Structural comparison of B. burgdorferi CheA₁ and CheA₂

To investigate why CheA₁ is dispensable for chemotaxis in vitro while CheA₂ is required [11], we generated AlphaFold [20] models of the two CheA proteins (BbCheA₁ and BbCheA₂) and compared them to the solved structure of CheA from Thermotoga maritima (Tm) [21,22] (Fig 1A). The models show high confidence (pLDDT scores >80) for all domains in each monomer, with pLDDT scores < 50 only for the residues that comprise the P1-P2 and P2-P3 linkers. The domain composition of TmCheA is similar to BbCheA₁ and BbCheA₂ with some notable differences (Fig 1B). BbCheA₁ has an analogous domain arrangement compared to TmCheA with the only major difference being an extended P2-P3 linker (73 residues-long compared to 42 residues of TmCheA). In contrast, BbCheA₂ differs from BbCheA₁ and TmCheA in several distinct ways. First, BbCheA₂ has two P2 domains (CheY binding domains, referred to here as P2^α and P2^β) compared to a single P2 domain in TmCheA and BbCheA₁. Superimposition of the P2^α and P2^β domains reveals a high degree of structural similarity despite a mere 26% identity (Fig 1C). Sequence alignment suggests that both P2 domains bind to CheY, which could potentially act to increase the local phospho-CheY concentration within the chemosensory arrays of B. burgdorferi (S1A Fig), as has been shown for the P2 domain of the canonical E. coli CheA [23]. The second major difference between BbCheA₁ and BbCheA₂ is that BbCheA₂ has an extended P3 domain (Fig 1B). CheA homodimerizes via P3-P3’ interactions, wherein two adjacent P3 domains come together to form a four-helix bundle [21]. In BbCheA₂, the 122 residues in P3 domain is significantly longer than the ~53 residues domain found in TmCheA and BbCheA₁ due to inserts between residues Ile446-Leu515 (Fig 1A and 1B).

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Fig 1. Structural comparison of B. burgdorferi CheA₁ and CheA₂.

(A) AlphaFold models of B. burgdorferi CheA₁ (BbCheA₁) and CheA₂ (BbCheA₂) subunits compared to Thermotoga maritima CheA (TmCheA). Protein structures are colored according to their pLDDT scores [20]. (B) Domain organization of BbCheA₁, BbCheA₂, and TmCheA. The first P2 domain of BbCheA2 (referred to here as P2^α) extends from Glu153 to Lys265 and is connected to the second P2 domain (P2^β) via an 18-residue long P2^α -P2^β linker. P2^β is comprised of residues Glu283 to Val387 and is connected to the dimerization domain (P3) via a 32-residue long P2^β -P3 linker. Overall, the P1-P2^α and P2^β -P3 linkers consist of approximately 50 residues, similar to the 43-residue long P2-P3 linker of TmCheA. (C) Superimposition of BbCheA P2^α (RMSD: 0.48 Å, cyan, C150-V267) and P2^β (gray, F279-V387). (D) Superimposition of phospho-transfer domain (P1), CheY-binding domain (P2), dimerization domain (P3), kinase domain (P4), and regulatory domain (P5) of TmCheA (tan), BbCheA₁ (pink), and BbCheA₂ (green). RMSD of each alignment is indicated in the lower region of each panel. All structural superimposition and figures were generated in PyMol [105].

https://doi.org/10.1371/journal.ppat.1011752.g001

To gain more insight into the overall differences in the domain architecture of CheA₁ and CheA₂, we superimposed domains P1-P5 from BbCheA₁ (pink) and BbCheA₂ (green) to the analogous domains of TmCheA (tan, Fig 1D). Overall, the P1, P4, and P5 domains are quite similar in all three proteins (RMSDs < 1), whereas the analogous P2 and P3 domains vary substantially (RMSD ranging 0.71–10.2 Å) among the three CheAs. For the P2 domain superimposition, the domain cores are similar with most of the structural deviations arising from the loops connecting the β-strands, and the N- and C-termini connecting to the P1-P2 and P2-P3 linkers, respectively. Analysis of the putative CheY binding site for BbCheA₁ P2 and BbCheA₂ P2 (α and β) reveals that the structures of the P2 domains are generally well-conserved in this region, supporting sequence alignment data that indicates conservation in the CheY:P2 binding interface residues (S1A–S1B Fig). Superimposition of the P3 domains from TmCheA, BbCheA₁, and BbCheA₂ AlphaFold models reveals the extent by which BbCheA₂ P3 domain differs from that of TmCheA and BbCheA₁, whose P3 domains align with an RMSD of 0.7 Å. Compared to TmCheA P3, BbCheA₂ P3 aligns with an RMSD of 3.7 Å and is approximately 43 Å longer. The extended P3 domain may be important for maintaining the proper protein-protein interactions between CheA subunits within a homodimer and/or between CheA and the chemosensory receptors in the arrays [21,24].

To better contextualize the differences in P2 and P3 predicted for the BbCheAs, we performed multiple-sequence alignment analysis on discrete groups of CheA sequences within the spirochete phylum. We first examined all the CheA sequences in spirochete species that contained only one CheA gene, assuming that this single CheA would be associated with chemotaxis signaling. Within this category, we examined only those members from the order Treponematales. Multiple sequence alignments of these CheA sequences reveal they have a single P2 domain and an extended P3 domain (S2 Fig). These results indicate that while the extra P2 domain may or may not play a role in chemotaxis for other spirochete species, the extended P3 domain is a conserved structural motif for CheAs associated with chemotaxis in spirochetes. We next looked at all CheA₁ and CheA₂ sequences from species in the Borreliaceae family. Multiple alignment data from these sequences reveal that in species with two distinct CheA isoforms, all the CheA₁ proteins encode a single P2 domain and have a short P3 domain similar to BbCheA₁ and TmCheA (S3 Fig), and all CheA₂ proteins encode for a second P2 domain and an extended P3 domain (S3 Fig). These data suggest that while an extended P3 domain appears to be important for CheA proteins associated with chemotaxis in diverse spirochete species, the addition of a second P2 domain may be a Borrelia spp. and Borreliella spp. specific modification.

Isolation of cheA₁ mutant and its complemented strain

To investigate the role of CheA₁ in the infectious cycle of B. burgdorferi, we have attempted to inactivate cheA₁ in the virulent B31 A3-68 Δbbe02 strain [25]. pGA₁kan, a previously constructed vector [11], was linearized and electro-transformed into wild-type competent cells. A previously described PCR analysis was first carried out to screen for clones with the desired targeted mutagenesis [11]. One clone (referred to as cheA₁^mut) was selected for further analysis. For the complementation, the plasmid CheA₁/pBBE22 (Fig 2A) was electro-transformed into the cheA₁^mut competent cells. The presence of CheA₁/pBBE22 in antibiotic-resistant colonies was confirmed by immunoblots using anti-CheA₁ antibody and anti-DnaK antibody as a control. As shown in Fig 2B, a band of approximately 80 kDa was detected in the wild type and the cheA₁^com strain but was absent in cheA₁^mut, indicating that the cognate gene product was abrogated in the mutant and restored in the complemented strain (Fig 2B). B. burgdorferi B31 strain contains 21 linear and circular plasmids [26] of which some are essential for its infectivity but are easily lost during in vitro cultivation [27]. To check for the plasmid profile of our isolated strains, a previously developed PCR method was used to determine if the obtained positive clones contain the same plasmids as their parental strains [28]. Our results showed that cheA₁^mut (S4B Fig) and its isogenic complemented strain (S4C Fig) contained the same plasmid profile as the wild-type strain (S4A Fig). Growth analysis indicated that deletion of cheA₁ did not affect the fitness of the mutant at 23°C,34°C or 37°C (Fig 2C–2E) indicating that depletion of CheA₁ has no impact on B. burgdorferi growth in vitro.

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Fig 2. Characterizations of cheA₁ mutant (cheA1mut) and its isogenic complemented strain (cheA1com).

(A) Deletion of cheA₁ has no impact on B. burgdorferi growth. Construction of CheA₁/pBBE22 for the complementation of cheA₁^mut. bb0566-cheA₁ fragment was PCR amplified using primer pair P₁/P₂ and cloned into pBBE22 [17,99], a shuttle vector of B. burgdorferi, yielding CheA₁/pBBE22. (B) Immunoblot analysis of WT, cheA₁^mut and cheA₁^com strains. The same amounts of WT, cheA₁^mut and cheA₁^com whole-cell lysates were analyzed by SDS-PAGE and then probed with CheA₁ and DnaK antibodies, as previously documented [11,97]. Growth analysis of WT, cheA₁^mut and cheA₁^com under routine laboratory condition (C) (34°C/pH7.6), (D) tick-like condition (23°C/pH7.6), and (E) host condition (37°C/pH6.8). Growth curve analysis was carried out to determine if CheA₁ affects cell growth. 10⁴ or 10⁵ cells/ml of bacteria were inoculated into 10 ml BSK-II media and cultivated under 34°C/pH7.6, 23°C/pH7.6, or 37°C/pH6.8. Cell numbers were enumerated every 1–4 days until cells entered stationary phase. Cell counting was repeated in triplicate with at least two independent samples, and the results are expressed as means ± SEM.

https://doi.org/10.1371/journal.ppat.1011752.g002

The cellular localization of CheA₁ is temporally and spatially regulated

As mentioned earlier, the genome of B. burgdorferi encodes for multiple copies of chemotaxis genes. It was speculated that B. burgdorferi may possess two chemotaxis pathways that function in different hosts during the infection cycle [8,11,13,18,29], i.e., CheA₂, CheW₃, and CheY₃ form a pathway that executes chemotaxis in mammalian hosts, whereas CheA₁, CheW₂, and CheY₂ form a distinct pathway that is activated in the tick vector. Consistent with this speculation, our previous work showed that inactivation of cheA₂ impaired the ability of the spirochete to establish infection in mice but not in ticks [17]. In order to decipher the function of CheA₁ in the enzootic cycle of B. burgdorferi, a GFP reporter construct (S5A Fig) was created and used to complement cheA₁^mut. The reporter strain was cultivated under laboratory conditions as well as conditions mimicking the tick milieu in vitro and observed microscopically. Surprisingly, we noticed that CheA₁-GFP was localized to one cell pole when cultivated under the tick-phase condition but appeared diffused at elevated temperature (Fig 3A). The diffused pattern was not due to degradation of the GFP-fusion protein at elevated temperature as full-length CheA₁-GFP fusion protein was detected with no excessive degradation (S5C Fig). This pattern was not observed in a strain carrying only the empty GFP vector (Fig 3A) or in a strain expressing CheA₂-GFP fusion protein (S5C–S5D Fig). This result indicates that the polar localization pattern is unique to CheA₁ (Fig 3A). In addition, the localization of CheA₁ appeared to be connected with cell division. During early log phase, the CheA₁-GFP signal was localized to one cell pole. As the cells entered mid log phase, the signal of CheA₁-GFP was seen distributed to the mid cell as well as both cell poles (Fig 3B Day 7 onward). As the cells entered late log to stationary phase, CheA₁-GFP signal was seen distributed along the cell body in specific puncta as indicated by the white arrows in Fig 3B Day 13–17 (Fig 3B). When the cells were cultivated under elevated temperature, the polar localization of CheA₁-GFP disappeared (S6 Fig). At elevated temperature, during the early growth phase, CheA₁-GFP signal appeared dispersed along the cell body and only when the cells entered stationary phase single pole localization reappeared as was the case with cells cultivated at 23°C (S6B Fig), suggesting that the localization of CheA₁ is regulated spatially and temporally. In contrast to CheA₁-GFP, the localization of GFP and CheA₂-GFP remained evenly distributed along the cells under all culture conditions and growth phases (S6A and S6C Fig). Collectively, these results strongly suggest that the localization of CheA₁ is dynamic throughout the growth of B. burgdorferi and that CheA₁ function may be more prominent during the tick phase of the enzootic cycle. In addition, pH (acidic when cell density increases and enters stationary phase [30]) or temperature may have an impact on the localization and function of CheA₁.

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Fig 3. CheA₁ has a unique polar localization when cultivated under tick-like condition.

(A) In vitro localization of CheA₁. To localize CheA₁, a CheA₁-GFP reporter construct (S3 Fig) was used to complement cheA₁^mut. The obtained cells were cultivated under unfed tick (UF) condition at 23°C/pH 7.6 or routine laboratory culture condition at 34°C/pH 7.6. Cells were monitored for the presence of GFP signal and images were taken at early log phase at ×200 magnification using a Zeiss Axiostar Plus microscope. Scale bars represent 10 μm. A cheA₁^mut carrying a GFP reporter construct was used as a control to confirm that the polar localization seen in CheA₁-GFP complemented strain is due to CheA₁ and not an artifact from GFP protein. (B) Time course images of CheA₁-GFP strain cultivated under UF condition. 10⁵ cells/ml of CheA₁-GFP strain was inoculated into 10 ml fresh BSK-II and cultivated under UF condition. Images were taken every 2 days to monitor the localization of CheA₁-GFP signals. As cells entered late log to early stationary phase, increasing numbers of discrete bright puncta were observed evenly distributed along the cell body, coincided with the zones of new peptidoglycan formation that mark the division sites for daughter cells [106].

https://doi.org/10.1371/journal.ppat.1011752.g003

CheA₁ has no impact on B. burgdorferi swimming behavior and chemotaxis in vitro

B. burgdorferi has three different swimming modes: run, flex, and reverse [11,12]. Our previous work showed that deletion of cheA₁ did not have impacts on the swimming behavior of B. burgdorferi in vitro [11]. All of these studies, however, were conducted using a high-passage avirulent B31A strain. To test whether this is the case in the virulent strain background, the swimming behaviors of the wild-type, cheA₁^mut, and cheA₁^com cells were analyzed using a computer-assisted cell tracker as documented previously [11]. Similar to the WT, cheA₁^mut showed three swimming modes–runs interrupted by reverses/flexes, with a mean swimming velocity of 12.5 ± 0.5 μm/sec compared to 13.7 ± 0.3 and 12.9 ± 0.3 μm/sec of WT and cheA₁^com, respectively (Fig 4A). In addition, capillary tube assays using N-acetyl-D-glucosamine (NAG) as a chemoattractant [31] (Fig 4B) and swimming plate analysis (Fig 4C–4D) did not reveal significant differences among WT, cheA₁^mut, and cheA₁^com, further supporting that CheA₁ is dispensable for B. burgdorferi motility and chemotaxis in vitro.

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Fig 4. CheA₁ is dispensable for spirochete motility and chemotaxis in vitro.

(A) Bacteria tracking analysis. The swimming velocities of WT, cheA₁^mut, and cheA₁^com were measured using a computer-based motion tracking system as previously described [11]. At least 25 cells were tracked and the swimming velocities were measured. (B) Capillary tube chemotaxis assay. This experiment was carried out using 100 mM N-acetyl-D-glucosamine (NAG) as an attractant according to a previous report [31]. The results are expressed as fold increases in cell number entering the capillary tubes containing the attractant relative to the number entering control tubes without attractant (buffer alone). Results are expressed as means ± SEM for five tubes. A 2-fold increase is considered the threshold for a positive response. (C) and (D) Swimming plate assays. The assay was carried out on 0.35% agarose containing 1:10-diluted BSK-II medium as described previously [100]. The flgE mutant, a previously constructed nonmotile mutant [103], was included to determine the sizes of the inocula. The data are presented as mean diameters (in millimeters) of rings ± SEM for twelve plates. All assays were repeated at least twice with independent replicates and representative data are shown here. * significant difference (P < 0.05).

https://doi.org/10.1371/journal.ppat.1011752.g004

cheA₁^mut fails to establish systemic infection in immunocompetent mice by needle inoculation

To determine if CheA₁ is essential for the virulence of B. burgdorferi BALB/c mice were infected intradermally with 1 x 10⁵ cells of the WT, cheA₁^mut, and cheA₁^com strains (3–7 mice per strain). Tissues from the ear, skin, heart, and joint were harvested three weeks post-infection and transferred to BSK-II medium for re-isolation. The wild-type strain was successfully recovered from all of the infected animals (7/7) while cheA₁^com was successfully recovered from some tissues of five infected mice (5/5), indicating a partial complementation. Interestingly, cheA₁^mut was only recovered from the skin at the site of inoculation and not from distal organs (Table 1). To rule out the potential of a spontaneous mutation in our mutant, a new deletion construct was produced to replace the entire cheA₁ gene in-frame with a kanamycin (kan) cassette (S4D Fig) and the mouse infection study was repeated. The newly constructed mutant (cheA₁^IFD) was able to survive at the inoculation site but failed to establish systemic infection in immunocompetent mice (Table 1), similar to cheA₁^mut. Taken together, these results suggest that CheA₁ is not essential for B. burgdorferi colonization of skin, but is required for dissemination from the initial inoculation site and/or to establish a systemic infection in mice.

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Table 1. cheA1mut is unable to cause systemic infection in immunocompetent mice.^a

https://doi.org/10.1371/journal.ppat.1011752.t001

cheA₁^mut can persist and proliferate at the site of inoculation

Intradermal infection of the mutant failed to cause systemic infection but the mutant could be re-isolated from the initial inoculation site, suggesting that the mutant is able to survive and persist at the site of injection and evade the host immune system. To determine how long the mutant can persist at the inoculation site, 1 x 10⁵ cells of WT or cheA₁^mut were injected subcutaneously into BALB/c mice and the inoculation site was marked for bacterial re-isolation at different time points as indicated in the Material and Methods. qRT-PCR analysis showed that the mutant was not only able to persist but replicate at the inoculation site to the same extent as the WT as indicated by the increase in flaB transcript copies for up to 28 days (Fig 5).

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Fig 5. cheA1^mut is able to persist at the site of inoculation.

To determine if CheA₁ is required for B. burgdorferi persistence at the inoculation site, a time point infection study was performed. 18 immunocompetent mice were subcutaneously infected with 10⁵ of WT or cheA₁^mut and mice were sacrificed at the indicated time points. Skin tissue from the inoculation sites were recovered to assess for borrelial burden using qRT-PCR as described [17]. The data are presented as mean flaB transcript copies over 10⁵ of mouse β-actin ± SEM for each group of mice.

https://doi.org/10.1371/journal.ppat.1011752.g005

cheA₁^mut is defective in hematogenous dissemination

We reasoned that the failure of the mutant to cause systemic infection could be due to the following three possibilities: 1) the mutant failed to disseminate from the injection site; 2) the mutant successfully disseminated from the injection site into the bloodstream but failed to spread to distal organs due to a defect in tissue tropism; or 3) the mutant successfully disseminated into the bloodstream but was cleared by the host immune system. To test these possibilities, the above animal study was repeated using severe combined immuno-deficiency (SCIDs) mice. Based on the tissue re-isolation results in Table 2, WT and cheA₁^com were successfully recovered from all of the harvested tissues. In contrast, the cheA₁^mut mutant was able to disseminate and cause systemic infection in 50% of the injected SCID mice. Consistent with the result obtained using immunocompetent mice, cheA₁^mut was able to colonize and persist in the skin better than in other tissues (Table 2). qRT-PCR analysis indicated that of those 50% SCID mice with systemic infection, no statistically significant difference in tissue spirochetal burdens was detected between the wild type, cheA₁^mut, and cheA₁^com with the exception of the ear and heart tissues (Fig 6). This result strongly suggests that in the absence of an adaptive immune response, cheA₁^mut can disseminate from the injection site and cause systemic infection albeit to an attenuated degree. In the immunocompetent mice, cheA₁^mut most likely failed to evade the host adaptive immune response and was cleared from the animal before it was able to colonize and establish infection in distal organs. Furthermore, blood culture showed that 3 out of 6 mice were positive for cheA₁^mut when injected subcutaneously into SCID mice (Table 2), further substantiating that the mutant is able to disseminate from the inoculation site and spread systemically through the bloodstream. Taken together, the mouse infection studies indicate that in the absence of CheA₁, B. burgdorferi is attenuated in both innate and adaptive immune evasion and tissue colonization.

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Table 2. cheA₁^mut is still attenuated in distal organ colonization in SCID mice.^b

https://doi.org/10.1371/journal.ppat.1011752.t002

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Fig 6. cheA₁^mut is attenuated in causing systemic infection in immunodeficient mice.

To determine if CheA₁ is required for B. burgdorferi immune evasion, needle infection study was repeated using severe combined immunodeficiency (SCIDs) mice. For this study, 10⁵ of WT, cheA₁^mut and cheA₁^com strains were subcutaneously inoculated into SCID mice and sacrificed three weeks after infection. Skin tissues around and distal from the injection site were harvested along with tissues from the ear, joint, heart and spleen to assess for borrelial burden using qRT-PCR as described [17]. The data are presented as mean flaB transcript copies over 10⁵ of mouse β-actin ± SEM. *, significant difference (P < 0.05).

https://doi.org/10.1371/journal.ppat.1011752.g006

cheA₁^mut is attenuated in colonization and adaptive immune evasion

The above study showed that the mutant has reduced infectivity but is still able to disseminate into the bloodstream to cause systemic infection in SCID mice but not in immunocompetent mice. It is possible that in the presence of adaptive immunity, the low number of mutant cells that survived was unable to disseminate and cause systemic infection. To rule out this possibility, a 10-fold higher number (1 x 10⁶ cells) of spirochetes were directly introduced into the bloodstream of BALB/c mice via tail-vein injection to determine if high doses of bacteria can overcome the dissemination and colonization barrier. As a control, a non-motile, non-infectious flaB^mut strain was included [32]. 1 x 10⁶ of WT, cheA₁^mut or flaB^mut cells in 100 μl of PBS were injected into the tail-vein of BALB/c mice. Tissues were harvested two weeks post-injection to check for dissemination. All mice injected with the WT strain showed disseminated infection (3/3) whereas the flaB^mut control was non-infectious as expected (0/3). For mice injected with the cheA₁^mut strain, live spirochetes were successfully recovered from the skin of only 1 out of 5 mice infected, indicating that the mutant is attenuated in tissue colonization even at a high inoculum introduced directly into the bloodstream (Table 3). In addition, no mutant cells were recovered from the blood of any mice injected with cheA₁^mut. This result clearly indicates that CheA₁ is essential for the survival of B. burgdorferi in the blood. Consistent with live culture recovery, high levels of flaB transcripts were detected from mice infected with the WT strain whereas cheA₁^mut-infected mice had less than one copy of flaB transcript per 10⁵ of mouse β-actin transcript overall (most likely due to residual B. burgdorferi that was initially introduced into the bloodstream (Fig 7A)). These findings further substantiate the key role of CheA₁ in B. burgdorferi immune evasion and tissue colonization.

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Table 3. cheA₁^mut has defects in dissemination and immune evasion.^c

https://doi.org/10.1371/journal.ppat.1011752.t003

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Fig 7. cheA₁^mut has defects in dissemination and serum resistance.

(A) To determine if high dose of inoculum can restore cheA₁^mut immune evasion and colonization barriers, 10⁶ of WT, cheA₁^mut and non-motile flaB^mut [32] strains were injected into wild type mice via tail-vein and sacrificed two weeks after infection. Tissues from the skin, ear, joint, heart and spleen were harvested to assess for borrelial burden using qRT-PCR as described [17]. The data are presented as mean flaB transcript copies over 10⁵ of mouse β-actin ± SEM. (B) Serum bactericidal activity. 10⁶ cells of WT, cheA₁^mut and cheA₁^com were incubated with 20% normal human serum (NHS) or heat-inactivated serum (hiNHS) for 2 hours. The percentage of viable cells in NHS treated cells were determined after normalization to their respective hiNHS-treated groups. The data shown here are means of three independent replicates ± SEM. *, significant difference (P < 0.05).

https://doi.org/10.1371/journal.ppat.1011752.g007

cheA₁^mut is more susceptible to serum bactericidal activity

B. burgdorferi is able to resist the bactericidal activity of serum [33,34] as it produces several virulence factors that protects the spirochetes from complement mediated killing, such as BBK32, OspC, and CRASP proteins [see recent reviews [35–38]]. The attenuated infectivity of cheA₁^mut in both the SCID mice and tail-vein infection model suggests that CheA₁ is important for the immune evasion of B. burgdorferi. To determine if deletion of CheA₁ renders the spirochete susceptible to serum bactericidal activity, WT, cheA₁^mut and cheA₁^com were treated with normal human serum (NHS) or heat-inactivated NHS (hiNHS) and the percentage of viable cells after treatment was normalized against the input cells. Our data showed that cheA₁^mut was more susceptible to serum killing with ~25% survival rate as compared to ~60% survival rate of the WT and cheA₁^com in NHS (Fig 7B). This observation is in congruence with the SCID mice study where only 50% of cheA₁^mut was able to survive in the blood of infected mice and the complemented strain fully restored the survival rate to WT level (Table 2). The reduced survival of the mutant in the bloodstream can likely be attributed to its susceptibility to serum bactericidal activity.

CheA₁ is not required for colonization of Ixodes scapularis ticks

As cheA₁^mut failed to cause disseminated infection in wild-type mice, we used a microinjection-based infection procedure to artificially infect ticks [39]. For this study, Naïve I. scapularis nymphs were microinjected with equal amounts of WT, cheA₁^mut, or cheA₁^com strains. After the infection, ticks were allowed to feed to repletion on naïve C3H mice (5 ticks/ mouse and 3 mice for each strain). The spirochete burdens in engorged ticks were measured by qRT-PCR as described previously [17,40,41]. As shown in Fig 8, the bacterial burden of cheA₁^mut in fed ticks was not significantly different from that of the WT and cheA₁^com (Fig 8A). This result indicates that CheA₁ is not required for B. burgdorferi colonization and survival in the tick vector.

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Fig 8. CheA₁ is not required for B. burgdorferi acquisition and survival in tick but necessary for transmission.

(A) Detection of spirochete burdens in microinjected nymphal ticks after feeding. RNA samples were extracted from whole fed ticks (after repletion; 5 to 7 days) and subjected to qRT-PCR analysis. The bacterial burdens in ticks were measured by the number of copies of flaB transcript compared to the number of copies of tick β-actin transcript as previously described [17]. The data are presented as the means of relative levels of flaB transcript ± SEM for each strain (WT, cheA₁^mut, and cheA₁^com). (B) Detection of spirochete burdens in mice infected via tick bite. At day 14 after tick feeding, mice were sacrificed and tissues from the skin, heart, joint, and bladder were harvested for qRT-PCR analysis as previously documented [17,107]. No trace of flaB transcript was detected in the mouse tissues fed upon by cheA₁^mut infected ticks while 1 in 3 mice fed upon by cheA₁^com showed positive results at the skin and heart tissues. *, significant difference (P < 0.05). (C) Detection of spirochete burdens in naïve nymphal ticks fed on infected mice. C3H mice were artificially infected with WT or cheA₁^mut strain via needle inoculation. Naïve nymphal ticks were confined to the injection site and allowed to feed to repletion. After 72 hours, fed nymphs were collected and individually tested for the presence of flaB via qPCR. Experiments were repeated twice and data are presented as mean flaB copies per fed nymph from both data sets ± SEM.

https://doi.org/10.1371/journal.ppat.1011752.g008

cheA₁^mut fails to be transmitted to mice via tick bite

The infectivity of cheA₁^mut in mice was first evaluated via needle inoculation. However, this method is different from the natural route of mammalian infection through tick bite. As cheA₁^mut is still able to establish infection in ticks (Fig 8A), we examined whether the mutant can be transmitted to mice when fed upon by infected ticks. Groups of naïve C3H mice were allowed to be parasitized by artificially infected ticks as detailed in the Material and Methods. Seven days after tick feeding, the mice were sacrificed and specimens from the skin, ear, heart, bladder and blood were collected and subjected to qRT-PCR analysis to determine the pathogen level. Consistent with the results obtained via needle inoculation (Table 1), only the tissue specimens from mice fed on by WT- and cheA₁^com-infected ticks were positive for flaB transcripts via qRT-PCR analysis (Table 4). flaB transcripts were only detected from the skin and heart of one mouse fed upon by cheA₁^com-infected ticks, consistent with the results observed by needle inoculation of mice in which only a partial complementation was achieved with cheA₁^com (Table 1). No flaB transcript was detected in the specimens from mice fed upon by cheA₁^mut-infected ticks (Table 4), indicating that the mutant failed to be transmitted to mice even though it could colonize the tick vector. Collectively, these results suggest that CheA₁ is essential for the transmission of B. burgdorferi to mice via tick bite.

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Table 4. cheA₁^mut has defects in tick transmission.^d

https://doi.org/10.1371/journal.ppat.1011752.t004

cheA₁^mut can be acquired by naïve tick from infected mice

The tick transmission experiment revealed that cheA₁^mut was able to survive in ticks via artificial infection but failed to transmit to naïve mammalian hosts (Fig 8A and Table 4). To test the possible role of CheA₁ in the acquisition phase, mice were infected with WT or cheA₁^mut via needle inoculation. Two weeks post-injection, infection was confirmed via culturing of the skin biopsy from the injection site. Nymphs were placed and confined to the injection site and allowed to feed for 72 hours. Fed ticks were then collected for qPCR to determine the spirochete acquisition efficiency. As shown in Fig 8B, the flaB gene was successfully detected in nymphs that parasitized mutant-infected mice and its copy number was comparable to that detected in nymphs that fed on WT-infected mice (Fig 8C). It should be noted that in the majority of cheA₁^mut-infected ticks, acquisition was greatly attenuated relative to wild type-infected nymphs with 3 out of 12 nymphs showing no detectable flaB gene. This result suggests that CheA₁ is not absolutely required for spirochete acquisition by ticks from mice, but may play some role in the process.

CheA₁ affects expression of the RpoS-regulon and RpoS stability

B. burgdorferi encodes several proteins that are essential for host tissue binding/interaction and immune evasion in mammalian hosts, e.g., OspC, BBK32, and DbpB/A [42–45]. The attenuated phenotype of cheA₁^mut observed in the mouse infection studies could be the result of altered expression of virulence-associated proteins. To test this possibility, B. burgdorferi cells were cultivated under elevated temperature and reduced pH (37°C/pH 6.8) to mimic the host environment. Total protein profiles of WT, cheA₁^mut and cheA₁^com were analyzed on SDS-PAGE (Fig 9A) and a number of B. burgdorferi virulence determinants were measured by immunoblotting analysis and compared to the level of DnaK, a loading control. The protein level of RpoS was significantly reduced in the cheA₁^mut mutant as compared to the WT and cheA₁^com strains (Fig 9B). Consistent with this finding, the levels of several RpoS-regulated proteins (i.e., BBK32, OspC, and DbpA) were also significantly reduced in the mutant and restored in the complemented strain. By contrast, the level of P66, a non-RpoS regulated protein [46], was not affected by the deletion of cheA₁, suggesting that CheA₁ specifically affects RpoS and RpoS-regulon expression (Fig 9B). qRT-PCR analysis revealed that the transcript level of rpoS was slightly reduced in the mutant but not significant while the transcript levels of ospC and dbpA were significantly reduced by 65% and 75%, respectively (Fig 9C). In contrast, no significant change was observed in the mutant for ospA transcript, a RpoD-regulated gene [47]. In sum, deletion of cheA₁ leads to reduced expression of RpoS and RpoS-regulated virulence factors, which in turn could explain the attenuated infectivity observed with cheA₁^mut. Depletion of CheA₁ affects RpoS post-transcriptionally as no significant reduction in rpoS transcript was detected in cheA₁^mut (Fig 9C). To determine how CheA₁ affects RpoS protein expression, protein turnover assays were performed using WT and mutant cells cultivated at the condition mimicking the host environment to induce RpoS expression. Compared to the wild type, RpoS turnover was increased in the absence of CheA₁. The level of RpoS was relatively stable in the stationary phase of the wild-type culture whereas over 50% turnover of RpoS was observed in cheA₁^mut as early as one hour after protein synthesis was arrested (Fig 9D). Complementation of CheA₁ successfully restored the stability of RpoS (S7 Fig). Thus, the reduced RpoS protein level observed in cheA₁^mut is due to reduced protein stability in the absence of CheA₁.

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Fig 9. Depletion of CheA₁ negatively affects the level of RpoS, BBK32, OspC, and DbpA.

B. burgdorferi strains (WT, cheA₁^mut, and cheA₁^com) were cultivated at 37°C/pH 6.8 to mimic the mammalian host condition and harvested at stationary phase (∼10⁸ cells/ml) for (A,B) immunoblotting analysis, and (C) qRT-PCR analysis. (A) Similar amounts of whole-cell lysates were analyzed by stain-free SDS-PAGE. Gel image showed the total protein of WT, cheA₁^mut, and cheA₁^com analyzed on SDS-PAGE. (B) Immunoblot analysis of WT, cheA₁^mut, and cheA₁^com samples using specific antibodies against CheA₂, CheA₁ [11], P66 [101], BBK32 [102], RpoS, OspC, and DbpA [86]. DnaK was used as an internal control, as previously described [97]. (C) Detection of rpoS, ospC, dbpA, and ospA transcript level in WT, cheA₁^mut, and cheA₁^com using qRT-PCR. The dnaK transcript was used as an internal control. Experiments were repeated at least twice with independent replicates. Data from two replicates are expressed as mean fold change relative to WT ± SEM. *, significant difference (P < 0.05). (D) RpoS protein turnover in WT and cheA₁^mut. The stability of RpoS protein was examined in stationary phase WT B31 A3-68 and B31 A3-68 cheA₁^mut upon protein synthesis arrest using spectinomycin. Samples were harvested at the indicated time points and probed using antibody against RpoS and DnaK (as a loading control). Experiments were repeated at least three times with independent biological replicates. A representative image is shown here.

https://doi.org/10.1371/journal.ppat.1011752.g009

Depletion of CheA₁ leads to dysregulated expression of proteases

The homeostasis and stability of proteins are governed by a set of proteases responsible for the regulated degradation of a given protein target [48,49]. The genome of B. burgdorferi encodes several proteases, including Lon-1 [50], Lon-2 [51], HtrA [52–55], CtpA [56], and Clp [9]. Lon-1 protease was shown to negatively affect RpoS expression at the transcriptional level [50] while Lon-2 protease positively regulates RpoS expression at the post-transcriptional level [51]. In other bacteria, such as E. coli, the protein stability of RpoS is governed by the ClpXP protease [57–59]. Since the stability of RpoS protein was affected in cheA₁^mut, the expression of these known proteases was examined in the mutant. qRT-PCR data from two independent biological replicates consistently showed that the transcripts of clpX, lon-1, and lon-2 were significantly reduced in cheA₁^mut as compared to the WT, which was successfully restored in the complemented strain (Fig 10A). Preliminary RNA-seq analysis also revealed a significant down-regulation of clpX, lon-1 and lon-2 expression in cheA₁^mut (S8 Fig). Our data suggest that depletion of CheA₁ leads to dysregulated expression of B. burgdorferi proteases, such as clpX and lon, which subsequently affects the expression and protein stability of RpoS in a negative manner. In congruence with this, deletion of clpX significantly impaired the expression of RpoS along with the various RpoS-regulated proteins (Fig 10B) while deletion of lon-1 led to a high level of RpoS expression as shown by Thompson et al. [50], further supporting the regulatory role of ClpX and Lon proteases on RpoS expression.

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Fig 10. Depletion of CheA₁ leads to dysregulated expression of proteases.

(A) Detection of clpX, lon-1, and lon-2 transcript level in WT, cheA₁^mut, and cheA₁^com using qRT-PCR. The dnaK transcript was used as an internal control. Experiment was repeated with two independent replicates. Data are expressed as mean fold change relative to WT ± SEM. *, significant difference (P < 0.05). (B) Immunoblot analysis of WT, clpX^mut, and lon-1^mut samples using specific antibodies against P66 [101], BBK32 [102], RpoS, OspC, and DbpA [86]. DnaK was used as an internal control, as previously described [97]. Experiments were repeated twice with independent replicates. Lon-1 mutant (lon-1^mut) was kindly provided by the Ouyang’s lab as a positive control to show the up-regulation of RpoS as previously described [50].

https://doi.org/10.1371/journal.ppat.1011752.g010

cheA₁^mut fails to elicit mouse il-10, tnf-α, ccl2, and il-1β expression

The attenuated phenotype of cheA₁^mut in establishing systemic infection even in immunodeficient mice implicates a defect in both innate and adaptive immune evasion. Interleukin 10 (IL-10) is a key mediator in innate immune responses during Lyme disease infection [60]. Timely induction of IL-10 expression is critical for the disease progression by dampening the host inflammatory response to favor the survival of B. burgdorferi during early infection. To dissect the cause behind cheA₁^mut failure to establish systemic infection in mice, RNA-seq analysis was performed using infected mouse skin tissues. Briefly, 100 μl of 1 x 10⁷ WT or cheA₁^mut were needle inoculated into the dorsal side of BALB/c mice at three separate locations. Skin tissues were harvested from the inoculation sites at 72-hour post-injection (p.i.) for RNA extraction and RNA-seq analysis. Scatter plot of enriched KEGG pathway showed that distinct host genes were differentially induced between Sham vs WT and Sham vs MT (cheA₁^mut) infected samples at 72 hrs p.i. (Fig 11A). In WT-infected samples, significant changes of numerous immune system related genes were observed, such as genes involved in leukocyte, granulocyte, and neutrophil migration. These genes were unchanged in the mutant infected samples. The transcript levels of several important cytokines and chemokines that have been reported to contribute to Lyme disease progression, such as IL-10, IL-1β, TNF-α, and CCL2 [61–68], were upregulated in the WT- but not MT-infected tissues (Fig 11B), which was validated by qRT-PCR (Fig 11C). Consistent with the RNA-seq analysis, qRT-PCR showed that the transcript levels of il-10, il-1β, and ccl-2 were significantly lower in the cheA₁^mut-infected mouse skin tissues as compared to those infected by WT (Fig 11C). The data obtained from RNA-seq analysis suggests that the inability of cheA₁^mut to establish systemic infection is partly due to its failure in eliciting timely expression of key host cytokines and chemokines to aid in dissemination and colonization.

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Fig 11. RNA-seq analysis of BALB/c mouse skin tissues infected by WT and cheA₁^mut.

(A) Scatter plot of enriched KEGG pathway of differentially expressed genes between Sham and infected samples at 72hr p.i. Vertical coordinates represent pathway name, and horizontal coordinates represent Rich factor. The size and color of point represent the number of differential genes in the pathway and the range of different Q value, respectively. (B) Volcano plot of differentially expressed genes (DEG) between MT (cheA₁^mut) vs WT samples at 72hr p.i.. Each dot represents a gene, and its position is determined by the log2 fold change (x-axis) and the -log10 (p-value) (y-axis). Genes with significant upregulation (red) or downregulation (green) are highlighted, while non-significant genes are shown in gray. (C) Detection of il-10, tnf, il-1β, and ccl-2 transcript levels in WT and cheA₁^mut infected mouse skin tissues using qRT-PCR. The β-actin transcript was used as an internal control. Data are expressed as mean fold change relative to WT ± SEM. *, significant difference (P < 0.05).

https://doi.org/10.1371/journal.ppat.1011752.g011

Ethics statement

All animal experimentation was conducted following the NIH guidelines for housing and care of laboratory animals and performed in accordance with the Virginia Commonwealth University institutional regulations after review and approval by the Institutional Animal Care and Use Committees [IACUC approval number: #AD10001779].

Bacterial strains and growth conditions

Infectious clone A3-68 and A3-68∆bbe02 (wild type), two derivative strains from the B. burgdorferi sensu stricto B31-A3, were used in this study [25]. They were a kind gift from P. Rosa (Rocky Mountain Laboratories, NIAID, NIH). Cells were grown in Barbour-Stoenner-Kelly (BSK-II) medium as previously described [97] in appropriate antibiotic(s) for selective pressure as needed: streptomycin (50 μg/ml), kanamycin (300 μg/ml), and/or gentamicin (40 μg/ml). To determine the expression of CheA₁, 10⁵ stationary phase wild type cells were inoculated into 10 ml of fresh BSK-II medium and then cultured at 23°C/pH 7.6 (unfed tick, UF) or 34°C/pH 7.6 (a routine laboratory cultural condition). To examine the expression of RpoS and RpoS-regulon, 10⁵ stationary phase wild type, mutants, and complemented strains were inoculated into 10 ml of fresh BSK-II medium and then cultivated at 37°C/pH 6.8 with 5% CO₂ to mimic the host condition. Cells were harvested for immunoblot analysis upon entry into stationary phase at ~10⁸ cells/ml.

CheA₁, CheA₂, and TmCheA AlphaFold model generation and analysis

To build CheA₁, CheA₂, and TmCheA models, protein sequences for TmCheA (AAA96387.1), CheA₁ (AAC66935.1), and CheA₂ (AAC67024.1) were retrieved from the NCBI database and submitted for automated model-building using AlphaFold2 [20]. All parameters were kept at their default values for model building.

Construction of CheA₁/pBBE22 for complementation of cheA₁ mutant

pGA₁kan, a previously constructed vector with an internal 393-bp deletion in cheA₁ gene (nucleotides 1,016–1,409) [11], was used to inactivate the cheA₁ in B31 A3-68 and A3-68∆bbe02 strains via allelic exchange mutagenesis. All the experiments performed in this report was done using mutants generated in the A3-68∆bbe02 background except for RpoS protein turnover analysis in which the cheA₁ mutant was generated in the B31 A3-68 background because A3-68∆bbe02 carries a streptomycin resistant cassette. To complement the cheA₁ mutant, CheA₁/pBBE22 (Fig 2A), was constructed via PCR ligation using primer pair P₁/P₂ to amplify bb0566-cheA₁ region and cloned into pBBE22G vector at BamHI restriction site [17,98,99]. All primers used in this study are listed in Table 5.

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Table 5. Oligonucleotide primers used in this study.^e

https://doi.org/10.1371/journal.ppat.1011752.t005

Construction of CheA₁IFD for in-frame deletion of cheA₁

To create an alternative cheA₁ mutant, an in-frame deletion construct was made using PCR ligation method to in-frame replace the entire open reading frame (orf) of cheA₁ with kanamycin (kan) cassette. The left and right arm of the deletion construct was PCR amplified using P₂₇/P₂₈ and P₂₉/P₃₀, respectively, and the kan cassette was amplified using P₃₁/P₃₂ (Table 5). The resulting three amplicons were then PCR ligated using P₂₇/P₃₀ and ligated to pJET1.2/blunt cloning vector (Thermo Scientific, Waltham, MA) yielding CheA₁IFD (S4B Fig).

Localization of CheA₁ and CheA₂ in B. burgdorferi

For the localization of CheA₁ and CheA₂, cheA₁-gfp/pBSV2G (S3A Fig) and cheA₂-gfp/pBSV2G (S3B Fig) constructs were used to complement cheA₁^mut and a previously constructed cheA₂ mutant strain [11], respectively. A control plasmid with gfp driven by the flgB promoter was included as a control for the localization study [100]. To determine the localization of CheA₁ and CheA₂, GFP construct expressing cells were inoculated into 10 ml of fresh BSK-II medium and then cultured at 23°C/pH 7.6 or 34°C/pH 7.6. Cells were observed every 48 hours for up to 2 weeks. Images were taken using a Zeiss Axiostar plus microscope and processed using Axiovision software (Zeiss, Germany) as previously described [100].

SDS-PAGE and immunoblots

10 to 20 μg of B. burgdorferi whole cell lysates were separated on 10 or 12% Stain-Free SDS-PAGE gel and transferred to PVDF membrane (Bio-Rad Laboratories, Hercules, CA). Stain-Free SDS-PAGE was first activated and imaged using ChemiDoc MP Imaging System (Bio-Rad Laboratories) prior to membrane transferring to visualize total proteins on the gel. The immunoblots were probed with antibodies against B. burgdorferi CheA₁, CheA₂ [11], P66 [101], BBK32 [102], RpoS, OspC, and DbpA [86]. DnaK was used as an internal control, as previously described [97]. Membranes were developed using horseradish peroxidase secondary antibody with an ECL luminol assay or with fluorescently labelled secondary antibodies. Signals were imaged using the ChemiDoc MP Imaging System and quantified with the Image Lab software (Bio-Rad Laboratories).

Bacterial motion tracking analysis, swimming plate, and capillary tube-based chemotaxis assays

The swimming velocity of B. burgdorferi cells was measured using a computer-based motion tracking system as previously described [31]. Swimming plate assays were conducted as previously documented [11,32,103]. The diameters of swimming rings were recorded in millimeters. Wild-type A3-68 Δbbe02 was used as a positive control. A non-motile strain, flgE mutant, [103] was used as a negative control to monitor the initial inoculum size. For the capillary tube assay, N-acetyl-D-glucosamine (NAG) was used as an attractant as previously reported [31]. The spirochete cells accumulated in the capillary tubes were enumerated using Petroff-Hausser counting chambers. The mean of three replicates was determined and the data are expressed as the mean relative increase over a buffer control containing no attractant for each group. A two- or more fold increase in the number of spirochetes in comparison to buffer control is considered as significant.

Quantitative reverse transcription PCR (qRT-PCR)

Ticks and mouse tissue samples for qRT-PCR analysis were prepared as described [17,97]. Briefly, total RNA from ticks or mouse tissues was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA) and contaminating genomic DNA was removed using Turbo DNase (Ambion, Austin, TX). The DNase-treated RNAs were re-purified and converted to cDNA using SuperScript IV VILO Master Mix (Invitrogen) according to the manufacturer’s instructions. Quantitative PCR was then performed using Fast SYBR Green Master Mix (Applied Biosystems, Foster City, CA). The spirochete burdens within infected mice and ticks were expressed as the flaB transcript levels relative to the copy number of the mouse or tick β-actin transcript levels as described previously [17]. Primers used in qRT-PCR are summarized in Table 5. Data are expressed as the mean fold change of two replicates relative to WT ± SEM. The significance of the difference between different experimental groups was evaluated with ANOVA (P value < 0.05).

Protein turnover assay

To determine the stability of RpoS protein, protein turnover was performed as described previously [104] using wild-type A3-68 and a cheA₁ mutant constructed in the same background. The mutant was confirmed to have the same antigen profile as cheA₁^mut [103,104]. Briefly 10⁵ cells/ml of B. burgdorferi cells were inoculated into 50 ml of BSK-II medium, pH 6.8, and cultivated at 37°C to mid log phase (5x10⁶ cells/ml) or stationary phase (10⁸ cells/ml). 100 μg/ml of spectinomycin was added to the culture to arrest protein synthesis. 5 ml of cells were harvested at 0, 1, 2, 3, 4, 6, 8, and 24 hours post-protein arrest for SDS-PAGE followed by immunoblots against RpoS and DnaK.

Mouse infection studies

BALB/c or BALB/c SCID mice at 6–8 weeks of age (Jackson Laboratory, Bar Harbor, MN) were used in the needle infection study. The animal studies were carried out as previously described [17,97]. Briefly, mice were given a single subcutaneous injection of 10⁵ spirochetes and sacrificed 3 weeks post-infection. For time point inoculation site study, mice were similarly infected with WT or cheA₁^mut and the initial inoculation sites were marked for harvesting at 96 hour, 14 day, 21 day, and 28 day post-infection. Skin specimens were subjected to qRT-PCR analysis as described above.

For tail-vein infection, 10⁶ spirochetes re-suspended in 100 μl PBS was injected into the tail-vein of BALB/c mice. The non-motile non-infectious flaB mutant was included as a negative control [32]. Mice were sacrificed two weeks after infection. Tissues from the ear, skin, joint, and heart were harvested and placed into 1 ml BSK-II medium containing 5 μg/ml of rifampicin. The samples were incubated at 34°C for up to one month and microscopically monitored for the presence of spirochetes. Tissues were similarly collected for qRT-PCR analysis to assess for spirochete burdens. For bulk RNA-seq analysis, 10⁷ spirochetes in 100 μl of BSK-II was injected into the dorsal side of BALB/c mice at three separate locations. For sham control, equal volume of BSK-II medium was injected instead. Mice were sacrificed at 72-hour post-injection. Skin tissues from the injection sites were harvested for RNA extraction as described above.

Serum bactericidal assays

Serum bactericidal assay was performed as previously described [43]. Normal human serum was purchased from BioIVT (Westbury, NY). (Briefly, wild-type A3-68∆bbe02, cheA₁^mut and cheA₁^com were grown to mid log phase at 34°C/pH 7.6. 10⁶ cells in 80 μl of BSK-II medium were inoculated into a 96-well plate followed by the addition of 20 μl of normal human serum (NHS) or heat inactivated NHS (hiNHS) to get a final concentration of 20% serum in 100 μl. Cells were incubated at 34°C for 2 h. The percentage of viable cells after incubation were enumerated under dark field microscope using Petroff-Hausser counting chambers based on immobilization, cell lysis, and loss of cell membrane integrity. Experiments were repeated at least three times. Data is presented as percent viable cells relative to input cells.

Tick-mouse transmission study

The tick-mouse transmission study was carried out as previously described [17,41]. Briefly, naïve Ixodes scapularis nymph (Oklahoma State University, Stillwater, OK) were artificially infected by microinjection as previously described [39]. Two days after injection, infected nymphs were allowed to parasitize on naïve C3H mice (5 ticks/mouse and 3 mice for each B. burgdorferi strain) for 5 to 7 days and allowed to fall off. Engorged ticks were collected and subjected to qRT-PCR analysis to determine spirochete burdens. At day 14 after the tick feeding, mice were sacrificed and tissues from the skin, heart, joint, bladder and blood were harvested for quantification of spirochete burden via qRT-PCR analysis to determine transmission rate as described previously [41]. Spirochetal burden in fed ticks is presented as log copies of flaB / 10³ tick β-actin copies while transmission data is shown as flaB copies per 10⁶ mouse β-actin.

Tick acquisition studies

To examine the ability of cheA₁^mut to be acquired by feeding ticks, 1 x 10⁴ of either WT or cheA₁^mut was injected subcutaneously into two naïve C3H mice each. Two weeks post-infection, skin biopsy was obtained from the site of injection to confirm present of live spirochetes via culturing. 2–3 naïve I. scapularis nymphs were placed onto the infected mice at the site of inoculation and allowed to feed to repletion. After 72 hours, fed nymphs were collected and tested individually for the presence of flaB via qPCR. Tick acquisition experiments were repeated twice and the mean of both data sets are presented as flaB copies per fed nymph.

Construction of an in-frame deletion mutant of clpX

An in-frame deletion construct of clpX was made using the same PCR ligation method as described for CheA₁IFD. The orf of clpX was replaced with kan cassette. The left and right arm of the deletion construct was PCR amplified using P₂₃/P₂₄ and P₂₅/P₂₆, respectively and kan cassette was amplified using P₃₁/P₃₂ (Table 5). The resulting three amplicons were then PCR ligated using P₃₉/P₄₂ and ligated to pJET1.2/blunt cloning vector (Thermo Scientific) and used for transformation into wild-type A3-68 strain.

Mouse RNA-seq analysis

Total RNAs were extracted from mouse skin tissues using TRIzol reagent as described and treated with DNase to remove contaminating genomic DNA. RNA samples were sent to CD Genomics (CD Genomics, Shirley, NY) for RNA-seq analysis. Briefly, rRNA was depleted using Illumina Ribo-Zero Plus rRNA Depletion Kit. Library preparation was performed with NEBNext Ultra II RNA (non-directional) Kit. Paired-end reads with a length of ~150 nt were generated with NovaSeq 6000 platform. Quality control was performed with Fastp with low-quality reads filtered. Reads were aligned to the host reference genome (GRCm39). Quantification was then performed based on the alignment results, followed by differentially expressed gene analyses with DESeq2. Significant differentially expressed genes (DEG) between pairwise comparisons were identified with threshold |log2FC| > 0.5, and p < 0.01. All the significant DEGs were annotated with eggNOG, COG, Pfam, Swiss-Port, and KEGG databases. GO enrichment analysis and KEGG pathway analysis were performed with ClusterProfiler in R.

Statistical analysis

For the swimming plates, motion tracking, capillary tube assays, and the mouse and tick infection studies, the results are expressed as means ± standard error of the mean (SEM). The significance of the difference between different experimental groups was evaluated with an unpaired Student t test or ANOVA (P value < 0.05). For serum bactericidal assay, statistical significance was determined using multiple t test (P value < 0.05) followed by the Holm–Bonferroni method to correct for multiple comparisons. Data is depicted as the mean of all replicates ± SEM.