E-64

The bacterium Enterococcus faecalis is a leading cause of catheter-associated urinary tract infection (CAUTI), whose treatment is increasingly challenged by antibiotic resistance. In examining alternative therapies, we previously found that the cysteine protease inhibitor E64 dramatically reduced bladder inflammation and bacterial dissemination in a murine model of E. faecalis CAUTI. However, the role of cysteine proteases in pathogenesis and the target(s) of E64 are unknown. Here, we found that while it did not affect E. faecalis growth in vitro , in a murine CAUTI model, E64 reduced host caspase-1-dependent cellular apoptosis and necrosis, levels of several pro-inflammatory cytokines, bladder epithelial damage, and formation of renal abscesses. Analysis of host cell transcription and inflammatory cell populations revealed that E64 did not affect neutrophil numbers but did enhance the expression of C-C chemokine receptor type 3 (CCR3), a receptor for eosinophil-specific chemokines, with a concomitant increase in eosinophil numbers. Treatments that reduced or increased the eosinophil response (anti-interleukin 5 antibody or eotaxin, respectively) confirmed a role for eosinophils in controlling bacterial burdens. Analysis of a panel of host cysteine proteases in vivo demonstrated that E64 decreased the activation of cathepsin L; subsequently, we found that the infection of cathepsin L-deficient mice yielded lower catheter and bladder colonization compared to wild-type mice. In total, we have shown that host cysteine proteases exacerbate CAUTI pathogenesis, suggesting that cysteine protease modulation may represent a novel approach for the treatment of persistent CAUTI.

Catheter-associated urinary tract infections (CAUTIs) are the most prevalent healthcare-associated infection globally, with Enterococcus faecalis posing a significant threat due to widespread antibiotic resistance. This study identifies host cysteine proteases—particularly cathepsin L and caspase-1—as unrecognized drivers of CAUTI pathogenesis and renal fibrosis. Pharmacologic inhibition of these proteases using E64 reduces bladder inflammation, epithelial disruption, and kidney abscesses, while restoring fibrinogen and collagen homeostasis. Strikingly, E64 treatment unmasks a protective eosinophil response via CCR3 signaling that enhances bacterial clearance. Genetic deletion of cathepsin L recapitulates these protective effects, establishing it as a key host factor in E. faecalis persistence. These findings reveal host cysteine proteases as viable therapeutic targets for CAUTI and provide proof-of-concept for host-directed strategies that bypass antibiotic resistance.