Human caspase-4 and mouse caspase-11 detect bacterial lipopolysaccharide (LPS) in the cytoplasm, triggering gasdermin D-mediated pyroptosis to remove infected cells. Pathogens counter this through various effectors, such as Shigella IpaH7.8, which degrades GSDMD/GSDMB, OspC3 blocks caspase-4/11, and E. coli NleL inhibits epithelial extrusion by degrading Rho-associated coiled-coil containing protein kinase (ROCK)2, caspase-4, and ROCK1. This demonstrates a host-pathogen arms race over epithelial defense. A recent study published in Nature aimed to identify how pathogens block intestinal epithelial cell extrusion, which is a key defense against infection.
All mouse experiments were conducted according to approved ethical guidelines of Genentech and Oregon Health and Science University institutional animal care and use committee (IACUC) in association with the assessment and accreditation of laboratory animal care (AAALAC)-accredited facilities. Mice were housed under controlled humidity (30-70%), light (light-dark cycle of 14h-10h), and temperature (20-26oC) conditions. Female Rock1fl/flRock2fl/fl villin.creERT2 mice were bred on C57BL/6J mice and treated with tamoxifen injection before C. rodentium infection. Bacteria were cultured under standard conditions, quantified, and administered via oral gavage. Infected mice were euthanized for tissue analysis, such as bacterial enumeration and intestinal permeability analysis. Additionally, tests like imaging, ELISA, cell assay, and protein analysis, as well as mass spectrometric analysis, were used in this study. All Statistical analyses were done through GraphPadPrism v.9 and V.10.4.2.
In this animal experiment, a total of 63 E. coli effectors were screened for blocking LPS-induced cell death in human cells. NleL and NleF improved survival and reduced lactate dehydrogenase (LDH) release. Wild-type NIeL suppressed LPS-induced pyroptosis but not its inactive mutant called C735A. This indicates its ubiquitin ligase activity. Since NleL did not affect caspase-1- or GSDMD-dependent pyroptosis, GSDMD was not its target.
Mass spectrometry identified NleL substrates like ROCK2, caspase-4, and ROCK 1. An ubiquitin ligase activity of NleL drives their proteasomal degradation and reduces the levels of ROCK2, caspase-4, and p66β in EA.hy926 cells. This degradation was blocked by proteasome or ubiquitin-activating enzyme 1 (UAE1) inhibitors, which confirms the proteasomal targeting. Catalytically inactive NleL or SoPA (close homologue of NleL) failed to degrade these proteins. In-vitro study confirmed that direct ubiquitylation of NleL substrates. This suggested that its inhibition activity of LPS-induced pyroptosis in mice and human cells.
NleL selectively ubiquitylated LPS-sensing N-terminal caspase activation and recruitment domains (CARDs) of caspases-4, -5, and -11, targeting them for degradation. Exchange of CARDs between caspase-4 and caspase-1 altered the susceptibility of NIeL and implicated residues from 6 to 20. The Caspase-1 CARD model showed the six antiparallel helices features of death proteins compared to the Caspase-4 CARD model.
The C-terminal PH domain of ROCK2 was sufficient for NleL-mediated degradation, whereas deleting its last 20 residues stabilized it. In-vitro study reveals that NleL ubiquitylates the PH domain but not the kinase domain. N-lobe and pentapeptide repeat (PPR) domains of NleL mediate binding to ROCK2, caspase-4, and ROCK1, which enabled substrate recognition.
During infection, NleL degraded ROCK2 and caspase-4 through the proteasome, which suppressed ROCK-MLC2 signaling and limited infected intestinal epithelial cell (IEC) extrusion. ΔnleL was an E. coli bacterium that lacks the NleL protein, showing increased extrusion and reduced colonization, effects absent in ROCK1/ROCK2-deficient IECs. NleL-mediated degradation of ROCK promoted bacterial colonization without compromising intestinal barrier integrity.
In conclusion, this study examines how ROCK1/ROCK2 emerge as critical NleL targets for promoting productive intestinal infection. NleL combats pyroptotic clearance of infected IECs by degrading caspases 4, 5, 11, and ROCK1/ROCK2. Caspase loss blocks LPS-induced pyroptosis, whereas ROCK degradation hinders cell extrusion.
Reference: Luchetti G, Miner MV, Peterson RM, et al. Enteropathogenic bacteria evade ROCK-driven epithelial cell extrusion. Nature. 2025. doi:10.1038/s41586-025-09645-0
