second messenger, diguanylate cyclase, phosphodiesterase, effector, TetR, leptospirosis

Leptospira form biofilms in vitro and in natural environments. Biofilm formation is driven by signaling systems and, amongst signaling molecules, cyclic di-GMP (c-di-GMP) is an important second messenger involved in biofilm formation. Studies in cell signaling in Leptospira biofilm are scarce and little is known about the c-di-GMP signaling pathway during biofilm formation. In this work, we performed a wide genomic study on the c-di- GMP signaling system in Leptospira biflexa. We executed similarity searches and accessed databases to identify c-di-GMP-related genes, predicted enzymatic and c-di-GMP-binding functions, and investigated the expression of the identified genes based on the L. biflexa biofilms transcriptome dataset. We identified 22 genes encoding putative diguanylate cyclases (DGCs), phosphodiesterases (PDEs) and hybrid proteins. From those, 16 genes were potentially functional. Two genes encoding the DGC LEPBI_I2876 and the PDE LEPBI_I0099 presented expression levels that suggested they modulated c-di-GMP concentration during biofilm development. We also detected nine putative effector genes, including a pnp ortholog that is more expressed in mature biofilms and four genes harbouring potentially functional PilZ domains. We also showed that csrA, bolA, hfq and fliA, known c- di-GMP regulators, were expressed in L. biflexa biofilms. Furthermore, we found five TetR family genes that were more expressed during biofilm dispersal in L. biflexa biofilm, and we suggested these genes could induce biofilm dispersal by repressing c-di-GMP signaling. Additionally, we proposed a coordinated action between Pnp, FliA and the DGC LEPBI_I2876 during biofilm development in L. biflexa. Finally, we were able to suggest a biological pathway for c-di-GMP signaling in L. biflexa. The present work, conducted in the saprophytic L. biflexa, pointed out components of a c-di-GMP signaling pathway that is in its early stages of study in Leptospira.