Characterization of differential expression of vlsE and a conjugal macrolide-lincosamide-streptogramin type B (MLS(B)) resistance determinant from Borrelia burgdorferi.

摘要

A mRNA subtractive hybridization technique was used to identify genes which were differentially expressed when virulent, low-passage strain B31 cells were exposed to human endothelial or human neurological tissue cells. Using Southern analysis, the majority of hybridizing chromosomal DNA was identified as ribosomal RNA and was not pursued further. The subtractive probes also hybridized to two bands (8.2 and 10 kb) in an EcoRI restriction digest of plasmid DNA. The 8.2-kb EcoRI fragment localized to the 54-kb linear (lp54) plasmid and the 10-kb EcoRI fragment localized to the 28-kb linear plasmid (lp28). Further analysis of the 8.2-kb EcoRI fragment indicated that it contained the 3' end of the gene encoding outer surface protein A (ospA), ospB, the decorin binding proteins (dbpA, dbpB) and several open reading frames encoding hypothetical proteins. The 10-kb EcoRI fragment contained the variable membrane protein-like sequence ( vls) which includes an expression locus, vlsE, and 15 silent cassettes. Sequencing of vlsE indicated that it was 93.6% identical at the nucleotide level and 87.8% identical at the amino acid level to the previously published sequence. Using RT-PCR, human tissue culture cells or purified membranes from these cells were found to induce expression of vlsE.;A resistance determinant in B. burgdorferi which confers constitutive resistance to the macrolide, lincosamide, and streptogramin type B (MLSB) antibiotics in high-passage, avirulent strain B31 and inducible resistance to the macrolides and lincosamides in low passage, virulent strain B31 was also identified. The MLSB-resistance determinant did not inactivate erythromycin and did not actively transport erythromycin from the resistant strains indicating that it is most likely functioning as a rRNA methylase. In broth matings, the MLSB-resistance determinant was transferred from B. burgdorferi to E. faecalis at a frequency of 10-5--10-6 and to B. subtilis at a higher frequency of 10-2--10 -3. Transfer occurred in the presence of DNase and filtered culture supernatant did not transfer resistance, suggesting that neither transformation nor transduction were the mechanisms of transfer. This information represents the first report of a functional antibiotic-resistance determinant in B. burgdorferi and the existence of a conjugal transfer mechanism in the spirochetes.