Overview

One important subject of our study is to elucidate the mechanisms of specific properties of bacterial spores which species of both the genus Bacillus and the genus Clostridium produce. They are highly resistant to various physical and chemical agents such as heat, dryness, radiation. disinfectants and antibiotics. However, one spores germinate, they lose their resistance. Germination is initiated by specific amino acid, sugar, nucleoside or inorganic ion alone, or in combination depending on species. Bacillus subtilis can be effectively germinated by L-alanine or its analogs. In genetic studies of germination mutants of B. subtilis, the gerA operon was demonstrated to be involved in L-alanine-initiated germination. The gerA operon encodes three membrane-associated proteins(GerAA, GerAB and GerAC) which make up the alanine receptor complex. In our laboratory, for the purpose of elucidating the germination mechanism, the localization of GerAA, GerAB and GerAC proteins in the spore has been investigated by synthesizing a peptide of each protein, which is probable to be an epitope, and by utilizing immunoelectron microscopy. So far it was mad clear that a peptide( amin oacids 61 to 80) of GreAB was present in the boundary region of a peptide of the other proteins is being examined in the same manner. Moreover we have been investigating the role of dipicolinic acid present much in a spore, the mechanism of heat resistance and a permeability barrier of a spore to small molecules.

Another subject of our study is to develop mucosal vaccines for various pathogens. Cholera toxin B subunit(CTB) of Vibrio cholerae is a good carrier protein and an effecive adjuvant which can stimulate the importnat sIgA production when administered orally or intranasally with other antigens. Therefore we constructed a very efficient synthesis and secretion system for CTB using Baccillus brevis. Now we have bee measuring antibody levels in serum and saliva, and in intestinal, bronchoalveolar and vaginal washings of mice which were immunized orally with a commercial cholera vaccine, a commercial adsorbed diphtheria-purified pertussis-tetanus combined vaccine or a commercial pneumococcal capsular polysaccharide vaccine in addition to recombinant CTB. In the near future we will prepare a conjugate vaccine consisting of CTB and a domain peptide of various protective antigens or their gene fusion protein, and examine the antigenicity of each conjugate vaccine and fusion protein.