Direct and Indirect Interactions Promote Complexes of the Lipoprotein LbcA, the CtpA Protease and Its Substrates, and Other Cell Wall Proteins in Pseudomonas aeruginosa.

The Pseudomonas aeruginosa lipoprotein LbcA was discovered because it copurified with and promoted the activity of CtpA, a carboxyl-terminal processing protease (CTP) required for type III secretion system function and virulence in a mouse model of acute pneumonia. In this study, we explored the role of LbcA by determining its effect on the proteome and its participation in protein complexes. lbcA- and ctpA-null mutations had strikingly similar effects on the proteome, suggesting that assisting CtpA might be the most impactful role of LbcA in the bacterial cell. Independent complexes containing LbcA and CtpA, or LbcA and a substrate, were isolated from P. aeruginosa cells, indicating that LbcA facilitates proteolysis by recruiting the protease and its substrates independently. An unbiased examination of proteins that copurified with LbcA revealed an enrichment for proteins associated with the cell wall. One of these copurification partners was found to be a new CtpA substrate and the first substrate that is not a peptidoglycan hydrolase. Many of the other LbcA copurification partners are known or predicted peptidoglycan hydrolases. However, some of these LbcA copurification partners were not cleaved by CtpA, and an in vitro assay revealed that while CtpA and all of its substrates bound to LbcA directly, these nonsubstrates did not. Subsequent experiments suggested that the nonsubstrates might copurify with LbcA by participating in multienzyme complexes containing LbcA-binding CtpA substrates. IMPORTANCE Carboxyl-terminal processing proteases (CTPs) are widely conserved and associated with the virulence of several bacteria, including CtpA in Pseudomonas aeruginosa. CtpA copurifies with the uncharacterized lipoprotein LbcA. This study shows that the most impactful role of LbcA might be to promote CtpA-dependent proteolysis and that it achieves this as a scaffold for CtpA and its substrates. It also reveals that LbcA copurification partners are enriched for cell wall-associated proteins, one of which is a novel CtpA substrate. Some of the LbcA copurification partners are not cleaved by CtpA but might copurify with LbcA because they participate in multienzyme complexes containing CtpA substrates. These findings are important because CTPs and their associated proteins affect peptidoglycan remodeling and virulence in multiple species.