BB0744 Affects Tissue Tropism and Spatial Distribution of Borrelia burgdorferi

Abstract

Borrelia burgdorferi, the etiologic agent of Lyme disease, produces a variety of proteins that promote survival and colonization in both the Ixodes species vector and various mammalian hosts. We initially identified BB0744 (also known as p83/100) by screening for B. burgdorferi strain B31 proteins that bind to α1β1 integrin and hypothesized that, given the presence of a signal peptide, BB0744 may be a surface-exposed protein. In contrast to this expectation, localization studies suggested that BB0744 resides in the periplasm. Despite its subsurface location, we were interested in testing whether BB0744 is required for borrelial pathogenesis. To this end, a bb0744 deletion was isolated in a B. burgdorferi strain B31 infectious background, complemented, and queried for the role of BB0744 following experimental infection. A combination of bioluminescent imaging, cultivation of infected tissues, and quantitative PCR (qPCR) demonstrated that Δbb0744 mutant B. burgdorferi bacteria were attenuated in the ability to colonize heart tissue, as well as skin locations distal to the site of infection. Furthermore, qPCR indicated a significantly reduced spirochetal load in distal skin and joint tissue infected with Δbb0744 mutant B. burgdorferi. Complementation with bb0744 restored infectivity, indicating that the defect seen in Δbb0744 mutant B. burgdorferi was due to the loss of BB0744. Taken together, these results suggest that BB0744 is necessary for tissue tropism, particularly in heart tissue, alters the ability of B. burgdorferi to disseminate efficiently, or both. Additional studies are warranted to address the mechanism employed by BB0744 that alters the pathogenic potential of B. burgdorferi.

FIG 1

Determination of the cell envelope localization of BB0744. (A) BB0744 is a soluble protein. B. burgdorferi 5A4 cells were partitioned into detergent (DET) and aqueous (AQ) phases with Triton X-114. Precipitated protein samples were resolved by SDS-PAGE and analyzed by Western blotting. OspA was included as a positive control for a membrane-localized protein (DET lane), and BB0796 was used as a positive control as an aqueous-phase protein (AQ lane). BB0744 was detected only in the aqueous phase. (B) BB0744 is not protease accessible in intact borrelial cells. B. burgdorferi 5A4 cells were suspended in PBS alone (Intact lanes) or PBS containing Triton X-100 (Triton X-100 lanes). Minus and plus signs signify the presence of added proteinase K (PK). The resulting B. burgdorferi samples were resolved by SDS-PAGE and analyzed by Western blotting. FlaB and OspC were used as controls for subsurface and surface-exposed proteins, respectively. No decrease in BB0744 was observed after proteinase K treatment. Lanes MW contained prestained protein molecular size markers (sizes are in kilodaltons).

FIG 2

Surface exposure of BB0744. B. burgdorferi 5A4 cells were either washed or permeabilized by heat fixation prior to incubation with antibodies to the proteins listed on the right. Nonpermeabilized cells were then fixed to silylated slides with formaldehyde, and all cells were incubated with appropriate fluorescent secondary antibodies (anti-mouse Alexa 594 for mouse antibody to OspA, anti-chicken Alexa 647 for chicken antibody to FlaB, and anti-rat Alexa 488 for rat antibody to BB0744). FlaB served as a periplasmic protein control, and OspA was used as an outer surface protein control. BB0744 was labeled only in permeabilized cells. Scale bars, 10 μm.

FIG 3

Construction of bb0744 knockout and complemented strains. (A) Construction of the Δbb0744::Str^r mutant strain and cis complementation strategies. Representation of the bb0744 locus in the B. burgdorferi B31 strain ML23 (parent) chromosome and subsequent replacement of intact bb0744 with a streptomycin resistance (Str^r) cassette (P_flgB-aadA) by homologous recombination with linearized p744KO, resulting in strain SH100 (Δbb0744). For complementation, the Str^r cassette of the bb0744 knockout mutant is replaced with an intact copy of bb0744 linked to a gentamicin resistance cassette (P_flgB-aacC) to yield strain SH200 (bb0744 Comp). Lines with arrows depict the locations of primers P1 and P2 (Table 2), which were used to confirm the deletion mutation and subsequent complemented (Comp) strain. Note that the gene depictions shown are not to scale. (B) Confirmation of the Δbb0744::Str^r strain and cis-complemented strains. The configuration of the bb0744 locus was determined by PCR for the ML23/pBBE22luc, SH100/pBBE22luc, and SH200 strains with the oligonucleotide primers depicted in panel A. The values to the left are DNA size markers (sizes are in kilobases). (C, D). The Δbb0744::Str^r strain does not produce BB0744 protein. B. burgdorferi whole-cell lysates were separated by SDS-PAGE, immunoblotted, and probed with antisera to BB0744. The Coomassie-stained gel used as a loading control is shown in panel C. The presence of BB0744 was detected with antiserum specific for BB0744 in panel D.

FIG 4

Bioluminescent temporal and spatial tracking of a B. burgdorferi Δbb0744 mutant strain in living mice. BALB/c mice were infected with a dose of 10³ (A) or 10⁵ (B) cells of ML23/pBBE22luc (parent), SH100/pBBE22luc (Δbb0744), or SH200/pBBE22luc (bb0744 complemented [Comp]). Mice indicated by plus signs were treated with d-luciferin (d-Luc) at the time points indicated (d, day). Mice indicated by minus signs were not given a substrate and were used to assess the background. The images obtained are 10-min exposures, and all of the images shown are adjusted to the same scale, as indicated by the color spectrum scale on the right.

FIG 5

Loss of BB0744 affects the B. burgdorferi load in distal tissues. Quantitative PCR analysis of joint, lymph node, skin, and ear tissues was performed. Circles, ML23/pBBE22luc; triangles, SH100/pBBE22luc; squares, SH200/pBBE22luc. Copies of recA were quantified and represent the number of total B. burgdorferi genomes. To normalize the data to host tissue, β-actin copy numbers were also enumerated and data points are expressed as numbers of copies of B. burgdorferi genomes per 10⁶ copies of β-actin. Bars indicate the average values of the samples tested. *, P < 0.05; **, P < 0.01; ***, P < 0.001.