RESUMEN
PURPOSE: To determine the importance of surfactant protein D in Pseudomonas keratitis. METHODS: The surfactant D status of wild-type and surfactant D-deficient Black Swiss mice was confirmed by PCR reactions and immunoblot assay. Mouse corneas were infected with one of three strains of P. aeruginosa. At 1, 2, 3, and 6 days postinfection, eyes were scored by slit-lamp examination and bacteria per cornea quantified. RESULTS: Infected wild-type mice had slit-lamp scores on 3 and 6 days postinfection that were significantly lower than those of surfactant D-deficient mice (p Asunto(s)
Queratitis/metabolismo
, Queratitis/microbiología
, Infecciones por Pseudomonas
, Pseudomonas aeruginosa
, Proteína D Asociada a Surfactante Pulmonar/metabolismo
, Animales
, Córnea/metabolismo
, Córnea/microbiología
, Immunoblotting
, Queratitis/patología
, Ratones
, Ratones Endogámicos C57BL
, Ratones Noqueados
, Reacción en Cadena de la Polimerasa
, Pseudomonas aeruginosa/crecimiento & desarrollo
, Proteína D Asociada a Surfactante Pulmonar/deficiencia
, Factores de Tiempo
RESUMEN
PURPOSE: Pseudomonas aeruginosa PAO1 deficient in LasA protease was reported to be ocularly avirulent. However, the avirulence of this mutant could not attributed to the loss of LasA protease. The purpose of this study was to define the mechanism for such a mutant's inability to cause corneal disease. METHODS: A LasA protease--deficient mutant of P. aeruginosa PAO1 was constructed by allelic exchange. Virulence of this mutant in mouse and rabbit models of keratitis was assessed by scoring for ocular disease and quantitating viable bacteria from infected corneas. Adherence to scarified mouse corneal tissue was determined with an organ culture assay. RESULTS: In the mouse eye, the LasA protease--deficient mutant was not virulent, despite being as adherent as its parent strain. Virulence of the mutant was also significantly reduced in the rabbit eye. Complementation with lasA did not restore virulence in either model of infection. Neither the mutant nor the mutant complemented with lasA grew well in ocular tissue. An analysis of the mutant showed that it was auxotrophic for leucine. CONCLUSION: These data show that the mutant's avirulence in the eye is caused by poor growth in the ocular environment and not the loss of a functional lasA gene.
Asunto(s)
Proteínas Bacterianas , Córnea/microbiología , Úlcera de la Córnea/microbiología , Infecciones Bacterianas del Ojo/microbiología , Metaloendopeptidasas/deficiencia , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/patogenicidad , Animales , Femenino , Ratones , Ratones Endogámicos C57BL , Técnicas de Cultivo de Órganos , Pseudomonas aeruginosa/enzimología , Conejos , VirulenciaRESUMEN
PURPOSE: A mutant strain of Pseudomonas aeruginosa deficient in LasA protease (staphylolytic protease) has been described as having reduced ocular virulence, suggesting that LasA is a major virulence factor. This study was undertaken to provide further genetic analysis of the role of P. aeruginosa LasA protease in ocular infections. METHODS: LasA protease-deficient mutants of P. aeruginosa PAO1-V and ATCC 19660 were constructed by allelic replacement. Mutants and their respective wild type parent strains were evaluated for virulence and growth in the eye using mouse scarification and rabbit intrastromal injection models of keratitis. RESULTS: LasA protease-deficient mutants of both strains were as virulent as wild type strains, growing to 4 to 6 log10 CFU/cornea and causing significant ocular pathology in the mouse (P > 0.42) and rabbit (P > 0.53). CONCLUSIONS: These data show that LasA protease is not a major corneal virulence factor, suggesting that the main mechanism of corneal damage has yet to be definitively identified.
Asunto(s)
Proteínas Bacterianas , Sustancia Propia/microbiología , Úlcera de la Córnea/microbiología , Infecciones Bacterianas del Ojo/microbiología , Metaloendopeptidasas/fisiología , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/patogenicidad , Animales , Recuento de Colonia Microbiana , Sustancia Propia/patología , Úlcera de la Córnea/patología , Infecciones Bacterianas del Ojo/patología , Femenino , Metaloendopeptidasas/deficiencia , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Infecciones por Pseudomonas/patología , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/aislamiento & purificación , Conejos , VirulenciaRESUMEN
Pseudomonas aeruginosa secretes multiple proteases that have been implicated as virulence factors and the detection of each specific enzyme can be difficult to determine. Unlike the three Pseudomonas enzymes that have been well characterized (elastase A, elastase B, and alkaline protease), the activity of protease IV in multiple assays has yet to be described. This study defines new assays for Pseudomonas proteases and compares protease IV activity to the activities of elastase A, elastase B, and alkaline protease. Six in vitro assays were studied: zymography, elastin congo red assay, staphylolytic assay, colorimetric peptide assay, solid-phase colorimetric peptide assay, and poly-l-lysine degradation. Casein zymography distinguished protease IV from elastase B and alkaline protease, and gelatin zymography differentiated all four proteases. The elastin congo red assay detected mainly elastase B while the staphylolytic assay was specific for elastase A. Protease IV activity was assayed specifically by the colorimetric assay and two new assays, the solid-phase colorimetric assay and degradation of poly-L-lysine in the presence of EDTA. Alkaline protease could be specifically assayed by poly-L-lysine degradation in the presence of N-alpha-p-tosyl-L-lysine chloromethyl ketone. The results identified three specific assays for protease IV, a new assay specific for alkaline protease, and showed that protease IV has a distinct enzymatic specificity relative to the three other Pseudomonas proteases.
Asunto(s)
Colorimetría/métodos , Péptido Hidrolasas/análisis , Pseudomonas aeruginosa/enzimología , Rojo Congo/química , Elastina/química , Elastasa Pancreática/análisis , Péptidos/química , Polilisina/química , Pseudomonas aeruginosa/química , Serina Endopeptidasas/análisisRESUMEN
PURPOSE: To determine the effectiveness of prophylactic antibiotic treatment prior to intra-corneal infection with Staphylococcus aureus. METHODS: One topical drop of Tobrex (0.3% tobramycin), tobramycin (0.3%) in the Tobrex vehicle with 0.05% dodecyl maltoside (DDM)/4.0% hydroxypropylmethycellulose (HPMC), Ocuflox (0.3% ofloxacin) or DDM/HPMC vehicle were applied to rabbit eyes at one or five hours prior to injection of bacteria. Approximately 500 colony-forming units (CFU) of S. aureus strain 8325-4 were injected into the corneal stroma. Rabbits were sacrificed five hours after infection and corneal homogenates were cultured to determine the number of colony forming units (CFU) per cornea. RESULTS: Rabbits treated at five hours prior to infection with tobramycin-DDM/HPMC reduced the bacterial load by approximately 2.4 log CFU/cornea as compared to the untreated control (3.47 +/- 0.98 vs. 5.71 +/- 0.14 log CFU/cornea, respectively; P = 0.0010); however, Ocuflox, Tobrex, or DDM/HPMC vehicle did not significantly reduce the log CFU (P >or= 0.4837). Rabbits treated at 1 hour prior to infection with Ocuflox or tobramycin-DDM/HPMC had significantly reduced CFU/cornea (1.31 +/- 0.86 and 0.48 +/- 0.31 log CFU/cornea, respectively) as compared to the untreated group (5.71 +/- 0.14 log CFU/cornea; P
Asunto(s)
Antibacterianos/uso terapéutico , Antiinfecciosos/uso terapéutico , Profilaxis Antibiótica , Infecciones Bacterianas del Ojo/tratamiento farmacológico , Queratitis/tratamiento farmacológico , Ofloxacino/uso terapéutico , Infecciones Estafilocócicas/tratamiento farmacológico , Tobramicina/uso terapéutico , Animales , Antibacterianos/administración & dosificación , Antiinfecciosos/administración & dosificación , Recuento de Colonia Microbiana , Sustancia Propia/microbiología , Infecciones Bacterianas del Ojo/microbiología , Queratitis/microbiología , Pruebas de Sensibilidad Microbiana , Ofloxacino/administración & dosificación , Soluciones Oftálmicas , Conejos , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/aislamiento & purificación , Tobramicina/administración & dosificaciónRESUMEN
PURPOSE: To determine the efficacy of lysostaphin treatment of methicillin-sensitive and methicillin-resistant Staphylococcus aureus (MRSA) keratitis in a rabbit model. METHODS: The sensitivity to lysostaphin and vancomycin were compared for 34 MRSA and 12 methicillin-sensitive strains. Methicillin-resistant S. aureus strain 301 (MRSA 301) or a methicillin-sensitive strain of low virulence, ISP546, was intrastromally injected into rabbit corneas. Rabbit eyes were treated topically every 30 minutes from 4 to 9 or 10 to 15 hours postinfection with 0.28% lysostaphin or 5.0% vancomycin. Rabbits were killed and corneas were excised and cultured to determine the number of colony forming units (CFU) per cornea. RESULTS: Ninety percent minimal inhibitory concentrations were at least 19-fold lower for lysostaphin than for vancomycin. With early therapy (4 -9 hours postinfection) lysostaphin sterilized all MRSA 301-infected corneas, whereas untreated corneas contained 6.52 log CFU/cornea (P < or = 0.0001). Corneas infected with MRSA 301 and treated similarly with vancomycin retained 2.3 +/-0.85 log CFU/cornea, and none were sterile. When therapy was begun later (10-15 hours postinfection) the residual bacteria in lysostaphin-treated eyes were significantly less numerous than in vancomycin-treated eyes (0.58 +/- 0.34 vs. 5.83 +/- 0.16 log CFU/cornea, respectively; P < or = 0.0001). Three experiments were performed to demonstrate that lysostaphin penetrated the cornea to kill bacteria in vivo; lysostaphin-treated eyes were found to recover from infection, bacteria that did not cause epithelial defects (ISP546) were susceptible to lysostaphin, and inhibition of lysostaphin when harvesting corneas did not alter the observed therapeutic values of lysostaphin. CONCLUSIONS: Lysostaphin is very effective in treating keratitis mediated by methicillin-sensitive or methicillin-resistant S. aureus.
Asunto(s)
Antiinfecciosos Locales/uso terapéutico , Córnea/microbiología , Infecciones Bacterianas del Ojo/tratamiento farmacológico , Queratitis/tratamiento farmacológico , Lisostafina/uso terapéutico , Resistencia a la Meticilina , Infecciones Estafilocócicas/tratamiento farmacológico , Staphylococcus aureus/aislamiento & purificación , Animales , Recuento de Colonia Microbiana , Infecciones Bacterianas del Ojo/microbiología , Queratitis/microbiología , Meticilina/uso terapéutico , Conejos , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/efectos de los fármacos , Vancomicina/uso terapéuticoRESUMEN
Streptokinases secreted by nonhuman isolates of group C streptococci (Streptococcus equi, S. equisimilis, and S. zooepidemicus) have been shown to bind to different mammalian plasminogens but exhibit preferential plasminogen activity. The streptokinase genes from S. equisimilis strains which activated either equine or porcine plasminogen were cloned, sequenced, and expressed in Escherichia coli. The streptokinase secreted by the equine isolate had little similarity to any known streptokinases secreted by either human or porcine isolates. The streptokinase secreted by the porcine isolate had limited structural and functional similarities to streptokinases secreted by human isolates. Plasminogen activation studies with immobilized (His)(6)-tagged recombinant streptokinases indicated that these recombinant streptokinases interacted with plasminogen in a manner similar to that observed when streptokinase and plasminogen interact in the fluid phase. Analysis of the cleavage products of the streptokinase-plasminogen interaction indicated that human, equine, and porcine plasminogens were all cleaved at the same highly conserved site. The site at which streptokinase was cleaved to form altered streptokinase (Sk*) was also determined. This study confirmed not only the presence of streptokinases in nonhuman S. equisimilis isolates but also that these proteins belong to a family of plasminogen activators more diverse than previously thought.
Asunto(s)
Enfermedades de los Caballos/microbiología , Streptococcus/enzimología , Estreptoquinasa/genética , Estreptoquinasa/metabolismo , Enfermedades de los Porcinos/microbiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Caballos , Humanos , Datos de Secuencia Molecular , Plasminógeno/aislamiento & purificación , Plasminógeno/metabolismo , Activadores Plasminogénicos/metabolismo , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , Infecciones Estreptocócicas/microbiología , Infecciones Estreptocócicas/veterinaria , Streptococcus/genética , Estreptoquinasa/química , PorcinosRESUMEN
Comparisons of virulence between a Pseudomonas parent strain and an isogenic mutant devoid of protease IV have demonstrated a significant role for this enzyme during infection. We have characterized purified Pseudomonas aeruginosa protease IV in terms of its biochemical and enzymatic properties, and found it to be a unique extracellular protease. The N-terminal decapeptide sequence of protease IV is not homologous with any published protein sequence. Protease IV has a molecular mass of 26 kDa, an isoelectric point of 8.70, and optimum enzymatic activity at pH 10.0 and 45 degreesC. Purified protease IV demonstrates activity for the carboxyl side of lysine-containing peptides and can digest a number of biologically important proteins, including immunoglobulin, complement components, fibrinogen, and plasminogen. Protease IV is not inhibited by thiol-, carboxyl-, or metalloproteinase inhibitors. The total loss of enzyme activity in the presence of N-p-tosyl-L-chloromethyl ketone and the partial inhibition of enzyme activity by diisopropyl fluorophosphate or phenylmethylsulfonyl fluoride imply that protease IV is a serine protease. Inhibition by dithiothreitol and beta-mercaptoethanol suggests that intramolecular disulfide bonds are essential for enzyme activity. The characteristics of this enzyme suggest that inhibitors of serine proteases could be developed into a medication designed to arrest tissue damage during Pseudomonas infection.