Dataset Information

MxiN Differentially Regulates Monomeric and Oligomeric Species of the Shigella Type Three Secretion System ATPase Spa47.

ABSTRACT: Shigella rely entirely on the action of a single type three secretion system (T3SS) to support cellular invasion of colonic epithelial cells and to circumvent host immune responses. The ATPase Spa47 resides at the base of the Shigella needle-like type three secretion apparatus (T3SA), supporting protein secretion through the apparatus and providing a likely means for native virulence regulation by Shigella and a much needed target for non-antibiotic therapeutics to treat Shigella infections. Here, we show that MxiN is a differential regulator of Spa47 and that its regulatory impact is determined by the oligomeric state of the Spa47 ATPase, with which it interacts. In vitro and in vivo characterization shows that interaction of MxiN with Spa47 requires the six N-terminal residues of Spa47 that are also necessary for stable Spa47 oligomer formation and activation. This interaction with MxiN negatively influences the activity of Spa47 oligomers while upregulating the ATPase activity of monomeric Spa47. Detailed kinetic analyses of monomeric and oligomeric Spa47 in the presence and absence of MxiN uncover additional mechanistic insights into the regulation of Spa47 by MxiN, suggesting that the MxiN/Spa47 species resulting from interaction with monomeric and oligomeric Spa47 are functionally distinct and that both could be involved in Shigella T3SS regulation. Uncovering regulation of Spa47 by MxiN addresses an important gap in the current understanding of how Shigella controls T3SA activity and provides the first description of differential T3SS ATPase regulation by a native T3SS protein.

SUBMITTER: Case HB

PROVIDER: S-EPMC5910026 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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MxiN Differentially Regulates Monomeric and Oligomeric Species of the Shigella Type Three Secretion System ATPase Spa47.

Case Heather B HB Dickenson Nicholas E NE

Biochemistry 20180403 15

Shigella rely entirely on the action of a single type three secretion system (T3SS) to support cellular invasion of colonic epithelial cells and to circumvent host immune responses. The ATPase Spa47 resides at the base of the Shigella needle-like type three secretion apparatus (T3SA), supporting protein secretion through the apparatus and providing a likely means for native virulence regulation by Shigella and a much needed target for non-antibiotic therapeutics to treat Shigella infections. Her ...[more]

PMID: 29595954

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Project description: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.

Dataset Information

MxiN Differentially Regulates Monomeric and Oligomeric Species of the Shigella Type Three Secretion System ATPase Spa47.

Publications

MxiN Differentially Regulates Monomeric and Oligomeric Species of the Shigella Type Three Secretion System ATPase Spa47.

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