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

ABSTRACT 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. IMPORTANCE Our findings establish Tsp as an important envelope-associated protease that contributes to beta-lactam persistence by modulating peptidoglycan remodeling through proteolytic control. Loss of Tsp disrupts the balance between synthesis and degradation of cell envelope components, leading to envelope destabilization, induction of envelope-stress response, and loss of beta-lactam tolerance. By identifying multiple Tsp-regulated envelope determinants and linking envelope proteostasis to persistence, this work expands the conceptual framework of bacterial tolerance beyond cytosolic stress pathways to include periplasmic proteolytic regulation as an important layer of persistence control.