Structural and Mechanistic Insights into a Specialized Secretion System Necessary for M. tuberculosis Virulence

Mycobacterium tuberculosis utilizes a specialized secretion system, known as Type VII Secretion (T7S) to translocate virulence factors into the host cell. T7S is absolutely necessary for virulence of M. tuberculosis, as well as other pathogens such as S. aureus. Intriguingly, T7S systems are broadly conserved amongst all Gram-positive bacteria, and likely play diverse roles in bacterial physiology. In this work, we focused on the two conserved components present in all T7S systems: the FtsK-like ATPase EccC and the WXG-100 substrate EsxB. We developed a biochemical model system to answer questions about the structure and molecular mechanisms of these two protein components, and confirm our results using genetics in M. tuberculosis. Using this system, we i) identified the substrate-binding pocket on EccC, ii) identified the non-canonical "signal sequence" on the EsxB substrate, iii) determined the molecular basis of substrate specificity, iv) solved the structure of the EccC ATPase both bound and unbound to signal sequence, as well as the EssC homolog from Geobacillus thermodenitrificans, v) determined a molecular mechanism of EccC autoinhibition, and vi) uncovered a mechanism by which substrates control activity of EccC through multimerization. Overall, this work generated the first biochemical model for T7S