Oligonucleotide primers used in this study.^<b>e</b>

As an enzootic pathogen, the Lyme disease bacterium Borrelia burgdorferi possesses multiple copies of chemotaxis proteins, including two chemotaxis histidine kinases (CHK), CheA1 and CheA2. Our previous study showed that CheA2 is a genuine CHK that is required for chemotaxis; however, the role of CheA1 remains mysterious. This report first compares the structural features that differentiate CheA1 and CheA2 and then provides evidence to show that CheA1 is an atypical CHK that controls the virulence of B. burgdorferi through modulating the stability of RpoS, a key transcriptional regulator of the spirochete. First, microscopic analyses using green-fluorescence-protein (GFP) tags reveal that CheA1 has a unique and dynamic cellular localization. Second, loss-of-function studies indicate that CheA1 is not required for chemotaxis in vitro despite sharing a high sequence and structural similarity to its counterparts from other bacteria. Third, mouse infection studies using needle inoculations show that a deletion mutant of CheA1 (cheA1mut) is able to establish systemic infection in immune-deficient mice but fails to do so in immune-competent mice albeit the mutant can survive at the inoculation site for up to 28 days. Tick and mouse infection studies further demonstrate that CheA1 is dispensable for tick colonization and acquisition but essential for tick transmission. Lastly, mechanistic studies combining immunoblotting, protein turnover, mutagenesis, and RNA-seq analyses reveal that depletion of CheA1 affects RpoS stability, leading to reduced expression of several RpoS-regulated virulence factors (i.e., OspC, BBK32, and DbpA), likely due to dysregulated clpX and lon protease expression. Bulk RNA-seq analysis of infected mouse skin tissues further show that cheA1mut fails to elicit mouse tnf-α, il-10, il-1β, and ccl2 expression, four important cytokines for Lyme disease development and B. burgdorferi transmigration. Collectively, these results reveal a unique role and regulatory mechanism of CheA1 in modulating virulence factor expression and add new insights into understanding the regulatory network of B. burgdorferi.</div

Additional file 2: Table S1. of Public health impact of strain specific immunity to Borrelia burgdorferi

Frequency of the different OspC types that were cultured from the skin of 200 patients with erythema migrans (17, 18) and expected percentage increase in total reinfections due to particular OspC types, if there were no strain specific immunity based on the deterministic probability model. Bold font indicates invasive OspC types, comprising 76.5 % of the total cases. (DOC 35 kb

<i>cheA₁^mut</i> 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, cheA1mut and cheA1com 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).</p

Transcriptional analysis of selected genes at different stages of the enzootic cycle.

<p>qRT-PCR was performed with RNA isolated from strain B31-A3-infected ticks and mouse joints as described in Methods. Statistical analysis was determined by Kruskal-Wallis multiple comparison Z- value test; * significant difference (<i>P</i>≤.05) between samples. Error bars indicate standard error of the mean (SEM).</p

Multiple sequence alignment analysis (MSA) of <i>Treponema</i> spp. that contain a single CheA isoform.

(A) MSA of Treponema spp. CheA P2 domain sequences. BbCheA1 and BbCheA2 sequences are included for comparison. The location of BbCheA2 P2α is marked with a black box. (B) MSA of Treponema spp. CheA P3 domain sequences. BbCheA1 and BbCheA2 sequences are included for comparison. The location of BbCheA2 P3 domain is marked with a black box. Sequences collected using Annotree [1], MSA files generated using Clustal Omega [6]. (TIFF)</p

Oligonucleotide primers used in this study.

As an enzootic pathogen, the Lyme disease bacterium Borrelia burgdorferi possesses multiple copies of chemotaxis proteins, including two chemotaxis histidine kinases (CHK), CheA1 and CheA2. Our previous study showed that CheA2 is a genuine CHK that is required for chemotaxis; however, the role of CheA1 remains mysterious. This report first compares the structural features that differentiate CheA1 and CheA2 and then provides evidence to show that CheA1 is an atypical CHK that controls the virulence of B. burgdorferi through modulating the stability of RpoS, a key transcriptional regulator of the spirochete. First, microscopic analyses using green-fluorescence-protein (GFP) tags reveal that CheA1 has a unique and dynamic cellular localization. Second, loss-of-function studies indicate that CheA1 is not required for chemotaxis in vitro despite sharing a high sequence and structural similarity to its counterparts from other bacteria. Third, mouse infection studies using needle inoculations show that a deletion mutant of CheA1 (cheA1mut) is able to establish systemic infection in immune-deficient mice but fails to do so in immune-competent mice albeit the mutant can survive at the inoculation site for up to 28 days. Tick and mouse infection studies further demonstrate that CheA1 is dispensable for tick colonization and acquisition but essential for tick transmission. Lastly, mechanistic studies combining immunoblotting, protein turnover, mutagenesis, and RNA-seq analyses reveal that depletion of CheA1 affects RpoS stability, leading to reduced expression of several RpoS-regulated virulence factors (i.e., OspC, BBK32, and DbpA), likely due to dysregulated clpX and lon protease expression. Bulk RNA-seq analysis of infected mouse skin tissues further show that cheA1mut fails to elicit mouse tnf-α, il-10, il-1β, and ccl2 expression, four important cytokines for Lyme disease development and B. burgdorferi transmigration. Collectively, these results reveal a unique role and regulatory mechanism of CheA1 in modulating virulence factor expression and add new insights into understanding the regulatory network of B. burgdorferi.</div

CheA₁ is not required for <i>B</i>. <i>burgdorferi</i> 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, cheA1mut, and cheA1com). (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 cheA1mut infected ticks while 1 in 3 mice fed upon by cheA1com showed positive results at the skin and heart tissues. *, significant difference (P (C) Detection of spirochete burdens in naïve nymphal ticks fed on infected mice. C3H mice were artificially infected with WT or cheA1mut 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.</p

lp36 contributes to the association of <i>B. burgdorferi</i> with specific populations of dendritic cells.

<p>Human PBMCs (4×10⁶) were co-incubated with 4×10⁷ GFP-tagged B31 (black bars), A3-M9 lp36− (white bars) or A3-M9 lp36−/lp36+ (cross-hatched bars) <i>B. burgdorferi</i> for 6 hours at 4°C or 37°C. The percentages of GFP⁺ mDC1s (CD19⁻CD3⁻BDCA2⁻BDCA1⁺) (<b><i>A</i></b>), pDCs (CD19⁻CD3⁻BDCA2⁺BDCA1⁻) or (<b><i>B</i></b>) mDC2s (CD19⁻CD3⁻BDCA3⁺BDCA2⁻) (<b><i>C</i></b>) were determined by multiparameter flow cytometry. Dot plots representing 500,000 collected events are provided to illustrate gating strategies (left). Column graphs represent the mean and standard deviation of three biological replicates (right). Statistical analysis was performed using a one-way ANOVA with a Tukey’s post-test for multiple comparisons.</p

Oligonucleotides used for plasmid content analysis.

<p>Oligonucleotides used for plasmid content analysis.</p

Additional file 1: Figure S1. of Public health impact of strain specific immunity to Borrelia burgdorferi

The proportion of cases occurring in patients with a prior infection and the proportion of averted cases increases exponentially with higher incidence rates in both the equilibrium dynamic (left column, panels A, C) and individual stochastic models (right column, panels B, D). The proportion of reinfections that are averted due to strain-specific immunity (bottom row, panels E, F) is constant across incidence rates in both models. The dashed lines describe the data output when strain-specific immunity is assumed to last 5 years; the black lines describe the data output when strain-specific immunity is assumed to last 30 years. (DOC 311 kb