MOLECULAR CHARACTERIZATION OF A 6.6-KILODALTON BORRELIA-BURGDORFERI OUTER MEMBRANE-ASSOCIATED LIPOPROTEIN (1P6.6) WHICH APPEARS TO BE DOWN-REGULATED DURING MAMMALIAN INFECTION

Isolated outer membranes of Borrelia burgdorferi 297 were utilized to obtain partial amino acid sequence information for a low-molecular-wei ght, outer membrane-associated polypeptide. Degenerate oligonucleotide primers based upon this information were used to amplify a 100-bp pro be for detection of the corresponding full-length gene within a B. bur gdorferi total genomic library, The relevant open reading frame (ORF) encoded a polypeptide comprised of a 17-amino-acid putative signal pep tide terminated by LFVAC, a probable consensus sequence for lipoprotei n modification, and a mature protein of 51 amino acids (predicted mole cular mass of 5.8 kDa). The DNA sequences of the corresponding ORFs in B. burgdorferi 297 and B31 were identical; the corresponding ORF in s train N40 differed by only one nucleotide. Assuming conventional proce ssing and acylation, the molecular weight of the lipoprotein, designat ed lp6.6, is about 6,600, The lp6.6 gene, which was localized to the 4 9-kb linear plasmid of B. burgdorferi, subsequently was cloned and exp ressed in Escherichia coli as a fusion protein with glutathione S-tran sferase. Immunoblot analysis with monoclonal antibody 240.7 revealed t hat lp6,6 was identical to a low-molecular-weight, highly conserved B. burgdorferi lipoprotein reported previously (L. I. Katona, G. Beck, a nd G. S. Habicht, Infect. Immun. 60: 1995-5003, 1992), Results of indi rect immunofluorescence assays, growth inhibition assays, passive immu nizations, and active immunizations indicated that this outer membrane -associated antigen is not surface exposed in B. burgdorferi, Particul arly interesting was the finding that mice and rhesus monkeys chronica lly infected with B. burgdorferi failed to develop antibodies against this antigen, We propose that high-level expression of lp6.6 is associ ated with the arthropod phase of the spirochetal life cycle and that e xpression of the gene is downregulated during mammalian infection.