Borrelia burgdorferi transcriptome in the central nervous system of non-human primates

Abstract

Neurological symptoms are common manifestations of Lyme disease; however, the paucibacillary nature of the spirochete in this environment has precluded a molecular analysis of the spirochete in the CNS. We have now adapted differential expression analysis by using a custom-amplified library (DECAL) in conjunction with Borrelia burgdorferi whole-genome and subgenome arrays to examine in vivo gene expression by B. burgdorferi in a non-human primate (NHP) model of neuroborreliosis. The expression profile of B. burgdorferi was examined in the CNS and heart of steroid-treated and immunocompetent NHPs. Eighty-six chromosomal genes and 80 plasmid-encoded genes were expressed at similar levels in the CNS and heart tissue of both immunocompetent and steroid-treated NHPs. The expression of 66 chromosomal genes and 32 plasmid-encoded genes was increased in the CNS of both immunocompetent and steroid-treated NHPs. It is likely that the expression of these genes is governed by physiological factors specific to the CNS milieu. However, 83 chromosomal and 114 plasmid-encoded genes showed contrasting expression profiles in steroid-treated and immunocompetent NHPs. The effect of dexamethasone on the immune status of the host as well as on the host metabolic pathways could contribute to these differences in the B. burgdorferi transcriptome. Results obtained herein underscore the complex interplay of host factors on B. burgdorferi gene expression in vivo. The results provide a global snapshot of the spirochetal transcriptome in the CNS and should spur the design of experiments aimed at understanding the molecular basis of neuroborreliosis.

Fig. 1.

Schematic representation of DECAL. (A) Construction of Bb-CAL. A ZAP II B. burgdorferi genomic library was screened with radioactively labeled B. burgdorferi rRNA probes and hybridizing clones discarded. The nonribosomal clones were pooled and restriction digested with EcoRV and SmaI. The 200- to 2,000-bp fragments were gel-purified, ligated to PCR adapters, and PCR-amplified. (B) Positive selection and amplification. Total RNA isolated from heart and CNS of B. burgdorferi-infected NHPs was reverse transcribed in the presence of biotin-dATP. The biotinylated cDNA samples were hybridized to Bb-CAL under stringent conditions. The cDNA-Bb-CAL hybrids were bound to streptavidin-coated magnetic beads and then washed to remove unhybridized Bb-CAL. (C) The bound genomic Bb-CAL (representing the B. burgdorferi transcripts in the CNS/heart) was eluted by boiling and PCR-amplified. The PCR products were radioactively labeled and hybridized to a replicate genomic array.

Fig. 2.

Array hybridization. The CNS and Heart Bb-DECAL probes were normalized to the flaB amplicon obtained by PCR (A) and by their hybridization intensity to an equal amount of (25 ng) of flaB PCR product spotted on a nitrocellulose membrane (B). B. burgdorferi whole-genome arrays (C) and glass slide subarrays representing a subset of B. burgdorferi genes (D) were probed with CNS-Bb-DECAL and Heart-Bb-DECAL as described in Materials and Methods. A representative field of each array and the visual scores assigned to the differentially hybridizing elements are shown.

Fig. 3.

Confirmation of the expression profiles. (A) cDNA prepared from steroid treated B. burgdorferi- (N40) infected NHPs and enriched by DECAL was used as template in PCRs by using gene-specific primers, as outlined in Materials and Methods. The amounts of template DNA from CNS and heart tissues in the PCR were normalized against the expression levels of flaB (bb0147). (B) Relative pixel intensities of the amplicons were measured by using scion image, an image processing and analysis software program (www.scioncorp.com).