Abstract
Some strains of Streptococcus pyogenes isolated from infected human tissues were shown to bind laminin, a major component of basement membranes. Binding of 125I-laminin to bacteria was time dependent and functionally irreversible. Of several unlabeled proteins tested in competition experiments, laminin and fibrinogen inhibited binding of the radiolabeled protein. The inhibitory effect exerted by fibrinogen was apparently not caused by a binding to the laminin receptors. The number of receptors available for laminin on cells of the strain examined ranged from 0 to 10(3) depending on the media used to grow the bacteria and an apparent KD of 4 X 10(-8)M was calculated for the reaction. Bacterial cells incubated with proteolytic enzymes lose the ability to bind laminin, and a trypsin digest contained active receptors capable of competing with intact cells for 125I-laminin. Active receptors may be adsorbed on a column of laminin-Sepharose but not on Sepharose gels substituted with fibrinogen or fibronectin. After radiolabeling the proteins in the trypsin digest a laminin-binding 125I-labeled protein (Mr greater than 10(6] was isolated by affinity chromatography from a receptor positive strain. Similar components could not be isolated from a strain apparently lacking laminin receptors. Therefore, this protein was tentatively identified as a laminin receptor of streptococci.
Highlights
Some strains of Streptococcus pyogenes isolated Many bacterial infections follow on an initial viral infection from infected humantissues were shown to bind lami- or trauma thatmay damage the protective epithelial lining of nin, amajor component of basement membranes
Affinity Chromatography-Samples (300 pl) of material released from the bacteria by trypsin were applied to columns containing 0.5 mlof unsubstituted or protein-substituted Sepharose equilibrated with PBS
Screening of 40 randomly selected strains of S. pyogenes representing clinical isolates showed that 21 strains bound lZ51-lamininS. trainsbinding less than 2%of the added labeled protein (ix. less than 150 cpm over the background level) were considered as nonbinders
Summary
One hundred mg (wet weight) of bacteria was heated at 80 "C for 10 min, centrifuged (3000 X g, 15 min), and resuspended in 0.5 ml of PBS without azide This suspension was digested with trypsin (25 pg/ml) for 1 h a t 37 "C.The reaction was stopped by the addition of soybean trypsininhibitor (50 pg/ml), the cells were pelleted by centrifugation, and the supernatant was incubated at 80 "C for 10 min and stored a t -20 "C. Affinity Chromatography-Samples (300 pl) of material released from the bacteria by trypsin were applied to columns containing 0.5 mlof unsubstituted or protein-substituted Sepharose equilibrated with PBS. Samples consisting of 2 X lo' cpm of radiolabeled proteins or a mixture of reference proteins (4 pg of each protein; Pharmacia Fine Chemicals, Uppsala, Sweden) were applied per lane after boiling in the presence of SDS-containing buffer. Following electrophoresis the gels were stained with Coomassiebrilliant blue R, destained, dried under vacuum and subjected to autoradiography using X-Omat AR film (Kodak, Rochester, NY)
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