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Bacillus anthracis , the causal agent of anthrax, synthesizes two surface layer (S-layer) proteins, EA1 and Sap, which account for 5 to 10% of total protein and are expressed in vivo. A recombinant B. anthracis strain was constructed by integrating into the chromosome a translational fusion harboring the DNA fragments encoding the cell wall-targeting domain of the S-layer protein EA1 and tetanus toxin fragment C (ToxC). This construct was expressed under the control of the promoter of the S-layer component gene. The hybrid protein was stably expressed on the cell surface of the bacterium. Mice were immunized with bacilli of the corresponding strain, and the hybrid protein elicited a humoral response to ToxC. This immune response was sufficient to protect mice against tetanus toxin challenge. Thus, the strategy developed in this study may make it possible to generate multivalent live veterinary vaccines, using the S-layer protein genes as a cell surface display system.

Fully virulent Bacillus anthracis bacilli are encapsulated and toxinogenic. These bacteria contain two plasmids, pXO1 and pXO2, carrying genes coding for toxins (pag, lef, and cya) and for capsule synthetic enzymes (capB, capC, capA, and dep), respectively. A transcriptional fusion between the capB regulatory region and the lacZ reporter gene was constructed to study the regulation of capsule synthesis. A single copy of this fusion was inserted into the cap region of pXO2. The influence of environmental factors on the capB-lacZ fusion expression was initially analyzed in a pXO1-negative background: bicarbonate but not temperature induced the transcription from the capB promoter. A strain carrying the recombinant pXO2 and (delta)pag pXO1 was constructed for transregulatory studies. The pXO1 plasmid strongly enhanced capsule formation without modifying the bicarbonate-dependent induction level. A (delta)cap pXO2 was transduced into a strain containing pXO1 harboring a pag-lacZ transcriptional fusion (19). pXO2 showed no influence on the toxin gene transcription.