Clustering of Tir during enteropathogenic E. coli infection triggers calcium influx-dependent pyroptosis in intestinal epithelial cells.

Clustering of the enteropathogenic Escherichia coli (EPEC) type III secretion system (T3SS) effector translocated intimin receptor (Tir) by intimin leads to actin polymerisation and pyroptotic cell death in macrophages. The effect of Tir clustering on the viability of EPEC-infected intestinal epithelial cells (IECs) is unknown. We show that EPEC induces pyroptosis in IECs in a Tir-dependent but actin polymerisation-independent manner, which was enhanced by priming with interferon gamma (IFNγ). Mechanistically, Tir clustering triggers rapid Ca2+ influx, which induces lipopolysaccharide (LPS) internalisation, followed by activation of caspase-4 and pyroptosis. Knockdown of caspase-4 or gasdermin D (GSDMD), translocation of NleF, which blocks caspase-4 or chelation of extracellular Ca2+, inhibited EPEC-induced cell death. IEC lines with low endogenous abundance of GSDMD were resistant to Tir-induced cell death. Conversely, ATP-induced extracellular Ca2+ influx enhanced cell death, which confirmed the key regulatory role of Ca2+ in EPEC-induced pyroptosis. We reveal a novel mechanism through which infection with an extracellular pathogen leads to pyroptosis in IECs.

Impaired neutrophil migration underpins host susceptibility to infectious colitis.

Citrobacter rodentium models infection with enteropathogenic E. coli and ulcerative colitis (UC). While C57BL/6 (C57) mice recover, C3H/HeN (C3H) mice succumb to infection, partially due to increased colonic neutrophil elastase activity, also seen in UC patients; however, the underlying cause was unknown. Here we found that bone marrow, blood and colonic C57 neutrophils expressed CD11bHi and reached the infected colonic lumen where they underwent productive NETosis. In contrast, while the number of C3H neutrophils increased in the bone marrow, blood, and colon, they remained CD11bLo and got trapped in the submucosa, away from C. rodentium, where they underwent harmful NETosis. CD11bLo neutrophils in C3H mice infected with CRi9, which triggers expression of neutrophil chemoattractants, reached the colonisation site resulting in host survival. UC patient neutrophils also displayed decreased levels of the activation/differentiation markers CD16/CXCR4. These results, suggesting that neutrophil malfunction contributes to exacerbated colitis, provide insight for future therapeutic prospects.

Cancer-associated FBXW7 loss is synthetic lethal with pharmacological targeting of CDC7.

The F-box and WD repeat domain containing 7 (FBXW7) tumour suppressor gene encodes a substrate-recognition subunit of Skp, cullin, F-box (SCF)-containing complexes. The tumour-suppressive role of FBXW7 is ascribed to its ability to drive ubiquitination and degradation of oncoproteins. Despite this molecular understanding, therapeutic approaches that target defective FBXW7 have not been identified. Using genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 screens, focussed RNA-interference screens and whole and phospho-proteome mass spectrometry profiling in multiple FBXW7 wild-type and defective isogenic cell lines, we identified a number of FBXW7 synthetic lethal targets, including proteins involved in the response to replication fork stress and proteins involved in replication origin firing, such as cell division cycle 7-related protein kinase (CDC7) and its substrate, DNA replication complex GINS protein SLD5 (GINS4). The CDC7 synthetic lethal effect was confirmed using small-molecule inhibitors. Mechanistically, FBXW7/CDC7 synthetic lethality is dependent upon the replication factor telomere-associated protein RIF1 (RIF1), with RIF1 silencing reversing the FBXW7-selective effects of CDC7 inhibition. The delineation of FBXW7 synthetic lethal effects we describe here could serve as the starting point for subsequent drug discovery and/or development in this area.

Insights from multi-omic modeling of neurodegeneration in xeroderma pigmentosum using an induced pluripotent stem cell system.

Xeroderma pigmentosum (XP) is caused by defective nucleotide excision repair of DNA damage. This results in hypersensitivity to ultraviolet light and increased skin cancer risk, as sunlight-induced photoproducts remain unrepaired. However, many XP patients also display early-onset neurodegeneration, which leads to premature death. The mechanism of neurodegeneration is unknown. Here, we investigate XP neurodegeneration using pluripotent stem cells derived from XP patients and healthy relatives, performing functional multi-omics on samples during neuronal differentiation. We show substantially increased levels of 5',8-cyclopurine and 8-oxopurine in XP neuronal DNA secondary to marked oxidative stress. Furthermore, we find that the endoplasmic reticulum stress response is upregulated and reversal of the mutant genotype is associated with phenotypic rescue. Critically, XP neurons exhibit inappropriate downregulation of the protein clearance ubiquitin-proteasome system (UPS). Chemical enhancement of UPS activity in XP neuronal models improves phenotypes, albeit inadequately. Although more work is required, this study presents insights with intervention potential.