Fig 3.

BB0238 influences proteolytic processing of BB0323, and their interaction does not involve the TPR-like/HTH motif. (A) BB0238-BB0323 interaction assessed by a far-western assay. Recombinant proteins rBB0238^WT, rY37A, and rP65A (red arrow) and control protein BBA57 (blue arrows) were separated via SDS-PAGE, transferred to nitrocellulose membrane and probed with 1 µg of BB0323, and interactions were detected via BB0323 antibody. Comparable loading of proteins was evidenced by Ponceau S staining (lower panels). The image is representative of three separate experiments. (B) Kinetics of BB0238-BB0323 interaction assessed by microscale thermophoresis (MST). The MST analysis depicts a titration of fluorescently labeled rBB0238^WT, rY37A, and rP65A with bound BB0323. K _D values were indicated as 1,001.3 ± 306.8 nM, 658.4 ± 245.7 nM, and 702.1 ± 326.1 nM, respectively, displaying no difference between the groups. (C) BB0238 stabilization of BB0323 was depicted via a protein turnover assay at 37°C. Protein synthesis was inhibited via the addition of erythromycin to B. burgdorferi culture, and levels were detected via immunoblotting using specific antibodies. (D) bb0323 transcript levels in wild-type or mutant B. burgdorferi were compared by RT-qPCR. The red arrow highlights no significant change in bb0323 levels (P > 0.05), even though protein levels were diminished in protein turnover analysis (panel C). (E) Far-western analysis (upper panel). Indicated proteins were probed with BbHtrA and bound protein detected using anti-BbHtrA antibody. Comparable loading of proteins was evidenced by Ponceau S staining (lower panel). (F) BB0238 influences BB0323 proteolytic processing via BbHtrA. The upper panel shows an in vitro digestion assay using BB0323 and catalytically active or inactive BbHtrA and/or BB0238, revealing that the latter protects BB0323 from proteolytic cleavage by BbHtrA (upper panels). BB0323 full-length (FL) protein was incubated either alone, with BbHtrA or with inactive BbHtrA, or in the presence of both BbHtrA and BB0238. Reactions were stopped at the indicated time intervals, and proteolytic processing of BB0323 was detected via immunoblot analysis using anti-BB0323 antibody showing full-length (BB0323FL, 42 kDa, red arrow) and processed protein (BB0323N, 27 kDa, blue arrow). The lower panels denote a repetition of the upper panel experiment, where wild-type BB0238 is replaced by either interaction-deficient BB0238 (BB0238_∆IM) or TPR-like/HTH motif mutants.