Michael V. Norgard

Cell-mediated immune processes play a prominent role in the clinical manifestations of syphilis, a sexually transmitted disease of humans caused by spirochetal bacteriumTreponema pallidum. The immune cell type that initiates the early immune response toT. pallidumthus far has not been identified. However, dendritic cells (DCs) are the first immune-competent cells to encounter antigens within skin or mucous membranes, the principal sites of early syphilitic infection. In the present study, immature DC line XS52, derived from murine skin, was utilized to examineT. pallidum-DC interactions and subsequent DC activation (maturation). Electron microscopy revealed thatT. pallidumwas engulfed by DCs via both coiling and conventional phagocytosis and was delivered to membrane-bound vacuoles. The XS52 DC line expressed surface CD14 and mRNA for Toll-like receptors 2 and 4, molecules comprising important signaling components for immune cell activation by bacterial modulins. BothT. pallidumand a synthetic lipopeptide (corresponding to the 47-kDa major membrane lipoprotein) activated the XS52 DC line, as indicated by the secretion of interleukin-12 (IL-12), IL-1β, tumor necrosis factor alpha, and IL-6 and elevated surface expression of CD54. The combined data support the contention that DCs stimulated byT. pallidumand/or its proinflammatory membrane lipoproteins are involved in driving the cellular immune processes that typify syphilis.

Previous studies showed that decorin-binding protein A (DbpA) of Borrelia burgdorferi was a protective immunogen in the murine model of Lyme borreliosis when mice were challenged (needle inoculated) intradermally with in vitro-cultivated spirochetes. In the present study, DbpA-immunized C3H/HeJ mice were not protected from infection when infested with Ixodes scapularis nymphs harboring virulent B. burgdorferi 297. This lack of protection correlated with the failure to detect DbpA on B. burgdorferi in ticks, suggesting that DbpA is not available as a target for bactericidal antibodies in serum when B. burgdorferi -infected ticks take their blood meal from an immunized host. The failure of DbpA immunization to protect tick-challenged mice contradicts the results of earlier needle inoculation vaccination experiments and suggests that DbpA may not be suitable as a Lyme disease vaccine.

Treponema pallidum, its membrane lipoproteins, and synthetic lipoprotein analogues (lipopeptides) were each examined to determine whether they induce

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°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°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°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°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.

Isolated outer membranes ofBorrelia burgdorferiwere used in immunoblotting experiments with sera from immune mice to identify new putative Lyme disease vaccine candidates. One immunoreactive polypeptide migrated on polyacrylamide gels just proximal to outer surface protein C and comigrated with [³H]palmitate-labeled polypeptides. A degenerate oligonucleotide primer based upon internal amino acid sequence information was used to detect the corresponding gene within aB. burgdorferitotal genomic library. The relevant open reading frame (ORF) encoded a polypeptide comprised of a 24-amino-acid putative signal peptide terminated by LLISC, a probable consensus sequence for lipoprotein modification, and a mature protein of 163 amino acids. Immunoblots of a recombinant fusion protein corresponding to this ORF supported the idea that the encoded protein was a previously reported decorin-binding protein (DBP) ofB. burgdorferiN40 (B. P. Guo, S. J. Norris, L. C. Rosenberg, and M. Höök, Infect. Immun. 63:3467–3472, 1995). However, further DNA sequencing revealed the presence of a second ORF, designated ORF-1, whose termination codon was 119 bp upstream of thedbpgene. ORF-1 also encoded a putative lipoprotein with a mature length of 167 amino acids. Northern blots, Southern blots, and primer extension analyses indicated that ORF-1 anddbpcomprised a two-gene operon located on the 49-kb linear plasmid. Both proteins, which were 40% identical and 56% similar, partitioned into Triton X-114 detergent extracts ofB. burgdorferiisolated outer membranes. Mice infected withB. burgdorferiproduced high titers of antibodies against the ORF-1-encoded protein and DBP during both early and later stages of chronic infection. Both DBP and the ORF-1-encoded protein were sensitive to proteinase K treatment of intact borreliae, suggesting that they were surface exposed. In active immunization experiments, 78% of mice immunized with recombinant DBP were immune to challenge. While it is not clear whether the two lipoproteins encoded by the ORF-1-dbpoperon have analogous decorin-binding functions in vivo, the combined studies implicate DBP as a new candidate for a human Lyme disease vaccine.

Syphilitic genital ulcers are cofactors for the bidirectional transmission of human immunodeficiency virus (HIV). U937 human promonocytic cells chron