Characterization of Pneumococcal Purpura-Producing Principle

Abstract

Purpura was grossly observable in albino mice 6-8 h after the i.p. injection of sterile, DNase-treated, cell-free extracts prepared by sodium deoxycholate-induced lysis, sonic disruption, Parr bomb treatment, autolysis without sodium deoxycholate or alternate freezing and thawing of washed suspensions of Streptococcus pneumoniae type I. Cell-free extracts obtained from sonically disrupted, heat-killed cells (100.degree. C for 20 min) did not contain purpurogenic activity. The reaction was maximal at approximately 24 h post-injection, started to fade slowly after 24-48 h and usually was not grossly observable by 4-6 days post-injection. The purpura-producing principle (PPP) in the cell-free extract was purified by sequential (NH4)2SO4 precipitation, protamine sulfate precipitation, Sepharose 6B gel filtration, wheat germ lectin-Sepharose 6MB affinity chromatography, RNase and trypsin treatment and a 2nd Sepharose 6B gel filtration step. The final preparation contained glucosamine (5.6%), muramic acid (8.0%), neutral carbohydrate (12.8%), phosphate (8.0%), orcinol-reactive material (6.0%) and Lowry-reactive material (1.6%) and was free of detectable amounts of DNA, capsular polysaccharide, neuraminidase, cytolysin and hyaluronidase. The isoelectric point and MW of the PPP were approximately pI 3.0 and several million daltons, respectively, and activity remaining in the supernatant fluid after centrifugation for 1 day at 105,000 .times. g. PPP activity was destroyed by incubation with egg white lysozyme and sodium metaperiodate but was resistant to trypsin, pronase, .alpha.-amylase, DNase, RNase, alkaline phosphatase, pancreatic lipase, 7% trichloroacetic acid, 6 M urea, autoclaving (121.degree. C) for 30 min and mild acid and alkali exposure. The PPP may require intact .beta.-1,4-glucosidic linkages for activity. Activity may be associated with pneumococcal peptidoglycan solubilized by the bacterium''s autolysin.