RESUMEN
The statin family of cholesterol-lowering drugs is known to have pleiotropic properties which include anti-inflammatory and immunomodulatory effects. Statins exert their pleiotropic effects by altering expression of human immune regulators including pro-inflammatory cytokines. Previously we found that statins modulate virulence phenotypes of the human pathogen Pseudomonas aeruginosa, and sought to investigate if simvastatin could alter the host response to this organism in lung epithelial cells. Simvastatin increased the expression of the P. aeruginosa target genes KLF2, KLF6, IL-8 and CCL20. Furthermore, both simvastatin and P. aeruginosa induced alternative splicing of KLF6. The novel effect of simvastatin on wtKLF6 expression was found to be responsible for induction of the KLF6 regulated genes CCL20 and iNOS. Simvastatin also increased the adhesion of P. aeruginosa to host cells, without altering invasion or cytotoxicity. This study demonstrated that simvastatin had several novel effects on the pulmonary cellular immune response.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Infecciones por Pseudomonas/tratamiento farmacológico , Pseudomonas aeruginosa/efectos de los fármacos , Simvastatina/administración & dosificación , Empalme Alternativo/efectos de los fármacos , Línea Celular , Quimiocina CCL20/biosíntesis , Humanos , Inmunidad Celular/efectos de los fármacos , Interleucina-8/biosíntesis , Factor 6 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/biosíntesis , Pulmón/efectos de los fármacos , Pulmón/inmunología , Pulmón/patología , Proteínas Proto-Oncogénicas/biosíntesis , Infecciones por Pseudomonas/genética , Infecciones por Pseudomonas/patología , Pseudomonas aeruginosa/patogenicidadAsunto(s)
Antibacterianos/farmacología , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Lovastatina/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/patogenicidad , Técnicas In Vitro , Lovastatina/análogos & derivados , Pruebas de Sensibilidad Microbiana , Fenotipo , Pseudomonas aeruginosa/genética , Virulencia/genéticaRESUMEN
Sensitive sensory mechanisms are instrumental in affording Pseudomonas aeruginosa the capacity to establish diverse yet severe human infections, which can manifest themselves in long-term untreatable disease. The ability of P. aeruginosa to tightly regulate gene expression and virulence factor production, in response to activation of these sensory components, enables the pathogen to sustain infection despite the host immune response and aggressive antibiotic treatment. Although a number of factors are recognized as playing a role in early infection, very little is known regarding the sensors involved in this process. In this study, we identified P. aeruginosa PA3191 as a novel host-responsive sensor that plays a key role during P. aeruginosa-host interactions and is required for optimum colonization and dissemination in a mouse model of infection. We demonstrated that PA3191 contributed to modulation of the type III secretion system (T3SS) in response to host cells and T3SS-inducing conditions in vitro. PA3191 (designated GtrS) acted in concert with the response regulator GltR to regulate the OprB transport system and subsequently carbon metabolism. Through this signal transduction pathway, T3SS activation was mediated via the RsmAYZ regulatory cascade and involved the global anaerobic response regulator Anr.
Asunto(s)
Proteínas Bacterianas/metabolismo , Sistemas de Secreción Bacterianos , Interacciones Huésped-Patógeno , Pseudomonas aeruginosa/genética , Virulencia , Animales , Proteínas Bacterianas/genética , Células CHO , Carbono/metabolismo , Línea Celular , Cricetinae , Regulación Bacteriana de la Expresión Génica , Humanos , Ratones , Ratones Endogámicos BALB C , Proteínas de Transporte de Monosacáridos/metabolismo , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidad , Transducción de Señal , Transactivadores/metabolismoRESUMEN
A steep oxygen gradient within the mucus of the cystic fibrosis (CF) lung combined with the biofilm mode of bacterial growth forces respiratory pathogens to adapt to varying oxygen availability. This study presents the novel finding that the Pseudomonas aeruginosa response to limiting oxygen stress includes induction of its type III secretion system (T3SS), which subsequently contributes towards host cell cytotoxicity. In P. aeruginosa, the global anaerobic response regulator Anr perceives low oxygen and subsequently triggers gene expression of a range of target genes, including the response regulator narL. Here we demonstrate that microaerobic induction of the T3SS is dependent on Anr, and that this is mediated through direct NarL transcriptional repression of the sRNAs rsmY and rsmZ, allowing free RsmA protein to positively regulate the T3SS. This study reveals a novel interplay between the Anr-NarL and RsmAYZ regulatory circuits, and introduces RsmA as an important regulator during P. aeruginosa adaptation to a low-oxygen environment.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Proteínas de Transporte de Membrana/biosíntesis , MicroARNs/metabolismo , Oxígeno/metabolismo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Adaptación Fisiológica , Aerobiosis , Proteínas Bacterianas/metabolismo , Humanos , Transactivadores/metabolismoRESUMEN
The dipeptide transport operon in bacteria comprises genes for the transport and metabolism of amino acids and dipeptides, as well as haem and haem precursors such as aminolaevulinic acid. Such nutrient and mineral sources are vital for bacteria to survive in and colonize a range of niches. In silico analysis of the dipeptide transport systems in sequenced Pseudomonas species identified the presence of two genes in P. aeruginosa strains that were absent in other sequenced pseudomonads. These genes encode a putative metallopeptidase, PA4498, and a putative transcriptional regulator, PA4499. Proteomic profiling of wild-type PAO1 and a PA4499 mutant strain indicated that PA4499 negatively regulated the putative peptidase, PA4498. Transcriptional fusion analysis verified that expression of PA4498 (mdpA, metallo-dipeptidase aeruginosa) was negatively regulated by the downstream putative transcriptional regulator PA4499 (psdR, Pseudomonas dipeptide regulator). Transcriptional fusion analysis also showed that the dppABCDF operon was under the negative control of psdR. Functional genomic analysis of mdpA indicated that it is required for the metabolism of a range of dipeptides and that it contributes to the cytotoxicity of PAO1 on an epithelial cell line.
Asunto(s)
Proteínas Bacterianas/metabolismo , Dipeptidasas/metabolismo , Dipéptidos/metabolismo , Regulación Bacteriana de la Expresión Génica , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/metabolismo , Factores de Transcripción/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Transporte Biológico , Línea Celular Transformada , Dipeptidasas/química , Dipeptidasas/genética , Regulación hacia Abajo , Electroforesis en Gel Bidimensional , Células Epiteliales/microbiología , Humanos , Operón , Proteómica , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidad , Factores de Transcripción/química , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
The ability of Pseudomonas aeruginosa to cause a broad range of infections in humans is due, at least in part, to its adaptability and its capacity to regulate the expression of key virulence genes in response to specific environmental conditions. Multiple two-component response regulators have been shown to facilitate rapid responses to these environmental conditions, including the coordinated expression of specific virulence determinants. RsmA is a posttranscriptional regulatory protein which controls the expression of a number of virulence-related genes with relevance for acute and chronic infections. Many membrane-bound sensors, including RetS, LadS, and GacS, are responsible for the reciprocal regulation of genes associated with acute infection and chronic persistence. In P. aeruginosa this is due to sensors influencing the expression of the regulatory RNA RsmZ, with subsequent effects on the level of free RsmA. While interactions between an rsmA mutant and human airway epithelial cells have been examined in vitro, the role of RsmA during infection in vivo has not been determined yet. Here the function of RsmA in both acute and chronic models of infection was examined. The results demonstrate that RsmA is involved in initial colonization and dissemination in a mouse model of acute pneumonia. Furthermore, while loss of RsmA results in reduced colonization during the initial stages of acute infection, the data show that mutation of rsmA ultimately favors chronic persistence and results in increased inflammation in the lungs of infected mice.
Asunto(s)
Neumonía/microbiología , Neumonía/patología , Infecciones por Pseudomonas/microbiología , Infecciones por Pseudomonas/patología , Pseudomonas aeruginosa/patogenicidad , Factores de Transcripción/fisiología , Factores de Virulencia/fisiología , Animales , Femenino , Eliminación de Gen , Ratones , Ratones Endogámicos BALB C , Pseudomonas aeruginosa/genética , Factores de Transcripción/genética , Virulencia/genética , Factores de Virulencia/genéticaRESUMEN
Pseudomonas aeruginosa is an important opportunistic pathogen which is capable of causing both acute and chronic infections in immunocompromised patients. Successful adaptation of the bacterium to its host environment relies on the ability of the organism to tightly regulate gene expression. RsmA, a small RNA-binding protein, controls the expression of a large number of virulence-related genes in P. aeruginosa, including those encoding the type III secretion system and associated effector proteins, with important consequences for epithelial cell morphology and cytotoxicity. In order to examine the influence of RsmA-regulated functions in the pathogen on gene expression in the host, we compared global expression profiles of airway epithelial cells in response to infection with P. aeruginosa PAO1 and an rsmA mutant. The RsmA-dependent response of host cells was characterized by significant changes in the global transcriptional pattern, including the increased expression of two Kruppel-like factors, KLF2 and KLF6. This increased expression was mediated by specific type III effector proteins. ExoS was required for the enhanced expression of KLF2, whereas both ExoS and ExoY were required for the enhanced expression of KLF6. Neither ExoT nor ExoU influenced the expression of the transcription factors. Additionally, the increased gene expression of KLF2 and KLF6 was associated with ExoS-mediated cytotoxicity. Therefore, this study identifies for the first time the human transcription factors KLF2 and KLF6 as targets of the P. aeruginosa type III exoenzymes S and Y, with potential importance in host cell death.
Asunto(s)
ADP Ribosa Transferasas/genética , Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Regulación de la Expresión Génica , Glucosiltransferasas/genética , Factores de Transcripción de Tipo Kruppel/genética , Proteínas Proto-Oncogénicas/genética , Pseudomonas aeruginosa/patogenicidad , Mucosa Respiratoria/microbiología , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Regulación Bacteriana de la Expresión Génica , Humanos , Factor 6 Similar a Kruppel , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/genética , Proteínas Represoras/metabolismo , Mucosa Respiratoria/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética , Virulencia/genéticaRESUMEN
Posttranscriptional regulation of certain virulence-related genes in Pseudomonas aeruginosa is brought about by RsmA, a small RNA-binding protein. During interaction with airway epithelial cells, RsmA promoted actin depolymerization, cytotoxicity, and anti-internalization of P. aeruginosa by positively regulating the virulence-associated type III secretion system.