Molecular analysis of the cytolytic protein ClyA (SheA) from Escherichia coli

Abstract

SummaryEscherichia coli K-12 carries a gene for a protein denoted ClyA or SheA that can mediate a cytolytic phenotype. The ClyA protein is not expressed at detectable levels in most strains of E. coli, but overproduction suitable for purification was accomplished by cloning the structural gene in an hns mutant strain. Highly purified ClyA protein was cytotoxic to macrophage cells in culture and caused detachment and lysis of the mammalian cells. Results from osmotic protection assays were consistent with the suggestion that the protein formed pores with a diameter of up to 3 nm. Using Acholeplasma laidlawii cells and multilamellar liposomes, we studied the effect of ClyA on membranes with varying compositions and in the presence of different ions. ClyA induced cytolytic release of the fluorescent tracer from carboxyfluorescein-loaded liposomes, and the release was stimulated if cholesterol was present in the membranes whereas addition of calcium had no effect. Pretreatment of the ClyA protein with cholesterol inhibited the pore formation, suggesting that ClyA could bind to cholesterol. Efficient coprecipitation of ClyA with either cholesterol or 1,2,3-trioctadecanoylglycerol in aqueous solutions showed that ClyA directly interacted with the hydrophobic molecular aggregates. We tested the possible functional importance of selected ClyA protein regions by site-directed mutagenesis. Defined mutants of ClyA were obtained with alterations in postulated transmembrane structures in the central part and in a postulated membrane-targeting domain in the C-terminal part. Our results were consistent with the suggestion that particular amphiphilic segments are required for ClyA activity. We propose that these domains are necessary for ClyA to form pores.

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

“…6a, e), positioning the Cterminal residues (which are only partially ordered in the crystal structure of the monomer) in the groove. This feature of the model is congruent with the deleterious effects on pore assembly of the deletion of aG and the Cterminal residues Oscarsson et al, 1999). It is also notable that the only proteolytic cleavage point that is seen in the multimer, but not in the monomer, is at Asp21 (Fig.…”

“…At the end containing the N-and C-terminal regions of the protein a shorter [30 Å (3 nm)] helix (aG) packs against the four long helices, forming a five-helix bundle for about one-third of the length of the molecule (the tail domain). Random and site-directed mutagenesis has revealed that residues in the aG region play important roles in HlyE activity Oscarsson et al, 1999). HlyE possesses only two cysteine residues and these are housed in the tail domain (Fig.…”

“…1a; Wallace et al, 2000). Site-directed mutagenesis has shown that the hydrophobic nature of the b-tongue has to be maintained to allow HlyE to bind to and lyse target cells (Oscarsson et al, 1999;Wallace et al, 2000). In the crystal structure, HlyE is a head-to-tail dimer in which the hydrophobic b-tongue of one protomer packs against a second hydrophobic surface in the tail domain of the second protomer ( Fig.…”

Mecanismos de virulencia de Escherichia coli enteropatógena

“…Según estudios, inhibe la síntesis de ADN en células de cáncer de colon, que depende de los niveles de calcio intracelulares (Figura 3) 79,80 . La citolisina A (ClyA: cytolysin A) expresada del gen clyA también ha sido descrita como citotoxina de ECET cuyas funciones son la formación de poros, inducción de apoptosis en los macrófagos y actividad hemolítica [81][82][83] .…”

A novel PhoP-regulated locus encoding the cytolysin ClyA and the secreted invasin TaiA of Salmonella enterica serovar Typhi is involved in virulence

“…ClyA monomers are exported in outer membrane vesicles (OMVs). E. coli cells expressing clyA are cytotoxic for mammalian cells (Lai et al, 2000;Oscarsson et al, 1999). A specific antibody response toward ClyA during human infection by Typhi or Paratyphi was recently reported, which indicates that ClyA is expressed in vivo (von Rhein et al, 2006).…”