RESUMO
Five analogs of cyclic di-nucleotidic acid including c-di-GMP were synthesized and evaluated for their biological activities on Slr1143, a diguanylate cyclase of Synechocystis sp. Slr1143 was overexpressed from the recombinant plasmid which contained the gene of interest and subsequently purified by affinity chromatography. A new HPLC method capable of separating the compound and product peaks with good resolution was optimized and applied to the analysis of the compounds. Results obtained show that cyclic di-inosinylic acid 1b demonstrates a stronger inhibition on Slr1143 than c-di-GMP and is a potential inhibitor for biofilm formation.
Assuntos
Proteínas de Bactérias/antagonistas & inibidores , GMP Cíclico/análogos & derivados , Fósforo-Oxigênio Liases/antagonistas & inibidores , Nucleotídeos de Purina/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes/efeitos dos fármacos , Cromatografia de Afinidade , GMP Cíclico/síntese química , GMP Cíclico/química , GMP Cíclico/farmacologia , Proteínas de Escherichia coli , Fósforo-Oxigênio Liases/genética , Fósforo-Oxigênio Liases/metabolismo , Nucleotídeos de Purina/síntese química , Nucleotídeos de Purina/farmacologia , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Synechocystis/enzimologiaRESUMO
Cyclic diguanylic acid (c-di-GMP) is an intracellular signaling molecule involved in regulation of cellular functions such as motility, biofilm formation and virulence. Intracellular level of c-di-GMP is controlled through opposing diguanylate cyclase (DGC) and phosphodiesterase (PDE) activities of GGDEF and EAL domain proteins, respectively. We report the identification and characterization of cdpA, a gene encoding a protein containing an EAL domain in the Gram-negative soil bacillus and human pathogen Burkholderia pseudomallei KHW. Purified recombinant CdpA protein exhibited PDE activity in vitro. Evidence that CdpA is a major c-di-GMP-specific PDE in B. pseudomallei KHW was shown by an 8-fold-higher c-di-GMP level in the cdpA-null mutant as compared to the wild type and the complemented cdpA mutant. The presence of higher intracellular c-di-GMP levels in the cdpA-null mutant was associated with increased production of exopolysaccharides, increased cell-to-cell aggregation, absence of flagella and swimming motility, and increased biofilm formation. The relevance of CdpA in B. pseudomallei virulence was demonstrated by a 3-fold reduction in invasion of human lung epithelial cells and a 6-fold reduction in cytotoxicity on human macrophage cells infected with the cdpA mutant.
Assuntos
3',5'-GMP Cíclico Fosfodiesterases/fisiologia , Proteínas de Bactérias/fisiologia , Burkholderia pseudomallei/enzimologia , Burkholderia pseudomallei/fisiologia , Fatores de Virulência/fisiologia , 3',5'-GMP Cíclico Fosfodiesterases/deficiência , Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Burkholderia pseudomallei/patogenicidade , Linhagem Celular , Sobrevivência Celular , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Células Epiteliais/microbiologia , Flagelos/fisiologia , Técnicas de Inativação de Genes , Humanos , Locomoção , Macrófagos/microbiologia , Virulência , Fatores de Virulência/deficiênciaRESUMO
A traceless solid-phase route to substituted xanthines, based on the late-stage pyrimidine ring closure, has been developed. This method is especially useful for the preparation of xanthines containing a variety of substituents at the N1, N3, N7, and C8 positions in an unambiguous manner. A representative set of 22 compounds was prepared.
