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. 2021 Jul 15;17(7):e1009725.
doi: 10.1371/journal.ppat.1009725. eCollection 2021 Jul.

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

Ashley M Groshong  1   2 , André A Grassmann  1 , Amit Luthra  1 , Melissa A McLain  1 , Anthony A Provatas  3 , Justin D Radolf  1   2   4   5   6 , Melissa J Caimano  1   2   4
Affiliations

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

Ashley M Groshong et al. PLoS Pathog. .

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.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Binding of c-di-GMP by PlzA is essential for survival of B. burgdorferi during tick feeding.
(A) Viable spirochete burdens for larvae immersion-fed with B31 A3-68 Δbbe02 (wt), ΔplzA, plzA-R145D and plzAcomp strains as determined by colony forming units (CFU). (B) Representative immunofluorescence images of immersion-fed larvae using FITC-conjugated anti-Borrelia antibody. (C) Burdens in immersion-fed larvae determined by qPCR using a TaqMan assay for flaB. Data points in panels A and C represent individual pools of larvae. Error bars indicate the mean ± standard error of the mean for each strain normalized per tick. Asterisks (*) indicate statistical significance (p < 0.05) of all pairwise comparisons determined by unpaired Student’s t-test.
Fig 2
Fig 2. PlzA deficiency does not impair the RpoN/RpoS pathway in vitro or during cultivation in DMCs.
Whole-cell lysates from B31 A3-68 Δbbe02 (wt), ΔplzA and plzAcomp strains following temperature-shift in vitro (A) or cultivation within rat peritoneal dialysis membrane chambers (B) were separated by SDS-PAGE and stained with silver or immunoblotted using antisera against PlzA, GlpD, RpoS, OspC, OspA, Lp6.6 or FlaB (loading control). Molecular weight markers (MWM) are shown on the left.
Fig 3
Fig 3. Constitutive synthesis of c-di-GMP functionally complements GlpD expression and motility in Δrrp1 spirochetes in vitro.
(A) Cartoon depiction of the PflaB-slr1143-HA construct encoding a B. burgdorferi codon-optimized, constitutively active Slr1143 diguanylate cyclase (cDGC) from Synechocystis sp. [85]. TSS (+1), transcriptional start site; PflaB, flaB promoter; RBS, ribosome binding site; HA, hemagglutinin tag. (B) Whole-cell lysates of B31 5A18 NP1 (wt), Δrrp1 and cDGC strains grown in vitro following temperature-shift were separated by SDS-PAGE and stained with silver or immunoblotted with antibodies against HA or GlpD. Molecular weight markers (MWM) are shown on the left. (C) c-di-GMP measurements for B31 5A18 NP1 (wt), Δrrp1, and cDGC strains as determined by LC-MS/MS. Bars represent the mean ± standard error of the mean for three independent cultures per strain. Statistical significance was determined by unpaired Student’s t-test. Asterisks (*) indicate statistical significance (p ≤ 0.05) of all pairwise comparisons. (D) Growth curves of 5A18 NP1 (wt), Δrrp1 and cDGC strains (in quadruplicate) from a starting density of 1 × 103 spirochetes per ml at 37°C. Statistical significance was determined using the CGGC permutation test [122]. Asterisks (*) indicate statistical significance (p ≤ 0.05) of all pairwise comparisons; ns, not significant.
Fig 4
Fig 4. Constitutive synthesis of c-di-GMP functionally complements Δrrp1 in feeding ticks.
(A) Viable spirochete burdens in larvae immersion-fed with B31 5A18 NP1 wild-type (wt), Δrrp1 and cDGC strains as determined by colony forming units (CFUs) following semi-solid plating. (B) Representative immunofluorescence images of immersion-fed larvae using FITC-conjugated anti-Borrelia antibody. (C) DNA burdens in immersion fed larvae determined by qPCR using a TaqMan assay for flaB. Data points in panels A and C represent individual pools of ticks. Error bars indicate the mean ± standard error of the mean for each strain normalized per tick. Asterisks (*) indicate statistical significance (p ≤ 0.05) of all pairwise comparisons, as determined by unpaired Student’s t-test.
Fig 5
Fig 5. Constitutive synthesis of c-di-GMP antagonizes the RpoN/RpoS pathway in mammalian host-adapted spirochetes.
(A) Whole-cell lysates from B31 5A18 NP1 wild-type (wt), Δrrp1, and cDGC strains cultivated in DMCs were separated by SDS-PAGE and stained with silver or immunoblotted with antisera against GlpD, PlzA, RpoS, OspC, OspA, Lp6.6 and FlaB (loading control). Molecular weight markers (MWM) are shown on the left. (B) Expression of rpoS determined by qRT-PCR using RNA extracted from wt, Δrrp1 and cDGC strains cultivated in DMCs. Transcript copy numbers for rpoS were normalized using flaB. Statistical significance was determined by unpaired Student’s t-test. No significance difference (p ≥ 0.05) was observed for any pairwise comparison.
Fig 6
Fig 6. c-di-GMP requires PlzA to antagonize RpoS-dependent mammalian host-adaptation.
Whole-cell lysates from wild-type B31 A3-68 Δbbe02 wild-type (wt), ΔplzA, plzA-R145D, ΔplzA+cDGC and cDGC strains cultivated within DMCs, separated by SDS-PAGE and stained with silver or immunoblotted with antisera against PlzA, GlpD, RpoS, OspC, OspA, Lp6.6, HA, and FlaB (loading control). Molecular weight markers (MWM) are shown on the left.
Fig 7
Fig 7. Structural analysis of PlzA.
(A) Kratky plots for liganded (orange) and unliganded PlzA (blue). (B) Superposition of the SAXS envelope and refined structural model for liganded-PlzA. The PilZN3 and PilZ β-barrels are colored in salmon and yellow, respectively. The α helices in the PilZN3 and PilZ domains are highlighted in light green and cyan, respectively, while the C-terminal α-helix in PilZN3 is shown in purple. c-di-GMP binding residues in RXXXR and DXSXXG motifs are highlighted in dark green. c-di-GMP (red) was docked into the model using HADDOCK [75].
Fig 8
Fig 8. Proposed model for the function(s) of liganded- and unliganded-PlzA throughout the enzootic cycle.
Global gene expression by B. burgdorferi throughout the enzootic cycle is modulated in large part by the ON and OFF states of two major regulatory networks–the Hk1/Rrp1 two component system and the RpoN/RpoS pathway [3,20,42]. During acquisition, exogenous as yet unidentified small molecules generated during tick feeding activate an Hk1-dependent signaling cascade that culminates in phosphorylation of Rrp1 and synthesis of c-di-GMP [–,–46]. Binding of c-di-GMP by PlzA (liganded-PlzA) promotes transcription of the glpKFD operon and other tick-phase genes, presumably by binding to an upstream “PlzA box” [38,41,42,53]. At the same time, transcription of rpoS by the BosR/Rrp2/RpoN complex ceases and spirochetes transition from an RpoS-ON to -OFF state [3,20,42]. To expedite the switch from an RpoS-ON to -OFF state, liganded-PlzA interferes with transcription by residual RpoS allosterically by, in essence, acting as a ‘brake’ on RNAP-RpoS holoenzyme. The absence of rpoS/RpoS within fed larval midguts allows for unfettered expression of RpoD-dependent tick-phase genes (i.e., glps, ospA, lp6.6) that are repressed by RpoS in mammals [36,42,55,61]. During transmission, c-di-GMP signaling also is activated (i.e., ON) in response to tick-specific signals, again allowing for expression of the glp operon and other tick-phase genes that are upregulated by liganded-PlzA [36,38,41,42,45,53]. In contrast to acquisition, however, during transmission, BosR/Rrp2/RpoN-dependent transcription of rpoS is induced [3,20,42], making this the only point within the enzootic cycle when both the c-di-GMP and RpoN/RpoS pathways are ON. Based on findings presented herein, we postulate that the ‘RpoS-brake’ function of liganded-PlzA prevents RpoS-mediated repression of tick phase genes, which are required for survival within fed midguts, while at the same time allowing transcription of RpoS-upregulated genes required for transmission (i.e., bba64) and/or early infection (i.e., ospC, dbpBA) [36,82,95]. Once within the mammal, loss of c-di-GMP signaling (i) restores RpoS’s Gatekeeper function and (ii) enables unliganded-PlzA, working directly or indirectly via an unidentified interactive partner, to assume its alternative, c-di-GMP-independent virulence-related function(s).
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