RESUMEN
ApoE is a well-known lipid-binding protein that plays a main role in the metabolism and transport of lipids. More recently, apoE-derived peptides have been shown to exert antimicrobial effects. Here, we investigated the antibacterial activity of apoE using in vitro assays, advanced imaging techniques, and in vivo mouse models. The formation of macromolecular complexes of apoE and endotoxins from Gram-negative bacteria was explored using gel shift assays, transmission electron microscopy, and CD spectroscopy followed by calculation of the α-helical content. The binding affinity of apoE to endotoxins was also confirmed by fluorescent spectroscopy detecting the quenching and shifting of tryptophan intrinsic fluorescence. We showed that apoE exhibits antibacterial activity particularly against Gram-negative bacteria such as Pseudomonas aeruginosa and Escherichia coli. ApoE protein folding was affected by binding of bacterial endotoxin components such as lipopolysaccharide (LPS) and lipid A, yielding similar increases in the apoE α-helical content. Moreover, high-molecular-weight complexes of apoE were formed in the presence of LPS, but not to the same extent as with lipid A. Together, our results demonstrate the ability of apoE to kill Gram-negative bacteria, interact with their endotoxins, which leads to the structural changes in apoE and the formation of aggregate-like complexes.
Asunto(s)
EndotoxinasRESUMEN
Myxothiazol is a respiratory chain complex III (CIII) inhibitor that binds to the ubiquinol oxidation site Qo of CIII. It blocks electron transfer from ubiquinol to cytochrome b and thus inhibits CIII activity. It has been utilized as a tool in studies of respiratory chain function in in vitro and cell culture models. We developed a mouse model of biochemically induced and reversible CIII inhibition using myxothiazol. We administered myxothiazol intraperitoneally at a dose of 0.56 mg/kg to C57Bl/J6 mice every 24 h and assessed CIII activity, histology, lipid content, supercomplex formation, and gene expression in the livers of the mice. A reversible CIII activity decrease to 50% of control value occurred at 2 h post-injection. At 74 h only minor histological changes in the liver were found, supercomplex formation was preserved and no significant changes in the expression of genes indicating hepatotoxicity or inflammation were found. Thus, myxothiazol-induced CIII inhibition can be induced in mice for four days in a row without overt hepatotoxicity or lethality. This model could be utilized in further studies of respiratory chain function and pharmacological approaches to mitochondrial hepatopathies.
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Antifúngicos/efectos adversos , Complejo III de Transporte de Electrones/antagonistas & inhibidores , Hígado/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/patología , Animales , Modelos Animales de Enfermedad , Complejo III de Transporte de Electrones/metabolismo , Hígado/metabolismo , Hígado/patología , Metacrilatos/efectos adversos , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Tiazoles/efectos adversosRESUMEN
Infant and adult MLL1/KMT2A-rearranged (MLLr) leukemia represents a disease with a dismal prognosis. Here, we present a functional and proteomic characterization of in utero-initiated and adult-onset MLLr leukemia. We reveal that fetal MLL::ENL-expressing lymphomyeloid multipotent progenitors (LMPPs) are intrinsically programmed towards a lymphoid fate but give rise to myeloid leukemia in vivo, highlighting a complex interplay of intra- and extracellular factors in determining disease subtype. We characterize early proteomic events of MLL::ENL-mediated transformation in fetal and adult blood progenitors and reveal that whereas adult pre-leukemic cells are mainly characterized by retained myeloid features and downregulation of ribosomal and metabolic proteins, expression of MLL::ENL in fetal LMPPs leads to enrichment of translation-associated and histone deacetylases signaling proteins, and decreased expression of inflammation and myeloid differentiation proteins. Integrating the proteome of pre-leukemic cells with their secretome and the proteomic composition of the extracellular environment of normal progenitors highlights differential regulation of Igf2 bioavailability, as well as of VLA-4 dimer and its ligandome, upon initiation of fetal- and adult-origin leukemia, with implications for human MLLr leukemia cells' ability to communicate with their environment through granule proteins. Our study has uncovered opportunities for targeting ontogeny-specific proteomic vulnerabilities in in utero-initiated and adult-onset MLLr leukemia.
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Proteína de la Leucemia Mieloide-Linfoide , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Humanos , Ratones , Animales , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Reordenamiento Génico , Proteómica/métodos , Feto/metabolismo , Adulto , Femenino , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Leucemia/genética , Leucemia/patología , Leucemia/metabolismoRESUMEN
The process of hematopoiesis is subject to substantial ontogenic remodeling that is accompanied by alterations in cellular fate during both development and disease. We combine state-of-the-art mass spectrometry with extensive functional assays to gain insight into ontogeny-specific proteomic mechanisms regulating hematopoiesis. Through deep coverage of the cellular proteome of fetal and adult lympho-myeloid multipotent progenitors (LMPPs), common lymphoid progenitors (CLPs), and granulocyte-monocyte progenitors (GMPs), we establish that features traditionally attributed to adult hematopoiesis are conserved across lymphoid and myeloid lineages, whereas generic fetal features are suppressed in GMPs. We reveal molecular and functional evidence for a diminished granulocyte differentiation capacity in fetal LMPPs and GMPs relative to their adult counterparts. Our data indicate an ontogeny-specific requirement of myosin activity for myelopoiesis in LMPPs. Finally, we uncover an ontogenic shift in the monocytic differentiation capacity of GMPs, partially driven by a differential expression of Irf8 during fetal and adult life.
Asunto(s)
Linaje de la Célula , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Proteómica , Células Madre Adultas/citología , Células Madre Adultas/metabolismo , Animales , Diferenciación Celular , Feto/citología , Granulocitos/citología , Células HEK293 , Humanos , Inmunofenotipificación , Factores Reguladores del Interferón/metabolismo , Cinética , Células Progenitoras Linfoides/citología , Células Progenitoras Linfoides/metabolismo , Ratones Endogámicos C57BL , Monocitos/citología , Monocitos/metabolismo , Células Progenitoras Mieloides/citología , Células Progenitoras Mieloides/metabolismo , Proteoma/metabolismo , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/metabolismoRESUMEN
In the present study, we investigate degradable anionic dendritic nanogels (DNG) as carriers for antimicrobial peptides (AMPs). In such systems, the dendritic part contains carboxylic acid-based anionic binding sites for cationic AMPs, whereas linear poly(ethylene glycol) (PEG) chains form a shell for promotion of biological stealth. In order to clarify factors influencing membrane interactions of such systems, we here address effects of nanogel charge, cross-linking, and degradation on peptide loading/release, as well as consequences of these factors for lipid membrane interactions and antimicrobial effects. The DNGs were found to bind the AMPs LL-37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES) and DPK-060 (GKHKNKGKKNGKHNGWKWWW). For the smaller DPK-060 peptide, loading was found to increase with increasing nanogel charge density. For the larger LL-37, on the other hand, peptide loading was largely insensitive to nanogel charge density. In line with this, results on the secondary structure, as well as on the absence of stabilization from proteolytic degradation by the nanogels, show that the larger LL-37 is unable to enter into the interior of the nanogels. While 40-60% nanogel degradation occurred over 10â¯days, promoted at high ionic strength and lower cross-linking density/higher anionic charge content, peptide release at physiological ionic strength was substantially faster, and membrane destabilization not relying on nanogel degradation. Ellipsometry and liposome leakage experiments showed both free peptide and peptide/DNG complexes to cause membrane destabilization, indicated also by antimicrobial activities being comparable for nanogel-bound and free peptide. Finally, the DNGs were demonstrated to display low toxicity towards erythrocytes even at peptide concentrations of 100⯵M.
Asunto(s)
Antibacterianos/administración & dosificación , Péptidos Catiónicos Antimicrobianos/administración & dosificación , Portadores de Fármacos/química , Nanogeles/química , Aniones/síntesis química , Aniones/química , Antibacterianos/química , Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/farmacología , Sistemas de Liberación de Medicamentos , Escherichia coli/efectos de los fármacos , Infecciones por Escherichia coli/tratamiento farmacológico , Humanos , Polietilenglicoles/químicaRESUMEN
In this study, the use of cubosomes for topical delivery of the antimicrobial peptide (AMP) LL-37 was investigated. Topical delivery of AMPs is of great interest for treatment of skin infections caused by bacteria, such as Staphylococcus aureus. AMP containing cubosomes were produced by three different preparation protocols and compared: (i) pre-loading, where LL-37 was incorporated into a liquid crystalline gel, which thereafter was dispersed into nanoparticles, (ii) post-loading, where LL-37 was let to adsorb onto pre-formed cubosomes, and (iii) hydrotrope-loading, where LL-37 was incorporated during the spontaneously formed cubosomes in an ethanol/glycerol monooleate mixture. Particle size and size distribution were analyzed using dynamic light scattering (DLS), liquid crystalline structure by small angle x-ray scattering (SAXS) and release of LL-37 by a fluorescamine assay. Proteolytic protection of LL-37 as well as bactericidal effect after enzyme exposure was investigated. The skin irritation potential of cubosomes was examined by an in vitro epidermis model. Finally, the bacterial killing property of the cubosomes was examined by an ex vivo pig skin wound infection model with Staphylococcus aureus. Data showed that a high loading of LL-37 induced formation of vesicles in case of cubosomes prepared by sonication (pre-loading). No release of LL-37 was observed from the cubosomes, indicating strong association of the peptide to the particles. Proteolysis studies showed that LL-37 was fully protected against enzymatic attacks while associated with the cubosomes, also denoting strong association of the peptide to the particles. As a consequence, bactericidal effect after enzyme exposure remained, compared to pure LL-37 which was subjected to proteolysis. No skin irritation potential of the cubosomes was found, thus enabling for topical administration. The ex vivo wound infection model showed that LL-37 in pre-loaded cubosomes killed bacteria most efficient.
Asunto(s)
Antiinfecciosos/administración & dosificación , Catelicidinas/administración & dosificación , Sistemas de Liberación de Medicamentos/métodos , Infecciones Cutáneas Estafilocócicas/tratamiento farmacológico , Infección de Heridas/tratamiento farmacológico , Administración Tópica , Animales , Antiinfecciosos/efectos adversos , Antiinfecciosos/farmacocinética , Péptidos Catiónicos Antimicrobianos , Catelicidinas/efectos adversos , Catelicidinas/farmacocinética , Modelos Animales de Enfermedad , Liberación de Fármacos , Epidermis/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Etanol/química , Glicéridos/química , Humanos , Cristales Líquidos/química , Pruebas de Sensibilidad Microbiana , Nanopartículas/química , Dispersión del Ángulo Pequeño , Pruebas de Irritación de la Piel/métodos , Infecciones Cutáneas Estafilocócicas/microbiología , Staphylococcus aureus/efectos de los fármacos , Porcinos , Resultado del Tratamiento , Infección de Heridas/microbiología , Difracción de Rayos XRESUMEN
Synthetic peptides composed of multiples of the consensus heparin-binding Cardin and Weintraub sequences AKKARA and ARKKAAKA are antimicrobial. Replacement of lysine and arginine by histidine in these peptides completely abrogates their antimicrobial and heparin-binding activities at neutral pH. However, the antibacterial activity against Gram-negative (Escherichia coli, Pseudomonas aeruginosa) and Gram-positive bacteria (Bacillus subtilis and Staphylococcus aureus) as well as the fungus Candida albicans, was restored at acidic conditions (pH 5.5). Fluorescence microscopy and FACS analysis showed that the binding of the histidine-rich peptides to E. coli and Candida was significantly enhanced at pH 5.5. Likewise, fluorescence studies for assessment of membrane permeation as well as electron microscopy analysis of peptide-treated bacteria, paired with studies of peptide effects on liposomes, demonstrated that the peptides induce membrane lysis only at acidic pH. No discernible hemolysis was noted for the histidine-rich peptides. Similar pH-dependent antimicrobial activities were demonstrated for peptides derived from histidine-rich and heparin-binding regions of human kininogen and histidine-rich glycoprotein. The results demonstrate that the presence of an acidic environment is an important regulator of the activity of histidine-rich antimicrobial peptides.
Asunto(s)
Antibacterianos/farmacología , Proteínas/farmacología , Secuencia de Aminoácidos , Antibacterianos/química , Membrana Celular/metabolismo , Dicroismo Circular , Heparina/metabolismo , Concentración de Iones de Hidrógeno , Liposomas , Datos de Secuencia Molecular , Proteínas/químicaRESUMEN
Microgels are interesting as potential delivery systems for antimicrobial peptides. In order to elucidate membrane interactions of such systems, we here investigate effects of microgel charge density on antimicrobial peptide loading and release, as well as consequences of this for membrane interactions and antimicrobial effects, using ellipsometry, circular dichroism spectroscopy, nanoparticle tracking analysis, dynamic light scattering and z-potential measurements. Anionic poly(ethyl acrylate-co-methacrylic acid) microgels were found to incorporate considerable amounts of the cationic antimicrobial peptides LL-37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES) and DPK-060 (GKHKNKGKKNGKHNGWKWWW) and to protect incorporated peptides from degradation by infection-related proteases at high microgel charge density. As a result of their net negative z-potential also at high peptide loading, neither empty nor peptide-loaded microgels adsorb at supported bacteria-mimicking membranes. Instead, membrane disruption is mediated almost exclusively by peptide release. Mirroring this, antimicrobial effects against several clinically relevant bacteria (methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli, and Pseudomonas aeruginosa) were found to be promoted by factors facilitating peptide release, such as decreasing peptide length and decreasing microgel charge density. Microgels were further demonstrated to display low toxicity towards erythrocytes. Taken together, the results demonstrate some interesting opportunities for the use of microgels as delivery systems for antimicrobial peptides, but also highlight several key factors which need to be controlled for their successful use.
Asunto(s)
Antibacterianos/química , Antibacterianos/metabolismo , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/metabolismo , Membrana Celular/metabolismo , Geles/química , Bacterias/efectos de los fármacos , Propiedades de SuperficieRESUMEN
Tuberculosis has been reaffirmed as the infectious disease causing most deaths in the world. Co-infection with HIV and the increase in multi-drug resistant Mycobacterium tuberculosis strains complicate treatment and increases mortality rates, making the development of new drugs an urgent priority. In this study we have identified a promising candidate by screening antimicrobial peptides for their capacity to inhibit mycobacterial growth. This non-toxic peptide, NZX, is capable of inhibiting both clinical strains of M. tuberculosis and an MDR strain at therapeutic concentrations. The therapeutic potential of NZX is further supported in vivo where NZX significantly lowered the bacterial load with only five days of treatment, comparable to rifampicin treatment over the same period. NZX possesses intracellular inhibitory capacity and co-localizes with intracellular bacteria in infected murine lungs. In conclusion, the data presented strongly supports the therapeutic potential of NZX in future anti-TB treatment.
Asunto(s)
Antituberculosos/farmacología , Pulmón/efectos de los fármacos , Macrófagos/efectos de los fármacos , Mycobacterium tuberculosis/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Péptidos/farmacología , Tuberculosis Pulmonar/tratamiento farmacológico , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Farmacorresistencia Bacteriana Múltiple , Femenino , Humanos , Pulmón/microbiología , Pulmón/ultraestructura , Macrófagos/microbiología , Ratones Endogámicos BALB C , Mycobacterium tuberculosis/crecimiento & desarrollo , Factores de Tiempo , Tuberculosis Pulmonar/microbiología , Tuberculosis Pulmonar/patologíaRESUMEN
Successful use of microgels as delivery systems of antimicrobial peptides (AMPs) requires control of factors determining peptide loading and release to/from the microgels as well as of membrane interactions of both microgel particles and released peptides. Addressing these, we here investigate effects of microgel charge density and conformationally induced peptide amphiphilicity on AMP loading and release using detailed nuclear magnetic resonance (NMR) structural studies combined with ellipsometry, isothermal titration calorimetry, circular dichroism, and light scattering. In parallel, consequences of peptide loading and release for membrane interactions and antimicrobial effects were investigated. In doing so, poly(ethyl acrylate-co-methacrylic acid) microgels were found to incorporate the cationic AMPs EFK17a (EFKRIVQRIKDFLRNLV) and its partially d-amino acid-substituted variant EFK17da (E(dF)KR(dI)VQR(dI)KD(dF)LRNLV). Peptide incorporation was found to increase with increasing with microgel charge density and peptide amphiphilicity. After microgel incorporation, which appeared to occur preferentially in the microgel core, NMR showed EFK17a to form a helix with pronounced amphiphilicity, while EFK17da displayed a folded conformation, stabilized by a hydrophobic hub consisting of aromatic/aromatic and aliphatic/aromatic interactions, resulting in much lower amphiphilicity. Under wide ranges of peptide loading, the microgels displayed net negative z-potential. Such negatively charged microgels do not bind to, nor lyse, bacteria-mimicking membranes. Instead, membrane disruption in these systems is mediated largely by peptide release, which in turn is promoted at higher ionic strength and lower peptide amphiphilicity. Analogously, antimicrobial effects against Escherichia coli were found to be dictated by peptide release. Taken together, the findings show that peptide loading, packing, and release strongly affect the performance of microgels as AMP delivery systems, effects that can be tuned by (conformationally induced) peptide amphiphilicity and by microgel charge density.
Asunto(s)
Polímeros/química , Antiinfecciosos , Geles , Interacciones Hidrofóbicas e Hidrofílicas , Conformación MolecularRESUMEN
Novel antibiotics, such as antimicrobial peptides (AMPs), have recently attended more and more attraction. In this work, dispersed cubic liquid crystalline gel (cubosomes) was used as drug delivery vehicles for three AMPs (AP114, DPK-060 and LL-37). Association of peptides onto cubosomes was studied at two cubosome/peptide ratios using high performance liquid chromatography, ζ-potential and circular dichroism measurements. AMPs impact on the cubosome structure was investigated using small angle x-ray scattering and cryogenic transmission electron microscopy. The antimicrobial effect of the AMP loaded cubosomes was studied in vitro by minimum inhibitory concentration and time-kill assays. Proteolytic protection was investigated by incubating the formulations with two elastases and the antimicrobial effect after proteolysis was studied using radial diffusion assay. Different association efficacy onto the cubosomes was observed among the AMPs, with LL-37 showing greatest association (>60%). AP114 loaded cubosomes displayed a preserved antimicrobial effect, whereas for LL-37 the broad spectrum bacterial killing was reduced to only comprise Gram-negative bacteria. Interestingly, DPK-060 loaded cubosomes showed a slight enhanced effect against S. aureus and E. coli strains. Moreover, the cubosomes were found to protect LL-37 from proteolytic degradation, resulting in a significantly better bactericidal effect after being subjected to elastase, compared to unformulated peptide.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/administración & dosificación , Sistemas de Liberación de Medicamentos , Escherichia coli , Geles , Pruebas de Sensibilidad Microbiana , Microscopía Electrónica de Transmisión , Staphylococcus aureusRESUMEN
Antimicrobial peptides (AMP) are effector molecules of the innate immune system. A cross-functionality exists between AMPs and heparin-binding peptides. Here, we show that the peptides QPTRRPRPGTGPGRRPRPRPRP (QPT22), derived from proline arginine-rich end leucine-rich repeat protein (PRELP) and KRFKQDGGWSHWSPWSS (KRF17) from thrombospondin exert antimicrobial activities against the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, as well as against the Gram-positive Bacillus subtilis. Analysis by fluorescence microscopy demonstrated that QPT22 and KRF17 bind to bacterial membranes. Particularly QPT22 was found to induce membrane leakage of liposomes. The peptides displayed no hemolytic activities, nor did they exert membrane permeabilising effects on human epithelial cells. Additional peptides derived from heparin-binding regions of laminin, vitronectin, and fibronectin exerted similar antibacterial effects. Several peptides also showed activity against Staphylococcus aureus. Thus, the data disclose a novel antimicrobial activity of heparin-binding regions of matrix glycoproteins. The findings can be utilized in the development of novel AMPs for therapeutic use.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/farmacología , Bacterias/efectos de los fármacos , Proteínas Sanguíneas/química , Proteínas Portadoras/química , Proteínas de la Matriz Extracelular/química , Glicoproteínas/química , Fragmentos de Péptidos/farmacología , Trombospondinas/química , Secuencia de Aminoácidos , Bacillus subtilis/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Matriz Extracelular/química , Humanos , Técnicas In Vitro , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Pseudomonas aeruginosa/efectos de los fármacosRESUMEN
The COX7A2L (Supercomplex Assembly Factor I, SCAFI) protein has been proposed to be a mitochondrial supercomplex assembly factor required for respirasome (supercomplex containing complexes I, III, and IV) formation. In the C57BL/6 mouse strain a homozygous in-frame 6-base-pair deletion in the COX7a2l/SCAF1 gene resulting in unstable protein and suggesting loss of function was previously identified. The loss of SCAFI was shown to impede respirasome formation, a major concern for the use of C57BL mouse strains in mitochondrial research. In contradiction, another recent study suggested that supercomplex formation is independent of SCAFI isoforms. We investigated whether SCAFI isoform status affected the disease severity and supercomplex formation in the liver of Bcs1lc.232A>G knock-in mice with incomplete complex III assembly. In homozygotes (Bcs1lG/G) of mixed (C57BL/6:129/Sv) genetic background, the lifespan was similar in mice with wild-type SCAFI allele and in those homozygous (SCAFIshort/short) for the deleted SCAF1 variant (34±3 days; n = 6 vs. 32±2 days; n = 7, respectively). SCAFI heterozygosity (SCAFIlong/short) resulted in decreased SCAFI protein but respirasome assembly was unaffected. Congenic (C57BL/6) mice were of the genotype SCAFIshort/short and had no detectable SCAFI protein. In their liver mitochondria, respirasome composition was altered as compared to mixed background mice. Complex IV was mainly present as monomers and dimers, and only low amounts were found in combination with complex I and complex III or with precomplex III. The main supercomplex in the liver mitochondria of C57BL/6 mice comprised only complexes I and III. In conclusion, in liver mitochondria of C57BL/6 mice, supercomplexes had markedly reduced amount of, but were not completely depleted of, complex IV, supporting a role for COX7A2L/SCAFI in supercomplex assembly. However, the disease progression of the Bcs1l mutant mice was unrelated to SCAFI isoforms and supercomplex composition, suggesting that other genetic factors contribute to the different survival in the different genetic backgrounds.
Asunto(s)
Complejo III de Transporte de Electrones/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , Mitocondrias Hepáticas/metabolismo , Proteínas Mitocondriales/metabolismo , Chaperonas Moleculares/metabolismo , Mutación , ATPasas Asociadas con Actividades Celulares Diversas , Animales , Complejo III de Transporte de Electrones/genética , Complejo IV de Transporte de Electrones/genética , Ratones , Ratones Mutantes , Mitocondrias Hepáticas/genética , Proteínas Mitocondriales/genética , Chaperonas Moleculares/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Especificidad de la EspecieRESUMEN
Membrane interactions are critical for the successful use of mesoporous silica nanoparticles as delivery systems for antimicrobial peptides (AMPs). In order to elucidate these, we here investigate effects of nanoparticle charge and porosity on AMP loading and release, as well as consequences of this for membrane interactions and antimicrobial effects. Anionic mesoporous silica particles were found to incorporate considerable amounts of the cationic AMP LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES (LL-37), whereas loading is much lower for non-porous or positively charged silica nanoparticles. Due to preferential pore localization, anionic mesoporous particles, but not the other particles, protect LL-37 from degradation by infection-related proteases. For anionic mesoporous nanoparticles, membrane disruption is mediated almost exclusively by peptide release. In contrast, non-porous silica particles build up a resilient LL-37 surface coating due to their higher negative surface charge, and display largely particle-mediated membrane interactions and antimicrobial effects. For positively charged mesoporous silica nanoparticles, LL-37 incorporation promotes the membrane binding and disruption displayed by the particles in the absence of peptide, but also causes toxicity against human erythrocytes. Thus, the use of mesoporous silica nanoparticles as AMP delivery systems requires consideration of membrane interactions and selectivity of both free peptide and the peptide-loaded nanoparticles, the latter critically dependent on nanoparticle properties.
Asunto(s)
Antibacterianos/administración & dosificación , Portadores de Fármacos/química , Nanopartículas/química , Dióxido de Silicio/química , Adsorción , Antibacterianos/farmacología , Supervivencia Celular/efectos de los fármacos , Eritrocitos/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Humanos , Pruebas de Sensibilidad Microbiana , Tamaño de la Partícula , Porosidad , Propiedades de SuperficieRESUMEN
Functional oxidative phosphorylation requires appropriately assembled mitochondrial respiratory complexes and their supercomplexes formed mainly of complexes I, III and IV. BCS1L is the chaperone needed to incorporate the catalytic subunit, Rieske iron-sulfur protein, into complex III at the final stage of its assembly. In cell culture studies, this subunit has been considered necessary for supercomplex formation and for maintaining the stability of complex I. Our aim was to assess the importance of fully assembled complex III for supercomplex formation in intact liver tissue. We used our transgenic mouse model with a homozygous c.232A>G mutation in Bcs1l leading to decreased expression of BCS1L and progressive decrease of Rieske iron-sulfur protein in complex III, resulting in hepatopathy. We studied supercomplex formation at different ages using blue native gel electrophoresis and complex activity using high-resolution respirometry. In isolated liver mitochondria of young and healthy homozygous mutant mice, we found similar supercomplexes as in wild type. In homozygotes aged 27-29 days with liver disorder, complex III was predominantly a pre-complex lacking Rieske iron-sulfur protein. However, the main supercomplex was clearly detected and contained complex III mainly in the pre-complex form. Oxygen consumption of complex IV was similar and that of complex I was twofold compared with controls. These complexes in free form were more abundant in homozygotes than in controls, and the mRNA of complex I subunits were upregulated. In conclusion, when complex III assembly is deficient, the pre-complex without Rieske iron-sulfur protein can participate with available fully assembled complex III in supercomplex formation, complex I function is preserved, and respiratory chain stability is maintained.
Asunto(s)
Complejo III de Transporte de Electrones/deficiencia , Complejo I de Transporte de Electrón/metabolismo , Mitocondrias Hepáticas/metabolismo , Chaperonas Moleculares/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas , Animales , Western Blotting , Transporte de Electrón , Complejo III de Transporte de Electrones/metabolismo , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Ratones , Ratones Transgénicos , Chaperonas Moleculares/genética , Consumo de Oxígeno/fisiologíaRESUMEN
BACKGROUND & AIMS: Liver is a target organ in many mitochondrial disorders, especially if the complex III assembly factor BCS1L is mutated. To reveal disease mechanism due to such mutations, we have produced a transgenic mouse model with c.232A>G mutation in Bcs1l, the causative mutation for GRACILE syndrome. The homozygous mice develop mitochondrial hepatopathy with steatosis and fibrosis after weaning. Our aim was to assess cellular mechanisms for disease onset and progression using metabolomics. METHODS: With mass spectrometry we analyzed metabolite patterns in liver samples obtained from homozygotes and littermate controls of three ages. As oxidative stress might be a mechanism for mitochondrial hepatopathy, we also assessed H(2)O(2) production and expression of antioxidants. RESULTS: Homozygotes had a similar metabolic profile at 14 days of age as controls, with the exception of slightly decreased AMP. At 24 days, when hepatocytes display first histopathological signs, increases in succinate, fumarate and AMP were found associated with impaired glucose turnover and beta-oxidation. At end stage disease after 30 days, these changes were pronounced with decreased carbohydrates, high levels of acylcarnitines and amino acids, and elevated biogenic amines, especially putrescine. Signs of oxidative stress were present in end-stage disease. CONCLUSIONS: The findings suggest an early Krebs cycle defect with increases of its intermediates, which might play a role in disease onset. During disease progression, carbohydrate and fatty acid metabolism deteriorate leading to a starvation-like condition. The mouse model is valuable for further investigations on mechanisms in mitochondrial hepatopathy and for interventions.
Asunto(s)
Complejo III de Transporte de Electrones/deficiencia , Hígado/metabolismo , Chaperonas Moleculares/genética , ATPasas Asociadas con Actividades Celulares Diversas , Adenosina Monofosfato/metabolismo , Animales , Antioxidantes/metabolismo , Complejo III de Transporte de Electrones/genética , Fumaratos/metabolismo , Peróxido de Hidrógeno/metabolismo , Espectrometría de Masas , Ratones , Enfermedades Mitocondriales/genética , Enfermedades Mitocondriales/metabolismo , Mutación , Estrés Oxidativo/genética , Estrés Oxidativo/fisiología , Ácido Succínico/metabolismoRESUMEN
BACKGROUND: Antimicrobial peptides (AMP) are important effectors of the innate immune system. Although there is increasing evidence that AMPs influence bacteria in a multitude of ways, bacterial wall rupture plays the pivotal role in the bactericidal action of AMPs. Structurally, AMPs share many similarities with endogenous heparin-binding peptides with respect to secondary structure, cationicity, and amphipathicity. FINDINGS: In this study, we show that RQA21 (RQAREHSERKKRRRESECKAA), a cationic and hydrophilic heparin-binding peptide corresponding to the C-terminal region of extracellular superoxide dismutase (SOD), exerts antimicrobial activity against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis and Candida albicans. The peptide was also found to induce membrane leakage of negatively charged liposomes. However, its antibacterial effects were abrogated in physiological salt conditions as well as in plasma. CONCLUSION: The results provide further evidence that heparin-binding peptide regions are multifunctional, but also illustrate that cationicity alone is not sufficient for AMP function at physiological conditions. However, our observation, apart from providing a link between heparin-binding peptides and AMPs, raises the hypothesis that proteolytically generated C-terminal SOD-derived peptides could interact with, and possibly counteract bacteria. Further studies are therefore merited to study a possible role of SOD in host defence.
RESUMEN
A novel approach for boosting antimicrobial peptides through end tagging with hydrophobic oligopeptide stretches is demonstrated. Focusing on two peptides derived from kininogen, GKHKNKGKKNGKHNGWK (GKH17) and HKHGHGHGKHKNKGKKN (HKH17), tagging resulted in enhanced killing of Gram-positive Staphylococcus aureus, Gram-negative Escherichia coli, and fungal Candida albicans. Microbicidal potency increased with tag length, also in plasma, and was larger for Trp and Phe stretches than for aliphatic ones. The enhanced microbicidal effects correlated to a higher degree of bacterial wall rupture. Analogously, tagging promoted peptide binding to model phospholipid membranes and liposome rupture, particularly for anionic and cholesterol-void membranes. Tagged peptides displayed low toxicity, particularly in the presence of serum, and resisted degradation by human leukocyte elastase and by staphylococcal aureolysin and V8 proteinase. The biological relevance of these findings was demonstrated ex vivo and in vivo in porcine S. aureus skin infection models. The generality of end tagging for facile boosting of antimicrobial peptides without the need for post-synthesis modification was also demonstrated.
Asunto(s)
Antibacterianos/farmacología , Antifúngicos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Candida albicans/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Oligopéptidos/farmacología , Staphylococcus aureus/efectos de los fármacos , Animales , Proteínas Bacterianas/metabolismo , Candida albicans/crecimiento & desarrollo , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Dicroismo Circular , Escherichia coli/crecimiento & desarrollo , Femenino , Hemólisis/efectos de los fármacos , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Quininógenos/química , L-Lactato Deshidrogenasa/metabolismo , Elastasa de Leucocito/metabolismo , Metaloendopeptidasas/metabolismo , Pruebas de Sensibilidad Microbiana , Serina Endopeptidasas/metabolismo , Staphylococcus aureus/enzimología , Staphylococcus aureus/crecimiento & desarrollo , PorcinosRESUMEN
Growth factors, comprising diverse protein and peptide families, are involved in a multitude of developmental processes, including embryogenesis, angiogenesis, and wound healing. Here we show that peptides derived from HB-EGF, amphiregulin, hepatocyte growth factor, PDGF-A and PDGF-B, as well as various FGFs are antimicrobial, demonstrating a previously unknown activity of growth factor-derived peptides. The peptides killed the Gram-negative bacteria Escherichia coli, Pseudomonas aeruginosa, and the Gram-positive Bacillus subtilis, as well as the fungus Candida albicans. Several peptides were also active against the Gram-positive S. aureus. Electron microscopy analysis of peptide-treated bacteria, paired with analysis of peptide effects on liposomes, showed that the peptides exerted membrane-breaking effects similar to those seen after treatment with the "classical" human antimicrobial peptide LL-37. Furthermore, HB-EGF was antibacterial per se, and its epitope GKRKKKGKGLGKKRDPCLRKYK retained its activity in presence of physiological salt and plasma. No discernible hemolysis was noted for the growth factor-derived peptides. Besides providing novel templates for design of peptide-based antimicrobials, our findings demonstrate a previously undisclosed link between the family of growth factors and antimicrobial peptides, both of which are induced during tissue remodelling and repair.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/aislamiento & purificación , Sustancias de Crecimiento/química , Secuencia de Aminoácidos , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/genética , Péptidos Catiónicos Antimicrobianos/farmacología , Bacillus subtilis/efectos de los fármacos , Bacterias/efectos de los fármacos , Candida albicans/efectos de los fármacos , Factor de Crecimiento Epidérmico/química , Factor de Crecimiento Epidérmico/genética , Escherichia coli/efectos de los fármacos , Sustancias de Crecimiento/genética , Factor de Crecimiento Similar a EGF de Unión a Heparina , Humanos , Técnicas In Vitro , Péptidos y Proteínas de Señalización Intercelular , Pruebas de Sensibilidad Microbiana , Microscopía Electrónica , Modelos Moleculares , Datos de Secuencia Molecular , Pseudomonas aeruginosa/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacosRESUMEN
Bacterial lipopolysaccharides (LPS) are important triggers of the widespread inflammatory response, which contributes to the development of multiple organ failure during sepsis. The helical 37-amino-acid-long human antimicrobial peptide LL-37 not only possesses a broad-spectrum antimicrobial activity but also binds and neutralizes LPS. However, the use of LL-37 in sepsis treatment is hampered by the fact that it is also cytotoxic. To find a less toxic analog of LL-37, we used in silico analysis to identify amphipathic helical regions of LL-37. A 21-amino-acid fragment (GKE) was synthesized, the biological actions of which were compared to those of two equally long peptides derived from the N and C termini of LL-37 as well as native LL-37. GKE displayed antimicrobial activity against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans, and Candida parapsilosis that was similar to or even stronger than LL-37. GKE, as well as the equally long control peptides, attracted granulocytes in a fashion similar to that of LL-37, while only GKE was as potent as LL-37 in inhibiting LPS-induced vascular nitric oxide production. GKE caused less hemolysis and apoptosis in human cultured smooth muscle cells than LL-37. In summary, we have identified an active domain of LL-37, GKE, which displays antimicrobial activity in vitro and LPS-binding activity similar to those of LL-37 but is less toxic. GKE therefore holds promise as a template for the development of peptide antibiotics for the treatment of sepsis.