Abstract
Actinobacillus pleuropneumoniae is a mucosal respiratory pathogen causing contagious porcine pleuropneumonia. Pathogenesis studies have demonstrated a major role for the capsule, exotoxins and outer membrane proteins. Actinobacillus pleuropneumoniae can also glycosylate proteins, using a cytoplasmic N-linked glycosylating enzyme designated NGT, but its transcriptional arrangement and role in virulence remains unknown. We investigated the NGT locus and demonstrated that the putative transcriptional unit consists of rimO, ngt and a glycosyltransferase termed agt. From this information we used the A. pleuropneumoniae glycosylation locus to decorate an acceptor protein, within Escherichia coli, with a hexose polymer that reacted with an anti-dextran antibody. Mass spectrometry analysis of a truncated protein revealed that this operon could add up to 29 repeat units to the appropriate sequon. We demonstrated the importance of NGT in virulence, by creating deletion mutants and testing them in a novel respiratory cell line adhesion model. This study demonstrates the importance of the NGT glycosylation system for pathogenesis and its potential biotechnological application for glycoengineering.
Highlights
Actinobacillus pleuropneumoniae is a Gram-negative bacterium and the causative agent of porcine pleuropneumonia, a severe respiratory disease responsible for significant losses to the pig industry worldwide
We report the generation of A. pleuropneumoniae ngt and agt deletion mutants, and demonstrate a biological role for this N-linked glycosylation system using a human adenocarcinoma lung epithelial cell adhesion assay
NGT and a6GlcT are required for adhesion of Actinobacillus pleuropneumoniae HS143 to
Summary
Actinobacillus pleuropneumoniae is a Gram-negative bacterium and the causative agent of porcine pleuropneumonia, a severe respiratory disease responsible for significant losses to the pig industry worldwide. This disease has a huge impact on the pig industry, costing an average E6.4 per fattened pig in an affected herd in Europe [1]. Actinobacillus pleuropneumoniae enters the lungs and colonizes tissues by binding to mucus proteins and cells of the lower respiratory tract, including ciliated cells of the terminal bronchioli and alveolar epithelial cells [2,3]. Several surface structures have been identified as being involved in adhesion, including fimbriae [6] and lipopolysaccharide (LPS) [7].
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