(183d) Role of Tail-Specific Protease in Ampicillin Tolerance

Antimicrobial resistance is a critical global health issue, causing millions of deaths each year. While antibiotics remain central to modern medicine, resistant (e.g., mutants) and tolerant cells (e.g., persisters) pose significant challenges. Persisters are phenotypic variants that survive antibiotic exposure without heritable resistance, serving as reservoirs for resistant strains. Despite their prevalence, the mechanisms behind their reversible, antibiotic-tolerant state remain poorly understood. Our previous work suggested that intracellular degradation by proteases and other enzymes, triggered by environmental stresses, promotes persister formation. This degradation enables cells to recycle resources under starvation but also induces intracellular damage, impairing growth while enhancing antibiotic tolerance¹. To explore this further, here, we investigated late-stationary-phase Escherichia coli cells using a promoter reporter library and identified 16 candidate genes upregulated during this phase. Subsequent deletion analysis revealed that lon deletion reduced persistence under ofloxacin, while tsp deletion reduced persistence under ampicillin. These results indicate distinct, antibiotic-specific roles for lon and tsp in persister formation. Since we characterized lon-mediated persistence in a separate study², this project focuses on elucidating the role of tsp.

Tsp is a periplasmic serine protease that cleaves substrates at their C-terminus, often in coordination with the adaptor protein NlpI. It participates in the post-translational processing of penicillin-binding protein 3 (PBP3, encoded by ftsI), which is essential for septal peptidoglycan synthesis during cell division. Tsp also regulates peptidoglycan remodeling by degrading MepS, an endopeptidase that cleaves crosslinks in the bacterial cell wall. In addition, Tsp degrades proteins tagged by the SsrA system, which marks proteins for degradation, linking Tsp to protein quality control and envelope homeostasis. To test whether reduced persistence in the tsp knockout strain was due to impaired PBP3 processing, we overexpressed a truncated version of ftsI from an IPTG-inducible plasmid in the tsp knockout. This did not restore persister levels, suggesting that Tsp-mediated PBP3 processing is not responsible for the observed phenotype. Likewise, deletion of nlpI, alone or in combination with tsp, had minimal impact on persistence, indicating that NlpI is not required for the Tsp-associated effect. Although deletion of mepS or ssrA alone did not significantly reduce persister levels, their combined perturbation with tsp deletion led to an approximately 100-fold reduction in persistence compared to the wild-type strain. These findings suggest that Tsp may function in coordination with the SsrA-tagging system to regulate MepS degradation. Disruption of this pathway could compromise cell wall integrity—an essential target of ampicillin—and contribute to reduced survival under antibiotic stress. Targeting Tsp-mediated proteolytic pathways may thus provide a novel approach for eliminating persister cells and combating antibiotic resistance.

References:

1. Orman, M.A. and Brynildsen, M.P., 2015. Inhibition of stationary phase respiration impairs persister formation in E. coli. Nature communications, 6(1), p.7983.
2. Mohiuddin, S.G., Massahi, A. and Orman, M.A., 2022. lon deletion impairs persister cell resuscitation in Escherichia coli. MBio, 13(1), pp.e02187-21.