RESUMEN
The virulence behaviors of many Gram-negative bacterial pathogens are governed by quorum-sensing (QS), a hierarchical system of gene regulation that relies on population density by producing and detecting extracellular signaling molecules. Although extensively studied under in vitro conditions, adaptation of QS system to physiologically relevant host environment is not fully understood. In this study, we investigated the influence of lung environment on the regulation of Pseudomonas aeruginosa virulence factors by QS in a mouse model of acute pneumonia. When cultured under laboratory conditions in lysogeny broth, wild-type P. aeruginosa strain PAO1 began to express QS-regulated virulence factors elastase B (LasB) and rhamnolipids (RhlA) during transition from late-exponential into stationary growth phase. In contrast, during acute pneumonia as well as when cultured in mouse bronchial alveolar lavage fluids (BALF), exponential phase PAO1 bacteria at low population density prematurely expressed QS regulatory genes lasI-lasR and rhlI-rhlR and their downstream virulence genes lasB and rhlA. Further analysis indicated that surfactant phospholipids were the primary components within BALF that induced the synthesis of N-(3-oxododecanoyl)-L-homoserine lactone (C12-HSL), which triggered premature expression of LasB and RhlA. Both phenol extraction and phospholipase A2 digestion abolished the ability of mouse BALF to promote LasB and RhlA expression. In contrast, provision of the major surfactant phospholipid dipalmitoylphosphatidylcholine (DPPC) restored the expression of both virulence factors. Collectively, our study demonstrates P. aeruginosa modulates its QS to coordinate the expression of virulence factors during acute pneumonia by recognizing pulmonary surfactant phospholipids.
Asunto(s)
Fosfolípidos/metabolismo , Pseudomonas aeruginosa/patogenicidad , Percepción de Quorum , Factores de Virulencia/genética , Animales , Proteínas Bacterianas/genética , Estudios de Cohortes , Femenino , Regulación Bacteriana de la Expresión Génica , Masculino , Ratones , Neumonía Bacteriana/microbiología , Pseudomonas aeruginosa/genética , Surfactantes Pulmonares/metabolismo , Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
Pseudomonas aeruginosa is a major bacterial pathogen commonly associated with chronic lung infections in cystic fibrosis (CF). Previously, we have demonstrated that the type IV pilus (Tfp) of P. aeruginosa mediates resistance to antibacterial effects of pulmonary surfactant protein A (SP-A). Interestingly, P. aeruginosa strains with group I pilins are O-glycosylated through the TfpO glycosyltransferase with a single subunit of O-antigen (O-ag). Importantly, TfpO-mediated O-glycosylation is important for virulence in mouse lungs, exemplified by more frequent lung infection in CF with TfpO-expressing P. aeruginosa strains. However, the mechanism underlying the importance of Tfp glycosylation in P. aeruginosa pathogenesis is not fully understood. Here, we demonstrated one mechanism of increased fitness mediated by O-glycosylation of group 1 pilins on Tfp in the P. aeruginosa clinical isolate 1244. Using an acute pneumonia model in SP-A+/+ versus SP-A-/- mice, the O-glycosylation-deficient ΔtfpO mutant was found to be attenuated in lung infection. Both 1244 and ΔtfpO strains showed equal levels of susceptibility to SP-A-mediated membrane permeability. In contrast, the ΔtfpO mutant was more susceptible to opsonization by SP-A and by other pulmonary and circulating opsonins, SP-D and mannose binding lectin 2, respectively. Importantly, the increased susceptibility to phagocytosis was abrogated in the absence of opsonins. These results indicate that O-glycosylation of Tfp with O-ag specifically confers resistance to opsonization during host-mediated phagocytosis.
Asunto(s)
Fimbrias Bacterianas/inmunología , Antígenos O/inmunología , Fagocitosis/inmunología , Pseudomonas aeruginosa/inmunología , Proteína A Asociada a Surfactante Pulmonar/inmunología , Animales , Línea Celular , Fimbrias Bacterianas/genética , Fimbrias Bacterianas/metabolismo , Glicosilación , Glicosiltransferasas/metabolismo , Evasión Inmune , Pulmón/inmunología , Pulmón/microbiología , Enfermedades Pulmonares/inmunología , Enfermedades Pulmonares/microbiología , Macrófagos/inmunología , Lectina de Unión a Manosa/inmunología , Ratones , Ratones Endogámicos C3H , Ratones Noqueados , Antígenos O/metabolismo , Infecciones por Pseudomonas/inmunología , Infecciones por Pseudomonas/patología , Pseudomonas aeruginosa/patogenicidad , Proteína A Asociada a Surfactante Pulmonar/genética , Proteína D Asociada a Surfactante Pulmonar/inmunologíaRESUMEN
Aberrant mucin secretion and accumulation in the airway lumen are clinical hallmarks associated with various lung diseases such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis. Mycoplasma pneumoniae, long appreciated as one of the triggers of acute exacerbations of chronic pulmonary diseases, has recently been reported to promote excessive mucus secretion. However, the mechanism of mucin overproduction induced by M. pneumoniae remains unclear. This study aimed to determine the mechanism by which M. pneumoniae induces mucus hypersecretion by using M. pneumoniae infection of mouse lungs, human primary bronchial epithelial (NHBE) cells cultured at the air-liquid interface, and the conventionally cultured airway epithelial NCI-H292 cell line. We demonstrated that M. pneumoniae induced the expression of mucins MUC5AC and MUC5B by activating the STAT6-STAT3 and epidermal growth factor receptor (EGFR) signal pathways, which in turn downregulated FOXA2, a transcriptional repressor of mucin biosynthesis. The upstream stimuli of these pathways, including interleukin-4 (IL-4), IL-6, and IL-13, increased dramatically upon exposure to M. pneumoniae. Inhibition of the STAT6, STAT3, and EGFR signaling pathways significantly restored the expression of FOXA2 and attenuated the expression of airway mucins MUC5AC and MUC5B. Collectively, these studies demonstrated that M. pneumoniae induces airway mucus hypersecretion by modulating the STAT/EGFR-FOXA2 signaling pathways.
Asunto(s)
Interacciones Huésped-Patógeno , Mucinas/metabolismo , Mycoplasma pneumoniae/fisiología , Transducción de Señal , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Receptores ErbB/metabolismo , Perfilación de la Expresión Génica , Factor Nuclear 3-beta del Hepatocito/metabolismo , Humanos , Ratones Endogámicos C57BL , Mucina 5AC/metabolismo , Mucina 5B/metabolismo , Neumonía por Mycoplasma/microbiología , Neumonía por Mycoplasma/patología , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción STAT6/metabolismoRESUMEN
Pseudomonas aeruginosa(PA) is a Gram-negative bacterial pathogen commonly associated with chronic lung infections. Previously, we have identified several PA virulence factors that are important for resistance to the surfactant protein-A (SP-A), a pulmonary innate immunity protein that mediates bacterial opsonization and membrane permeabilization. In this study, we demonstrate that the type IV pilus (Tfp) is important in the resistance of PA to the antibacterial effects of SP-A. The Tfp-deficient mutant ΔpilA is severely attenuated in an acute pneumonia model of infection in the lungs of wild-type mice, but is virulent in the lungs of SP-A(-/-) mice. The ΔpilA bacteria are more susceptible to SP-A-mediated aggregation and opsonization. In addition, the integrity of the outer membranes of ΔpilA bacteria is compromised, rendering them more susceptible to SP-A-mediated membrane permeabilization. By comparing Tfp extension and retraction mutants, we demonstrate that the increased susceptibility of ΔpilA to SP-A-mediated opsonization requires the total absence of Tfp from PA cells. Finally, we provide evidence of increased expression of nonpilus adhesin OprH that may serve as an SP-A ligand, resulting in increased phagocytosis and preferential pulmonary clearance of ΔpilA.
Asunto(s)
Proteínas Fimbrias/inmunología , Pulmón/inmunología , Pseudomonas aeruginosa/inmunología , Proteína A Asociada a Surfactante Pulmonar/inmunología , Animales , Antiinfecciosos/inmunología , Antiinfecciosos/metabolismo , Antiinfecciosos/farmacología , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas de la Membrana Bacteriana Externa/metabolismo , Línea Celular , Modelos Animales de Enfermedad , Farmacorresistencia Bacteriana/genética , Farmacorresistencia Bacteriana/inmunología , Proteínas Fimbrias/genética , Proteínas Fimbrias/metabolismo , Fimbrias Bacterianas/inmunología , Fimbrias Bacterianas/metabolismo , Fimbrias Bacterianas/ultraestructura , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Humanos , Ligandos , Pulmón/metabolismo , Pulmón/microbiología , Macrófagos/inmunología , Ratones Endogámicos C3H , Ratones Noqueados , Viabilidad Microbiana/efectos de los fármacos , Viabilidad Microbiana/genética , Viabilidad Microbiana/inmunología , Microscopía Electrónica de Transmisión , Mutación , Fagocitosis/inmunología , Neumonía/genética , Neumonía/inmunología , Neumonía/microbiología , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/fisiología , Proteína A Asociada a Surfactante Pulmonar/genética , Proteína A Asociada a Surfactante Pulmonar/metabolismo , Factores de Virulencia/genética , Factores de Virulencia/inmunología , Factores de Virulencia/metabolismoRESUMEN
Aerosolized or aspirated manufactured carbon nanotubes have been shown to be cytotoxic, cause pulmonary lesions, and demonstrate immunomodulatory properties. CD-1 mice were used to assess pulmonary toxicity of helical carbon nanotubes (HCNTs) and alterations of the immune response to subsequent infection by Pseudomonas aeruginosa in mice. HCNTs provoked a mild inflammatory response following either a single exposure or 2X/week for three weeks (multiple exposures) but were not significantly toxic. Administering HCNTs 2X/week for three weeks resulted in pulmonary lesions including granulomas and goblet cell hyperplasia. Mice exposed to HCNTs and subsequently infected by P. aeruginosa demonstrated an enhanced inflammatory response to P. aeruginosa and phagocytosis by alveolar macrophages was inhibited. However, clearance of P. aeruginosa was not affected. HCNT exposed mice depleted of neutrophils were more effective in clearing P. aeruginosa compared to neutrophil-depleted control mice, accompanied by an influx of macrophages. Depletion of systemic macrophages resulted in slightly inhibited bacterial clearance by HCNT treated mice. Our data indicate that pulmonary exposure to HCNTs results in lesions similar to those caused by other nanotubes and pre-exposure to HCNTs inhibit alveolar macrophage phagocytosis of P. aeruginosa. However, clearance was not affected as exposure to HCNTs primed the immune system for an enhanced inflammatory response to pulmonary infection consisting of an influx of neutrophils and macrophages.
Asunto(s)
Macrófagos Alveolares/microbiología , Nanotubos de Carbono , Fagocitosis/efectos de los fármacos , Pseudomonas aeruginosa/inmunología , Animales , Pulmón/inmunología , Pulmón/microbiología , Ratones , Neutrófilos/inmunologíaRESUMEN
The redox-active pyocyanin (PCN) secreted by the respiratory pathogen Pseudomonas aeruginosa generates reactive oxygen species (ROS) and causes oxidative stress to pulmonary epithelial cells. Nuclear factor (erythroid-derived 2)-like 2 (NRF2) confers protection against ROS-mediated cell death by inducing the expression of detoxifying enzymes and proteins via its binding to the cis-acting antioxidant response element (ARE). However, a clear relationship between NRF2 and PCN-mediated oxidative stress has not been established experimentally. In this study, we investigated the induction of NRF2-ARE response by PCN in the pulmonary epithelial cells. We analyzed the effect of PCN on NRF2 expression and nuclear translocation in cultured human airway epithelial cells, and in a mouse model of chronic PCN exposure. NRF2-dependent transcription of antioxidative enzymes was also assessed. Furthermore, we used inhibitors to examine the involvement of EGFR and its downstream signaling components that mediate NRF2-ARE-activation in response to PCN. PCN enhances the nuclear NRF2 accumulation and activates the transcription of ARE-mediated antioxidant genes. Furthermore, PCN activates NRF2 by inducing the EGFR-phosphoinositide-3-kinase (PI3K) signaling pathway and its main downstream effectors, AKT and MEK1/2-ERK1/2 MAP kinases. Inhibition of the EGFR-PI3K signaling markedly attenuates PCN-stimulated NRF2 accumulation in the nucleus. We demonstrate for the first time that PCN-mediated oxidative stress activates the EGFR-PI3K-AKT/MEK1/2-ERK1/2 MAP kinase signaling pathway, leading to nuclear NRF2 translocation and ARE responsiveness in pulmonary epithelial cells.
Asunto(s)
Elementos de Respuesta Antioxidante , Núcleo Celular/metabolismo , Células Epiteliales/metabolismo , Receptores ErbB/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Pulmón/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Factor 2 Relacionado con NF-E2/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Pseudomonas aeruginosa/química , Piocianina/farmacología , Especies Reactivas de Oxígeno/metabolismo , Transcripción Genética/efectos de los fármacos , Transporte Activo de Núcleo Celular/efectos de los fármacos , Animales , Línea Celular , Células Epiteliales/citología , Humanos , Pulmón/citología , Ratones , Estrés Oxidativo/efectos de los fármacos , Piocianina/químicaRESUMEN
BACKGROUND: The redox-active pyocyanin (PCN) is a toxic, secondary metabolite secreted by the respiratory pathogen Pseudomonas aeruginosa (PA). Previously, we have shown that mouse lungs chronically exposed to PCN develop goblet cell hyperplasia and metaplasia (GCHM) and mucus hypersecretion, fibrosis and emphysema. These pathological features are commonly found in the airways of several chronic lung diseases, including cystic fibrosis (CF), as well as in mouse airways deficient in the forkhead box A2 (FOXA2), a transcriptional repressor of goblet GCHM and mucus biosynthesis. Furthermore, PCN inhibits FOXA2 by activating the pro-GCHM signaling pathways Stat6 and EGFR. However, it is not known whether PCN-generated reactive oxygen (ROS) and nitrogen (RNS) species posttranslationally modify and inactivate FOXA2. METHODS: We examined the posttranslational modifications of FOXA2 by PCN using specific antibodies against oxidation, nitrosylation, acetylation and ubiquitination. Electrophoretic mobility shift assay (EMSA) was used to examine the ability of modified FOXA2 to bind the promoter of MUC5B mucin gene. In addition, we used quantitative real time PCR, ELISA, immunofluorescence and mouse lung infection to assess whether the loss of FOXA2 function caused GCHM and mucin overexpression. Finally, we examined the restoration of FOXA2 function by the antioxidant glutathione (GSH). RESULTS: We found that PCN-generated ROS/RNS caused nitrosylation, acetylation, ubiquitination and degradation of FOXA2. Modified FOXA2 had reduced ability to bind the promoter of the MUC5B gene. The antioxidant GSH alleviated the modification of FOXA2 by PCN, and inhibited the overexpression of MUC5AC and MUC5B mucins. CONCLUSION: These results suggest that PCN-mediated posttranslational modifications of FOXA2 are positively correlated with GCHM and overexpression of airway mucins. Furthermore, antioxidant treatment restores the function of FOXA2 to attenuate GCHM and mucus hypersecretion.
Asunto(s)
Factor Nuclear 3-beta del Hepatocito/metabolismo , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Mucina 5AC/metabolismo , Mucina 5B/metabolismo , Piocianina/farmacología , Animales , Antioxidantes/farmacología , Secuencia de Bases , Carcinoma Mucoepidermoide/metabolismo , Carcinoma Mucoepidermoide/patología , Línea Celular , Línea Celular Tumoral , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Glutatión/farmacología , Factor Nuclear 3-beta del Hepatocito/análisis , Humanos , Pulmón/citología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Datos de Secuencia Molecular , Oxidación-Reducción , Especies de Nitrógeno Reactivo/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The redox-active exotoxin pyocyanin (PCN) can be recovered in 100 µM concentrations in the sputa of bronchiectasis patients chronically infected with Pseudomonas aeruginosa (PA). However, the importance of PCN within bronchiectatic airways colonized by PA remains unrecognized. Recently, we have shown that PCN is required for chronic PA lung infection in mice, and that chronic instillation of PCN induces goblet cell hyperplasia (GCH), pulmonary fibrosis, emphysema and influx of immune cells in mouse airways. Many of these pathological features are strikingly similar to the mouse airways devoid of functional FoxA2, a transcriptional repressor of GCH and mucus biosynthesis. In this study, we postulate that PCN causes and exacerbates GCH and mucus hypersecretion in bronchiectatic airways chronically infected by PA by inactivating FoxA2. We demonstrate that PCN represses the expression of FoxA2 in mouse airways and in bronchial epithelial cells cultured at an air-liquid interface or conventionally, resulting in GCH, increased MUC5B mucin gene expression and mucus hypersecretion. Immunohistochemical and inhibitor studies indicate that PCN upregulates the expression of Stat6 and EGFR, both of which in turn repress the expression of FoxA2. These studies demonstrate that PCN induces GCH and mucus hypersecretion by inactivating FoxA2.
Asunto(s)
Células Caliciformes/microbiología , Factor Nuclear 3-beta del Hepatocito/genética , Pulmón/patología , Pseudomonas aeruginosa/fisiología , Piocianina/metabolismo , Animales , Línea Celular Tumoral , Regulación hacia Abajo , Receptores ErbB/metabolismo , Células Caliciformes/metabolismo , Células Caliciformes/patología , Factor Nuclear 3-beta del Hepatocito/metabolismo , Interacciones Huésped-Patógeno , Humanos , Hiperplasia , Pulmón/microbiología , Metaplasia , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mucina 5B/genética , Mucina 5B/metabolismo , Moco/metabolismo , Pseudomonas aeruginosa/metabolismo , Piocianina/farmacología , Factor de Transcripción STAT6/genética , Factor de Transcripción STAT6/metabolismo , Transducción de Señal , Activación TranscripcionalRESUMEN
Pseudomonas aeruginosa is an opportunistic pathogen that causes both acute pneumonitis in immunocompromised patients and chronic lung infections in individuals with cystic fibrosis and other bronchiectasis. Over 75% of clinical isolates of P. aeruginosa secrete elastase B (LasB), an elastolytic metalloproteinase that is encoded by the lasB gene. Previously, in vitro studies have demonstrated that LasB degrades a number of components in both the innate and adaptive immune systems. These include surfactant proteins, antibacterial peptides, cytokines, chemokines and immunoglobulins. However, the contribution of LasB to lung infection by P. aeruginosa and to inactivation of pulmonary innate immunity in vivo needs more clarification. In this study, we examined the mechanisms underlying enhanced clearance of the ΔlasB mutant in mouse lungs. The ΔlasB mutant was attenuated in virulence when compared to the wild-type strain PAO1 during lung infection in SP-A+/+ mice. However, the ΔlasB mutant was as virulent as PAO1 in the lungs of SP-Aâ»/â» mice. Detailed analysis showed that the ΔlasB mutant was more susceptible to SP-A-mediated opsonization but not membrane permeabilization. In vitro and in vivo phagocytosis experiments revealed that SP-A augmented the phagocytosis of ΔlasB mutant bacteria more efficiently than the isogenic wild-type PAO1. The ΔlasB mutant was found to have a severely reduced ability to degrade SP-A, consequently making it unable to evade opsonization by the collectin during phagocytosis. These results suggest that P. aeruginosa LasB protects against SP-A-mediated opsonization by degrading the collectin.
Asunto(s)
Proteínas Bacterianas/metabolismo , Pulmón/microbiología , Metaloendopeptidasas/metabolismo , Fagocitosis/fisiología , Infecciones por Pseudomonas/metabolismo , Pseudomonas aeruginosa/enzimología , Proteína A Asociada a Surfactante Pulmonar/fisiología , Animales , Proteínas Bacterianas/genética , Western Blotting , Permeabilidad de la Membrana Celular , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Pulmón/inmunología , Pulmón/metabolismo , Macrófagos/citología , Macrófagos/metabolismo , Metaloendopeptidasas/genética , Ratones , Ratones Endogámicos C3H , Ratones Noqueados , Mutación/genética , Proteínas Opsoninas/metabolismo , Infecciones por Pseudomonas/patología , Infecciones por Pseudomonas/prevención & controlRESUMEN
Surfactant protein-A (SP-A) is an important antimicrobial protein that opsonizes and permeabilizes membranes of microbial pathogens in mammalian lungs. Previously, we have shown that Pseudomonas aeruginosa flagellum-deficient mutants are preferentially cleared in the lungs of wild-type mice by SP-A-mediated membrane permeabilization, and not by opsonization. In this study, we report a flagellum-mediated mechanism of P. aeruginosa resistance to SP-A. We discovered that flagellum-deficient (ΔfliC) bacteria are unable to produce adequate amounts of exoproteases to degrade SP-A in vitro and in vivo, leading to its preferential clearance in the lungs of SP-A(+/+) mice. In addition, ΔfliC bacteria failed to degrade another important lung antimicrobial protein lysozyme. Detailed analyses showed that ΔfliC bacteria are unable to upregulate the transcription of lasI and rhlI genes, impairing the production of homoserine lactones necessary for quorum-sensing, an important virulence process that regulates the production of multiple exoproteases. Thus, reduced ability of ΔfliC bacteria to quorum-sense attenuates production of exoproteases and limits degradation of SP-A, thereby conferring susceptibility to this major pulmonary host defence protein.
Asunto(s)
Proteínas Bacterianas/inmunología , Exopeptidasas/inmunología , Flagelos/inmunología , Pulmón/inmunología , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa/enzimología , Proteína A Asociada a Surfactante Pulmonar/inmunología , Percepción de Quorum , Animales , Proteínas Bacterianas/genética , Exopeptidasas/genética , Femenino , Flagelos/genética , Humanos , Pulmón/microbiología , Masculino , Ratones , Ratones Noqueados , Infecciones por Pseudomonas/genética , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/inmunología , Pseudomonas aeruginosa/fisiología , Proteína A Asociada a Surfactante Pulmonar/genéticaRESUMEN
The cystic fibrosis (CF) airway bacterial pathogen Pseudomonas aeruginosa secretes multiple virulence factors. Among these, the redox active exotoxin pyocyanin (PCN) is produced in concentrations up to 100 mumol/L during infection of CF and other bronchiectatic airways. However, the contributions of PCN during infection of bronchiectatic airways are not appreciated. In this study, we demonstrate that PCN is critical for chronic infection in mouse airways and orchestrates adaptive immune responses that mediate lung damage. Wild-type FVBN mice chronically exposed to PCN developed goblet cell hyperplasia and metaplasia, airway fibrosis, and alveolar airspace destruction. Furthermore, after 12 weeks of exposure to PCN, mouse lungs down-regulated the expression of T helper (Th) type 1 cytokines and polarized toward a Th2 response. Cellular analyses indicated that chronic exposure to PCN profoundly increased the lung population of recruited macrophages, CD4(+) T cells, and neutrophils responsible for the secretion of these cytokines. PCN-mediated goblet cell hyperplasia and metaplasia required Th2 cytokine signaling through the Stat6 pathway. In summary, this study establishes that PCN is an important P. aeruginosa virulence factor capable of directly inducing pulmonary pathophysiology in mice, consistent with changes observed in CF and other bronchiectasis lungs.
Asunto(s)
Fibrosis Quística/microbiología , Infecciones por Pseudomonas/complicaciones , Piocianina/toxicidad , Infecciones del Sistema Respiratorio/patología , Animales , Fibrosis Quística/inmunología , Citocinas/biosíntesis , Citocinas/inmunología , Ensayo de Inmunoadsorción Enzimática , Células Caliciformes/patología , Hiperplasia , Inmunohistoquímica , Ratones , Infecciones por Pseudomonas/inmunología , Infecciones por Pseudomonas/patología , Pseudomonas aeruginosa , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/inmunología , Fibrosis Pulmonar/patología , Infecciones del Sistema Respiratorio/inmunología , Infecciones del Sistema Respiratorio/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/inmunología , Células TH1/inmunología , Células Th2/inmunología , Factores de Virulencia/toxicidadRESUMEN
BACKGROUND: To study the full length L and M sequence of Hantavirus Q32 strain gene and explore its molecular characters. METHODS: The L and M segment cDNA of Hantavirus Q32 strain was amplified by RT-PCR. The purified PCR products were sequenced directly or cloned into pGEM-T Vector and then sequenced. RESULTS: The L genome segment of Q32 virus was found to be 6533 nucleotides in length. One large open reading frame was found located at bases 38 to 6493. This was predicted to encode an L protein 2151 amino acids in length with a molecular mass of 2.46 x 10(5). The M genome segment was 3616 nucleotides in length. One open reading frame was located at bases 41 to 3488. This was predicted to encode an M protein 1135 amino acids with a molecular mass of 1.26 x 10(5). CONCLUSION: The nucleotides sequence of M and L segments of strain Q32 was similar to that of other Hantavirus M and L segments. Deduced amino acid sequences of glycoprotein and RNA polymerase revealed high homologue to other Hantavirus.