RESUMEN
BACKGROUND: The Salmonella enterica serovar Typhimurium PhoPQ two component system (TCS) is activated by low Mg2+ levels, low pH and by antimicrobial peptides (AP). Under Mg2+ limitation, the PhoPQ system induces pmrD expression, which post-translationally activates the PmrAB TCS. PhoPQ and PmrAB control many genes required for intracellular survival and pathogenesis. These include the polymyxin resistance (pmr) operon, which is required for aminoarabinose modification of LPS and protecting the outer membrane from antimicrobial peptide disruption and killing. Extracellular DNA is a ubiquitous polymer in the matrix of biofilms and accumulates in some infection sites. Extracellular DNA chelates cations and thus activates the Pseudomonas aeruginosa PhoPQ/PmrAB systems, leading to expression of the orthologous arn (pmr) operon. RESULTS: Here we show that extracellular DNA induces expression of the S. Typhimurium pmr antimicrobial peptide resistance operon in a PhoPQ and PmrAB-dependent manner. Induction of the pmr genes by DNA was blocked when present with excess Mg2+. Exogenous DNA led to increased resistance of planktonic cultures to aminoglycosides, antimicrobial peptides (AP) and ciprofloxacin, but only AP resistance was PhoPQ/PmrAB-dependent. Extracellular DNA was shown to be a matrix component of S. Typhimurium biofilms cultivated in flow chambers and on glass surfaces. A pmrH-gfp fusion was highly expressed in flow chamber biofilms cultivated in medium with repressing levels of 10 mM Mg2+ and co-localized with eDNA. Expression of pmrH-lux was monitored in plastic peg biofilms and shown to require PhoPQ and PmrAB. Biofilms had higher levels of pmrH expression compared to planktonic cultures. We propose that DNA accumulation in biofilms contributes to the increased pmrH-lux expression in biofilms. CONCLUSIONS: The Salmonella PhoPQ/PmrAB systems and antimicrobial peptide resistance are activated by the cation chelating properties of extracellular DNA. DNA-induced AP resistance may allow immune evasion and increased survival of S. Typhimurium biofilms formed during extracellular growth stages of an infection or outside the host.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/farmacología , ADN/química , Operón , Salmonella typhimurium/crecimiento & desarrollo , Proteínas Bacterianas/metabolismo , Biopelículas , Quelantes/química , Medios de Cultivo/química , Farmacorresistencia Bacteriana , Matriz Extracelular/química , Matriz Extracelular/microbiología , Magnesio/química , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/genética , Factores de Transcripción/metabolismoRESUMEN
Extracellular DNA acts as a cation chelator and induces the expression of antibiotic resistance genes regulated by Mg(2+) levels. Here we report the characterization of novel DNA-induced genes in Pseudomonas aeruginosa that are annotated as homologs of the spermidine synthesis genes speD (PA4773) and speE (PA4774). The addition of sublethal concentrations of DNA and membrane-damaging antibiotics induced expression of the genes PA4773 to PA4775, as shown using transcriptional lux fusions and quantitative RT-PCR. Exogenous polyamine addition prevented DNA- and peptide-mediated gene induction. Mutation of PA4774 resulted in an increased outer membrane (OM) susceptibility phenotype upon polymyxin B, CP10A, and gentamicin treatment. When the membrane-localized fluorescent probe C(11)-BODIPY(581/591) was used as an indicator of peroxidation of membrane lipids, the PA4774::lux mutant demonstrated an increased susceptibility to oxidative membrane damage from H(2)O(2) treatment. Addition of exogenous polyamines protected the membranes of the PA4774::lux mutant from polymyxin B and H(2)O(2) treatment. Polyamines from the outer surface were isolated and shown to contain putrescine and spermidine by using high-performance liquid chromatography and mass spectrometry. The PA4774::lux mutant did not produce spermidine on the cell surface, but genetic complementation restored surface spermidine production as well as the antibiotic and oxidative stress resistance phenotypes of the membrane. We have identified new functions for spermidine on the cell surface and propose that polyamines are produced under Mg(2+)-limiting conditions as an organic polycation to bind lipopolysaccharide (LPS) and to stabilize and protect the outer membrane against antibiotic and oxidative damage.
Asunto(s)
Antibacterianos/farmacología , Membrana Celular/metabolismo , Farmacorresistencia Bacteriana/genética , Estrés Oxidativo , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/metabolismo , Espermidina/metabolismo , Péptidos Catiónicos Antimicrobianos/farmacología , Membrana Celular/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica , Gentamicinas/farmacología , Peróxido de Hidrógeno/farmacología , Lipopolisacáridos/metabolismo , Magnesio/metabolismo , Pruebas de Sensibilidad Microbiana , Estrés Oxidativo/genética , Polimixina B/farmacología , Pseudomonas aeruginosa/genética , Putrescina/metabolismo , Espermidina Sintasa/genéticaRESUMEN
Pseudomonas aeruginosa is an opportunistic pathogen capable of causing both acute and chronic infections in susceptible hosts. Chronic P. aeruginosa infections are thought to be caused by bacterial biofilms. Biofilms are highly structured, multicellular, microbial communities encased in an extracellular matrix that enable long-term survival in the host. The aim of this research was to develop an animal model that would allow an in vivo study of P. aeruginosa biofilm infections in a Drosophila melanogaster host. At 24 h post oral infection of Drosophila, P. aeruginosa biofilms localized to and were visualized in dissected Drosophila crops. These biofilms had a characteristic aggregate structure and an extracellular matrix composed of DNA and exopolysaccharide. P. aeruginosa cells recovered from in vivo grown biofilms had increased antibiotic resistance relative to planktonically grown cells. In vivo, biofilm formation was dependent on expression of the pel exopolysaccharide genes, as a pelB::lux mutant failed to form biofilms. The pelB::lux mutant was significantly more virulent than PAO1, while a hyperbiofilm strain (PAZHI3) demonstrated significantly less virulence than PAO1, as indicated by survival of infected flies at day 14 postinfection. Biofilm formation, by strains PAO1 and PAZHI3, in the crop was associated with induction of diptericin, cecropin A1 and drosomycin antimicrobial peptide gene expression 24 h postinfection. In contrast, infection with the non-biofilm forming strain pelB::lux resulted in decreased AMP gene expression in the fly. In summary, these results provide novel insights into host-pathogen interactions during P. aeruginosa oral infection of Drosophila and highlight the use of Drosophila as an infection model that permits the study of P. aeruginosa biofilms in vivo.
Asunto(s)
Biopelículas/crecimiento & desarrollo , Drosophila melanogaster/microbiología , Interacciones Huésped-Patógeno , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/patogenicidad , Animales , Antiinfecciosos/metabolismo , Péptidos Catiónicos Antimicrobianos/metabolismo , ADN Bacteriano/metabolismo , Drosophila melanogaster/fisiología , Farmacorresistencia Bacteriana , Expresión Génica , Masculino , Modelos Animales , Polisacáridos Bacterianos/metabolismo , VirulenciaRESUMEN
Biofilm formation is a conserved strategy for long-term bacterial survival in nature and during infections. Biofilms are multicellular aggregates of cells enmeshed in an extracellular matrix. The RetS, GacS and LadS sensors control the switch from a planktonic to a biofilm mode of growth in Pseudomonas aeruginosa. Here we detail our approach to identify environmental triggers of biofilm formation by investigating environmental conditions that repress expression of the biofilm repressor RetS. Mg(2+) limitation repressed the expression of retS leading to increased aggregation, exopolysaccharide (EPS) production and biofilm formation. Repression of retS expression under Mg(2+) limitation corresponded with induced expression of the GacA-controlled small regulatory RNAs rsmZ and rsmY and the EPS biosynthesis operons pel and psl. We recently demonstrated that extracellular DNA sequesters Mg(2+) cations and activates the cation-sensing PhoPQ two-component system, which leads to increased antimicrobial peptide resistance in biofilms. Here we show that exogenous DNA and EDTA, through their ability to chelate Mg(2+), promoted biofilm formation. The repression of retS in low Mg(2+) was directly controlled by PhoPQ. PhoP also directly controlled expression of rsmZ but not rsmY suggesting that PhoPQ controls the equilibrium of the small regulatory RNAs and thus fine-tunes the expression of genes in the RetS pathway. In summary, Mg(2+) limitation is a biologically relevant environmental condition and the first bonafide environmental signal identified that results in transcriptional repression of retS and promotes P. aeruginosa biofilm formation.
Asunto(s)
Biopelículas/crecimiento & desarrollo , Microbiología Ambiental , Magnesio/metabolismo , Pseudomonas aeruginosa/crecimiento & desarrollo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Biopelículas/efectos de los fármacos , Quelantes/farmacología , ADN/metabolismo , ADN/farmacología , Relación Dosis-Respuesta a Droga , Ácido Edético/farmacología , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Magnesio/farmacología , Microscopía Electrónica de Rastreo , Microscopía Fluorescente , Polisacáridos Bacterianos/metabolismo , Polisacáridos Bacterianos/ultraestructura , Regiones Promotoras Genéticas/genética , Unión Proteica , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , ARN Bacteriano/genéticaRESUMEN
Pseudomonas aeruginosa is an opportunistic pathogen that occupies a wide variety of environmental niches. Extracellular DNA is ubiquitous in various environments and is a rich source of carbon, nitrogen and phosphate. Here we show that P. aeruginosa is capable of using DNA as a nutrient source. Under phosphate-limiting conditions, or when DNA is supplied as a source of phosphate, expression of PA3909 is induced. PA3909 encodes an extracellular deoxyribonuclease (DNase), which is required for degradation of DNA and utilization of DNA as a source of carbon, nitrogen and phosphate. Stabilization of PA3909 by the addition of excess Mg(2+) and Ca(2+) was required for DNase activity in culture supernatants. Extracellular DNase activity was seen in multiple P. aeruginosa strains and isolates from cystic fibrosis patients. The primary Xcp type II secretion system but not the Hxc type II secretion system is required for DNase activity and the ability to use DNA as a source of nutrients. This study identifies an extracellular DNase produced by P. aeruginosa that enables degradation of extracellular DNA into an accessible source of carbon, nitrogen and phosphate. DNase production by P. aeruginosa also has important implications for virulence and biofilm formation.
Asunto(s)
Proteínas Bacterianas/metabolismo , ADN/metabolismo , Desoxirribonucleasas/metabolismo , Pseudomonas aeruginosa , Proteínas Bacterianas/genética , Calcio/metabolismo , Desoxirribonucleasas/genética , Exocitosis/fisiología , Genoma Bacteriano , Magnesio/metabolismo , Fosfatos/metabolismo , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/genéticaRESUMEN
Biofilms are surface-adhered bacterial communities encased in an extracellular matrix composed of DNA, bacterial polysaccharides and proteins, which are up to 1000-fold more antibiotic resistant than planktonic cultures. To date, extracellular DNA has been shown to function as a structural support to maintain Pseudomonas aeruginosa biofilm architecture. Here we show that DNA is a multifaceted component of P. aeruginosa biofilms. At physiologically relevant concentrations, extracellular DNA has antimicrobial activity, causing cell lysis by chelating cations that stabilize lipopolysaccharide (LPS) and the outer membrane (OM). DNA-mediated killing occurred within minutes, as a result of perturbation of both the outer and inner membrane (IM) and the release of cytoplasmic contents, including genomic DNA. Sub-inhibitory concentrations of DNA created a cation-limited environment that resulted in induction of the PhoPQ- and PmrAB-regulated cationic antimicrobial peptide resistance operon PA3552-PA3559 in P. aeruginosa. Furthermore, DNA-induced expression of this operon resulted in up to 2560-fold increased resistance to cationic antimicrobial peptides and 640-fold increased resistance to aminoglycosides, but had no effect on beta-lactam and fluoroquinolone resistance. Thus, the presence of extracellular DNA in the biofilm matrix contributes to cation gradients, genomic DNA release and inducible antibiotic resistance. DNA-rich environments, including biofilms and other infection sites like the CF lung, are likely the in vivo environments where extracellular pathogens such as P. aeruginosa encounter cation limitation.
Asunto(s)
Biopelículas , Cationes/farmacología , ADN Bacteriano/farmacología , Farmacorresistencia Microbiana , Pseudomonas aeruginosa/fisiología , Péptidos Catiónicos Antimicrobianos , Proteínas Bacterianas , ADN Bacteriano/ultraestructura , Lipopolisacáridos , Factores de TranscripciónRESUMEN
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
A novel vector has been constructed for the constitutive luminescent tagging of gram-negative bacteria by site-specific integration into the 16S locus of the bacterial chromosome. A number of gram-negative pathogens were successfully tagged using this vector, and the system was validated during murine infections of living animals.
Asunto(s)
Vectores Genéticos , Bacterias Gramnegativas/genética , Proteínas Luminiscentes/metabolismo , Animales , Cromosomas Bacterianos , Bacterias Gramnegativas/aislamiento & purificación , Ratones , ARN Ribosómico 16S/metabolismo , Proteínas RecombinantesRESUMEN
Diverse pathogenic bacteria have developed similar mechanisms to subvert host cell responses. In this Progress article, we focus on bacterial virulence factors with different enzymatic activities that can increase the expression of the Kruppel-like factor (KLF) family of mammalian transcriptional regulators through their ability to modify the activity of a common host-cell target - the Rho protein family. By using a common virulence strategy, both Gram-negative and Gram-positive pathogens exploit the KLF regulatory cascade to modulate nuclear factor kappaB activation, pro-inflammatory cytokine expression, actin cytoskeletal dynamics and phagocytosis.
Asunto(s)
Exotoxinas/farmacología , Regulación de la Expresión Génica , Bacterias Gramnegativas/patogenicidad , Bacterias Grampositivas/patogenicidad , Factores de Transcripción de Tipo Kruppel/metabolismo , Factores de Virulencia/farmacología , Animales , Línea Celular , Humanos , Factor 6 Similar a Kruppel , Proteínas Proto-Oncogénicas/metabolismo , Transducción de SeñalRESUMEN
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.