PlzA is a bifunctional c-di-GMP biosensor that promotes tick and mammalian host-adaptation of Borrelia burgdorferi

Abstract

In this study, we examined the relationship between c-di-GMP and its only known effector protein, PlzA, in Borrelia burgdorferi during the arthropod and mammalian phases of the enzootic cycle. Using a B. burgdorferi strain expressing a plzA point mutant (plzA-R145D) unable to bind c-di-GMP, we confirmed that the protective function of PlzA in ticks is c-di-GMP-dependent. Unlike ΔplzA spirochetes, which are severely attenuated in mice, the plzA-R145D strain was fully infectious, firmly establishing that PlzA serves a c-di-GMP-independent function in mammals. Contrary to prior reports, loss of PlzA did not affect expression of RpoS or RpoS-dependent genes, which are essential for transmission, mammalian host-adaptation and murine infection. To ascertain the nature of PlzA’s c-di-GMP-independent function(s), we employed infection models using (i) host-adapted mutant spirochetes for needle inoculation of immunocompetent mice and (ii) infection of scid mice with in vitro-grown organisms. Both approaches substantially restored ΔplzA infectivity, suggesting that PlzA enables B. burgdorferi to overcome an early bottleneck to infection. Furthermore, using a Borrelia strain expressing a heterologous, constitutively active diguanylate cyclase, we demonstrate that ‘ectopic’ production of c-di-GMP in mammals abrogates spirochete virulence and interferes with RpoS function at the post-translational level in a PlzA-dependent manner. Structural modeling and SAXS analysis of liganded- and unliganded-PlzA revealed marked conformational changes that underlie its biphasic functionality. This structural plasticity likely enables PlzA to serve as a c-di-GMP biosensor that in its respective liganded and unliganded states promote vector- and host-adaptation by the Lyme disease spirochete.

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

“…Twenty of these tick-phase genes, including bba15/ospA, bba16/ospB, bba62/lp6.6, bba68/BbCRASP1 and the glp operon (bb0240-0243), were shown previously to be repressed by RpoS in mammals (11,15,25).…”

“…While a large majority (78%) of RpoS-independent genes upregulated in DMCs encode hypothetical proteins, 14 encode gene products related to DNA replication (bb0455, bb0552/ligA, bb0632/recD), influx/efflux of small molecules (bb0642/potA and bb0641/potB), biosynthesis of metabolic cofactors (bb0782/nadD, bb0589/pta), purine salvage (bb0384/bmpC, bb0467, bb0524 and bbb23) and maintenance of the cell envelope (bb0304/murF, bb0586/femA, and bb0721/pgsA) (49)(50)(51)(52)(53)(54). Also noteworthy, bb0733/plzA, which has a virulence-related function in mice unrelated to binding of c-di-GMP (23)(24)(25), was upregulated in DMCs compared to fed nymphs. With the exception of rrp1, which was slightly higher in mammals, all other known or putative regulatory factors (7) were expressed at comparable levels in both milieus (Supplemental Table 4).…”

Potential Regulatory Role in Mammalian Host Adaptation for a Small Intergenic Region of Lp17 in the Lyme Disease Spirochete

“…In B. burgdorferi , c-di-GMP serves as a cyclic nucleotide second messenger that promotes expression of tick phase-specific genes through activation of the HK1-Rrp1 regulon, which is critical for spirochete survival in ticks ( Novak et al., 2014 ). More recently, PlzA was shown to contribute to mammalian host adaptation in the absence of c-di-GMP ( Groshong et al., 2021 ). Considering the combination of previous observations and those reported herein, it appears that the overall outcomes of SR0726 activity are diverse and impactful on the virulence of B. burgdorferi .…”

TonB-dependent receptor epitopes expressed in M. bovis BCG induced significant protection in the hamster model of leptospirosis

“…Immunoblots were performed as previously described, with small changes (Groshong et al 2021 ). Briefly, samples containing 1 µg of each recombinant protein (rTBDRchi, rLIC10896, rLIC10964, and rLIC12374) or L. interrogans st. Fiocruz L1-130 whole-cell lysate (10 8 /lane) were analyzed by SDS-PAGE and transferred to nitrocellulose membranes (GE Healthcare, IL, USA) at 20 V for 25 min using a semi-dry transfer (Bio-Rad).…”