Pathogenecity of Anthrax
Infection commences once the spore enters a susceptible host and germinates. This key event in the disease process represents a critical point at which if we inhibit germination we prevent subsequent disease development. My group has been working for a number of years to characterize the biology of the spore surface and to determine the role that it plays in initiating infection. The outer layer of the spore, the exosporium, is the primary contact surface between the spore and environment or host. Analysis reveals it to be composed of a surprisingly complex mixture of constitutents including enzymes such immune inhibitor A, a zinc metalloprotease, which degrades components of the insects immune system, alanine racemase which regulates germination and superoxide dismutases which contribute to intracellular survival within the macrophage (Redmond et al., 2004). Indeed we recently reported that the exosporium contributed directly to intracellular survival within murine macrophages (Kang et al., 2005).
Aims of Project
In the context of mammalian infection the interaction of the spore with the macrophage is the key first step in the disease process. In their quiescent state, macrophages are metabolically subdued. Yet upon encountering a microorganism, these cells become stimulated, resulting in sequestration of the invading microbe into an enclosed vacuole, the phagosome, into which superoxide (O2•) is secreted and proteins and proteases are released following fusion of lysosomes to form the phagolysosome. Mouse studies have shown that spores of anthrax need to be taken up by a macrophage to trigger germination and that this happens within the phagolysosome. Thus to survive within this harsh environment the bacterium must possess mechanisms, such as superoxide dismutase (SOD) and catalase which enable it to circumvent the antibacterial attentions of O2• and hydrogen peroxide generated by the oxidative burst. We recently reported that exposure of B.anthracis endspores to fluxes of O2• typically found in stimulated phagocytes had no effect on viability and that the endospores were able to scavenge O2• (Baillie et al., 2005). This observation lead us to hypothesis that SOD located in the exosporium may play a role in protecting the germinating bacilli from oxidative injury during a sensitive period until the bacillus fully matures and it able to express it own scavangers. Studies are in progress to test this hypothesis.
Superoxide is not the only antibacterial radical generated by macrophages and in a recent study we demonstrated susceptibility of vegetative B.anthracis to nitric oxide (Raines et al., 2006). Macrophages generate nitric oxide (NO•) from the oxidative metabolism of L-arginine using the enzyme nitric oxide synthase (NOS 2). Inhibition of NOS 2 activity by L-NIL or by culturing macrophages in L-arginine depleted media decreased B.anthracis specific antibacterial activity. At least one human pathogen, H.pylori a causative agent of gastric ulcers, produces a microbial arginase which by converts arginine to ornithine deprives NOS 2 of its substrate thus preventing NO• production. B.anthracis also expresses an arginase during vegetative growth and in recent studies we have shown that spore has arginase activity associated with the exosporium (Raines et al., 2006). Again it is tempting to speculate that arginase on the surface of the spore competes with the macrophages NOS 2 for its substrate, L-arginine, and as a consequences down regulates NO mediated killing. As macrophage-generated NO B. anthracis to regulate the production of this free radical has important implications in the control B. anthracis-mediated infection. Studies are in progress to define the precise role of bacterial arginase in this process.
While the presence of enzymes on the surface of the spore may explain the ability of the organism to subvert the macrophages antibacterial killing mechanisms it does not explain the central role that macrophages play in triggering germination. We recently reported that low fluxes of O2• actually promote germination (Baillie et al., 2005). The presence on the spore surface of a well known negative regulation of germination, alanine racemase an enzyme which converts L-alanine to D-alanine, an isomer that is not recognized by the spores germination receptors, may have some role to play in this mechanism. It is fascinating to speculate that this enzyme may play a role in regulating germination by preventing the access of L-alanine to its receptor and that their activities are influenced by the conditions found inside the macrophage, principally radical production. Studies are in progress to define the roles of alanine racemase in germination.
In addition to characterizing bacterial virulence factors we have also been working to determine how the interaction of the spore with the macrophage triggers host cell responses. Of particular interest is the role of pattern recognition receptors such as Toll like Receptors and NOD 2 in stimulating inflammatory immune response.