Activation of the envelope stress-responsive two-component system AmgRS compensates for depletion of the essential lipoprotein signal peptidase LspA in Pseudomonas aeruginosa

Bacterial lipoproteins play crucial roles in cell envelope biogenesis, signaling, transport, and virulence, making the enzymes responsible for their maturation attractive targets for antibacterial drug development. Among these, the type II signal peptidase LspA is a particularly promising candidate, as several LspA inhibitors have been identified that exert potent antibacterial effects in some Gram-negative species. However, despite predictions that LspA is essential in Pseudomonas aeruginosa, these inhibitors show poor or no activity against this Gramnegative pathogen. To assess the essentiality of P. aeruginosa LspA and its potential as a drug target, here we generated and characterized an arabinose-dependent lspA conditional mutant. LspA depletion completely inhibited bacterial growth, progressively reduced cell viability, and caused severe defects in outer membrane integrity, leading to increased susceptibility to multiple antibiotics, including those that are normally inactive against P. aeruginosa. Selection of revertant clones, whole genome sequencing, and allelic replacement mutagenesis revealed that a gain-of-function mutation in amgS, encoding the sensor kinase of the envelope stressresponsive two-component system AmgRS, can support growth under LspA-limiting conditions and partially restore membrane integrity and antibiotic resistance. Functional analyses further showed that the AmgRSregulated inner membrane proteins HtpX and YccA are required for this compensatory effect, although the underlying mechanism remains unclear. Together, these findings confirm the essentiality of LspA in P. aeruginosa, establish it as a promising antibacterial target, and uncover a role for the AmgRS-mediated stress response in mitigating the consequences of defective lipoprotein maturation.