Analysis of LPS released from Salmonella abortus equi in human serum

We investigated in which form lipopolysaccharide (LPS) is released from live bacteria incubated with human serum and whether the released LPS can interact with high density lipoprotein (HDL), the main transport protein for purified LPS in circulation. Live biotinylated Salmonella abortus equi bacteria were incubated with fresh serum (37 degrees C; 2 h). The released LPS was isolated by immunoprecipitation or immunoabsorption using specific anti-O antibodies. It was analysed and compared with purified LPS, also incubated with serum under identical conditions. Immunoprecipitation led to a 35% recovery and immunoabsorption to quantitative recovery of released or purified LPS. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent immunoblot analysis revealed that all molecular species present in the purified LPS were present in the released LPS. The rough fraction, which was co-isolated from serum together with the true smooth (O-polysaccharide-containing) molecules, exhibited S. minnesota rough mutant Rb antigenic specificity. In the immunoprecipitated material two forms of released LPS were identified. One represented LPS associated with a biotinylated bacterial component with an apparent molecular mass of 35-36 kDa, which was identified as OmpA, a major outer membrane protein. The OmpA-associated LPS was free of HDL. Another part of the released LPS was free of biotinylated bacterial components. This portion of LPS was associated with HDL, indicating that the interaction with HDL may also proceed with a part of LPS released from bacteria.