Identification, recombinant expression, immunolocalization in macrophages, and T-cell responsiveness of the major extracellular proteins of Francisella tularensis

Abstract

A safer and more effective vaccine than the previously developed live attenuated vaccine is needed for combating Francisella tularensis, a highly infectious bacterial pathogen. To search for potential candidates for inclusion in a new vaccine, we characterized the proteins present in the culture filtrates of a virulent recent clinical isolate and the attenuated live vaccine strain of F. tularensis using a proteomic approach. We identified a total of 12 proteins; among these, catalase-peroxidase was much more abundant in the culture filtrate of the virulent clinical isolate, whereas bacterioferritin was more abundant in the culture filtrate of the live vaccine strain. Streptolysin O treatment of infected human macrophages indicated that catalase-peroxidase and the heat shock protein GroEL are released intracellularly by actively growing F. tularensis. Mice immunized with F. tularensis developed significant cell-mediated immune responses to catalase-peroxidase, the heat shock protein GroEL, and bacterioferritin as measured by splenic lymphocyte proliferation and gamma interferon production. Finally, we expressed the major culture filtrate proteins that are promising vaccine candidates in Escherichia coli at high levels in soluble form to facilitate study of their immunobiology and potential role in vaccines.

FIG. 1.

Growth and culture filtrate protein profiles of F. tularensis in Chamberlain medium. The growth of F. tularensis LVS and RCI strains was monitored by measurement of culture density (absorbance at 540 nm) over a 24-h period. Culture filtrates harvested from bacterial cultures at the indicated time points were concentrated, and the concentrate derived from 10 ml of original culture for each sample was analyzed by SDS-PAGE on a 12.5% gel and stained with Coomassie blue. This experiment was performed twice with similar results.

FIG. 2.

Growth of F. tularensis in defined and complex media. The growth of F. tularensis LVS and RCI in four different media was monitored by assaying the culture density over an 8-h period. The media are abbreviated as follows: CM, Chamberlain medium; BHI, brain heart infusion broth; MH, Mueller-Hinton broth; TS, tryptic soy broth. This experiment was performed twice with similar results.

FIG. 3.

Protein profiles of the culture filtrates and the corresponding bacterial lysates of F. tularensis LVS and RCI. An equal amount (30 μg) of culture filtrate (CF) and sonicated bacterial lysate (BL) obtained from F. tularensis LVS and RCI grown in Chamberlain medium for 5 h or 17 h was analyzed by SDS-PAGE on a 12.5% gel and stained with Coomassie blue. Lanes M contain molecular mass markers (in kilodaltons). This experiment was performed twice with similar results.

FIG. 4.

2-DE separation of radiolabeled F. tularensis culture filtrate proteins. Early culture filtrate proteins of F. tularensis RCI radiolabeled with [³⁵S]methionine were mixed with nonradiolabeled early culture filtrate proteins of RCI and separated by 2-DE. Coomassie blue-stained protein spots on PVDF membrane (A) and the corresponding radiolabeled spots on X-ray film (B) are indicated by the white arrowheads. This experiment was performed twice with similar results.

FIG. 5.

Comparative 2-DE of F. tularensis LVS and RCI culture filtrate proteins. Equal amounts of culture filtrate proteins (200 μg) harvested from F. tularensis LVS and RCI grown in Chamberlain medium for 4 h or 14 h were separated by 2-DE and stained with Coomassie blue. The spots corresponding to KatG, DnaK, GroEL, and Bfr are indicated by the black arrowheads. Three independent experiments for LVS and RCI at each time point were performed with similar results.

FIG. 6.

Recombinant expression in E. coli of genes corresponding to F. tularensis culture filtrate proteins. E. coli BL21 CodonPlus(DE3)-RIL expressing F. tularensis culture filtrate proteins with a N-terminal histidine tag were disrupted by sonication. (A) The released soluble proteins were analyzed by subjecting them to SDS-PAGE, staining with Coomassie blue and by immunoblotting using an anti-histidine tag monoclonal antibody. Lanes 1, vector alone; lanes 2, Bfr; lanes 3, SodB; lanes 4, FabD; lanes 5, GroEL; lane M, molecular mass markers. (B) Culture filtrate proteins from the recombinant E. coli expressing F. tularensis KatG without (−) or with (+) IPTG induction were analyzed by subjecting them to SDS-PAGE and staining with Coomassie blue and by immunoblotting using an anti-histidine tag monoclonal antibody. The protein band indicated by the small black arrowhead was subjected to N-terminal amino acid sequencing. The first seven amino acids of the protein band corresponded to those in the N-terminal sequence of the mature KatG of F. tularensis. (C) The sequence of the first 40 amino acid residues of the full-length KatG protein is shown. The cleavage site of the KatG signal peptide is indicated by the black arrow pointing down.

FIG. 7.

Immunodetection of the intracellular release of culture filtrate proteins by F. tularensis. F. tularensis RCI (10⁸), THP-1 cells (10⁷), or RCI-infected THP-1 cells were treated with streptolysin O (+), and the soluble supernatant released from each sample was divided equally for analysis by immunoblotting with antibodies specific to KatG, GroEL, or Bfr.

FIG. 8.

T-cell responses to culture filtrate proteins in mice immunized with F. tularensis. BALB/c mice were injected with PBS or with heat-killed or live F. tularensis LVS. Proliferation of T cells to individual protein antigens was measured, and the stimulation index (SI) was calculated as described in Materials and Methods. Data are the mean SI values ± standard errors (SE) (error bars) for nine animals in a group. The frequency of IFN-γ-secreting CD4⁺ and CD8⁺ T cells specific for individual protein antigens was determined by flow cytometry analysis. Data are presented as means ± SE for three mice per group. Values for animals immunized with live LVS and sham-immunized animals that were significantly different (P value of less than 0.05 by Student's t test) are indicated by an asterisk.