Molecular characterization of the oafA locus responsible for acetylation of Salmonella typhimurium O-antigen: oafA is a member of a family of integral membrane trans-acylases

Abstract

Lipopolysaccharide (LPS) coats the surface of gram-negative bacteria and serves to protect the cell from its environment. The O-antigen is the outermost part of LPS and is highly variable among gram-negative bacteria. Strains of Salmonella are partly distinguished by serotypic differences in their O-antigen. In Salmonella typhimurium, the O-antigen is acetylated, conferring the O5 serotype. We have previously provided evidence that this modification significantly alters the structure of the O-antigen and creates or destroys a series of conformational epitopes. Here we report the detailed mapping, cloning, and DNA sequence of the oafA gene. The locus contains one open reading frame that is predicted to encode an inner membrane protein, consistent with its role in modification of the O-antigen subunit. The OafA protein shows homology to proteins in a number of prokaryotic and one eukaryotic species, and this defines a family of membrane proteins involved in the acylation of exported carbohydrate moieties. In many of these instances, acylation defines serotype or host range and thus has a profound effect on microbe-host interaction.Lipopolysaccharide (LPS) forms the outer surface of gramnegative bacteria and protects the cell from the environment. LPS is composed of lipid A, core oligosaccharide, and O-antigen (23). The O-antigen polymer of Salmonella enterica serovar Typhimurium (Salmonella typhimurium) LPS has a branchedtetrasaccharide repeating subunit with galactose-rhamnosemannose in the main chain and abequose attached to the mannose residue (13). The 2-hydroxyl group of the abequose moiety is acetylated, conferring the O5 serotype (13). We have previously characterized a series of monoclonal immunoglobulin A antibodies directed against S. typhimurium O-antigen and have shown that the epitopes of all of these antibodies are affected by this acetylation reaction. We proposed that the acetylation affected the three-dimensional structure of the molecule and that many of the antibodies recognize conformational epitopes that are created or destroyed depending on the acetylation state of the molecule (28).In pathogenic bacteria, O-antigen is clearly important for infection. It is the dominant antigen in many immune responses and has been shown in a number of species to be critical for protection (see reference 20 for a review). Indeed, we have shown that one of our monoclonal immunoglobulin A antibodies is capable of protecting mice against oral infection with S. typhimurium and that this protection was completely dependent on the acetylation and therefore on the conformation of the O-antigen (19, 28).The acetylation reaction is dependent on the oafA locus (13, 28), which was originally mapped by Hfr crosses between naturally O5-positive and O5-negative strains of Salmonella (14). Here we report the detailed mapping, cloning, and DNA sequence analysis of the oafA gene. The results suggest that a single protein is responsible for the acetylation reaction. The putative OafA protein shows homology to a number of prote...

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