Mutations Affecting Export and Activity of Cytolysin A from Escherichia coli

Abstract

Cytolysin A (known as ClyA, HlyE, and SheA) is a cytolytic pore-forming protein toxin found in several Escherichia coli and Salmonella enterica strains. The structure of its water-soluble monomeric form and that of dodecameric ClyA pores is known, but the mechanisms of ClyA export from bacterial cells and of pore assembly are only partially understood. Here we used site-directed mutagenesis to study the importance of different regions of the E. coli ClyA protein for export and activity. The data indicate that ClyA translocation to the periplasm requires several protein segments located closely adjacent to each other in the "tail" domain of the ClyA monomer, namely, the N-and C-terminal regions and the hydrophobic sequence ranging from residues 89 to 101. Deletion of most of the "head" domain of the monomer (residues 181 to 203), on the other hand, did not strongly affect ClyA secretion, suggesting that the tail domain plays a particular role in export. Furthermore, we found that the N-terminal amphipathic helix ␣A1 of ClyA is crucial for the formation and the properties of the transmembrane channel, and hence for hemolytic activity. Several mutations affecting the C-terminal helix ␣G, the "␤-tongue" region in the head domain, or the hydrophobic region in the tail domain of the ClyA monomer strongly impaired the hemolytic activity and reduced the activity toward planar lipid bilayer membranes but did not totally prevent formation of wild-type-like channels in these artificial membranes. The latter regions thus apparently promote membrane interaction without being directly required for pore formation in a lipid bilayer.

Haemolysin E- and enterohaemolysin-derived haemolytic activity of O55/O157 strains and other Escherichia coli lineages

“…1a). This finding is particularly important because HlyE has long been regarded as a 'silent haemolysin' due to its wide distribution among E. coli strains, even though its expression is repressed by H-NS (Ludwig et al, 1999;Westermark et al, 2000;Wyborn et al, 2004). Currently, we do not fully understand the exact reasons why the HlyE-derived haemolysis can be detected under these assay conditions.…”

A Comparative Genomic Analysis of Diverse Clonal Types of EnterotoxigenicEscherichia coliReveals Pathovar-Specific Conservation

“…However, even laboratory-adapted E. coli strains, such as K-12, have been shown to express a cytolytic phenotype (48). Research has shown that clyA is regulated by a complex system of regulation, including HN-S, cyclic AMP, and slyA (38,48), which are present in all sequenced ETEC genomes.…”

Basic mechanism of pore-forming toxins

“…closely adjacent to each other in the "tail" domain of the ClyA monomer-namely, the N-and C-terminal regions and the hydrophobic sequence of ClyA ranging from residues 89-101 [18]. Only a little periplasmic ClyA is found in the supernatant of E. coli cultures, which means that the cytolysin is presumably leaking out of the cells through outer membrane lesions [19].…”

“…The oligomer has approximately a length of about 13 nm from which 3-4 nm are inserted into the membrane [24]. The yellow part at the bottom of the ClyA oligomer shows the amino acids 1-15 that are important for channel formation [18]. (B) The right panel shows the view on top of the ClyA oligomer (N-terminal ends) of the ClyA monomers.…”

“…The diameter of the outer shell of the oligomer is about 9 to 10 nm [24]. [18] and the ClyA dodecamer, modified after 2WCD.pdb [24]. (A) The ClyA monomer, where the amino acids 1-15 are given in white (yellow in e-version) and the amino acids 40 to 80 in dark gray (pink in e-version).…”