Deciphering Borrelia burgdorferi RpoS regulatory networks in ticks and mammals

Abstract Borrelia burgdorferi (Bb) is maintained in nature within an enzootic cycle involving a mammalian reservoir host and a tick vector. To sustain this dual-host lifestyle, Bb must remodel its transcriptome as it shuttles between these two host milieus. The RpoN/RpoS pathway is central to borrelial gene regulation. In addition to upregulating genes required for tick transmission and mammalian infection, this pathway represses tick-phase genes, hence its designation as the `gatekeeper'. Activation of the spirochete's other major pathway, Hk1/Rrp1, results in synthesis of the second messenger c-di-GMP, which is required for survival in ticks. c-di-GMP exerts its regulatory effect via the effector protein PlzA. Using a novel transcriptomic approach, TBDCapSeq, we compared the RpoS regulons during the nymphal blood meal and mammalian host-adaptation. These analyses established unequivocally that the contours of the RpoS regulon differ dramatically during the tick and mammalian phases and that RpoS-mediated repression occurs exclusively within mammals. Unexpectedly, we found that deletion of BosR in an IPTG-inducible rpoS strain diminished expression of RpoS-upregulated genes and abrogated RpoS-mediated repression. Thus, in addition to its well-recognized function as a cofactor for RpoN-dependent transcription of rpoS, BosR also plays an essential downstream role modulating promoter recognition by RNAP-RpoS. Using a Bb strain that synthesizes c-di-GMP constitutively, we made the intriguing observation that liganded-PlzA (PlzALig) exerts a `brake' effect on RNAP-RpoS, antagonizing RpoS-mediated repression and diminishing transcription of RpoS-upregulated genes. Remarkably, the effect of PlzALig on RNAP- RpoS mirrors BosR-deficiency. These findings lead to our central hypothesis that PlzALig and BosR determine the contours of the RpoS regulon by exerting reciprocal effects on RNAP-RpoS. In Aim 1, we will determine how DNA binding by BosR shapes the RpoS regulon and investigate the mechanism underlying RpoS-mediated repression. Experiments in Aim 2 will investigate two possible mechanisms to explain the PlzALig brake. Lastly, in Aim 3, we will assess the contributions of other regulatory factors to modulation of the tick- and mammalian-phase RpoS regulons. Our long-term objective is to develop a holistic understanding of how the RpoN/RpoS regulatory networks orchestrate gene expression to sustain Bb in nature.