Poster Presentation Second Round 16th Lorne Infection and Immunity 2026

Evolution of a pyroptosis-apoptosis crosstalk drives non-canonical inflammasome signalling. (#130)

Seb Grant 1 2 3 , Ellis Nicholson 2 , Callum Robson 2 , Adam Sung 2 , Timur Ozdenya 2 , Barwin Visawanathan 2 , Pranav Patel 2 , Yunjia Chang 2 , Zoe Chen 4 , James Kleppen 3 , Kaiwen Chen 4 , Chris Spicer 3 , Dave Boucher 2
  1. The Univeristy of Queensland, St Lucia, QUEENSLAND, Australia
  2. Department of Biology, The University of York, York, United Kingdom
  3. Department of Chemistry, The Univeristy of York, York, United Kingdom
  4. Life science institute, Univerisity of Singapore, Singapore

The coordination of efficient immune responses to infection is essential to enable pathogen clearance and host survival. Caspases are a family of cysteine proteases which drive cellular responses by cleaving and activating a defined set of proteins. Different caspases have distinct activation stimuli and substrate repertoire, meaning the response generated is dependent on which caspases are activated in a cell. Human caspase-4 is an inflammatory caspase activated upon intracellular detection of bacterial lipopolysaccharide (LPS), and triggers pro-inflammatory responses via cytokine release, and pyroptotic cell death. Previous literature has also demonstrated the importance of apoptotic executioner caspase-3 in gram-negative bacterial immune responses and has linked its activation to caspase-4, but the molecular basis of how this crosstalk is orchestrated is unknown. Here, we demonatrate that caspase-3 is directly cleaved and activated by caspase-4, and that this specificty is not regulated by traditional active site interactions. Instead, specificity was found to be entirely mediated by an exosite, a molecular interface distal from the caspase-4 active site. Using AlphaFold, we generated an interaction model between caspase-4 and caspases-3, and experimentally validated the importance of a hydrophobic interface on caspase-3 that was engaged by the exosite, enabling its cleavage and activation by caspase-4. Strikingly, this interface is not used other caspases, such as apopotiic initiator caspase-8, which targeted caspase-3 via active-site interactions. Finally, alligntment of residues involved at the exosite interface revealed stronge conservation only within the mammals, suggesting that this may be a recently evolved signalling axis exclusive to this lineage. Together, our work elucidates the molcular underpinnings of a novel signalling axis, which has only recently evolved in the mammlain lineage to enable efficient clearance of intracellular bacterial infections.  

 

 

 

  1. Grant, S., Nicholson, E., Chen, Z.H.X., Robson, C., Sung, A., Viswanathan, B., Chang, Y., Patel, P., Kleppen, J.P., Ozdenya, T. and Chen, K.W., 2025. Evolution of a Pyroptosis-Apoptosis crosstalk drives non-canonical inflammasome signalling. bioRxiv, pp.2025-11.
  2. Kulkarni M, Bourne CM, Mahale AB, Exconde PM, Murphy C, Cervantes S, Kardhashi M, Kambayashi M, Yoo W, Wrong TJ, Patio RC, Discher BM, Taabazuing CY. Human non-canonical inflammasomes activate CASP3 to limit intracellular Salmonella replication in macrophages. bioRxiv [Preprint]. 2025 Nov 8:2025.10.31.685927. doi: 10.1101/2025.10.31.685927. PMID: 41279788; PMCID: PMC12637516.