Abstract
Protein acylation is critical for many cellular functions across all domains of life. In bacteria, lipoproteins have important roles in virulence and are targets for the development of antimicrobials and vaccines. Bacterial lipoproteins are secreted from the cytosol via the Sec pathway and acylated on an N-terminal cysteine residue through the action of three enzymes. In Gram-negative bacteria, the Lol pathway transports lipoproteins to the outer membrane. Here, we demonstrate that the Aat secretion system is a composite system sharing similarity with elements of a type I secretion systems and the Lol pathway. During secretion, the AatD subunit acylates the substrate CexE on a highly conserved N-terminal glycine residue. Mutations disrupting glycine acylation interfere with membrane incorporation and trafficking. Our data reveal CexE as the first member of a new class of glycine-acylated lipoprotein, while Aat represents a new secretion system that displays the substrate lipoprotein on the cell surface.
Highlights
Protein acylation by the covalent attachment of fatty acids occurs for hundreds of proteins in eukaryotic and prokaryotic organisms
We propose that Aap and CexE are members of a novel class of lipoprotein that are secreted through the Aat system, which is a conglomeration of the Lol pathway and a T1SS
The Aat system was first identified in enteroaggregative Escherichia coli (EAEC), where it corresponds to the molecular probe (CVD432) used to define this E. coli pathovar (Baudry et al, 1990; Nishi et al, 2003)
Summary
Protein acylation by the covalent attachment of fatty acids occurs for hundreds of proteins in eukaryotic and prokaryotic organisms. The mature triacylated lipoprotein remains embedded in the inner membrane or is localised to the inner leaflet of the outer membrane by the essential Lol pathway. Aap/CexE is translocated across the inner membrane into the periplasm by the Sec pathway (Pilonieta et al, 2007). We reveal that AatD is both necessary and sufficient for monoacylation of CexE and Aap. in contrast to Lol lipoprotein substrates, CexE lacks an N-terminal cysteine and instead an invariant glycine is the site of acylation. We demonstrate that the addition of an N-terminal glycine to the coding sequence of a heterologous protein was sufficient for this novel AatD-catalysed acylation event. We propose that Aap and CexE are members of a novel class of lipoprotein that are secreted through the Aat system, which is a conglomeration of the Lol pathway and a T1SS. We reveal a new function for acylation-protein secretion
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