YebC regulates variable surface antigen VlsE expression and is required for host immune evasion in Borrelia burgdorferi.

Borrelia burgdorferi, the Lyme disease pathogen causes persistent infection by evading the host immune response. Differential expression of the surface-exposed lipoprotein VlsE that undergoes antigenic variation is a key immune evasion strategy employed by B. burgdorferi. Most studies focused on the mechanism of VlsE antigen variation, but little is known about VlsE regulation and factor(s) that regulates differential vlsE expression. In this study, we investigated BB0025, a putative YebC family transcriptional regulator (and hence designated BB0025 as YebC of B. burgdorferi herein). We constructed yebC mutant and complemented strain in an infectious strain of B. burgdorferi. The yebC mutant could infect immunocompromised SCID mice but not immunocompetent mice, suggesting that YebC plays an important role in evading host adaptive immunity. RNA-seq analyses identified vlsE as one of the genes whose expression was most affected by YebC. Quantitative RT-PCR and Western blot analyses confirmed that vlsE expression was dependent on YebC. In vitro, YebC and VlsE were co-regulated in response to growth temperature. In mice, both yebC and vlsE were inversely expressed with ospC in response to the host adaptive immune response. Furthermore, EMSA proved that YebC directly binds to the vlsE promoter, suggesting a direct transcriptional control. These data demonstrate that YebC is a new regulator that modulates expression of vlsE and other genes important for spirochetal infection and immune evasion in the mammalian host.

Introduction

Lyme disease is the most commonly reported arthropod-borne infection in the United States and Europe, and is also found in Asia [1]. The causative agents of Lyme disease are members of the genus Borrelia (including B. burgdorferi, B. garinii, and B. afzelii), which are transmitted to mammals via the bite of Ixodes ticks. During the transmission and colonization in both tick vectors and mammalian hosts, B. burgdorferi dramatically regulates its gene expression [2-6]. In the past two decades, several key regulators/pathways have been identified that govern differential expression of B. burgdorferi during the tick-mammal transmission. These include two sets of two-component systems, with each modulating the adaptation to each of the two hosts [5]. Hk1/Rrp1, a c-di-GMP producing system, controls spirochete’s adaptation to the tick vector [7-13], whereas Hk2/Rrp2 is essential for B. burgdorferi to establish infection in the mammalian host [14-17]. Rrp2, along with transcriptional activator BosR and repressor BadR, activates the RpoN-RpoS (σ54-σS) sigma cascade, which in turn controls the production of OspC and several virulence factors [14-28]. Several additional regulators have been identified that differentially regulate gene expression during the tick-mammal transmission including DsrA [29], Hfq [30], Hbb [31,32], CsrA [33-35], BpuR [36], EbfC [37], BpaB [38], SpoVG [39], BBD18 [40], LptA [41], BadP [42] and DksA [43,44].

Differential gene expression of B. burgdorferi is vital to its transition from the early phase to the persistent phase of mammalian infection. In the first week of infection, expression of ospC, which is essential for the early stage of spirochetal infection, is high [45-47]. As a highly immunogenic surface lipoprotein, OspC levels become downregulated as the host adaptative immune response is activated [48, 49]. Meanwhile, another surface-exposed lipoprotein, VlsE, which is structurally similar to OspC but antigenically variable, is upregulated [47,49,50]. Antigen variation of VlsE is achieved via gene conversion that occurs between the expressing vlsE locus and the adjacent 15 silent cassettes, each of which contains six VlsE antigenic variable regions [50-52]. Spirochetes lacking vlsE are able to maintain persistent infection in immunocompromised mice, but are unable to sustain infection in immunocompetent mice [53-57]. Despite the importance of VlsE in immune evasion, very little is known about the mechanism of differential expression of vlsE [51,52].

B. burgdorferi has a reduced genome with relatively few known or predicted transcriptional regulators [58]. BB0025, originally assigned as a hypothetical protein, recently was annotated as a putative YebC/PmpR family DNA-binding transcriptional regulator (TACO1 family, pfam PF01709) in the UniProt database (). Herein, we designated BB0025 as YebC of B. burgdorferi. Although functions of this group of proteins are largely unknown, several studies suggest that they play important roles in gene regulation and pathogenesis. In Pseudomonas aeruginosa, PmpR is involved in regulation of the quinolone signal (PQS) system and of pyocyanin production [59]. Disruption of yebC in Escherichia coli resulted in reduced survival upon on exposure to extreme ionizing radiations [60]. In Lactobacillus, YebC was shown to regulate proteolytic activity by binding to the promoter region of genes involved in proteolysis [61]. More recently, YebC of Edwardsiella piscicida was shown to control virulence by directly binding to the promoter and activating its Type III secretion system [62]. In current study, we discovered that YebC of B. burgdorferi is a new regulator that controls expression of vlsE and other genes important for mammalian infection of B. burgdorferi.

Alignment of YebC of B. burgdorferi (BB0025) with other members of YebC/PmpR family transcriptional regulators.

(A), YebC of B. burgdorferi was aligned with three other YebC/RmpR family proteins (TACO1 family, pfam PF01709), YebC from E. coli (Ec), PmpR_Ps, PmpR from P. aeruginosa and YebC from Edwardsiella piscicida (Ep). Multiple alignment was generated with CLUSTALW (https://www.genome.jp/tools-bin/clustalw) and the figure was draw with BoxShade (https://embnet.vital-it.ch/software/BOX_form.html). (B), Homology model for B. burgdorferi YebC. The model was generated using the crystal structure of E. coli YebC (1KON) as template and SWISS-MODEL program (https://swissmodel.expasy.org/interactive/HvXRyP).

Results

Construction of a yebC mutant and the complemented strain

Sequence analyses revealed the BB0025 belongs to YebC/RmpR transcriptional regulator family (). Structural modeling of B. burgdorferi YebC based on the crystal structure of E.coli YebC demonstrated strong structural similarity to E.coli YebC (). To investigate the function of YebC, we constructed a yebC mutant by allelic exchange in the low-passage, infectious strain of B. burgdorferi strain B31 clone 5A4NP1 [63]. A suicide vector pCT007 was constructed with an aadA gene (which confers streptomycin-resistance) with flanking upstream and downstream regions of yebC (), and transformed into 5A4NP1. Streptomycin-resistant Borrelia transformants were analyzed by PCR for confirming yebC deletion (). One of the yebC mutant clones that had plasmid profiles identical to that of 5A4NP1 was chosen for complementation with pCT016, a pBSV2-derived shuttle vector with a gentamicin-resistance cassette carrying a wild-type copy of yebC with its native promoter (). A complemented clone that had an identical endogenous plasmid profiles to that of the yebC mutant was selected for further study (). The RT-PCR result confirmed that the yebC mutant no longer expressed yebC, and the complemented strain restored yebC expression (). In addition, RT-PCR confirmed that deletion of yebC did not affect expression of the adjacent genes bb0024 and bb0026 ().

Construction of the yebC mutant and the complemented strain.

(A) Strategy for constructing the yebC mutant. WT: genomic context of yebC in the parent strain, 5A4NP1 (referred to here as WT). pCT007: the suicide vector used for inactivation of yebC. ΔyebC: the yebC mutant. Arrows indicate the primers used for PCR analyses. (B) PCR analyses of the wild-type (W) and the yebC mutant (M) strains. The specific primer pairs used are indicated at the top. (C) Endogenous plasmid profiles of each strain by multiplex PCR analyses as previously described [82]. cp, circular plasmid; lp, linear plasmid. Letters on the left indicate the bands corresponding to each endogenous plasmid that was defined previously for the B. burgdorferi strain B31genome [58, 84]. ★ indicates the band corresponding to plasmid C (cp9) that is missing in all three strains.

RT-PCR analyses of expressions of yebC, bb0024 and bb0026.

Wild-type 5A4NP1 (WT), the yebC mutant (Mut) and the complemented strain (Com) were cultured in BSK-II medium, and then were collected at the mid-log phase (~3×107 spirochetes/ml). RNA was extracted and subjected to RT-PCR analyses. flaB serves as an internal control. “-RT”, RT-PCR reaction without of reverse transcriptase. “DNA”: PCR control using the genomic DNA as a template.

The yebC mutant was not able to infect immunocompetent mice

During in vitro cultivation, the yebC mutant did not show any significant difference in growth when compared to parental strain. To examine the yebC mutant’s phenotype in vivo, groups of C3H/HeN mice were needle inoculated with the 5A4NP1 parent strain (hereafter referred to as wild-type strain), the yebC mutant, or the complemented strain. Ear punch biopsies were collected at 2, 3, 4 weeks post-infection and cultured in BSK-II medium for the presence of spirochetes. At 4-weeks post-infection, all mice were sacrificed and several mouse tissues including ear, joint, heart, skin, and bladder were collected and cultured. As shown in , mice inoculated with the wild type or the complemented strain were culture-positive at each time point of post-infection with two different doses (1×105 or 1×106 spricochetes/mouse). No mice were infected with 1×105 of the yebC mutant (). However, at high dose of infection (1×106), after a prolonged culture of mouse tissues in BSK-II medium (> 2 weeks), two mice infected with the yebC mutant showed culture positive for some tissues, while remaining eight were culture negative (). These data indicate that the yebC mutant has a defect in establishing infection in immunocompetent mice.

We further examined whether the yebC mutant could infect immunocompromised mice. Groups of SCID mice were needle inoculated with wild-type strain, the yebC mutant, or complemented strain with a dose of 1×106 spirochetes per mouse. All mice infected with wild type or the complemented strain were culture positive at all time points of post infection (). No mice yielded positive cultures at 2 weeks post-infection with the yebC mutant, 3 out of 6 mice were culture positive at 3 weeks post infection, and all mice were culture positive at 4 weeks post infection. These observations suggest that the yebC mutant was able to establish infection in immunocompromised mice despite a delayed infectious course.

Transcriptome analyses of the yebC mutant

To investigate the molecular mechanisms underlying the requirement of YebC for mammalian infection, we sought to identify YebC-regulated genes by RNA sequencing analyses. Wild-type 5A4NP1 and the yebC mutant were cultivated in BSK-II at 37°C and harvested at the mid-logarithmic growth. Comparison of the transcriptomes of the wild type and yebC mutant revealed a total of 33 genes whose expressions were either up- or down-regulated by YebC (> 2.5-fold) under standard in vitro culture conditions. Among these, 21 genes were positively regulated by YebC (), whereas 11 genes were negatively regulated by YebC (). Most of differentially regulated genes are located on various endogenous plasmids, especially on the redundant cp32 circular plasmids. Genes having >2.5-fold changes of expression were highlighted in the volcano plot, and genes with > 3-fold changes were highlighted and labeled (). One of the genes most positively regulated by YebC was vlsE, which showed 6.43-fold reduction in the yebC mutant.

Volcano plots of differentially expressed genes.

The volcano plot depicts log2 fold change on the x-axis and False Discovery Rate adjusted p value (q-value) on the y-axis. Single genes are depicted as dots. The transcript levels of 33 genes had greater than 2.5-fold change in the yebC mutant relative to the parental strain (p < 0.05). 22 genes were upregulated (in red) in the parent relative to the mutant, and 11 genes were downregulated (in blue). Genes with greater than 3-fold change are labeled with their gene IDs.

Verification of genes positively or negatively regulated by YebC

qRT-PCR analyses were performed to validate the expression of YebC-regulated genes identified by RNA-seq. Two positively regulated genes that are important for infection, vlsE and rpoS (bb0771), were examined. Consistent with RNA-seq results, qRT-PCR analyses also showed that vlsE expression was under the control of YebC (). Even though rpoS showed 3.14-fold decrease of expression in the yebC mutant in RNA-seq, no significant difference was observed in qRT-PCR analysis. Three top negatively regulated genes, bbi16, bba49, and bba51, were assessed by qRT-PCR. The result confirmed that all the three genes were negatively regulated by YebC ().

qRT-PCR verification of positively or negatively regulated genes by YebC identified by RNA-seq.

Wild-type B. burgdorferi strain 5A4NP1 (WT), the yebC mutant (Mut) and the complemented strain (Com) were cultured in BSK-II medium at pH 7.5 and harvested at the mid-log phase. RNAs were extracted and subjected to qRT-PCR analyses for expression of vlsE, rpoS, bbi16, bba49, bba51. The expression levels for each gene in wild-type strain are set as 1.0. The bars represent the mean values of three independent experiments, and the error bars represent the standard deviation. ***, p < 0.001 using one-way Anova.

YebC controls VlsE production in vitro

To determine whether YebC regulates VlsE protein levels, immunoblot analysis was performed with cells lysates from wild type, the yebC mutant, the complemented strain, and a B. burgdorferi strain lacking the entire plasmid lp28-1 that harbors vlsE. The result showed that the VlsE protein was dramatically reduced in the yebC mutant and levels were restored in the complemented strain. (). Previous reports showed that environmental cues can influence vlsE expression, although disparate results were observed [64-66]. We thus further investigated VlsE and YebC levels under various growth conditions. The result showed that cell density did not dramatically affect VlsE expression at the protein level, and elevated pH (pH 8.0) moderately increased VlsE production (). Consistent with previous reports, ambient temperature (23°C) dramatically increased the level of VlsE, which is inversely correlated with the level of OspC () [64,65]. The level of YebC displayed a similar pattern of temperature-dependent regulation as that of VlsE, showing an increased production when grown at ambient temperature ().

Immunoblot analysis of VlsE and YebC protein levels.

Wild-type B. burgdorferi strain 5A4NP1 (WT), the yebC mutant (Mut), the complemented strain (Com), or a B31 clone lacking the entire plasmid lp28-1 (Δlp28-1) were cultured in BSK-II medium at pH 7.5, 37°C, and harvested at the mid-log phase (M) (A), or cultured under various pH conditions (pH 7.5, or 8.0) at 37°C, and harvested at either mid-log (M) or stationary phase (S) (B), or cultured at 23°C (pH 7.5) and harvested at stationary phase (C). Cell lysates were probed with antibodies against VlsE, YebC, OspC, or FlaB (loading control).

Correlative expression of yebC and vlsE in mice

vlsE is differentially expressed during mammalian infection: at the early stage of spirochetal infection, vlsE level is low whereas ospC is high. As the host adaptative immune response is activated, expression of vlsE increases when ospC becomes downregulated [45-50,53]. To gather further evidence that YebC modulates vlsE expression, vlsE, ospC, and yebC transcript levels were examined during the course of mammalian infection. Similar to what was reported previously, ospC expression was high at 1 week post-infection in skin at the site of inoculation, and then became undetectable in heart and joint tissues during persistent infection (1, 2, 3 months post infection) (). On the other hand, vlsE expression was low in skin at the site of inoculation at 1 week post-infection, and then dramatically increased (5 to 10-fold increase) at 1 to 3 months of post-infection (). The result showed that yebC displayed a similar pattern of differential expression as vlsE during infection (), supporting the hypothesis that YebC modulates vlsE expression during spirochete’ transition from early to persistent infection in mammals.

Analyses of yebC, vlsE and ospC expression in mouse tissues by qRT-PCR.

Groups of C3H/HeN mice (n = 3 for each data point) were inoculated with 106 of wild-type spirochetes (5A4NP1) via the intradermal dorsal route. Mice were euthanized at 1-week, and 1, 2, 3 months post infection and skin (site of infection), heart (top) and joint (bottom) tissues were collected and then were subjected to RNA extraction followed by qRT-PCR analyses. The ospC (A), vlsE (B), and yebC (C) transcripts were analyzed in mouse samples by qRT-PCR via absolute quantification. The values represent the average copy number of ospC, vlsE or yebC normalized relative to 100 copies of flaB. All data are collected from three independent experiments, and the bars represent the mean values, and the error bars represent the standard deviation. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

YebC directly binds to the vlsE promoter

Since YebC is predicted to be a transcriptional regulator, we postulate that YebC may directly bind to vlsE promoter to regulate its expression. Accordingly, recombinant YebC proteins were purified and electrophoretic mobility shift assays (EMSA) were performed using various concentrations of YebC proteins (ranging from 50 nM to 750 nM) and with 50 nM of 200-bp DNA fragment of the vlsE promoter region (). There is a 100-bp inverted repeat sequence overlapping the -35 region of the vlsE promoter in B. burgdorferi B31 [67] (). The inverted repeat DNA may form cruciform and higher form structures [68]. Interestingly, in the absence of YebC, two DNA bands were observed with the purified PCR DNA fragment from vlsE promoter under EMSA conditions (, first lane), but not with the control DNA fragment from the bosR promoter (, first lane). The lower band corresponded to the 200 bp linear form of the DNA fragment (Lin-DNA). We labeled the higher DNA band as High-DNA. Whether it is cruciform remains to be determined. A protein-DNA interaction was observed at 50 nM of protein concentration (1:1 of protein/DNA ratio) (). A larger protein-DNA complex was formed when protein concentrations increased (100 nM to 750 nM). No binding was evident when YebC protein was incubated with a DNA fragment from the bosR promoter (), nor when BosR protein incubated with the vlsE promoter (), indicating specific binding of YebC to vlsE promoter.

YebC binds to the vlsE promoter.

A. Promoter sequence of the vlsE promoter. P1 and P2 are the primers used for PCR amplification of the DNA fragment for electrophoretic mobility shift analyses (EMSA). The inverted repeat sequences are labeled IR. -35 box of the vlsE promoter overlaps with the right arm of the IR. EMSA were performed using various concentrations of recombinant YebC (B and C) or BosR (D) proteins incubated with 50 nM of DNA fragments from 200 bp upstream of the vlsE promoter region or 300 bp upstream of the bosR promoter region (serving as a negative control). All the EMSA reactions include 0.01mg/ml BSA to prevent non-specific binding. Binding reactions were carried out for 30 min at 25°C, and then separated on a 5% polyacrylamide gel and stained with ethidium bromide. The band corresponding to the linear form DNA fragment (Lin-DNA), the higher form DNA (High-DNA), and the DNA-YebC protein complex (DNA-YebC), are labelled on the right.

Discussion

One of the unique features of B. burgdorferi infection is the ability of the spirochete to evade immune responses and maintain a persistent infection in the mammalian host [1,52]. Antigen variation and differential expression of VlsE plays a critical role in this process [46,50-52]. Over the past two decades, while much information regarding Borrelia gene regulation has been revealed, limited is known about regulation of vlsE expression [2,6,49,51,65-67]. In this study, we provide several lines of evidences to support the notion that YebC, a member of YebC/PmpR transcription factor, modulates differential expression of vlsE. First, genome-wide RNA-seq analysis showed that vlsE is one of the most regulated genes and its expression was dramatically reduced upon inactivation of yebC (). Second, both vlsE and yebC expression were induced by ambient temperature when grown in vitro (). YebC directly binds to the vlsE promoter (). Third, the expression of vlsE and yebC correlated well during the course of infection in mice, consistent with the hypothesis that YebC regulates vlsE expression (). Forth, YebC directly binds to the vlsE promoter (). Lastly, the infection data showed that the yebC mutant could not infect immunocompetent mice efficiently but was still able to infect immunocompromised mice (Tables and ), indicating the essential role of yebC in evading the host adaptive immune system.

The YebC/PmpR family of proteins are widespread in all prokaryotes and eukaryotes [69]. Several recent structural and functional studies indicate that these proteins function as transcriptional regulators [69,70]. Bioinformatic analyses reveal that YebC family proteins can be divided into two subgroups [69]. Subgroup I yebC genes are adjacent to ruvABC, which encode for Holliday junction branch migrases and resolvase, suggesting that this subgroup of YebC proteins may be involved in regulating the process of Holliday junction resolution during DNA recombination. B. burgdorferi has a subgroup I YebC, and the yebC gene (bb0025) is adjacent to ruvAB (bb0022-bb0023). Interestingly, two groups independently reported that the mechanism underlying VlsE antigen variation is unique: unlike other bacteria, B. burgdorferi does not require RecA or other proteins generally involved in DNA recombination, repair, or replication, rather, it requires RuvAB for efficient vlsE recombination [71, 72]. Thus, along with the finding on YebC in present study, we postulate that the ruvAB-yebC gene cluster of B. burgdorferi plays a key role in modulating both processes of gene recombination and differential expression of vlsE.

Limited knowledge is known about the mechanism underlying differential expression of vlsE. Jutras et al. reported that SpoVG, a DNA-binding protein, binds to a sequence in the vlsE coding region of B. burgdorferi B31 [39]. As the binding sequence is located near the recombination site within the coding region of the vlsE gene, it may be involved in vlsE recombination rather than vlsE expression [39]. Drecktrah et. al, performed RNA-seq analysis and showed that RelBbu, a bifunctional synthetase/hydrolase (RelA/SpoT homolog) that is responsible to synthesize and hydrolyze the alarmones guanosine tetraphosphate and guanosine pentaphosphate [(p)ppGpp], upregulates vlsE expression. They also demonstrated that vlsE was upregulated upon starvation [66]. However, how RelBbu regulates vlsE expression remains to be elucidated. In this study, we showed that YebC directly binds to the vlsE promoter (). Hudson et al. first reported the presence of a long inverted repeat sequence overlapping with the -35 sequence of the promoter in B. burgdorferi B31 genome [67]. Recently, more IR sequences are reported in other B. burgdorferi strains [52]. The Function of this IR in vlsE expression remains unknown. These inverted repeats can form cruciform structures that have been recognized to play important biological roles [73]. Interestingly, we observed two bands of the vlsE promoter DNA under native conditions, and a single band on an agarose gel for the purified PCR product of the vlsE promoter fragment. Given that the lower band ran at 200-bp position and was the linear form of DNA, we postulate that the higher band (High-DNA) observed under the native condition could be cruciform DNA. Obviously, further direct structural evidence such as electron microscopy is needed to demonstrate that it is indeed the cruciform DNA. Nevertheless, one important observation is that both linear form and higher form of the DNA fragment were diminished when incubated with YebC (). We envision three scenarios based on this observation. 1, YebC is capable of binding to both linear form and cruciform of the vlsE promoter; 2, YebC can only bind to the linear form but stabilize it such that the equilibrium between linear and cruciform structures favors the linear form; 3, YebC can only bind to the cruciform structure and the linear form is eventually converted to cruciform. Given that YebC is a putative transcriptional activator that positively regulates vlsE expression, the second scenario makes sense: YebC binds and stabilizes the linear form of the vlsE promoter, drives conversion of cruciform to linear form, releases the -35 promoter region available for transcriptional activation. Further biochemical analysis is warranted to determine whether YebC has the activity to alter the thermodynamics of between linear and cruciform DNA structure.

Several factors, such as anti-OspC antibody, have been implied involving in regulation of vlsE expression [51]. The expression of vlsE increases as B. burgdorferi transitions from the early to persistent stages of mammalian infection, coinciding with the downregulation of ospC [47]. However, in SCID mice or B-cell-deficient mice, the transcript levels of both vlsE and ospC are high [47]. Furthermore, treatment of SCID mice with monoclonal antibodies against OspC resulted in dramatic decreases in ospC mRNA levels and increases in vlsE transcripts. These observations suggest that the induction of antibody responses against OspC may lead to the reciprocal regulation of ospC and vlsE observed in mice [47]. Co-culture of B. burgdorferi with endothelial cells was also shown to increase vlsE expression [67]. In addition, interferon γ expression was also reported to promote vlsE recombination, and possibly vlsE expression [74]. Whether yebC expression is in some manner linked to these host factors remain to be determined. In present study, RNA-seq analysis showed no influence of yebC deletion on ospC expression, suggesting that although YebC controls vlsE expression, it may not involve regulation of ospC under the in vitro culture conditions. Whether YebC regulates downregulation of ospC in vivo remains to be determined.

The role of VlsE of B. burgdorferi in evading host innate immune response was evidenced with the fact that a B. burgdorferi strain lacking the entire lp28-1 plasmid or an intact vls locus cannot sustain infection in wild-type mice at 2 week post-infection, but can maintain persistent infection in SCID mice [55-57,75,76]. The present study also showed that the yebC mutant had similar difference in infectivity in wild-type mice and SCID mice as that of the vlsE mutant (Tables and ), suggesting that an defect in vlsE expression is the key contributor to the yebC mutant’s infection phenotype. The yebC mutant could not establish infection in immunocompetent mice even at a dose of 1 X 105 spirochetes/mouse, suggesting that YebC is essential for mammalian infection. We did observe that very few tissue samples became positive in mice inoculated with 1 X 106 spirochetes/mouse, likely due to that fact that yebC deletion does not completely abolish vlsE expression and recombination during infection, as shown in vitro (). The avirulent phenotype prevents us to examine vlsE expression in the yebC mutant in immunocompetent mice. The yebC mutant was infectious in SCID mice. Of note, the yebC mutant had delayed infection in SCID mice (). It is possible at lower dose of inoculation that the yebC mutant may show avirulent phenotype even in SCID mice since YebC may other functions in addition to VlsE. Regardless, because differential expression of vlsE and ospC does not occur in SCID mice, these mice are not suited for studying vlsE regulation. Therefore, we relied on the correlation of yebC and vlsE expression in wild-type spirochetes during the course of infection, which supports the hypothesis that YebC regulates vlsE expression ().

It is reported that the vlsE-lacking spirochetes are capable of establishing infection in the early stage of infection in immunocompetent mice, and spirochetes can be detected in blood samples in first 1–2 weeks of infection [45-50,53]. One caveat of present study is that the infectivity of the yebC mutant was not examined at early time point of post-infection. We speculate that the yebC mutant may be deficient in establishing infection in mice even at this early time point, given that YebC regulates additional functions important for infection. In addition to vlsE, RNA-seq results showed that YebC regulates expression more than 33 genes (Tables and ). Many of these genes are located on the plasmids and have unknown functions. One gene whose function is known to be important for pathogenesis is rpoS, which showed over three-fold reduction in the yebC mutant. RpoS controls expression of ospC, dbpBA and several other genes important for mammalian infection [2,6,17,18]. However, this result could not be confirmed by qRT-PCR analysis (). RNA-seq also did not show that the yebC deletion affected ospC and dbpBA expression. Thus, it is unlikely that YebC involves in regulation of the RpoS regulon. Other top positively regulated genes by YebC, including bbq37, bbp29, bbl36 and bbr32, are members of paralogous families whose sequences are highly identical and could not be verified by qRT-PCR. Three top negatively regulated genes, bbi16, bba49, and bba51, were confirmed by qRT-PCR to be negatively regulated genes by YebC (). bbi16 is located on lp28-4, encoding VraA (virulent strain-associated repetitive antigen A), a surface antigen that shows partial protection with active immunization [77]. bba49 and bba51 are located on linear plasmid lp54, encoding conserved hypothetical proteins with unknown function [58]. Whether increased expressions of bbi16, bba49 and bba51 in the yebC mutant contributed to the avirulence phenotype remain to be determined.

Recently, Ramsey et al. used transposon insertion sequencing (Tn-seq) for a genome-wide screen and identified several factors important for resistance to NO, H2O2, and TBHP in vitro and YebC (BB0025) was identified as one of the genes that may be involved in H2O2 resistance [78]. Ramsey et al. also performed animal study with groups of pooled Tn mutants and found that bb0025 Tn mutant was attenuated in both mouse strains. Our results using yebC deletion mutant was consistent with previous infection data using pooled Tn mutants. YebC’s role in defending host ROS may also explain why the yebC mutant had a delayed infection in SCID mice observed in this study (), as SCID mice have both macrophages and neutrophils that produce ROS. In addition, Ramsey et al., also showed that the gp91phox-/- mice lacking phagocyte superoxide production could not rescue the infectivity of the bb0025 Tn mutant [78]. Given that YebC controls vlsE expression discovered herein, the phenotype of the yebC Tn mutant in the gp91phox-/- mice becomes evident.

In summary, this work identified YebC as the transcriptional factor that regulates vlsE gene expression, and it plays a vital role in mammalian infection of B. burgdorferi. This finding opens many exciting opportunities to study the mechanism of host immune evasion by B. burgdorferi. For example, how is YebC itself regulated, and what activates YebC during mammalian infection? How does IR influence YebC to regulate vlsE expression? Does YebC also modulate vlsE antigenic variation and contribute to the avirulent phenotype observed in the yebC mutant? In addition, given that YebC is also important for ROS response and that spirochetes encounter ROS during tick feeding in tick gut and salivary gland, it will be interesting to investigate whether YebC is important for the tick part of enzootic cycle of B. burgdorferi.

Materials and methods

Ethics statement

All animal experiments were approved by the IACUC committee of Indiana University School of Medicine under the protocol number # 11339. All experiments were in accordance with the institutional guidelines.

B. burgdorferi strains and culture conditions

Low-passage, virulent B. burgdorferi strain 5A4NP1 (a gift from Drs. H. Kawabata and S. Norris, University of Texas Health Science Center at Houston) was used in this study. It was derived from wild-type B. burgdorferi strain B31 by inserting a kanamycin resistance marker in a restriction modification gene bbe02 on plasmid lp25 [63]. Spirochetes were cultivated in Barbour-Stoenner-Kelly (BSK-II) medium supplemented with 6% normal rabbit serum (Pel-Freez Biologicals, Rogers, AR) [79] at 37°C with 5% CO2. At the time of growth, appropriate antibiotics were added to the cultures with the following final concentrations: 300 μg/ml for kanamycin, 50 μg/ml for streptomycin, and 50 μg/ml for gentamicin. The constructed suicide vector (pCT007) and shuttle vector (pCT016) were maintained in Escherichia coli strain DH5α. The antibiotic concentrations used for E. coli selection were as follows: kanamycin, 50 μg/ml; streptomycin, 50 μg/ml; and gentamicin, 10 μg/ml.

Immunoblot analysis

Spirochetes from mid-log cultures were harvested by centrifuging at 8,000 × g for 10 min and followed by three time washing with PBS (pH 7.4) at 4°C. Pellets were suspended in SDS buffer containing 50 mM Tris-HCl (pH 8.0), 0.3% sodium dodecyl sulfate (SDS) and 10 mM dithiothreitol (DTT). Cell lysates (108 cells per lane) were separated by 12% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose membranes (GE-Healthcare, Milwaukee, WI). Membranes were blotted with mouse polyclonal antibody against VlsE (1:1,000 dilution) [76] and monoclonal antibody against FlaB (1:1,000 dilution) [41], and then incubated with goat anti-mouse lgG-HRP secondary antibody (1:1,000; Santa Cruz Biotechnology). Detection of horseradish peroxidase activity was determined by the enhanced chemiluminescence method (Thermo Pierce ECL Western Blotting Substrate) with subsequent exposure to X-ray film.

Generation of the yebC mutant and the complemented strain

To inactivate the yebC in parent strain 5A4NP1, a suicide vector pCT007 was constructed for homologous recombination as following: the regions of DNA corresponding to 1.4 kb upstream and 1.4 kb downstream regions of yebC () were PCR amplified from 5A4NP1 genomic DNA with primer pairs PRCT017/PRCT018 and PRCT019/PRCT020 (Supplemental ), respectively. The resulting PCR fragments were then cloned into upstream and downstream of an aadA streptomycin-resistant marker within the suicide vector pMP025 to generate the suicide vector pCT007. pCT007 was confirmed by both restriction enzyme digestion and sequencing, and then was transformed into 5A4NP1 as described previously [80,81]. Streptomycin-resistant Borrelia transformants were analyzed by PCR to confirm the correct yebC deletion (). Plasmid profiles of the confirmed yebC mutant clones were determined by multiplex PCR analyses with twenty pairs of primers specific for each of the endogenous plasmids as reported by Bunikis et al.[82]. One of the yebC mutant clones (BbCT006) that had plasmid profiles identical to those of 5A4NP1 was selected for complementation (). For trans complementation, a shuttle plasmid carrying a native promoter-driven yebC was generated as follows. The yebC regions flanked by BamHI and PstI restriction sites were amplified using primers PRCT092 and PRCT096 and then cloned into the shuttle vector pBSVG [83], resulting in in complementation plasmid named as pCT016. One complementation clone (BbCT009) that had an identical endogenous plasmid profiles to that of 5A4NP1 was selected for further study ().

Mouse infection studies

Four-week-old C3H/HeN mice and C3H/SCID mice (Harlan, Indianapolis, IN) were subcutaneously inoculated with two doses of spirochetes (1×105 and 1×106) respectively. Ear punch biopsy samples were taken at 2 and 3 weeks post-injection. At 4 weeks post-injection, mice were euthanized, and multiple tissues (i.e. ear, joint, heart, skin and bladder tissues from each mouse) were harvested. All tissues were cultivated in 2 ml of the BSK-II medium (Sigma-Aldrich, St. Louis, MO) containing an antibiotic mixture of phosphomycin (2 mg/ml), rifampin (5 mg/ml), and amphotericin B (250 mg/ml) (Sigma-Aldrich) to inhibit bacterial and fungal contamination. All cultures were maintained at 37°C and examined for the presence of spirochetes by dark-field microscopy beginning from 5 days after inoculation. A single growth-positive culture was used as the criterion to determine positive mouse infection.

qRT-PCR analyses

For verification of differentially expressed genes by YebC, wild-type B. burgdorferi strain 5A4NP1 (WT), the yebC mutant (Mut) and the complemented strain (Com) were cultured in BSK-II medium at pH 7.5 and harvested at the mid-log phase. RNAs were extracted and subjected to qRT-PCR analyses for expression of vlsE, rpoS, bbi16, bba49, bba51. Primers used were listed in Supplemental . For yebC, vlsE and ospC expression in infected mice, four-week-old C3H/HeN mice were injected with wild-type strain 5A4NP1 at a dose of 1×106 spirochetes per mouse. Mice were euthanized at different time points as indicated and mouse tissues were harvested and homogenized using the FastPrep-24 (MP Biomedicals). Total RNA was isolated using the TRIzol reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. To reduce trace amounts of DNA contamination, samples were further digested with RNase-free DNaseI (Qiagen), purified using the RNeasy mini kit (Qiagen) and analyzed with NanoDrop Spectrophotometer (Thermo Fisher Scientific). DNA-free RNA was confirmed by PCR amplification for the B. burgdorferi flaB gene. cDNA was synthesized using the PrimeScript 1st strand cDNA Synthesis Kit (TaKaRa). Given the low levels of bacterial RNA in mouse tissues, the specific primers for each gene target were used for cDNA synthesis instead of random primers. To quantify the transcript levels of genes of interest, an absolute quantitation method was used to create a standard curve for the qPCR assay according to the manufacturer’s protocol (Strategene, La Jolla, CA). Briefly, the PCR product of the flaB gene served as a standard template. A series of tenfold dilutions (102-107copies/ml) of the standard template was prepared, and qPCR was performed to generate a standard curve by plotting the initial template quantity against the Ct values for the standards. The quantity of the targeted genes in the cDNA samples was calculated using their Ct values and the standard curve. The samples were assayed in triplicate using a ABI 7000 Sequence Detection System and PowerUp SYBR Green Master Mix (Applied Biosystems). The levels of the target gene transcript were reported as per 100 copies of flaB.

RNA Sequencing (RNA-seq)

Wild-type 5A4NP1 and the yebC mutant were cultivated in BSK-II at 37°C and harvested at the mid-logarithmic growth. RNA samples were extracted using the RNeasy mini kit (Qiagen, Valencia, CA) according to the manufacturer's protocol. Three independent culture samples were used for each strain. Removal of contaminated genomic DNA in the RNA samples was performed using RNase-free DNase I (Promega), and then confirmed by PCR amplification for the flaB gene. The concentration and quality of total RNA samples were first assessed using Agilent 2100 Bioanalyzer. A RIN (RNA Integrity Number) of 5 or higher was required to pass the quality control. rRNA removal was performed for each RNA sample (2 μg/sample) using Ribo-Zero rRNA Removal Kit (Bacterial, Illumina), and the RNA samples were then subjected to dual-indexed strand-specific cDNA library synthesis using TruSeq Stranded Total RNA Library Prep Kit (Illumina). Synthesized libraries were assessed for the quantity and size distribution using Qubit and Agilent 2100 Bioanalyzer. Two hundred picomolar pooled libraries were utilized per flow cell for clustering amplification on cBot using HiSeq 3000/4000 PE Cluster Kit and sequenced with 2×75bp paired-end configuration on HiSeq4000 (Illumina) using HiSeq 3000/4000 PE SBS Kit. A Phred quality score (Q score) was used to measure the quality of sequencing. More than 90% of the sequencing reads reached Q30 (99.9% base call accuracy). The sequencing data were first assessed using FastQC (Babraham Bioinformatics, Cambridge, UK) for quality control. Then all sequenced libraries were mapped to the Borrelia burgdorferi B31 genome (NCBI, GCA_000008685.2) using STAR RNA-seq aligner with the following parameter: “—outSAMmapqUnique 60”. The reads distribution across the genome was assessed using bamUtils software (from ngsutils). Uniquely mapped sequencing reads were assigned to Borrelia burgdorferi B31 refSeq genes using featureCounts (from subread) with the following parameters: “-s 2 -p–Q 10”. Quality control of sequencing and mapping results was summarized using MultiQC. Genes with read count per million (CPM) > 0.4 in more than 4 of the samples were kept. The data was normalized using TMM (trimmed mean of M values) method. Differential expression analysis was performed using edgeR. False discovery rate (FDR) was computed from p values using the Benjamini-Hochberg procedure. Genes with fold changes greater than 2.5 are listed in Tables and . All genes are listed in supplemental file The RNA-seq data set was plotted on a volcano plot with the negative log of the q value on the y axis and the log2 of the fold change between wild type and the yebC mutant on the x axis using GraphPad Prism software ().

Electrophoretic mobility shift analyses (EMSA)

200-bp upstream to vlsE ORF and 350-bp region upstream to bosR were PCR amplified using specific sets of primers as listed in Supplementary . All the EMSA reactions were carried out in a 20 μL reaction mixture containing PCR amplified promoter DNA (50 nM), and various concentrations of recombinant YebC or BosR proteins (50 nM to 750 nM). Final 1 X EMSA buffer used for the reaction contained 10 mM Tris (pH 7.5), 1 mM EDTA, 100 mM KCl, 5% glycerol, 0.1 mM dithiothreitol and 0.01mg/ml BSA. Binding reactions were carried out for 30 min at 25°C. The reaction mix was then loaded on a 5% polyacrylamide gel and electrophoresis was carried out at 35 V at 4°C and later stained using ethidium bromide. Image was captured using a DigiDoc- Imaging System by UVP.

Protein modeling

For molecular modeling of B. burgdorferi YebC, online tools Iterative Threading Assessment Server9 and SWISS MODEL were used. The pair-wise sequence alignment threshold was set to 70% by default. The template for generating model, E. coli YebC (PDB ID: 1KON) was taken from RCSB PDB (https://www.rcsb.org/). Further validation of the model was carried out employing RAMPAGE (http://mordred.bioc.cam.ac.uk/~rapper/rampage.php) and ERRAT (http://services.mbi.ucla.edu/ERRAT/). The model was further tested for all structural parameters which includes: MolProbity Score, 2.06; Clash score, 10.47; Ramachandran favored, 94.14%; Ramachandran outlayers, 1.46%; Rotamer outlayers, 1.46%; ERAT score, 7.

Strains, plasmids, and primers used in this study.

(DOCX)

Click here for additional data file.

Complete RNA-seq data.

(XLSX)

Click here for additional data file.

Table 1

Infection of the yebC mutant in immunocompetent mice (C3H/HeN).

Strains and infection duration	No. cultures positive/No. tested					No. infected/Total No. of mice
	Ear	Joint	Heart	Skin	Bladder
5A4NP1 (1 × 105, 1 × 106)
2 weeks	5/5					5/5
3 weeks	5/5					5/5
4 weeks	5/5	5/5	5/5	5/5	5/5	5/5
△yebC (1 × 105)
2 weeks	0/5					0/5
3 weeks	0/5					0/5
4 weeks	0/5	0/5	0/5	0/5	0/5	0/5
△yebC (1 × 106)
2 weeks	0/10					0/10
3 weeks	0/10					0/10
4 weeks	0/10	0/10	1/10	2/10	2/10	2/10
yebCcom (1 × 105, 1 × 106)
2 weeks	3/5					3/5
3 weeks	5/5					5/5
4 weeks	5/5	5/5	5/5	5/5	5/5	5/5

Table 2

Infection of the yebC mutant in immunodeficient mice (SCID).

Strains and infection duration*	No. cultures positive/No. tested					No. infected/Total No. of mice
	Ear	Joint	Heart	Skin	Bladder
5A4NP1 (parent strain)
2 weeks	6/6					6/6
3 weeks	6/6					6/6
4 weeks	6/6	6/6	6/6	6/6	6/6	6/6
△yebC
2 weeks	0/6					0/6
3 weeks	3/6					3/6
4 weeks	6/6	6/6	6/6	6/6	6/6	6/6
yebCcom
2 weeks	3/3					3/3
3 weeks	3/3					3/3
4 weeks	3/3	3/3	3/3	3/3	3/3	3/3

* Dose of infection: 1 × 106 spirochetes per mouse

Table 3

RNA-seq analysis identification of genes that are positively regulated by YebC.

Locus	Common Name	Location	WT/ΔyebC*
BB_Q37	hypothetical protein	lp56	7.36
BB_F0041	outer surface protein VlsE1	lp28-1	6.43
BB_P29	hypothetical protein	cp32-1	4.64
BB_L36	protein BppA	cp32-8	3.92
BB_R32	hypothetical protein	cp32-4	3.70
BB_0844	lipoprotein	chromosome	3.39
BB_0771	RNA polymerase sigma factor RpoS	chromosome	3.14
BB_L31	hypothetical protein	cp32-8	3.08
BB_0799	hypothetical protein	chromosome	3.01
BB_L32	PF-32 protein	cp32-8	2.99
BB_K07	lipoprotein	lp36	2.96
BB_N02	hypothetical protein	cp32-9	2.85
BB_L29	hypothetical protein	cp32-8	2.81
BB_0438	DNA polymerase III subunit beta	chromosome	2.77
BB_0800	transcription termination/antitermination protein NusA	chromosome	2.74
BB_0801	translation initiation factor IF-2	chromosome	2.72
BB_F01	hypothetical protein	lp28-1	2.71
BB_F20	BBF20	lp28-1	2.67
BB_L27	protein BdrP	cp32-8	2.61
BB_S34	hypothetical protein	cp32-3	2.59
BB_R33	plasmid partition protein, putative	cp32-4	2.57
BB_N03	hypothetical protein	cp32-9	2.52

*Fold changes of gene expression of the wild-type strain with respect to the yebC mutant (the genes with fold change > 2.50). q (adjusted p value) < 0.05.

Table 4

RNA-seq analysis identification of genes that are negatively regulated by YebC.

Locus	Common Name	Location	WT/ΔyebC*
BB_I16	repetitive antigen A, VraA	lp28-4	-6.17
BB_R13	hypothetical protein BB_R13	cp32-4	-3.85
BB_A51	hypothetical protein BB_A51	lp54	-3.47
BB_A49	hypothetical protein BB_A49	lp54	-2.91
BB_N14	hypothetical protein BB_N14	cp32-9	-2.90
BB_0603	integral outer membrane protein p66	chromosome	-2.70
BB_S17	hypothetical protein BB_S17	cp32-3	-2.70
BB_0240	glycerol uptake facilitator	chromosome	-2.68
BB_G02	hypothetical protein	lp28-2	-2.67
BB_t31	NA		-2.65
BB_0001	hypothetical protein BB_0001	chromosome	-2.57

*Fold changes of gene expression of the wild-type strain with respect to the yebC mutant (the genes with fold change <-2.50). q (adjusted p value) < 0.05.