RESUMEN
Here we show that bis(3'-5') cyclic diadenylic acid (c-di-AMP) and a diadenylate cyclase (DAC) domain protein involved in the biosynthesis of c-di-AMP were identified in Streptococcus pyogenes. The matrix-assisted laser desorption ionization (MALDI)-time of flight (TOF) mass spectrum of the cell extract of S. pyogenes, which showed a fragment pattern very similar to that of the authentic sample of c-di-AMP, revealed that S. pyogenes produces c-di-AMP in the cell. Subsequently, we confirmed by an in vitro experiment that the production of c-di-AMP in the cell is due to the action of Spy1036 gene encoding a DAC domain protein named spyDAC, which is a new protein different from a well-known diadenylate cyclase. Moreover, the experiment gave a product with a molecular weight of 657.021, which is consistent with the molecular weight of c-di-AMP. Furthermore, the mass spectral fragment pattern of the product obtained by the in vitro biosynthesis is quite similar to that of the product produced by the above in vivo experiment. This in vitro production of c-di-AMP indicated that spyDAC in S. pyogenes actually catalyzes the in vivo biosynthesis of c-di-AMP from ATP.
Asunto(s)
Fosfatos de Dinucleósidos/biosíntesis , Genes Bacterianos , Streptococcus pyogenes/genética , Streptococcus pyogenes/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , ADN Bacteriano/genética , Fosfatos de Dinucleósidos/análisis , Humanos , Datos de Secuencia Molecular , Liasas de Fósforo-Oxígeno/genética , Liasas de Fósforo-Oxígeno/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Streptococcus pyogenes/patogenicidadRESUMEN
Cyclic bis(3'-5')diguanylic acid (cyclic-di-GMP) functions as a second messenger in diverse species of bacteria to trigger wide-ranging physiological changes. We measured cyclic-di-GMP and its structural analogs such as cyclic bis(3'-5')guanylic/adenylic acid (cyclic-GpAp), cyclic bis(3'-5')guanylic/inosinic acid (cyclic-GpIp) and monophosphorothioic acid of cyclic-di-GMP (cyclic-GpGps) for effects on the biofilm formation of Staphylococcus aureus and Pseudomonas aeruginosa. We constructed a knockout mutant of SA0701, which is a GGDEF motif protein relevant to diguanylate cyclase from S. aureus 2507. We confirmed that the biofilm formation of this mutant (MS2507 Delta SA0701) was reduced. Cyclic-di-GMP corresponding to physiological intracellular levels given in the culture recovered the biofilm formation of MS2507 Delta SA0701, whereas its analogs did not, indicating that unlike a previous suggestion, cyclic-di-GMP was involved in the positive regulation of the biofilm formation of S. aureus and its action was structurally specific. At a high concentration (200 microM), cyclic-di-GMP and its analogs showed suppression effects on the biofilm formation of S. aureus and P. aeruginosa, and according to the quantification study using costat analysis, the suppression potential was in the order of cyclic-di-GMP, cyclic-GpGps, cyclic-GpAp and cyclic-GpIp, suggesting that the suppression effect was not strictly specific and the change of base structure quantitatively affected the suppression activity.
Asunto(s)
Biopelículas/crecimiento & desarrollo , GMP Cíclico/análogos & derivados , Sustancias de Crecimiento/farmacología , Pseudomonas aeruginosa/crecimiento & desarrollo , Staphylococcus aureus/crecimiento & desarrollo , Biopelículas/efectos de los fármacos , Biomasa , GMP Cíclico/farmacología , Técnicas de Inactivación de Genes , Estructura Molecular , Pseudomonas aeruginosa/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacosRESUMEN
BACKGROUND: Helicobacter pylori survival in a hostile acidic environment is known to be caused by its production of urease, which is not released by known secretion pathways. It has been proposed that H. pylori cells undergo spontaneous autolysis during cultivation and that urease becomes surface-associated only concomitant with bacterial autolysis. The aim of this study was to elucidate mechanisms by which H. pylori cells undergo autolysis during cultivation. MATERIALS AND METHODS: Autolysis of H. pylori KZ109 cells was estimated by measuring the turbidity of the culture, by detection of cytoplasmic protein release into the culture supernatant and by scanning electron microscopic observation of H. pylori cells during cultivation. An autolysis-inducing factor (AIF) was partially purified from the culture supernatant by a partition method using ethyl acetate. RESULTS: Bacterial turbidity of KZ109 cells was drastically decreased after late-log phase accompanying release of urease and HspB into the extracellular space. Concomitantly, cell lytic activity was detected in the culture supernatant. Scanning electron microscopic observation suggested that partially purified AIF induced cell lysis. It was also shown that the AIF is different from other autolytic enzymes or substances so far reported. CONCLUSIONS: This study demonstrated the presence of the peptidergic autolytic substances in the culture supernatant of H. pylori KZ109 cells. The results of this study should be useful for further studies aimed at elucidation of the strategy of survival of H. pylori in the gastric environment and elucidation of the mechanisms of pathogenesis induced by H. pylori.