Figure 1.

Expression of Tp0954 in B. burgdorferi B314 strain and detection on the surface of transformed spirochetes. (A) Schematic of the construct expressing the Tp0954 coding region driven by the B. burgdorferi ospC promoter (pTp-Bb). (B) Tp0954 expression in B314(pTp-Bb) and not vector transformed B314(V) strain was detected by RT-PCR using Tp0954 primers. Positive control RT-PCR is also shown in which primers were used to amplify the ospC gene of B. burgdorferi. RT-PCR products were absent when reverse transcriptase was not included in the reaction indicting specificity of RT-PCR. (C) Detection of three Tp0954 protein bands (marked by horizontal lines) in T. pallidum (Tp) Nichols strain as well as Tp0954 transformed B314 strain, B314(pTp-Bb) using antibodies against 281 N-terminal amino acids of mature Tp0954 protein (anti-Tp0954N) suggest differential post-translational processing of full size Tp0954 protein of ~50 kD size. *Non-specific binding with cross reactivity of antibodies with unrelated B314 proteins. (D) Limited proteinase K treatment of intact spirochetes digested only surface Tp0954 versions and B. burgdorferi OspC protein but not periplasmic flagellar protein. (E,F) IFA using anti-Tp0954N primary antibodies followed by TRITC-labeled secondary antibodies using unpermeabilized spirochetes confirmed the surface localization of Tp0954 only in Tp0954 transformed B314 and not in the control, B314(V), strain. (G,H) The lack of staining of flagella in unpermeabilized spirochetes by anti-FlaB monoclonal antibodies treatment followed by anti-mouse Alexa fluor 488 antibodies indicate that integrity of outer membrane of B314 was maintained during IFA procedure. (I,J) Staining of B. burgdorferi flagella after permeabilization with methanol indicates the specific reactivity of anti-FlaB antibodies. All spirochetes present in each microscopic field are detected by co-labeling with DNA stain, DAPI and superimposed images are shown in (E–J). Bar indicates 20 μ size.