A Proteolytic Complex Targets Multiple Cell Wall Hydrolases in Pseudomonas aeruginosa

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• Bacterial carboxy-terminal processing proteases (CTPs) are conserved but their roles, especially in virulence, remain poorly understood.
• Few direct substrates of bacterial CTPs have been identified, limiting our understanding of their functions.
• The CtpA protease of *Pseudomonas aeruginosa* is crucial for type III secretion system (T3SS) function and virulence, yet its substrates and regulation are unclear.

Purpose of the Study:

• To investigate the function of the *Pseudomonas aeruginosa* CtpA protease.
• To identify direct proteolytic substrates of CtpA.
• To elucidate the regulatory partners and mechanisms controlling CtpA activity.

Main Methods:

• Protease activity assays in vitro and in vivo.
• Identification of protein-protein interactions using complex formation.
• Proteolytic cleavage site mapping and substrate identification.
• Analysis of mutant phenotypes, including T3SS function and cell wall integrity.

Main Results:

• CtpA forms a complex with a novel protein, LbcA, essential for CtpA activity.
• Four uncharacterized CtpA substrates were identified, all predicted peptidoglycan cross-link hydrolases.
• A *ctpA* null mutant exhibits reduced peptidoglycan cross-links and impaired growth in salt-free media.
• Accumulation of a single CtpA substrate is sufficient to cause some Δ*ctpA* mutant phenotypes, including defective T3SS.

Conclusions:

• The LbcA-CtpA complex functions in the *P. aeruginosa* cell envelope to regulate peptidoglycan hydrolase activity through substrate degradation.
• This mechanism highlights a widespread strategy for controlling peptidoglycan hydrolases in bacteria, potentially involving partner proteins.
• Understanding CTPs and their partners offers insights into bacterial virulence and cell wall maintenance.