Novel evidence for the specific interaction between cholesterol and α-haemolysin of Escherichia coli

Abstract

Several toxins that act on animal cells present different, but specific, interactions with cholesterol or sphingomyelin. In the present study we demonstrate that HlyA (α-haemolysin) of Escherichia coli interacts directly with cholesterol. We have recently reported that HlyA became associated with detergent-resistant membranes enriched in cholesterol and sphingomyelin; moreover, toxin oligomerization, and hence haemolytic activity, diminishes in cholesterol-depleted erythrocytes. Considering these results, we studied the insertion process, an essential step in the lytic mechanism, by the monolayer technique, finding that HlyA insertion is favoured in cholesterol- and sphingomyelin-containing membranes. On the basis of this result, we studied the direct interaction with either of the lipids by lipid dot blotting, lysis inhibition and SPR (surface plasmon resonance) assays. The results of the present study demonstrated that an interaction between cholesterol and HlyA exists that seems to favour a conformational state of the protein that allows its correct insertion into the membrane and its further oligomerization to form pores.

Structure and function of RTX toxins

“…They further showed that a peptide corresponding to CRAC336 inhibited the LtxA cytotoxic activity and demonstrated that a point mutation in the CRAC336 site abolished LtxA toxicity. The conservation of CRAC336 among RTX toxins suggests that this mechanism may be conserved among RTX toxins, and indeed a specific interaction between cholesterol and E. coli HlyA has recently been reported [381]. DiFranco et al [382] reported that binding of LtxA to LFA-1 results in the internalization of both LtxA and LFA-1.…”

The conserved tyrosine residue 940 plays a key structural role in membrane interaction of Bordetella adenylate cyclase toxin

“…The presence of cholesterol in the membrane was, indeed, previously found to enhance the binding and lytic capacity of CyaA on erythrocytes and artificial membranes 30 , 40 . Moreover, two other RTX toxins, the Aggregatibacter actinomycetemcomitans leukotoxin LtxA and the Escherichia coli α-hemolysin HlyA, were previously shown to specifically bind cholesterol 41 – 43 . These toxins possess in their pore-forming domains the so called cholesterol recognition/interaction amino acid consensus (CRAC) motifs.…”

Aggregatibacter actinomycetemcomitansleukotoxin causes activation of lymphocyte function-associated antigen 1

“…The interaction of LtxA with its human host is multifaceted and involves both the membrane lipids (cholesterol; Brown et al, 2013) and a cell-surface glycoprotein lymphocyte function-associated antigen 1 (LFA-1), a β 2 integrin family member (Lally et al, 1997). LtxA contains a conserved cholesterol recognition amino acid consensus motif (Brown et al, 2013;Vazquez et al, 2014) that is defined by the sequence -L/V-X 1-5 -Y-X 1-5 -R/K- (Epand, 2006;Li, Yao, Degenhardt, Teper, & Papadopoulos, 2001). The presence of the binding sequence ( 334 LEEYSKR 339 ) enhances the maximal affinity (minimal K d ) of the toxin for membranes containing 40% cholesterol, which is approximately four orders of magnitude (10 −12 M) greater than the affinity for cholesterol-free membranes (10 −8 M).…”

“…The KIM185 epitope (residues 581-621) is located at the distal end of CRR-4 and extends onto the stalk to a point 57 amino acids from the LFA-1 transmembrane segment (Lu et al, 2001). The ability of LtxA to bind cholesterol (Brown et al, 2013;Vazquez et al, 2014), interact with extra cellular domains of LFA-1 (Lally et al, 1997), and modify bilayer structure (Brown et al, 2012) suggests that the LtxA/LFA-1 interaction is not confined to the ectodomains of LFA-1 but rather is providing a clear pathway for LtxA to enter the cytosol.…”