RpoS-independent gene regulation by the Borrelia burgdorferi Fur ortholog BosR

About This Grant

Abstract Borrelia burgdorferi (Bb), the Lyme disease (LD) spirochete, cycles between an Ixodes spp. tick vector and a reservoir host, typically small rodents. To sustain its dual-host lifestyle, Bb employs sophisticated, yet parsimonious, regulatory mechanisms to reshape its transcriptome in response to tick and mammalian host signals. BosR–Bb's sole member of the FUR (ferric uptake regulator) family of regulators was initially described for its putative role in regulation of genes involved in Bb's oxidative stress response in vitro. It subsequently gained prominence from the discovery that it binds to the rpoS promoter and is required for RpoN-dependent expression of rpoS as well as RNAP-RpoS function in mammals. For the past two decades, the LD field has focused almost exclusively on BosR's role in the expression of rpoS and RpoS-regulated genes. BosR's Fur-like regulatory functions outside the RpoN/RpoS pathway remain a major knowledge gap. Our preliminary studies with our dialysis membrane chamber (DMCs) system for generating mammalian host-adapted Bb revealed that BosR regulates 82 genes independently of RpoS in mammals. We also found that BosR regulates 90 genes in vitro independently of RpoS, but only three of these are regulated by BosR in DMCs. Notably, none of the oxidative stress genes previously linked to BosR were dysregulated in ΔbosR under either condition. We further engineered an otherwise WT Bb strain expressing a BosR point mutant (R39A) with impaired DNA-binding activity and found that DNA-binding by BosR is essential for transcription of rpoS as well as its RpoS- independent function in DMCs. Collectively, these findings support our central hypothesis: Bb expresses distinct BosR regulons in ticks and mammals as a product of its Fur-like function that (i) is unrelated to the RpoN/RpoS pathway and (ii) involves differential promoter recognition in response to environmental signals. In our first Aim, we will define the BosR regulon in ticks using TBDCapSeq, a Bb-specific transcript enrichment methodology that we recently developed to investigate Bb gene expression in feeding ticks. Experiments in larvae are particularly relevant as they could prove that BosR has gene regulatory functions during a phase of the enzootic cycle when RpoS is OFF. Studies in this Aim will also establish whether BosR regulates distinct regulons in ticks and mammals. In our second Aim, we will examine the mechanism used by BosR to regulate gene expression independently of RpoS. Accordingly, we seek to establish that bind of BosR to a degenerate DNA motif gives rise to the BosR regulon(s). Unlike other Furs, BosR lacks a clearly defined regulatory metal binding site. Therefore, we will also investigate whether allosteric regulation of BosR, either by heme binding as suggested by AlphaFold3 structural modelling or interactions of BosR with c-di-GMP-liganded PlzA dictate distinct functions of BosR in ticks and mammals.