Acylation ofEscherichia coliHemolysin: A Unique Protein Lipidation Mechanism Underlying Toxin Function

Abstract

SUMMARY The pore-forming hemolysin (HlyA) of Escherichia coli represents a unique class of bacterial toxins that require a posttranslational modification for activity. The inactive protoxin pro-HlyA is activated intracellularly by amide linkage of fatty acids to two internal lysine residues 126 amino acids apart, directed by the cosynthesized HlyC protein with acyl carrier protein as the fatty acid donor. This action distinguishes HlyC from all bacterial acyltransferases such as the lipid A, lux-specific, and nodulation acyltransferases, and from eukaryotic transferases such as N-myristoyl transferases, prenyltransferases, and thioester palmitoyltransferases. Most lipids directly attached to proteins may be classed as N-terminal amide-linked and internal ester-linked acyl groups and C-terminal ether-linked isoprenoid groups. The acylation of HlyA and related toxins does not equate to these but does appear related to a small number of eukaryotic proteins that include inflammatory cytokines and mitogenic and cholinergic receptors. While the location and structure of lipid moieties on proteins vary, there are common effects on membrane affinity and/or protein-protein interactions. Despite being acylated at two residues, HlyA does not possess a “double-anchor” motif and does not have an electrostatic switch, although its dependence on calcium binding for activity suggests that the calcium-myristoyl switch may have relevance. The acyl chains on HlyA may provide anchorage points onto the surface of the host cell lipid bilayer. These could then enhance protein-protein interactions either between HlyA and components of a host signal transduction pathway to influence cytokine production or between HlyA monomers to bring about oligomerization during pore formation.

Palmitoylation of the P2X7 receptor, an ATP‐gated channel, controls its expression and association with lipid rafts

“…Thus, it has been previously demonstrated that palmitoylation of 3 cysteines of the cytoplasmic tail of influenza virus hemagglutinin was required for its oligomerization and for virus assembly (46). Moreover, Escherichia coli hemolysin is palmitoylated on 2 lysine residues, and this acylation is essential for its ability to form a pore in the host membrane (47)(48)(49). Removal of one acyl group results in an almost complete loss of cytolytic activity of the hemolysin.…”

SiiA and SiiB are novel type I secretion system subunits controlling SPI4-mediated adhesion ofSalmonella enterica

“…There are several examples of accessory components which can specifically modulate T1SS substrate activity. HlyC of pathogenic E. coli is involved in post-translational acylation of the α-haemolysin HlyA (reviewed in Stanley et al, 1998). In P. fluorescens, the c-di-GMP-binding protein LapD regulates the activity of the periplasmic protease LapG which controls surface localization of the adhesin LapA (Newell et al, 2011).…”

Why Escherichia coli α‐hemolysin induces calcium oscillations in mammalian cells‐the pore is on its own

“…Common to all are the presence of a Ca 2ϩ binding nonapeptide repeat sequence and the requirement for posttranslational fatty acylation at one or two lysine residues for the acquisition of pore-forming activity (1,2). In the absence of Ca 2ϩ or fatty acylation, binding capacity is retained but the toxins are unable to adopt the pore-forming configuration (3,4).…”

“…The bindability of labeled mutant S177C and the hemolytically inactive mutant S177C/K564R/ K690R was identical. Protein purification of toxins was carried out as described previously (1,4).…”