C3 binds preferentially to long-chain lipopolysaccharide during alternative pathway activation by Salmonella...

We studied the population of LPS molecules on Salmonella montevideo that bind C3 during alternative pathway activation in serum. LPS molecules of Salmonella are composed of lipid A:core oligosaccharide (one copy per molecule), substituted by an O-polysaccharide (O-PS) side chain, which is a linear polymer of 0 to greater than 60 O-antigen repeat units containing mannose. A mutant of S. montevideo called SL5222 that inserts galactose only into core oligosaccharide and mannose only into O-antigen subunits was grown with [3H]mannose and [14C]galactose, so that LPS molecules bearing large numbers of O-antigen subunits have high 3H to 14C ratios, whereas molecules with few O-antigen subunits have lower 3H to 14C ratios. Double-labeled SL5222 was incubated in C8-deficient (C8D) serum or C8D serum with 2 mM Mg++Cl2 and 10 mM ethylene glycoltetraacetic acid (MgEGTA C8D). LPS molecules with covalently attached C3 were identified by binding to anti-C3. LPS molecules that bound C3 under both incubation conditions had O chains seven to eight times longer than the average LPS molecule. SL5222 was then grown in suboptimal concentrations of mannose in order to decrease the number of LPS molecules with long O-PS side chains. C3 attached to progressively shorter chain molecules of LPS as the mannose input was lowered, but still chose the longest available molecules. This finding and recently published observations indicate that C3 can bind to LPS molecules with short O-PS side chains. We postulate that preferential attachment of C3 to long-chain LPS in SL5222 results because long-chain LPS molecules sterically hinder shorter chain LPS molecules from macromolecules. This study provides direct proof that the O-PS of LPS sterically hinders access of large molecules to the outer membrane and indicates that the LPS coat of these bacteria functions as a barrier against large protein molecules.