On the role of membrane active toxins in Bacillus anthracis pathogenesis

Anthrax is a disease that proceeds through discrete stages. While B. anthracis is considered an extracellular pathogen at later stages of disease, its brief intracellular phase during the establishment of anthrax is believed to be critical. Since the infectious lifecycle of B. anthracis transitions through professional phagocytes at the onset of infection, I hypothesized that B. anthracis utilizes membrane active toxins to disrupt the integrity of phagosomal membranes and permit the passage of the bacterium into the host cell cytosol.; Three genes were identified on the B. anthracis genome that encode phospholipases C (PLCs). Defined deletions of the PLC genes, both individually and in combination, were generated in the Sterne background resulting in the respective loss of their activities. Disruption of all three PLC genes was required for attenuation in a murine model of anthrax. This attenuation could be attributed to a growth deficiency within macrophages as a consequence of the PLC null strains' inefficient escape from phagosomal compartments.; Although the loss of PLC activity impedes the ability of B. anthracis to escape from vacuoles, the bacterium still possessed additional means to gain access to the cytosol. Vegetative bacilli were observed growing in association with the macrophages, albeit to a lesser extent, in the absence of all three PLCs implicating that additional factors are involved in the process of phagosomal escape. Two such factors were investigated; the anthrax lethal factor (LF) and the cholesterol dependent cytolysin anthrolysin O (ALO). Combinatorial deletions of the ALO and LF genes with the PLCs demonstrated that ALO cooperates with the PLCs during both murine and macrophage challenges while LF has a distinct role from that of the PLCs in anthrax pathogenesis. Altogether the results presented in this dissertation demonstrate that B. anthracis utilizes four membrane active toxins to disrupt phagosomal membranes, thus permitting the passage of the bacterium into the host cell cytosol to ensure its survival during the early interactions with phagocytes. Identification of the toxins that mediate phagosomal escape is an important first step towards understanding how B. anthracis transitions from an intracellular to extracellular pathogen.