Anthrax is a severe disease caused by the gram-positive bacterium Bacillus anthracis. The organism infects humans and many other animals and the only identified virulence factors are anthrax toxin and a glutamic acid capsule, both of which are encoded on large plasmids. Inhalation anthrax, the most severe form of the disease, is a systemic infection in which the organism spreads to the lymph nodes and then into the blood where it is able to replicate to levels as high as 100 million bacteria / ml. Factors encoded on the chromosome have also been implicated in virulence, since certain strains lacking the virulence plasmids are still capable of causing disease in immunized animals. The future development of these """"""""vaccine resistant"""""""" strains as bioterrorism weapons is of major concern, and stresses the importance of establishing a fundamental understanding of the molecular mechanisms involved in the pathogenesis of this bacterium. For many bacterial pathogens, the ability to survive in the human host requires the acquisition of the essential element iron. Mechanisms involved in the acquisition of iron have been shown to be important for the virulence of numerous bacterial pathogens, including organisms that replicate in the blood, where much of the available iron is sequestered by host iron compounds such as transferrin and hemoglobin (in erythrocytes). No investigations have examined the mechanism by which B. anthracis acquires iron during growth in vivo or in vitro. This is an important area of study that has not been explored, and in this proposed research, I intend to test the hypothesis that virulence factors involved in the transport and utilization of host iron compounds are present in B. anthracis. Our long-term interest is to identify surface proteins that are involved in the utilization of host iron compounds, and determine if these proteins may be useful vaccine candidates. We have developed a low-iron minimal medium to cultivate B. anthracis and have used this medium to show that the bacterium could use a variety of host compounds as essential iron sources, including heme, hemoglobin, ferritin, transferrin, and lactoferrin. In the past year we have independently developed a method for constructing site directed mutations in the chromosome of B. anthracis. This technique has been used to construct mutations in genes involved in the biosynthesis of the B. anthracis siderophore. The siderophore mutations resulted in strains that had diminished production of iron chelating activity, but nevertheless still made detectable quantities of an extracellular iron-chelating compound. These findings suggested that B. anthracis may produce multiple siderophores. In an attempt to identify proteins involved in the utilization of transferrin and lactoferrin as iron sources, we have used affinity chromatography to enrich for iron-regulated membrane proteins that bind to transferrin and lactoferrin. Several proteins that have the ability to bind to transferrin and lactoferrin were partially purified using this technique and the amino acid sequence of their N-terminal is currently being determined. The N-terminal sequence obtained from these proteins will be used to identify the genes that encode these various proteins. Genes and their associated products that appear to be directly involved in the utilization of either transferrin or lactoferrin as iron sources will be examined in greater detail.
