RESUMEN
How the microbiome and mucosal environment mediate homeostatic immunity in the skin is not well understood. In this issue of Cell, Lima-Junior and colleagues report that skin commensal bacteria induce endogenous retrovirus (ERV) expression and antiviral immune responses in keratinocytes, favoring the priming of beneficial commensal-specific T cell subsets that promote tissue homeostasis.
Asunto(s)
Retrovirus Endógenos , Microbiota , Bacterias , Retrovirus Endógenos/genética , Membrana Mucosa , Subgrupos de Linfocitos TRESUMEN
Phage therapy is a therapeutic approach to treat multidrug-resistant (MDR) infections that employs lytic bacteriophages (phages) to eliminate bacteria. Despite the abundant evidence for its success as an antimicrobial in Eastern Europe, there is scarce data regarding its effects on the human host. Here, we aimed to understand how lytic phages interact with cells of the airway epithelium, the tissue site that is colonized by bacterial biofilms in numerous chronic respiratory disorders. Using a panel of Pseudomonas aeruginosa phages and human airway epithelial cells (AECs) derived from a person with cystic fibrosis (CF), we determined that interactions between phages and epithelial cells depend on specific phage properties as well as physiochemical features of the microenvironment. Although poor at internalizing phages, the airway epithelium responds to phage exposure by changing its transcriptional profile and secreting antiviral and proinflammatory cytokines that correlate with specific phage families. Overall, our findings indicate that mammalian responses to phages are heterogenous and could potentially alter the way that respiratory local defenses aid in bacterial clearance during phage therapy. Thus, besides phage receptor specificity in a particular bacterial isolate, the criteria to select lytic phages for therapy should be expanded to include mammalian cell responses.
Asunto(s)
Fibrosis Quística , Citocinas , Células Epiteliales , Pseudomonas aeruginosa , Humanos , Pseudomonas aeruginosa/virología , Células Epiteliales/virología , Células Epiteliales/metabolismo , Células Epiteliales/inmunología , Citocinas/metabolismo , Fibrosis Quística/terapia , Fibrosis Quística/inmunología , Fibrosis Quística/metabolismo , Terapia de Fagos , Bacteriófagos/fisiología , Bacteriófagos/genética , Mucosa Respiratoria/virología , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/inmunología , Infecciones por Pseudomonas/terapia , Infecciones por Pseudomonas/inmunología , Fagos Pseudomonas/metabolismo , BiopelículasRESUMEN
Laboratory models are central to microbiology research, advancing the understanding of bacterial physiology by mimicking natural environments, from soil to the human microbiome. When studying host-bacteria interactions, animal models enable investigators to examine bacterial dynamics associated with a host, and in the case of human infections, animal models are necessary to translate basic research into clinical treatments. Efforts toward improving animal infection models are typically based on reproducing host genotypes/phenotypes and disease manifestations, leaving a gap in how well the physiology of microbes reflects their behavior in a human host. Understanding bacterial physiology is vital because it dictates host response and bacterial interactions with antimicrobials. Thus, our goal was to develop an animal model that accurately recapitulates bacterial physiology in human infection. The system we chose to model was a chronic Pseudomonas aeruginosa respiratory infection in cystic fibrosis (CF). To accomplish this goal, we leveraged a framework that we recently developed to evaluate model accuracy by calculating the percentage of bacterial genes that are expressed similarly in a model to how they are expressed in their infection environment. We combined two complementary models of P. aeruginosa infection-an in vitro synthetic CF sputum model (SCFM2) and a mouse acute pneumonia model. This combined model captured the chronic physiology of P. aeruginosa in CF better than the standard mouse infection model, showing the power of a data-driven approach to refining animal models. In addition, the results of this work challenge the assumption that a chronic infection model requires long-term colonization.
Asunto(s)
Fibrosis Quística , Modelos Animales de Enfermedad , Infecciones por Pseudomonas , Pseudomonas aeruginosa , Fibrosis Quística/microbiología , Fibrosis Quística/complicaciones , Pseudomonas aeruginosa/fisiología , Pseudomonas aeruginosa/patogenicidad , Animales , Infecciones por Pseudomonas/microbiología , Ratones , Humanos , Infecciones del Sistema Respiratorio/microbiología , Interacciones Huésped-Patógeno , Esputo/microbiologíaRESUMEN
Mucosa-associated biofilms are associated with many human disease states, but the host mechanisms promoting biofilm remain unclear. In chronic respiratory diseases like cystic fibrosis (CF), Pseudomonas aeruginosa establishes chronic infection through biofilm formation. P. aeruginosa can be attracted to interspecies biofilms through potassium currents emanating from the biofilms. We hypothesized that P. aeruginosa could, similarly, sense and respond to the potassium efflux from human airway epithelial cells (AECs) to promote biofilm. Using respiratory epithelial co-culture biofilm imaging assays of P. aeruginosa grown in association with CF bronchial epithelial cells (CFBE41o-), we found that P. aeruginosa biofilm was increased by potassium efflux from AECs, as examined by potentiating large conductance potassium channel, BKCa (NS19504) potassium efflux. This phenotype is driven by increased bacterial attachment and increased coalescence of bacteria into aggregates. Conversely, biofilm formation was reduced when AECs were treated with a BKCa blocker (paxilline). Using an agar-based macroscopic chemotaxis assay, we determined that P. aeruginosa chemotaxes toward potassium and screened transposon mutants to discover that disruption of the high-sensitivity potassium transporter, KdpFABC, and the two-component potassium sensing system, KdpDE, reduces P. aeruginosa potassium chemotaxis. In respiratory epithelial co-culture biofilm imaging assays, a KdpFABCDE deficient P. aeruginosa strain demonstrated reduced biofilm growth in association with AECs while maintaining biofilm formation on abiotic surfaces. Furthermore, we determined that the Kdp operon is expressed in vivo in people with CF and the genes are conserved in CF isolates. Collectively, these data suggest that P. aeruginosa biofilm formation can be increased by attracting bacteria to the mucosal surface and enhancing coalescence into microcolonies through aberrant AEC potassium efflux sensed by the KdpFABCDE system. These findings suggest host electrochemical signaling can enhance biofilm, a novel host-pathogen interaction, and potassium flux could be a therapeutic target to prevent chronic infections in diseases with mucosa-associated biofilms, like CF.
Asunto(s)
Biopelículas , Fibrosis Quística , Células Epiteliales , Operón , Potasio , Infecciones por Pseudomonas , Pseudomonas aeruginosa , Biopelículas/crecimiento & desarrollo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/fisiología , Humanos , Fibrosis Quística/microbiología , Fibrosis Quística/metabolismo , Células Epiteliales/microbiología , Células Epiteliales/metabolismo , Potasio/metabolismo , Infecciones por Pseudomonas/microbiología , Infecciones por Pseudomonas/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/microbiologíaRESUMEN
Laboratory models are critical to basic and translational microbiology research. Models serve multiple purposes, from providing tractable systems to study cell biology to allowing the investigation of inaccessible clinical and environmental ecosystems. Although there is a recognized need for improved model systems, there is a gap in rational approaches to accomplish this goal. We recently developed a framework for assessing the accuracy of microbial models by quantifying how closely each gene is expressed in the natural environment and in various models. The accuracy of the model is defined as the percentage of genes that are similarly expressed in the natural environment and the model. Here, we leverage this framework to develop and validate two generalizable approaches for improving model accuracy, and as proof of concept, we apply these approaches to improve models of Pseudomonas aeruginosa infecting the cystic fibrosis (CF) lung. First, we identify two models, an in vitro synthetic CF sputum medium model (SCFM2) and an epithelial cell model, that accurately recapitulate different gene sets. By combining these models, we developed the epithelial cell-SCFM2 model which improves the accuracy of over 500 genes. Second, to improve the accuracy of specific genes, we mined publicly available transcriptome data, which identified zinc limitation as a cue present in the CF lung and absent in SCFM2. Induction of zinc limitation in SCFM2 resulted in accurate expression of 90% of P. aeruginosa genes. These approaches provide generalizable, quantitative frameworks for microbiological model improvement that can be applied to any system of interest.
Asunto(s)
Infecciones Bacterianas , Fibrosis Quística , Infecciones por Pseudomonas , Humanos , Ecosistema , Infecciones por Pseudomonas/microbiología , Transcriptoma , Células Epiteliales/microbiología , Medios de Cultivo/metabolismo , Fibrosis Quística/microbiología , Pseudomonas aeruginosa/genética , Esputo/microbiologíaRESUMEN
Clinical studies report that viral infections promote acute or chronic bacterial infections at multiple host sites. These viral-bacterial co-infections are widely linked to more severe clinical outcomes. In experimental models in vitro and in vivo, virus-induced interferon responses can augment host susceptibility to secondary bacterial infection. Here, we used a cell-based screen to assess 389 interferon-stimulated genes (ISGs) for their ability to induce chronic Pseudomonas aeruginosa infection. We identified and validated five ISGs that were sufficient to promote bacterial infection. Furthermore, we dissected the mechanism of action of hexokinase 2 (HK2), a gene involved in the induction of aerobic glycolysis, commonly known as the Warburg effect. We report that HK2 upregulation mediates the induction of Warburg effect and secretion of L-lactate, which enhances chronic P. aeruginosa infection. These findings elucidate how the antiviral immune response renders the host susceptible to secondary bacterial infection, revealing potential strategies for viral-bacterial co-infection treatment.
Asunto(s)
Infecciones Bacterianas , Coinfección , Virosis , Virus , Humanos , Interferones/metabolismo , Virus/metabolismoRESUMEN
Viral-bacterial coinfections of the respiratory tract have long been associated with worsened disease outcomes. Clinical and basic research studies demonstrate that these infections are driven via complex interactions between the infecting pathogens, microbiome, and host immune response, although how these interactions contribute to disease progression is still not fully understood. Research over the last decade shows that the gut has a significant role in mediating respiratory outcomes, in a phenomenon known as the "gut-lung axis." Emerging literature demonstrates that acute respiratory viruses can modulate the gut-lung axis, suggesting that dysregulation of gut-lung cross talk may be a contributing factor during respiratory coinfection. This review will summarize the current literature regarding modulation of the gut-lung axis during acute respiratory infection, with a focus on the role of the microbiome, secondary infections, and the host immune response.
Asunto(s)
Coinfección , Microbioma Gastrointestinal , Microbiota , Infecciones del Sistema Respiratorio , Humanos , Microbioma Gastrointestinal/fisiología , Pulmón/microbiología , Infecciones del Sistema Respiratorio/microbiología , Bacterias/genéticaRESUMEN
Pseudomonas aeruginosa grows as a biofilm under many environmental conditions, and the bacterium can disperse from biofilms via highly regulated, dynamic processes. However, physiologic triggers of biofilm dispersal remain poorly understood. Based on prior literature describing dispersal triggered by forms of starvation, we tested bacterial respiratory inhibitors for biofilm dispersal in two models resembling chronic airway infections. Our underlying hypothesis was that respiratory inhibitors could serve as a model for the downstream effects of starvation. We used two experimental conditions. In the first condition, biofilms were grown and dispersed from the surface of airway epithelial cells, and the second condition was a model where biofilms were grown on glass in cell culture media supplemented with host-relevant iron sources. In both biofilm models, the respiratory inhibitors potassium cyanide and sodium azide each triggered biofilm dispersal. We hypothesized that cyanide-induced dispersal was due to respiratory inhibition rather than signaling via an alternative mechanism, and, indeed, if respiration was supported by overexpression of cyanide-insensitive oxidase, dispersal was prevented. Dispersal required the activity of the cyclic-di-GMP regulated protease LapG, reinforcing the role of matrix degradation in dispersal. Finally, we examined the roles of individual phosphodiesterases, previously implicated in dispersal to specific triggers, and found signaling to be highly redundant. Combined deletion of the phosphodiesterases dipA, bifA, and rbdA was required to attenuate the dispersal phenotype. In summary, this work adds insight into the physiology of biofilm dispersal under environmental conditions in which bacterial respiration is abruptly limited. IMPORTANCE The bacterium Pseudomonas aeruginosa grows in biofilm communities that are very difficult to treat in human infections. Growing as a biofilm can protect bacteria from antibiotics and the immune system. Bacteria can leave a biofilm through a process called "dispersal." Dispersed bacteria seed new growth areas and are more susceptible to killing by antibiotics. The triggers for biofilm dispersal are not well understood, and if we understood dispersal better it might lead to the development of new treatments for infection. In this paper, we find that inhibiting P. aeurginosa's ability to respire (generate energy) can trigger dispersal from a biofilm grown in association with human respiratory epithelial cells in culture. The dispersal process requires a protease which is previously known to degrade the biofilm matrix. These findings give us a better understanding of how the biofilm dispersal process works so that future research can discover better ways of clearing bacteria growing in biofilms.
Asunto(s)
Biopelículas , Pseudomonas aeruginosa , Humanos , Pseudomonas aeruginosa/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Antibacterianos/farmacología , Péptido Hidrolasas/metabolismo , Cianuros/metabolismo , Cianuros/farmacología , Regulación Bacteriana de la Expresión Génica , Proteínas Bacterianas/metabolismo , GMP Cíclico/metabolismoRESUMEN
BACKGROUND: Shotgun sequencing of cultured microbial isolates/individual eukaryotes (whole-genome sequencing) and microbial communities (metagenomics) has become commonplace in biology. Very often, sequenced samples encompass organisms spanning multiple domains of life, necessitating increasingly elaborate software for accurate taxonomic classification of assembled sequences. RESULTS: While many software tools for taxonomic classification exist, SprayNPray offers a quick and user-friendly, semi-automated approach, allowing users to separate contigs by taxonomy (and other metrics) of interest. Easy installation, usage, and intuitive output, which is amenable to visual inspection and/or further computational parsing, will reduce barriers for biologists beginning to analyze genomes and metagenomes. This approach can be used for broad-level overviews, preliminary analyses, or as a supplement to other taxonomic classification or binning software. SprayNPray profiles contigs using multiple metrics, including closest homologs from a user-specified reference database, gene density, read coverage, GC content, tetranucleotide frequency, and codon-usage bias. CONCLUSIONS: The output from this software is designed to allow users to spot-check metagenome-assembled genomes, identify, and remove contigs from putative contaminants in isolate assemblies, identify bacteria in eukaryotic assemblies (and vice-versa), and identify possible horizontal gene transfer events.
Asunto(s)
Metagenoma , Microbiota , Bacterias/genética , Metagenómica , Microbiota/genética , Programas InformáticosRESUMEN
Severe asthma exacerbations are a major cause of school absences and healthcare costs in children, particularly those in high-risk racial/ethnic groups.To identify susceptibility genes for severe asthma exacerbations in Latino children and adolescents, we conducted a meta-analysis of genome-wide association studies (GWAS) in 4010 Latino youth with asthma in four independent cohorts, including 1693 Puerto Ricans, 1019 Costa Ricans, 640 Mexicans, 256 Brazilians and 402 members of other Latino subgroups. We then conducted methylation quantitative trait locus, expression quantitative trait locus and expression quantitative trait methylation analyses to assess whether the top single nucleotide polymorphism (SNP) in the meta-analysis is linked to DNA methylation and gene expression in nasal (airway) epithelium in separate cohorts of Puerto Rican and Dutch children and adolescents.In the meta-analysis of GWAS, an SNP in FLJ22447 (rs2253681) was significantly associated with 1.55 increased odds of severe asthma exacerbation (95% CI 1.34-1.79, p=6.3×10-9). This SNP was significantly associated with DNA methylation of a CpG site (cg25024579) at the FLJ22447 locus, which was in turn associated with increased expression of KCNJ2-AS1 in nasal airway epithelium from Puerto Rican children and adolescents (ß=0.10, p=2.18×10-7).SNP rs2253681 was significantly associated with both DNA methylation of a cis-CpG in FLJ22447 and severe asthma exacerbations in Latino youth. This may be partly explained by changes in airway epithelial expression of a gene recently implicated in atopic asthma in Puerto Rican children and adolescents (KCNJ2-AS1).
Asunto(s)
Asma , Estudio de Asociación del Genoma Completo , Adolescente , Asma/genética , Brasil , Niño , Hispánicos o Latinos/genética , Humanos , Puerto RicoRESUMEN
Recurrent Pseudomonas aeruginosa infections coupled with robust, damaging neutrophilic inflammation characterize the chronic lung disease cystic fibrosis (CF). The proresolving lipid mediator, 15-epi lipoxin A4 (15-epi LXA4), plays a critical role in limiting neutrophil activation and tissue inflammation, thus promoting the return to tissue homeostasis. Here, we show that a secreted P. aeruginosa epoxide hydrolase, cystic fibrosis transmembrane conductance regulator inhibitory factor (Cif), can disrupt 15-epi LXA4 transcellular biosynthesis and function. In the airway, 15-epi LXA4 production is stimulated by the epithelial-derived eicosanoid 14,15-epoxyeicosatrienoic acid (14,15-EET). Cif sabotages the production of 15-epi LXA4 by rapidly hydrolyzing 14,15-EET into its cognate diol, eliminating a proresolving signal that potently suppresses IL-8-driven neutrophil transepithelial migration in vitro. Retrospective analyses of samples from patients with CF supported the translational relevance of these preclinical findings. Elevated levels of Cif in bronchoalveolar lavage fluid were correlated with lower levels of 15-epi LXA4, increased IL-8 concentrations, and impaired lung function. Together, these findings provide structural, biochemical, and immunological evidence that the bacterial epoxide hydrolase Cif disrupts resolution pathways during bacterial lung infections. The data also suggest that Cif contributes to sustained pulmonary inflammation and associated loss of lung function in patients with CF.
Asunto(s)
Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Proteínas Bacterianas/metabolismo , Lipoxinas/metabolismo , Activación Neutrófila/inmunología , Neutrófilos/inmunología , Pseudomonas aeruginosa/metabolismo , Factores de Virulencia/metabolismo , Ácido 8,11,14-Eicosatrienoico/metabolismo , Líquido del Lavado Bronquioalveolar/química , Línea Celular , Cristalografía por Rayos X , Fibrosis Quística/microbiología , Fibrosis Quística/patología , Humanos , Inflamación/inducido químicamente , Enfermedades Pulmonares/microbiología , Enfermedades Pulmonares/patología , Infecciones por Pseudomonas/microbiología , Infecciones por Pseudomonas/patología , Pseudomonas aeruginosa/patogenicidad , Estudios RetrospectivosRESUMEN
Clinical observations link respiratory virus infection and Pseudomonas aeruginosa colonization in chronic lung disease, including cystic fibrosis (CF) and chronic obstructive pulmonary disease. The development of P. aeruginosa into highly antibiotic-resistant biofilm communities promotes airway colonization and accounts for disease progression in patients. Although clinical studies show a strong correlation between CF patients' acquisition of chronic P. aeruginosa infections and respiratory virus infection, little is known about the mechanism by which chronic P. aeruginosa infections are initiated in the host. Using a coculture model to study the formation of bacterial biofilm formation associated with the airway epithelium, we show that respiratory viral infections and the induction of antiviral interferons promote robust secondary P. aeruginosa biofilm formation. We report that the induction of antiviral IFN signaling in response to respiratory syncytial virus (RSV) infection induces bacterial biofilm formation through a mechanism of dysregulated iron homeostasis of the airway epithelium. Moreover, increased apical release of the host iron-binding protein transferrin during RSV infection promotes P. aeruginosa biofilm development in vitro and in vivo. Thus, nutritional immunity pathways that are disrupted during respiratory viral infection create an environment that favors secondary bacterial infection and may provide previously unidentified targets to combat bacterial biofilm formation.
Asunto(s)
Biopelículas/crecimiento & desarrollo , Inmunidad , Fenómenos Fisiológicos de la Nutrición , Pseudomonas aeruginosa/fisiología , Infecciones por Virus Sincitial Respiratorio/patología , Virus Sincitiales Respiratorios/fisiología , Animales , Antivirales/farmacología , Bronquios/patología , Líquido del Lavado Bronquioalveolar , Fibrosis Quística/microbiología , Fibrosis Quística/patología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/microbiología , Células Epiteliales/virología , Homeostasis/efectos de los fármacos , Humanos , Interferón beta/farmacología , Hierro/farmacología , Ratones , Interacciones Microbianas/efectos de los fármacos , Modelos Biológicos , Pseudomonas aeruginosa/efectos de los fármacos , Virus Sincitiales Respiratorios/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transferrina/metabolismoRESUMEN
The opportunistic pathogen Pseudomonas aeruginosa colonizes the lungs of susceptible individuals by deploying virulence factors targeting host defenses. The secreted factor Cif (cystic fibrosis transmembrane conductance regulator inhibitory factor) dysregulates the endocytic recycling of CFTR and thus reduces CFTR abundance in host epithelial membranes. We have postulated that the decrease in ion secretion mediated by Cif would slow mucociliary transport and decrease bacterial clearance from the lungs. To test this hypothesis, we explored the effects of Cif in cultured epithelia and in the lungs of mice. We developed a strategy to interpret the "hurricane-like" motions observed in reconstituted cultures and identified a Cif-mediated decrease in the velocity of mucus transport in vitro. Presence of Cif also increased the number of bacteria recovered at two time points in an acute mouse model of pneumonia caused by P. aeruginosa. Furthermore, recent work has demonstrated an inverse correlation between the airway concentrations of Cif and 15-epi-lipoxin A4, a proresolving lipid mediator important in host defense and the resolution of pathogen-initiated inflammation. Here, we observe elevated levels of 15-epi-lipoxin A4 in the lungs of mice infected with a strain of P. aeruginosa that expresses only an inactive form of cif compared with those mice infected with wild-type P. aeruginosa. Together these data support the inclusion of Cif on the list of virulence factors that assist P. aeruginosa in colonizing and damaging the airways of compromised patients. Furthermore, this study establishes techniques that enable our groups to explore the underlying mechanisms of Cif effects during respiratory infection.
Asunto(s)
Proteínas Bacterianas/metabolismo , Bronquios/patología , Células Epiteliales/patología , Neumonía/etiología , Infecciones por Pseudomonas/complicaciones , Pseudomonas aeruginosa/patogenicidad , Factores de Virulencia/metabolismo , Animales , Transporte Biológico , Bronquios/enzimología , Bronquios/microbiología , Células Cultivadas , Modelos Animales de Enfermedad , Células Epiteliales/enzimología , Células Epiteliales/microbiología , Humanos , Lipoxinas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Depuración Mucociliar , Neumonía/metabolismo , Neumonía/patología , Infecciones por Pseudomonas/microbiologíaRESUMEN
Sodium nitrite inhibits bacterial respiration and is in development as an antimicrobial for chronic bacterial infections associated with cystic fibrosis. The goal of the current study was to investigate the interaction between nitrite and ciprofloxacin. Using liquid culture killing assays and a biotic biofilm model, we observed that nitrite induces tolerance of ciprofloxacin.
Asunto(s)
Antibacterianos/farmacología , Biopelículas/efectos de los fármacos , Ciprofloxacina/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Nitrito de Sodio/farmacología , Fibrosis Quística/microbiología , Pruebas de Sensibilidad MicrobianaRESUMEN
Respiratory virus infections are common but generally self-limiting infections in healthy individuals. Although early clinical studies reported low detection rates, the development of molecular diagnostic techniques by PCR has led to an increased recognition that respiratory virus infections are associated with morbidity and acute exacerbations of chronic lung diseases, such as cystic fibrosis (CF). The airway epithelium is the first barrier encountered by respiratory viruses following inhalation and the primary site of respiratory viral replication. Here, we describe how the airway epithelial response to respiratory viral infections contributes to disease progression in patients with CF and other chronic lung diseases, including the role respiratory viral infections play in bacterial acquisition in the CF patient lung.
Asunto(s)
Fibrosis Quística/complicaciones , Infecciones Oportunistas/etiología , Infecciones del Sistema Genital/etiología , Mucosa Respiratoria/virología , Virosis/etiología , Animales , Infecciones Bacterianas/etiología , Infecciones Bacterianas/metabolismo , Infecciones Bacterianas/patología , Enfermedad Crónica , Coinfección , Progresión de la Enfermedad , Humanos , Enfermedades Pulmonares/complicaciones , Infecciones Oportunistas/metabolismo , Infecciones Oportunistas/patología , Infecciones del Sistema Genital/metabolismo , Infecciones del Sistema Genital/patología , Virosis/metabolismo , Virosis/patologíaRESUMEN
Pseudomonas aeruginosa is an opportunistic pathogen that can cause nosocomial and chronic infections in immunocompromised patients. P. aeruginosa secretes a lipoxygenase, LoxA, but the biological role of this enzyme is currently unknown. LoxA is poorly similar in sequence to both soybean LOX-1 (s15-LOX-1) and human 15-LOX-1 (37 and 39%, respectively) yet has kinetics comparably fast versus those of s15-LOX-1 (at pH 6.5, Kcat = 181 ± 6 s(-1) and Kcat/KM = 16 ± 2 µM(-1) s(-1)). LoxA is capable of efficiently catalyzing the peroxidation of a broad range of free fatty acid (FA) substrates (e.g., AA and LA) with high positional specificity, indicating a 15-LOX. Its mechanism includes hydrogen atom abstraction [a kinetic isotope effect (KIE) of >30], yet LoxA is a poor catalyst against phosphoester FAs, suggesting that LoxA is not involved in membrane decomposition. LoxA also does not react with 5- or 15-HETEs, indicating poor involvement in lipoxin production. A LOX high-throughput screen of the LOPAC library yielded a variety of low-micromolar inhibitors; however, none selectively targeted LoxA over the human LOX isozymes. With respect to cellular activity, the level of LoxA expression is increased when P. aeruginosa undergoes the transition to a biofilm mode of growth, but LoxA is not required for biofilm growth on abiotic surfaces. However, LoxA does appear to be required for biofilm growth in association with the host airway epithelium, suggesting a role for LoxA in mediating bacterium-host interactions during colonization.
Asunto(s)
Araquidonato 15-Lipooxigenasa/química , Araquidonato 15-Lipooxigenasa/metabolismo , Ácidos Hidroxieicosatetraenoicos/metabolismo , Inhibidores de la Lipooxigenasa/metabolismo , Pseudomonas aeruginosa/enzimología , Secuencia de Aminoácidos , Animales , Formación de Anticuerpos , Araquidonato 15-Lipooxigenasa/inmunología , Humanos , Cinética , Conejos , Especificidad por SustratoRESUMEN
OBJECTIVES: Chronic infections with the opportunistic pathogen Pseudomonas aeruginosa are responsible for the majority of the morbidity and mortality in patients with cystic fibrosis (CF). While P. aeruginosa infections may initially be treated successfully with standard antibiotics, chronic infections typically arise as bacteria transition to a biofilm mode of growth and acquire remarkable antimicrobial resistance. To address the critical need for novel antimicrobial therapeutics that can effectively suppress chronic bacterial infections in challenging physiological environments, such as the CF lung, we have rationally designed a de novo engineered cationic antimicrobial peptide, the 24-residue WLBU2, with broad-spectrum antibacterial activity for pan-drug-resistant P. aeruginosa in liquid culture. In the current study, we tested the hypothesis that WLBU2 also prevents P. aeruginosa biofilm growth. METHODS: Using abiotic and biotic biofilm assays, co-culturing P. aeruginosa with polarized human airway epithelial cells, we examined the ability of WLBU2 to prevent biofilm biogenesis alone and in combination with currently used antibiotics. RESULTS: We observed a dose-dependent reduction in biofilm growth on an abiotic surface and in association with CF airway epithelial cells. WLBU2 prevented P. aeruginosa biofilm formation when co-cultured with mucus-producing primary human CF airway epithelial cells and using CF clinical isolates of P. aeruginosa, even at low pH and high salt conditions that mimic the CF airway. When used in combination, WLBU2 significantly increases killing by the commonly used antibiotics tobramycin, ciprofloxacin, ceftazidime and meropenem. CONCLUSIONS: While other studies have demonstrated the ability of natural and synthetic antimicrobial peptides to prevent abiotic bacterial biofilm formation, the current studies for the first time demonstrate the effective peptide treatment of a biotic bacterial biofilm in a setting similar to the CF airway, and without negative effects on human airway epithelial cells, thus highlighting the unique potential of this engineered cationic antimicrobial peptide for treatment of human respiratory infections.
Asunto(s)
Antiinfecciosos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Biopelículas/efectos de los fármacos , Células Epiteliales/microbiología , Pseudomonas aeruginosa/efectos de los fármacos , Proteínas Recombinantes/farmacología , Péptidos Catiónicos Antimicrobianos/genética , Biopelículas/crecimiento & desarrollo , Línea Celular , Técnicas de Cocultivo , Humanos , Ingeniería de Proteínas , Pseudomonas aeruginosa/fisiología , Proteínas Recombinantes/genéticaRESUMEN
Endocytic recycling of the cystic fibrosis transmembrane conductance regulator (CFTR) is blocked by the CFTR inhibitory factor (Cif). Originally discovered in Pseudomonas aeruginosa, Cif is a secreted epoxide hydrolase that is transcriptionally regulated by CifR, an epoxide-sensitive repressor. In this report, we investigate a homologous protein found in strains of the emerging nosocomial pathogens Acinetobacter nosocomialis and Acinetobacter baumannii ("aCif"). Like Cif, aCif is an epoxide hydrolase that carries an N-terminal secretion signal and can be purified from culture supernatants. When applied directly to polarized airway epithelial cells, mature aCif triggers a reduction in CFTR abundance at the apical membrane. Biochemical and crystallographic studies reveal a dimeric assembly with a stereochemically conserved active site, confirming our motif-based identification of candidate Cif-like pathogenic EH sequences. Furthermore, cif expression is transcriptionally repressed by a CifR homolog ("aCifR") and is induced in the presence of epoxides. Overall, this Acinetobacter protein recapitulates the essential attributes of the Pseudomonas Cif system and thus may facilitate airway colonization in nosocomial lung infections.
Asunto(s)
Acinetobacter baumannii/metabolismo , Acinetobacter/enzimología , Proteínas Bacterianas/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Epóxido Hidrolasas/metabolismo , Acinetobacter/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Cristalografía por Rayos X , Endocitosis , Epóxido Hidrolasas/genética , Eliminación de Gen , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Plásmidos/metabolismo , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Transcripción Genética , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
Cif (PA2934), a bacterial virulence factor secreted in outer membrane vesicles by Pseudomonas aeruginosa, increases the ubiquitination and lysosomal degradation of some, but not all, plasma membrane ATP-binding cassette transporters (ABC), including the cystic fibrosis transmembrane conductance regulator and P-glycoprotein. The goal of this study was to determine whether Cif enhances the ubiquitination and degradation of the transporter associated with antigen processing (TAP1 and TAP2), members of the ABC transporter family that play an essential role in antigen presentation and intracellular pathogen clearance. Cif selectively increased the amount of ubiquitinated TAP1 and increased its degradation in the proteasome of human airway epithelial cells. This effect of Cif was mediated by reducing USP10 deubiquitinating activity, resulting in increased polyubiquitination and proteasomal degradation of TAP1. The reduction in TAP1 abundance decreased peptide antigen translocation into the endoplasmic reticulum, an effect that resulted in reduced antigen available to MHC class I molecules for presentation at the plasma membrane of airway epithelial cells and recognition by CD8(+) T cells. Cif is the first bacterial factor identified that inhibits TAP function and MHC class I antigen presentation.