Comparative transcriptomics of Leptospira interrogans serovar Manilae strain L495 wild-type, PerR1 and PerR2 single and double mutants

Leptospirosis is a neglected tropical disease with a worldwide distribution caused by pathogenic spirochetes of Leptospira genus. The ability of Leptospira spp. to sense and respond to environmental signals encountered within mammals is generally believed to be critical for the bacteriums zoonotic lifestyle. To gain better insight into the transcriptomic and antigenic changes the L. interrogans undergoes within mammals, the proximal renal tubules in particular, we developed an in vivo model in which leptospires are cultivated within dialysis membrane chambers (DMCs) implanted into the peritoneal cavities of rats, a natural reservoir host. In a previous study, we showed a number of genes differentially expressed in L. interrogans in response to host signals, including PerR1, a peroxide stress regulator (PerR). In this study, we compare isogenic single and double mutants in LIMLP10155 (perR1-KO) and LIMLP05620 (perR2-KO) using complementary animal models. Unlike the perR1-KO and perR2-KO single mutants, both of which are fully infectious in C3H/HeJ mice, the perR1-perR2-KO double mutant is avirulent. Trans-complementation of the perR1-perR2-KO mutant with perR2 alone restored its ability to infect mice. To investigate the molecular basis for the phenotypic differences between single and double mutants and identify genes dysregulated by the loss of both PerR-like regulators, we performed comparative RNA-Seq on single and double mutants cultivated within rat peritoneal cavities. These studies identified a large cohort of genes that are dysregulated only in the double mutant. Of particular note, the perR1-perR2-KO-only regulon includes at least four virulence-associated genes, ligA and ligB, encoding Leptospiral Immunoglobulin-like proteins LigA and B, and lvrAB, encoding tandem hybrid histidine kinases. The perR1-perR2-KO-only regulon also contains at least 14 additional genes related to environmental sensing and/or gene regulation, including nine putative hybrid HKs and five putative DNA binding proteins. Surprisingly, we saw minimal overlap between the single mutant regulons. Taken together, these data lead us to hypothesize that PerR1 and PerR2 work cooperatively as part of a complex signaling network that regulates the expression of genes required by L. interrogans for adaptation to and survival within reservoir and incidental (i.e., human) hosts.