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Research Article

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

  • Ashley M. Groshong,

    Roles Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing

    Current address: Laboratory of Bacteriology, Rocky Mountain Laboratories, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, United States of America

    Affiliations Department of Medicine, UConn Health, Farmington, Connecticut, United States of America, Department of Pediatrics, UConn Health, Farmington, Connecticut, United States of America

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  • André A. Grassmann,

    Roles Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review & editing

    Affiliation Department of Medicine, UConn Health, Farmington, Connecticut, United States of America

    ⨯
  • Amit Luthra,

    Roles Data curation, Formal analysis, Investigation, Methodology, Visualization, Writing – original draft

    Affiliation Department of Medicine, UConn Health, Farmington, Connecticut, United States of America

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  • Melissa A. McLain,

    Roles Data curation, Visualization

    Affiliation Department of Medicine, UConn Health, Farmington, Connecticut, United States of America

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  • Anthony A. Provatas,

    Roles Data curation, Investigation, Validation, Visualization, Writing – original draft, Writing – review & editing

    Affiliation Center for Environmental Sciences and Engineering, University of Connecticut, Storrs, Connecticut, United States of America

    ⨯
  • Justin D. Radolf,

    Roles Conceptualization, Funding acquisition, Methodology, Project administration, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing

    Affiliations Department of Medicine, UConn Health, Farmington, Connecticut, United States of America, Department of Pediatrics, UConn Health, Farmington, Connecticut, United States of America, Department of Molecular Biology and Biophysics, UConn Health, Farmington, Connecticut, United States of America, Department of Genetics and Genome Science, UConn Health, Farmington, Connecticut, United States of America, Department of Immunology, UConn Health, Farmington, Connecticut, United States of America

    ⨯
  • Melissa J. Caimano

    Roles Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing

    Mcaima@uchc.edu

    Affiliations Department of Medicine, UConn Health, Farmington, Connecticut, United States of America, Department of Pediatrics, UConn Health, Farmington, Connecticut, United States of America, Department of Molecular Biology and Biophysics, UConn Health, Farmington, Connecticut, United States of America

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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.

Author summary

Herein, we examined the roles of c-di-GMP and PlzA throughout the Borrelia burgdorferi lifecycle. Using a plzA point mutant that is unable to bind c-di-GMP, we confirmed that the protective function(s) of PlzA in feeding ticks and mammals are c-di-GMP-dependent and -independent, respectively. Contrary to previous studies, loss of PlzA did not affect expression of rpoS or RpoS-dependent genes, which are important for host-adaptation and persistence in the mammal. Host-adaptation prior to needle-inoculation of C3H mice or infection of scid mice substantially restored infectivity in the absence of PlzA. Using a Borrelia strain that synthesizes c-di-GMP constitutively, this secondary messenger was shown to be tick-phase specific and detrimental to host-adaptation via PlzA-mediated antagonism of RpoS function. Structural modeling and SAXS analysis of liganded- and unliganded-PlzA provide insight into the dramatic conformational changes upon c-di-GMP binding that likely underlie its differential function: liganded-PlzA supports survival within feeding ticks, while unliganded-PlzA promotes an RpoS-independent facet of host adaptation, enabling spirochetes to evade early immune clearance by the host.

Citation: Groshong AM, Grassmann AA, Luthra A, McLain MA, Provatas AA, Radolf JD, et al. (2021) PlzA is a bifunctional c-di-GMP biosensor that promotes tick and mammalian host-adaptation of Borrelia burgdorferi. PLoS Pathog 17(7): e1009725. https://doi.org/10.1371/journal.ppat.1009725

Editor: Jenifer Coburn, Medical College of Wisconsin, UNITED STATES

Received: May 3, 2021; Accepted: June 18, 2021; Published: July 15, 2021

This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

Data Availability: All relevant data are within the manuscript and its Supporting Information files.

Funding: This work was supported by grants from the National Institutes of Health/National Institute for Allergy and Infectious Diseases (R01AI029735 and R21AI39940 to MJC and JDR; R21AI126146 to MJC) and the Global Lyme Alliance (AMG). MJC and JDR are supported in part by Connecticut Children’s Medical Center. NIH/NIAID: https://www.niaid.nih.gov/ Global Lyme Alliance:https://globallymealliance.org/ CCMC: https://www.connecticutchildrens.org/ The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Lyme disease, a multisystem disorder characterized by cutaneous, neurologic, cardiac, and rheumatologic manifestations [1,2], is caused by the spirochete Borrelia burgdorferi [3]. With ~35,000 confirmed cases reported to the Centers for Disease Control and Prevention annually [4,5], Lyme disease is the most prevalent arthropod-borne infection in the United States [6]. Based on insurance claims data, Kugeler et al. [5] estimated that during 2010 to 2018 the incidence of Lyme disease was ≈476,000 cases per year.

In nature, B. burgdorferi cycles between a hard tick vector and a vertebrate reservoir host, usually small rodents [3,7–9]; the generalist feeding behavior of Ixodes spp. is responsible for transmission of B. burgdorferi to humans, an incidental host [10,11]. Because transmission of B. burgdorferi is transstadial, larvae must acquire spirochetes by feeding on an infected reservoir host. Following acquisition, spirochetes enter a quiescent state within the midguts of flat nymphs [3,12]. The subsequent nymphal blood meal stimulates a replicative burst during which spirochetes replicate exponentially, traverse the midgut epithelium, migrate through the hemocoel to the salivary glands, and, following penetration of salivary acini, access the next host via the salivary stream [7,13,14]. To maintain its complex bi-phasic life cycle, B. burgdorferi must coordinate the expression of colonization factors and protective surface molecules and adjust its physiologic state to contend with vastly different environmental threats and nutrient profiles encountered in mammals and arthropods [3,15,16].

Two-component systems (TCS) are important mechanisms by which bacteria can adapt globally to their surroundings [17,18]. B. burgdorferi encodes only two TCSs, Hk1/Rrp1 and Hk2/Rrp2 [3,19,20]. While the sensory function(s) of Hk2 remains unclear [21,22], Rrp2 is part of a regulatory pathway in which the alternative sigma (σ54) factor RpoN transcribes the ‘effector’ alternative σ factor RpoS (σ38) [3,20,23–25]. Transcription of rpoS by Rrp2/RpoN also requires binding by the Borrelia oxidative stress Regulator (BosR), which is a member of the Ferric Uptake Regulator (FUR) superfamily [20,26–32]. Genes within the RpoS regulon are required for tick transmission as well as to establish and sustain infection within the reservoir host [23,25,33–36]. The hk1/rrp1 TCS encodes a membrane-associated hybrid sensory histidine kinase and response regulator with guanylate cyclase activity, respectively [37–39]. Upon binding of as yet unidentified exogenous ligand(s) (i.e., amino acids or their derivatives [40]), Hk1 initiates a signal transduction cascade that culminates in phosphorylation of Rrp1 and synthesis of bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) [37–39,41–46]. Spirochetes lacking either Hk1 or Rrp1 host-adapt normally within dialysis membrane chambers (DMCs) and are fully virulent in mice but are destroyed within feeding larvae and nymphs [38,39, 41–43,47].

Production of c-di-GMP has pleiotropic effects in Lyme disease spirochetes, affecting both motility and gene expression [38,39,42–45,48–53]. With respect to the latter, it promotes utilization of alternate carbon sources (i.e., glycerol, chitobiose and N-acetylglucosamine), upregulation of surface lipoproteins to ward off complement-mediated lysis, and protection against osmotic stress during tick feeding [38,41–43,45,54]. While we observed only limited overlap between the RpoS and Hk1/Rrp1 regulons in vitro [42], there is evidence for some degree of “cross-talk” between these two regulatory pathways. For example, within mammals, RpoS represses the expression of c-di-GMP-upregulated glycerol uptake and metabolism (glp) operon, which is essential for spirochete fitness in ticks [36,41,42,45,53,55,56]. Transcriptomic analyses of RpoS- and Rrp1-deficient spirochetes also identified genes that are upregulated by both regulatory pathways in vitro [38,41,42]. There are conflicting data, however, regarding the influence of c-di-GMP on expression of ospC, the prototypical RpoS-upregulated borrelial gene [38,41,42,45].

Efforts to elucidate how c-di-GMP exerts it effector function(s) in B. burgdorferi to date have centered about PlzA (BB0733), the sole PilZ-domain containing protein in strain B31 [42,44,46,49,51,53]. PlzA is monomeric and undergoes structural rearrangements upon binding of c-di-GMP to its PilZ domain via RxxxR and (D/N)hSxxG motifs [44,46,51]. As with Hk1/Rrp1 [39,41,42,47], PlzA has been shown to modulate expression of the glp operon in vitro [42,53] and spirochete survival in ticks [49,57]. Consistent with these data, expression of plzA is increased during tick feeding [46]. Paradoxically, in mice, an environment that does not stimulate Hk1/Rrp1-dependent synthesis of c-di-GMP, infectivity of PlzA-deficient organisms is markedly attenuated [49], an observation that contrasts with findings that Δhk1 and Δrrp1 strains are fully virulent [39,41–43]. The reduced virulence of ΔplzA spirochetes in mice has been attributed to abnormal growth and motility, altered cell envelope biogenesis and/or decreased levels of BosR [45,49,52,57]. Collectively, these studies point to multiple effector functions and, likely, different interaction partners for liganded- and unliganded-PlzA in ticks and mice, respectively [49,53,57].

The unresolved issues surrounding the relationship between PlzA, c-di-GMP signaling and RpoS-dependent gene regulation prompted us to re-examine the role of this novel c-di-GMP effector throughout the enzootic cycle. Herein, we establish unequivocally that PlzA function in ticks is c-di-GMP-dependent. Conversely, binding of c-di-GMP by PlzA is not required by B. burgdorferi to establish or sustain infection in mice following needle-inoculation. Unliganded-PlzA, however, promotes an RpoS-independent facet of mammalian host adaptation that enables spirochetes to overcome an early infection bottleneck. Using a B. burgdorferi strain that synthesizes c-di-GMP constitutively at measurable amounts in vitro, we show that the presence of this second messenger is detrimental to murine infectivity due, at least in part, to PlzA-mediated antagonism of RpoS function at the post-translational level. Using small angle X-ray scattering (SAXS) analysis and computational modeling, we generated the first three-dimensional structural model for liganded-PlzA. Consistent with FRET studies by Mallory et al. [51], SAXS analysis revealed substantial conformational changes in PlzA upon c-di-GMP binding. PlzA’s structural plasticity likely underlies its ability to serve as both a c-di-GMP biosensor within feeding ticks and a c-di-GMP-independent regulatory protein in mammals.

Results

Protection of spirochetes by PlzA during tick feeding is c-di-GMP-dependent

Prior studies by us [36,39] and others [38,41,43,49,57] demonstrating that both the Hk1/Rrp1 TCS and PlzA are required for survival of B. burgdorferi during the blood meal strongly suggest that PlzA functions as a c-di-GMP biosensor in feeding ticks. To confirm this experimentally, we took advantage of a finding by Mallory et al. [51] demonstrating that recombinant PlzA containing an arginine to aspartic acid substitution at residue 145 (R145D), the first position of the RxxxR motif, completely abolished c-di-GMP binding. At the outset, we confirmed this finding by comparing the ability of recombinant wild-type and PlzA-R145D His-tagged proteins to bind a fluorescent c-di-GMP analog (2’-O-(N’-methylanthraniloyl)-cyclic diguanosine monophosphate [MANT-c-di-GMP]) [58] (S1A Fig). As shown in S1B Fig, at the emission wavelength of 450 nm (MANT λemmax = 448 nm), PlzA-R145D and the lysozyme negative control bound significantly less (p < 0.0001) MANT-c-di-GMP than wild-type PlzA.

To evaluate the consequence of the R145D substitution on PlzA function in ticks, we inserted the mutated allele into the native plzA locus of BbP1473, a wild-type (wt) strain B31 A3-68 isolate (S1 Table). The resulting mutant, designated plzA-R145D, was tested in parallel with isogenic wt, ΔplzA and plzA complemented (plzAcomp) strains in larvae infected via immersion and then fed to repletion on naïve C3H/HeJ mice as previously described [39,42]. In all comparisons, the plzA-R145D mutant was indistinguishable from its ΔplzA counterpart. By semi-solid phase plating, no viable plzA-R145D or ΔplzA spirochetes were recovered from replete larvae (Fig 1A). Immunofluorescence assay (IFA) of larval midguts revealed only sparse spirochete remnants for both of the PlzA mutant strains (Fig 1B). By qPCR, we detected ~1-log10 lower burdens for the plzA-R145D and ΔplzA mutants in replete larvae compared to the wt and plzAcomp strains (Fig 1C). Based on prior studies [39,41,42,45,54] and the IFA results herein, we attribute the decreased spirochete burdens for both mutants to their destruction during tick feeding. Collectively, these data establish unequivocally that the protection afforded by PlzA during the blood meal is c-di-GMP-dependent.

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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.

https://doi.org/10.1371/journal.ppat.1009725.g001

Loss of PlzA markedly impairs spirochete infectivity in a c-di-GMP-independent manner

Unlike Δhk1 and Δrrp1 strain, which display wt infectivity in mice [39,41,42], ΔplzA organisms are highly attenuated [49,57]. Thus, while the phenotypes for all three mutants are highly similar, if not identical, in ticks (Fig 1), their phenotypes in mice are dichotomous. Importantly, these data suggest that PlzA function in mammals is c-di-GMP-independent. To garner support for this notion, we compared infectivity of plzA-R145D and plzA strains in C3H/HeJ mice in parallel with wt and plzAcomp strains. Consistent with prior studies [49,57], infectivity of the ΔplzA mutant was markedly impaired compared to wt at two-weeks post-infection (Table 1). Of the ten mice infected with the ΔplzA strain, only two seroconverted (S2 Fig). Though not every tissue from the two ΔplzA-infected mice was culture positive (Table 1), in these animals, the mutant clearly disseminated from the site of inoculation. In contrast, all mice infected with the plzA-R145D mutant seroconverted and nearly all tissues were culture-positive at two-weeks post needle-inoculation (Table 1 and S2 Fig), thereby confirming that PlzA function in the mammal does not require binding of c-di-GMP. Complementation restored infectivity of the ΔplzA strain to wt levels (Table 1).

As noted earlier, attenuation of PlzA-deficient strains has been attributed to abnormal growth and/or motility in vitro in standard BSK medium [49,57]. However, in side-by-side growth curves with wt, Δrrp1, ΔplzA and plzAcomp strains, the virulent plzA-R145D mutant exhibited a more pronounced growth defect than the attenuated ΔplzA null mutant (p = 0.025) (S3 Fig). Moreover, we saw no significant difference in growth between Δrrp1 spirochetes, which are fully virulent ([41,42] and below), and the ΔplzA mutant (S3 Fig). Thus, while the ΔplzA strain grows more slowly than its wt and plzAcomp counterparts in vitro (p<0.05), this phenotype does not explain its markedly reduced virulence in mice. By darkfield microscopy, we also compared the motility of wt, Δrrp1 and ΔplzA strains in BSK-II medium. As previously reported [43], Rrp1-deficient spirochetes display a faster run speed and significantly decreased flexing compared to wt, essentially locking them in “run” mode (S1 and S2 Movies). In contrast, the swimming behaviors of wt and ΔplzA (S1 and S3 Movies, respectively) were indistinguishable, as previously noted [49,57].

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Table 1. c-di-GMP binding by PlzA is not required for murine infectivity.

https://doi.org/10.1371/journal.ppat.1009725.t001

Attenuation of ΔplzA spirochetes is RpoS-independent

The reduced virulence of PlzA-deficient spirochetes has been attributed to loss of BosR expression with subsequent ablation of the RpoN/RpoS pathway [52]. In our hands, however, wt, ΔplzA and plzAcomp strains express comparable levels of RpoS and OspC, the prototypical RpoS-dependent gene product, in vitro following temperature-shift (Fig 2A). As a control, lysates were immunoblotted for GlpD, a known c-di-GMP-dependent downstream target of PlzA [36,41,55,56]. Because in vitro cultivation does not recapitulate the full spectrum of transcriptional changes associated mammalian-host specific signals [36,55,59,60], we examined whether loss of PlzA impaired RpoS-dependent function(s) that are specific to mammalian host-adaptation. Fig 2B shows that this is not the case. In spirochetes lacking PlzA, RpoS and OspC were produced at essentially wt levels, while RpoS-repressed tick-phase lipoproteins OspA and BBA62/Lp6.6 were appropriately downregulated [36,55,61–63].

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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.

https://doi.org/10.1371/journal.ppat.1009725.g002

PlzA overcomes an RpoS-independent immune bottleneck during early infection

To examine whether host-adaptation prior to needle-inoculation enables spirochetes to overcome the early infection defect caused by loss of PlzA, wt, ΔplzA, plzA-R145D and plzAcomp strains were cultivated in DMCs and then immediately used to inoculate (1 × 104 spirochetes) naïve C3H/HeJ mice. Analysis of whole cell lysates confirmed that all strains had properly host-adapted prior to inoculation (S4A Fig). In contrast to mice inoculated with in vitro-cultivated ΔplzA organisms (Table 1), all mice needle-inoculated with DMC-cultivated ΔplzA organisms seroconverted (Table 2 and S4B Fig) and were culture positive at three or more tissue sites at two weeks post-infection (Table 2). As expected, plzA-R145D and plzAcomp strains displayed infectivity comparable to that of the wt parent (Table 2). To determine whether adaptive immune pressure contributes to the attenuation of the ΔplzA mutant, we assessed infectivity of wt, ΔplzA and plzAcomp strains (1 × 104 spirochetes) in immunodeficient Prkdcscid (scid) mice four-weeks post-inoculation. As shown in Table 3, the ΔplzA strain infected scid mice at near wt levels. Collectively, these data suggest that PlzA promotes the expression of one or more virulence-related gene products required to evade host adaptive immunity.

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Table 2. Prior host-adaptation enhances infectivity of PlzA-deficient spirochetes.

https://doi.org/10.1371/journal.ppat.1009725.t002

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Table 3. PlzA-deficient spirochetes show increased infectivity in immunodeficient (scid) mice.

https://doi.org/10.1371/journal.ppat.1009725.t003

Constitutive production of c-di-GMP ablates spirochete virulence

Kostick et al. [43] previously reported that overexpression of Rrp1 in a wild-type B. burgdorferi background had no effect on motility or chemotaxis in vitro but substantially attenuated virulence in mice infected by needle-inoculation. These results suggest that c-di-GMP is deleterious to spirochetes during the mammalian host phase of the enzootic cycle. However, because the authors were unable to measure c-di-GMP levels in either the wild-type or Rrp1-overexpressing strains, it is unclear how the levels of this secondary messenger compare in in the two strains. Thus, to further investigate the effect of c-di-GMP on infectivity in the mammal, we took advantage of studies by Ryjenkov et al. [37] demonstrating that Slr1143, a diguanylate cyclase from an oxygenic phototroph Synechocystis sp., constitutively synthesizes c-di-GMP. As described in Methods, we generated a cp26-based suicide-vector containing a Borrelia-codon-optimized, hemagglutinin (HA)-tagged slr1143 construct expressed under the constitutive borrelial flaB promoter (Fig 3A) and transformed it into a strain B31 Δrrp1 mutant. Constitutive expression of HA-tagged Slr1143 in this strain (designated cDGC) was confirmed by immunoblot using anti-HA antibodies (Fig 3B). cDGC grown at 37°C to late-logarithmic phase harbored concentrations of c-di-GMP, measured by LS-MS/MS, only slightly (3-4-fold) greater than those in the wt; as expected, no c-di-GMP was detected in the Δrrp1 control (Fig 3C). During in vitro growth, constitutive synthesis of c-di-GMP by Slr1143 functionally complemented loss of Rrp1 based on restoration of GlpD expression (Fig 3B), wt growth kinetics (Fig 3D), and normal motility (S4 Movie). Lastly, we examined the survival of the cDGC strain in ticks (Fig 4). As expected [39,41–43,45], and similar to the ΔplzA and plzA-R145D mutant phenotypes (Fig 1), the Δrrp1 mutant did not survive the blood meal (Fig 4A and 4B), though qPCR detected residual spirochete DNA (Fig 4C). Importantly, Slr1143 functionally complemented the Δrrp1 mutation, restoring the mutant’s ability to survive during the larval blood meal (Fig 4).

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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.

https://doi.org/10.1371/journal.ppat.1009725.g003

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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.

https://doi.org/10.1371/journal.ppat.1009725.g004

We next used the cDGC strain to assess how continued synthesis of c-di-GMP affects the ability of Lyme disease spirochetes to establish infection, disseminate and persist in mice. Naïve C3H/HeJ mice were needle-inoculated (1 × 104 spirochetes) with wt, Δrrp1 and cDGC strains and infection was assessed by tissue culturing at two weeks post-inoculation. In contrast to mice infected with the wt and Δrrp1 strains, all of which were culture positive at each site tested, none of the mice inoculated with the cDGC strain yielded positive tissue cultures or seroconverted (S2 Table).

Production of c-di-GMP antagonizes the RpoN/RpoS pathway through PlzA

We postulated that antagonism of RpoS-dependent gene regulation by c-di-GMP might explain the avirulence of the cDGC strain. To investigate this possibility, we cultivated wild-type, Δrrp1 and cDGC strains in DMCs. Whereas wt and Δrrp1 spirochetes host-adapted normally, the cDGC strain demonstrated a markedly abnormal protein profile with significant reduction of OspC and strong expression of OspA and Lp6.6, both of which are RpoS-repressed during mammalian host adaptation [36,55,61] (Fig 5A). Notably, we also detected GlpD in the cDGC strain (Fig 5A), indicating that c-di-GMP is able to promote glp gene expression when RpoS-mediated repression of glp gene transcription [20,36,55,62] is antagonized. Surprisingly, by both immunoblot (Fig 5A) and qRT-PCR (Fig 5B), rpoS/RpoS levels in all three strains were equivalent in DMCs, confirming c-di-GMP interferes with RpoS function at the post-translational level. Of note, constitutive synthesis of c-di-GMP had no effect on OspC expression in vitro, suggesting this phenomenon is unique to host-adaptation (S5 Fig).

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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.

https://doi.org/10.1371/journal.ppat.1009725.g005

To determine whether the interference of c-di-GMP on RpoS function in DMCs is mediated via PlzA, we inserted the constitutive-expressed/-active diguanylate cyclase (PflaB-slr1143-HA) cassette into the Δplz background, generating ΔplzA+cDGC, and evaluated the protein profiles of wt, ΔplzA, plzA-R145D, ΔplzA+cDGC and cDGC strains cultivated in DMCs by SDS-PAGE and immunoblot (Fig 6). Whereas the presence of c-di-GMP (cDGC) interfered with both positive and negative aspects of RpoS-dependent gene regulation in DMCs, the absence of PlzA (ΔplzA+cDGC) restored normal RpoS function in vivo. Consistent with results above (S4 Fig), wt, ΔplzA and plzA-R145D strains all host-adapted normally (Fig 6). From these data, we conclude that ectopic expression of c-di-GMP in vivo dampens RpoS activity at the post-translational level in a PlzA-dependent manner.

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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.

https://doi.org/10.1371/journal.ppat.1009725.g006

PlzA has a flexible bipartite domain structure that potentially explains its differential function

Galperin and Chou [64] recently categorized bipartite ‘xPilZ-PilZ’ proteins from diverse bacteria into nine classes based on their N-terminal non-canonical PilZ-related domains. PlzA belongs to the ‘PilZN3-PilZ’ class based on the presence of a newly described N-terminal PilZN3 domain, which is predicted to form a six-stranded PilZ-like β-barrel. To date, crystal structures have been reported for only three xPilZ-PilZ proteins in c-di-GMP-liganded and -unliganded states (S6A Fig): (i) MrkH, a PilZN2-PilZ DNA binding transcriptional factor that promotes biofilm formation in Klebsiella pneumoniae [65,66]; (ii) FlgZ, a PilZN-PilZ protein, which functions as a flagellar brake in Pseudomonas putida [67]; and (iii) PlzD, a PilZNR-PilZ protein, which regulates virulence and motility in Vibrio cholerae [68,69]. In all three, binding of c-di-GMP by the C-terminal PilZ domain induces a large rotational change that brings their N- and C-terminal β-barrels into proximity, with c-di-GMP intercalated at xPilZ-PilZ domain interface. Root-mean-square deviation (RMSD) values for liganded and unliganded MrkH, FlgZ and PlzD (S6B Fig) confirm that the major conformational changes occur at the xPilZ-PilZ domain interface (11.8–13.0 Å), with essentially no structural changes in the individual β-barrels (0.2–0.5 Å).

Using a FRET-based approach, Mallory et al. [51] demonstrated that recombinant PlzA undergoes ligand-induced rearrangement. To elucidate the structural changes that PlzA undergoes upon binding of c-di-GMP, we performed small angle X-ray scattering (SAXS) on recombinant monomeric His-tagged PlzA (S7A Fig) in the presence and absence of c-di-GMP (S7B Fig). Shapes of the Kratky plots (Fig 7A) revealed that liganded-PlzA is well-ordered, while the unliganded protein is unfolded and/or highly flexible, confirming a major structural rearrangement upon binding of c-di-GMP. We next used trRosetta [70], I-TASSER [71,72] and SWISS-MODEL [73] to generate structural models for liganded-PlzA and then screened twenty of the resulting models against our experimental SAXS data (S4 Table). One model, generated using trRosetta, produced the lowest χ2 value (7.14) which, after SREFLEX refinement [74], improved to 6.66 (S7C Fig). Of note, refinement did not require structural changes within the N- or C-terminal domains, just reorientation of the β-barrels. Next, the refined model was docked with c-di-GMP using HADDOCK [75] and fit into the SAXS envelope. As shown in Figs 6D and S7D, the RxxxR c-di-GMP binding motif is located within the extended interdomain linker, while the (D/N)hSxxG motif is located within the C-terminal PilZ β-barrel. Helices α1 and α2 in the N-terminal PilZN3 domain are positioned in proximity to its unique C-terminal α-helix (CT-α) and RxxxR motif. Electrostatics analysis of liganded-PlzA indicates three positively charged surface regions; one contains the c-di-GMP binding site, while the other two are located in grooves within the N- and C-terminal domains (S7E Fig, dashed lines) and could be available for interactions with DNA or other proteins. Due to its high ambiguity score, an envelope for unliganded PlzA could not be generated from the SAXS data, further confirming that c-di-GMP locks both domains of PlzA into a static, condensed conformation.

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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].

https://doi.org/10.1371/journal.ppat.1009725.g007

Note added in proof: Singh et al. [123] recently reported a 1.6 Å high resolution crystal structure for Borrelia (Borreliella) burgdorferi PlzA in complex with two molecules of c-di-GMP (PDB ID: 7MIE). The structural model and domain organization for PlzA presented herein are highly similar to the solved structure reported by Singh et al.

Discussion

In many bacteria, c-di-GMP inhibits motility [76] and, by a variety of allosteric mechanisms, regulates the transition from a motile planktonic to a biofilm-associated, communal lifestyle [77–81]. In B. burgdorferi, c-di-GMP production is tied to environmental sensing by the Hk1/Rrp1 TCS in response to unknown biochemical cues generated during tick feeding [38–44]. Spirochetes unable to produce c-di-GMP cannot withstand the onslaught of nutritional and/or biophysical stressors unleashed by the blood meal and, consequently, are destroyed in the midguts of both larvae and nymphs [39,41–43]. Since the signals driving Hk1/Rrp1 activation emanate from the blood meal [38–43], production of and signaling by c-di-GMP is restricted to the tick phase of the enzootic cycle. Indeed, not only is c-di-GMP not required during the reservoir phase [39,41,42], herein, we present evidence that it is inimical, at least in part, because it antagonizes RpoS function(s) required for the establishment and maintenance of mammalian infection by B. burgdorferi. A conundrum the spirochete faces is that it must produce c-di-GMP during two ‘opposing’ tick phases—acquisition, the RpoS-OFF state when spirochetes colonize the vector, and transmission, the RpoS-ON state when spirochetes regain motility and exit the midgut [20,34,35,42,55,82]. B. burgdorferi appears to have partially resolved this dilemma by integrating the adaptive changes mediated by c-di-GMP-liganded PlzA into the ON and OFF states of the RpoS genetic program in ticks along with divergent motility responses during the acquisition and transmission blood meals. Moreover, in PlzA, the spirochete has evolved a novel c-di-GMP effector protein [64] that serves as a biosensor for the presence or absence of c-di-GMP to promote, respectively, vector- or mammalian host-adaptation.

In vitro and in vivo studies of PlzA function yield widely disparate results. In vitro, the consequences of PlzA deficiency are negligible (i.e., minor growth and motility defects), whereas the in vivo effects related to the loss of PlzA are dramatic [38,44,49,57]. As shown here and elsewhere [49,57], ΔplzA spirochetes are destroyed in ticks and, in mice, they exhibit marked attenuation of infectivity; importantly, neither phenotype can readily be related to the modest motility defect observed in vitro by others [49,57]. As noted by Novak et al. [44], B. burgdorferi mutants defective in motility (e.g., cheA2 and pdeA) not only survive but replicate exponentially within feeding ticks [50,83]. Rather, the bulk of available evidence indicates that lysis of Δhk1 and Δrrp1 spirochetes during the blood meal is due to decreased expression of permeases for the uptake and utilization of alternative carbon sources (e.g., glycerol, N-acetylglucosamine (NAG) and chitobiose) with consequent inability to support both energy generation and cell envelope biogenesis [16,41,42,45,54]. Lysis of ΔplzA and plzA-R145D strains under these same conditions implies that these transcriptional effects of c-di-GMP are, at least in part, PlzA-dependent. Other investigators previously have raised the possibility that PlzA function in mammals might be c-di-GMP-independent [44,52,53,57]. We confirmed this by demonstrating that the virulence of the plzA-R145D strain is comparable to wild-type. In other bacteria, the regulatory effect of PilZ domain proteins on the flagellar motor requires c-di-GMP [84–86]; results with the plzA-R145D strain also argue that PlzA’s role in borrelial virulence is unrelated to motility. While non-canonical PilZ domain proteins (e.g., Vibrio cholerae PlzB and Xanthamonas campestris Xcc6021) that are unable to bind c-di-GMP have been linked to virulence [64,68,87], to our knowledge, PlzA is the first example of a c-di-GMP biosensor with dual functionality. Because the effector functions of c-di-GMP-liganded and -unliganded PlzA, respectively, are strictly segregated to the tick and mammalian stages of the enzootic cycle (Fig 8), it seems almost certain that they reflect discrete interaction partners and downstream effector mechanisms [49,53,57].

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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 [37–39,41–46]. 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).

https://doi.org/10.1371/journal.ppat.1009725.g008

In independent studies, Sze et al. [45] and He et al. [52] reported that inactivation of rrp1 and plzA, respectively, resulted in reduced levels of BosR in vitro, leading to the conclusion that reduced transcription of rpoS is responsible for the virulence defect of the ΔplzA mutant. Our data are not in accord with this reasoning, as we observed no effect on rpoS/RpoS levels either in vitro or in DMC-cultivated ΔplzA spirochetes. Moreover, we detected comparable levels of rpoS/RpoS in wt, Δrrp1 and cDGC strains in DMCs ([42] and herein). Collectively, these results indirectly confirm that the levels of RpoS are not adversely affected by loss of either liganded- or unliganded-PlzA and that, consequently, one must look elsewhere to understand how PlzA interfaces with RpoS. They also strongly infer that the early infection defect of ΔplzA spirochetes is both RpoS- and c-di-GMP-independent and, by extrapolation, that PlzA promotes mammalian host-adaptation via a mechanism(s) that is extrinsic to the RpoN/RpoS pathway. Although this RpoS-/c-di-GMP-independent function of PlzA is cryptic at present, one can glean insights into its general features from three facets of the ΔplzA murine phenotype uncovered herein: (i) The ΔplzA defect is overcome by a small subpopulation of organisms; in 2 of 10 C3H/HeJ mice inoculated with the ΔplzA strain cultivated in vitro, the mutant spirochetes survived the inoculation, disseminated and persisted in metastatic sites. Consistent with these findings, Pitzer et al. [49] found that they could increase the proportion of mice with disseminated infection by increasing the ΔplzA inoculum. (ii) The ΔplzA defect can be bypassed to a substantial degree if the mutant is host-adapted in DMCs prior to inoculation. (iii) The ΔplzA defect involves, at least in part, evasion of adaptive immunity since ΔplzA infectivity is substantially greater in scid mice. A body of evidence indicates that, once inoculated, spirochetes must overcome stochastic bottlenecks created by host barriers to cause systemic infection [88,89]. Unliganded-PlzA appears to participate in one or more regulatory pathways that increase the probability that an infecting population will contain a sufficient number of organisms with the ‘appropriate’ transcriptional profile to surmount these bottlenecks.

Fig 8 presents our working model for how gene regulation by PlzA, c-di-GMP and RpoS interdigitates to sustain B. burgdorferi within its dual-host lifecycle. In mammals, when c-di-GMP signaling is normally OFF, one sees RpoS-mediated repression of tick-phase genes [36,55,56,62,90]. When this ‘gatekeeper’ function of RpoS was first noted [55,61], predating the discovery of c-di-GMP signaling in B. burgdorferi [37–39,41,43,46], we postulated that RpoS-mediated repression was induced by mammalian host-specific signals [55,61]. However, two subsequent, closely related lines of evidence recently led us to propose an alternative hypothesis, namely, that antagonism of RpoS within feeding ticks is alleviated in the mammal by the cessation of c-di-GMP synthesis [36,42,82]: (i) RpoS-mediated repression of tick-phase genes does not occur concurrently with the RpoS-ON state in feeding nymphs and (ii) in engorged nymphs, ΔrpoS organisms express significantly higher levels of RpoS-repressed genes than wt [42,82]. Herein, we assessed this counterintuitive idea by engineering a B. burgdorferi strain (cDGC) that constitutively synthesizes c-di-GMP at levels comparable to those produced by wt strains. The results were striking and, once again, underscore the dichotomy between in vitro and in vivo gene expression by B. burgdorferi. In vitro, ‘ectopic’ expression of c-di-GMP had no discernible effect on the RpoN/RpoS pathway, while in vivo we saw a dramatic PlzA-dependent dampening of RpoS activity that affected both RpoS-upregulated and RpoS-repressed gene products. Our finding that this antagonism occurred without diminution of either rpoS transcript or protein, suggesting that liganded-PlzA directly or indirectly interacts with RNA polymerase-RpoS holoenzyme (RNAP-RpoS). In conjunction with these new insights, one can envision how liganded-PlzA could act as a ‘brake’ for RpoS-dependent gene regulation in ticks. During acquisition, transcription of rpoS by the Rrp2/BosR/RpoN complex is quickly shut OFF in the midgut in response to unknown signals [16,20]. Conceivably, to expedite the switch from an RpoS-ON to -OFF, liganded-PlzA also could interfere with transcription by residual RNAP-RpoS holoenzyme allosterically, while BBD18, which is RpoS-repressed and c-di-GMP-induced [36,42], would target free RpoS for proteolytic degradation [91–94]. During transmission, partial restriction of RNAP-RpoS holoenzyme by liganded-PlzA could enable spirochetes to sustain expression of RpoS-repressed tick phase genes (i.e., ospA, glps) while at the same time allowing reduced expression of RpoS-upregulated genes required for transmission (i.e., bba64) or early infection (i.e., ospC, dbpBA) [36,82,95]. Once within the mammal, loss of c-di-GMP signaling, presumably hastened by the spirochete’s two phosphodiesterases, PdeA and PdeB [44,48,50], removes impedance of the RpoS gatekeeper and enables unliganded-PlzA to assume its alternative, c-di-GMP-independent function(s) either directly or indirectly via an as yet unidentified interactive partner.

The crystal structures of MrkH [66], FlgZ [67], and PlzD [96] in their liganded and unliganded states show that c-di-GMP binding causes large spatial rotations in the N- and C-terminal domains and reorganization with their inter-domain linkers, with minimal structural changes in the individual domains. c-di-GMP-induced conformational changes in PlzA [51,57], confirmed herein by SAXS, also likely underlie this novel effector protein’s dual functionality in ticks and mice. In its liganded state, PlzA forms a stable, compact protein, while the apo-protein is partially unfolded and/or significantly flexible; our analysis further suggests that apo-PlzA requires an interaction partner to promote structural stabilization and functionality. The extended length of the PlzA linker region likely contributes to even greater flexibility between its PilZN3 and PilZ domains [51]. Reorientation of the three α-helices of the PilZN3 domain in liganded PlzA would create a new interface for interactions distinct from those of the unliganded protein. Expression data for the glp operon indicate that liganded-PlzA directly or indirectly promotes expression of this RpoS-repressed locus by RNAP-RpoD (σ70) [42,53]; this could occur by two possible mechanisms (Fig 8). One, analogous to MrkH, is by DNA binding as a transcription factor [66,97]. As with MrkH [66,97], the surface electrostatics of our PlzA model indicate large positively charged regions, including major grooves in N- and C-terminal β-barrels. The other, as proposed by Zhang et al. [53], is by allosteric interaction with RNAP-RpoD. The possibility that PlzA can act as a positive or negative regulator for RNAP-RpoD and RNAP-RpoS holoenzymes, respectively, presents a unifying and, therefore, appealing mechanism for PlzA’s transcriptional effects.

Efficient migration between the vector and reservoir hosts is essential for perpetuation of B. burgdorferi in nature [3,7–9]; once within a reservoir or incidental human host, motility becomes critical for dissemination and tissue invasion [98–100]. In other bacteria, there is an inverse relationship between intracellular c-di-GMP concentrations and motility, with low levels promoting motility and high levels stimulating adherence and biofilm formation [76,101]. In Gram-negatives, YcgR orthologs acts as a ‘clutch’ to slow flagellar rotation in response to c-di-GMP by directly interacting with the flagellar motor at the rotor-stator interface [76,86,102]. Results presented herein show that B. burgdorferi deviates from this motility control paradigm. Comparison of the swimming behaviors of wt, Δrrp1 and cDGC strains in vitro revealed that elevated c-di-GMP exerts a potent inhibitory effect on motility; however, as shown here and elsewhere [49,57], inhibition by c-di-GMP appears to be largely independent of PlzA given that neither ΔplzA nor plzA-R145D exhibited discernably aberrant motility in BSK-II. Along similar lines, using a ΔpdeAΔplzA double mutant, Pitzer et al. [49] demonstrated that elevated c-di-GMP regulates motility in B. burgdorferi by a PlzA-independent mechanism. Thus, B. burgdorferi appears to contain an as yet unidentified c-di-GMP-responsive regulator of motility that does not contain a recognizable PilZ domain. The broader implication is that the spirochete has, at least in part, separated c-di-GMP control of motility from PlzA-mediated gene expression which is needed for survival during the acquisition and transmission blood meals. Previously, we reported that spirochete transmission during the nymphal blood meal is biphasic, occurring initially via a non-motile, replicative process termed ‘adherence-mediated migration’ followed by an invasive, motile phase [13]. In support of this theory, we demonstrated that the contents of engorged midguts inhibit B. burgdorferi motility, now attributable to luminal tick factor(s) that stimulates synthesis of c-di-GMP via activation of the Hk1 sensor. Presumably, in the small number of spirochetes that reach the midgut basement membrane, these stimulatory cues diminish, allowing c-di-GMP levels to drop low enough to restore motility. Spirochetes lacking RpoS survive within the nymphal midgut, indicating indirectly that Hk1/Rrp1 pathway is active, but remain confined within the luminal space [82]. Thus, the ON/OFF state of the RpoN/RpoS pathway likely determines whether spirochetes colonize or penetrate the midgut epithelium in concert with precise, spatiotemporal regulation of c-di-GMP production [42,82].

Methods

Ethics statement

All experiments involving animals conducted at UConn Health were performed in accordance with The Guide for the Care and Use of Laboratory Animals (8th Edition) [103] using protocols reviewed and approved by the UConn Health Institutional Animal Care and Use Committee [Animal Welfare Assurance (AWA) number A347-01].

Bacterial strains and culture conditions

S1 Table contains detailed descriptions of strains and plasmids used in these studies. Escherichia coli Top10 (Life Technologies, Grand Island, NY) or Stellar (TaKaRa, Mountain View, CA) strains were maintained in Lysogeny broth (LB) or LB agar supplemented with the appropriate antibiotics (ampicillin, 100 μg/ml; spectinomycin, 100 μg/ml; kanamycin, 100 μg/ml; and/or gentamycin, 5 μg/ml). B. burgdorferi strains were maintained in Barbour-Stoenner-Kelly (BSK)-II medium [104] supplemented with 6% rabbit serum (Pel-Freeze Biologicals, Rogers, AR); when appropriate, antibiotics were added (kanamycin, 400 μg/ml; streptomycin, 100 μg/ml; gentamycin, 50 μg/ml). Plasmid content of B. burgdorferi strains was monitored as previously described [105,106]. Temperature-shifts were performed as previously described [107]. For growth curves, B. burgdorferi cultures were inoculated in quadruplicate at a starting density of 1 × 103 spirochetes/ml in BSK-II containing appropriate antibiotics and grown at 37°C for up to 10 days. Mammalian host-adapted spirochetes were generated by cultivation in rat peritoneal dialysis membrane chambers (DMCs) as previously described [108,109]. Spirochetes were enumerated daily by darkfield microscopy using a Petroff-Hausser counting chamber (Hausser Scientific Co., Horsham, PA). Spirochete motility was assessed by darkfield microscopy on an Olympus BX41 epifluorescence microscope using a ×100 UPlanFI objective (total magnification ×1000). Images were acquired for 10 sec per field using a QImaging Retiga R6 CCD camera (Teledyne Photometrics, Tucson, AZ).

Routine DNA manipulation and cloning

Oligonucleotide primers used these studies are described in S3 Table. Plasmids were purified from E. coli using QIAprep spin and midi kits (Qiagen, Valencia, CA) or NucleoBond PC2000 (TaKaRa, Mountain View, CA). Bacterial genomic DNA was extracted using the Gentra Puregene Yeast/Bacteria kit (Qiagen). Except where noted, routine cloning was performed using the In-Fusion HD Cloning Plus kit (Takara Bio USA Inc., Mountain View, CA). Routine and high-fidelity PCR amplifications were performed using RedTaq (Denville Scientific, Metuchen, NJ, United States) and CloneAmp HiFi (Takara Bio USA Inc., Mountain View, CA), respectively. DNA sequencing was performed by Genewiz, Inc. (Cambridge, MA) and analyzed using MacVector v17.0.1 (MacVector, Inc., Cary, NC, United States). Oligonucleotide primers were purchased from Sigma-Aldrich (St. Louis, MO); S4 Table provides primer sequences.

SDS-PAGE and immunoblot analyses

Whole-cell lysates were prepared from spirochetes (~2 × 107 cells/lane) cultivated to late logarithmic phase at 37°C following temperature-shift were separated on 12.5% SDS-PAGE mini-gels and stained with silver as previously described [55]. For immunoblotting, whole cell lysates and/or recombinant proteins were transferred to nitrocellulose and incubated overnight with antiserum against RpoS/BB0771 [110], FlaB/BB0147 [61], GlpD/BB0243 [56], Lp6.6/BBA62 [63], OspC/BBB19 [36], OspA/BBA15 [36], or PlzA/BB0733, each diluted 1:1000–1:6000, followed by horseradish peroxidase (HRP)-conjugated secondary antibody (Southern Biotechnology Associates, Birmingham, AL) diluted 1:20,000. Anti-HA antibody (Sigma-Aldrich, St. Louis, MO) was used to track expression of Slr1143-HA. Immunoblots were developed using the SuperSignal West Pico chemiluminescence substrate (Pierce, Rockford, IL).

Construction of B. burgdorferi plzA site-directed mutant

To distinguish between the c-di-GMP-dependent and -independent effector functions for PlzA, an arginine to aspartic acid point mutation was introduced at residue 145 (R145D) by site-directed mutagenesis [51]. Briefly, an AgeI site was introduced into the pbb0733-Easy suicide vector [49] in the plzA-bb0734 intergenic region using the QuikChange II site-directed mutagenesis kit (Agilent Technologies Inc., Santa Clara, CA) and primers BB0733AgeImut-F and BB0733AgeImut-R. A PflgB-GentR cassette [111] was inserted into the AgeI site of pbb0733-Easy in the same orientation as plzA. A single point mutation (R145D) was introduced in the plzA coding region using primers BB0733-R145D-F and BB0733-R145D-R. The resulting suicide vector (pbb0733R145DGenta-Easy) was linearized by digestion with NotI, electroporated into B. burgdorferi strain B31 A3-68 Δbbe02 as previously described [112]. Transformants were screened for the gentamycin-resistance cassette by PCR using PlessGent-F and PlessGent-R. The R145D point mutation was confirmed by sequencing. A single transformant containing the same plasmid profile as the parent was selected for further analysis and designated plzA-R145D.

Generation of B. burgdorferi strain expressing a constitutively active diguanylate cyclase

A Δrrp1 mutant was generated by transforming B31 5A18 NP1 [113] with pΔBB0419 [38]. A single streptomycin-resistant clone containing the same plasmid profile as the parent was selected for further analysis. A codon-optimized version of slr1143, encoding a constitutively active diguanylate cyclase from Synechocystis sp. [37] and C-terminal hemagglutinin (HA) tag, was synthesized by Genewiz (South Plainfield, NJ). slr1143opt-HA was fused to the B. burgdorferi flaB promoter via overlapping PCR using the primers listed in S4 Table. The PflaB-slr1143opt-HA cassette was cloned into the AatII site of EcAG265 [106], a modified version of the cp26-based E. coli-B. burgdorferi shuttle vector pMC2498 [60] in which the promoterless gfp cassette has been replaced with an AatII site. The resulting plasmid (EcAG284) was confirmed by sequencing and then electroporated into Δrrp1 as previously described [112]. Transformants were screened for slr1143opt-HA by PCR using primers 5’ bb0733 ORF and 3’ bb0733 ORF. A single gentamycin-resistant clone containing the same plasmid profile as the parent was selected and designated cDGC. To generate a ΔplzA strain that constitutively synthesizes c-di-GMP, the cassette encoding PflaB-slr1143opt-HA followed by GentR was sub-cloned into pUC19 containing ~1200 bp framing the rrp1 coding region, so that PflaB-slr1143opt-HA + GentR replaced rrp1 (pEcAG391). This plasmid was transformed into ΔplzA and a single gentamycin-resistant clone containing the same plasmid profile as the parent and designated ΔplzA+cDGC.

Recombinant protein expression and purification

His-tagged PlzA-R145D was generated by site-directed mutagenesis as described above using plzA/pTrc-His [49] as a template. His-tagged PlzA and PlzA-R145D were overexpressed in E. coli Rosetta 2 (DE3) pRare (MilliporeSigma, Burlington, MA) and purified by nickel affinity chromatography using HisTrap Column (GE Healthcare Life Sciences Pittsburgh, PA) followed by size-exclusion chromatography using Superdex 200 10/300 GL column (GE Healthcare Life Sciences Pittsburgh, PA). Recombinant proteins were assessed for purity by SDS-PAGE followed by staining with GelCode Blue (Thermo Fisher Scientific, Waltham, MA) and/or immunoblot using HRP-conjugated anti-His antibody (ThermoFisher). Purified recombinant PlzA-His protein (40–60 μg) was used to generate polyclonal antisera in female Sprague-Dawley rats (Envigo, South Easton, MA) as previously described [108].

Murine infection studies

Five to eight-week-old female C3H/HeJ or NOD.Cg-Prkdcscid/J (scid; Jackson Laboratories, Bar Harbor, ME) mice were needle-inoculated with 1 × 104 organisms cultivated in vitro or from freshly harvested DMCs. At designated time points, animals were sacrificed, and blood and tissues collected for serology and culturing in BSK-II containing Borrelia antibiotic cocktail (0.05 mg/ml sulfamethoxazole, 0.02 mg/ml phosphomycin, 0.05 mg/ml rifampicin, 0.01 mg/ml trimethoprim and 2.5 μg/ml amphotericin B). Cultures were examined weekly by dark-field microscopy for up to 4 weeks. Seroconversion was determined by immunoblotting B. burgdorferi whole cell lysates with infected mouse serum, diluted 1:1000, followed by incubation with HRP-conjugated secondary antibody (Southern Biotechnology Associates, Birmingham, AL) diluted 1:20,000 and detection using SuperSignal West Pico chemiluminescence substrate (Pierce, Rockford, IL).

Tick experiments

Pathogen-free Ixodes scapularis larvae were purchased from Oklahoma State University Tick Rearing Facility (Stillwater, OK). Immersion feeding of naïve larvae was performed as previously described [90] using the method established by Policastro et al. [114]. Pools of 10 replete larvae per strain were processed for DNA extraction and semi-solid phase plating. A separate pool of 10 larvae was processed for immunofluorescence using FITC-conjugated anti-Borrelia antibody (Kirkegaard and Perry Laboratories, Gaithersburg, MD) as previously described [90]. Replete larvae used to assess borrelial gene expression were infected by whole body infestation of needle-inoculate mice as previously described [90]. To assess gene expression during transmission, infected I. scapularis nymphs were fed for at least 72 h on naïve C3H/HeJ mice as previously described [90]. Fed larvae (~100–150 per pool) and nymphs (~20–25 per pool) were crushed into TRIzol reagent (Invitrogen) and stored at -80°C until RNA was extracted.

qRT-PCR

Total RNA was isolated from triplicate pools of replete larvae, unfed and fed nymphs, and DMC-cultivated organisms) as previously described [90]. Following DNase treatment, RNA was converted to cDNA using SuperScript III (Life Technologies), including a negative control with no reverse transcriptase. cDNAs were assayed in quadruplicate using SsoAdvanced Universal SYBR (rpoS) or Probe (flaB) Mix (Bio-Rad) using the primers described in S3 Table. Transcript copy numbers were calculated using the iCycler post-run analysis software based on internal standard curves and then normalized against copies of flaB as previously described [90].

Measurement of c-di-GMP levels

Cultures (50 mls) of wt B31 5A18 NP1, Δrrp1, and cDGC were grown to late-logarithmic phase and cells were harvested for ethanol extraction as previously described [42]. Extracted supernatants were filtered through a 0.22 μm syringe-filter, concentrated via SpeedVac, and resuspended to a final volume of 0.1 ml in HPLC grade water. c-di-GMP was detected by ultraperformance liquid chromatography (UPLC) in tandem with mass spectrometry (MS) using an Acquity UPLC system coupled to an Acquity TQD mass spectrometer (Waters Corporation, Milford, MA). The separation of c-di-GMP was achieved using a High Strength Silica (HSS) reversed-phase UPLC column. Briefly, the eluent system was composed of 0.1% formic acid in water (pH 2.9) (eluent A) and 0.1% formic acid in acetonitrile (eluent B). 98% eluent A was held for 0.5 min followed by a gradient to 100% eluent B in 4 min, held for 0.5 min, then switched back to 98% eluent A at a flow rate of 0.4 ml/min. An Acquity HSS T3 column (2.1 by 100 mm; 1.8 μm particle size; Waters) was used with a sample injection volume of 10 ul. The column and autosampler were maintained at 35°C and 20°C, respectively. Detection of c-di-GMP was performed in electrospray ionization (ESI) negative-ion mode using the multiple-reaction monitoring mode. For ESI-MS/MS analysis, the following ion transition, cone voltage (CV), and collision energy (CE) were used: c-di-GMP m/z 689.1 (precursor ion) and 150.0 (product ion); CV, 66 V; and CE, 56 eV. The ESI capillary voltage was 3 kV, the source temperature was set at 150°C, and the desolvation temperature was set at 400°C. The flow rate of the desolvation gas (N2) was set at 650 liters/h. The Waters IntelliStart software was utilized for analyte signal optimization. Statistical analysis for obtaining calibration and quantification results for c-di-GMP. was performed using Waters QuanLynx, which is included in the MassLynx software v.4.2. The concentration of c-di-GMP was calculated by interpolation of the observed analyte peak area with the corresponding calibration curve. Concentrations were determined as c-di-GMP (nmol/μg) of wet cell pellet weight.

c-di-GMP binding assay

A qualitative assessment of c-di-GMP binding by PlzA and PlzA-R145D was performed using 2’-O-(N’-Methylanthraniloyl)-c-di-GMP (MANT-c-di-GMP) (Biolog Life Science Institute GmbH & Co., Germany) [58]. Recombinant PlzA and PlzA-R145D, described above, and lysozyme (negative control) were diluted to 10 μM and incubated with 5 μM MANT-c-di-GMP in a 96 well plate, 200 μl reactions for 5 min. Binding was detected by monitoring the relative increase in fluorescence of MANT-c-di-GMP (λex = 355 nm, λem = 448 nm) in the presence and absence of protein using a SpectraMax M2 spectrofluorometer (Molecular Devices, USA).

Small angle X-ray scattering (SAXS) data acquisition and analysis, structural modeling, and c-di-GMP docking

Prior to SAXS, recombinant PlzA (135, 67 and 33 μM) was incubated with or without 270 μM of c-di-GMP in 500 mM NaCl and 20 mM Na2HPO4 (pH 7.4). SAXS data were acquired on the Bio-SAXS beamline BL4-2 at the Stanford Synchrotron Research Laboratory using a Rayonix MX225-HE CCD detector. All scattering data (to a maximum q of 0.5 Å−1) were collected at a wavelength of 1.3 Å for ten consecutive 2-second exposures. Results from the buffer alone, with or without c-di-GMP, were subtracted from the liganded and unliganded scattering, respectively. Data were analyzed using the ATSAS package [115]. Kratky plots and radii of gyration (Rg), extrapolated from the Guinier region of the Guinier plot, were computed using PRIMUS [116]. Scattering curves for liganded-PlzA were scaled and merged in PRIMUS primarily using the low q data for PlzA at 33 μM and the high q range data for PlzA at 67 μM. Unliganded-PlzA was analyzed using data set collected from PlzA at 67 μM only. P(r) functions were calculated using GNOM and ambiguity scores by AMBIMETER [117]. Ab initio shape determination was performed using DAMMIN [118] followed by DAMAVER [119]. Twenty three-dimensional models of PlzA were predicted using trRosetta [70], I-TASSER [71,72] and Swiss-Model [73] (S4 Table). Theoretical scattering curves were computed from different structural models and compared to the experimental scattering curves using FoXS [120]. The best-fitted model was refined by normal mode analysis from SAXS data using SREFLEX [74]. Coordinates for c-di-GMP were extracted from the crystal structure of Vibrio cholerae VCA0042/PlzD (PDB 2RDE) [96]. HADDOCK v2.2 [75] was used to dock c-di-GMP into the SREFLEX refined PlzA model. In the docking protocol, PlzA residues R145, R149, D182, A184 and G187 were designated as active residues to apply distance restraints. Superimposition of the PlzA model into the SAXS envelope structure was performed by SUPCOMB [121]. PyMOL Molecular Graphics System v2.3.2 (Schrödinger, LLC, New York, NY) was used for structure visualization, calculation of RMSD and surface electrostatics, and image rendering.

Statistical analysis

Growth curves were compared using the CGGC permutation test [122], with 1000 permutations. All other pairwise comparisons were evaluated by unpaired Student’s t-test with two-tailed p values and a 95% confidence interval using Prism v8.4.3 (GraphPad Software, San Diego, CA).

Supporting information

S1 Table. Bacterial strains and plasmids used in these studies.

https://doi.org/10.1371/journal.ppat.1009725.s001

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S2 Table. Constitutive synthesis of c-di-GMP abrogates virulence of B. burgdorferi in mice.

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S3 Table. Oligonucleotide primers used in these studies.

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S4 Table. Comparison of the liganded experimental SAXS data to modeled PlzA structures.

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S1 Fig. A single point mutation within the canonical C-terminal PilZ domain abrogates binding of c-di-GMP by PlzA.

A qualitative assessment of c-di-GMP binding by recombinant PlzA and PlzA-R145D His-tagged proteins was performed in triplicate using 5 μM 2’-O-(N’-Methylanthraniloyl)-c-di-GMP (MANT-c-di-GMP) [58] and 10 μM PlzA and PlzA-R145D proteins. Lysozyme (10 μM) was used as a negative control and MANT-c-di-GMP alone was used as a reference fluorescence. (A) Samples were excited at 355 nm and emission was measured between 400 to 500 nm at 5 nm intervals using a SpectraMax M2 spectrofluorometer (Molecular Devices, USA). Each point represents the average fluorescence at each interval following background subtraction (buffer alone). (B) Bars represent the changes in fluorescence (ΔF) in arbitrary units (au) for PlzA, PlzA-R145D and lysozyme compared to MANT-c-di-GMP alone at 450 nm (MANT λemmax = 448 nm) following background subtraction. Statistical significance was determined using unpaired Student’s t-test. Error bars indicate the mean ± standard error of the mean for three replicates. Asterisks (*) indicate p ≤ 0.05; ns, not significant.

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S2 Fig. Immunoblot analysis of sera collected from C3H/HeJ mice inoculated with in vitro-cultivated B31 A3-68 Δbbe02 (wt), ΔplzA, plzA-R15D and plzAcomp strains two weeks post-infection.

Sera (diluted 1:1000) were immunoblotted against B. burgdorferi strain B31 whole cell lysates. Wild-type and ΔplzA strains were compared to plzAcomp (A) and plzA-R145D (B) strains in separate experiments (5 mice per strain, per experiment).

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S3 Fig. B. burgdorferi lacking Rrp1 or PlzA or expressing a PlzA-R154D show reduced growth in vitro.

Growth curves of B31 A3-68 Δbbe02 (wt), ΔplzA, plzAcomp, plzA-R145D and Δrrp1 strains (in quadruplicate) from a starting density of 1 × 103 spirochetes/ml at 37°C. Statistical significance was determined by the CGGC permutation test [122]. Asterisks (*) indicate p ≤ 0.05; ns, not significant.

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S4 Fig. Host-adaptation prior to needle-inoculation restore infectivity of PlzA-deficient spirochetes in mice to near wild-type levels.

(A) Whole cell lysates of freshly harvested DMC-cultivated B31 A3-68 Δbbe02 (wt), ΔplzA, plzA-R145D and plzAcomp strains used to inoculate mice (Table 2) were separated by SDS-PAGE and stained with silver. Molecular weight markers (MWM) are shown on the left. (B) Immunoblot analysis of sera from mice needle-inoculated with DMC-cultivated B31 A3-68 Δbbe02 (wt), ΔplzA, plzA-R145D and plzAcomp strains two weeks post-infection. Sera (diluted 1:1,000) were immunoblotted against B. burgdorferi whole cell lysates. Wild-type and ΔplzA strains were compared to plzAcomp and plzA-R145D strains in separate experiments.

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S5 Fig. cDGC temperature-shifts normally in vitro.

Whole-cell lysates from B31 5A18 NP1 wild-type (wt), Δrrp1 and cDGC strains in vitro following temperature-shift were separated by SDS-PAGE and immunoblotted with antisera against PlzA, RpoS, OspC and FlaB (loading control). Molecular weight markers (MWM) are shown on the left.

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S6 Fig. Comparative structural analysis of ‘xPilZ-PilZ’ proteins.

(A) Conformational changes upon binding of c-di-GMP by Pseudomonas putida PP4397/FlgZ (PDBs: 2GJG-unliganded, 3KYF-liganded), Klebsiella pneumoniae MrkH (PDBs: 5KEC-unliganded, 5KGO-liganded), and Vibrio cholerae VCA0042/PlzD (PDBs: 1YLN-unliganded, 2RDE-liganded). The N- and C-terminal barrels are colored in salmon and yellow, respectively. The α-helices in the N-terminal degenerate PilZ-like domain and in the C-terminal PilZ domain are highlighted in light green and cyan, respectively. The RXXXR and (D/N)hSXXG c-di-GMP binding residues are represented with dark green sticks. c-di-GMP is shown in red. (B) Root-mean-square deviation (RMSD) values calculated from superimposition of PilZ domain from different xPilZ-PilZ proteins.

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S7 Fig. Experimental SAXS data.

(A) Analytical SEC elution profile for PlzA in the presence (orange) or absence (blue) of c-di-GMP. The inset shows SEC calibration curve calculated by linear fit of known molecular weight values as a function of the measured partition coefficient (Kav). The black, orange, and blue circles show the partition coefficients of calibration standards, liganded, and unliganded PlzA, respectively. (B) Raw logarithmic intensity plots of SAXS data collected from liganded- (orange) and unliganded- (blue) PlzA. (C) The best fits of the PlzA model in A before (green line) and after (black line) SREFLEX optimization [74] to the experimental scattering data for liganded PlzA (orange dots). The indicated χ2 values were calculated by FoXS [120]. (D) SREFLEX refined PlzA model with a docked c-di-GMP molecule. The PilZN3 and PilZ barrels are colored in salmon and yellow, respectively. The α-helices in the PilZN3 and in PilZ domains are highlighted in light green and cyan, respectively, with the exception of the unique PilZN3 C-terminal α-helix, in purple. The RXXXR and DXSXXG c-di-GMP binding residues are represented with dark green sticks, and c-di-GMP is labeled and shown in orange. (E) Surface electrostatics of the refined model in D as calculated in PyMol. In addition to the positively charged surface of the c-di-GMP binding region, two other large positively charged region are circled by dashed lines. The ribbon structure in D is in the same orientation as the surface electrostatics representation in E.

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S1 Movie. Motility in liquid medium using darkfield microscopy of wt spirochetes at mid-logarithmic growth with total magnification ×1000.

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S2 Movie. Motility in liquid medium using darkfield microscopy of Δrrp1 spirochetes at mid-logarithmic growth with total magnification ×1000.

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S3 Movie. Motility in liquid medium using darkfield microscopy of ΔplzA spirochetes at mid-logarithmic growth with total magnification ×1000.

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S4 Movie. Motility in liquid medium using darkfield microscopy of cDGC spirochetes at mid-logarithmic growth with total magnification ×1000.

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Acknowledgments

The authors would like to thank Md A. Motaleb (East Carolina University) for generously providing B. burgdorferi strains B31 A3-68 Δbbe02, ΔplzA, plzAcomp and plzA-R145D and Md A. Motaleb and Christopher Pappas (Manhattanville College) for providing the plasmid constructs used to express recombinant PlzA and GlpD, respectively. We also thank Bing Hao (UConn Health) for the use of the size exclusion column.

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