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Type three secretion systems (T3SS) are complex nano-machines that evolved to inject bacterial effector proteins directly into the cytoplasm of eukaryotic cells. Many high-priority human pathogens rely on one or more T3SSs to cause disease and evade host immune responses, underscoring the need to better understand the mechanisms through which T3SSs function and their role(s) in supporting pathogen virulence. We recently identified the  Shigella  protein Spa47 as an oligomerization-activated T3SS ATPase that fuels the T3SS and supports overall  Shigella  virulence. Here, we provide both  in vitro  and  in vivo  characterization of Spa47 oligomerization and activation in the presence and absence of engineered ATPase-inactive Spa47 mutants. The findings describe mechanistic details of Spa47-catalyzed ATP hydrolysis and uncover critical distinctions between oligomerization mechanisms capable of supporting ATP hydrolysis  in vitro  and those that support T3SS function  in vivo . Concentration-dependent ATPase kinetics and experiments combining wild-type and engineered ATPase inactive Spa47 mutants found that monomeric Spa47 species isolated from recombinant preparations exhibit low-level ATPase activity by forming short-lived oligomers with active site contributions from at least two protomers. In contrast, isolated Spa47 oligomers exhibit enhanced ATP hydrolysis rates that likely result from multiple preformed active sites within the oligomeric complex, as is predicted to occur within the context of the type three secretion system injectisome. High-resolution fluorescence microscopy, T3SS activity, and virulence phenotype analyses of  Shigella  strains co-expressing wild-type Spa47 and the ATPase inactive Spa47 mutants demonstrate that the N-terminus of Spa47, not ATPase activity, is responsible for incorporation into the injectisome where the mutant strains exhibit a dominant negative effect on T3SS function and  Shigella  virulence. Together, the findings presented here help to close a significant gap in our understanding of how T3SS ATPases are activated and define restraints with respect to how ATP hydrolysis is ultimately coupled to T3SS function  in vivo .

Dominant negative effects by inactive Spa47 mutants inhibit T3SS function and Shigella virulence