Biochemical characterization of Escherichia coli-binding proteins from avian air sacs and plasma

Weebadda, Wineeta K. C.
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University of Guelph

Avian pathogenic strains of 'Escherichia coli' serotypes O2, O35 and O78 cause air sac infections in turkeys, but the reason for the colonization of these serotypes is unknown. Air sac surface fluid (ASSF) from 8-12 weeks old turkeys was examined to identify proteins that bind surface polysaccharides of pathogenic respiratory 'E. coli' O2. A multimeric protein termed lipid-free polysaccharide binding protein-40 (LFPBP-40) composed of six covalently associated subunits of ~40 kDa was isolated by elution from LFP by EDTA or L-rhamnose. The N-terminal amino acid sequence of LFPBP-40 DINGGGATLPQHLYLTPDV was related only to the N-terminus of fragment 3 of partially characterized human protein possessing T-cell stimulation activity in synovial membrane of rheumatoid arthritis patients. Endogenous amino acid sequences were unrelated to other known proteins. LFPBP-40 was immunoreactively distinct from pulmonary collectins and ficolins. A 40 kDa (reduced) turkey serum protein immunoreactive with rabbit anti LFPBP-40 polyclonal serum and anti-peptide antibodies was eluted from 'E. coli' LFP affinity matrix with 100 mM sodium acetate (pH 4.5). The purified serum form consisted of equivalent immunoreactive subunits of ~40 kDa reduced, and ~240 kDa non-reduced forms as seen for the air sac form and contained similar immunoreactive isoforms in the pI range of 6.5-6.9 under reduced condition. Rabbit anti LFPBP-40 polyclonal serum identified a 40 kDa (reduced) protein band in air sac homogenates from turkeys. In affinity chromatographic experiments LFPBP-40 from turkey serum and air sac washings did not bind to D-mannose, N-acetylglucosamine, or ' E. coli' P13 LPS coupled to Epoxy-Toyopearl AF 650M matrix. LFPBP-40 from air sac washings bound to intact 'E. coli' O2 in a N-acetylglucosamine and acid-elutable manner, whereas the serum form could be eluted with L-rhamnose and N-acetylglucosamine from intact 'E. coli' O2 bacteria. A chicken serum protein immunoreactive with rabbit anti LFPBP-40 polyclonal serum was isolated by its calcium-dependent binding to 'E. coli' LFP. LFPBP-40 immunoreactive proteins were not detected in porcine serum or lung lavage fluids. These results demonstrate related multimeric lectins for ' E. coli' O2 with limited distribution in plasma and air sac of turkeys and chickens.

Escherichia coli, proteins, binding, surface polysaccharides, avian, air sacs, plasma