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Project 5: The Immune Response to Bacterial Spores It is clear that the marginal zone of the spleen is particularly involved in the antibody response to bacteria and bacterial products. We have chosen to study the immune response to the inert metabolically inactive spores of gram positive bacilli as model antigens since these are trapped very efficiently in the marginal zone and they do not express the various mitogenic components expressed by the vegetative forms. |
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Generation of Monoclonal Antibodies Panels of mouse monoclonal antibodies were made against B. subtilis, B. thuringiensis B. cereus and B. anthracis spores by immunization with intact native spores (or a mixture of 40kGy irradiated DAmes and Sterne strains in the case of B. anthracis). These antibodies were purified, conjugated with fluorochromes, and screened for specificity by ELISA and by flow cytometry. In each case multiple monoclonal antibodies were obtained which were specific for the immunizing spore species and did not cross-react with spores of other closely related organisms or vegetative forms of these bacilli. These antibodies also discriminate between mixtures of spores (Fig. 7). |
| Anthrax-Spore host Interactions The spore-forming bacterium Bacillus anthracis (BA) is the causative agent of anthrax in animals and humans. The use of B. anthracis as a bioweapon depends upon the spore’s entrance into the body, its uptake by and germination within host cells, and the host’s response to toxins expressed by the vegetative bacteria. Because the spore is the infectious form of B. anthracis, the interaction between its outermost layer, or exosporium, and the host receptors is considered a vital step in the initiation of disease. Although a previous paradigm maintained that successful infection involved a step of intracellular germination and outgrowth, macrophages and other cell types have been shown to play an important role in early control of the pathogen because they efficiently kill phagocytosed spores upon germination. The destruction of germinated spores is enhanced by factors that promote macrophage activation, including cytokine production and spore-opsonization. Of particular interest are the mechanisms governing initial contact, spore uptake, spore-induced activation, and intracellular spore termination, as these processes have yet to be elucidated fully. We recently showed that attachment and uptake of B. anthracis spores by macrophages occurs through a CD14-enhanced, Mac-1-dependent pathway mediated by the major exosporium protein BclA. Absence of BclA on spores results in indiscriminate entry into a variety of cell types in addition to macrophages, and, paradoxically, B. anthracis virulence. It appears, therefore, that BclA promotes the selective uptake of B. anthracis spores by host macrophages, wherein the spores are most susceptible to elimination. Despite the capacity of macrophages to kill spores efficiently, all phagocytosed spores are not always killed; the surviving spores lead to anthrax. In our studies using phagocytic cells, we have shown that engulfment of spores and the survival of spore-bearing cells depend on NF-kappaB signaling pathways that promote the production of inflammatory proteins by means of a PI3 kinase pathway. . A deeper understanding of these mechanisms will enable the development of strategies to enhance spore extermination prior to or during germination, thus preventing bacterial development, the elaboration of toxins, and disease. A deeper understanding of these mechanisms will enable the development of strategies to enhance spore extermination prior to or during germination, thus preventing bacterial development, the elaboration of toxins, and disease. |
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| References Lisanby, N., Swiecki M.K., Dizon, B., Pflughoeft. K., Koehler T., Kearney J.F. Cathelicidin administration protects mice from Bacillus anthracis spore challenge. J. Immunol: 181:4989-5000, 2008 McPherson, S.A., Li, M., Kearney, J.F., Turnbough, C.L., Jr. ExsB, an unusually highly phosphorylated protein required for the stable attachment of the exosporium of Bacillus anthracis. Mol Microbiol: 2010 Jun;76(6):1527-38. Epub 2010 Apr 23. Oliva, C., Turnbough, C. L. , Jr., and Kearney, J. F. 2009. CD14-Mac-1 interactions in Bacillus anthracis spore internalization by macrophages. Proc Natl Acad Sci U S A: 106:13957-13962, 2009. Oliva, C. R., Swiecki, M. K. , Griguer, C. E. , Lisanby, M. W. , Bullard, D. C. , Turnbough,C. L., Jr., and Kearney,J.F. The integrin Mac-1 (CR3) mediates internalization and directs Bacillus anthracis spores into professional phagocytes. Proc Natl Acad Sci U S A: 105:1261-1266, 2008. |
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