Abstract

Bacterial pore-forming toxins are potent secreted protein effectors that disrupt cellular ion homeostasis by integrating into the plasma membrane. Among them, the RTX toxin alphahemolysin (HlyA) from uropathogenic Escherichia coli (UPEC) has been extensively studied for its hemolytic activity, broad cell tropism, and role in virulence. HlyA was identified as the first type 1 secreted substrate, with β2 integrin CD18/LFA-1 proposed as its receptor. However, its diverse mechanisms of cellular toxicity and impact on outcomes of urinary tract infection (UTI) have remained incompletely defined. During pyelonephritis, renal epithelial cells are at the front line of infection, directly contacting ascending UPEC and playing an essential role in initiating innate immune responses. Importantly, these cells are highly sensitive to HlyA cytotoxicity during in vitro exposure. Here, leveraging an updated preclinical mouse model of ascending pyelonephritis, we demonstrate that HlyA functions as a renal damage factor, exacerbating renal tubular epithelial injury in vivo. As CD18 is expressed exclusively on hematopoietic cells, we leveraged a CRISPR-Cas9 loss-ofxii function screen to uncover an alternative HlyA targeting mechanism in renal epithelial cells. We found that HlyA enters epithelial cells via clathrin-mediated endocytosis (CME) and induces cytotoxicity through a mechanism distinct from the previously proposed plasma membrane poration model. Our findings show that internalized HlyA triggers permeabilization of the endolysosome, causing rapid cytoplasmic acidification, protease release, mitochondrial dysfunction, and caspase-independent cell death. Additionally, we identify the low-density lipoprotein receptor (LDLR) as an epithelial receptor for HlyA and demonstrate that targeting of LDLRHlyA interactions via peptide biologics neutralizes HlyA toxicity to renal epithelial cells. Further, we demonstrate a sex-biased response to HlyA-induced epithelial cell death, where testosterone exposure enhances HlyA-mediated killing of renal epithelial cells through an androgen receptor-independent mechanism. Given that renal damage during pyelonephritis contributes to scarring and chronic kidney disease, our findings reveal a novel HlyA mechanism of action and suggest therapeutic targeting of LDLR-HlyA interactions to mitigate UPECassociated renal injury. Beyond the context of UTI, E. coli is among the common causes of bacterial sepsis, commonly disseminating into the bloodstream during severe pyelonephritis or gastrointestinal tract infections. Sepsis is a life-threatening condition by which infectious organisms (predominantly bacteria) trigger a dysregulated proinflammatory response followed by subsequent immunosuppression. Despite intensive research on immune modulators of septic shock, bacterial sepsis continues to have a substantial mortality rate, with current host-targeted therapeutics exhibiting limited efficacy. Here, we establish a mouse model of E. coli-induced sepsis, using the urosepsis isolate CFT073, that yields nearly 80% mortality and significant manifestations of illness in the first few days. Leveraging this model, we identify that genetic knockout of HlyA in CFT073 diminishes sepsis-induced weight loss and increases survival rates 4-fold. Together, these findings uncover a previously unrecognized intracellular mechanism of HlyA cytotoxicity, establish LDLR as a functional epithelial receptor for HlyA, and demonstrate that HlyA is a critical virulence determinant in both localized and systemic E. coli infections. Our work provides a foundation for therapeutic strategies aimed at neutralizing HlyA and other bacterial toxins to reduce renal injury and improve sepsis outcomes.

Committee Chair

David Hunstad

Committee Members

David Rosen; Juliane Bubeck-Wardenburg; Regina Clemens; Scott Hultgren; Sean Whelan

Degree

Doctor of Philosophy (PhD)

Author's Department

Biology & Biomedical Sciences (Molecular Microbiology & Microbial Pathogenesis)

Author's School

Graduate School of Arts and Sciences

Document Type

Dissertation

Date of Award

7-23-2025

Language

English (en)

Author's ORCID

https://orcid.org/0000-0002-7876-3521

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