Abstract
Like many Gram-negative pathogens, Shigella rely on a complex type III secretion system (T3SS) to inject effector proteins into host cells, take over host functions, and ultimately establish infection. Despite these critical roles, the energetics and regulatory mechanisms controlling the T3SS and pathogen virulence remain largely unclear. In this study, we present a series of high resolution crystal structures of Spa47 and use the structures to model an activated Spa47 oligomer, finding that ATP hydrolysis may be supported by specific side chain contributions from adjacent protomers within the complex. Follow-up mutagenesis experiments targeting the predicted active site residues validate the oligomeric model and determined that each of the tested residues are essential for Spa47 ATPase activity, although they are not directly responsible for stable oligomer formation. Although N-terminal domain truncation was necessary for crystal formation, it resulted in strictly monomeric Spa47 that is unable to hydrolyze ATP, despite maintaining the canonical ATPase core structure and active site residues. Coupled with studies of ATPase inactive full-length Spa47 point mutants, we find that Spa47 oligomerization and ATP hydrolysis are needed for complete T3SS apparatus formation, a proper translocator secretion profile, and Shigella virulence. This work represents the first structure-function characterization of Spa47, uniquely complementing the multitude of included Shigella T3SS phenotype assays and providing a more complete understanding of T3SS ATPase-mediated pathogen virulence. Additionally, these findings provide a strong platform for follow-up studies evaluating regulation of Spa47 oligomerization in vivo as a much needed means of treating and perhaps preventing shigellosis.
Highlights
Like many Gram-negative pathogens, Shigella rely on a complex type III secretion system (T3SS) to inject effector proteins into host cells, take over host functions, and establish infection
Coupled with studies of ATPase inactive full-length Spa47 point mutants, we find that Spa47 oligomerization and ATP hydrolysis are needed for complete T3SS apparatus formation, a proper translocator secretion profile, and Shigella virulence
Sequence similarity to known ATPases including the catalytic -subunit of F1 ATP synthase and the Salmonella flagellar ATPase FliI suggested that the Shigella T3SS protein Spa47 is a T3SS-associated ATPase and that it could in turn provide the energy required to assemble and activate the T3SA
Summary
Like many Gram-negative pathogens, Shigella rely on a complex type III secretion system (T3SS) to inject effector proteins into host cells, take over host functions, and establish infection. Many Gram-negative pathogens rely on highly conserved type III secretion systems (T3SSs) to cause infection by supporting direct injection of effector proteins into the targeted host cell cytoplasm [1,2,3,4] One example from this class includes Shigella spp., which is the causative agent of bacillary dysentery in humans and a significant contributor to diarrheal disease. Some of the most highly studied virulence-associated T3SS ATPases are those from Escherichia coli and Salmonella, which have been shown to recognize T3SS chaperones [29, 30] and unfold the bound effector to facilitate secretion [31] These and related works have contributed significantly to the understanding of T3SS ATPases in general, the mechanism(s) driving enzyme activation and the specific role(s) that T3SS ATPases play in many pathogens remain unstudied and unclear
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