Title of Oral/Poster Presentation

Enzymatic Cooperativity of the Shigella flexneri Type Three Secretion System (T3SS) ATPase Spa47

Presenter Information

Jamie BurgessFollow

Class

Article

Graduation Year

2018

College

College of Science

Department

Chemistry and Biochemistry Department

Faculty Mentor

Dr. Nicholas Dickenson

Presentation Type

Oral Presentation

Abstract

Many Gram-negative pathogens, including Shigella flexneri, rely on a conserved type three secretion system (T3SS) as a primary virulence factor. The Shigella type three secretion apparatus (T3SA) penetrates the host cell membrane and provides a unidirectional conduit for injection of effectors into the host cell. The T3SS ATPase, Spa47, has been shown to reside at the base of the apparatus and is speculated to provide the mechanical energy necessary for T3SA formation and secretion. Using the crystal structure of Spa47 as a guide, we modeled an activated Spa47 oligomer. The structure and resulting model identify several residues likely involved in ATP hydrolysis and suggest that Spa47 oligomerization is required to complete the active site and support Spa47 activity. Mutation of each of the identified active site residues to alanine found that the Spa47 point mutants maintain oligomerization profiles similar to wild-type, however, ATPase activity was eliminated. In contrast, the N-terminal truncation construct used for crystallization purifies as a strictly monomeric species that is also catalytically inactive. Shigella strains expressing any of the ATPase inactive mutants are unable to properly secrete T3SS translocators, do not lyse red blood cells, form incomplete T3SA, and are ultimately unable to infect cultured host cells. Further enzymatic characterization of the Spa47 mutants reveal varying levels of cooperativity depending on the targeted residue. Together, these results begin to uncover the specific role(s) of Spa47 in Shigella and suggest that proper Spa47 oligomerization and cooperativity is required for ATPase activity, T3SS activation, and Shigella virulence.

Location

Room 101

Start Date

4-13-2017 12:00 PM

End Date

4-13-2017 1:15 PM

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Apr 13th, 12:00 PM Apr 13th, 1:15 PM

Enzymatic Cooperativity of the Shigella flexneri Type Three Secretion System (T3SS) ATPase Spa47

Room 101

Many Gram-negative pathogens, including Shigella flexneri, rely on a conserved type three secretion system (T3SS) as a primary virulence factor. The Shigella type three secretion apparatus (T3SA) penetrates the host cell membrane and provides a unidirectional conduit for injection of effectors into the host cell. The T3SS ATPase, Spa47, has been shown to reside at the base of the apparatus and is speculated to provide the mechanical energy necessary for T3SA formation and secretion. Using the crystal structure of Spa47 as a guide, we modeled an activated Spa47 oligomer. The structure and resulting model identify several residues likely involved in ATP hydrolysis and suggest that Spa47 oligomerization is required to complete the active site and support Spa47 activity. Mutation of each of the identified active site residues to alanine found that the Spa47 point mutants maintain oligomerization profiles similar to wild-type, however, ATPase activity was eliminated. In contrast, the N-terminal truncation construct used for crystallization purifies as a strictly monomeric species that is also catalytically inactive. Shigella strains expressing any of the ATPase inactive mutants are unable to properly secrete T3SS translocators, do not lyse red blood cells, form incomplete T3SA, and are ultimately unable to infect cultured host cells. Further enzymatic characterization of the Spa47 mutants reveal varying levels of cooperativity depending on the targeted residue. Together, these results begin to uncover the specific role(s) of Spa47 in Shigella and suggest that proper Spa47 oligomerization and cooperativity is required for ATPase activity, T3SS activation, and Shigella virulence.