J. LABRIE, Faculté de Médecine Vétérinaire, Université de Montréal M. JACQUES , Faculté de Médecine Vétérinaire, Université de Montréal
Title
Identification of an ABC transporter with probable implication in O-polysaccharide assembly of Actinobacillus pleuropneumoniae serotype 1
Abstract Text
Actinobacillus pleuropneumoniae (App) is the causative agent of porcine pleuropneumonia. Lipopolysaccharide (LPS) has been identified as an important adhesin of App involved in adherence to porcine respiratory tract cells. ABC transporters have been shown to be implicated in the translocation of exopolysaccharides in a variety of Gram-negative bacteria. In a previous study, we identified a region of 18 ORFs in App serotype 1 involved in O-antigen biosynthesis. The proteins encoded by two of these ORFs (ORF10 and ORF11) showed high homologies with Wzm/Wzt proteins belonging to the ABC superfamily of active transporters. The purpose of the present study was to characterize these two ORFs in order to learn more about the ABC transport system involved in O-antigen export in App serotype 1. ORF10 encoded a predicted polypeptide of 30.1 kDa and the deduced amino acid (aa) sequence showed 66% identity with an ORF of Actinobacillus actinomycetemcomitans. This protein is a predicted ATP-binding component of an ABC transporter required for LPS O-antigen translocation. Homologies were also observed with Wzm (formerly RfbA) protein of Escherichia coli (28%) and Klebsiella pneumoniae (27%). The hydrophobicity profile of ORF10 indicates a highly hydrophobic protein with six putative membrane-spanning domains. This profile is similar to those of other proteins considered to be integral membrane components of ABC transport systems in different bacteria. ORF11 encoded a predicted polypeptide of 24.5 kDa which showed homologies with Wzt protein of Aeromonas salmonicida (AbcA) (53%), Pseudomonas aeruginosa (RfbB) (49%) and Serratia marcescens (RfbB) (46%) which are all known ATP-binding components of ABC transporters. The deduced aa sequence of the protein encoded by ORF11 revealed an ATP-binding motif (GXXGXGKS, position 62 to 69) and an ABC transporter family signature (FSSGMAARLGFAIAT, position 150 to 164). ABC transporter-dependent pathway for O-polysaccharide assembly is found in enteric as well as nonenteric bacteria. However, this pathway appears to be restricted to homopolymers or to relatively simple O-antigen structures which is not the case for App serotype 1 as its O-antigen structure was previously reported to consist of branched tetrasaccharide repeating units. Using a single step transconjugation system, the generation of an App ORF10/11 null mutant is in progress to confirm the involvement of this ABC transporter in App serotype 1 O-antigen biosynthesis.
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