Haemophilus somnus immunoglobulin binding proteins and surface fibrils

Abstract

The high-molecular-weight (HMW) immunoglobulin binding proteins (IgBPs) of Haemophilus somnus and a 76-kDa surface protein (p76) are found in serum-resistant virulent strains but not in several serum-sensitive strains from asymptomatic carriers. For the first time, p76 was shown to be an IgBP also. This was done by competitive inhibition studies with affinity-purified antidinitrophenol (anti-DNP) and DNP to ensure that binding was not antigen specific. The HMW IgBPs, but not the p76 IgBP, were partially purified from concentrated culture supernatant in detergent by fluid-phase liquid chromatography with a gel filtration column. Membrane extraction studies showed that p76 predominated in the Sarkosyl-soluble fraction of the bacterial cell pellet. Since integral outer membrane (OM) proteins are Sarkosyl insoluble, this is consistent with our previous finding that implicated p76 as a peripheral OM protein. The HMW IgBPs were found predominantly in the Sarkosyl-soluble fraction of the culture supernatant. This suggests that they were not integral membrane proteins and that their presence in the supernatant was not due to OM blebbing. We then showed that two IgBP-positive serum-resistant virulent strains have a surface fibrillar network but that two IgBP-negative serum-sensitive H. somnus strains from asymptomatic preputial carriers do not. Fibrils on the surfaces of IgBP+ strains bound gold-labelled bovine immunoglobulin G2 (IgG2) anti-DNP, indicating that these fibrils have IgG2 binding activity. Therefore, this study shows that H. somnus has two IgBPs, including a peripheral membrane protein and a fibrillar surface network.