Abstract
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.
Highlights
Type three secretion systems (T3SS) are highly conserved complex multi-component nanomachines that inject proteins from the cytoplasm of pathogenic bacteria into eukaryotic host cells [1,2,3,4]
Recent insights into Spa47 activation suggest that oligomerization supports ATP hydrolysis by contributing amino acid sidechains from adjacent Spa47 protomers to form a single complete active site [21], supporting the observed enhancement of in vitro ATPase activity exhibited by oligomeric Shigella Spa47 and several related type three secretion system (T3SS) ATPases [22, 24, 25, 38,39,40]
Spa47 has served and continues to serve as a valuable model for studying T3SS ATPase activation and regulation as monomeric and oligomeric Spa47 are isolated via size exclusion chromatography (SEC) and isolated Spa47 fractions maintain their original stoichiometry for several weeks when stored at the concentrations collected directly from the sizing column (~1–20 μM)
Summary
Type three secretion systems (T3SS) are highly conserved complex multi-component nanomachines that inject proteins from the cytoplasm of pathogenic bacteria into eukaryotic host cells [1,2,3,4]. While the injected T3SS effector proteins are specific to each pathogen’s infection mechanism and replicative niche, they are all secreted through a highly-conserved syringe and needle-like injectisome [10, 11]. Like much of the actions supporting and regulating type three secretion, the specific contributions of the associated T3SS ATPase are not well understood It is clear, that most, if not all, T3SSs contain a highly conserved ATPase that is most active in oligomeric form and resides just below the apparatus export gate at the base of the T3SS [22,23,24,25,26]. Dissecting the specific mechanisms through which T3SS ATPases function and precisely how they facilitate protein secretion has proven exceptionally challenging due to long-standing difficulties expressing and purifying soluble/active forms of many of the isozymes as well as accessing and interrogating both the dormant monomeric and activated oligomeric forms of the enzymes
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