RESUMEN
OBJECTIVES: Pseudomonas aeruginosa quorum sensing (QS) circuits regulate virulence factors and co-ordinate bacterial pathogenicity. This study aimed to investigate the inhibitory activity of subinhibitory concentrations of curcumin with azithromycin and gentamicin against P. aeruginosa QS-related genes and virulence factors. METHODS: The minimum inhibitory concentrations (MICs) and synergistic activity of curcumin with azithromycin and gentamicin against P. aeruginosa PAO1 were determined using broth microdilution and checkerboard titration methods, respectively. The activity of sub-MICs (1/4× and 1/16× MIC) of curcumin on the QS signal molecules was assessed using a reporter strain assay. The influence of sub-MICs of curcumin, azithromycin and gentamicin alone and in combination on motility and biofilm formation was also determined and was confirmed by RT-PCR to test the expression of the QS regulatory genes lasI, lasR, rhlI and rhlR. RESULTS: Addition of curcumin drastically decreased the MIC of azithromycin and gentamicin. Curcumin showed synergistic effects with azithromycin and gentamicin. Treated PAO1 cultures in the presence of curcumin showed a significant reduction of signals C12-HSL and C4-HSL (P<0.05). Sub-MICs (1/4× and 1/16× MIC) of curcumin, azithromycin and gentamicin alone and in combination significantly reduced swarming and twitching motilities as well as biofilm formation. Expression of QS regulatory genes lasI, lasR, rhlI and rhlR using 1/4× MIC of curcumin, azithromycin and gentamicin alone and in combination was decreased significantly compared with untreated PAO1. CONCLUSIONS: These results indicate that a combination of sub-MIC of curcumin with azithromycin and gentamicin exhibited synergism against P. aeruginosa QS systems.
Asunto(s)
Azitromicina/farmacología , Curcumina/farmacología , Gentamicinas/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Percepción de Quorum/efectos de los fármacos , 4-Butirolactona/análogos & derivados , 4-Butirolactona/metabolismo , Proteínas Bacterianas/efectos de los fármacos , Proteínas Bacterianas/genética , Biopelículas/efectos de los fármacos , Combinación de Medicamentos , Sinergismo Farmacológico , Genes Reguladores/efectos de los fármacos , Genes Reguladores/genética , Ligasas/efectos de los fármacos , Ligasas/genética , Pruebas de Sensibilidad Microbiana , Pseudomonas aeruginosa/genética , Percepción de Quorum/genética , ARN Bacteriano/análisis , Transactivadores/efectos de los fármacos , Transactivadores/genética , Factores de Transcripción/efectos de los fármacos , Factores de Transcripción/genética , Factores de Virulencia/genéticaRESUMEN
Quorum sensing (QS) system in Pseudomonas aeruginosa may be an important target for pharmacological intervention. The present study aimed to investigate the synergetic activity of sub-MIC concentrations of curcumin (C) with ceftazidime (CAZ) and ciprofloxacin (CIP) against P. aeroginusa QS system. We determined the MIC and synergistic activity of C, CAZ and CIP against P. aeroginusa PAO1 using broth microdilution and checkerboard titration methods. The activity of sub-MIC (1/4 and 1/16 MIC) concentrations of C on the QS signal molecules was assessed using a reporter strain assay. The influence of sub-MIC of C, CAZ and CIP alone and in combination on motility and biofilm formation was also determined and confirmed by RT-PCR to test the expression of QS regulatory genes lasI, lasR, rhlI and rhlR. The addition of C decreased the MIC of CAZ and CIP. Curcumin showed synergistic effects with CAZ and additive activity with CIP. Treated PAO1 cultures in the presence of C showed significant reduction of signals C12-HSL and C4-HSL (P < 0.05). Sub-MIC concentrations (1/4 and 1/16 MIC) of C, CAZ and CIP alone and in combination significantly reduced swarming and twitching motilities and biofilm formation. Expression of QS regulatory genes lasI, lasR, rhlI, and rhlR using 1/4 MIC of C, CAZ and CIP alone and in combination was repressed significantly relative to untreated PAO1. Our results indicate that a combination of the sub-MIC concentration of C and CAZ exhibited synergism against P. aeroginusa QS system. This combination could lead to the development of a new combined therapy against P. aeruginosa.
Asunto(s)
Antibacterianos/farmacología , Ceftazidima/farmacología , Ciprofloxacina/farmacología , Curcumina/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Percepción de Quorum/efectos de los fármacos , Factores de Virulencia/genética , Biopelículas/efectos de los fármacos , Sinergismo Farmacológico , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Viabilidad Microbiana/efectos de los fármacos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidad , Virulencia/efectos de los fármacosRESUMEN
BACKGROUND: Hepatitis delta virus (HDV) is a subviral human pathogen that exploits host RNA editing activity to produce two essential forms of the sole viral protein, hepatitis delta antigen (HDAg). Editing at the amber/W site of HDV antigenomic RNA leads to the production of the large form (L-HDAg), which is required for RNA packaging. METHODS: In this study, PCR-based site-directed mutagenesis by the overlap extension method was used to create the point mutation converting the small-HDAg (S-HDAg) stop codon to a tryptophan codon through three stages. RESULTS: Sequencing confirmed the desirable mutation and integrity of the L-HDAg open reading frame. The amplicon was ligated into pcDNA3.1 and transfected to Huh7 and HEK 293 cell lines. Western blot analysis using enhanced chemiluminescence confirmed L-HDAg expression. The recombinant L-HDAg localized within the nuclei of cells as determined by immunofluorescence and confocal microscopy. CONCLUSION: Because L-HDAg requires extensive post-translational modifications, the recombinant protein expressed in a mammalian system might be fully functional and applicable as a tool in HDV molecular studies, as well as in future vaccine research.
