Borrelia burgdorferi gene expression in vivo and spirochete pathogenicity

Abstract

Borrelia burgdorferi spirochetes that do not cause arthritis or carditis were developed and used to investigate Lyme disease pathogenesis. A clonal isolate of B. burgdorferi N40 (cN40), which induces disease in C3H/HeN (C3H) mice, was repeatedly passaged in vitro to generate nonpathogenic spirochetes. The passage 75 isolate (N40-75) was infectious for C3H mice but did not cause arthritis or carditis, and spirochetes were at low levels or absent in the joints or hearts, respectively. N40-75 could, however, cause disease in severe combined immunodeficient (SCID) mice, suggesting that the response in immunocompetent mice prevented effective spirochete dissemination and the subsequent development of arthritis and carditis. Administration of immune sera at 4 days after spirochete challenge aborted N40-75, but not cN40, infection in SCID mice. A B. burgdorferi genomic expression library was differentially probed with sera from cN40- and N40-75-infected mice, to identify genes that may not be effectively expressed by N40-75 in vivo. N40-75 was defective in the up-regulation of several genes that are preferentially expressed during mammalian infection, including dbpAB, bba64, and genes that map to the cp32 family of plasmids. These data suggest that adaptation and gene expression may be required for B. burgdorferi to effectively colonize the host, evade humoral responses, and cause disease.

FIG. 1

cN40 and N40-75 do not differ substantially when grown in vitro. cN40, N40-75, and several N40-75 clones were grown in vitro to stationary phase and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to compare protein contents (A). Western blot analysis with cN40 rabbit hyperimmune sera was also performed to test the presence of OspA, -B, and -C in both passages (B). The immune response to cN40 and N40-75 was also tested by Western blotting (C), by using 21-day-infected mouse sera with cN40 and N40-75.

FIG. 2

DNA contents of cN40 and N40-75, as well as of several N40-75 clones. DNA PCR for several genes was performed to detect any possible loss of DNA as a result of in vitro passage. The plasmids in which the amplified genes are present are indicated.

FIG. 3

N40-75 grows faster than cN40 in vitro. Equal amounts of cN40 and N40-75 were grown in BSK II medium and analyzed at different time points over 1 week. Data represent the average of five individual experiments ±SE. An asterisk indicates significant difference between cN40 and N40-75 at that particular time point (t test, P < 0.05).

FIG. 4

N40-75 does not effectively disseminate in the murine host. Ear, joint, and heart tissue was used to quantitate B. burgdorferi by DNA PCR using flaB targets in mice infected for 3 weeks. Data represent the average ± SE of five individual mice. ■, cN40; □, N40-75.

FIG. 5

N40-75 disseminates efficiently in SCID mice. cN40 and N40-75 were quantitated in ears, joints, and hearts of C.B-17-scid mice after 2 weeks of infection as for Fig. 3. ■, cN40; □, N40-75. Data represent five individual mice.

FIG. 6

N40-75 does not up-regulate in vivo genes. cN40- and N40-75-infected mouse spleens were used to extract total RNA, and RT-PCR for B. burgdorferi dbpA, dbpB, bba64, bba65, bba66, p21, erpD, gene-1, and gene-2 was performed. As a control, flaB was used to identify spirochetes in spleens of the infected animals. N40-75 DNA was used as a positive control, demonstrating the presence of the genes in high-passage N40.