|FRYE, JONATHAN - SIDNEY KIMMEL CANCER CNTR
|QUINN, FREDERICK - CDC
|GHERARDINI, FRANK - UNIVERSITY OF GEORGIA
Submitted to: Molecular Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/27/2001
Publication Date: 10/1/2001
Citation: Jackson, C.R., Frye, J.G., Quinn, F.D., Gherardini, F.C. 2001. Increased expression of borrelia burgdorferi vise in response to human endothelial cell membranes. Molecular Microbiology. 41(1). P. 229-239.
Interpretive Summary: Vector-borne bacterial pathogens, such as Borrelia burgdorferi, encounter different conditions as they are transmitted to various hosts. Sensing environmental changes and modulating gene expression is likely to be important for adaptation and survival of B. burgdorferi within various hosts. In order to identify the changes in gene expression in Borrelia, gene probes were made by incubating B. burgdorferi cells with either human tissue cells or in media alone, extracting the RNA from all samples, and then subjecting the RNA to subtractive hybridization. The probes from the subtraction hybridized with a variable membrane protein- like sequence (vls) that encodes an outer surface protein that undergoes antigenic variation. This information will be useful for scientists and physicians in order to fully understand the interaction between B. burgdorferi and the human host and for future gene targets for vaccine development.
Technical Abstract: 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 EcoRI digestion of total plasmid DNA. The 10 kb EcoRI fragment localized to lp28-1 and was subsequently identified as the variable membrane protein-like sequence (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 50 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 **32P end-labeled 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.