title: Use of a promoter trap system in Bacillus anthracis and Bacillus subtilis for the development of recombinant protective antigen-based vaccines.
abstract: We have recently reported Bacillus anthracis attenuated 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 Bacillus promoters for a further increase in the production of rPA, we developed a promoter trap system based on various gfp reporter genes adapted for use in both Bacillus subtilis and B. anthracis backgrounds. Accordingly, a B. anthracis library of 6,000 clones harboring plasmids with chromosomal B. anthracis DNA fragments inserted upstream from gfpuv was constructed. Based on fluorescence intensity, 57 clones carrying potentially strong promoters were identified, some of which were DNA sequenced. The most potent B. anthracis promoter identified (Pntr; 271 bp) was 500 times more potent than the native pagA promoter and 70 times more potent than the alpha-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. subtilis or B. anthracis. The number of cell-associated pre-PA molecules in B. anthracis was 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. anthracis expressing rPA under the control of promoters more potent than Pamy was 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.
authors.affiliation: Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness-Ziona 74100, Israel. avidgor@iibr.gov.il
authors.forename: O
authors.initials: O
authors.lastname: Gat
authors.forename: I
authors.initials: I
authors.lastname: Inbar
authors.forename: R
authors.initials: R
authors.lastname: Aloni-Grinstein
authors.forename: E
authors.initials: E
authors.lastname: Zahavy
authors.forename: C
authors.initials: C
authors.lastname: Kronman
authors.forename: I
authors.initials: I
authors.lastname: Mendelson
authors.forename: S
authors.initials: S
authors.lastname: Cohen
authors.forename: B
authors.initials: B
authors.lastname: Velan
authors.forename: A
authors.initials: A
authors.lastname: Shafferman
mesh_terms: D000818:Animals; D000881:Anthrax / Q000517:prevention & control*; D000942:Antigens, Bacterial / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D001408:Bacillus anthracis / Q000235:genetics* / Q000276:immunology; D001412:Bacillus subtilis / Q000235:genetics / Q000276:immunology; D001428:Bacterial Vaccines / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D015195:Drug Design; D005260:Female; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006168:Guinea Pigs; D006801:Humans; D007114:Immunization; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D014613:Vaccines, Attenuated
chemical_list: D000942:Antigens, Bacterial; D001428:Bacterial Vaccines; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D014613:Vaccines, Attenuated; D049452:Green Fluorescent Proteins