Search for potential vaccine candidate open reading frames in the Bacillus anthracis virulence plasmid pXO1: in silico and in vitro screening

Abstract

A genomic analysis of the Bacillus anthracis virulence plasmid pXO1, aimed at identifying potential vaccine candidates and virulence-related genes, was carried out. The 143 previously defined open reading frames (ORFs) (R. T. Okinaka, K. Cloud, O. Hampton, A. R. Hoffmaster, K. K. Hill, P. Keim, T. M. Koehler, G. Lamke, S. Kumano, J. Mahillon, D. Manter, Y. Martinez, D. Ricke, R. Svensson, and P. J. Jackson, J. Bacteriol. 181:6509-6515, 1999) were subjected to extensive sequence similarity searches (with the nonredundant and unfinished microbial genome databases), as well as motif, cellular location, and domain analyses. A comparative genomics analysis was conducted with the related genomes of Bacillus subtilis, Bacillus halodurans, and Bacillus cereus and the pBtoxis plasmid of Bacillus thuringiensis var. israeliensis. As a result, the percentage of ORFs with clues about their functions increased from approximately 30% (as previously reported) to more than 60%. The bioinformatics analysis permitted identification of novel genes with putative relevance for pathogenesis and virulence. Based on our analyses, 11 putative proteins were chosen as targets for functional genomics studies. A rapid and efficient functional screening method was developed, in which PCR-amplified full-length linear DNA products of the selected ORFs were transcribed and directly translated in vitro and their immunogenicities were assessed on the basis of their reactivities with hyperimmune anti-B. anthracis antisera. Of the 11 ORFs selected for analysis, 9 were successfully expressed as full-length polypeptides, and 3 of these were found to be antigenic and to have immunogenic potential. The latter ORFs are currently being evaluated to determine their vaccine potential.

FIG. 1.

Translation and immunogenicity of selected pXO1 ORFs. [³⁵S]methionine-labeled proteins were obtained from PCR-derived DNA by using an in vitro transcription-translation TNT kit and were analyzed by SDS-PAGE followed by autoradiography (lanes TNT). The labeled TNT products were immunoprecipitated by using anti-B. anthracis hyperimmune antisera, including rabbit sera (lanes R), guinea pig sera (lanes G), and naöve sera (1:1 mixture of rabbit and guinea pig naöve sera) (lanes N). The apparent molecular masses determined for the ORF products are as follows: ORF13 product, ∼150 kDa (discrete band with the slowest migration); ORF53 product, 13 kDa; ORF54 product, 41 kDa; ORF65 product, 20 kDa; ORF67 product, 66 kDa; ORF76 product, 35 kDa; ORF79 product, no discrete band observed (see text); ORF79tr product (residues 423 to 1042 of ORF79 product), 65 kDa; ORF90 product, 73 kDa; ORF130 product, 28 kDa; and PA, 83 kDa. The predicted molecular weights of the full-length ORF products are shown in Table 1; the expected molecular mass of the ORF79tr product is 65 kDa.

FIG. 2.

Effect of truncation of the SLH domains on the antigenicity of the ORF90 product: SDS-PAGE autoradiography of the immunoprecipitation reactions for the full-length ORF90 product and the ORF90tr product (an N-terminal SLH-truncated TNT product; residues 211 to 652; calculated and observed molecular mass, 53 kDa) with anti-B. anthracis hyperimmune rabbit sera (lanes R) and guinea pig sera (lanes G). The control serum consisted of 1:1 mixture of rabbit and guinea pig naöve sera (lanes N). Equivalent counts of [³⁵S]methionine-containing polypeptide TNT products were used for the immunoprecipitation reactions for the ORF90 and ORF90tr products and PA.