Interfacial amino acids support Spa47 oligomerization and shigella type three secretion system activation

Area of Science:

• Microbiology
• Structural Biology
• Biochemistry

Background:

• Shigella, a Gram-negative pathogen, utilizes a type three secretion system (T3SS) to inject effector proteins into host cells, crucial for infection.
• The T3SS ATPase Spa47 powers protein secretion via the type three secretion apparatus (T3SA) and is a potential therapeutic target.

Purpose of the Study:

• To model the activated Spa47 homo-hexamer and identify regions critical for oligomerization and enzymatic activity.
• To investigate the role of specific interfacial residues in Spa47 function and Shigella virulence.

Main Methods:

• Development of a model for the activated Spa47 homo-hexamer.
• Site-directed mutagenesis and high-resolution crystal structure determination of Spa47 mutants.
• In vitro ATP hydrolysis assays and in vivo Shigella virulence assays.

Main Results:

• Two distinct interfacial regions in Spa47 were identified as crucial for homo-hexamerization and ATP hydrolysis.
• Mutations in these regions impaired oligomerization and ATPase activity in vitro.
• In vivo, these mutations correlated with reduced T3SS effector secretion, host cell membrane disruption, and bacterial invasion.

Conclusions:

• Specific interfacial interactions within the Spa47 homo-hexamer are essential for T3SS function and Shigella virulence.
• Targeting these conserved interactions offers a promising strategy for developing broad-spectrum therapeutics against T3SS-mediated pathogens.