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Increased expression of Borrelia burgdorferi vlsE in response to human endothelial cell membranes
Author(s) -
Hudson Charlene R.,
Frye Jonathan G.,
Quinn Frederick D.,
Gherardini Frank C.
Publication year - 2001
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1046/j.1365-2958.2001.02511.x
Subject(s) - biology , suppression subtractive hybridization , microbiology and biotechnology , borrelia burgdorferi , ecori , plasmid , gene , complementary dna , genetics , cdna library , antibody
RNA isolated from virulent Borrelia burgdorferi cells incubated with human endothelial or neurological tissue cells was subjected to subtractive hybridization using RNA from the same strain incubated in tissue culture medium alone. This RNA subtractive technique generated specific probes that hybridized to two restriction fragments (8.2 kb and 10 kb respectively) generated by Eco RI digestion of total plasmid DNA. The 10 kb Eco RI fragment localized to lp28‐1 and was subsequently identified as the v ariable membrane protein‐ l ike s equence ( vls ) region, which includes an expression locus ( vlsE ) and 15 silent cassettes. vlsE encodes a 36 kDa outer surface protein that undergoes antigenic variation during animal infections. Primer extension analysis identified the 5′ end of a transcript and a putative promoter for vlsE. Quantitative reverse transcription–polymerase chain reaction (RT–PCR) suggested that the expression of vlsE increased when virulent B. burgdorferi cells were incubated with human tissue cells or purified cell membranes isolated from those same cell lines. A 138 bp region upstream of the vlsE region that was not reported in the genome sequence was sequenced using specific 32 P end‐labelled primers in a DNA cycle sequencing system at high annealing temperatures. Analysis revealed that it contained a 51 bp inverted repeat, which could form an extremely stable cruciform structure. Southern blots probed with the vlsE promoter/operator region indicated that part or all of this sequence could be found on other B. burgdorferi plasmids.