Oral Presentation The 5th Prato Conference on Pore Forming Proteins 2021

Tripartite ClyA family alpha helical pore forming toxins. (#7)

Alicia M Churchill-Angus 1 , Jason S Wilson 2 , Svetlana E Sedelnikova 2 , Thomas H Schofield 3 , Thomas R Marlow 2 , Per A Bullough 2 , John B Rafferty 2 , Claudine Bisson 4 , Patrick J Baker 2
  1. University of Oxford, Oxford, UK
  2. University of Sheffield, Sheffield, UK
  3. University of Leeds , Leeds, UK
  4. Kings College, London, UK

The ClyA family of alpha helical pore forming toxins (α-PFTs) contains single, two, and three component members. Structures of the single component Escherichia coli ClyA1 and the two component Yersinia enterolytica YaxAB2 show both undergo conformational changes from soluble to pore forms, and oligomerization to produce the active pore. We have identified ClyA family tripartite α-PFTs in Gram negative pathogenic bacteria3 and together with studies showing the role of Gram positive ClyA tripartite α-PFTs in inducing septic shock in mice4, this has emphasised how understanding how these tripartite toxins function is an important step in combatting infection and designing virulence targeted therapies. As there is limited structural and biochemical knowledge on these tripartite toxins, we have expressed and purified tripartite α-PFTs from Serratia marcescens (SmhABC5,6) an important antibiotic resistant nosocomial human pathogen and Aeromonas hydrophila (AhlABC3), a commercially important fish pathogen. We have shown that these α-PFTs require all three components for maximal cell lysis and that chimeric pores between the two species retain toxic activity. We have determined the structures of the three components, which exhibit considerable structural similarity despite low sequence identity and have shown how they convert from the soluble to pore conformations, presenting a similar helix-turn-helix motif in each component that interacts with the membrane: the C-component is responsible for the initial single leaflet membrane attachment, the B-component forms a hydrophobic oligomeric pore and the A component provides the hydrophilic lining to the pore. These same functions are displayed on just one or two polypeptides in the single and two component ClyA family members, respectively. Using this structural data in combination with biochemical assays, and negative stain electron microscopy, we have proposed a model for the active Serratia and Aeromonas toxins as a funnel-shaped decameric A10B10C10 tripartite pore5.

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  3. Wilson, J. S. et al. Identification and structural analysis of the tripartite α-pore forming toxin of Aeromonas hydrophila. Nat. Commun. 10, 2900 (2019).
  4. Mathur, A. et al. A multicomponent toxin from Bacillus cereus incites inflammation and shapes host outcome via the NLRP3 inflammasome. Nat. Microbiol. 4, 362–374 (2019).
  5. Churchill-Angus, A. M. et al. Characterisation of a tripartite α‐pore forming toxin from Serratia marcescens. Sci Reports, 11, 6447 (2021).
  6. Churchill-angus, A. M., et al. The A component ( SmhA ) of a tripartite pore forming toxin from Serratia marcescens : expression , purification , and crystallographic analysis. Acta Crystallogr. Sect. F 76, 577 (2020).