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As a ubiquitous second messenger, cyclic dimeric GMP (c-di-GMP) has been studied in numerous bacteria. The oral spirochete Treponema denticola, a periodontal pathogen associated with human periodontitis, has a complex c-di-GMP signaling network. However, its function remains unexplored. In this report, a PilZ-like c-di-GMP binding protein (TDE0214) was studied to investigate the role of c-di-GMP in the spirochete. TDE0214 harbors a PilZ domain with two signature motifs: RXXXR and DXSXXG. Biochemical studies showed that TDE0214 binds c-di-GMP in a specific manner, with a dissociation constant (Kd) value of 1.73 μM, which is in the low range compared to those of other reported c-di-GMP binding proteins. To reveal the role of c-di-GMP in T. denticola, a TDE0214 deletion mutant (TdΔ214) was constructed and analyzed in detail. First, swim plate and single-cell tracking analyses showed that TdΔ214 had abnormal swimming behaviors: the mutant was less motile and reversed more frequently than the wild type. Second, we found that biofilm formation of TdΔ214 was substantially repressed (~6.0-fold reduction). Finally, in vivo studies using a mouse skin abscess model revealed that the invasiveness and ability to induce skin abscesses and host humoral immune responses were significantly attenuated in TdΔ214, indicative of the impact that TDE0214 has on the virulence of T. denticola. Collectively, the results reported here indicate that TDE0214 plays important roles in motility, biofilm formation, and virulence of the spirochete. This report also paves a way to further unveil the roles of the c-di-GMP signaling network in the biology and pathogenicity of T. denticola.
Bis-(3′,5′)-cyclic dimeric GMP (c-di-GMP) is a soluble molecule that functions as a second messenger in bacteria (1). As a ubiquitous signaling molecule, c-di-GMP controls a wide range of cellular functions and processes, including motility/sessility, biofilm formation, cell cycle progression, antibiotic production, virulence, and other cellular functions (for a review, see references 2, 3, and 4). Cyclic di-GMP is produced from GTP by diguanylate cyclases (DGCs) and is degraded by phosphodiesterases (PDEs). Genetic, biochemical, and structural studies have shown that DGC activity is associated with the GGD/EEF domain (5, 6), whereas PDE activity is associated with the EAL or HD-GYP domains (7, 8). Those conserved domains are essential for the enzymatic activities (4, 5, 9). In a signaling network of c-di-GMP, bacteria typically use DGC and PDE enzymes to dynamically modulate the intracellular level of c-di-GMP in response to internal and/or external environmental cues (2–4). The c-di-GMP in turn interacts with different effectors and subsequently regulates diverse downstream cellular processes, such as transcription (10), translation (11), protein activity (12, 13), and secretion and stability (14), at different levels (for a review, see references 2, 4, and 15). Numerous c-di-GMP effectors have been identified, such as PilZ, PelD, PlzA, FleQ, Clp, VpsT, and riboswitch RNA (16–21). Among those effectors, PilZ was first identified in silico as a putative c-di-GMP binding domain (Pfam 07238) based on the BcsA1 protein of Acetobacter xylinus (22) and named after the type IV pilus control protein from Pseudomonas aeruginosa (23). Proteins in this family often contain two conserved domains: RXXXR and DXSXXG (16). In several bacterial species, PilZ-containing proteins have been confirmed to bind c-di-GMP in a highly specific manner, with dissociation constants (Kd) in the submicromolar range. Those proteins play a pivotal role in the regulatory networks of c-di-GMP (12, 24–28).
Human periodontitis is a prevalent chronic inflammatory disease that damages the supporting connective tissues around teeth and ultimately leads to tooth loss (29, 30). In the United States, approximately 30% of adults have moderate or severe disease (31). Periodontitis is triggered by overgrowth of microbial biofilms (also known as plaques). In the oral flora, more than 50 different spirochetal species have been identified, and they all belong to the Treponema genus (32, 33). Due to their fastidious growth requirements, very few oral treponemes can be reliably cultivated (34, 35). During the past decade, Treponema denticola, one of the etiological agents strongly associated with human periodontitis, has emerged as a model organism to study oral spirochetes, as it is readily cultivated and more amenable than some other species to experimental manipulations (35, 36). Numerous virulence factors involved in cytotoxicity, adherence, invasion, and immunomodulation have been identified (for a review, see references 35 and 37–39). The T. denticola ATCC 35405 strain was recently sequenced (40). Its genome encodes five proteins (TDE0125, TDE1685, TDE2725, TDE2580, and TDE2582) that harbor a well-conserved GGD/EEF domain, two proteins (TDE0128 and TDE2075) with an EAL domain, four proteins (TDE1256, TDE1467, TDE2302, and TDE2659) with an HD-GYP domain, and two putative c-di-GMP binding proteins (TDE0214 and TDE1318) (41), suggesting the existence of c-di-GMP signaling pathways in T. denticola. The work reported here attempts to uncover the potential roles of c-di-GMP-mediated regulatory networks in T. denticola via study of TDE0214, a putative PilZ-containing protein.
T. denticola ATCC 35405 (wild type) and its derivative mutant strains were grown in oral bacterial growth medium (OBGM) containing 10% heat-inactivated rabbit serum at 37°C in an AS-580 anaerobic chamber (Anaerobe Systems, Morgan Hill, CA) with an atmosphere of 85% nitrogen, 5% carbon dioxide, and 10% hydrogen, as previously described (42, 43). Escherichia coli TOP10 strain (Invitrogen, Carlsbad, CA) was used for routine plasmid constructions and preparations, an E. coli dam−/dcm− strain (New England BioLabs, Ipswich, MA) was used to prepare unmethylated plasmids, and the E. coli BL21 Star (DE3) strain (Invitrogen) was used for preparing recombinant proteins. E. coli strains were cultured in Difco Luria-Bertani (LB) broth (BD Biosciences, Sparks, MD) supplemented with 100 μg/ml of ampicillin.
The entire open reading frame (ORF) of TDE0214 was PCR amplified with the primers P12/P13 using Platinum pfx DNA polymerase (Invitrogen). The resulting amplicon was cloned into the pET101/D-TOPO expression vector (Invitrogen) with a C-terminal His6 fusion tag. The obtained plasmid was transformed into E. coli BL21 Star (DE3). The overexpression of TDE0214 was induced with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG). The recombinant protein was purified using nickel-nitrilotriacetic acid (Ni-NTA) agarose (Qiagen, Valencia, CA) either under a native condition or a denature condition using 8.0 M urea as previously described (43). The purified recombinant protein (designated His6-TDE0214) was analyzed by SDS-PAGE to determine protein concentration and purity.
Protein-nucleotide binding affinity was measured using an equilibrium dialysis method as described before (28). The assay was carried out in DispoBiodialyzer cassettes (The Nest Group, Southborough, MA) that are separated into two chambers by a 5-kDa-cutoff membrane. Briefly, 60 μl of c-di-GMP (0 to 25 μM) (purchased from KeraFAST, Boston, MA) was injected into one chamber, and 60 μl (10 μM) of His6-TDE0214 or the same amount of denatured protein (negative control) was injected into the other chamber. The loaded cassettes were maintained for 24 h at room temperature to reach equilibrium with gentle rocking. Samples from both sides of the cassettes were taken, boiled for 4 min, centrifuged, and filtered through a 0.22-μm-pore-size microfilter (Millipore, Billerica, MA). Concentrations of c-di-GMP were quantified via reverse-phase high-performance liquid chromatography (HPLC) on an Agilent 1100 series LC-MSD Trap SL (Agilent, Santa Clara, CA) equipped with a Zobex XDB C18 column (Agilent Eclipse; 2.1 by 50 mm, 5 μm) under the same detection conditions as previously documented (44). The binding constants were calculated with GraphPad Prism 5 software (GraphPad Software, San Diego, CA).
The TDE0214 gene was inactivated by targeted mutagenesis mediated by allelic exchange as previously described (45). The vector for the mutagenesis was constructed by multiple-step PCR as illustrated in Fig. S1 in the supplemental material. In the first step, the TDE0214 upstream region (region 1), its downstream region (region 2), and a modified gentamicin resistance gene (aacCm, where “m” stands for “modified”) (45) were PCR amplified. In the second step, region 1 and aacCm were linked by PCR with primers P1/P4. In the third step, the region 1-aacCm and region 2 fragments were fused by PCR with primers P1/P6. The final fragment, region 1-aacCm-region 2, was cloned into pGEM-T Easy vector (Promega, Madison, WI), yielding TDE0214::aacCm, in which the entire TDE0214 gene was deleted and in-frame replaced with aacCm. The TDE0214::aacCm plasmid was purified from either the TOP10 or E. coli dam−/dcm− strain as previously described (46). To inactivate TDE0214, 10 μg of linearized TDE0214::aacCm was transformed into either the wild-type ATCC 35405 strain or TdΔ911, a genetically modified T. denticola strain, by electroporation. The TdΔ911 strain contains a mutation in TDE0911, a gene encoding an endonuclease, and it is more acceptable to unmethylated DNA (46). Mutants were selected on OBGM semisolid agar plates containing either 20 μg ml−1 gentamicin (for the wild type) or 20 μg ml−1 gentamicin and 60 μg ml−1 erythromycin (for TdΔ911).
Bacterial RNA isolation was carried out as previously described (43). A total of 100 ml of mid-logarithmic-phase T. denticola cultures (~1 × 108 cells ml−1) was harvested for RNA preparations. Total RNA was extracted using TRI reagent (Sigma-Aldrich, St. Louis, MO) by following the manufacturer's instructions. The resultant samples were treated with Turbo DNase I (Ambion, Austin, TX) at 37°C for 2 h to eliminate genomic DNA contamination. The obtained RNA samples were extracted using acid-phenol-chloroform (Ambion), precipitated in isopropanol, and washed with 70% ethanol. Finally, the RNA pellets were resuspended in RNase-free water. To generate cDNA, 1 μg of purified RNA was reversely transcribed using the iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA). The quantitative PCR was conducted using iQ SYBR green Supermix and an MyiQ thermal cycler (Bio-Rad). For quantitative analysis of gene expression, the target transcripts were normalized to the dnaK transcript, a housekeeping gene of T. denticola, as previously described (43). The results were expressed as threshold cycle (CT) values. In addition, the quantitative reverse transcription-PCR (qRT-PCR) products were directly detected by electrophoresis on a 1.5% agarose gel. The primers used for the qRT-PCR analysis are listed in Table S1 in the supplemental material.
To measure planktonic growth curves of T. denticola, 5 μl of late-logarithmic-phase T. denticola cultures (~5 × 108 cells ml−1) was inoculated into 5 ml of fresh OBGM and grown at 37°C in the anaerobic chamber. The cultures were enumerated every 24 h using the Petroff-Hausser counting chamber (Hausser Scientific, Horsham, PA) for up to 7 days. Counts were repeated in triplicate with at least three independent experiments. The results were expressed as means of cell numbers ± standard deviations (SD). Biofilm formation was measured as previously described (47, 48) with slight modifications. Briefly, 200 μl of mid-logarithmic-phase T. denticola cultures (1 × 108 cells ml−1) was added into 96-well flat-bottom polystyrene plates that were coated with or without heat-inactivated rabbit serum. The plates were incubated anaerobically at 37°C for 5 days, allowing for biofilms to develop. The biofilms were stained with 25 μl of 1% crystal violet for 15 min, washed with water three times, and then air-dried for 30 min. To quantify the amount of biofilms, 150 μl of 95% ethanol was added to each well and shaken for 15 min. The optical density at 570 nm (OD570) was measured with an xMark microplate spectrophotometer (Bio-Rad). Data are expressed as the average absorbance of six parallel samples.
These three assays were carried out as previously described with slight modifications (49–51). For the swim plate assay, 2 μl of mid-logarithmic-phase T. denticola cultures (1 × 108 cells ml−1) was spotted onto 0.35% agarose plates containing 1:1 Dulbecco's phosphate-buffered saline-diluted OBGM. Plates were incubated anaerobically at 35°C for 3 days. Diameters of swarm rings were measured and recorded in millimeters. A previously reported nonmotile Tap1 mutant, JS97 (52), was used as a negative control to determine the initial inoculum size. For the motion tracking analysis, 100 μl of mid-logarithmic-phase T. denticola cultures was first diluted (1:1) in fresh OBGM, and then 10 μl of diluted cultures was mixed with an equal volume of 2% methylcellulose with a viscosity of 4,000 cp (MC4000). T. denticola cells were videotaped and tracked using a computer-based bacterial tracking system as described before (51, 53). For each bacterial strain, at least 50 cells were recorded for up to 1 min. The average cell swimming velocities (μm/s) and reversal frequencies (times/min) of tracked cells were calculated. Capillary assays were carried out using glucose (0.1 mM) as an attractant as previously documented (50), and JS97 was used as a negative control in the assays. For the swim plate, motion tracking, and capillary assays, results are expressed as means ± SD. The significance of the difference between different strains was evaluated with one-way analysis of variance (ANOVA) (P < 0.01).
A previously documented mouse skin abscess model was used to assess the virulence of T. denticola (54). For the animal studies, 4-week-old BALB/c mice (Jackson Laboratory, Bar Harbor, ME) were subcutaneously injected with 100 μl of bacterial suspension (~1010 cells) on their posterior dorsolateral surfaces. For each T. denticola strain, at least three mice were included. Ten days after the injection, the mice were euthanized by CO2 asphyxiation. The abscess sizes under the skin were recorded, and blood samples were collected to measure seroconversion in infected mice by immunoblotting. The skin and muscle tissues around the abscesses were collected and subjected to either histopathology staining or qRT-PCR to detect bacterial RNA. The total RNA in mice tissues was extracted, and the flaA transcript (a gene encoding a flagellin protein) (40) and mouse β-actin transcript were used as surrogate genes to measure the relative bacterial burden in the specimens as previously described (55). Hematoxylin and eosin (H&E)-stained slides were prepared by standard pathological techniques. All animal experimentation was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocols for animal studies were approved by the Institutional Animal Care and Use Committee (permit number ORB 23068Y).
TDE0214 is annotated as a hypothetical protein in the genome of T. denticola (40), and it consists of 314 amino acids (aa) with a predicted molecular weight (MW) of 34.8. Frederick et al. recently predicted that TDE0214 is an effector protein of c-di-GMP (41). Along with this prediction, sequence alignment with the Vibrio cholerae PlzD (17), P. aeruginosa PilZ (23), and Borrelia burgdorferi PlzA (18, 28) proteins revealed that TDE0214 harbors a PilZ-like domain with two signature motifs, RXXXR and DXSXXG (Fig. 1). To determine if TDE0214 is a c-di-GMP binding protein, the binding affinity of purified His6-TDE0214 (Fig. 2a) to synthetic c-di-GMP was measured using a previously reported equilibrium dialysis method (28). As shown in Fig. 2b, His6-TDE0214 bound c-di-GMP, and its dissociation constant (Kd) to c-di-GMP reached 1.73 ± 0.24 μM, consistent with Kd values (ranging from 1 nM to 2 μM) reported for c-di-GMP binding proteins from other bacteria (2, 12, 26, 27). No binding activity was detected with a denatured His6-TDE0214 or with other cyclic nucleotides (i.e., cAMP and cGMP) (data not shown). Collectively, these results indicate that TDE0214 specifically binds c-di-GMP with a moderate binding affinity compared to those of its counterparts from other bacteria.
There are three ORFs located downstream of TDE0214 within the operon (40). Insertion of an antibiotic resistance cassette within the locus of TDE0214 may have a polar effect on its downstream gene expression. To limit this possibility, TDE0214::aacCm was constructed to replace in frame the entire TDE0214 gene with aacCm. We first attempted to inactivate TDE0214 in the wild-type ATCC 35405 strain (40). However, all of the attempts failed. Our recent study shows that a type II endonuclease (TDE0911) prevents the wild-type strain from accepting foreign DNA and that its isogenic mutant (TdΔ911) is more amenable to unmethylated DNA (46). As such, we attempted to inactivate TDE0214 in TdΔ911 using unmethylated TDE0214::aacCm. Seven days after the transformation, at least 20 gentamicin-resistant colonies appeared on the plates. Eight colonies were randomly selected and subjected to PCR to detect the presence of aacCm. All eight colonies were positive (data not shown), indicating that the antibiotic cassette was integrated into the chromosome of these colonies.
Two positive clones (designated TdΔ214-1 and TdΔ214-2) were selected for further characterizations. First, a previously described PCR analysis (45) was conducted to determine if the allelic exchange at TDE0214 in these two clones occurred as predicted. Three pairs of primers (P7/P8, P9/P10, and P11/P8) were used for the PCR analysis (Fig. 3a). The first pair of primers is for detecting the aacCm cassette, the second pair for detecting TDE0214, and the last pair for detecting the product of the TDE0214 flanking region and aacCm. The PCR results showed that TDE0214 in these two clones was deleted and replaced by aacCm, as expected (Fig. 3a and andb).b). Second, RT-PCR analysis showed that the transcript of TDE0214 was detected in the parental strains but not in TdΔ214-1 and TdΔ214-2 (Fig. 3c), indicating that the expression of TDE0214 was abolished in these two mutant clones. Finally, the expression level of TDE0213, a downstream gene of TDE0214, was measured by qRT-PCR. The results showed that the relative level of TDE0213 transcript to the dnaK (average ΔCT = 4.32 ± 0.54) in the TdΔ214 mutants is similar to that in the parental TdΔ911 strain (average ΔCT = 3.93 ± 0.16). The products from the qRT-PCR were further visualized by DNA electrophoresis, and a similar result was observed (Fig. 3c), indicating that replacing TDE0214 with aacCm in the mutant clones has no polar effect on its downstream gene expression.
Cyclic di-GMP and its effector proteins are often implicated in the control of bacterial motility (4, 12, 26, 27). To determine if a similar scenario occurs in T. denticola, the motility of the TdΔ214 mutant cells was analyzed using swim plate and bacterial motion tracking assays. As shown in Fig. 4a, on the soft agar plates, the TdΔ214-1 and TdΔ214-2 mutants formed substantially smaller (P < 0.01) swarm rings (average diameter of the rings = 5.7 ± 0.8 mm; n = 8 swarms) than the wild-type ATCC 35405 (17.0 ± 1.7 mm; n = 4 swarms) and their parental TdΔ911 (16.0 ± 0.7 mm; n = 4 swarms) strains, suggesting that inactivation of TDE0214 impaired T. denticola motility. This observation was further confirmed by the tracking analysis (Fig. 4b). For this analysis, the spirochete cells were tracked for at least 1 min in the presence of 1% methylcellulose at room temperature. Under this condition, although the TdΔ214 mutant cells ran, flexed, and reversed, as did the wild-type cells, they appeared to reverse more frequently than their parental strains (see Movies S1 and S2 in the supplemental material). This observation was further confirmed by the tracking analysis. The average reversal frequencies (times/min) of the wild-type and TdΔ911 strains were 8.5 ± 4.2 (n = 29 cells) and 7.7 ± 4.5 (n = 30 cells), respectively, and they were increased to 15.4 ± 8.4 (n = 30 cells) in TdΔ214-1 and 17.7 ± 7.8 (n = 30 cells) in TdΔ214-2. Consistent with the swim plate assay, the tracking analysis further confirmed that the motility was impaired in the TdΔ214 mutants. The average cell swimming velocities (μm/s) of the TdΔ214-1 (4.3 ± 0.6; n = 30 cells) and TdΔ214-2 (4.4 ± 1.1; n = 30 cells) cells were approximately 2-fold less (P < 0.01) than those of the wild-type (8.3 ± 1.5; n = 29 cells) and TdΔ911 (9.2 ± 1.8; n = 30 cells) strains. Collectively, these results indicate that the inactivation of TDE0214 affects the swimming behaviors of T. denticola, suggesting that c-di-GMP is implicated in the regulation of spirochete motility.
The above-described studies showed that two TdΔ214 mutant clones had altered swimming behaviors. We reasoned that these changes may affect the chemotaxis of T. denticola (e.g., the response to an attractant). To test this hypothesis, the chemotactic response to glucose, an attractant of T. denticola, was measured by capillary tube assays as previously described (50, 51). After a 2-h incubation, the numbers of wild-type and TdΔ911 cells in the tubes containing 0.1 M glucose were 8- to 10-fold greater than those in the control tubes, which contained only the buffer (Fig. 5), indicating that these two strains strongly responded to the attractant. Under the same condition, the numbers of TdΔ214 mutant cells in the glucose-containing tubes were increased only 2- to 3-fold, suggesting that the ability of responding to glucose was severely impaired in the TdΔ214 mutants. Of note, in the capillaries without glucose, the number of TdΔ214 mutant cells was almost the same as that of their parental strains (Fig. 5), implying that the mutations in TDE0214 does not affect the ability of the mutant cells randomly (independent of chemotaxis) swimming toward the capillaries. Rather, it specifically affects the mutants to sense and respond to the attractant, indicative of the regulatory role of c-di-GMP on the chemotaxis of T. denticola.
Besides motility, c-di-GMP is involved in the regulation of biofilm formation in many bacteria (24, 56–58). Previous studies indicate that T. denticola forms biofilm (47, 48). To determine if the inactivation of TDE0214 affects biofilm formation of T. denticola, the spirochete cells were grown in serum-coated static microtiter plates for 5 days, and the formed biofilms were first stained with crystal violet and then quantitated by absorbance measurement, as described in Materials and Methods. Compared to the wild-type and their parental TdΔ911 strains, the two TdΔ214 mutant clones formed biofilm poorly, and the amount of biofilms formed by the mutants was decreased by approximately 6-fold (Fig. 6a). Of note, parallel experiments were also carried out in the plates without serum coating, and a similar pattern was observed, albeit the overall biofilm formation was attenuated in both the wild-type and mutant strains (see Fig. S2 in the supplemental material), ruling out, at least in part, the possibility that decreased binding to the serum components impaired the mutants to form biofilms. In contrast to the biofilm growth, the two mutant clones had growth rates that were similar to those of the wild-type and their parental TdΔ911 strains when they were cultured in the OBGM (Fig. 6b). Collectively, these results indicate that inactivation of TDE0214 impairs the biofilm formation but not planktonic growth of T. denticola.
A previously described mouse skin abscess model (54) was used to evaluate the role of TDE0214 in the virulence of T. denticola. For this study, BALB/c mice received a single subcutaneous injection with 100 μl of bacterial suspension (~1010 cells/ml) on their posterior dorsolateral surfaces. Ten days after the injections, mice were sacrificed and the induced skin abscesses were measured. Even though demarcated subcutaneous abscesses were observed in all infected mice, the skin lesions induced by the TdΔ214 mutants were significantly smaller than those induced by the parental strains (Fig. 7a). The average sizes of skin abscesses induced by the wild-type and TdΔ911 strains were 59.9 mm2 (ranging from 49.5 to 77.8 mm2; n = 4 mice) and 52.4 mm2 (ranging from 44.0 to 62.8 mm2; n = 3 mice), respectively, and the average size was reduced to 14.8 mm2 (ranging from 7.1 to 23.6 mm2; n = 7 mice) in the TdΔ214 mutants.
We also observed that the skin lesions induced by the wild-type strains, but not by the TdΔ214 mutants, had extended to adjacent muscle tissues and caused prominent muscle necrosis. To confirm this observation, the inflamed muscle tissues were dissected and subjected to H&E staining to directly visualize the spirochetes and to qRT-PCR to detect spirochetal loads. For the qRT-PCR analysis, the flaA transcript was used as a surrogate gene, as the mutation in TDE0214 has no impact on the expression of flaA (see Fig. S3 in the supplemental material). As shown in Fig. 7b, H&E staining found abundant spiral-shaped organisms in the inflamed muscle tissue, indicating that the wild-type cells invaded the adjacent muscle tissues. In support of the histological examination, the qRT-PCR analysis showed that the flaA transcripts of T. denticola were detected in the muscle tissues infected by the wild-type strain but not in the tissues infected by the mutant (Fig. 7c). In addition, immunoblotting analysis using the antisera from infected mice showed that the wild-type strains induced strong humoral responses, which was evident by multiple spirochetal antigens being recognized by the antisera (Fig. 7d). In contrast, the humoral responses induced by the TdΔ214 mutants were poor (Fig. 7d). One possible explanation for the observed phenotype is that the TdΔ214 mutants are less invasive than their parental strains and are rapidly eliminated by local innate immunity before inducing adaptive immune responses. Collectively, these in vivo studies indicate that TDE0214 is a virulence factor that is implicated in the pathogenicity of T. denticola.
The turnover of intracellular c-di-GMP is controlled by DGCs and PDEs (2, 4). T. denticola has at least five DGCs with a GGD/EEF domain, two PDEs with an EAL domain, and four PDEs with an HD-GYP domain (40, 41) (see Fig. S4 in the supplemental material), suggesting existence of a complex c-di-GMP signaling network in the spirochete. In addition, bioinformatics analyses revealed that these proteins also harbor diverse input sensor domains (see Fig. S5 in the supplemental material). For instance, the N termini of TDE0128 and TDE2075 contain a conserved Cache domain (named for calcium channels and chemotaxis receptors). Cache domain proteins are widespread in both prokaryotic and eukaryotic organisms and are implicated in small molecule sensing (59, 60). The N termini of TDE2725 and TDE2726 contain a cNMP binding domain. This domain often binds cyclic nucleotide monophosphate (cAMP or cGMP) to activate protein functions (61–63). Both TDE0125 and TDE2659 harbor a GAF domain, which is named from the first three classes of proteins containing this domain: cGMP-specific phosphodiesterases, adenylyl cyclases, and FhlA (a σ54 activator) (64). GAF domains can bind different small molecules (i.e., amino acids, cNMP, tetrapyrrole, mononuclear nonheme iron, and nitric oxide) (65–67). TDE1467 harbors a Per-Arnt-Sim (PAS) domain that often senses secondary physical or chemical signals, such as gas molecules, redox potential, or photons (68, 69). The presence of these input sensor domains suggests that T. denticola is able to use a complex c-di-GMP signaling network to sense and integrate environmental and cellular stimuli and accordingly regulate various cellular functions and processes in response to the changes of the oral niche. In contrast to the presence of multiple DGCs and PDEs, T. denticola has only two recognizable c-di-GMP binding proteins, TDE0214 and TDE1318. Studies of these two effector proteins can help us to understand the roles of c-di-GMP signaling pathways in T. denticola. In this report, through studies of TDE0214, we experimentally uncovered that c-di-GMP is implicated at least in the regulation of motility (Fig. 4), chemotaxis (Fig. 5), biofilm formation (Fig. 6), and virulence (Fig. 7) of T. denticola.
PilZ domain proteins have been studied in several motile bacteria, and they are often implicated in regulation of cell motility (12, 13, 26–28). In these bacteria, mutations in the genes encoding those c-di-GMP effectors have different effects on cell motility. For instance, disruptions of B. burgdorferi plzA and V. cholerae plzB impair the cell motility (27, 28). In contrast, mutations of E. coli ycgR and Caulobacter crescentus dgrA or dgrB have no impact on the wild-type cell motility (12, 13, 70). Instead, mutations in these three genes can relieve the inhibition of motility that is caused by either deletions of PDE proteins or overexpression of DGC proteins, highlighting that these PilZ domain proteins affect cell motility only at conditions where the level of c-di-GMP is elevated (12, 13, 26). In this report, we found that inactivation of TDE0214 impaired the cell motility (Fig. 4; see also Movies S1 and S2 in the supplemental material), which is similar to the phenotypes of B. burgdorferi plzA and V. cholerae plzB mutants but different from that of E. coli ycgR and C. crescentus dgrA and dgrB mutants.
How do PilZ domain proteins affect bacterial cell motility? Recent studies revealed that the E. coli YcgR protein acts as a molecular brake of flagellar motors (71, 72). Upon binding to c-di-GMP, YcgR interacts with the flagellar motor, causing the motor to slow down. If TDE0214 adopts a mechanism similar to that of YcgR, we would expect that the swimming velocities of the TDE0214 mutant cells should be faster than those of the wild type or at least remain unchanged. However, this speculation is not supported by the phenotype of the TdΔ214 mutants (Fig. 4), suggesting that TDE0214 may affect the motility through different mechanism. Previous studies have shown that increased concentrations of c-di-GMP often repress cell motility (13, 15, 26). We reasoned that a similar mechanism may account for the abnormal swimming behavior of TdΔ214 mutants (e.g., the deletion of TDE0214 may increase the level of free c-di-GMP; the increased level of c-di-GMP activates TDE1318, the other c-di-GMP binding protein of T. denticola, which in turn binds to the flagellar motor to curb flagellar motor output or rotation, altering the swimming behaviors of the mutant cells). To test this hypothesis, we attempted to compare the intracellular c-di-GMP levels between the wild type and the TdΔ214 mutant strains using the HPLC detection method described in Materials and Methods. However, we were unable to obtain a measurement of the intracellular c-di-GMP level due to background noise under the tested condition. Due to this technical roadblock, at this point, we are unable to elucidate the underlying molecular mechanism. A similar issue also occurs in the analysis of PilZ-like mutants in other bacteria (27, 28). Interestingly, Kostick et al. recently reported that T. denticola produces detectable c-di-GMP (73). The reason that we were unable to detect c-di-GMP could be due to differences in the detection methods, bacterial strains, and cultivation conditions. We are currently trying to use more advanced methods to measure intracellular levels of c-di-GMP in different T. denticola strains under various cultivation conditions. In addition, we are also attempting to determine if TDE1318 is a c-di-GMP binding protein and its potential role in motility. Accomplishment of these studies will help us to unravel how TDE0214 affects the cell motility of T. denticola.
Besides the regulation of motility, one of other major roles of c-di-GMP is to control bacterial biofilm formation primarily by regulating the biosynthesis of adhesins and exopolysaccharide matrix substances (56–58). In this report, we found that the inactivation of TDE214 reduced biofilm formation (Fig. 6), indicating that c-di-GMP is involved in regulating the biofilm formation. T. denticola forms biofilm; however, the factors involved in biofilm formation remain largely unknown. Previous studies showed that mutations in the genes encoding the cytoplasmic filament protein CfpA, the flagellar hook protein FlgE, the leucine-rich repeat protein LrrA, or the major outer sheath protein Msp severely impaired the biofilm formation of T. denticola, albeit the underlying mechanisms remain unknown (47). We reasoned that TDE0214 may affect the biofilm formation via regulating one of these genes. However, qRT-PCR analysis showed that the expression of these four genes remained unchanged in the TdΔ214 mutants (data not shown). A body of studies has shown that motility often plays a pivotal role in the early stages (i.e., cell attachment and colonization) of biofilm formation and that nonmotile mutants are sometimes incapable of forming biofilms (74–76). As shown in Fig. 4, the TdΔ214 mutant cells swam slower and reversed more frequently than their parental strains (see Movies S1 and S2 in the supplemental material). Thus, it is possible that the observed defects in motility may account for the reduced biofilm formation in the mutants. This is supported by the fact that the nonmotile flgE mutant of T. denticola poorly forms biofilms (47).
The PilZ-like proteins are implicated in regulating virulence in several pathogenic bacteria (25, 27, 28, 77). For example, the plzD mutant of V. cholerae was 10-fold less infectious than its parental strains in the murine model (27), and the plzA mutant of B. burgdorferi reduced cell survivability in fed ticks and failed to complete the mouse-tick-mouse infection cycle (28). In this report, in vivo studies using a mouse skin abscess model showed that the skin lesions and seroconversion induced by the TdΔ214 mutants were significantly decreased compared to those of their parental wild-type strains (Fig. 7), indicating that the inactivation of TDE0214 reduces the virulence of T. denticola. Previous studies have shown that motility and chemotaxis are important virulent factors of T. denticola and that the ability to form biofilm is linked to the pathogenicity of the spirochete (49, 78). Thus, the reduced virulence can be due to the synergistic effects of defective motility, chemotaxis, and biofilm formation in the TdΔ214 mutants. T. denticola produces numerous virulence factors involved in cytotoxicity, adherence, invasion, and immunomodulation (38, 39). Alternatively, TDE0214 may also affect the pathogenicity of T. denticola via other virulence factors. A genome-wide transcriptomic analysis (e.g., microarray and RNA-Seq) of the TdΔ214 mutant will allow us to determine whether or not the expression levels of these virulence factors are affected.
In summary, this is the first report to experimentally investigate the roles of the c-di-GMP signaling network in T. denticola. In this report, we demonstrate that TDE0214, as a PilZ-like c-di-GMP binding protein, is implicated in the regulation of motility, chemotaxis, biofilm formation, and virulence of T. denticola. These observations will provide us a platform to further investigate the complex c-di-GMP signaling network and its roles in the biology and pathogenicity of T. denticola and probably in other oral spirochetes as well.
We thank M. Miller for providing a protocol for the equilibrium dialysis assay and R. Limberger for providing the nonmotile Tap1 mutant (JS97).
This research was supported by Public Health Service Grants DE019667 and DE023080 to C. Li.
Published ahead of print 21 June 2013
Supplemental material for this article may be found at http://dx.doi.org/10.1128/JB.00610-13.