B. anthracis is an agent of bioterrorism. Prevention, treatment and cure of anthrax disease must target key steps in the infectious cycle, including iron uptake. Recently, a novel iron import system, containing a putative sortase (SrtB) and its substrate BasK, were discovered in B. anthracis. The hypothesis of this application is that SrtB functions to anchor BasK to the cell wall, a process necessary for BasK to transport heme into B. anthracis during infection. There are two specific aims: 1. Investigate the role of SrtB and BasK in virulence and heme acquisition. 1.a. Determine the contribution of SrtB and BasK to B. anthracis virulence. The srtB and basK genes of B. anthracis strain Ames will be deleted and mutants tested for virulence in a Guinea pig model of infection. 1.b. Investigate the role of SrtB and BasK in iron sequestration and transport. The regulation of the expression of SrtB and BasK will be determined. The binding, transport, and specificity of/for heme will be measured in Wt, ?srtB and ?basK Sterne. 2. Determine the mechanism of SrtB-mediated anchoring of BasK in B. anthracis. 2.a. Determine if BasK is anchored to the peptidoglycan by SrtB. The localization of BasK in Wt and ?srtB cells will be determined. 2.b. Determine the topology and anchor structure of BasK to the peptidoglycan. Affinity-tagged BasK sorting signal will be purified and subjected to MS analysis. The long-term objectives of this project include, (i) defining the mechanism of extraction, binding, transport, and breakdown of heme by the isd-like system of B. anthracis, (ii) identifying inhibitors of SrtB or heme binding to BasK for antimicrobial development, and (iii) generating an anthrax vaccine composed of the B. anthracis surface proteins. Relevance: This application investigates the mechanism of heme-iron acquisition in B. anthracis, the causative agent of the disease anthrax. Knowledge generated herein will increase the understanding of iron acquisition in bacterial pathogens, facilitate the development of novel antibacterials targeting these systems, and generate reagents for the creation of a more safe and effective vaccine against a major bioterrorism threat. ? ? ?
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