Germination occurs as an upstream event during the life cycle of Bacillus anthracis. Germinating spores may be considered the weakest link during its life cycle because stopping their function would prevent further development of the disease. Therefore, the development of vaccines against specific proteins expressed during the germination stage of Bacillus anthracis may be an effective strategy to halt the growth of anthrax. Blocking the germination stage would prevent downstream events including the production of anthrax's three natural toxins, PA, LF, and EF, as well as any other foreign toxins that bioterrorists may insert into anthrax. We have recently characterized twenty-two germination-associated proteins from Bacillus anthracis by using proteomic techniques. Conventional proteomics studies proteins primarily using 2D electrophoresis and mass spectrometry. We have determined these germination proteins are differentially expressed from spore dormancy to germination. Eleven identified proteins have been identified. They include one secreted protein (Immune inhibitor A), four potential membrane-associated proteins (Camelysin, Alanine racemase, Larabinose transporter, and L-type calcium channel), two molecular chaperones (HSP60 and cpn60), two energy-related proteins (ATP synthase and Glyceraldehyde 3-phosphate dehydrogenase) and one novel anthrax specific protein. Immune inhibitor A and camelysin are virulent metalloproteinases and have been unreported in Bacillus anthracis. Dr. Tang, a Co-PI in this study, has well-established a noninvasive vaccination system based on vector based vaccines applied to the skin. That system will be used in this study to develop anthrax vaccines that will specifically target two of the germination-associated proteins: immune inhibitor A and camelysin. We will also compare current PA and LF targeted vaccines that have been constructed in our laboratory. In parallel, vaccines derived from immune inhibitor A and camelysin will be evaluated in vitro and in vivo to determine their efficacies in blocking spore germination and cytotoxicity to macrophage cells. This unprecedented study will elicit the pathological mechanisms and vaccination capabilities of immune inhibitor A and camelysin expressed in Bacillus anthracis.
