The Breadth and Molecular Basis of Hcp-Driven Type VI Secretion System Effector Delivery

Area of Science:

• Microbiology
• Bacterial Pathogenesis
• Molecular Biology

Background:

• The type VI secretion system (T6SS) is a critical bacterial weapon for inter-bacterial competition and host colonization.
• T6SS effectors are delivered via various components, including the Hcp inner tube, but Hcp-associated effectors are poorly characterized.
• Understanding T6SS effector loading is crucial for discovering new toxins and potential antimicrobial targets.

Purpose of the Study:

• To identify novel T6SS effector proteins associated with Hcp proteins in Pseudomonas aeruginosa.
• To investigate the role of different Hcp proteins (Hcp1-3) in T6SS cargo transport.
• To elucidate the mechanism and consequences of effector loading onto the Hcp component of the T6SS.

Main Methods:

• In vivo pulldown assays using Hcp1, Hcp2, and Hcp3 from Pseudomonas aeruginosa.
• Copurification of Hcp-effector complexes.
• Functional characterization of T6SS activity using a Tse1-Bla chimera.
• Toxicity assays against Escherichia coli.

Main Results:

• Confirmed known Hcp1-Tse interactions and identified potential novel Hcp1-bound effectors.
• Demonstrated that Hcp2 and Hcp3 can deliver toxic T6SS cargoes to Escherichia coli.
• Showed that loading large effectors onto Hcp can jam the T6SS, inhibiting its function.

Conclusions:

• Hcp proteins serve as crucial shuttles for diverse T6SS effectors, expanding the known repertoire of bacterial toxins.
• The size and loading site of effectors significantly influence T6SS functionality, highlighting a regulatory mechanism.
• This study provides a foundation for discovering uncharacterized T6SS toxins for antimicrobial drug development.