Spa47 is an oligomerization-activated type three secretion system (T3SS) ATPase from Shigella flexneri.

Publisher: Cold Spring Harbor Laboratory Press Country of Publication: United States NLM ID: 9211750 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1469-896X (Electronic) Linking ISSN: 09618368 NLM ISO Abbreviation: Protein Sci Subsets: MEDLINE

Publication: 2001- : Woodbury, NY : Cold Spring Harbor Laboratory Press
Original Publication: New York, N.Y. : Cambridge University Press, c1992-

Adenosine Triphosphatases/*chemistry ; Adenosine Triphosphatases/*metabolism ; Shigella flexneri/*pathogenicity; Adenosine Triphosphatases/genetics ; Catalytic Domain ; Chromatography, Gel ; HeLa Cells/microbiology ; Humans ; Point Mutation ; Protein Multimerization ; Shigella flexneri/genetics ; Shigella flexneri/metabolism ; Type III Secretion Systems/chemistry ; Type III Secretion Systems/genetics ; Type III Secretion Systems/metabolism

Gram-negative pathogens often use conserved type three secretion systems (T3SS) for virulence. The Shigella type three secretion apparatus (T3SA) penetrates the host cell membrane and provides a unidirectional conduit for injection of effectors into host cells. The protein Spa47 localizes to the base of the apparatus and is speculated to be an ATPase that provides the energy for T3SA formation and secretion. Here, we developed an expression and purification protocol, producing active Spa47 and providing the first direct evidence that Spa47 is a bona fide ATPase. Additionally, size exclusion chromatography and analytical ultracentrifugation identified multiple oligomeric species of Spa47 with the largest greater than 8 fold more active for ATP hydrolysis than the monomer. An ATPase inactive Spa47 point mutant was then engineered by targeting a conserved Lysine within the predicted Walker A motif of Spa47. Interestingly, the mutant maintained a similar oligomerization pattern as active Spa47, but was unable to restore invasion phenotype when used to complement a spa47 null S. flexneri strain. Together, these results identify Spa47 as a Shigella T3SS ATPase and suggest that its activity is linked to oligomerization, perhaps as a regulatory mechanism as seen in some related pathogens. Additionally, Spa47 catalyzed ATP hydrolysis appears to be essential for host cell invasion, providing a strong platform for additional studies dissecting its role in virulence and providing an attractive target for anti-infective agents.
(© 2016 The Protein Society.)

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R15 GM110671 United States GM NIGMS NIH HHS; R15GM110671 United States GM NIGMS NIH HHS

Keywords: ADP; ATP; ATPase; oligomerization; protein export; type III secretion system (T3SS); virulence factor

0 (Type III Secretion Systems)
EC 3.6.1.- (Adenosine Triphosphatases)
EC 3.6.1.- (Spa47 protein, Shigella flexneri)

Date Created: 20160308 Date Completed: 20161228 Latest Revision: 20181113

20240829

PMC4838647

10.1002/pro.2917

26947936