| While each branch of the immune system works cooperatively to defend its host, often the effectiveness of early, innate defenses, such as phagocytes, determines the overall success of the battles against pathogens. Bacillus anthracis has evolved means by which to survive this innate defense, permitting both the escape of the bacilli from the phagosome and the release from the phagocyte. While Lethal Toxin (LeTx) is the primary agent responsible for the progression of anthrax, we have previously shown that LeTx is not required for the escape/release phenomenon. We found that B. anthracis encodes four potential membrane-active proteins (MAPs), and one such protein, subsequently named anthrolysin O (ALO), is a putative member of the family of cholesterol-dependent cytolysins (CDCs). Because ALO is more than 60% similar at the amino acid level to the Listeria monocytogenes orthologue listeriolysin O (LLO) and because of the transient intracellular lifestyle of B. anthracis, I hypothesized that ALO functions as mediator cellular escape.; Through the use of erythrocyte hemolysis assays, ALO became firmly established as a member of the CDC family of cytotoxins, and gain-of-function studies illustrated that the expression of aloA by Bacillus subtilis and LLO-null L. monocytogenes was sufficient to permit phagosomal escape from these typically escape-incompetent strains.; Deletion studies were utilized to investigate the essentiality of ALO for B. anthracis in both in vitro and in vivo models of anthrax. B. anthracis Delta aloA was found to be indistinguishable from wildtype Sterne in bone marrow-derived macrophages and in DBA2/J mice. Because the presence of four putative membrane-active proteins made redundancy of function a possibility, knock-out strains of each of the MAPs in all combinations were constructed. Ultimately, the quadruple mutant, a strain deleted for all four MAPs, showed neither the ability to grow within nor cytotoxicity to macrophages. Furthermore, the quadruple mutant was nearly 40-fold less virulent than the wildtype strain in the mouse model.; With this body of work, I have shown that ALO is indeed a functional member of the CDC family of proteins, capable of mediating bacterial escape from host cells. And while aloA is not an essential element of anthrax pathogenesis in the models tested, it is part of a repertoire of MAPs required for full virulence. |
