Ethics statement

Murine infections and immunization protocols were performed in accordance with the Guide for the Care and Use of Laboratory Animals, the Public Health Science Policy on Humane Care and Use of Laboratory Animals, and the Animal Welfare Act, and the protocol used was approved by the University of Arkansas for Medical Sciences (UAMS) Institutional Animal Care and Use Committee (IACUC). UAMS is accredited by the International Association for Assessment and Accreditation of Laboratory Animals Care (AAALAC).

Bacterial strains and culture conditions

Bacterial strains used in this study are listed in Table 1. Escherichia coli strain Top10F’ (Life Technologies, Carlsbad, CA) was used for cloning and plasmid propagation and strain LOBSTR-BL21 (DE3)-RIL (Kerafast, Boston, MA) was used for recombinant protein expression. E. coli was grown in lysogeny broth (LB) medium supplemented with 100 μg/ml ampicillin, 5 μg/ml gentamicin, 100 μg/ml spectinomycin, 50 μg/ml kanamycin, or 30 μg/ml chloramphenicol where appropriate. The infectious Ornithodoros moubata tick isolate, B. duttonii strain 1120K3 (designated BdWT), was used in this study [53]. B. duttonii was cultured in liquid modified Barbour-Stoenner-Kelly (mBSK) medium supplemented with 6 or 12% rabbit serum at a pH of 7.6 at 35°C and 3% CO₂ [41,54]. B. duttonii was grown in the presence of 40 μg/ml gentamicin, 100 μg/ml streptomycin, or 150 μg/ml kanamycin when necessary.

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Table 1. Plasmids and strains used in this study.

https://doi.org/10.1371/journal.pntd.0012348.t001

Generation of B. duttonii shuttle vectors

Primers used in this study are listed in S1 Table. Plasmid and genomic DNA (gDNA) preparations were performed using the Wizard Plus SV Miniprep DNA Purification System (Promega Corp, Fitchburg, WI), and PCR amplifications for cloning were conducted using high-fidelity PrimeSTAR Max DNA Polymerase (TaKaRa Bio, Mountain View, CA). All plasmids and cloned fragments were Sanger sequenced to confirm no mutations were introduced during cloning. Primers and gene nomenclature are based on the B. duttonii strain Ly sequence from Lescot et al. available on NCBI (GenBank assembly accession: GCA_000019685.1) [58]. Multiple-sequence alignments of B. hermsii and B. duttonii plasmid sequences were generated using MUSCLE in MacVector 18.6.4 (MacVector Inc., Apex, North Carolina).

To make the PflaB-aphI resistance marker for the B. duttonii shuttle vector, the putative promoter region for the flaB gene (bdu_150) was amplified from BdWT gDNA (primers: 5’ BdPflaB and 3’ BdPflaB-SOE), and the aphI open reading frame (ORF) was amplified from pBSV2 (primers: 5’ aphI-SOE/Bd and 3’ UAMS-88 Kan AscI) [59]. The promoter and ORF amplicons were then fused by overlap extension PCR, generating the final PflaB-aphI resistance marker flanked by 5’ BglII and 3’ AscI restriction sites. The PflaB-aphI marker was then TA-cloned into pGEM-T Easy (Promega), and the resulting plasmid intermediate was designated pUAMS359. The PflaB-aphI resistance marker from pUAMS359 was subsequently ligated into the B. burgdorferi shuttle vector, pJD44, replacing the aph[3′]-IIIa gene, and generating the plasmid pUAMS363 [55,59,60]. The E. coli ColE1 origin of replication (ori) was amplified from pJD44, (primers: 5’ pJD44 ColE1 ori and 3’ pJD44 ColE1 ori) flanked by 5’ AscI and 3’ BglII restriction sites and TA-cloned into pGEM-T Easy, designated pUAMS353. The putative ori of the B. duttonii pl41 plasmid was amplified from BdWT gDNA (primers: 5’ Bd pl41 ori BamHI and 3’ Bd pl41 ori AscI) flanked by 5’ BamHI and 3’ AscI restriction sites and TA-cloned into pGEM-T Easy, designated pUAMS364. To generate the final B. duttonii shuttle vector, designated pBdSV, the AscI-flanked multiple cloning site (MCS) and PflaB-aphI cassette from pUAMS363, the AscI/BglII-flanked ColE1 ori from pUAMS353, and the BamHI/AscI-flanked putative B. duttonii pl41 ori from pUAMS364 were ligated together. Another version of the shuttle vector was additionally made in which the MCS and aphI resistance cassette from pUAMS363 were ligated into the shuttle vector in the opposite orientation of that in pBdSV, designated pBdSV^CCW.

To make a B. duttonii-adapted gfp cassette, the flaB promoter was amplified from BdWT gDNA (primers: 5’ BdflaB/GFP and 3’ BdflaB/GFP_SOE) and the gfp ORF was amplified from pGreenTIR (primers: 5’ GFPtir ORF and 3’ GFPtir ORF) [56, 57]. The flaB promoter and gfp ORF were then fused by overlap extension PCR, and the resulting product was TA-cloned into pGEM-T Easy, designated pUAMS369. The PflaB-gfp cassette was then excised from pUAMS369 with BamHI and HindIII and ligated into the MCS in pBdSV, generating the shuttle vector pBdSV::gfp.

Generation of the pl165-specific integration construct

To create the pl165 integration construct, 5’ (primers: 5’ F1 pl165 int_v2 and 3’ F1 pl165 int_v2.2) and 3’ (primers: 5’ F2 pl165 int_v2.2 and 3’ F2 pl165 int_v2) regions flanking the integration site (907- and 1096-bp in size, respectively) were first amplified and TA-cloned into pGEM-T Easy. The flanking regions were then ligated together with an AscI restriction site between them in pGEM-T Easy, generating the plasmid pUAMS376. To create the gfp integration construct, pBdgfp, a segment of pBdSV::gfp containing the PflaB-gfp and PflaB-aphI cassettes was excised with AscI and ligated into the AscI site in pUAMS376.

Generation of B. duttonii p66 mutational and complementation constructs

To generate the PflgB-aacC1 gentamicin-resistance marker, the putative promoter region for the flgB gene (bdu_297) was amplified from BdWT gDNA (primers: 5’ BdPflgB-AscI and 3’ BdPflgB-SOE), and the aacC1 ORF was amplified from pUAMS4 (primers: 5’ Gent-SOE/Bd and 3’ Gent-AscI/Bd) [46,47]. The promoter and marker amplicons were fused by overlap extension PCR, generating the PflgB-aacC1 resistance marker flanked by AscI restriction sites. The PflgB-aacC1 marker was subsequently ligated into pGEM-T Easy and designated pUAMS356.

The allelic exchange mutagenesis construct to inactivate the p66 homolog of B. duttonii (e.g., bdu_604) was made by first amplifying upstream (primers: 5’ F1 Bd P66 and 3’ F1 Bd P66_AscI; size: 1098 bp) and downstream (primers: 5’ F2 Bd P66_AscI and 3’ F2 P66_BssHII; size: 1032 bp) regions flanking the mutation site and TA-cloning the respective amplicons into pGEM-T Easy. Flanking regions were then ligated together with the PflgB-aacC1 resistance marker from pUAMS356 between them using the AscI restriction site to yield the final p66 mutational construct, designated pBdΔp66.

To create the BdΔp66 shuttle vector complementation construct, the p66 ORF and 540-bp upstream of the coding region were amplified from BdWT gDNA (primers: 5’ Bd P66 comp BamHI and 3’ Bd P66 comp BamHI), and the resulting amplicon was ligated into the pBdSV MCS at its unique BamHI site. The resulting clone was designated pBdSV::p66. To make the p66 cis integration complementation construct, an aadA resistance marker was first adapted for use in BdWT. To generate the PflaB-aadA streptomycin-resistance marker, a 284-bp region upstream of the flagellin (flaB) gene (bdu_150) was amplified from BdWT gDNA (primers: 5’ BdPflaB-AscI_long and 3’ BdPflaB-aadA SOE), and the aadA ORF was amplified from a derivative of the B. burgdorferi flgBp-aadA-trpLt streptomycin/spectinomycin resistant cassette described by Jackson-Litteken et al. and Drecktrah et al. (primers: 5’ BdPflaB-aadA SOE and 3’ aadA ORF term-AscI) [45,46,61]. The resulting amplicons were fused by overlap extension PCR to generate a PflaB-aadA resistance marker flanked by AscI restriction sites; this PflaB-aadA marker was then ligated into pGEM-T Easy and designated pUAMS521. The genomic regions corresponding to a portion of bdu_602 and all of bdu_603 (primers: 5’ F1 Bd P66 and 3’ F1 Bd P66 cis comp; size: 990 bp) or all of bdu_604/p66 and a portion of bdu_605 (primers: 5’ F2 Bd P66 cis comp and 3’ F2 Bd P66_BssHII; size: 2894 bp) were then amplified and cloned into pGEM-T Easy. The two fragments were ligated together in pGEM-T Easy to introduce an AscI site in the bdu_603 and bdu_604 intergenic region, and the PflaB-aadA marker from pUAMS521 was ligated into this unique AscI site. The resulting p66 cis integration complementation construct was designated pBdp66^Cis.

Transformation of B. duttonii and confirmation of transformants

Competent cell preparation and electroporation of B. duttonii were performed via previously described methods for B. hermsii and B. turicatae [41,47]. Briefly, the constructs of interest were electroporated into the appropriate strain. Following a 24-hr recovery period in mBSK medium without selection, transformants were selected using corresponding antibiotic treatment. Once viable spirochetes were observed in the post-transformation recovery cultures, individual clones were then isolated by serial-dilution plating. Transformants were confirmed by PCR, and products were separated by electrophoresis in a 0.8% agarose gel and visualized by ethidium bromide staining. GeneRuler DNA Ladder Mix (ThermoFisher Scientific, Waltham, MA) served as the molecular weight standard.

Clones transformed with shuttle vector (e.g., BdSV, BdSV^CCW, and BdSV::gfp) were initially confirmed by plasmid recovery in E. coli, restriction digest, and Sanger sequencing. Additionally, PCR was performed with gDNA from these clones to amplify internal regions of aphI (primers: 5’ aphI diag and 3’ aphI diag), gfp (primers: 5’ GFPtir diag and 3’ GFPtir diag), and flaB (primers: 5’ BdFlaB and 3’ BdFlaB). PCR was also performed to amplify an internal region of pl41 to confirm that the endogenous plasmid was not lost (primers: 5’ pl41 diag and 3’ pl41 diag) in shuttle vector transformants.

Successful integration of gfp into pl165 (e.g., Bdgfp) was confirmed by PCR to amplify internal regions of gfp, aphI, and flaB (see above). PCR was also performed to link a region upstream of the insertion site to the gfp allele (primers: 5’ pl165 IG diag and 3’ GFPtir diag) and a region downstream of the insertion site to the aphI allele (primers: 5’ aphI diag and 3’ pl165 IG diag).

Confirmation of the BdΔp66 mutant was performed by PCR to amplify an internal segment of p66 (primers: 5’ Bd P66 diag and 3’ Bd P66 diag), an internal region of aacC1 (primers: 5′ aacC1 diag and 3′ aacC1 diag), and an internal region of flaB (see above). To confirm the pBdSV::p66-transformed BdΔp66 (e.g., BdΔp66^{SV comp}), clones were initially confirmed by plasmid recovery in E. coli, restriction digest, and Sanger sequencing. Additionally, PCR with gDNA from shuttle vector-complemented clones was performed to amplify internal regions of p66, aacC1, and flaB (see above). To confirm BdΔp66 clones transformed with pBdp66^Cis (e.g., BdΔp66^{Cis comp}), PCR with gDNA from transformants was performed to amplify internal regions of p66, aacC1, and flaB (see above), as well as aadA (primers: 5’ aadA diag and 5’ aadA diag).

Imaging of B. duttonii

Imaging of gfp-expressing bacteria was performed as previously described [45]. Briefly, strains of interest were grown to late-exponential phase, centrifuged at ~9,400 x g for 5 min, and resuspended in PBS-MgCl₂. This step was repeated one more time, after which bacteria were spotted on a 1% agarose pad and covered with a coverslip [62]. Brightfield and fluorescent images were then captured using an Eclipse Ti2 microscope (Nikon Corporation, Tokyo, Japan) under 60X magnification with oil immersion.

Production of recombinant B. duttonii P66 protein

The coding region for the B. duttonii p66 homolog (BDU_604) was amplified using primers that introduced BamHI and SpeI restriction sites on the 5’ and 3’ ends, respectively (primers: 5’ Bd P66 ORF BamHI and 3’ Bd P66 ORF SpeI). The primers amplified a region of B. duttonii P66 corresponding to amino acids 21–612 of the predicted coding region, which was then ligated into pProEX-HTb (Life Technologies) to generate pUAMS481. To produce recombinant P66 protein, pUAMS481 was transformed in E. coli LOBSTR-BL21(DE3)-RIL. Expression and purification of recombinant P66 were performed as previously described, except all buffers were adjusted to a final pH 9.0 [45]. Final elution samples were buffer exchanged to 1X phosphate buffered saline (PBS), 2M urea, 5% SDS and concentrated with a 50-kDa molecular weight cutoff (MWCO) Amicon Ultra Centrifugal Filter (MilliporeSigma, Burlington, MA). The concentration of purified recombinant P66 was determined with a DC protein assay kit (Bio-Rad Laboratories, Hercules, CA).

Generation of P66-specific antisera

The antigen-adjuvant emulsion was generated by combining 25 μg of recombinant P66 in 200 μL of sterile 1X PBS with an equal volume of AddaVax adjuvant (InvivoGen, San Diego, CA). Three- to four-week-old, female Sprague-Dawley rats (Envigo, Indianapolis, IN) were injected subcutaneously at two sites with 200 μL of emulsion per injection. Rats were boosted twice at four-week intervals with 25 μg of recombinant protein and AddaVax as described above. Rats were euthanized and serum was collected two weeks after the second boost [45].

Murine infections

The murine model of B. duttonii infection was adapted from a previous model used for B. turicatae and B. hermsii [45,46,63–65]. BdWT, BdΔp66, BdΔp66^{SV comp}, and BdΔp66^{Cis comp} were grown to mid-log phase, enumerated by dark-field microscopy, and diluted in fresh mBSK media to a concentration of 10³ spirochetes/mL. Four- to six-week-old Swiss Webster mice (Charles River Laboratories, Wilmington, MA) were injected intradermally/subcutaneously in the thoracic region with 100 μL of suspension containing a total of 10² bacteria. On days 3–14 post-infection, 2.5 μL blood samples were collected via tail vein nick, combined with 47.5 μL of SideStep Lysis & Stabilization Buffer (Agilent Technologies, Santa Clara, CA), and stored at -80°C until quantitative PCR (qPCR) was performed (see below). 2.5 μL of blood was also added to 1 ml of mBSK medium containing 1X Borrelia antibiotic mixture (BAM) (Monserate, San Diego, CA) to culture bacteria from the blood. On day 14 post-infection, mice were euthanized, and serum was collected for seroconversion analyses (see below). Naïve mice were also sacrificed, and blood was added to lysis/stabilization buffer at a 1:18 ratio for use in qPCR standards (see below). To assess bacterial outgrowth from the blood, blood cultures were analyzed at two-weeks post-collection by dark-field microscopy. 20 fields of view were scanned, and the presence of one or more spirochetes was considered culture positive.

qPCR for bloodstream bacterial burdens

The method for qPCR to quantify bloodstream bacterial burden was adapted from previous experiments with B. turicatae using flaB as a target [45,46,63–65]. To generate a qPCR standard, BdWT was cultured to late-exponential growth phase and pelleted by centrifugation at 6,000 x g for 15 min at room temperature. The cells were then resuspended in PBS containing 5 mM MgCl₂ (PBS-MgCl₂). This wash step was repeated two more times. After the final resuspension in PBS-MgCl₂, bacteria were quantified by dark-field microscopy. Tenfold serial dilutions were then made from this resuspension from 10⁴−10⁸ bacteria/ml in PBS-MgCl₂, and dilutions were combined with naïve blood in lysis/stabilization buffer at a 1:19 ratio. For use as a no-template control, nuclease-free water was diluted tenfold in PBS-MgCl₂ and added to naïve blood in lysis/stabilization buffer at a 1:19 ratio. In a 96-well real-time PCR reaction plate, 17 μL of master mix consisting of 1X SsoAdvanced Universal Probes Supermix (Bio-Rad Laboratories), 400 nM of each primer (Bd FlaB IDT FWD and Bd FlaB IDT REV) and 300 nM of probe (Bd FlaB IDT PRB) was added to each well. 3 μL of murine blood sample in lysis/stabilization buffer or standard preparation were then added in a 20 μL reaction. qPCR reactions for blood samples and standards were performed in triplicate. The QuantStudio 6 Pro Real-Time PCR System (ThermoFisher Scientific) was used for real-time PCR, and reaction conditions included an initial 2 min, 50°C step followed by a 10 min, 95°C hold for polymerase activation. Amplification was then achieved by 40 cycles of DNA denaturation at 95°C for 15 sec and primer annealing/DNA extension at 60°C for 60 sec. Following completion of the qPCR reaction, data was imported into GraphPad Prism version 9 (GraphPad Software, San Diego, CA) for analysis. Mean peak bacterial burdens (first peak at ~8 days and second peak at ~12 days) and the area under the curve (AUC) for each group of mice were compared using the Kruskal-Wallis test. Difference with P values <0.05 were considered statistically significant.

SDS-PAGE and immunoblotting

SDS-PAGE and immunoblot analyses were performed as previously described [45,66]. Briefly, whole lysates containing the equivalent of 2 x 10⁷ bacteria were separated by SDS-PAGE and transferred to nitrocellulose. Membranes were subsequently probed with rat antiserum recognizing P66 (see above), and horseradish peroxidase (HRP)-conjugated goat anti-rat IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) was used as a secondary antibody. To assess equal loading, membranes were additionally probed for FlaB using chicken anti-B. burgdorferi FlaB IgY as the primary antibody and HRP-conjugated donkey anti-chicken IgY (Jackson ImmunoResearch Laboratories) as a secondary antibody [66]. Colorimetric detection of protein was achieved using 4-chloro-1-naphthol as a substrate. To evaluate seroconversion, whole cell lysates were prepared from BdWT bacteria grown to the late exponential growth phase, and volumes containing the equivalent of 2 x 10⁷ cells were separated by SDS-PAGE and transferred to PVDF. Membranes were probed with serum from mice collected at 14 days post-infection (1:1000 dilution), and HRP-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories) served as the secondary antibody (1:1000 dilution). Reactivity was detected using SuperSignal West Dura extended duration substrate (ThermoFisher Scientific). For seroconversion immunoblots, all images were manually captured for 3 sec without image optimization. Pooled serum from naïve mice and secondary-only exposed controls were also included for the comparison. Precision Plus Protein All Blue Prestained Protein Standard (Bio-Rad Laboratories) was used for the molecular weight standard.

Development of a shuttle vector for use in B. duttonii

Despite the public health relevance of Old World RF spirochetes, our knowledge regarding the pathogenesis of these bacteria is limited. This is due in part to the lack of techniques to genetically manipulate these pathogens. To begin addressing this gap in the TBRF Borrelia field, we sought to develop the requisite genetic tools for use in the Old World RF spirochete, B. duttonii. First, we aimed to make a shuttle vector that is competent for replication in both E. coli and B. duttonii. To identify a putative B. duttonii ori for the shuttle vector, the plasmid sequences of strain Ly were scanned for regions homologous to the B. hermsii ori used for generation of pBhSV-2 [41]. The cloned origin of replication, derived from B. duttonii pl41, included a portion of bdu_4015, all of bdu_4016 and bdu_4017, and 137 bp upstream of bdu_4017. A kanamycin resistance cassette was generated for B. duttonii by fusing the putative promoter for the flagellin gene (flaB; bdu_150) to the aphI start codon [45,59]. This aphI resistance cassette was then ligated together with the MCS from pJD44, an E. coli ColE1 ori, and the putative ori from B. duttonii pl41, generating the shuttle vector, pBdSV (Fig 1A) [41,55,59,60]. An alignment of the ori regions of B. hermsii strain DAH lp27 and the pl41 analogous region amplified from B. duttonii strain 1120K3 is shown in S1 Fig (67.7% identical). A second version of the shuttle vector was additionally made in which the aphI cassette was oriented in the opposite direction of that in pBdSV, designated pBdSV^CCW. To assess if a gene could be successfully expressed from the shuttle vector, a gfp reporter was made by fusing the B. duttonii flaB promoter to a gfp allele and ligating this cassette into the MCS of pBdSV, generating pBdSV::gfp [56,57].

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Fig 1. Generation of the B. duttonii shuttle vector.

(A) Diagram illustrating the two B. duttonii shuttle plasmids, pBdSV and pBdSV^CCW, developed in this study. In pBdSV^CCW, the MCS and aphI resistance cassette from pUAMS363 were ligated in the opposite orientation of this region in pBdSV. (B) Genotypic confirmation of shuttle vector transformants. PCR was performed with BdWT gDNA, the relevant shuttle vectors, and gDNA from B. duttonii electroporated with each of the pBdSV constructs to amplify internal regions of aphI (624 bp), gfp (456 bp), pl41 (326 bp), and flaB (531 bp). PCR was also performed with no template (H₂O) as a contamination control. “MW” denotes the DNA standard, and numbers to the left indicate molecular weight in kb.

https://doi.org/10.1371/journal.pntd.0012348.g001

To determine if pBdSV, pBdSV^CCW, and pBdSV::gfp were capable of autonomous replication in B. duttonii, the three shuttle vectors were transformed into BdWT, creating the strains, BdSV, BdSV^CCW, and BdSV::gfp, respectively. Clones were first confirmed to harbor the shuttle vectors by plasmid recovery in E. coli, followed by restriction digest and sequencing of the respective plasmids. PCR was then performed with gDNA from each clone to amplify internal regions of aphI, gfp, and flaB (Fig 1B). As expected, all B. duttonii transformants were positive for aphI, whereas only BdSV::gfp was positive for gfp. PCR for flaB, which served as an amplification control, resulted in amplicons of the appropriate size in all B. duttonii strains. Next, it was possible that transformation of a pl41-derived shuttle vector into B. duttonii could result in plasmid incompatibility and cause loss of the endogenous pl41 plasmid [48,59,67–73]. Therefore, a PCR was performed to amplify an internal region of pl41 that was not part of the ori sequence used for generation of pBdSV. All shuttle vector-containing clones screened positive for the endogenous copy of pl41, demonstrating that pBdSV did not displace pl41 in these B. duttonii transformants (Fig 1B). Finally, to determine that a heterologous gene could be expressed from pBdSV, BdSV::gfp was imaged by fluorescence microscopy (Fig 2). As expected, BdSV::gfp exhibited a bright green fluorescence, but BdWT did not. In all, these results demonstrate successful generation of a shuttle vector capable of autonomous replication in B. duttonii that can be used for genetic complementation and ectopic expression experiments (see below).

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Fig 2. Fluorescent imaging of gfp-expressing B. duttonii.

BdWT, BdSV::gfp, and Bdgfp strains were visualized by either brightfield (left) or fluorescence (right) microscopy. Two biological replicate cultures were examined and shown are representative images from one replicate. Scale bar = 10 μm.

https://doi.org/10.1371/journal.pntd.0012348.g002

Development of a trans integration approach for B. duttonii

While the intrinsically higher transformation efficiency of shuttle vectors makes them easy to use for in vitro expression and complementation experiments, shuttle vectors are not always ideal for in vivo applications because the lack of antibiotic selection makes them less stable. Therefore, we sought to develop a trans integration approach for use in B. duttonii that would recombine gene(s) of interest into the genome [18,74–76], which could prove more optimal for use in animal infection studies. A site on pl165, which is analogous to the large linear plasmids of New World RF Borrelia, between the genes bdu_1105 and bdu_1106 was chosen as a prospective integration site. This site was selected because it is a large intergenic region (248 bp) between convergently transcribed genes, decreasing the possibility that integration at this site will result in polar mutation effects. To test if a gene could be inserted at this region in pl165, regions flanking the prospective integration site were first amplified, and a segment of pBdSV::gfp containing the aphI and gfp cassettes was excised and ligated between them, creating the integration construct, pBdgfp (Fig 3A). This construct was then transformed into BdWT, generating Bdgfp. Genotypic confirmation of integration in Bdgfp was achieved by PCR to amplify internal regions of gfp and aphI, as well as by a PCR to amplify from upstream of the 5’ flanking region used for the integration construct to a site within the gfp gene and a PCR to amplify from within the aphI gene to a site downstream of the 3’ flanking region used for the integration construct (Fig 3B). As expected, Bdgfp screened positive for gfp and aphI, whereas BdWT did not. Additionally, PCR linking sites on pl165 flanking the segments used for the integration construct to sites within the gfp and aphI ORFs yielded amplicons of the appropriate size in Bdgfp, but failed to generate products when BdWT gDNA or pBdgfp was used as a template. Finally, a PCR for flaB, which served as an amplification control, resulted in products of the appropriate size for BdWT and Bdgfp. Fluorescence microscopy was then used to demonstrate expression of gfp in Bdgfp (Fig 2). As expected, Bdgfp fluoresced green, whereas BdWT exhibited no fluorescence. In all, these results confirm that the integration site selected within pl165 can be used for insertion of genes, validating use of this approach for genetic expression and complementation experiments.

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Fig 3. Use of the trans integration approach to generate gfp-expressing B. duttonii.

(A) Generation of Bdgfp. BdWT was transformed with an allelic exchange construct (pBdgfp) to insert PflaB-gfp and PflaB-aphI cassettes into pl165, generating Bdgfp. Primers used for PCR in Fig 3B are denoted with numbered smaller arrows. (B) Genotypic confirmation of Bdgfp. PCR was performed with BdWT gDNA, pBdgfp plasmid, and Bdgfp gDNA to amplify an internal segment of gfp (P1+P2; 456 bp), an internal segment of aphI (P3+P4; 624 bp), a segment spanning regions of bdu_1105 and PflaB-gfp (P5+P2; 1967 bp), a segment spanning regions of PflaB-aphI and bdu_1106 (P3+P6; 1937 bp), and an internal segment of flaB (531 bp). PCR was also performed with no template (H₂O) as a contamination control. “MW” denotes DNA standard, and numbers to the left indicate molecular weight in kb.

https://doi.org/10.1371/journal.pntd.0012348.g003

Allelic exchange mutagenesis in B. duttonii

The above data confirmed successful transformation of B. duttonii with the shuttle vector and pl165-targeting integration construct. As future work will aim to characterize bacterial factors required for the B. duttonii enzootic cycle, it was also necessary to develop and confirm methods for allelic exchange mutagenesis in B. duttonii. As a proof of principle, we chose to mutate a homolog of a gene that is known to be required for mammalian infection by the related LD spirochete, B. burgdorferi. Specifically, P66 can function as both an adhesin and a porin in B. burgdorferi, so the gene encoding the B. duttonii P66 homolog (BDU_604) was targeted for allelic exchange mutagenesis [51,52,77–85]. BDU_604 and P66 of B. burgdorferi strain B31 are 53.7% identical and their genes are colinear in the chromosome. A gentamicin resistance marker was first adapted for use in B. duttonii by amplifying the putative promoter region for the flagellar basal body rod (flgB) gene and fusing it to the start codon of the aacC1 ORF [41,46,47]. The B. duttonii-adapted aacC1 marker was subsequently ligated between regions flanking bdu_604, replacing a 1750-bp region of the ORF and generating the mutational construct, pBdΔp66. This construct was then electroporated into BdWT, and transformants were selected with gentamicin (Fig 4A).

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Fig 4. Mutation and complementation of B. duttonii p66.

(A) Schematic representation of the relevant regions of the chromosomes and relevant constructs used to generate BdΔp66 mutant and BdΔp66^{Cis comp}. BdWT was transformed with an allelic exchange construct, pBdΔp66, to replace an internal region of p66 with PflgB-aacC1 marker, generating BdΔp66. To restore p66 in BdΔp66, the mutant was transformed with pBdp66^Cis to replace the PflgB-aacC1 with an intact copy of p66 and PflaB-aadA, generating BdΔp66^{Cis comp}. Numbered arrows represent approximate locations of primers used in panel B. (B) Genotypic confirmation of BdΔp66, BdΔp66^{SV comp}, and BdΔp66^{Cis comp}. PCR was performed with gDNAs isolated from BdWT, BdΔp66, BdΔp66^{SV comp}, and BdΔp66^{Cis comp}. Plasmids pBdΔp66, pBdSV::p66, and pBdp66^Cis were included as positive amplification controls, and PCR was also performed with no template (H₂O) as a purity control. Diagnostic PCR, identified on right, was designed to amplify internal regions of p66 (P1+P2; 565 bp), aacC1 (P3+P4; 489 bp), aadA (P5+P6; 463 bp), or flaB (531 bp). “MW” denotes the DNA standard, and numbers to the left indicate molecular weight in kb. (C) Immunoblot confirmation of BdΔp66, BdΔp66^{SV comp}, and BdΔp66^{Cis comp}. Whole-cell lysates, prepared from bacteria in late-exponential phase cultures, were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Membranes were then probed with antiserum or antibody against P66 or FlaB, respectively. Antiserum/antibodies used to detect the respective proteins are indicated to the right. “MW” denotes the protein standard, and numbers to the left indicate molecular weight in kDa.

https://doi.org/10.1371/journal.pntd.0012348.g004

A B. duttonii p66 mutant clone, designated BdΔp66, was confirmed by PCR with primer pairs that amplified a region within the deleted portion of the p66 ORF or an internal region of aacC1 (Fig 4B). An internal region of flaB was also amplified as a positive control. As expected, PCR for the internal region of bdu_604 resulted in an amplicon of the appropriate size for BdWT, while no amplicon was observed for BdΔp66. Alternatively, BdΔp66 screened positive for the aacC1 marker, while BdWT did not. Additionally, PCR for a region flanking the site of mutation resulted in products of the appropriate size for BdWT and BdΔp66. Finally, PCR for an internal region of flaB resulted in amplicons for both BdWT and BdΔp66 strains (Fig 4B).

Genetic complementation was used to restore expression of p66 in BdΔp66. There are three commonly used methods for genetic complementation; i) complementation in cis by replacing the mutated gene with a wild-type allele, ii) complementation in trans by transforming a plasmid that harbors a wild-type copy of the gene into the mutant strain, and iii) complementation in trans by inserting a wild-type copy of the gene into a stable genomic element (e.g., endogenous plasmid or chromosome) in the mutant strain [19]. We encountered complications cloning the p66 region into the pl165-targeting trans integration construct (e.g., pUAMS376), so cis complementation and shuttle-vector-based trans complementation were pursued in this study. To generate the p66 shuttle vector complementation construct, the p66 ORF and 540-bp upstream of the coding region were amplified from BdWT and cloned into pBdSV to generate pBdSV::p66. For the cis integration p66 complementation construct, an aadA resistance marker was first adapted for B. duttonii by fusing the promoter region of BdWT flaB to the aadA ORF to generate PflaB-aadA [41,46,61,86]. The genomic regions flanking bdu_604/p66, corresponding to a portion of bdu_602 with all of bdu_603 and all of bdu_604 with a portion of bdu_605 were then amplified, and the PflaB-aadA marker was then ligated into a unique AscI site introduced downstream of the p66 ORF. The resulting p66 cis complementation construct was designated pBdp66^Cis (Fig 4A). pBdSV::p66 and pBdp66^Cis were electroporated into BdΔp66, and transformants were designated BdΔp66^{SV comp} and BdΔp66^{Cis comp}, respectively. BdΔp66^{SV comp} and BdΔp66^{Cis comp} were initially confirmed by PCR, and p66 was detected in both complemented strains (Fig 4B). In BdΔp66^{Cis comp}, the aadA marker was detected instead of aacC1, whereas aacC1 was detected in BdΔp66^{SV comp}. The pBdSV::p66 shuttle vector was also recovered from BdΔp66^{SV comp} and confirmed by restriction digestion and sequencing. Finally, loss of P66 production in BdΔp66 and restoration of P66 in BdΔp66^{SV comp} and BdΔp66^{Cis comp} was confirmed by immunoblot (Fig 4C). In all, these results indicate the first successful mutagenesis of a gene in an Old World RF spirochete and confirm that allelic exchange is a viable option for targeted mutagenesis in B. duttonii.

The BdΔp66 mutant is attenuated in a murine model of RF

Murine infection models are commonly used to determine if RF spirochete mutants are competent for mammalian infection, [41–50,65]. We therefore adapted the previously described murine infection model for the New World RF Borrelia, B. turicatae, for use with B. duttonii [44–47,50]. Groups of four mice were intradermally/subcutaneously inoculated with 10² spirochetes of BdWT, BdΔp66, BdΔp66^{SV comp}, and BdΔp66^{Cis comp}. Blood samples were collected on days 3–14 and analyzed by qPCR to quantify the bacterial number in the bloodstream. Mice infected with BdWT experienced two recurring bouts of high bacteremia with peaks of ~10⁸ bacteria/ml of blood (Fig 5A). In contrast, two mice infected with BdΔp66 failed to reach detectable levels of bacteremia on any day post-infection. The other two BdΔp66-infected mice showed two bacteremia peaks, but the bacteria reached levels lower than BdWT (Fig 5B). In these latter two mice, the first bacteremic peaks reached 10⁵−10⁶ bacteria/ml and ~10⁶ bacteria/ml were detected during the second peak. Statistical analyses comparing the mean peak bacterial burdens or AUC values between BdWT- and BdΔp66-infected mice confirmed statistically significant differences (S2 Fig). To fulfill molecular Koch’s postulates and confirm the requirement for P66 during B. duttonii mammalian infection, genetic complementation was used to restore expression of p66 in BdΔp66 and determine if the infection defect could be reversed. For the genetically complemented strains, bacterial burdens were comparable to BdWT in three of the four mice infected with BdΔp66^{SV comp} (Fig 5C) and in all four mice infected with BdΔp66^{Cis comp} (Fig 5D). Statistical comparison of the data from the BdΔp66^{Cis comp}- and BdΔp66-infected mice confirmed significant differences only in the mean peak bacterial burdens. Neither the AUC values or peak burdens between the BdΔp66^{SV comp}- and BdΔp66-infected groups were statistically significant (S2 Fig). Daily blood samples were also collected and inoculated into mBSK medium to culture B. duttonii from the bloodstream. This approach is predicted to be more sensitive with an estimated limit of infection (LOD) of ~4 x 10² live bacteria/ml [45,46]. BdWT was cultured from the blood of infected mice on multiple days post-infection, but BdΔp66 spirochetes could only be cultured from the two mice that showed the bacteremia peaks. Furthermore, all the mice infected with BdΔp66^{SV comp} and BdΔp66^{Cis comp} were culture positive on at least one day during infection. Seroconversion results agreed with the qPCR results (S3 Fig). Specifically, the two BdΔp66-infected and one BdΔp66^{SV comp}-infected mice that didn’t show detectable levels of bacteremia after infection also did not produce antibodies that reacted against BdWT lysates. Taken together, these results support the notion that P66 is required for optimal mammalian infection and demonstrate our ability to adapt our murine model of RF used for the New World RF bacterium, B. turicatae, to study pathogenesis of the Old World RF spirochete, B. duttonii.

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Fig 5. Murine infection phenotype of the B. duttonii p66 mutant and complemented clones.

Groups of four mice were intradermally inoculated with 10² (A) BdWT, (B) BdΔp66, (C) BdΔp66^{SV comp}, and (D) BdΔp66^{Cis comp} spirochetes. On days 3–14 post-infection, bacterial levels in the bloodstream of each mouse were quantified by qPCR. The dashed line indicates the LOD for this assay (10⁴ spirochetes/ml). Daily blood samples were also collected and inoculated into mBSK to culture B. duttonii from the bloodstream. Cultures were analyzed up to two-weeks post-collection by dark-field microscopy and results for each day are indicated below the X-axis; +, positive for spirochetes and -, no spirochetes observed. Numbers above the graphs indicate individual mice in each experimental group, and error bars represent standard error of the mean (SEM).

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