Identification of in vivo-expressed immunogenic proteins by serological proteome analysis of the Bacillus anthracis secretome

Abstract

In a previous comparative proteomic study of Bacillus anthracis examining the influence of the virulence plasmids and of various growth conditions on the composition of the bacterial secretome, we identified 64 abundantly expressed proteins (T. Chitlaru, O. Gat, Y. Gozlan, N. Ariel, and A. Shafferman, J. Bacteriol. 188:3551-3571, 2006). Using a battery of sera from B. anthracis-infected animals, in the present study we demonstrated that 49 of these proteins are immunogenic. Thirty-eight B. anthracis immunogens are documented in this study for the first time. The relative immunogenicities of the 49 secreted proteins appear to span a >10,000-fold range. The proteins eliciting the highest humoral response in the course of infection include, in addition to the well-established immunogens protective antigen (PA), Sap, and EA1, GroEL (BA0267), AhpC (BA0345), MntA (BA3189), HtrA (BA3660), 2,3-cyclic nucleotide diesterase (BA4346), collagen adhesin (BAS5205), an alanine amidase (BA0898), and an endopeptidase (BA1952), as well as three proteins having unknown functions (BA0796, BA0799, and BA0307). Of these 14 highly potent secreted immunogens, 11 are known to be associated with virulence and pathogenicity in B. anthracis or in other bacterial pathogens. Combining the results reported here with the results of a similar study of the membranal proteome of B. anthracis (T. Chitlaru, N. Ariel, A. Zvi, M. Lion, B. Velan, A. Shafferman, and E. Elhanany, Proteomics 4:677-691, 2004) and the results obtained in a functional genomic search for immunogens (O. Gat, H. Grosfeld, N. Ariel, I. Inbar, G. Zaide, Y. Broder, A. Zvi, T. Chitlaru, Z. Altboum, D. Stein, S. Cohen, and A. Shafferman, Infect. Immun. 74:3987-4001, 2006), we generated a list of 84 in vivo-expressed immunogens for future evaluation for vaccine development, diagnostics, and/or therapeutic intervention. In a preliminary study, the efficacies of eight immunogens following DNA immunization of guinea pigs were compared to the efficacy of a PA DNA vaccine. All eight immunogens induced specific high antibody titers comparable to the titers elicited by PA; however, unlike PA, none of them provided protection against a lethal challenge (50 50% lethal doses) of virulent B. anthracis strain Vollum spores.

FIG. 1.

SERPA of B. anthracis secreted proteins: comparison of Coomassie blue-stained 2-DE (gels 1, 3, and 5) with the corresponding Western blots (gels 2, 4, and 6). (Gels 1 and 2) FAG medium secretome of the Vollum strain. (Gels 3 and 4) BHI medium secretome of the Vollum strain. (Gels 5 and 6) FAG medium secretome of the Δ14185 strain. Blots were probed with the sera indicated (see Materials and Methods for a description of the sera). See Table 1 for the identities of the marked protein spots and a complete list of immunogenic proteins. The protein spot numbering is the numbering on the proteomic maps reported previously (16). Also see Fig. S1 in the supplemental material for an extended overview. MW, molecular weight.

FIG. 2.

ISs of B. anthracis secreted proteins. The immunogenicities of proteins identified by SERPA were determined by quantitative comparison of the levels of the various proteins with the intensities of the corresponding Western signals, as described in Materials and Methods. (A) ISs calculated from Western blots probed with rabbit antisera R-1 and R-3 and guinea pig antiserum G-3. (B) Average ISs calculated by probing with all six antisera used in this study. Proteins are arranged in descending order according to their R-1 ISs and are categorized arbitrarily as strong immunogens (IS, > 1,000) (solid bars), medium immunogens (1,000 > IS > 100) (dark gray bars), and weak immunogens (IS, <100) (light gray bars). Immunogens which were also detected by IP assays of in vitro products, as detected in the genomic serologic screen described by Gat et al. (28, 29), are indicated by dots at the bottom and, by definition, belong to group I described in Fig. 3.

FIG. 3.

Segregation of immunogenic proteins identified by SERPA and by the genomic-serologic screens into various groups. The genomic screen (left circle) of 199 ORFs (28) allowed identification of 54 immunogenic proteins (group I and group II). The combined proteomic screens described in the present study and by previous SERPA (6, 15) allowed identification of 88 proteins (small circle), 58 of which are immunogenic proteins and 26 of which are included in group I.

FIG. 4.

Humoral immune responses and protective values of selected ORF products as assessed by DNA-mediated immunization. (Upper left panel) Description of the immunogenic proteins selected for analysis. S, export signal. (Upper middle panel) Immunogenicities of the proteins as determined by SERPA and/or by IP of their ³⁵S-radiolabeled T&T products (see Materials and Methods). (Upper right panel) Specific antibody responses following DNA immunization of guinea pigs by gene gun vaccination using the administration regimen described in Materials an Methods. Immune responses were determined quantitatively by IP of tertiary serial serum dilutions of ³⁵S-labeled translation products. (Lower panel) Survival of guinea pigs (10 guinea pigs per group) and mean times to death (MTD) following a challenge with 50 LD₅₀ of fully virulent spores of the B. anthracis Vollum strain injected subcutaneously.