Serum Opacity Factor, a Streptococcal Virulence Factor That Binds to Apolipoproteins A-I and A-II and Disrupts High Density Lipoprotein Structure

Harry S Courtney,Yong-Mei Zhang,Matthew W Frank,Charles O Rock

doi:10.1074/jbc.m512538200

Abstract

Serum opacity factor (SOF) is a virulence determinant of group A streptococci that opacifies mammalian sera. We analyzed the specificity and mechanism of the opacity reaction using a recombinant form of the amino-terminal opacification domain of SOF, rSOF. Our data indicate that rSOF is neither a protease nor a lipase, but rather it is the binding of rSOF to high density lipoprotein (HDL) that triggers the opacity reaction. rSOF did not opacify plasma from apoA-I(-/-) mice or purified low or very low density lipoproteins but readily opacified HDL. rSOF binding to HDL was characterized by two high affinity binding sites; it bound to apoA-I (K(d) = 6 nm) and apoA-II (K(d) = 30 nm), and both apoA-I and apoA-II blocked the binding of rSOF to HDL. Electron microscopic examination and biochemical analyses of HDL treated with rSOF revealed the formation of lipid droplets devoid of apolipoproteins. Thus, SOF interacts with HDL in human blood by binding to apoA-I and apoA-II and causing the release of HDL lipid cargo, which coalesces to form lipid droplets, resulting in opacification. The disruption of HDL may attenuate its anti-inflammatory functions and contribute to the pathogenesis of group A streptococcal infections.

Highlights

Serum opacity factor (SOF) is a virulence determinant of group A streptococci that opacifies mammalian sera
Our data indicate that recombinant SOF (rSOF) is neither a protease nor a lipase, but rather it is the binding of rSOF to high density lipoprotein (HDL) that triggers the opacity reaction. rSOF did not opacify plasma from apolipoprotein A-I (apoA-I)؊/؊ mice or purified low or very low density lipoproteins but readily opacified HDL. rSOF binding to HDL was characterized by two high affinity binding sites; it bound to apoA-I (Kd ‫ ؍‬6 nM) and apoA-II (Kd ‫ ؍‬30 nM), and both apoA-I and apoA-II blocked the binding of rSOF to HDL
These findings suggested that rSOF induced changes in HDL that resulted in the formation of lipid particles that were essentially free of apolipoproteins, and it is these protein-deficient particles that are responsible for turning serum opaque because of their insolubility in aqueous media

Summary

EXPERIMENTAL PROCEDURES

Materials—Purified human very low density lipoproteins (VLDL), low density lipoproteins (LDL), HDL, apoA-I, and apoA-II were purchased from Calbiochem. Opacification Assays—The capacity of SOF to opacify horse and human serum was determined by adding 50 ␮l of rSOF at various concentrations to 2 ml of serum and recording the absorbance at 405 nm at timed intervals. The human lipoproteins, HDL, VLDL, and LDL, were diluted in 0.05 M Tris-HCl, 0.15 M NaCl, pH 7.2, to ϳ2 mg/ml, and 200 ␮l of this mixture was added to microtiter wells. Ten ␮l of rSOF was added to the wells to obtain 0, 0.1, 1.0, and 10 ␮g/ml final concentrations. The microtiter plate was incubated at 37 °C, and the absorbance at 405 nm was recorded at timed intervals.

Mechanism of SOF Action

Effect of enzyme inhibitors on opacification of serum by rSOF

Carboxypeptidase inhibitor

RESULTS

DISCUSSION