Molecular and Structural Basis of Anthrax Lethal Toxin Pore Formation and Translocation
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Issue Date
2020-08-31Author
Machen, Alexandra
Publisher
University of Kansas
Format
163 pages
Type
Dissertation
Degree Level
Ph.D.
Discipline
Biochemistry & Molecular Biology
Rights
Copyright held by the author.
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The morbidity and mortality of anthrax disease are associated with the anthrax toxin, which is generated by the gram-positive bacterium Bacillus anthracis. The anthrax toxin is an AB toxin composed of two components: an active (A) moiety named lethal factor (LF) and a binding (B) moiety termed protective antigen (PA). In order for the anthrax toxin to elicit its cytotoxic effect, the LF component must first enter the cell. To accomplish this, the PA component binds to a target host cell receptor and forms a pore (PApore) that translocates LF into the cytosol. In this work, we explore PA binding the host cell receptor, the interactions between PA and LF during pore formation, and the translocation of LF through the PApore. We hypothesize PA dissociates from its cellular receptor to facilitate pore formation. To test this, in Chapter 2, we investigated the anthrax toxin receptor CMG2 binding capabilities to the PApore under endosomal conditions. Our results provide evidence for receptor release prior to pore formation. In Chapter 3, we hypothesized the LF N-terminal tail travels down the pore lumen and interacts with the narrowest part of the pore. To test this, we characterized the structure of three LFN bound to PApore at neutral pH. Our results indicate the N-terminal tail of LF remains flexible in the translocation incompetent neutral pH environment and underscores the necessity of using physiologically relevant conditions. In Chapter 4, we hypothesize LF begins to refold inside the PApore during translocation. To test this, we captured intermediates of LF translocation through PApore using cryoEM. Our results support the hypothesis that initial refolding of LF structural elements occurs in the PApore beta barrel during translocation. Cumulatively, we have made significant contributions to our understanding of the anthrax intoxication mechanism at multiple biologically relevant steps.
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