CdaR-DAC signaling systems regulating the synthesis of a second messenger c-di-AMP in Leptospira interrogans

Abstract

Objective To analyze the activity of diadenylate cyclase (DAC) encoded by LA3304 gene of Leptospira interrogans (L.interrogans) and to investigate the influence of CdaR encoded by LA3303 gene on DAC activity. Methods The LA3304 gene in L. interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai was amplified by PCR and inserted into a prokaryotic expression system for expressing DAC. The expressed recombinant protein, rDAC, was purified by Ni-NTA affinity chromatography. High Performance Liquid Chromatography (HPLC) was used to measure the synthesis of c-di-AMP from ATP by rDAC in vitro. Bacterial two-hybrid analysis was used to detect the interaction between CdaR and DAC. Prokaryotic co-expression system was constructed and used in combination with HPLC to analyze the role of CdaR in activating DAC. Results The constructed prokaryotic expression system for LA3304 gene of L. interrogans strain Lai could highly express the rDAC upon the induction of IPTG. The purified rDAC showed high purity with a single protein band in gel as indicated by SDS-PAGE. rDAC could synthesize c-di-AMP from ATP in vitro. CdaR interacted with DAC and enhanced the activity of DAC (P<0.05). Conclusion DAC encoded by LA3304 gene was a diadenylate cyclase that could convert ATP into c-di-AMP. CdaR promoted the activation of DAC and formed a CdaR-DAC system with DAC. The system was involved in the synthesis of c-di-AMP in L. interrogans. Key words: Leptospira; c-di-AMP; DAC; CdaR