of compounds against planktonic bacteria, microdilution assays to determine the minimal inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and time-kill studies were conducted. The anti-biofilm activity of the agents was assessed on biofilms grown under static (on microplates) and dynamic (in a CDC-reactor) flow regimes. RESULTS: The antimicrobial activity of lipopeptides differed from that of non-acylated peptides in their killing mechanisms on planktonic and biofilm-forming cells. Thus, acylation enhanced the bactericidal activity of the parental peptides and resulted in lipopeptides that were uniformly bactericidal at their MIC. In contrast, acylation of the most potent anti-biofilm peptides resulted in compounds with lower anti-biofilm activity. Both peptides and lipopeptides displayed very rapid killing kinetics and all of them required less than 21 min to reduce 1,000 times the viability of planktonic cells when tested at 2 times their MBC. The peptides, LF11-215 (FWRIRIRR) and LF11-227 (FWRRFWRR), displayed the most potent anti-biofilm activity causing a 10,000 fold reduction in cell viability after 1 h of treatment at 10 times their MIC. At that concentration, these two compounds exhibited low citotoxicity on human cells. In addition to its bactericidal activity, LF11-227 removed more that 50 % of the biofilm mass in independent assays. Peptide LF11-215 and two of the shortest and least hydrophobic lipopeptides, DI-MB-LF11-322 (2,2-dimethylbutanoyl-PFWRIRIRR) and DI-MB-LF11-215, penetrated deep into the biofilm structure and homogenously killed biofilm-forming bacteria. CONCLUSION: We identified peptides derived from human lactoferricin with potent antimicrobial activity against P. aeruginosa growing either in planktonic or in biofilm mode. Although further structure-activity relationship analyses are necessary to optimize the anti-biofilm activity of these compounds, the results indicate that lactoferricin derived peptides are promising anti-biofilm agents.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/farmacología , Biopelículas/efectos de los fármacos , Lactoferrina/genética , Lipopéptidos/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Péptidos Catiónicos Antimicrobianos/genética , Humanos , Pruebas de Sensibilidad Microbiana , Pseudomonas aeruginosa/fisiología , Relación Estructura-ActividadRESUMEN
This paper describes the design, implementation and validation of a sensitive and integral technology solution for endotoxin detection. The unified and portable platform is based on the electrochemical detection of endotoxins using a synthetic peptide immobilized on a thin-film biosensor. The work covers the fabrication of an optimized sensor, the biofunctionalization protocol and the design and implementation of the measuring and signalling elements (a microfluidic chamber and a portable potentiostat-galvanostat), framed ad hoc for this specific application. The use of thin-film technologies to fabricate the biosensing device and the application of simple immobilization and detection methods enable a rapid, easy and sensitive technique for in situ and real time LPS detection.
Asunto(s)
Técnicas Biosensibles/métodos , Técnicas Electroquímicas/métodos , Lipopolisacáridos/análisis , Electrodos , Escherichia coli/patogenicidad , Técnicas Analíticas Microfluídicas/métodosRESUMEN
Bacterial infections are known to cause severe health-threatening conditions, including sepsis. All attempts to get this disease under control failed in the past, and especially in times of increasing antibiotic resistance, this leads to one of the most urgent medical challenges of our times. We designed a peptide to bind with high affinity to endotoxins, one of the most potent pathogenicity factors involved in triggering sepsis. The peptide Pep19-2.5 reveals high endotoxin neutralization efficiency in vitro, and here, we demonstrate its antiseptic/anti-inflammatory effects in vivo in the mouse models of endotoxemia, bacteremia, and cecal ligation and puncture, as well as in an ex vivo model of human tissue. Furthermore, we show that Pep19-2.5 can bind and neutralize not only endotoxins but also other bacterial pathogenicity factors, such as those from the Gram-positive bacterium Staphylococcus aureus. This broad neutralization efficiency and the additive action of the peptide with common antibiotics makes it an exceptionally appropriate drug candidate against bacterial sepsis and also offers multiple other medication opportunities.
Asunto(s)
Lipopolisacáridos/antagonistas & inhibidores , Péptidos/farmacología , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/patogenicidad , Factores de Virulencia/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Antibacterianos/farmacología , Bacteriemia/tratamiento farmacológico , Bacteriemia/metabolismo , Bacteriemia/microbiología , Bacteriemia/mortalidad , Modelos Animales de Enfermedad , Sinergismo Farmacológico , Endotoxemia/tratamiento farmacológico , Endotoxemia/metabolismo , Endotoxemia/microbiología , Endotoxemia/mortalidad , Femenino , Humanos , Lipopolisacáridos/biosíntesis , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Péptidos/síntesis química , Sepsis/tratamiento farmacológico , Sepsis/metabolismo , Sepsis/microbiología , Sepsis/mortalidad , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/metabolismo , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/mortalidad , Staphylococcus aureus/crecimiento & desarrollo , Análisis de Supervivencia , Factores de Virulencia/biosíntesisRESUMEN
Acne is a common chronic skin condition with serious physical and psychosocial consequences. In some cases, the appearance of pimples, whiteheads, or blackheads on the face, neck, and back may lead to scarring, disfiguring, depression, frustration, and anxiety in patients. Current treatments rely on antibiotics to eradicate Cutibacterium acnes (C. acnes), the bacterium responsible for this skin condition. However, these approaches do not scavenge the reactive oxidative species (ROS) generated during disease development and raise concerns about the increase in antimicrobial resistance. In this study, an environmentally friendly and cost-effective self-assembly nanoencapsulation technology based on zein, a bio-based hydrophobic protein, was employed to produce multifunctional essential oil (EO)-loaded nanocapsules (NCs) with superior antioxidant and bactericidal activity toward C. acnes. The NCs displayed "smart" release of the active cargo only under the conditions that were conducive to acne proliferation on skin. Once incorporated into creams, the EO-loaded NCs led to a complete inhibition of C. acnes and demonstrated the capacity to scavenge ROS, thus preventing damage to human skin cells. The in vitro permeation studies revealed that the nanoformulated EO was able to penetrate through the epidermis, indicating its potential for the treatment of skin diseases, such as acne.
RESUMEN
Pseudomonas aeruginosa is naturally resistant to many antibiotics, and infections caused by this organism are a serious threat, especially to hospitalized patients. The intrinsic low permeability of P. aeruginosa to antibiotics results from the coordinated action of several mechanisms, such as the presence of restrictive porins and the expression of multidrug efflux pump systems. Our goal was to develop antimicrobial peptides with an improved bacterial membrane-permeabilizing ability, so that they enhance the antibacterial activity of antibiotics. We carried out a structure activity relationship analysis to investigate the parameters that govern the permeabilizing activity of short (8- to 12-amino-acid) lactoferricin-derived peptides. We used a new class of constitutional and sequence-dependent descriptors called PEDES (peptide descriptors from sequence) that allowed us to predict (Spearman's ρ = 0.74; P < 0.001) the permeabilizing activity of a new peptide generation. To study if peptide-mediated permeabilization could neutralize antibiotic resistance mechanisms, the most potent peptides were combined with antibiotics, and the antimicrobial activities of the combinations were determined on P. aeruginosa strains whose mechanisms of resistance to those antibiotics had been previously characterized. A subinhibitory concentration of compound P2-15 or P2-27 sensitized P. aeruginosa to most classes of antibiotics tested and counteracted several mechanisms of antibiotic resistance, including loss of the OprD porin and overexpression of several multidrug efflux pump systems. Using a mouse model of lethal infection, we demonstrated that whereas P2-15 and erythromycin were unable to protect mice when administered separately, concomitant administration of the compounds afforded long-lasting protection to one-third of the animals.
Asunto(s)
Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Lactoferrina/química , Péptidos/farmacología , Péptidos/uso terapéutico , Infecciones por Pseudomonas/tratamiento farmacológico , Pseudomonas aeruginosa/efectos de los fármacos , Animales , Antibacterianos/química , Sinergismo Farmacológico , Femenino , Ratones , Pruebas de Sensibilidad Microbiana , Péptidos/química , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/patogenicidadRESUMEN
Systemic bacterial infections are associated with high mortality. The access of bacteria or constituents thereof to systemic circulation induces the massive release of immunomodulatory mediators, ultimately causing tissue hypoperfusion and multiple-organ failure despite adequate antibiotic treatment. Lipid A, the "endotoxic principle" of bacterial lipopolysaccharide (LPS), is one of the major bacterial immunostimuli. Here we demonstrate the biological efficacy of rationally designed new synthetic antilipopolysaccharide peptides (SALPs) based on the Limulus anti-LPS factor for systemic application. We show efficient inhibition of LPS-induced cytokine release and protection from lethal septic shock in vivo, whereas cytotoxicity was not observed under physiologically relevant conditions and concentrations. The molecular mechanism of LPS neutralization was elucidated by biophysical techniques. The lipid A part of LPS is converted from its "endotoxic conformation," the cubic aggregate structure, into an inactive multilamellar structure, and the binding affinity of the peptide to LPS exceeds those of known LPS-binding proteins, such as LPS-binding protein (LBP). Our results thus delineate a novel therapeutic strategy for the clinical management of patients with septic shock.
Asunto(s)
Antiinfecciosos/farmacología , Antiinfecciosos/uso terapéutico , Péptidos/farmacología , Péptidos/uso terapéutico , Choque Séptico/prevención & control , Animales , Antiinfecciosos/síntesis química , Antiinfecciosos/química , Bacterias/efectos de los fármacos , Calorimetría , Células Cultivadas , Citocinas/metabolismo , Femenino , Hemólisis/efectos de los fármacos , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Lipopolisacáridos/química , Lipopolisacáridos/toxicidad , Ratones , Ratones Endogámicos C57BL , Pruebas de Sensibilidad Microbiana , Péptidos/síntesis química , Péptidos/química , Choque Séptico/tratamiento farmacológico , Choque Séptico/inmunologíaRESUMEN
Targeted bactericide nanosystems hold significant promise to improve the efficacy of existing antimicrobials for treatment of severe bacterial infections, minimizing the side effects and lowering the risk of the development of antibiotic resistance. In this work, we developed antibody-functionalized nanocapsules (NCs) containing antibacterial essential oil (EO) for selective and effective eradication of Staphylococcus aureus. Antibacterial EO NCs were produced via self-assembly nanoencapsulation in the plant-derived protein zein. The obtained EO NCs were decorated with aminocellulose to provide more reactive surface groups for carboxyl-to-amine immobilization of a antibody that is specific against S. aureus. The antibody-enabled EO NCs (Ab@EO NCs) demonstrated 2-fold higher bactericidal efficacy against the targeted bacterium compared to the pristine EO NCs at the same concentrations. The improved antibacterial effect of the Ab@EO NCs toward S. aureus was also confirmed in a real-time assay by monitoring bacterial cells elimination using a quartz crystal microbalance. Furthermore, the Ab@EO NCs selectively decreased the load and changed the cell morphology of the targeted S. aureus in a mixed inoculum with nontargeted Pseudomonas aeruginosa. Applying the nanoformulated antibacterial actives to an in vitro coculture model of the bacteria and skin fibroblasts resulted in suppression of S. aureus growth while preserving the human cells viability. The novel antibody-enabled antibacterial NCs showed potential for improving the treatment efficacy of staphylococcal infections, minimally affecting the beneficial microbial and human cells.
Asunto(s)
Antiinfecciosos/química , Nanocápsulas/química , Aceites Volátiles/química , Staphylococcus aureus/efectos de los fármacos , Antiinfecciosos/farmacología , Anticuerpos/química , Farmacorresistencia Microbiana , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Humanos , Pruebas de Sensibilidad Microbiana , Terapia Molecular Dirigida , Aceites Volátiles/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Infecciones Estafilocócicas/metabolismo , Proteína Estafilocócica A/metabolismoRESUMEN
OBJECTIVES: ß-lactamases are the major resistance determinant for ß-lactam antibiotics in Gram-negative bacteria. Although there are ß-lactamase inhibitors (BLIs) available, ß-lactam-BLI combinations are increasingly being neutralised by diverse mechanisms of bacterial resistance. This study hypothesised that permeability-increasing antimicrobial peptides (AMPs) could lower the amount of BLIs necessary to sensitise bacteria to antibiotics that are ß-lactamase substrates. METHODS: To test this hypothesis, checkerboard assays were performed to measure the ability of several AMPs to synergise with piperacillin, ticarcillin, amoxicillin, ampicillin, and ceftazidime in the presence of either tazobactam, clavulanic acid, sulbactam, aztreonam, phenylboronic acid (PBA), or oxacillin. Assays were performed using planktonic and biofilm-forming cells of Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae overexpressing ß-lactamases. RESULTS: Synergy between polymyxin B nonapeptide (PMBN) and tazobactam boosted piperacillin activity by a factor of 128 in Escherichia coli (from 256 to 2 mg/L, fractional inhibitory concentration index (FICI) ≤ 0.02) and by a factor of at least 64 in Klebsiella pneumoniae (from 1024 mg/L to 16 mg/L, FICI ≤ 0.05). Synergy between PMBN and PBA enhanced ceftazidime activity 133 times in Pseudomonas aeruginosa (from 16 mg/L to 0.12 mg/L, FICI ≤ 0.03). As a consequence, MICs of all the tested antibiotics were brought down to therapeutic range. In addition, the combinations also reduced several orders of magnitude the amount of inhibitor needed for antibiotic sensitisation. Ceftazidime/PBA/PMBN at 50 times the planktonic MIC caused a 10 million-fold reduction in the viability of mature biofilms. CONCLUSION: This study proved that AMPs can synergise with BLIs and that this phenomenon can be exploited to sensitise bacteria to antibiotics.
Asunto(s)
Antibacterianos/farmacología , Enterobacteriaceae Resistentes a los Carbapenémicos/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Klebsiella pneumoniae/efectos de los fármacos , Proteínas Citotóxicas Formadoras de Poros/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Inhibidores de beta-Lactamasas/farmacología , Enterobacteriaceae Resistentes a los Carbapenémicos/genética , Enterobacteriaceae Resistentes a los Carbapenémicos/metabolismo , Ceftazidima/farmacología , Sinergismo Farmacológico , Quimioterapia Combinada , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/metabolismo , Pruebas de Sensibilidad Microbiana , Polimixina B/farmacología , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Tazobactam/farmacología , beta-Lactamasas/genética , beta-Lactamasas/metabolismoRESUMEN
Resistance to antibiotics poses a major global threat according to the World Health Organization. Restoring the activity of existing drugs is an attractive alternative to address this challenge. One of the most efficient mechanisms of bacterial resistance involves the expression of efflux pump systems capable of expelling antibiotics from the cell. Although there are efflux pump inhibitors (EPIs) available, these molecules are toxic for humans. We hypothesized that permeability-increasing antimicrobial peptides (AMPs) could lower the amount of EPI necessary to sensitize bacteria to antibiotics that are efflux substrates. To test this hypothesis, we measured the ability of polymyxin B nonapeptide (PMBN), to synergize with antibiotics in the presence of EPIs. Assays were performed using planktonic and biofilm-forming cells of Pseudomonas aeruginosa strains overexpressing the MexAB-OprM efflux system. Synergy between PMBN and EPIs boosted azithromycin activity by a factor of 2,133 and sensitized P. aeruginosa to all tested antibiotics. This reduced several orders of magnitude the amount of inhibitor needed for antibiotic sensitization. The selected antibiotic-EPI-PMBN combination caused a 10 million-fold reduction in the viability of biofilm forming cells. We proved that AMPs can synergize with EPIs and that this phenomenon can be exploited to sensitize bacteria to antibiotics.
Asunto(s)
Antibacterianos/farmacología , Proteínas de la Membrana Bacteriana Externa/metabolismo , Farmacorresistencia Bacteriana Múltiple , Proteínas de Transporte de Membrana/metabolismo , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/metabolismo , Proteínas de la Membrana Bacteriana Externa/genética , Biopelículas , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Humanos , Proteínas de Transporte de Membrana/genética , Pruebas de Sensibilidad Microbiana , PermeabilidadRESUMEN
BACKGROUND: Growing concerns about bacterial resistance to antibiotics have prompted the development of alternative therapies like those based on cationic antimicrobial peptides (APs). These compounds not only are bactericidal by themselves but also enhance the activity of antibiotics. Studies focused on the systematic characterization of APs are hampered by the lack of standard guidelines for testing these compounds. We investigated whether the information provided by methods commonly used for the biological characterization of APs is comparable, as it is often assumed. For this purpose, we determined the bacteriostatic, bactericidal, and permeability-increasing activity of synthetic peptides (n = 57; 9-13 amino acid residues in length) analogous to the lipopolysaccharide-binding region of human lactoferricin by a number of the most frequently used methods and carried out a comparative analysis. RESULTS: While the minimum inhibitory concentration determined by an automated turbidimetry-based system (Bioscreen) or by conventional broth microdilution methods did not differ significantly, bactericidal activity measured under static conditions in a low-ionic strength solvent resulted in a vast overestimation of antimicrobial activity. Under these conditions the degree of antagonism between the peptides and the divalent cations differed greatly depending on the bacterial strain tested. In contrast, the bioactivity of peptides was not affected by the type of plasticware (polypropylene vs. polystyrene). Susceptibility testing of APs using cation adjusted Mueller-Hinton was the most stringent screening method, although it may overlook potentially interesting peptides. Permeability assays based on sensitization to hydrophobic antibiotics provided overall information analogous - though not quantitatively comparable- to that of tests based on the uptake of hydrophobic fluorescent probes. CONCLUSION: We demonstrate that subtle changes in methods for testing cationic peptides bring about marked differences in activity. Our results show that careful selection of the test strains for susceptibility testing and for screenings of antibiotic-sensitizing activity is of critical importance. A number of peptides proved to have potent permeability-increasing activity at subinhibitory concentrations and efficiently sensitized Pseudomonas aeruginosa both to hydrophilic and hydrophobic antibiotics.
Asunto(s)
Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Bacterias/efectos de los fármacos , Permeabilidad de la Membrana Celular/efectos de los fármacos , Lactoferrina/farmacología , Pruebas de Sensibilidad Microbiana/normas , Bacterias/crecimiento & desarrollo , Medios de Cultivo/química , Viabilidad MicrobianaRESUMEN
Bacteria-mediated diseases are a global healthcare concern due to the development and spread of antibiotic-resistant strains. Cationic compounds are considered membrane active biocidal agents having a great potential to control bacterial infections, while limiting the emergence of drug resistance. Herein, the versatile and simple layer-by-layer (LbL) technique is used to coat alternating multilayers of an antibacterial aminocellulose conjugate and the biocompatible hyaluronic acid on biocompatible polymer nanoparticles (NPs), taking advantage of the nanosize of these otherwise biologically inert templates. Stable polyelectrolyte-decorated particles with an average size of 50 nm and ζ potential of +40.6 mV were developed after five LbL assembly cycles. The antibacterial activity of these NPs against the Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli increased significantly when the polycationic aminocellulose was in the outermost layer. The large number of amino groups available on the particle surface, together with the nanosize of the multilayer conjugates, improved their interaction with bacterial membrane phospholipids, leading to membrane disruption, as confirmed by a Langmuir monolayer model, and the 10 logs reduction for both bacteria. The biopolymer decorated NPs were also able to inhibit the biofilm formation of S. aureus and E. coli by 94 and 40%, respectively, without affecting human cell viability. The use of LbL-coated NPs appears to be a promising antibiotic-free alternative for controlling bacterial infections using a low amount of antimicrobial agent.
Asunto(s)
Nanopartículas , Antibacterianos , Escherichia coli , Humanos , Staphylococcus aureusRESUMEN
The most potent cell wall-derived inflammatory toxins ("pathogenicity factors") of Gram-negative and -positive bacteria are lipopolysaccharides (LPS) (endotoxins) and lipoproteins (LP), respectively. Despite the fact that the former signals via toll-like receptor 4 (TLR4) and the latter via TLR2, the physico-chemistry of these compounds exhibits considerable similarity, an amphiphilic molecule with a polar and charged backbone and a lipid moiety. While the exterior portion of the LPS (i.e., the O-chain) represents the serologically relevant structure, the inner part, the lipid A, is responsible for one of the strongest inflammatory activities known. In the last years, we have demonstrated that antimicrobial peptides from the Pep19-2.5 family, which were designed to bind to LPS and LP, act as anti-inflammatory agents against sepsis and endotoxic shock caused by severe bacterial infections. We also showed that this anti-inflammatory activity requires specific interactions of the peptides with LPS and LP leading to exothermic reactions with saturation characteristics in calorimetry assays. Parallel to this, peptide-mediated neutralization of LPS and LP involves changes in various physical parameters, including both the gel to liquid crystalline phase transition of the acyl chains and the three-dimensional aggregate structures of the toxins. Furthermore, the effectivity of neutralization of pathogenicity factors by peptides was demonstrated in several in vivo models together with the finding that a peptide-based therapy sensitizes bacteria (also antimicrobial resistant) to antibiotics. Finally, a significant step in the understanding of the broad anti-inflammatory function of Pep19-2.5 was the demonstration that this compound is able to block the intracellular endotoxin signaling cascade.
Asunto(s)
Antiinflamatorios/uso terapéutico , Inflamación/tratamiento farmacológico , Inflamación/etiología , Lipopolisacáridos/efectos adversos , Lipoproteínas/efectos adversos , Péptidos/uso terapéutico , Animales , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Antiinflamatorios/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Péptidos Catiónicos Antimicrobianos/uso terapéutico , Endotoxinas/efectos adversos , Endotoxinas/antagonistas & inhibidores , Endotoxinas/química , Humanos , Inflamación/metabolismo , Péptidos/farmacologíaRESUMEN
Biofilm-associated infections constitute a daunting threat to human health, since these pathologies increase patient mortality and morbidity, resulting in prolonged hospitalization periods and heavy economic losses. Moreover, these infections contribute to the increasing emergence and dissemination of antibiotic resistance in hospitals and in the community. Although biofilm-associated microorganisms can proliferate in healthy tissue, abiotic surfaces like those of medical implants greatly increase the likelihood of biofilm formation in the host. Due to their broad spectrum of bactericidal activity against multi-drug resistant microorganisms including metabolically inactive cells, antimicrobial peptides (AMPs) have great potential as anti-biofilm agents. In fact, a clinically available AMP, polymyxin E (colistin), frequently constitutes the drug of last recourse in biofilm-associated infections (e.g. cystic fibrosis) when resistance to all the other drugs arises. In this article, we outline the main strategies under development to combat biofilm-associated infections with an emphasis in the prevention of microbial colonization of medical implants. These approaches include the use of AMPs both for the development of anti-adhesive surface coatings and to kill biofilm-forming cells either on contact or via controlled release (leaching surfaces). Although in vitro results for all these applications are very encouraging, further research is needed to improve the anti-biofilm activity of these coatings in vivo. The possibility of exploiting the antibiotic potentiating activity of some AMPs and to combine several anti-biofilm mechanisms in tandem targeting the biofilm formation process at different stages is also discussed.
Asunto(s)
Antiinfecciosos/farmacología , Biopelículas/efectos de los fármacos , Péptidos/farmacología , Prótesis e Implantes/microbiologíaRESUMEN
Sepsis, a life-threatening syndrome with increasing incidence worldwide, is triggered by an overwhelming inflammation induced by microbial toxins released into the bloodstream during infection. A well-known sepsis-inducing factor is the membrane constituent of Gram-negative bacteria, lipopolysaccharide (LPS), signalling via Toll-like receptor-4. Although sepsis is caused in more than 50% cases by Gram-positive and mycoplasma cells, the causative compounds are still poorly described. In contradicting investigations lipoproteins/-peptides (LP), lipoteichoic acids (LTA), and peptidoglycans (PGN), were made responsible for eliciting this pathology. Here, we used human mononuclear cells from healthy donors to determine the cytokine-inducing activity of various LPs from different bacterial origin, synthetic and natural, and compared their activity with that of natural LTA and PGN. We demonstrate that LP are the most potent non-LPS pro-inflammatory toxins of the bacterial cell walls, signalling via Toll-like receptor-2, not only in vitro, but also when inoculated into mice: A synthetic LP caused sepsis-related pathological symptoms in a dose-response manner. Additionally, these mice produced pro-inflammatory cytokines characteristic of a septic reaction. Importantly, the recently designed polypeptide Aspidasept(®) which has been proven to efficiently neutralize LPS in vivo, inhibited cytokines induced by the various non-LPS compounds protecting animals from the pro-inflammatory activity of synthetic LP.
Asunto(s)
Antibacterianos/farmacología , Endotoxinas/efectos adversos , Endotoxinas/antagonistas & inhibidores , Lipoproteínas/efectos adversos , Lipoproteínas/antagonistas & inhibidores , Péptidos/farmacología , Sepsis/etiología , Animales , Antibacterianos/síntesis química , Citocinas/biosíntesis , Modelos Animales de Enfermedad , Endotoxemia/tratamiento farmacológico , Endotoxemia/etiología , Endotoxemia/metabolismo , Endotoxemia/mortalidad , Femenino , Bacterias Gramnegativas/inmunología , Células HEK293 , Humanos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Lipopolisacáridos/efectos adversos , Lipopolisacáridos/antagonistas & inhibidores , Lipopolisacáridos/química , Lipoproteínas/química , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Péptidos/síntesis química , Peptidoglicano/efectos adversos , Sepsis/tratamiento farmacológico , Sepsis/metabolismo , Sepsis/mortalidad , Staphylococcus aureus/inmunología , Ácidos Teicoicos/efectos adversosRESUMEN
The bacterial cell wall represents the primary target for antimicrobial agents. Microbial destruction is accompanied by the release of potent immunostimulatory membrane constituents. Both Gram-positive and Gram-negative bacteria release a variety of lipoproteins and peptidoglycan fragments. Gram-positive bacteria additionally provide lipoteichoic acids, whereas Gram-negative bacteria also release lipopolysaccharide (LPS, endotoxin), essential component of the outer leaflet of the bacterial cell wall and one of the most potent immunostimulatory molecules known. Immune activation therefore can be considered as an adverse effect of antimicrobial destruction and killing during anti-infective treatment. In contrast to antibiotics, the use of cationic amphiphilic antimicrobial peptides allows both effective bacterial killing and inhibition of the immunostimulatory effect of the released bacterial membrane constituents. The administration of antimicrobial peptides alone or in combination with antibiotic agents thus represents a novel strategy in the antiinfective treatment with potentially important beneficial aspects. Here, data are presented which describe immunological and clinical aspects of the use of antimicrobial peptides (AMPs) as therapeutic agents to treat bacterial infection and neutralize the immunostimulatory activity of released cell wall constituents.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Antiinfecciosos/farmacología , Bacterias/efectos de los fármacos , Pared Celular/efectos de los fármacos , Bacterias/inmunología , Bacterias/metabolismo , Pared Celular/inmunología , Pared Celular/metabolismo , HumanosRESUMEN
The first barrier that an antimicrobial agent must overcome when interacting with its target is the microbial cell wall. In the case of Gram-negative bacteria, additional to the cytoplasmic membrane and the peptidoglycan layer, an outer membrane (OM) is the outermost barrier. The OM has an asymmetric distribution of the lipids with phospholipids and lipopolysaccharide (LPS) located in the inner and outer leaflets, respectively. In contrast, Gram-positive bacteria lack OM and possess a much thicker peptidoglycan layer compared to their Gram-negative counterparts. An additional class of amphiphiles exists in Gram-positives, the lipoteichoic acids (LTA), which may represent important structural components. These long molecules cross-bridge the entire cell envelope with their lipid component inserting into the outer leaflet of the cytoplasmic membrane and the teichoic acid portion penetrating into the peptidoglycan layer. Furthermore, both classes of bacteria have other important amphiphiles, such as lipoproteins, whose importance has become evident only recently. It is not known yet whether any of these amphiphilic components are able to stimulate the immune system under physiological conditions as constituents of intact bacteria. However, all of them have a very high pro-inflammatory activity when released from the cell. Such a release may take place through the interaction with the immune system, or with antibiotics (particularly with those targeting cell wall components), or simply by the bacterial division. Therefore, a given antimicrobial agent must ideally have a double character, namely, it must overcome the bacterial cell wall barrier, without inducing the liberation of the pro-inflammatory amphiphiles. Here, new data are presented which describe the development and use of membrane-active antimicrobial agents, in particular antimicrobial peptides (AMPs) and lipopolyamines. In this way, essential progress was achieved, in particular with respect to the inhibition of deleterious consequences of bacterial infections such as severe sepsis and septic shock.