Fig 5. R. prowazekii Madrid E Adr2 confers acquisition of vitronectin and resistance in serum.

(A) E. coli BL21(DE3) harboring the empty vector pET22b or the plasmid encoding R. prowazekii Madrid E His₆-tagged Adr2 (pRP828) were incubated with normal human serum. Western immunoblot analysis revealed a reactive band at approximately 75 kDa in the lane with R. prowazekii Adr2 only when probed with antibody raised against vitronectin. Expression of Adr2 was confirmed by anti-His₆, and equal loading was demonstrated with anti-E. coli RNA Polymerase alpha (RNAP). (B) Western immunoblot analysis of multimeric vitronectin binding to E. coli expressing Adr1 derivative mutant (pAF17) or R. conorii Adr2. Expression of both the Adr1 derivative and Adr2 was confirmed by anti-His₆, and equal loading was verified using anti-E. coli RNA polymerase alpha (RNAP). (C) Expression of R. prowazekii Adr2 in E. coli BL21(DE3) is sufficient to mediate resistance in the presence of serum. Results are shown as the mean ± SD (P value: ** = 0.0024) and E. coli harboring R. conorii Adr1 (pJP01) was used as a positive control. (D) OM preparations or WCL from E. coli transformed with the empty vector (pET22b) or the plasmid encoding for R. prowazekii His₆-tagged Adr2 are separated by SDS-PAGE. Subsequent silver staining demonstrates similar protein loading and migration. Anti-His₆ was used to demonstrate expression of R. prowazekii Adr2. A reactive band at approximately 25 kDa was observed when probed with anti-R. conorii Adr2.