BosR and PlzA reciprocally regulate RpoS function to sustain Borrelia burgdorferi in ticks and mammals

Abstract

The alternative sigma factor RpoS in Borrelia burgdorferi (Bb), the Lyme disease pathogen, is responsible for programmatic positive and negative gene regulation essential for the spirochete's dual-host enzootic cycle. RpoS is expressed during tick-to-mammal transmission and throughout mammalian infection.Although the mammalian-phase RpoS regulon is well described, its counterpart during the transmission blood meal is unknown. Here, we used Bb-specific transcript enrichment by TBDCapSeq to compare the transcriptomes of wild-type and ΔrpoS Bb in engorged nymphs and following mammalian host-adaptation within dialysis membrane chambers. TBDCapSeq revealed dramatic changes in the contours of the RpoS regulon within ticks and mammals and further confirmed that RpoS-mediated repression is specific to the mammalian-phase of Bb's enzootic cycle. We also provide evidence that RpoS-dependent gene regulation, including repression of tick-phase genes, is required for persistence in mice. Comparative transcriptomics of engineered Bb strains revealed that BosR, a non-canonical Fur family regulator, and the c-di-GMP effector PlzA reciprocally regulate the function of RNA polymerase complexed with RpoS. BosR is required for RpoS-mediated transcription activation and repression in addition to its well-defined role promoting RpoN-dependent transcription of rpoS. During transmission, liganded-PlzA antagonizes RpoSmediated repression, presumably acting through BosR.

Borrelia burgdorferiPlzA is a cyclic-di-GMP dependent DNA and RNA binding protein

“…During vertebrate infection, no c-di-GMP is produced, favoring apo-PlzA functions such as RNA chaperone activity (Van Gundy et al ., 2023). When c-di-GMP is present, holo-PlzA can also anneal RNAs and potentially interact with RNAP (Grassmann et al ., 2023; Van Gundy et al ., 2023). At lower levels of c-di-GMP, PlzA nucleic-acid binding affinity is low for certain target genes such as glpFKD .…”

“…Mutation of plzA has been implicated in many B. burgdorferi phenotypes, including mis-regulation of alternative carbohydrate utilization, decreased motility, and virulence defects (Pitzer et al ., 2011; He et al ., 2014; Mallory et al ., 2016; Zhang et al ., 2018; Groshong et al ., 2021). Binding of c-di-GMP induces conformational changes in PlzA structure, which has led to the hypothesis that c-di-GMP binding acts as a switching mechanism providing the holo and apo-forms of PlzA with distinct functions (Mallory et al ., 2016; Groshong et al ., 2021; Grassmann et al ., 2023). Specifically, c-di-GMP bound PlzA is thought to regulate genes required for survival in the tick host, while apo-PlzA is speculated to play a role in regulation during vertebrate infection (Groshong et al ., 2021; Grassmann et al ., 2023).…”

“…Binding of c-di-GMP induces conformational changes in PlzA structure, which has led to the hypothesis that c-di-GMP binding acts as a switching mechanism providing the holo and apo-forms of PlzA with distinct functions (Mallory et al ., 2016; Groshong et al ., 2021; Grassmann et al ., 2023). Specifically, c-di-GMP bound PlzA is thought to regulate genes required for survival in the tick host, while apo-PlzA is speculated to play a role in regulation during vertebrate infection (Groshong et al ., 2021; Grassmann et al ., 2023).…”

“…A common mechanism of action for c-di-GMP effector proteins is to bind nucleic acids (Wilksch et al ., 2011; Tschowri et al ., 2014; Tan et al ., 2015; Wang et al ., 2016; Schäper et al ., 2017; Hsieh et al ., 2018; Skotnicka et al ., 2020). It has been hypothesized that PlzA could bind DNA and regulate genes through impacts on transcription, such as directly interacting with RNA polymerase (RNAP), but this has yet to be experimentally tested (Groshong et al ., 2021; Grassmann et al ., 2023). Conversely, others have hypothesized that PlzA cannot bind DNA, as it does not contain an obvious, conventional DNA binding motif (Caimano et al ., 2015; Zhang et al ., 2018).…”

Evolution of cyclic di-GMP signalling on a short and long term time scale

“…citri [94, 95]. While the C-terminal domain binds cyclic di-GMP, the N-terminal domain of PlzA, a tandem xPilZ-PilZ domain protein of the Lyme disease spirochete Borrelia burgdorferi , has been shown to be involved in DNA and RNA binding [96, 97]. In tandem xPilZ-PilZ domain proteins, such as in the flagellar motor break protein YcgR, the non-canonical xPilZ domain is often highly degraded giving rise to novel domain subclasses (Table 1; [98]).…”