A 9.0-KILOBASE-PAIR CIRCULAR PLASMID OF BORRELIA-BURGDORFERI ENCODES AN EXPORTED PROTEIN - EVIDENCE FOR EXPRESSION ONLY DURING INFECTION

In this study, we report the cloning, sequencing, and molecular analys is of a gene located on a 9.0-kbp circular plasmid of virulent Borreli a burgdorferi B31 designated eppA (exported plasmid protein A). This g ene encodes a precursor protein of 174 amino acids including a signal peptide of 20 amino acids and a type I signal peptidase cleavage site. The mature EppA protein of 154 amino acids has a calculated molecular weight of 17,972. Several lines of evidence suggest that eppA is not expressed by B. burgdorferi B31 during in vitro cultivation. Immunoblo t analysis using hyperimmune rabbit antiserum to recombinant EppA (rEp pA) did not detect the presence of EppA in B. burgdorferi B31 cultivat ed in vitro. Northern blot analysis using total RNA isolated from in v itro cultivated virulent B. burgdorferi B31 failed to detect an eppA t ranscript. EppA was not detected in culture supernatants of virulent B . burgdorferi B31 in 9 sensitive antigen-capture enzyme-linked immunos orbent assay. In contrast, evidence for expression of eppA during infe ction,vas based on the observation that patients with Lyme disease as well as rabbits experimentally infected with B. burgdorferi B31 produc ed antibodies that recognized rEppA. Because the cellular location of EppA in B. burgdorferi cannot be determined in vivo because of very sm all numbers of organisms present in vertebrate infection, we examined the cellular location of rEppA expressed in Escherichia coli. In E. co li, rEppA is targeted to the outer membrane. In addition, purified E. coli outer membranes containing rEppA treated with chaotrophic agents did not result in rEppA release. These findings are consistent with th e idea that EppA is not peripherally associated with the outer membran e of E. coli but rather has an integral outer membrane association.