EPEC-induced activation of the Ca2+ transporter TRPV2 leads to pyroptotic cell death.

The enteropathogenic Escherichia coli (EPEC) type III secretion system effector Tir, which mediates intimate bacterial attachment to epithelial cells, also triggers Ca2+ influx followed by LPS entry and caspase-4-dependent pyroptosis, which could be antagonized by the effector NleF. Here we reveal the mechanism by which EPEC induces Ca2+ influx. We show that in the intestinal epithelial cell line SNU-C5, Tir activates the mechano/osmosensitive cation channel TRPV2 which triggers extracellular Ca2+ influx. Tir-induced Ca2+ influx could be blocked by siRNA silencing of TRPV2, pre-treatment with the TRPV2 inhibitor SET2 or by growing cells in low osmolality medium. Pharmacological activation of TRPV2 in the absence of Tir failed to initiate caspase-4-dependent cell death, confirming the necessity of Tir. Consistent with the model implicating activation on translocation of TRPV2 from the ER to plasma membrane, inhibition of protein trafficking by either brefeldin A or the effector NleA prevented TRPV2 activation and cell death. While infection with EPECΔnleA triggered pyroptotic cell death, this could be prevented by NleF. Taken together this study shows that while integration of Tir into the plasma membrane activates TRPV2, EPEC uses NleA to inhibit TRPV2 trafficking and NleF to inhibit caspase-4 and pyroptosis.

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.

Cyclic di-adenosine monophosphate (c-di-AMP) is required for osmotic regulation in Staphylococcus aureus but dispensable for viability in anaerobic conditions.

Cyclic di-adenosine monophosphate (c-di-AMP) is a recently discovered signaling molecule important for the survival of Firmicutes, a large bacterial group that includes notable pathogens such as Staphylococcus aureus However, the exact role of this molecule has not been identified. dacA, the S. aureus gene encoding the diadenylate cyclase enzyme required for c-di-AMP production, cannot be deleted when bacterial cells are grown in rich medium, indicating that c-di-AMP is required for growth in this condition. Here, we report that an S. aureus dacA mutant can be generated in chemically defined medium. Consistent with previous findings, this mutant had a severe growth defect when cultured in rich medium. Using this growth defect in rich medium, we selected for suppressor strains with improved growth to identify c-di-AMP-requiring pathways. Mutations bypassing the essentiality of dacA were identified in alsT and opuD, encoding a predicted amino acid and osmolyte transporter, the latter of which we show here to be the main glycine betaine-uptake system in S. aureus. Inactivation of these transporters likely prevents the excessive osmolyte and amino acid accumulation in the cell, providing further evidence for a key role of c-di-AMP in osmotic regulation. Suppressor mutations were also obtained in hepS, hemB, ctaA, and qoxB, coding proteins required for respiration. Furthermore, we show that dacA is dispensable for growth in anaerobic conditions. Together, these findings reveal an essential role for the c-di-AMP signaling network in aerobic, but not anaerobic, respiration in S. aureus.

The pathogen-encoded signalling receptor Tir exploits host-like intrinsic disorder for infection.

The translocated intimin receptor (Tir) is an essential type III secretion system (T3SS) effector of attaching and effacing pathogens contributing to the global foodborne disease burden. Tir acts as a cell-surface receptor in host cells, rewiring intracellular processes by targeting multiple host proteins. We investigated the molecular basis for Tir's binding diversity in signalling, finding that Tir is a disordered protein with host-like binding motifs. Unexpectedly, also are several other T3SS effectors. By an integrative approach, we reveal that Tir dimerises via an antiparallel OB-fold within a highly disordered N-terminal cytosolic domain. Also, it has a long disordered C-terminal cytosolic domain partially structured at host-like motifs that bind lipids. Membrane affinity depends on lipid composition and phosphorylation, highlighting a previously unrecognised host interaction impacting Tir-induced actin polymerisation and cell death. Furthermore, multi-site tyrosine phosphorylation enables Tir to engage host SH2 domains in a multivalent fuzzy complex, consistent with Tir's scaffolding role and binding promiscuity. Our findings provide insights into the intracellular Tir domains, highlighting the ability of T3SS effectors to exploit host-like protein disorder as a strategy for host evasion.