RESUMEN
Because most of animal viruses are enveloped, cytoplasmic entry of these viruses via fusion with cellular membrane initiates their invasion. However, the strategies in which host cells counteract cytoplasmic entry of such viruses are incompletely understood. Pore-forming toxin aerolysin-like proteins (ALPs) exist throughout the animal kingdom, but their functions are mostly unknown. In this study, we report that ßγ-crystallin fused aerolysin-like protein and trefoil factor complex (ßγ-CAT), an ALP and trefoil factor complex from the frog Bombina maxima, directly blocks enveloped virus invasion by interfering with cytoplasmic entry. ßγ-CAT targeted acidic glycosphingolipids on the HSV type 1 (HSV-1) envelope to induce pore formation, as indicated by the oligomer formation of protein and potassium and calcium ion efflux. Meanwhile, ßγ-CAT formed ring-like oligomers of â¼10 nm in diameter on the liposomes and induced dye release from liposomes that mimic viral envelope. Unexpectedly, transmission electron microscopy analysis showed that the ßγ-CAT-treated HSV-1 was visibly as intact as the vehicle-treated HSV-1, indicating that ßγ-CAT did not lyse the viral envelope. However, the cytoplasmic entry of the ßγ-CAT-treated HSV-1 into HeLa cells was totally hindered. In vivo, topical application of ßγ-CAT attenuated the HSV-1 corneal infection in mice. Collectively, these results uncovered that ßγ-CAT possesses the capacity to counteract enveloped virus invasion with its featured antiviral-acting manner. Our findings will also largely help to illustrate the putative antiviral activity of animal ALPs.
Asunto(s)
Proteínas Anfibias/metabolismo , Antivirales/metabolismo , Córnea/patología , Herpes Simple/inmunología , Herpesvirus Humano 1/fisiología , Complejos Multiproteicos/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Factores Trefoil/metabolismo , Proteínas Anfibias/genética , Animales , Anuros , Toxinas Bacterianas/genética , Córnea/virología , Femenino , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Ratones , Microscopía Electrónica de Transmisión , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/genética , Envoltura Viral/metabolismo , Envoltura Viral/ultraestructura , Internalización del Virus , gamma-Cristalinas/químicaRESUMEN
Bacterial pore-forming toxin aerolysin-like proteins are widely distributed in animals and plants. Emerging evidence supports their roles in host innate immunity, but their direct actions in adaptive immunity remain elusive. In this study, we found that ßγ-CAT, an aerolysin-like protein and trefoil factor complex identified in the frog Bombina maxima, modulated several steps of endocytic pathways during dendritic cell antigen presentation. The protein augmented the antigen uptake of dendritic cells and actively neutralized the acidification of cellular endocytic organelles to favor antigen presentation. In addition, the release of functional exosome-like extracellular vesicles was largely enhanced in the presence of ßγ-CAT. The cellular action of ßγ-CAT increased the number of major histocompatibility complex (MHC) I-ovalbumin and MHC II molecules on dendritic cell surfaces and the released exosome-like extracellular vesicles. An enhanced antigen presentation capacity of dendritic cell for priming of naive T cells was detected in the presence of ßγ-CAT. Collectively, these effects led to strong cytotoxic T lymphocyte responses and antigen-specific antibody responses. Our findings provide evidence that a vertebrate-secreted pore-forming protein can augment antigen presentation by directly modulating cellular endocytic and exocytic pathways, leading to robust activation of adaptive immunity.
Asunto(s)
Presentación de Antígeno/efectos de los fármacos , Células Dendríticas , Endosomas , Proteínas Citotóxicas Formadoras de Poros/farmacología , Linfocitos T , Inmunidad Adaptativa , Animales , Anuros/metabolismo , Células Cultivadas , Células Dendríticas/citología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Endosomas/efectos de los fármacos , Endosomas/inmunología , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunologíaRESUMEN
Endolysosomes are key players in cell physiology, including molecular exchange, immunity, and environmental adaptation. They are the molecular targets of some pore-forming aerolysin-like proteins (ALPs) that are widely distributed in animals and plants and are functionally related to bacterial toxin aerolysins. ßγ-CAT is a complex of an ALP (BmALP1) and a trefoil factor (BmTFF3) in the firebelly toad (Bombina maxima). It is the first example of a secreted endogenous pore-forming protein that modulates the biochemical properties of endolysosomes by inducing pore formation in these intracellular vesicles. Here, using a large array of biochemical and cell biology methods, we report the identification of BmALP3, a paralog of BmALP1 that lacks membrane pore-forming capacity. We noted that both BmALP3 and BmALP1 contain a conserved cysteine in their C-terminal regions. BmALP3 was readily oxidized to a disulfide bond-linked homodimer, and this homodimer then oxidized BmALP1 via disulfide bond exchange, resulting in the dissociation of ßγ-CAT subunits and the elimination of biological activity. Consistent with its behavior in vitro, BmALP3 sensed environmental oxygen tension in vivo, leading to modulation of ßγ-CAT activity. Interestingly, we found that this C-terminal cysteine site is well conserved in numerous vertebrate ALPs. These findings uncover the existence of a regulatory ALP (BmALP3) that modulates the activity of an active ALP (BmALP1) in a redox-dependent manner, a property that differs from those of bacterial toxin aerolysins.
Asunto(s)
Proteínas Anfibias/química , Disulfuros/química , Proteínas Citotóxicas Formadoras de Poros/química , Multimerización de Proteína , Animales , Anuros , Oxidación-Reducción , Dominios ProteicosRESUMEN
Naturally occurring monoterpenes are known for their various pharmacological activities including anti-inflammation. KV1.3 ion channel is a voltage-gated potassium channel and has been validated as a drug target for autoimmune and chronic inflammatory diseases like psoriasis. Here we experimentally test the direct interaction between monoterpenes and KV1.3 ion channel. Our electrophysiological analysis determined that monoterpenes (geraniol, nerol, ß-citronellol, citral and linalool) have inhibitory effects on KV1.3 ion channel. Representatively, geraniol reversibly blocked KV1.3 currents in a voltage-dependent manner with an IC50 of 490.50⯱â¯1.04⯵M at +40â¯mV in HEK293T cells. At the effective concentrations, geraniol also inhibited cytokine secretion of activated human T cells, including IL-2, TNF-α and IFN-γ. In an imiquimod-induced psoriasis-like animal model, geraniol administration significantly reduced psoriasis area and severity index scores, ameliorated the deteriorating histopathology and decreased the degree of splenomegaly. Together, our findings not only suggest that monoterpenes may serve as lead molecules for the development of KV1.3 inhibitors, but also indicate that geraniol could be considered as a promising therapeutic candidate to treat autoimmune diseases.
Asunto(s)
Monoterpenos Acíclicos/farmacología , Antiinflamatorios/farmacología , Canal de Potasio Kv1.3/antagonistas & inhibidores , Bloqueadores de los Canales de Potasio/farmacología , Animales , Citocinas/metabolismo , Modelos Animales de Enfermedad , Células HEK293 , Humanos , Masculino , Ratones Endogámicos BALB C , Psoriasis/tratamiento farmacológico , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismoRESUMEN
Bacterial pore-forming toxin aerolysin-like proteins (ALPs) are widely distributed in animals and plants. However, functional studies on these ALPs remain in their infancy. ßγ-CAT is the first example of a secreted pore-forming protein that functions to modulate the endolysosome pathway via endocytosis and pore formation on endolysosomes. However, the specific cell surface molecules mediating the action of ßγ-CAT remain elusive. Here, the actions of ßγ-CAT were largely attenuated by either addition or elimination of acidic glycosphingolipids (AGSLs). Further study revealed that the ALP and trefoil factor (TFF) subunits of ßγ-CAT bind to gangliosides and sulfatides, respectively. Additionally, disruption of lipid rafts largely impaired the actions of ßγ-CAT. Finally, the ability of ßγ-CAT to clear pathogens was attenuated in AGSL-eliminated frogs. These findings revealed a previously unknown double binding pattern of an animal-secreted ALP in complex with TFF that initiates ALP-induced endolysosomal pathway regulation, ultimately leading to effective antimicrobial responses.
Asunto(s)
Glicoesfingolípidos Acídicos/química , Proteínas Anfibias/inmunología , Toxinas Bacterianas/inmunología , Infecciones por Bacterias Gramnegativas/inmunología , Lisosomas/inmunología , Complejos Multiproteicos/inmunología , Proteínas Citotóxicas Formadoras de Poros/inmunología , Factor Trefoil-3/inmunología , Glicoesfingolípidos Acídicos/antagonistas & inhibidores , Glicoesfingolípidos Acídicos/biosíntesis , Aeromonas hydrophila/crecimiento & desarrollo , Aeromonas hydrophila/patogenicidad , Proteínas Anfibias/genética , Proteínas Anfibias/metabolismo , Animales , Anuros , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Ceramidas/antagonistas & inhibidores , Ceramidas/biosíntesis , Ceramidas/química , Cerebrósidos/antagonistas & inhibidores , Cerebrósidos/biosíntesis , Cerebrósidos/química , Gangliósidos/antagonistas & inhibidores , Gangliósidos/biosíntesis , Gangliósidos/química , Expresión Génica , Infecciones por Bacterias Gramnegativas/genética , Infecciones por Bacterias Gramnegativas/microbiología , Humanos , Interleucina-1beta/biosíntesis , Lisosomas/efectos de los fármacos , Lisosomas/microbiología , Microdominios de Membrana/efectos de los fármacos , Microdominios de Membrana/inmunología , Microdominios de Membrana/microbiología , Meperidina/análogos & derivados , Meperidina/farmacología , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Esfingosina/antagonistas & inhibidores , Esfingosina/biosíntesis , Esfingosina/química , Células THP-1 , Factor Trefoil-3/genética , Factor Trefoil-3/metabolismoRESUMEN
Tissue repair is a highly dynamic process, and the immediate onset of acute inflammation has been considered necessary for repair. Pore-forming proteins are important, both in pathogen invasion and host immunity. However, their roles in wound healing and tissue repair are unclear. ßγ-crystallin fused aerolysin-like protein (α-subunit) and trefoil factor (ß-subunit) complex (ßγ-CAT) is a complex of a bacterial pore-forming toxin aerolysin-like protein and trefoil factor identified in the frog Bombina maxima. In this study, we established mouse cutaneous wound models to explore the effects of ßγ-CAT on skin wound healing. ßγ-CAT accelerated the healing of full-thickness wounds by improving re-epithelialization. This complex relieved dermal edema and promoted scarless healing. ßγ-CAT treatment resulted in a rapid release of IL-1ß, which initiated an acute inflammation response in the early stage of healing. Meanwhile, the expression levels of TGF-ß1, VEGF, and bFGF and the recruitment of M2 macrophages around the wound significantly increased after ßγ-CAT treatment. ßγ-CAT protected skin wounds against methicillin-resistant Staphylococcus aureus by improving neutrophil recruitment at the site of the wound. Overall, our results suggest that ßγ-CAT can promote tissue repair and protect skin wounds against antibiotic-resistant bacterial infection by triggering the acute inflammatory response. This is the first example that aerolysin-like pore-forming proteins widely existing in plants and animals may act in wound healing and tissue repair.-Gao, Z.-H., Deng, C.-J., Xie, Y.-Y., Guo, X.-L., Wang, Q.-Q., Liu, L.-Z., Lee, W.-H., Li, S.-A., Zhang, Y. Pore-forming toxin-like protein complex expressed by frog promotes tissue repair.
Asunto(s)
Proteínas Citotóxicas Formadoras de Poros/metabolismo , Toxinas Biológicas/metabolismo , Cicatrización de Heridas , Animales , Anuros , Línea Celular , Colágeno/metabolismo , Cristalinas/metabolismo , Células Epiteliales/citología , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Fibroblastos/citología , Humanos , Interleucina-1beta/metabolismo , Macrófagos/citología , Masculino , Staphylococcus aureus Resistente a Meticilina/patogenicidad , Ratones , Neutrófilos/citología , Conejos , Piel/lesiones , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/prevención & control , Factor de Crecimiento Transformador beta1/metabolismo , Factores Trefoil/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
BACKGROUND: Nonscar wound healing is a desirable treatment for cutaneous wounds worldwide. Peptide OH-CATH30 (OH30) from king cobra can selectively regulate the innate immunity and create an anti-inflammatory micro-environment which might benefit nonscar wound healing. PURPOSE: To overcome the enzymatic digestion and control release of OH30, OH30 encapsulated in carboxymethyl chitosan nanoparticles (CMCS-OH30 NP) were prepared and their effects on wound healing were evaluated. METHODS: CMCS-OH30 NP were prepared by mild ionic gelation method and properties of the prepared CMCS-OH30 NP were determined by dynamic light scattering. Encapsulation efficiency, stability and release profile of OH30 from prepared CMCS-OH30 NP were determined by HPLC. Cytotoxicity, cell migration and cellular uptake of CMCS-OH30 NP were determined by conventional methods. The effects of prepared CMCS-OH30 NP on the wound healing was investigated by full-thickness excision animal models. RESULTS: The release of encapsulated OH30 from prepared CMCS-OH30 NP was maintained for at least 24 h in a controlled manner. CMCSOH30 NP enhanced the cell migration but had no effects on the metabolism and proliferation of keratinocytes. In the full-thickness excision animal models, the CMCS-OH30 NP treatment significantly accelerated the wound healing compared with CMCS or OH30 administration alone. Histopathological examination suggested that CMCS-OH30 NP promoted wound healing by enhancing the granulation tissue formation through the re-epithelialized and neovascularized composition. CMCS-OH30 NP induced a steady anti-inflammatory cytokine IL10 expression but downregulated the expressions of several pro-inflammatory cytokines. CONCLUSION: The prepared biodegradable drug delivery system accelerates the healing and shows better prognosis because of the combined effects of OH30 released from the nanoparticles.
Asunto(s)
Quitosano/análogos & derivados , Cicatriz/patología , Nanopartículas/química , Péptidos/uso terapéutico , Cicatrización de Heridas/efectos de los fármacos , Animales , Antibacterianos/farmacología , Movimiento Celular/efectos de los fármacos , Quitosano/química , Colágeno/metabolismo , Citocinas/metabolismo , Sistemas de Liberación de Medicamentos , Liberación de Fármacos , Dispersión Dinámica de Luz , Endocitosis , Femenino , Humanos , Queratinocitos/efectos de los fármacos , Ratones , Ratones Desnudos , Pruebas de Sensibilidad Microbiana , Nanopartículas/ultraestructura , Péptidos/farmacología , Células RAW 264.7RESUMEN
Dimethyl sulfoxide (DMSO) is widely used in the laboratory and in clinical situations because it is soluble in both aqueous and organic media and can be used to treat many types of diseases. Thus, it is meaningful to assess the comprehensive and in-depth biological activities of DMSO. Here, we showed that a high concentration of DMSO induced pro-inflammatory cytokine interleukin-1ß (IL-1ß) secretion from the monocytic cell line THP-1. DMSO-induced IL-1ß secretion was dependent on intracellular caspase-1 activation. Further study revealed that the activation of caspase-1 by DMSO relied on NLRP3 inflammasome formation. It is generally accepted that the NLRP3 inflammasome is activated by reactive oxygen species generation or potassium efflux; however, the common NLRP3 inflammasome trigger remains controversial. Here, we showed that although DMSO is a ROS scavenger, this chemical increases membrane permeability and potassium efflux, and the formation of the NLRP3 inflammasome reflects the increased membrane permeability and potassium efflux induced by DMSO. The present study reveals a new characteristic of DMSO, which should be considered when using this chemical in either the laboratory or the clinic.
RESUMEN
Many intracellular pathogens invade cells via endocytic organelles and have adapted to the drop in pH along the endocytic pathway. However, the strategy by which the host cell counteracts this pathogen adaptation remains unclear. ßγ-CAT is an aerolysin-like pore-forming protein and trefoil factor complex in the frog Bombina maxima. We report here that ßγ-CAT, as a host-secreted factor with an intrinsic channel-forming property, is the first example of a molecule that actively neutralizes the acidification of endocytic organelles to counteract Listeria monocytogenes infection. Immunodepletion of endogenous ßγ-CAT largely impaired the control of L. monocytogenes by frog cells. ßγ-CAT elevates the pH of L. monocytogenes-containing vacuoles to limit the vacuole escape of L. monocytogenes to cytosol. Furthermore, ßγ-CAT promotes intracellular L. monocytogenes clearance via autophagy and by that the nonlytic expulsion of the bacteria from host cells. Finally, ßγ-CAT attenuated the dissemination of L. monocytogenes in vivo. These findings reveal a novel host strategy and effectors that combat pathogen adaptation to acidic conditions along the endocytic pathway.
Asunto(s)
Lisosomas/inmunología , Proteínas Citotóxicas Formadoras de Poros/inmunología , Factores Trefoil/inmunología , Animales , Anuros , Autofagia/inmunología , Listeria monocytogenes/inmunología , Listeriosis/inmunología , Listeriosis/microbiología , Lisosomas/microbiología , Proteínas Citotóxicas Formadoras de Poros/metabolismoRESUMEN
The protease-activated receptor 1 (PAR1) is a G-protein-coupled receptor that is irreversibly activated by either thrombin or metalloprotease 1. Due this irrevocable activation, activated internalization and degradation are critical for PAR1 signaling termination. Prohibitin (PHB) is an evolutionarily conserved, ubiquitously expressed, pleiotropic protein and belongs to the stomatin/prohibitin/flotillin/HflK/C (SPFH) domain family. In a previous study, we found that PHB localized on the platelet membrane and participated in PAR1-mediated human platelet aggregation, suggesting that PHB likely regulates the signaling of PAR1. Unfortunately, PHB's exact function in PAR1 internalization and degradation is unclear. In the current study, flow cytometry revealed that PHB expressed on the surface of endothelial cells (HUVECs) but not cancer cells (MDA-MB-231). Further confocal microscopy revealed that PHB dynamically associates with PAR1 in a time-dependent manner following induction with PAR1-activated peptide (PAR1-AP), though differently between HUVECs and MDA-MB-231 cells. Depletion of PHB by RNA interference significantly inhibited PAR1 activated internalization and led to sustained Erk1/2 phosphorylation in the HUVECs; however, a similar effect was not observed in MDA-MB-231 cells. For both the endothelial and cancel cells, PHB repressed PAR1 degradation, while knockdown of PHB led to increased PAR1 degradation, and PHB overexpression inhibited PAR1 degradation. These results suggest that persistent PAR1 signaling due to the absence of membrane PHB and decreased PAR1 degradation caused by the upregulation of intracellular PHB in cancer cells (such as MDA-MB-231 cells) may render cells highly invasive. As such, PHB may be a novel target in future anti-cancer therapeutics, or in more refined cancer malignancy diagnostics.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/genética , Receptor PAR-1/genética , Proteínas Represoras/genética , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Microscopía Confocal , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Especificidad de Órganos , Péptidos/farmacología , Prohibitinas , Transporte de Proteínas/efectos de los fármacos , Proteolisis/efectos de los fármacos , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptor PAR-1/antagonistas & inhibidores , Receptor PAR-1/metabolismo , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/metabolismo , Transducción de SeñalRESUMEN
Aerolysins are virulence factors belonging to the bacterial ß-pore-forming toxin superfamily. Surprisingly, numerous aerolysin-like proteins exist in vertebrates, but their biological functions are unknown. ßγ-CAT, a complex of an aerolysin-like protein subunit (two ßγ-crystallin domains followed by an aerolysin pore-forming domain) and two trefoil factor subunits, has been identified in frogs (Bombina maxima) skin secretions. Here, we report the rich expression of this protein, in the frog blood and immune-related tissues, and the induction of its presence in peritoneal lavage by bacterial challenge. This phenomena raises the possibility of its involvement in antimicrobial infection. When ßγ-CAT was administrated in a peritoneal infection model, it greatly accelerated bacterial clearance and increased the survival rate of both frogs and mice. Meanwhile, accelerated Interleukin-1ß release and enhanced local leukocyte recruitments were determined, which may partially explain the robust and effective antimicrobial responses observed. The release of interleukin-1ß was potently triggered by ßγ-CAT from the frog peritoneal cells and murine macrophages in vitro. ßγ-CAT was rapidly endocytosed and translocated to lysosomes, where it formed high molecular mass SDS-stable oligomers (>170 kDa). Lysosomal destabilization and cathepsin B release were detected, which may explain the activation of caspase-1 inflammasome and subsequent interleukin-1ß maturation and release. To our knowledge, these results provide the first functional evidence of the ability of a host-derived aerolysin-like protein to counter microbial infection by eliciting rapid and effective host innate immune responses. The findings will also largely help to elucidate the possible involvement and action mechanisms of aerolysin-like proteins and/or trefoil factors widely existing in vertebrates in the host defense against pathogens.
Asunto(s)
Interacciones Huésped-Patógeno/inmunología , Péptidos/inmunología , Proteínas Citotóxicas Formadoras de Poros/inmunología , Secuencia de Aminoácidos , Animales , Anuros/genética , Anuros/inmunología , Anuros/microbiología , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Toxinas Bacterianas/inmunología , Comamonas , Endocitosis , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/prevención & control , Infecciones por Bacterias Gramnegativas/inmunología , Infecciones por Bacterias Gramnegativas/prevención & control , Inmunidad Innata , Inflamasomas/inmunología , Interleucina-1beta/biosíntesis , Lisosomas/inmunología , Masculino , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/química , Péptidos/genética , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/genética , Conformación Proteica , Homología de Secuencia de Aminoácido , Piel/inmunología , Piel/microbiología , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/prevención & control , Factor Trefoil-2RESUMEN
The therapeutic potential of antimicrobial peptides (AMPs) has been evaluated in many infectious diseases. However, the topical application of AMPs for ocular bacterial infection has not been well investigated. The AMP OH-CATH30, which was identified in the king cobra, exhibits potent antimicrobial activity. In this study, we investigated the therapeutic potential of OH-CATH30 for Pseudomonas aeruginosa keratitis. Ten isolates of P. aeruginosa from individuals with keratitis were susceptible to OH-CATH30 but not to cefoperazone, ciprofloxacin, gentamicin, and levofloxacin. The microdilution checkerboard assay showed that OH-CATH30 exhibited synergistic activity with ciprofloxacin and levofloxacin against antibiotic-resistant P. aeruginosa. Meanwhile, P. aeruginosa did not develop resistance to OH-CATH30, even after exposure at 0.5× the MIC for up to 25 subcultures. Furthermore, treatment with OH-CATH30, alone or in combination with levofloxacin, significantly improved the clinical outcomes of rabbit keratitis induced by antibiotic-resistant P. aeruginosa. Taken together, our data indicate that the topical application of OH-CATH30 is efficacious against drug-resistant P. aeruginosa keratitis. In addition, our study highlights the potential application of AMPs in treating ocular bacterial infections.
Asunto(s)
Antiinfecciosos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Infecciones Bacterianas del Ojo/tratamiento farmacológico , Queratitis/tratamiento farmacológico , Infecciones por Pseudomonas/tratamiento farmacológico , Pseudomonas aeruginosa/efectos de los fármacos , Administración Tópica , Animales , Antibacterianos/farmacología , Ciprofloxacina/farmacología , Modelos Animales de Enfermedad , Farmacorresistencia Bacteriana , Sinergismo Farmacológico , Infecciones Bacterianas del Ojo/microbiología , Gentamicinas/farmacología , Humanos , Queratitis/microbiología , Levofloxacino/farmacología , Pruebas de Sensibilidad Microbiana , Infecciones por Pseudomonas/microbiología , ConejosRESUMEN
Sepsis, which is a systemic inflammatory response that follows a bacterial infection, has a high mortality rate and limited therapeutic options. Here we show that the antimicrobial peptide OH-CATH30, which naturally occurs in snake, selectively regulates the innate immune response to protect mice from lethal sepsis. The administration of OH-CATH30 significantly improves the survival rate of mice infected by antibiotic-susceptible and -resistant pathogens, including Escherichia coli , Pseudomonas aeruginosa , and Staphylococcus aureus . OH-CATH30 selectively up-regulates the production of chemokines and cytokines without harmful immune response. Recruitment of monocytes, macrophages, and neutrophils to the infection site is pivotal to the protective capacity of OH-CATH30. Furthermore, the alternative activation of the innate immune response by OH-CATH30 depends on p38 mitogen-activated protein kinase signaling. Taken together, our study demonstrates that OH-CATH30, a naturally occurring antimicrobial peptide, selectively stimulates the innate immune response to protect against sepsis.
Asunto(s)
Antibacterianos/farmacología , Productos Biológicos/farmacología , Inmunidad Innata/efectos de los fármacos , Inmunomodulación/efectos de los fármacos , Péptidos/farmacología , Sepsis/inmunología , Secuencia de Aminoácidos , Animales , Antibacterianos/química , Productos Biológicos/química , Línea Celular , Quimiocina CXCL2/metabolismo , Farmacorresistencia Bacteriana/efectos de los fármacos , Femenino , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Datos de Secuencia Molecular , Monocitos/efectos de los fármacos , Monocitos/inmunología , Monocitos/metabolismo , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Neutrófilos/metabolismo , Péptidos/química , Sepsis/prevención & control , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
It has been established that Adenosine-5'-triphosphate (ATP) can activate the NLRP3 inflammasome. However, the physiological effect of extracellular ATP on NLRP3 inflammasome activation has not yet been investigated. In the present study, we found that ATP was indeed released during bacterial infection. By using a murine peritonitis model, we also found that ATP promotes the fight against bacterial infection in mice. ATP induced the secretion of IL-1ß and chemokines by murine bone marrow-derived macrophages in vitro. Furthermore, the intraperitoneal injection of ATP elevated the levels of IL-1ß and chemokines in the mouse peritoneal lavage. Neutrophils were rapidly recruited to the peritoneum after ATP injection. In addition, the effects on cytokine and chemokine secretion and neutrophil recruitment were markedly attenuated by the pre-administration of the caspase-1 inhibitor Ac-YVAD-cho. Ac-YVAD-cho also significantly attenuated the protective effect of ATP against bacterial infection. In the present study, we demonstrated a protective role for ATP during bacterial infection and this effect was related to NLRP3 inflammasome activation. Together, these results suggest a role for ATP in initiating the immune response in hosts suffering from infections.
Asunto(s)
Adenosina Trifosfato/inmunología , Adenosina Trifosfato/metabolismo , Infecciones Bacterianas/prevención & control , Proteínas Portadoras/metabolismo , Inflamasomas/inmunología , Infiltración Neutrófila/inmunología , Animales , Infecciones Bacterianas/inmunología , Infecciones Bacterianas/metabolismo , Proteínas Portadoras/inmunología , Caspasa 1/inmunología , Caspasa 1/metabolismo , Quimiocina CXCL2/inmunología , Quimiocina CXCL2/metabolismo , Inflamasomas/metabolismo , Interleucina-1beta/inmunología , Interleucina-1beta/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Ratones , Proteína con Dominio Pirina 3 de la Familia NLR , Neutrófilos/inmunología , Neutrófilos/metabolismo , Peritonitis/inmunología , Peritonitis/metabolismoRESUMEN
Antimicrobial peptides (AMPs) have been considered alternatives to conventional antibiotics for drug-resistant bacterial infections. However, their comparatively high toxicity toward eukaryotic cells and poor efficacy in vivo hamper their clinical application. OH-CATH30, a novel cathelicidin peptide deduced from the king cobra, possesses potent antibacterial activity in vitro. The objective of this study is to evaluate the efficacy of OH-CATH30 and its analog OH-CM6 against drug-resistant bacteria in vitro and in vivo. The MICs of OH-CATH30 and OH-CM6 ranged from 1.56 to 12.5 µg/ml against drug-resistant clinical isolates of several pathogenic species, including Escherichia coli, Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus. The MICs of OH-CATH30 and OH-CM6 were slightly altered in the presence of 25% human serum. OH-CATH30 and OH-CM6 killed E. coli quickly (within 60 min) by disrupting the bacterial cytoplasmic membrane. Importantly, the 50% lethal doses (LD(50)) of OH-CATH30 and OH-CM6 in mice following intraperitoneal (i.p.) injection were 120 mg/kg of body weight and 100 mg/kg, respectively, and no death was observed at any dose up to 160 mg/kg following subcutaneous (s.c.) injection. Moreover, 10 mg/kg OH-CATH30 or OH-CM6 significantly decreased the bacterial counts as well as the inflammatory response in a mouse thigh infection model and rescued infected mice in a bacteremia model induced by drug-resistant E. coli. Taken together, our findings demonstrate that the natural cathelicidin peptide OH-CATH30 and its analogs exhibit relatively low toxicity and potent efficacy in mouse models, indicating that they may have therapeutic potential against the systemic infections caused by drug-resistant bacteria.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/farmacología , Animales , Péptidos Catiónicos Antimicrobianos/síntesis química , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/uso terapéutico , Infecciones Bacterianas/tratamiento farmacológico , Células Cultivadas , Farmacorresistencia Bacteriana , Escherichia coli/efectos de los fármacos , Hemólisis , Humanos , Masculino , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Ratones , Pruebas de Sensibilidad Microbiana , Pseudomonas aeruginosa/efectos de los fármacos , CatelicidinasRESUMEN
Animal models are essential for the development of new anti-infectious drugs. Although some bacterial infection models have been established in rodents, small primate models are rare. Here, we report on two bacterial infection models established in tree shrew (Tupaia belangeri chinensis). A burnt skin infection model was induced by dropping 5×10(6) CFU of Staphylococcus aureus on the surface of a wound after a third degree burn. This dose of S. aureus caused persistent infection for 7 days and obvious inflammatory response was observed 4 days after inoculation. A Dacron graft infection model, 2×10(6) CFU of Pseudomonas aeruginosa also caused persistent infection for 6 days, with large amounts of pus observed 3 days after inoculation. These models were used to evaluate the efficacy of levofloxacin (LEV) and cefoperazone (CPZ), which reduced the viable bacteria in skin to 4log10 and 5log10 CFU/100 mg tissue, respectively. The number of bacteria in graft was significantly reduced by 4log10 CFU/mL treatment compared to the untreated group (P<0.05). These results suggest that two bacterial infection models were successfully established in tree shrew using P. aeruginosa and S. aureus. In addition, tree shrew was susceptible to P. aeruginosa and S. aureus, thus making it an ideal bacterial infection animal model for the evaluation of new antimicrobials.