The BB0345 Hypothetical Protein of Borrelia burgdorferi Is Essential for Mammalian Infection

Abstract

During the natural enzootic life cycle of Borrelia burgdorferi (also known as Borreliella burgdorferi), the bacteria must sense conditions within the vertebrate and arthropod and appropriately regulate expression of genes necessary to persist within these distinct environments. bb0345 of B. burgdorferi encodes a hypothetical protein of unknown function that is predicted to contain an N-terminal helix-turn-helix (HTH) domain. Because HTH domains can mediate protein-DNA interactions, we hypothesized that BB0345 might represent a previously unidentified borrelial transcriptional regulator with the ability to regulate events critical for the B. burgdorferi enzootic cycle. To study the role of BB0345 within mammals, we generated a bb0345 mutant and assessed its virulence potential in immunocompetent mice. The bb0345 mutant was able to initiate localized infection and disseminate to distal tissues but was cleared from all sites by 14 days postinfection. In vitro growth curve analyses revealed that the bb0345 mutant grew similar to wild-type bacteria in standard Barbour-Stoenner-Kelley II (BSK-II) medium; however, the mutant was not able to grow in dilute BSK-II medium or dialysis membrane chambers (DMCs) implanted in rats. Proteinase K accessibility assays and whole-cell partitioning indicated that BB0345 was intracellular and partially membrane associated. Comparison of protein production profiles between the wild-type parent and the bb0345 mutant revealed no major differences, suggesting BB0345 may not be a global transcriptional regulator. Taken together, these data show that BB0345 is essential for B. burgdorferi survival in the mammalian host, potentially by aiding the spirochete with a physiological function that is required by the bacterium during infection.

Keywords: Borrelia; Borrelia burgdorferi; Borreliella; Lyme disease; molecular genetics; pathogenesis; spirochetes.

FIG 1

Construction and confirmation of BbΔbb0345. (A) Schematic illustrating inactivation of bb0345 in Bb297 and genetic complementation in BbΔbb0345. pJSB436A, relevant region of suicide vector used to inactivate bb0345; Bb297, bb0345 and flanking regions in B. burgdorferi chromosome; BbΔbb0345, predicated genomic structure of bb0345::Kan mutation. Solid black arrows represent relative positions of primers used to confirm BbΔbb0345 by PCR. The complementation constructs introduced into BbΔbb0345 are shown at the right. pJSB450A, bb0345 ORF and 420 bp immediately upstream of bb0345 start codon; pJSB654A, bb0345 ORF and putative bb0346 promoter region. (B) PCR analysis of Bb297-, BbΔbb0345-, and pJSB654A-complemented bb0345 (Bbbb0345^C). PCR with primers P1 and P2, shown in panel A, produced different product sizes, 828 bp (Bb297 and Bbbb0345^C) and 1,578 bp (BbΔbb0345), respectively. PCR with H₂O served as a contamination control. pJSB436A served as a positive amplification control for the disrupted bb0345 allele. DNA-size standard values in kilobases (kb) are indicated on the left. (C) Bb297, BbΔbb0345, BbJSB450, and Bbbb0345^C were cultivated in vitro to late-exponential phase at pH 7.5 and 37°C. Whole-cell lysates were separated by SDS-PAGE, transferred to nitrocellulose, and assessed by immunoblot. Antibodies used to detect BB0345 and FlaB are indicated at the right. Molecular weight standard (kDa) values are indicated on the left. FlaB was included as a loading control. Samples from three independent biological replicates were analyzed and shown to yield similar results. Depicted is a representative immunoblot from one of these biological replicates.

FIG 2

Analysis of bb0345 operon organization by RT-PCR and 5′ RACE. (A) Bb297 RNA was extracted, and cDNA was synthesized using random priming. Primer pairs for RT-PCR were designed to amplify individual ORFs or across the bb0345-bb0346 intergenic region. G, genomic DNA; −, negative-control reaction mixture lacking RT; +, presence of RT in the reaction. Primer pairs are indicated above each set, and arrows on the schematic represent relative positions of primers. DNA-size standard values (kb) are indicated on the left. (B) A schematic of the TSS mapped by 5′ RACE and predicted promoter elements. −35 and −10, putative promoter region; SD, putative Shine-Dalgarno site; bent arrow, first codon of the bb0346 ORF. The TSS, mapped to 73 bp upstream of the bb0346 start codon, is indicated by the asterisk. The red bracket indicates the position of a predicted promoter 69 to 96 nucleotides upstream of the bb0346 start codon.

FIG 3

Spirochete burdens from BbΔbb0345-infected mice. Mice were needle inoculated with a dose of 10⁵ spirochetes of Bb297, BbΔbb0345, and Bbbb0345^C. DNA was analyzed by qPCR from tissue samples collected at 3, 5, 9, 11, and 14 days postinfection. The results from inoculation site skin, dorsal skin, tibiotarsal, and heart represent a single infection experiment (n = 5 mice per strain). The results represent the mean values from two independent assays, with each sample measured in triplicate during each assay. The error bars represent SEM. Statistical significance was determined using ANOVA with Dunnett’s procedure for multiple comparisons. *, statistically significant (P < 0.05) difference relative to the Bb297 strain.

FIG 4

In vitro growth analysis of BbΔbb0345 in BSK-II. (A) Bb297, BbΔbb0345, and Bbbb0345^C were inoculated into BSK-II medium at a starting density of 10³ bacteria/ml and incubated for 8 days at 35°C. Starting at 3 days postinoculation, cultures were counted daily using dark-field microscopy. (B) Bb297, BbΔbb0345, and Bbbb0345^C were inoculated in 25% BSK-II diluted in PBS at a starting density of 10³ bacteria/ml and incubated for 17 days at 35°C. Starting at 6 days postinoculation, cultures were counted daily using dark-field microscopy. Values in the growth curves represent the mean cell counts ± SEM. Shown are representative data from one of two independently repeated growth curve experiments that generated equivalent results. Statistical significance was determined using ANOVA with Dunnett’s procedure for multiple comparisons. *, statistically significant (P < 0.05) decrease relative to the Bb297 strain.

FIG 5

Regulation of bb0345 during in vitro growth. Bb297 was cultivated in vitro at pH 7.5 to early exponential phase at 23°C (unfed tick conditions) or pH 6.8 to late exponential phase at 37°C (fed tick/mammalian conditions). (A) qRT-PCR analysis of bb0345 expression. Detection of ospC was used as a control for in vitro culture adaptation. The data represent two biological replicates with measurements performed in duplicate, and error bars represent SEM. Statistical significance was determined using an unpaired Student's t test. *, statistically significant (P < 0.05) difference relative to the 37°C/pH 6.8 condition. (B) Immunoblot analysis of BB0345 protein levels. Whole-cell lysates were separated by SDS-PAGE, transferred to nitrocellulose membrane, and assessed by immunoblot. Antibodies used to detect BB0345, OspC, and FlaB production are indicated at the right. Molecular weight standard (kDa) values are indicated on the left. FlaB and OspC were used as negative and positive controls, respectively. Samples from three independent biological replicates were analyzed and shown to yield similar results. Depicted is an immunoblot from one of these biological replicates.

FIG 6

Expression of bb0345 by spirochetes in infected murine tissues. RNA was isolated from tissues collected 2 weeks postinfection from mice needle inoculated with Bb297 at a dose of 10⁵ spirochetes. bb0345 expression by Bb297 in mouse tissues was quantified by qRT-PCR. The results are derived from a single-infection experiment (n = 5 mice). The results represent the mean values from two independent assays, with each sample measured in triplicate during each assay. The error bars represent SEM. Statistical significance was determined using ANOVA with Tukey’s procedure for multiple comparisons. *, statistically significant (P < 0.05) difference relative to heart tissue.

FIG 7

Assessing the impact of BB0345 on protein expression. (A) Whole-cell lysates of Bb297, BbΔbb0345, and Bbbb0345^C, grown to the late exponential growth phase at pH 7.5 and 37°C, were separated by SDS-PAGE and stained with Coomassie brilliant blue. A representative result is shown. The 30-, 20-, and 17-kDa bands noted in the text are indicated to the right by black, red, and blue asterisks, respectively. Molecular weight standard (kDa) values are indicated on the left. (B and C) Cultures of Bbibb0345 were grown to the mid-exponential growth phase and induced with 0.05 mM IPTG for 12 h; a representative result from two biological replicates is shown. Whole-cell lysates were prepared, and proteins were separated by SDS-PAGE for Coomassie brilliant blue (B) or transferred to nitrocellulose membranes for Western blot (C). Levels of BB0345, OspC, and FlaB, identified to the right of each panel, were assessed by Western blotting. Positions of the molecular weight standards (kDa) are indicated on the left. (D) Whole-cell lysates of Bb297 and BbΔbb0345 were separated by SDS-PAGE gel and transferred to nitrocellulose membranes. The membrane was then probed with pooled serum samples from the 42-day postinfection group of mice to evaluate for seroreactivity. Bands with stronger intensity in Bb297 are indicated with green asterisks, and the band with stronger intensity in BbΔbb0345 is indicated by the orange asterisk. Molecular weight standard (kDa) values are indicated on the left.

FIG 8

Cellular localization of BB0345. (A) Proteinase K accessibility assay. Bb297 was incubated with (+) or without (−) proteinase K in the presence or absence of Triton X-100 for 1 h. Proteins were separated by SDS-PAGE, transferred to nitrocellulose membrane, and assessed by immunoblot. FlaB and OspC were included as periplasmic and extracellular controls, respectively. Molecular weight standard (kDa) values are indicated on the left. (B) Whole-cell partitioning. Bb297 was grown to exponential growth phase. Cells were collected, sonicated, and incubated overnight at 4°C with Triton X-114. The aqueous (AQ) and detergent (DET) phases were to enrich for soluble and membrane-associated proteins, respectively. Proteins were separated by SDS-PAGE, transferred to nitrocellulose or PVDF membranes, and assessed by immunoblot. OspC and BB0796 were included as DET- and AQ-phase controls, respectively. Molecular weight standard (kDa) values are indicated on the left. Samples from three independent biological replicates were analyzed and shown to yield similar results. Depicted are immunoblots from one of these biological replicates.

FIG 9

Bioinformatics assessment of BB0345. (A) I-TASSER homology model of full-length BB0345 against SMU1 (PDB: 5O9Z) with the C-terminal WD40 domain. BB0345 is shown in green and SMU1 in red. (B) I-TASSER homology model of N-terminal^1-143 BB0345 against T. maritima RodZ (PDB: 2WUS) with the characteristic cytoplasmic 5-helices domain. BB0345 is shown in green and T. maritima RodZ in red. (C) Scale prediction of protein topology for E. coli (Ec) RodZ, T. maritima (Tm) RodZ, and B. burgdorferi (Bb) BB0345. N-terminal secondary structure of Tm RodZ was constructed from crystal structure (PDB: 2WUS). N-terminal structure for Ec RodZ and Bb BB0345 and the C-terminal region for all three proteins were predicted by JPred. Transmembrane regions were predicted with TMpred. Alpha helices are represented as red barrels, beta sheets as yellow arrows, and transmembrane helices as blue barrels.