Use of a Promoter Trap System in Bacillus anthracisand Bacillus subtilisfor the Development of Recombinant Protective Antigen-Based Vaccines

ABSTRACTWe have recently reported Bacillus anthracisattenuated live vaccine strains efficiently expressing recombinant protective antigen (rPA) and have shown a direct correlation between the level of rPA secreted by these cells and efficacy (S. Cohen, I. Mendelson, Z. Altboum, D. Kobiler, E. Elhanany, T. Bino, M. Leitner, I. Inbar, H. Rosenberg, Y. Gozes, R. Barak, M. Fisher, C. Kronman, B. Velan, and A. Shafferman, Infect. Immun. 68:4549-4558, 2000). To isolate more potent Bacilluspromoters for a further increase in the production of rPA, we developed a promoter trap system based on various gfpreporter genes adapted for use in both Bacillus subtilisand B. anthracisbackgrounds. Accordingly, a B. anthracislibrary of 6,000 clones harboring plasmids with chromosomal B. anthracisDNA fragments inserted upstream from gfpuvwas constructed. Based on fluorescence intensity, 57 clones carrying potentially strong promoters were identified, some of which were DNA sequenced. The most potent B. anthracispromoter identified (Pntr; 271 bp) was 500 times more potent than the native pagApromoter and 70 times more potent than the α-amylase promoter (Pamy). This very potent promoter was tested along with the other promoters (which are three, six, and eight times more potent than Pamy) for the ability to drive expression of rPA in either B. subtilisor B. anthracis. The number of cell-associated pre-PA molecules in B. anthraciswas found to correlate well with the strength of the promoter. However, there appeared to be an upper limit to the amount of mature PA secreted into the medium, which did not exceed that driven by Pamy. Furthermore, the rPA constructs fused to the very potent promoters proved to be deleterious to the bacterial hosts and consequently led to genetic instability of the PA expression plasmid. Immunization with attenuated B. anthracisexpressing rPA under the control of promoters more potent than Pamywas less efficient in eliciting anti-PA antibodies than that attained with Pamy. The results are consistent with the notion that overexpression of PA leads to severe secretion stress and have practical implications for the design of second-generation rPA-based vaccines.