Identification, characterization, and expression of three new members of the Borrelia burgdorferi mlp (2.9) lipoprotein gene family

We previously reported on the existence of a family of lipoprotein genes, d esignated 2.9 lipoprotein genes, encoded in at least seven versions on the circular (supercoiled) cp32 and cp18 plasmids of Borrelia burgdorferi 297, A distinguishing feature of the 2.9 lipoproteins mere highly similar signal sequences but variable mature polypeptides that segregated into two antige nic classes. Further screenings of B. burgdorferi 297 genomic libraries led to the identification of three additional 2.9 lipoprotein genes, renamed h erein mlp, for multicopy lipoprotein genes. Computer analyses and immunoblo tting revealed that Mlp-9 segregated,vith the antigenic class I lipoprotein s, whereas MIp-8 and Mlp-10 were members of class II. Northern blotting sho wed that all three of the mtp genes were expressed when B. burgdorferi was cultivated in vitro at 34 degrees C, although mlp-9 and mlp-10 transcripts were expressed at very low levels. Additional combined immunoblotting and c omparative reverse transcription-PCR analyses performed on borreliae cultiv ated in vitro at 23, 34, or 37 degrees C indicated that: although Mlp-8 was substantially more abundant than Mlp-9 or Mlp-10, all three of the mlp gen es were upregulated during B. burgdorferi replication at 37 degrees C. Expr ession of the same three lipoproteins was further enhanced upon growth of t he spirochetes within dialysis membrane chambers (DMCs) implanted intraperi toneally in rats (i.e,, spirochetes in a mammalian host-adapted state), sug gesting that temperature alone did not account for maximal upregulation of the mlp genes. That certain mlp genes are likely expressed during the growt h of B. burgdorferi in mammalian tissues was supported by findings of antib odies against all three Mlp lipoproteins in mice after challenge with Ixode s scapularis nymphs harboring B. burgdorferi 297. The combined data suggest that as opposed to being differentially expressed in any reciprocal fashio n (e.g., OspA/OspC), at least three mlp genes are simultaneously upregulate d by temperature (37 degrees C) and some other mammalian host factor(s), Th e findings have importance not only for understanding alternative modes of differential antigen expression by B. burgdorferi but also for assessing wh ether one or more of the Mlp lipoproteins represent new candidate vaccinoge ns for Lyme disease.