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.

FIG. 1.

Schematic representation of the promoter-trapping vector used in the present study. pXX_MCS-5 is a versatile plasmid conveniently allowing cloning of either promoter cassettes or reporter genes. This plasmid was used for (i) evaluation of various promoters and reporter genes, (ii) construction and screening of a genomic B. anthracis library, and (iii) expression of the pagA gene. Restriction sites for insertion of the various promoters and different reporter genes, as well as the respective promoter cassettes, are indicated. The linker DNA sequence between the promoter and the reporter gene insertion sites contains the RBS.

FIG.2.

Fluorescence emitted by B. anthracis Δ14185 and B. subtilis WB600 expressing different GFP reporter genes from various promoters. Strains expressing the three versions of the gfp gene in conjunction with the indicated promoter cassettes were cultured in FAG liquid media. GFP expression was monitored from a bacterial culture drawn at an A₅₅₀ of 5.0 by scanning the emission between 450 and 550 nm at excitations of 410 nm for wt GFP and GFPuv and 488 nm for EGFP. (A) Emission scans of B. subtilis and B. anthracis cultures expressing wt gfp, driven by the Pamy, Ptms, and P43 promoters. (B) Emission scans of cultures expressing the wt gfp, gfpuv, and egfp genes driven by the P43 promoter. Note the lack of EGFP fluorescence (driven by the P43 promoter) in B. anthracis as opposed to the high fluorescence level in B. subtilis. (C) Fluorescence-activated cell sorter-based detection of fluorescent B. anthracis vegetative cells expressing gfpuv from the P43 promoter (shaded curve) versus nonfluorescent cells (solid curve). FL1-Height, fluorescence intensity per cell; counts, normalized fractions of cells.

FIG.3.

Measurements of GFPuv expression driven by a series of promoters in B. anthracis Δ14185 cells. (A and B) Cultures were monitored during growth for optical density at 550 nm (OD₅₅₀) (A) and whole-cell fluorescence emission (510 nm) following excitation at 410 nm (B). FU, fluorescence units. B. anthracis strains expressed gfpuv from the following promoter cassettes: •, no promoter; ▴, Pamy; ♦, P43; ▪, Psap_short; and □, Psap_long. (C and D) Specific fluorescence spectra measured during logarithmic (C) and stationary (D) phases. For measurement of specific activities, whole-cell samples were washed in PBS and resuspended to equal densities (A₅₅₀ = 1.0), and emission scans were inspected in the range of 450 to 600 nm. (E) Amounts of intracellular GFP were also determined by Western blot analysis (with anti-GFP specific antibodies) of B. anthracis bacterial extracts obtained from the culture samples 7 h after initiation of growth. (F) Direct UV-mediated visualization of fluorescence emitted by cells expressing the gfpuv gene with the indicated promoters.

FIG. 4.

Comparison of GFPuv fluorescence emitted by selected individual B. anthracis library isolates. Fluorescence analysis of cultures from 10 clones, which exhibited high fluorescence, compared to cultures expressing gfpuv under the control of the Pamy, Psap_short, and Psap_long promoters is depicted by the bar diagrams. All measurements were determined in cultures of identical densities. The growth rates (determined by growth curves similar to those shown in Fig. 3) and insert size (determined by PCR analysis) are indicated in the table. Four library isolates, L-19, L-25, L-32, and L-51, indicated by bold-frame histograms, were selected for sequence analysis. ND, not detected. The error bars indicate standard deviations.

FIG. 5.

Chromosomal gene arrangement and promoter locations of the insert fragments from the four selected library clones. The numbers of contigs and open reading frames (orfs) are according to the February 2001 version of the B. anthracis Ames draft genome (The Institute for Genomic Research). The putative RBSs contain the core sequence with flanking nucleotides on both sides, with the highest sequence complementary to the 3′ end of the B. subtilis 16S rRNA (2). The solid boxes represent the various B. anthracis DNA fragment inserts; hatched boxes represent the region not sequenced in clone L-25. Clones L-25 and L-51 originate from ligation of two chromosomal fragments. For clones L-19 and L-32, the first and last base pairs are indicated according to the contig sequence. The darkly shaded rectangles represent promoter noncoding regions, and transcription directions are marked by open arrows.

FIG. 6.

Analysis of recombinant PA expression in B. anthracis by various promoters. (A) SDS-PAGE of extracellular samples taken from liquid cultures at identical cell densities. Migration of the mature secreted PA (83 kDa) is indicated. The upper protein band (94 kDa), which appears only in culture supernatants of strains harboring pA_MCS-5 (no promoter) and pA_pag, represents the S-layer protein Sap, as determined by specific antibodies (not shown). The growth rates are averaged from five independent experiments carried out for each clone. (B) Western blots of cell lysates probed with two anti-PA antibodies targeted to both premature and mature forms of PA (B-11) and to the mature PA form only (AI-10) (see Materials and Methods).

FIG. 7.

Expression of rPA driven by Pamy in B. subtilis WB600. (A) SDS-PAGE of extracellular samples collected at the indicated growth times. (B) Growth curves of B. subtilis WB600 host strain (dashed line) and recombinant strain harboring pA_amy (solid circles). (C) Western blot analysis with anti-PA antibodies (B-11) of control mature B. anthracis rPA (I), secreted (II), or cell-associated (III) rPA collected from 8-h cell cultures of a B. subtilis strain harboring pA_amy.