RESUMEN
Cystic fibrosis (CF) patients are extremely vulnerable to Burkholderia cepacia complex (Bcc) infections. However, the underlying etiology is poorly understood. We tested the hypothesis that short palate lung and nasal epithelial clone 1 (SPLUNC1)-epithelial sodium channel (ENaC) interactions at the plasma membrane are required to reduce Bcc burden in normal airways. To determine if SPLUNC1 was needed to reduce Bcc burden in the airways, SPLUNC1 knockout mice and their wild-type littermates were infected with B. cenocepacia strain J2315. SPLUNC1 knockout mice had increased bacterial burden in the lungs compared to wild-type littermate mice. SPLUNC1-knockdown primary human bronchial epithelia (HBECs) were incubated with J2315, which resulted in increased bacterial burden compared to non-transduced HBECs. We next determined the interaction of the SPLUNC1-ENaC complex during J2315 infection. SPLUNC1 remained at the apical plasma membrane of normal HBECs but less was present at the apical plasma membrane of CF HBECs. Additionally, SPLUNC1-ßENaC complexes reduced intracellular J2315 burden. Our data indicate that (i) secreted SPLUNC1 is required to reduce J2315 burden in the airways and (ii) its interaction with ENaC prevents cellular invasion of J2315.
Asunto(s)
Infecciones por Burkholderia/microbiología , Burkholderia cenocepacia/patogenicidad , Fibrosis Quística/microbiología , Células Epiteliales/microbiología , Canales Epiteliales de Sodio/metabolismo , Glicoproteínas/metabolismo , Pulmón/microbiología , Fosfoproteínas/metabolismo , Infecciones del Sistema Respiratorio/microbiología , Adolescente , Adulto , Animales , Carga Bacteriana , Infecciones por Burkholderia/genética , Infecciones por Burkholderia/metabolismo , Estudios de Casos y Controles , Fibrosis Quística/genética , Fibrosis Quística/metabolismo , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Canales Epiteliales de Sodio/genética , Femenino , Glicoproteínas/genética , Células HEK293 , Interacciones Huésped-Patógeno , Humanos , Pulmón/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Fosfoproteínas/genética , Infecciones del Sistema Respiratorio/genética , Infecciones del Sistema Respiratorio/metabolismo , Adulto JovenRESUMEN
C109 is a potent but poorly soluble FtsZ inhibitor displaying promising activity against Burkholderia cenocepacia, a high-risk pathogen for cystic fibrosis (CF) sufferers. To harness C109 for inhalation, we developed nanocrystal-embedded dry powders for inhalation suspension consisting in C109 nanocrystals stabilized with D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) embedded in hydroxypropyl-ß-cyclodextrin (CD). The powders could be safely re-dispersed in water for in vitro aerosolization. Owing to the presence of a PEG shell, the rod shape and the peculiar aspect ratio, C109 nanocrystals were able to diffuse through artificial CF mucus. The promising technological features were completed by encouraging in vitro/in vivo effects. The formulations displayed no toxicity towards human bronchial epithelial cells and were active against planktonic and sessile B. cenocepacia strains. The efficacy of C109 nanosuspensions in combination with piperacillin was confirmed in a Galleria mellonella infection model, strengthening their potential for combined therapy of B. cenocepacia lung infections.
Asunto(s)
Antibacterianos , Proteínas Bacterianas/antagonistas & inhibidores , Bronquios/microbiología , Infecciones por Burkholderia/tratamiento farmacológico , Burkholderia cenocepacia/crecimiento & desarrollo , Fibrosis Quística/tratamiento farmacológico , Proteínas del Citoesqueleto/antagonistas & inhibidores , Sistemas de Liberación de Medicamentos , Células Epiteliales/microbiología , Nanopartículas , Antibacterianos/química , Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Bronquios/metabolismo , Bronquios/patología , Infecciones por Burkholderia/metabolismo , Infecciones por Burkholderia/patología , Línea Celular Tumoral , Fibrosis Quística/metabolismo , Fibrosis Quística/microbiología , Fibrosis Quística/patología , Proteínas del Citoesqueleto/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/patología , Humanos , Nanopartículas/química , Nanopartículas/uso terapéuticoRESUMEN
The cytosolic pathogen Burkholderia pseudomallei and causative agent of melioidosis has been shown to regulate IL-1ß and IL-18 production through NOD-like receptor NLRP3 and pyroptosis via NLRC4. Downstream signalling pathways of those receptors and other cell death mechanisms induced during B. pseudomallei infection have not been addressed so far in detail. Furthermore, the role of B. pseudomallei factors in inflammasome activation is still ill defined. In the present study we show that caspase-1 processing and pyroptosis is exclusively dependent on NLRC4, but not on NLRP3 in the early phase of macrophage infection, whereas at later time points caspase-1 activation and cell death is NLRC4- independent. In the early phase we identified an activation pathway involving caspases-9, -7 and PARP downstream of NLRC4 and caspase-1. Analyses of caspase-1/11-deficient infected macrophages revealed a strong induction of apoptosis, which is dependent on activation of apoptotic initiator and effector caspases. The early activation pathway of caspase-1 in macrophages was markedly reduced or completely abolished after infection with a B. pseudomallei flagellin FliC or a T3SS3 BsaU mutant. Studies using cells transfected with the wild-type and mutated T3SS3 effector protein BopE indicated also a role of this protein in caspase-1 processing. A T3SS3 inner rod protein BsaK mutant failed to activate caspase-1, revealed higher intracellular counts, reduced cell death and IL-1ß secretion during early but not during late macrophage infection compared to the wild-type. Intranasal infection of BALB/c mice with the BsaK mutant displayed a strongly decreased mortality, lower bacterial loads in organs, and reduced levels of IL-1ß, myeloperoxidase and neutrophils in bronchoalveolar lavage fluid. In conclusion, our results indicate a major role for a functional T3SS3 in early NLRC4-mediated caspase-1 activation and pyroptosis and a contribution of late caspase-1-dependent and -independent cell death mechanisms in the pathogenesis of B. pseudomallei infection.
Asunto(s)
Infecciones por Burkholderia/inmunología , Burkholderia pseudomallei/inmunología , Inflamasomas/inmunología , Macrófagos/microbiología , Transducción de Señal/inmunología , Animales , Sistemas de Secreción Bacterianos/fisiología , Western Blotting , Infecciones por Burkholderia/metabolismo , Burkholderia pseudomallei/metabolismo , Caspasa 1/metabolismo , Muerte Celular/fisiología , Modelos Animales de Enfermedad , Citometría de Flujo , Células HEK293 , Humanos , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutagénesis Sitio-Dirigida , TransfecciónRESUMEN
Burkholderia cenocepacia, the causative agent of cepacia syndrome, primarily affects cystic fibrosis patients, often leading to death. In the lung, epithelial cells serve as the initial barrier to airway infections, yet their responses to B. cenocepacia have not been fully investigated. Here, we examined the molecular responses of human airway epithelial cells to B. cenocepacia infection. Infection led to early signaling events such as activation of Erk, Akt, and NF-κB. Further, TNFα, IL-6, IL-8, and IL-1ß were all significantly induced upon infection, but no IL-1ß was detected in the supernatants. Because caspase-1 is required for IL-1ß processing and release, we examined its expression in airway epithelial cells. Interestingly, little to no caspase-1 was detectable in airway epithelial cells. Transfection of caspase-1 into airway epithelial cells restored their ability to secrete IL-1ß following B. cenocepacia infection, suggesting that a deficiency in caspase-1 is responsible, at least in part, for the attenuated IL-1ß secretion.
Asunto(s)
Bronquios/metabolismo , Infecciones por Burkholderia/metabolismo , Burkholderia cenocepacia , Células Epiteliales/metabolismo , Interleucina-1beta/metabolismo , Mucosa Respiratoria/metabolismo , Bronquios/microbiología , Bronquios/patología , Infecciones por Burkholderia/genética , Infecciones por Burkholderia/microbiología , Infecciones por Burkholderia/patología , Caspasa 1/biosíntesis , Caspasa 1/genética , Línea Celular , Citocinas/biosíntesis , Citocinas/genética , Células Epiteliales/microbiología , Células Epiteliales/patología , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Interleucina-1beta/genética , FN-kappa B/genética , FN-kappa B/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Mucosa Respiratoria/microbiología , Mucosa Respiratoria/patología , TransfecciónRESUMEN
Cystic fibrosis is the most common inherited lethal disease in Caucasians. It is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), of which the cftr ΔF508 mutation is the most common. ΔF508 macrophages are intrinsically defective in autophagy because of the sequestration of essential autophagy molecules within unprocessed CFTR aggregates. Defective autophagy allows Burkholderia cenocepacia (B. cepacia) to survive and replicate in ΔF508 macrophages. Infection by B. cepacia poses a great risk to cystic fibrosis patients because it causes accelerated lung inflammation and, in some cases, a lethal necrotizing pneumonia. Autophagy is a cell survival mechanism whereby an autophagosome engulfs non-functional organelles and delivers them to the lysosome for degradation. The ubiquitin binding adaptor protein SQSTM1/p62 is required for the delivery of several ubiquitinated cargos to the autophagosome. In WT macrophages, p62 depletion and overexpression lead to increased and decreased bacterial intracellular survival, respectively. In contrast, depletion of p62 in ΔF508 macrophages results in decreased bacterial survival, whereas overexpression of p62 leads to increased B. cepacia intracellular growth. Interestingly, the depletion of p62 from ΔF508 macrophages results in the release of the autophagy molecule beclin1 (BECN1) from the mutant CFTR aggregates and allows its redistribution and recruitment to the B. cepacia vacuole, mediating the acquisition of the autophagy marker LC3 and bacterial clearance via autophagy. These data demonstrate that p62 differentially dictates the fate of B. cepacia infection in WT and ΔF508 macrophages.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Autofagia/genética , Infecciones por Burkholderia/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/genética , Proteínas de Choque Térmico/genética , Macrófagos/metabolismo , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Biomarcadores/metabolismo , Infecciones por Burkholderia/complicaciones , Infecciones por Burkholderia/metabolismo , Infecciones por Burkholderia/microbiología , Burkholderia cenocepacia/fisiología , Fibrosis Quística/complicaciones , Fibrosis Quística/metabolismo , Fibrosis Quística/microbiología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Expresión Génica , Proteínas de Choque Térmico/antagonistas & inhibidores , Proteínas de Choque Térmico/metabolismo , Humanos , Macrófagos/microbiología , Macrófagos/patología , Ratones , Ratones Transgénicos , Viabilidad Microbiana , Proteínas Asociadas a Microtúbulos/metabolismo , Fagosomas/metabolismo , Transporte de Proteínas , ARN Interferente Pequeño/genética , Proteína Sequestosoma-1 , Transfección , Ubiquitina/genética , Ubiquitina/metabolismoRESUMEN
Burkholderia cenocepacia H111, a strain isolated from a cystic fibrosis patient, has been shown to effectively kill the nematode Caenorhabditis elegans. We used the C. elegans model of infection to screen a mini-Tn5 mutant library of B. cenocepacia H111 for attenuated virulence. Of the approximately 5,500 B. cenocepacia H111 random mini-Tn5 insertion mutants that were screened, 22 showed attenuated virulence in C. elegans. Except for the quorum-sensing regulator cepR, none of the mutated genes coded for the biosynthesis of classical virulence factors such as extracellular proteases or siderophores. Instead, the mutants contained insertions in metabolic and regulatory genes. Mutants attenuated in virulence in the C. elegans infection model were also tested in the Drosophila melanogaster pricking model, and those also attenuated in this model were further tested in Galleria mellonella. Six of the 22 mutants were attenuated in D. melanogaster, and five of these were less pathogenic in the G. mellonella model. We show that genes encoding enzymes of the purine, pyrimidine, and shikimate biosynthesis pathways are critical for virulence in multiple host models of infection.
Asunto(s)
Proteínas Bacterianas/metabolismo , Infecciones por Burkholderia/metabolismo , Burkholderia cenocepacia/metabolismo , Factores de Virulencia/metabolismo , Animales , Proteínas Bacterianas/genética , Infecciones por Burkholderia/genética , Infecciones por Burkholderia/microbiología , Burkholderia cenocepacia/genética , Burkholderia cenocepacia/patogenicidad , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Regulación Bacteriana de la Expresión Génica/genética , Mutación , Virulencia/genética , Factores de Virulencia/genéticaRESUMEN
AIM: To investigate the clinical features of cystic fibrosis (CF) in adult patients with carbohydrate metabolic disturbances (CMD). MATERIAL AND METHODS: CF was diagnosed on the basis of its clinical picture and a positive sweat test, and/or genetic study. Clinical, anthropometric, functional, microbiological data were compared with the results of imaging diagnosis in CF patients with and without CMD. RESULTS: The data of 350 patients were retrospectively analyzed. An oral glucose tolerance test was randomly performed in 154 CF patients without CMD. There was normal carbohydrate metabolism in 92 (59.7%) patients with CF, impaired glucose tolerance (IGT) in 44 (28.6%), CF-dependent diabetes mellitus (CFDDM) in 18 (11.70%). The latter had been previously diagnosed in 37 (10.6%) other patients with CF. Three groups of patients were formed: 1) 92 patients without CMD; 2) 44 with IGT, and 3) 55 with CFDDM. The patients with CFDDM had lower stature, weight, and lung function, significantly more common bronchiectases, a lower Staphylococcus aureus colonization with a tendency toward a higher Burkholderia cepacia colonization than those without CMD. As compared with the patients without CMD, those with this disorder were found to have a high rate of severe mutations; mild mutations were absent in the patients with CFDDM. CONCLUSION: CMD in CF is characterized by its high rates and latent course. The patients with CMD have retarded physical development, more pronounced morphofunctional disorders in the bronchopulmonary system, lower lung functional parameters, and more aggressive sputum microbial composition.
Asunto(s)
Glucemia/metabolismo , Fibrosis Quística/complicaciones , Fibrosis Quística/metabolismo , Adulto , Infecciones por Burkholderia/complicaciones , Infecciones por Burkholderia/etiología , Infecciones por Burkholderia/metabolismo , Fibrosis Quística/microbiología , Diabetes Mellitus/etiología , Diabetes Mellitus/metabolismo , Diabetes Mellitus/microbiología , Femenino , Prueba de Tolerancia a la Glucosa , Humanos , Masculino , Enfermedades Metabólicas/etiología , Enfermedades Metabólicas/genética , Enfermedades Metabólicas/metabolismo , Estudios Retrospectivos , Infecciones Estafilocócicas/etiología , Infecciones Estafilocócicas/genética , Infecciones Estafilocócicas/metabolismo , Adulto JovenRESUMEN
BACKGROUND: Burkholderia cenocepacia is an opportunistic pathogen causing life-threatening infections in patients with cystic fibrosis. The bacterium survives within macrophages by interfering with endocytic trafficking and delaying the maturation of the B. cenocepacia-containing phagosome. We hypothesize that B. cenocepacia undergoes changes in gene expression after internalization by macrophages, inducing genes involved in intracellular survival and host adaptation. RESULTS: We examined gene expression by intracellular B. cenocepacia using selective capture of transcribed sequences (SCOTS) combined with microarray analysis. We identified 767 genes with significantly different levels of expression by intracellular bacteria, of which 330 showed increased expression and 437 showed decreased expression. Affected genes represented all aspects of cellular life including information storage and processing, cellular processes and signaling, and metabolism. In general, intracellular gene expression demonstrated a pattern of environmental sensing, bacterial response, and metabolic adaptation to the phagosomal environment. Deletion of various SCOTS-identified genes affected bacterial entry into macrophages and intracellular replication. We also show that intracellular B. cenocepacia is cytotoxic towards the macrophage host, and capable of spread to neighboring cells, a role dependent on SCOTS-identified genes. In particular, genes involved in bacterial motility, cobalamin biosynthesis, the type VI secretion system, and membrane modification contributed greatly to macrophage entry and subsequent intracellular behavior of B. cenocepacia. CONCLUSIONS: B. cenocepacia enters macrophages, adapts to the phagosomal environment, replicates within a modified phagosome, and exhibits cytotoxicity towards the host cells. The analysis of the transcriptomic response of intracellular B. cenocepacia reveals that metabolic adaptation to a new niche plays a major role in the survival of B. cenocepacia in macrophages. This adaptive response does not require the expression of any specific virulence-associated factor, which is consistent with the opportunistic nature of this microorganism. Further investigation into the remaining SCOTS-identified genes will provide a more complete picture of the adaptive response of B. cenocepacia to the host cell environment.
Asunto(s)
Infecciones por Burkholderia/microbiología , Burkholderia cenocepacia/genética , Burkholderia cenocepacia/metabolismo , Regulación Bacteriana de la Expresión Génica , Macrófagos/microbiología , Animales , Infecciones por Burkholderia/metabolismo , Línea Celular , Regulación hacia Abajo , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Fagocitosis , Fagosomas/metabolismo , Análisis de Secuencia de ADN , Transcriptoma , Regulación hacia ArribaRESUMEN
Burkholderia cenocepacia (B. cenocepacia) is an opportunistic bacterium; causing severe life threatening systemic infections in immunocompromised individuals including cystic fibrosis patients. The lack of gasdermin D (GSDMD) protects mice against endotoxin lipopolysaccharide (LPS) shock. On the other hand, GSDMD promotes mice survival in response to certain bacterial infections. However, the role of GSDMD during B. cenocepacia infection is not yet determined. Our in vitro study shows that GSDMD restricts B. cenocepacia replication within macrophages independent of its role in cell death through promoting mitochondrial reactive oxygen species (mROS) production. mROS is known to stimulate autophagy, hence, the inhibition of mROS or the absence of GSDMD during B. cenocepacia infections reduces autophagy which plays a critical role in the restriction of the pathogen. GSDMD promotes inflammation in response to B. cenocepacia through mediating the release of inflammasome dependent cytokine (IL-1ß) and an independent one (CXCL1) (KC). Additionally, different B. cenocepacia secretory systems (T3SS, T4SS, and T6SS) contribute to inflammasome activation together with bacterial survival within macrophages. In vivo study confirmed the in vitro findings and showed that GSDMD restricts B. cenocepacia infection and dissemination and stimulates autophagy in response to B. cenocepacia. Nevertheless, GSDMD promotes lung inflammation and necrosis in response to B. cenocepacia without altering mice survival. This study describes the double-edged functions of GSDMD in response to B. cenocepacia infection and shows the importance of GSDMD-mediated mROS in restriction of B. cenocepacia.
Asunto(s)
Infecciones por Burkholderia/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de Unión a Fosfato/metabolismo , Animales , Autofagia/fisiología , Infecciones por Burkholderia/prevención & control , Burkholderia cenocepacia/patogenicidad , Caspasas Iniciadoras/genética , Caspasas Iniciadoras/metabolismo , Muerte Celular , Femenino , Inflamasomas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/fisiología , Lipopolisacáridos/metabolismo , Macrófagos/citología , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/metabolismo , Proteínas de Unión a Fosfato/genética , Proteínas de Unión a Fosfato/fisiología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
It is questionable that how gut symbiont can be proliferated in the host symbiotic organs, such as host midgut region, which are known to be highly stressful and nutritional depleted conditions. Since Riptortus-Burkholderia symbiosis system is a good model to study this question, we hypothesized that Burkholderia symbiont will use host-derived bacterial growth factor(s) to colonize persistently in the host midgut 4 (M4) region, which is known as symbiotic organ. In this study, we observed that although gut-colonized symbiotic Burkholderia cells did not grow in the nutrient-limited media conditions, these symbionts were able to grow dose-dependent manner by addition of host naïve M4 lysate, supporting that host-derived growth factor molecule(s) may exist in the host M4 lysate. By further experiments, a host-derived growth factor(s) did not lose its biological activity in the conditions of high temperature, treatment of phenol-chloroform or ethyl alcohol precipitation, indicating that a growth factor molecule(s) is neither a protein nor a DNA. Also, based on the biochemical analyses data, molecular weight of the host-derived bacterial growth factor(s) was turned out to be less than 3 kDa molecular mass and to give the positive chemical response to the ninhydrin reagent on thin layer chromatography. Finally, we found that one specific peak showing ninhydrin positive signal was separated by gel filtration column and induced proliferative activity for Burkholderia gut symbiont cells.
Asunto(s)
Infecciones por Burkholderia/metabolismo , Burkholderia/fisiología , Proteínas de Insectos/metabolismo , Insectos/fisiología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/microbiología , Animales , Procesos de Crecimiento Celular , Microbioma Gastrointestinal , SimbiosisRESUMEN
Melioidosis is an often-severe tropical infection caused by Burkholderia pseudomallei (Bp) with high associated morbidity and mortality. Burkholderia thailandensis (Bt) is a closely related surrogate that does not require BSL-3 conditions for study. Lactoferrin is an iron-binding glycoprotein that can modulate the innate inflammatory response. Here we investigated the impact of lactoferrin on the host immune response in melioidosis. Lactoferrin concentrations were measured in plasma from patients with melioidosis and following ex vivo stimulation of blood from healthy individuals. Bt growth was quantified in liquid media in the presence of purified and recombinant human lactoferrin. Differentiated THP-1 cells and human blood monocytes were infected with Bt in the presence of purified and recombinant human lactoferrin, and bacterial intracellular replication and cytokine responses (tumor necrosis factor-α (TNF-α), interleukin-1ß and interferon-γ) were measured. In a cohort of 49 melioidosis patients, non-survivors to 28 days had significantly higher plasma lactoferrin concentrations compared to survivors (median (interquartile range (IQR)): 326 ng/ml (230-748) vs 144 ng/ml (99-277), p<0.001). In blood stimulated with heat-killed Bp, plasma lactoferrin concentration significantly increased compared to unstimulated blood (median (IQR): 424 ng/ml (349-479) vs 130 ng/ml (91-214), respectively; p<0.001). Neither purified nor recombinant human lactoferrin impaired growth of Bt in media. Lactoferrin significantly increased TNF-α production by differentiated THP-1 cells and blood monocytes after Bt infection. This phenotype was largely abrogated when Toll-like receptor 4 (TLR4) was blocked with a monoclonal antibody. In sum, lactoferrin is produced by blood cells after exposure to Bp and lactoferrin concentrations are higher in 28-day survivors in melioidosis. Lactoferrin induces proinflammatory cytokine production after Bt infection that may be TLR4 dependent.
Asunto(s)
Infecciones por Burkholderia/metabolismo , Infecciones por Burkholderia/microbiología , Burkholderia , Lactoferrina/metabolismo , Receptor Toll-Like 4/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Burkholderia pseudomallei , Células Cultivadas , Humanos , Melioidosis/metabolismo , Monocitos , Receptor Toll-Like 4/genética , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Burkholderia cenocepacia is an important respiratory pathogen in persons with cystic fibrosis (CF). Recent studies indicate that B. cenocepacia survives within macrophages and airway epithelial cells in vitro by evading endosome-lysosome fusion. We investigated the role of a plasmid-encoded type IV secretion system in the intracellular survival, replication, and processing of B. cenocepacia. Both a wild-type strain (K56-2) and its type IV secretion system mutant (designated LC101) entered and replicated in CF airway epithelial cells and monocyte-derived macrophages. However, significantly more intracellular K56-2 than LC101 bacteria were found in both cell types at 24 h postinfection. Colocalization of bacteria with markers of the classical endocytic pathway indicated that although both K56-2 and LC101 reside transiently in early endosomes, a greater proportion of the mutant bacteria are targeted to lysosomal degradation. In contrast, wild-type bacteria escape from the classical endocytic pathway and traffic to the endoplasmic reticulum, where they replicate. Our results show that the intracellular processing of B. cenocepacia is similar in both professional and nonprofessional phagocytes and that a functional plasmid-encoded type IV secretion system contributes to the survival and replication of B. cenocepacia in eukaryotic cells.
Asunto(s)
Infecciones por Burkholderia/metabolismo , Complejo Burkholderia cepacia/fisiología , Complejo Burkholderia cepacia/patogenicidad , Southern Blotting , Infecciones por Burkholderia/genética , Línea Celular , Fibrosis Quística/microbiología , Retículo Endoplásmico , Endosomas/microbiología , Células Epiteliales/microbiología , Técnica del Anticuerpo Fluorescente , Humanos , Lisosomas/microbiología , Macrófagos/microbiología , Microscopía Confocal , Mutagénesis Insercional , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Chronic infection with opportunistic pathogens including Burkholderia cepacia complex (Bcc) is a hallmark of cystic fibrosis (CF). We investigated the adaptive mechanisms facilitating chronic lung infection in sequential Bcc isolates from two siblings with CF (P1 and P2), one of whom also experienced intermittent blood-stream infections (P2). We previously showed increased lung cell attachment with colonisation time in both P1 and P2. WGS analysis confirmed that the isolates are closely related. Twelve genes showed three or more mutations, suggesting these were genes under selection. Single nucleotide polymorphisms (SNVs) in 45 regulatory genes were also observed. Proteomic analysis showed that the abundance of 149 proteins increased over 61-months in sputum isolates, and both time- and source-related alterations in protein abundance between the second patient's isolates. A consistent time-dependent increase in abundance of 19 proteins encoded by a low-oxygen-activated (lxa) locus was observed in both sets of isolates. Attachment was dramatically reduced in a B. cenocepacia K56-2Δlxa-locus deletion mutant, further indicating that it encodes protein(s) involved in host-cell attachment. Time-related changes in virulence in Galleria mellonella or motility were not observed. We conclude that the lxa-locus, associated with anoxic persistence in vitro, plays a role in host-cell attachment and adaptation to chronic colonization in the hypoxic niche of the CF lung.
Asunto(s)
Adaptación Fisiológica , Infecciones por Burkholderia , Burkholderia cenocepacia , Fibrosis Quística , Sitios Genéticos , Oxígeno/metabolismo , Neumonía Bacteriana , Secuencia de Bases , Infecciones por Burkholderia/genética , Infecciones por Burkholderia/metabolismo , Infecciones por Burkholderia/microbiología , Burkholderia cenocepacia/genética , Burkholderia cenocepacia/metabolismo , Fibrosis Quística/genética , Fibrosis Quística/metabolismo , Fibrosis Quística/microbiología , Femenino , Humanos , Pulmón/metabolismo , Pulmón/microbiología , Masculino , Neumonía Bacteriana/genética , Neumonía Bacteriana/metabolismo , Neumonía Bacteriana/microbiología , Polimorfismo de Nucleótido Simple , Eliminación de SecuenciaRESUMEN
CASP4/caspase-11-dependent inflammasome activation is important for the clearance of various Gram-negative bacteria entering the host cytosol. Additionally, CASP4 modulates the actin cytoskeleton to promote the maturation of phagosomes harboring intracellular pathogens such as Legionella pneumophila but not those enclosing nonpathogenic bacteria. Nevertheless, this non-inflammatory role of CASP4 regarding the trafficking of vacuolar bacteria remains poorly understood. Macroautophagy/autophagy, a catabolic process within eukaryotic cells, is also implicated in the elimination of intracellular pathogens such as Burkholderia cenocepacia. Here we show that CASP4-deficient macrophages exhibit a defect in autophagosome formation in response to B. cenocepacia infection. The absence of CASP4 causes an accumulation of the small GTPase RAB7, reduced colocalization of B. cenocepacia with LC3 and acidic compartments accompanied by increased bacterial replication in vitro and in vivo. Together, our data reveal a novel role of CASP4 in regulating autophagy in response to B. cenocepacia infection.
Asunto(s)
Autofagosomas/metabolismo , Autofagia/genética , Infecciones Bacterianas/inmunología , Burkholderia cenocepacia/inmunología , Caspasas/fisiología , Animales , Autofagosomas/microbiología , Autofagia/inmunología , Infecciones Bacterianas/genética , Infecciones Bacterianas/metabolismo , Infecciones por Burkholderia/genética , Infecciones por Burkholderia/inmunología , Infecciones por Burkholderia/metabolismo , Burkholderia cenocepacia/metabolismo , Caspasas/genética , Caspasas Iniciadoras , Células Cultivadas , Escherichia coli/inmunología , Escherichia coli/metabolismo , Inflamasomas/genética , Inflamasomas/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/microbiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fagosomas/genética , Fagosomas/metabolismo , Fagosomas/microbiología , Fagosomas/patologíaRESUMEN
Antibiotic drugs exhibit concentration dependence in their efficacy. Therefore, ensuring appropriate concentration of these drugs in the relevant body fluid is important for obtaining the desired therapeutic and physiological action. Until recently there had been no suitable method available to measure or estimate concentration of drugs in the human airways resulting from inhaled aerosols or to determine the amount of inhaled antibiotics required to ensure minimum inhibitory concentration of a drug in the airway surface liquid (ASL). In this paper a numerical method is used for estimating local concentration of inhaled pharmaceutical aerosols in different generations of the human tracheobronchial airways. The method utilizes a mathematical lung deposition model to estimate amounts of aerosols depositing in different lung generations, and a recent ASL model along with deposition results to assess the concentration of deposited drugs immediately following inhalation. Examples of concentration estimates for two case studies: one for the antibiotic tobramycin against Pseudomonas aeruginosa, and another for taurolidine against Burkholderia cepacia are presented. The aerosol characteristics, breathing pattern and properties of nebulized solutions were adopted from two recent clinical studies on efficacy of these drugs in cystic fibrosis (CF) patients and from other sources in the literature. While the clinically effective tobramycin showed a concentration higher than the required in vivo concentration, that for the ineffective taurolidine was found to be below the speculated required in vivo concentration. Results of this study thus show that the mathematical ASL model combined with the lung deposition model can be an effective tool for helping decide the optimum dosage of inhaled antibiotic drugs delivered during human clinical trials.
Asunto(s)
Antibacterianos/administración & dosificación , Infecciones por Burkholderia/tratamiento farmacológico , Fibrosis Quística/tratamiento farmacológico , Nebulizadores y Vaporizadores , Infecciones por Pseudomonas/tratamiento farmacológico , Sistema Respiratorio/metabolismo , Taurina/análogos & derivados , Tiadiazinas/administración & dosificación , Tobramicina/administración & dosificación , Administración por Inhalación , Aerosoles , Antibacterianos/química , Antibacterianos/metabolismo , Infecciones por Burkholderia/metabolismo , Infecciones por Burkholderia/microbiología , Infecciones por Burkholderia/fisiopatología , Burkholderia cepacia/efectos de los fármacos , Burkholderia cepacia/crecimiento & desarrollo , Burkholderia cepacia/aislamiento & purificación , Simulación por Computador , Fibrosis Quística/metabolismo , Fibrosis Quística/microbiología , Fibrosis Quística/fisiopatología , Relación Dosis-Respuesta a Droga , Humanos , Pruebas de Sensibilidad Microbiana , Modelos Biológicos , Tamaño de la Partícula , Infecciones por Pseudomonas/metabolismo , Infecciones por Pseudomonas/microbiología , Infecciones por Pseudomonas/fisiopatología , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/crecimiento & desarrollo , Pseudomonas aeruginosa/aislamiento & purificación , Proyectos de Investigación , Mecánica Respiratoria , Sistema Respiratorio/microbiología , Sistema Respiratorio/fisiopatología , Esputo/metabolismo , Esputo/microbiología , Taurina/administración & dosificación , Taurina/química , Taurina/metabolismo , Tiadiazinas/química , Tiadiazinas/metabolismo , Tobramicina/química , Tobramicina/metabolismoRESUMEN
Alcohol consumption has diverse and well-documented effects on the human immune system and its ability to defend against infective agents. One example is melioidosis, a disease caused by infection with Burkholderia pseudomallei, which is of public health importance in Southeast Asia and Northern Australia, with an expanding global distribution. While B. pseudomallei infections can occur in healthy humans, binge alcohol use is progressively being recognized as a major risk factor. Although binge alcohol consumption has been considered as a risk factor for the development of melioidosis, no experimental studies have investigated the outcomes of alcohol exposure on Burkholderia spp. infection. Therefore, we proposed the use of non-pathogenic B. thailandensis E264 as a useful BSL-1 model system to study the effects of binge alcohol exposure on bacteria and alveolar macrophage interactions. The MH-S alveolar macrophage (AMs) cell line was used to characterize innate immune responses to infection in vitro. Our results showed that alcohol exposure significantly suppressed the uptake and killing of B. thailandensis by AMs. Alveolar macrophages incubated in alcohol (0.08%) for 3 h prior to infection showed significantly lower bacterial uptake at 2 and 8 h post infection. Activated AMs with IFN-γ and pre and post-incubation in alcohol when exposed to B. thailandensis released lower nitric oxide (NO) concentrations, compared to activated AMs with IFN-γ from non-alcoholic controls. As a result, B. thailandensis survival and replication increased â¼2.5-fold compared to controls. The presence of alcohol (1%) also increased bacterial survival within AMs. Alcohol significantly decreased bacterial motility compared to non-alcoholic controls. Increased biofilm formation was observed at 3 and 6 h when bacteria were pre-incubated in (0.08%) alcohol. These results provide insights into binge alcohol consumption, a culturally prevalent risk factor, as a predisposing factor for melioidosis.
Asunto(s)
Infecciones por Burkholderia/metabolismo , Burkholderia/aislamiento & purificación , Etanol/toxicidad , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/microbiología , Animales , Consumo Excesivo de Bebidas Alcohólicas/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Relación Dosis-Respuesta a Droga , Macrófagos Alveolares/efectos de los fármacos , Ratones , Óxido Nítrico/metabolismoRESUMEN
Burkholderia cenocepacia is an opportunistic pathogen responsible for life-threatening infections in cystic fibrosis patients. B. cenocepacia is extremely resistant towards antibiotics and therapy is complicated by its ability to form biofilms. We investigated the efficacy of an alternative antimicrobial strategy for B. cenocepacia lung infections using in vitro and in vivo models. A screening of the NIH Clinical Collection 1&2 was performed against B. cenocepacia biofilms formed in 96-well microtiter plates in the presence of tobramycin to identify repurposing candidates with potentiator activity. The efficacy of selected hits was evaluated in a three-dimensional (3D) organotypic human lung epithelial cell culture model. The in vivo effect was evaluated in the invertebrate Galleria mellonella and in a murine B. cenocepacia lung infection model. The screening resulted in 60 hits that potentiated the activity of tobramycin against B. cenocepacia biofilms, including four imidazoles of which econazole and miconazole were selected for further investigation. However, a potentiator effect was not observed in the 3D organotypic human lung epithelial cell culture model. Combination treatment was also not able to increase survival of infected G. mellonella. Also in mice, there was no added value for the combination treatment. Although potentiators of tobramycin with activity against biofilms of B. cenocepacia were identified in a repurposing screen, the in vitro activity could not be confirmed nor in a more sophisticated in vitro model, neither in vivo. This stresses the importance of validating hits resulting from in vitro studies in physiologically relevant model systems.
Asunto(s)
Biopelículas/efectos de los fármacos , Infecciones por Burkholderia/tratamiento farmacológico , Burkholderia cenocepacia/fisiología , Econazol/farmacología , Miconazol/farmacología , Neumonía Bacteriana/tratamiento farmacológico , Tobramicina/farmacología , Células A549 , Animales , Biopelículas/crecimiento & desarrollo , Infecciones por Burkholderia/metabolismo , Infecciones por Burkholderia/patología , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Quimioterapia Combinada/métodos , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Neumonía Bacteriana/metabolismo , Neumonía Bacteriana/patologíaRESUMEN
Burkholderia is a genus within the ß-Proteobacteriaceae that contains at least 90 validly named species which can be found in a diverse range of environments. A number of pathogenic species occur within the genus. These include Burkholderia cenocepacia and Burkholderia multivorans, opportunistic pathogens that can infect the lungs of patients with cystic fibrosis, and are members of the Burkholderia cepacia complex (Bcc). Burkholderia pseudomallei is also an opportunistic pathogen, but in contrast to Bcc species it causes the tropical human disease melioidosis, while its close relative Burkholderia mallei is the causative agent of glanders in horses. For these pathogens to survive within a host and cause disease they must be able to acquire iron. This chemical element is essential for nearly all living organisms due to its important role in many enzymes and metabolic processes. In the mammalian host, the amount of accessible free iron is negligible due to the low solubility of the metal ion in its higher oxidation state and the tight binding of this element by host proteins such as ferritin and lactoferrin. As with other pathogenic bacteria, Burkholderia species have evolved an array of iron acquisition mechanisms with which to capture iron from the host environment. These mechanisms include the production and utilization of siderophores and the possession of a haem uptake system. Here, we summarize the known mechanisms of iron acquisition in pathogenic Burkholderia species and discuss the evidence for their importance in the context of virulence and the establishment of infection in the host. We have also carried out an extensive bioinformatic analysis to identify which siderophores are produced by each Burkholderia species that is pathogenic to humans.
Asunto(s)
Infecciones por Burkholderia/metabolismo , Infecciones por Burkholderia/microbiología , Burkholderia/metabolismo , Burkholderia/patogenicidad , Hierro/metabolismo , Virulencia , Animales , Burkholderia/clasificación , Burkholderia/genética , Burkholderia gladioli/metabolismo , Burkholderia gladioli/patogenicidad , Burkholderia mallei/metabolismo , Burkholderia mallei/patogenicidad , Burkholderia pseudomallei/metabolismo , Burkholderia pseudomallei/patogenicidad , Biología Computacional , Fibrosis Quística/microbiología , Ferritinas/metabolismo , Muermo , Hemo/metabolismo , Caballos , Humanos , Lactoferrina/metabolismo , Pulmón/microbiología , Melioidosis/microbiología , Sideróforos/metabolismoRESUMEN
Burkholderia cenocepacia is a member of the Burkholderia cepacia complex, a group of genetically similar species that inhabit a number of environmental niches, including the lungs of patients with cystic fibrosis (CF). To colonize the lung, this bacterium requires a source of iron to satisfy its nutritional requirements for this important metal. Because of the high potential for damage in lung tissue resulting from oxygen-iron interactions, this metal is sequestered by a number of mechanisms that render it potentially unavailable to invading micro-organisms. Such mechanisms include the intracellular and extracellular presence of the iron-binding protein ferritin. Ferritin has a highly stable macromolecular structure and may contain up to 4500 iron atoms per molecule. To date, there has been no known report of a pathogenic bacterial species that directly utilizes iron sequestered by this macromolecule. To examine the ability of ferritin to support growth of B. cenocepacia J2315, iron-deficient media were supplemented with different concentrations of ferritin and the growth kinetics characterized over a 40 h period. The results indicated that B. cenocepacia J2315 utilizes iron bound by ferritin. Further studies examining the mechanisms of iron uptake from ferritin indicated that iron utilization results from a proteolytic degradation of this otherwise stable macromolecular structure. Since it is known that the ferritin concentration is significantly higher in the CF lung than in healthy lungs, this novel iron-acquisition mechanism may contribute to infection by B. cenocepacia in people with CF.
Asunto(s)
Complejo Burkholderia cepacia/metabolismo , Ferritinas/metabolismo , Hierro/metabolismo , Animales , Infecciones por Burkholderia/etiología , Infecciones por Burkholderia/metabolismo , Infecciones por Burkholderia/microbiología , Complejo Burkholderia cepacia/efectos de los fármacos , Complejo Burkholderia cepacia/crecimiento & desarrollo , Complejo Burkholderia cepacia/patogenicidad , Medios de Cultivo , Fibrosis Quística/complicaciones , Fibrosis Quística/metabolismo , Fibrosis Quística/microbiología , Caballos , Humanos , Técnicas In Vitro , Pulmón/metabolismo , Pulmón/microbiología , Infecciones Oportunistas/etiología , Infecciones Oportunistas/metabolismo , Infecciones Oportunistas/microbiología , Inhibidores de Proteasas/farmacologíaRESUMEN
Burkholderia cenocepacia is an opportunistic pathogen of the cystic fibrosis lung that elicits a strong inflammatory response. B. cenocepacia employs a type VI secretion system (T6SS) to survive in macrophages by disarming Rho-type GTPases, causing actin cytoskeletal defects. Here, we identified TecA, a non-VgrG T6SS effector responsible for actin disruption. TecA and other bacterial homologs bear a cysteine protease-like catalytic triad, which inactivates Rho GTPases by deamidating a conserved asparagine in the GTPase switch-I region. RhoA deamidation induces caspase-1 inflammasome activation, which is mediated by the familial Mediterranean fever disease protein Pyrin. In mouse infection, the deamidase activity of TecA is necessary and sufficient for B. cenocepacia-triggered lung inflammation and also protects mice from lethal B. cenocepacia infection. Therefore, Burkholderia TecA is a T6SS effector that modifies a eukaryotic target through an asparagine deamidase activity, which in turn elicits host cell death and inflammation through activation of the Pyrin inflammasome.