Biochemical characterization of the sole Leptospira interrogans diadenylate cyclase

Leptospirosis, a widespread zoonosis impacting humans, companion animals, livestock, and wildlife, is caused by a diverse group of Leptospira species. Leptospires can infect virtually any vertebrate and can survive in and adapt to radically different environments. Adaptive responses are dependent on a sophisticated network of sensing and signaling systems. Bioinformatic analyses revealed that Leptospira species encode a single CdaA-type diadenylate cyclase (DAC). DACs catalyze the synthesis of cyclic di-adenosine monophosphate (c-di-AMP) from two molecules of ATP. The regulatory roles of c-di-AMP in Leptospira have not yet been explored. Herein, we present a biochemical analysis of the L. interrogans str. Fiocruz L1-130 CdaA-type diadenylate cyclase, LIC10844. Triton X-114 extraction, phase partitioning, and subsequent immunoblot analyses revealed that LIC10844 is an inner membrane-associated protein. Recombinant CdaA was produced and tested for DAC activity and cofactor specificity. Cobalt and manganese were competent divalent cations, whereas zinc, magnesium, copper, nickel, and calcium were not. Notably, the optimal concentrations of cobalt and manganese for maximal enzymatic activity differed significantly. Size exclusion chromatography and subsequent DAC assays of the eluate revealed the enzymatically active form of CdaA to be a homodimer. Amino acid residues involved in DAC activity were identified through site-directed mutagenesis and DAC assays. This report is the first to characterize a DAC in Leptospira . The data suggest that Leptospira utilizes c-di-AMP as a second messenger molecule and that its enzymatic activity is responsive to environmental divalent cation concentrations.