Dominant negative effects by inactive Spa47 mutants inhibit T3SS function and Shigella virulence

Area of Science:

• Microbiology
• Molecular Biology
• Structural Biology

Background:

• Type three secretion systems (T3SS) are crucial for virulence in many human pathogens.
• Understanding T3SS function is vital for developing strategies against infectious diseases.
• Spa47 is a key ATPase that fuels T3SS and supports Shigella virulence.

Purpose of the Study:

• To characterize Spa47 oligomerization and activation in vitro and in vivo.
• To elucidate the mechanistic details of Spa47-catalyzed ATP hydrolysis.
• To distinguish mechanisms of ATP hydrolysis supporting T3SS function in vitro versus in vivo.

Main Methods:

• ATPase kinetics assays with wild-type and mutant Spa47.
• Co-expression of wild-type and ATPase-inactive Spa47 mutants in Shigella.
• High-resolution fluorescence microscopy.
• T3SS activity and virulence phenotype analyses.

Main Results:

• Monomeric Spa47 shows low ATPase activity via transient oligomers.
• Oligomeric Spa47 exhibits enhanced ATP hydrolysis rates.
• Spa47's N-terminus, not ATPase activity, mediates injectisome incorporation.
• ATPase-inactive mutants exert a dominant negative effect on T3SS function and virulence.

Conclusions:

• Spa47 activation and ATP hydrolysis mechanisms differ between in vitro and in vivo settings.
• The N-terminus is critical for Spa47 localization to the injectisome.
• Understanding Spa47 regulation provides insights into T3SS energy coupling and virulence.