Isolation of heart- and kidney-binding protein from group A streptococci

Abstract

Tritium-labeled, water-soluble components of Streptococcus pyogenes type M6 adsorbed to cardiac tissue in vitro. Tissue binding was time dependent, saturable, and reversible. Chromatography of the crude bacterial extract on Bio-Gel P-300 indicated a molecular weight greater than 300,000 for the heart-binding component. Sodium dodecyl sulfate (SDS) dissociated this aggregate into a protein of 18,000 to 20,000 daltons as determined by Sephacryl S-200 chromatography and SDS-polyacrylamide disc gel electrophoresis. The tissue-binding protein was also purified from streptococcal extracts by adsorption to immobilized heart components. SDS-polyacrylamide gel electrophoresis of the protein desorbed from tissue revealed a radioactive band of 19,000 daltons. Indirect immunofluorescence tests on cardiac tissue treated with streptococcal extract showed an accumulation of a bacterial antigen on the sarcolemmal sheaths. Streptococcal components also adsorbed to basement membranes of kidney. Antisera prepared to isolated cytoplasmic membranes and water-soluble extracts of S. pyogenes type M6 were the most sensitive reagents for the detection of bacterial components bound to tissue. Antisera prepared to isolated cell walls and to intact bacteria were weakly reactive in these assays.

Heparin-inhibitable basement membrane-binding protein of Streptococcus pyogenes

“…We reported previously (23,24) on extracted surface proteins of S. pyogenes M serotypes 6 and 12 that bind specifically to basement membranes of muscle and kidney tissues in vitro. The proteins, which behaved as a large aggregate during chromatographic manipulations, were dis-* Corresponding author.…”

“…These substances, released by streptococci at remote sites of infection such as the nasopharynx or skin, may be carried via the blood stream to other tissues, where they accumulate on the basement membranes. Histopathology may then arise from direct toxicity of the streptococcal component, from its in situ reaction with antibodies and complement, or from a combination of these mechanisms (1,15,23).…”

“…Alkali extraction of S. pyogenes. Bacteria were extracted sequentially with organic solvents and alkali as previously described (23). After centrifugation at 4°C, the supernatant fluid was collected, dialyzed against water, and lyophilized.…”

“…IIF assay. Rabbit anti-S. pyogenes sera were titrated by indirect immunofluorescence (IIF) assay on 2-p,m-thick, cryostat-cut sections of fresh cynomolgus monkey tissue and on sections of tissue that were pretreated with bacterial cell extracts as previously described (23). Naturally occurring, heart-reactive antibodies (24, 25a) were removed from normal and immune rabbit sera by absorption with homogenized human heart.…”

“…Tissue preparation. Cardiac muscle which had been freshly obtained from cynomolgus monkeys was cut into 5-mm pieces and blended in cold PBS with a Sorvall Omnimixer (Ivan Sorvall, Inc., Norwalk, Conn.) as described elsewhere (23). The homogenate was passed through three layers of cotton gauze and centrifuged at 105,000 x g for 1 h. The pellet of cardiac muscle fragments was washed twice with PBS and stored at -20°C.…”