The formation and structure of Escherichia coli K-12 haemolysin E pores

Abstract

Some enteric bacteria synthesize a pore-forming toxin, HlyE, which is cytolytic and cytotoxic to host cells. Measurement of HlyE binding to erythrocyte ghosts and the kinetics of HlyE-mediated erythrocyte lysis suggests that interaction with target membranes is not the rate-limiting step in the formation of HlyE pores, but that there is a temperature-dependent lag phase before a functional pore is formed. Circular dichroism and fluorescence energy transfer analyses show that HlyE protomers retain an a-helical structure when oligomerized to form a pore consisting of parallel HlyE protomers. Comparison of the proteolytic sensitivities of the water-soluble and oligomeric forms of HlyE identifies inner and outer surfaces of the pore. This new information has been used to constrain a model of the HlyE pore, which allows a more detailed interpretation of previous low-resolution 3D reconstructions and suggests a novel mechanism for insertion of HlyE into target membranes.

A Non-classical Assembly Pathway of Escherichia coli Pore-forming Toxin Cytolysin A

“…The transmembrane pore (collected as peak 10 from GFC) only exhibited a background level of hemoglobin release, supporting the notion that this population had lost its ability to lyse cells. This result is in agreement with previous observations that transmembrane ClyA pores formed in detergent octyl-glucoside solution showed no pore-forming activity on cell membranes (37). Because both ClyA O9 and ClyA O8 were still functional in forming pores on erythrocyte membranes, we concluded that they might be oligomeric states with an ordered structure instead of disordered aggregates.…”

“…However, this contradicts two observations: (i) the pre-formed ClyA transmembrane pore lacks hemolytic activity as demonstrated by our study and previous data (37). A transmembrane ClyA pore embedded in the OMV membrane loses functionality for further attack.…”

Mutations Affecting Export and Activity of Cytolysin A from Escherichia coli

“…The ClyA residues 177 to 203, which have almost exclusively hydrophobic side chains, form a surface-exposed hydrophobic area in the head domain of the ClyA monomer. This area includes the ␤-tongue (residues 185 to 195), which has previously been suggested to play a crucial role in membrane interaction and pore formation (9,15,25,30,37,43,45,47). To further study the significance of this region, two ClyA mutants were constructed, one containing the substitution A187D in the first ␤-strand (␤1) of the ␤-tongue and the other lacking the residues 181 to 203, i.e., the complete ␤-tongue and the adjacent helix ␣E.…”

“…Site-directed mutagenesis has previously revealed that the ␤-tongue region of ClyA is important for its cytolytic activity (30,43,45,47), and different models for pore formation by ClyA suggested that this region is directly involved in target (9,15,37,45). Considering the structural differences between the monomeric and the protomeric form of ClyA, it has recently been hypothesized that the ␤-tongue of the monomer is particularly required for membrane association and that its insertion into the membrane triggers a series of conformational changes that result in generation of the protomeric form and hence in ClyA oligomerization and pore formation (25).…”

“…Exported ClyA most likely binds in monomeric form to target membranes and then forms ring-shaped oligomeric complexes that penetrate the lipid bilayer, resulting in the final, stable ClyA pores that are water permeable and cation selective (9,15,23,25,37,40,42,45). Pore reconstructions based on electron microscopy have indicated that the ClyA protein must undergo considerable structural rearrangements to form the pore (9,37).…”