Identification, characterization, and expression of three new members of the Borrelia burgdorferi Mlp (2.9) lipoprotein gene family - PubMed (original) (raw)

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

X Yang et al. Infect Immun. 1999 Nov.

Abstract

We previously reported on the existence of a family of lipoprotein genes, designated 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 were highly similar signal sequences but variable mature polypeptides that segregated into two antigenic classes. Further screenings of B. burgdorferi 297 genomic libraries led to the identification of three additional 2.9 lipoprotein genes, renamed herein mlp, for multicopy lipoprotein genes. Computer analyses and immunoblotting revealed that Mlp-9 segregated with the antigenic class I lipoproteins, whereas Mlp-8 and Mlp-10 were members of class II. Northern blotting showed that all three of the mlp 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 comparative reverse transcription-PCR analyses performed on borreliae cultivated 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 genes were upregulated during B. burgdorferi replication at 37 degrees C. Expression of the same three lipoproteins was further enhanced upon growth of the spirochetes within dialysis membrane chambers (DMCs) implanted intraperitoneally in rats (i.e., spirochetes in a mammalian host-adapted state), suggesting that temperature alone did not account for maximal upregulation of the mlp genes. That certain mlp genes are likely expressed during the growth of B. burgdorferi in mammalian tissues was supported by findings of antibodies against all three Mlp lipoproteins in mice after challenge with Ixodes scapularis nymphs harboring B. burgdorferi 297. The combined data suggest that as opposed to being differentially expressed in any reciprocal fashion (e.g., OspA/OspC), at least three mlp genes are simultaneously upregulated by temperature (37 degrees C) and some other mammalian host factor(s). The findings have importance not only for understanding alternative modes of differential antigen expression by B. burgdorferi but also for assessing whether one or more of the Mlp lipoproteins represent new candidate vaccinogens for Lyme disease.

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Figures

FIG. 1

Schematic representation of three new 2.9 loci of B. burgdorferi 297. ORFs are shown as boxed regions; the number above each box indicates the size of the polypeptide encoded by each ORF. Promoter-like elements are delineated as arrows in the direction of transcription; stem-loop structures (putative rho-independent terminators) are shown as inverted half arrows. Areas of close vertical lines represent repeat motifs. Positive and negative DNA strands are designated by + and −, respectively. rep, repeat-containing gene; mlp, multicopy lipoprotein; rev, an ORF in the opposite orientation as rep.

FIG. 2

Lipoproteins Mlp-8, Mlp-9, and Mlp-10 segregate into two distinct antigenic classes. Purified recombinant versions of the 10 lipoproteins were separated by SDS-PAGE (0.5 μg per gel lane). Numbers at the left represent protein molecular masses in kilodaltons. Lanes 1 through 5, Mlp-1, -4, -5, -7B, and -9 (antigenic class I), respectively; lanes 6 through 10, Mlp-2, -3, -7A, -8, and -10 (antigenic class II), respectively. (A) Gel stained with Coomassie brilliant blue; (B to D) immunoblots of panel A, using rat polyclonal antisera raised against recombinant Mlp-8, Mlp-9, and Mlp-10, respectively. Note that these antisera are cross-reactive with other Mlp members of the same antigenic class.

FIG. 3

Specificities of oligonucleotide probes for the mlp genes and localization of mlp-8, mlp-9, and mlp-10 to supercoiled plasmids of B. burgdorferi 297. (A) Dot blot hybridization assay; target (plasmid) DNAs encoding each mlp gene were spotted onto nylon membranes, cross-linked, and hybridized with radioactively labeled oligonucleotide probes for mlp-8, mlp-9, or mlp-10. Designations at the left denote each target DNA. (B) Supercoiled plasmids isolated from B. burgdorferi 297 were separated by electrophoresis on 0.4% agarose and hybridized with probes for ospC, mlp-8, mlp-9, or mlp-10. The left-most lane shows the ethidium bromide (EtBr)-stained plasmid profile. Circular plasmids are denoted at the left of the ethidium bromide-stained gel; cp26 encodes ospC. cp18 identity was confirmed by hybridization with probes specific for mlp-3 (29) and p21 (2) (not shown).

FIG. 4

Northern blot analysis of the mlp genes. RNA from B. burgdorferi 297 was hybridized with probes (indicated above the lanes) specific for flaB, mlp-8, mlp-9, and mlp-10. Lanes 1, 2, 4, and 6 contain 3 μg of RNA; lanes 3, 5, and 7 contain 30 μg of RNA. Arrows beside lane 2 show 0.6-, 0.9-, and 1.3-kb transcripts of mlp-8. Arrows beside lanes 5 and 7 show transcripts of 1.3 and 0.4 kb, respectively. Molecular size markers are shown at the left.

FIG. 5

Evidence that mlp-8 is cotranscribed with its upstream rep+ gene. (A) Schematic of the RT-PCR strategy used for assessing the sizes of mlp-8 transcripts. Four pairs of oligonucleotide primers were used; the 5′ primer for each pair was either primer B, C, D, or E, positioned downstream of putative promoter P3, P2, or P1 or upstream of P1, respectively. The 3′ primer for all amplifications was primer A, which was positioned near the end of the mlp-8 gene. Prospective transcripts are indicated by the solid lines below the schematic. (B) Ethidium bromide-stained amplicons. Lanes: 1, PCR products from B. burgdorferi genomic DNA (positive controls); 2, RT-PCR products from the four RT-PCR reaction combinations (i.e., primers A plus B, A plus C, A plus D, or A plus E); lanes 3, RT-PCRs lacking reverse transcriptase (negative controls).

FIG. 6

Specificities of antibodies directed against Mlp-8, Mlp-9, and Mlp-10. Ten individual purified recombinant Mlp proteins (0.5 μg of each per gel lane) were separated by SDS-PAGE, transferred to nitrocellulose membranes, and probed either with rat antisera directed against epitope-specific versions (see text) of Mlp-8 and Mlp-10 or with a monoclonal antibody (mAb; 17C3-73) directed against Mlp-9.

FIG. 7

Immunoblot analysis of B. burgdorferi. Whole-cell lysates from spirochetes cultivated in vitro at 23°C (lane 1), 34°C (lane 2), or 37°C (lane 3) or within DMCs in rat peritoneal cavities (lane 4) were probed with antibodies directed against the indicated antigens (left). Five micrograms of total protein per gel lane was loaded, except for OspA and OspC, in which case 0.1 μg of protein was used. Antibodies (indicated at the left) used to detect Mlp-8, Mlp-9, and Mlp-10 are those used for Fig. 6; α-Mlp-9 is monoclonal antibody 17C3-73. HA, mammalian host-adapted spirochetes from DMCs implanted in rat peritoneal cavities.

FIG. 8

Comparative RT-PCR analysis of mlp-8, mlp-9, and mlp-10 gene expression in spirochetes cultivated in vitro or within DMCs in rat peritoneal cavities. Total RNA was isolated from spirochetes grown in vitro either at 23, 34, or 37°C or within DMCs in rat peritoneal cavities (host adapted). Tenfold serial dilutions of RNA were used for amplification of each mRNA. The final amounts (nanograms) of RNA used in each reaction is indicated above each lane. -RT denotes reaction mixtures lacking reverse transcriptase. The column at the right (DNA) corresponds to reactions using B. burgdorferi genomic DNA as the template in place of RNA. Note that for the analyses of flaB and ospA, 100-fold-lower quantities of RNA were required to visualize endpoints.

FIG. 9

Tick-inoculated mice infected with B. burgdorferi 297 produce antibodies against the Mlp lipoproteins. Recombinant Mlp-8, -9, and -10 (lanes 1, 2, and 3, respectively) (0.5 μg of protein per gel lane) were separated by SDS-PAGE. The gel was either stained with Coomassie brilliant blue (CS) or transferred to nitrocellulose membranes for immunoblotting. Membranes were immunoblotted with sera harvested from _B. burgdorferi_-infected (tick-inoculated) C3H/HeJ mice at 0, 2, 4, 8, or 16 weeks post-infection. Note that antibodies directed against Mlp-8 were detectable as early as 2 weeks postinfection, whereas antibodies against Mlp-10 and Mlp-9 were detected at 8 and 16 weeks postinfection, respectively.

References

1. Akins D R, Bourell K W, Caimano M J, Norgard M V, Radolf J D. A new animal model for studying Lyme disease spirochetes in a mammalian host-adapted state. J Clin Investig. 1998;101:2240–2250. -PMC -PubMed
1. Akins D R, Caimano M J, Yang X, Cerna F, Norgard M V, Radolf J D. Molecular and evolutionary analysis of Borrelia burgdorferi 297 circular plasmid-encoded lipoproteins with OspE- and OspF-like leader peptides. Infect Immun. 1999;67:1526–1532. -PMC -PubMed
1. Akins D R, Porcella S F, Popova T G, Shevchenko D, Li M, Norgard M V, Radolf J D. Evidence for in vivo but not in vitro expression of a Borrelia burgdorferi outer surface protein F (OspF) homologue. Mol Microbiol. 1995;18:507–520. -PubMed
1. Aron L, Toth C, Godfrey H P, Cabello F C. Identification and mapping of a chromosomal gene cluster of Borrelia burgdorferi containing genes expressed in vivo. FEMS Microbiol Lett. 1996;145:309–314. -PubMed
1. Borrelia burgdorferi Genome Database. 7 July 1999, revision date. [Online.] http://www.tigr.org/tdb/mdb/bbdb/bbdb.html. The Institute for Genomic Research, Rockville, Md. [24 September 1999, last date accessed].

Identification, characterization, and expression of three new members of the Borrelia burgdorferi Mlp (2.9) lipoprotein gene family - PubMed (original) (raw)