Tail-specific protease (Tsp)-mediated envelope remodeling and beta-lactam tolerance in Escherichia coli

Bacterial persisters are transiently drug-tolerant cells that survive antibiotic treatment without genetic resistance. While certain cytosolic proteases are known to regulate persistence through toxin-antitoxin and stress pathways, the role of envelope-associated proteolysis remains poorly understood. Here, our high-throughput promoter-reporter screen identified several candidate proteases, and subsequent antibiotic treatment of knockout strains lacking these degradative genes revealed that Tsp plays a critical role in beta-lactam persistence. The deletion of tsp markedly reduced cell survival to multiple cell-wall-targeting antibiotics. This loss of tsp also compromised cell envelope integrity and caused the accumulation of periplasmic, membrane-associated, and cell-wall-related proteins, including the DD-endopeptidase MepS, as shown by quantitative proteomics. Comprehensive genetic deletion and overexpression analyses revealed that both loss and excessive activity of envelope-associated proteins, particularly MepS, compromise persistence. Moreover, cell envelope permeabilization and antibiotic sensitivity can be independent, as certain envelope perturbations increased permeability without reducing beta-lactam survival. Altogether, our findings support a model in which Tsp-mediated proteolysis helps prevent aberrant accumulation of peptidoglycan-remodeling proteins that would otherwise destabilize the envelope.