GONUTS has been updated to MW1.31 Most things seem to be working but be sure to report problems.

Have any questions? Please email us at ecoliwiki@gmail.com

PMID:26947936

From GONUTS
Jump to: navigation, search
Citation

Burgess, JL, Jones, HB, Kumar, P, Toth, RT 4th, Middaugh, CR, Antony, E and Dickenson, NE (2016) Spa47 is an oligomerization-activated type three secretion system (T3SS) ATPase from Shigella flexneri. Protein Sci. 25:1037-48

Abstract

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.

Links

PubMed PMC4838647 Online version:10.1002/pro.2917

Keywords

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

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

Notes

See also

References

See Help:References for how to manage references in GONUTS.