RESUMEN
Infections of the lung cause observable sickness thought to be secondary to inflammation. Signs of sickness are crucial to alert others via behavioral-immune responses to limit contact with contagious individuals. Gram-negative bacteria produce exopolysaccharide (EPS) that provides microbial protection; however, the impact of EPS on sickness remains uncertain. Using genome-engineered Pseudomonas aeruginosa (P. aeruginosa) strains, we compared EPS-producers versus non-producers and a virulent Escherichia coli (E. coli) lung infection model in male and female mice. EPS-negative P. aeruginosa and virulent E. coli infection caused severe sickness, behavioral alterations, inflammation, and hypothermia mediated by TLR4 detection of the exposed lipopolysaccharide (LPS) in lung TRPV1+ sensory neurons. However, inflammation did not account for sickness. Stimulation of lung nociceptors induced acute stress responses in the paraventricular hypothalamic nuclei by activating corticotropin-releasing hormone neurons responsible for sickness behavior and hypothermia. Thus, EPS-producing biofilm pathogens evade initiating a lung-brain sensory neuronal response that results in sickness.
Asunto(s)
Infecciones por Escherichia coli , Escherichia coli , Pulmón , Polisacáridos Bacterianos , Infecciones por Pseudomonas , Pseudomonas aeruginosa , Animales , Femenino , Masculino , Ratones , Biopelículas , Escherichia coli/fisiología , Hipotermia/metabolismo , Hipotermia/patología , Inflamación/metabolismo , Inflamación/patología , Pulmón/microbiología , Pulmón/patología , Neumonía/microbiología , Neumonía/patología , Pseudomonas aeruginosa/fisiología , Células Receptoras Sensoriales , Polisacáridos Bacterianos/metabolismo , Infecciones por Escherichia coli/metabolismo , Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/patología , Infecciones por Pseudomonas/metabolismo , Infecciones por Pseudomonas/microbiología , Infecciones por Pseudomonas/patología , Nociceptores/metabolismoRESUMEN
Many bacteria use the second messenger cyclic diguanylate (c-di-GMP) to control motility, biofilm production and virulence. Here, we identify a thermosensory diguanylate cyclase (TdcA) that modulates temperature-dependent motility, biofilm development and virulence in the opportunistic pathogen Pseudomonas aeruginosa. TdcA synthesizes c-di-GMP with catalytic rates that increase more than a hundred-fold over a ten-degree Celsius change. Analyses using protein chimeras indicate that heat-sensing is mediated by a thermosensitive Per-Arnt-SIM (PAS) domain. TdcA homologs are widespread in sequence databases, and a distantly related, heterologously expressed homolog from the Betaproteobacteria order Gallionellales also displayed thermosensitive diguanylate cyclase activity. We propose, therefore, that thermotransduction is a conserved function of c-di-GMP signaling networks, and that thermosensitive catalysis of a second messenger constitutes a mechanism for thermal sensing in bacteria.
Asunto(s)
Proteínas Bacterianas/metabolismo , GMP Cíclico/análogos & derivados , Proteínas de Escherichia coli/metabolismo , Liasas de Fósforo-Oxígeno/metabolismo , Pseudomonas aeruginosa/metabolismo , Sistemas de Mensajero Secundario/fisiología , Transducción de Señal/fisiología , Algoritmos , Proteínas Bacterianas/genética , Biopelículas/crecimiento & desarrollo , Cromatografía Liquida , GMP Cíclico/metabolismo , Proteínas de Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Espectrometría de Masas , Liasas de Fósforo-Oxígeno/genética , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/fisiología , TemperaturaRESUMEN
Pseudomonas aeruginosa colonizes the airways of cystic fibrosis (CF) patients, causing infections that can last for decades. During the course of these infections, P. aeruginosa undergoes a number of genetic adaptations. One such adaptation is the loss of swimming motility functions. Another involves the formation of the rugose small colony variant (RSCV) phenotype, which is characterized by overproduction of the exopolysaccharides Pel and Psl. Here, we provide evidence that the two adaptations are linked. Using random transposon mutagenesis, we discovered that flagellar mutations are linked to the RSCV phenotype. We found that flagellar mutants overexpressed Pel and Psl in a surface-contact dependent manner. Genetic analyses revealed that flagellar mutants were selected for at high frequencies in biofilms, and that Pel and Psl expression provided the primary fitness benefit in this environment. Suppressor mutagenesis of flagellar RSCVs indicated that Psl overexpression required the mot genes, suggesting that the flagellum stator proteins function in a surface-dependent regulatory pathway for exopolysaccharide biosynthesis. Finally, we identified flagellar mutant RSCVs among CF isolates. The CF environment has long been known to select for flagellar mutants, with the classic interpretation being that the fitness benefit gained relates to an impairment of the host immune system to target a bacterium lacking a flagellum. Our new findings lead us to propose that exopolysaccharide production is a key gain-of-function phenotype that offers a new way to interpret the fitness benefits of these mutations.
Asunto(s)
Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Interacciones Huésped-Patógeno/genética , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/genética , Proteínas Bacterianas/metabolismo , Biopelículas/crecimiento & desarrollo , Vías Biosintéticas/genética , Fibrosis Quística/complicaciones , Fibrosis Quística/microbiología , Flagelos/metabolismo , Humanos , Mutagénesis Sitio-Dirigida , Mutación , Polisacáridos Bacterianos/biosíntesis , Pseudomonas aeruginosa/patogenicidad , Selección GenéticaRESUMEN
The human pathogen Pseudomonas aeruginosa can cause both acute infections and chronic biofilm-based infections. Expression of acute virulence factors is positively regulated by cAMP, whereas biofilm formation is positively regulated by c-di-GMP. We provide evidence that increased levels of cAMP, caused by either a lack of degradation or increased production, inhibit P. aeruginosa biofilm formation. cAMP-mediated inhibition of P. aeruginosa biofilm formation required Vfr, and involved a reduction of the level of c-di-GMP, as well as reduced production of biofilm matrix components. A mutant screen and characterization of defined knockout mutants suggested that a subset of c-di-GMP-degrading phosphodiesterases is involved in cAMP-Vfr-mediated biofilm inhibition in P. aeruginosa.
Asunto(s)
Biopelículas/crecimiento & desarrollo , AMP Cíclico/metabolismo , GMP Cíclico/análogos & derivados , Pseudomonas aeruginosa/fisiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteína Receptora de AMP Cíclico/genética , Proteína Receptora de AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Matriz Extracelular de Sustancias Poliméricas/metabolismo , Mutación , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crecimiento & desarrollo , Pseudomonas aeruginosa/metabolismoRESUMEN
The second messenger, cyclic diguanylate (c-di-GMP), regulates a variety of bacterial cellular and social behaviors. A key determinant of c-di-GMP levels in cells is its degradation by c-di-GMP-specific phosphodiesterases (PDEs). Here, we describe an assay to determine c-di-GMP degradation rates in vitro using 2'-O-(N'-methylanthraniloyl)-cyclic diguanylate (MANT-c-di-GMP). Additionally, a protocol for the production and purification of recombinant Pseudomonas aeruginosa RocR, a c-di-GMP-specific PDE that may serve as a control in MANT-c-di-GMP assays, is provided. The use of the fluorescent MANT-c-di-GMP analogue can deliver fundamental information about PDE function, and is suitable for identifying and investigating c-di-GMP-specific PDE activators and inhibitors.
Asunto(s)
GMP Cíclico/análogos & derivados , Pruebas de Enzimas , Hidrolasas Diéster Fosfóricas/metabolismo , ortoaminobenzoatos , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , GMP Cíclico/química , GMP Cíclico/metabolismo , Activación Enzimática , Pruebas de Enzimas/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , ortoaminobenzoatos/químicaRESUMEN
Secreted polysaccharides are important functional and structural components of bacterial biofilms. The opportunistic pathogen Pseudomonas aeruginosa produces the cationic exopolysaccharide Pel, which protects bacteria from aminoglycoside antibiotics and contributes to biofilm architecture through ionic interactions with extracellular DNA. A bioinformatics analysis of genome databases suggests that gene clusters for Pel biosynthesis are present in >125 bacterial species, yet little is known about how this biofilm exopolysaccharide is synthesized and exported from the cell. In this work, we characterize PelC, an outer membrane lipoprotein essential for Pel production. Crystal structures of PelC from Geobacter metallireducens and Paraburkholderia phytofirmans coupled with structure-guided disulfide cross-linking in P. aeruginosa suggest that PelC assembles into a 12- subunit ring-shaped oligomer. In this arrangement, an aromatic belt in proximity to its lipidation site positions the highly electronegative surface of PelC toward the periplasm. PelC is structurally similar to the Escherichia coli amyloid exporter CsgG; however, unlike CsgG, PelC does not possess membrane-spanning segments required for polymer export across the outer membrane. We show that the multidomain protein PelB with a predicted C-terminal ß-barrel porin localizes to the outer membrane, and propose that PelC functions as an electronegative funnel to guide the positively charged Pel polysaccharide toward an exit channel formed by PelB. Together, our findings provide insight into the unique molecular architecture and export mechanism of the Pel apparatus, a widespread exopolysaccharide secretion system found in environmental and pathogenic bacteria.
Asunto(s)
Biología Computacional , Polisacárido Liasas/química , Polisacáridos Bacterianos/química , Pseudomonas aeruginosa/química , Biopelículas/crecimiento & desarrollo , Cristalografía por Rayos X , Regulación Bacteriana de la Expresión Génica , Genoma Bacteriano , Lipoproteínas/química , Lipoproteínas/genética , Periplasma/química , Periplasma/genética , Periplasma/metabolismo , Polisacárido Liasas/genética , Polisacáridos Bacterianos/genética , Pseudomonas aeruginosa/patogenicidadRESUMEN
Allelic exchange is an efficient method of bacterial genome engineering. This protocol describes the use of this technique to make gene knockouts and knock-ins, as well as single-nucleotide insertions, deletions and substitutions, in Pseudomonas aeruginosa. Unlike other approaches to allelic exchange, this protocol does not require heterologous recombinases to insert or excise selective markers from the target chromosome. Rather, positive and negative selections are enabled solely by suicide vector-encoded functions and host cell proteins. Here, mutant alleles, which are flanked by regions of homology to the recipient chromosome, are synthesized in vitro and then cloned into allelic exchange vectors using standard procedures. These suicide vectors are then introduced into recipient cells by conjugation. Homologous recombination then results in antibiotic-resistant single-crossover mutants in which the plasmid has integrated site-specifically into the chromosome. Subsequently, unmarked double-crossover mutants are isolated directly using sucrose-mediated counter-selection. This two-step process yields seamless mutations that are precise to a single base pair of DNA. The entire procedure requires â¼2 weeks.
Asunto(s)
Marcación de Gen/métodos , Genoma Bacteriano , Biología Molecular/métodos , Pseudomonas aeruginosa/genética , Ingeniería Genética , Vectores Genéticos , Recombinación HomólogaRESUMEN
UNLABELLED: The opportunistic human pathogen Pseudomonas aeruginosa expresses numerous acute virulence factors in the initial phase of infection, and during long-term colonization it undergoes adaptations that optimize survival in the human host. Adaptive changes that often occur during chronic infection give rise to rugose small colony variants (RSCVs), which are hyper-biofilm-forming mutants that commonly possess mutations that increase production of the biofilm-promoting secondary messenger cyclic di-GMP (c-di-GMP). We show that RSCVs display a decreased production of acute virulence factors as a direct result of elevated c-di-GMP content. Overproduction of c-di-GMP causes a decrease in the transcription of virulence factor genes that are regulated by the global virulence regulator Vfr. The low level of Vfr-dependent transcription is caused by a low level of its coactivator, cyclic AMP (cAMP), which is decreased in response to a high level of c-di-GMP. Mutations that cause reversion of the RSCV phenotype concomitantly reactivate Vfr-cAMP signaling. Attempts to uncover the mechanism underlying the observed c-di-GMP-mediated lowering of cAMP content provided evidence that it is not caused by inhibition of adenylate cyclase production or activity and that it is not caused by activation of cAMP phosphodiesterase activity. In addition to the studies of the RSCVs, we present evidence that the deeper layers of wild-type P. aeruginosa biofilms have high c-di-GMP levels and low cAMP levels. IMPORTANCE: Our work suggests that cross talk between c-di-GMP and cAMP signaling pathways results in downregulation of acute virulence factors in P. aeruginosa biofilm infections. Knowledge about this cross-regulation adds to our understanding of virulence traits and immune evasion by P. aeruginosa in chronic infections and may provide new approaches to eradicate biofilm infections.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteína Receptora de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , GMP Cíclico/análogos & derivados , Pseudomonas aeruginosa/metabolismo , Transducción de Señal/fisiología , Proteínas Bacterianas/genética , Proteína Receptora de AMP Cíclico/genética , GMP Cíclico/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Pseudomonas aeruginosa/genéticaRESUMEN
Clinical infections by Pseudomonas aeruginosa, a deadly Gram-negative, opportunistic pathogen of immunocompromised hosts, often involve the formation of antibiotic-resistant biofilms. Although biofilm formation has been extensively studied in vitro on glass or plastic surfaces, much less is known about biofilm formation at the epithelial barrier. We have previously shown that when added to the apical surface of polarized epithelial cells, P. aeruginosa rapidly forms cell-associated aggregates within 60 minutes of infection. By confocal microscopy we now show that cell-associated aggregates exhibit key characteristics of biofilms, including the presence of extracellular matrix and increased resistance to antibiotics compared to planktonic bacteria. Using isogenic mutants in the type III secretion system, we found that the translocon, but not the effectors themselves, were required for cell-associated aggregation on the surface of polarized epithelial cells and at early time points in a murine model of acute pneumonia. In contrast, the translocon was not required for aggregation on abiotic surfaces, suggesting a novel function for the type III secretion system during cell-associated aggregation. Supernatants from epithelial cells infected with wild-type bacteria or from cells treated with the pore-forming toxin streptolysin O could rescue aggregate formation in a type III secretion mutant, indicating that cell-associated aggregation requires one or more host cell factors. Our results suggest a previously unappreciated function for the type III translocon in the formation of P. aeruginosa biofilms at the epithelial barrier and demonstrate that biofilms may form at early time points of infection.
Asunto(s)
Sistemas de Secreción Bacterianos/inmunología , Biopelículas , Células Epiteliales/inmunología , Neumonía Bacteriana/inmunología , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa/fisiología , Animales , Sistemas de Secreción Bacterianos/genética , Modelos Animales de Enfermedad , Perros , Células Epiteliales/microbiología , Células Epiteliales/patología , Células de Riñón Canino Madin Darby , Ratones , Mutación , Neumonía Bacteriana/genética , Neumonía Bacteriana/patología , Infecciones por Pseudomonas/genética , Infecciones por Pseudomonas/patologíaRESUMEN
In the present review, we describe and compare the molecular mechanisms that are involved in the regulation of biofilm formation by Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas aeruginosa and Burkholderia cenocepacia. Our current knowledge suggests that biofilm formation is regulated by cyclic diguanosine-5'-monophosphate (c-di-GMP), small RNAs (sRNA) and quorum sensing (QS) in all these bacterial species. The systems that employ c-di-GMP as a second messenger regulate the production of exopolysaccharides and surface proteins which function as extracellular matrix components in the biofilms formed by the bacteria. The systems that make use of sRNAs appear to regulate the production of exopolysaccharide biofilm matrix material in all these species. In the pseudomonads, QS regulates the production of extracellular DNA, lectins and biosurfactants which all play a role in biofilm formation. In B.cenocepacia QS regulates the expression of a large surface protein, lectins and extracellular DNA that all function as biofilm matrix components. Although the three regulatory systems all regulate the production of factors used for biofilm formation, the molecular mechanisms involved in transducing the signals into expression of the biofilm matrix components differ between the species. Under the conditions tested, exopolysaccharides appears to be the most important biofilm matrix components for P.aeruginosa, whereas large surface proteins appear to be the most important biofilm matrix components for P.putida, P.fluorescens, and B.cenocepacia.