BB0238, a Presumed Tetratricopeptide Repeat-Containing Protein, Is Required during Borrelia burgdorferi Mammalian Infection

Abstract

The Lyme disease spirochete, Borrelia burgdorferi, occupies both a tick vector and mammalian host in nature. Considering the unique enzootic life cycle of B. burgdorferi, it is not surprising that a large proportion of its genome is composed of hypothetical proteins not found in other bacterial pathogens. bb0238 encodes a conserved hypothetical protein of unknown function that is predicted to contain a tetratricopeptide repeat (TPR) domain, a structural motif responsible for mediating protein-protein interactions. To evaluate the role of bb0238 during mammalian infection, a bb0238-deficient mutant was constructed. The bb0238 mutant was attenuated in mice infected via needle inoculation, and complementation of bb0238 expression restored infectivity to wild-type levels. bb0238 expression does not change in response to varying culture conditions, and thus, it appears to be constitutively expressed under in vitro conditions. bb0238 is expressed in murine tissues during infection, though there was no significant change in expression levels among different tissue types. Localization studies indicate that BB0238 is associated with the inner membrane of the spirochete and is therefore unlikely to promote interaction with host ligands during infection. B. burgdorferi clones containing point mutations in conserved residues of the putative TPR motif of BB0238 demonstrated attenuation in mice that was comparable to that in the bb0238 deletion mutant, suggesting that BB0238 may contain a functional TPR domain.

BB0744 Affects Tissue Tropism and Spatial Distribution of Borrelia burgdorferi

“…BB0744-specific polyclonal antiserum was generated in 3-to 4-week-old female Sprague-Dawley rats (Harlan, Indianapolis, IN) as described by Groshong et al (49). Briefly, 25 g of recombinant BB0744 protein was combined with complete Freund's adjuvant (Sigma, St. Louis, MO).…”

“…Immunofluorescence assays (IFAs) were performed as described by Groshong et al (49). Briefly, B. burgdorferi cells were grown to mid-log growth phase and harvested by centrifugation.…”

“…B. burgdorferi protein lysates were resolved by SDS-PAGE, and gels were either stained with Coomassie brilliant blue R-250 (Sigma-Aldrich, St. Louis, MO, USA) or transferred to polyvinylidene difluoride membranes and immunoblotted. Primary antibodies were used at the following dilutions: rat anti-BB0744 at 1:5,000, chicken anti-FlaB IgY at 1:25,000, rat anti-OspA at 1:25, mouse anti-OspC at 1:1,000, and rat anti-BB0796 at 1:1,500 (49). Appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (antimouse IgG-HRP, anti-rat IgG-HRP [Invitrogen, Carlsbad, CA], and donkey anti-chicken IgY antibodies at 1:2,500) were used to detect immune complexes.…”

“…Membrane protein phase partitioning was performed as previously described (49). B. burgdorferi cells were collected at the mid-log growth phase and washed with phosphate-buffered saline (PBS).…”

“…Proteinase K assays were performed as previously described (49). B. burgdorferi cells were pelleted following growth to mid-log phase and washed with PBS.…”