Shutting Down Shigella Secretion: Characterizing Small Molecule Type Three Secretion System ATPase Inhibitors

Area of Science:

• Microbiology
• Molecular Biology
• Drug Discovery

Background:

• Type three secretion systems (T3SSs) are crucial virulence factors for many human pathogens, enabling direct injection of effector proteins into host cells.
• The T3SS apparatus (T3SA) relies on a conserved ATPase, Spa47, making it a key target for anti-infective drug development.

Purpose of the Study:

• To kinetically analyze the inhibition of the Shigella T3SS ATPase, Spa47, using newly developed inhibitors.
• To evaluate the in vitro and in vivo efficacy and mechanism of action of these Spa47 inhibitors.

Main Methods:

• Purification of an active oligomeric Shigella T3SS ATPase (Spa47).
• Kinetic analysis of Spa47 ATPase activity in the presence of inhibitors.
• In silico docking simulations.
• In vivo assessment of effector secretion, bacterial growth, and host cell viability.
• In vitro characterization of Spa47 complexes and in vivo T3SA formation.

Main Results:

• Three Spa47 inhibitors demonstrated noncompetitive inhibition profiles, reducing ATPase activity with IC50s as low as 52 ± 3 μM.
• Two inhibitors effectively abolished effector protein secretion in vivo without impacting bacterial growth or host cell viability.
• Inhibitors did not disrupt Spa47 oligomerization or prevent T3SA formation, indicating a specific mechanism of action on ATPase activity.

Conclusions:

• Spa47 inhibitors represent a viable strategy for combating Shigella infections by disabling T3SS-mediated virulence.
• These inhibitors allow for the presentation of T3SA tip proteins, potentially aiding immune response and memory development.
• This study provides crucial insights into T3SS ATPase inhibition and establishes a foundation for developing novel non-antibiotic therapeutics targeting T3SS ATPases.