Purification of Staphylococcus aureus capsular polysaccharide serotype 5 /

Abstract

Staphylococcus aureus continues to pose a major health threat, most prevalent in the immunocompromised, despite the development ofantibiotics. Due to the increased antibiotic resistance seen with this pathogen another means oftreatment is warranted. Ninety-eight percent of the clinical isolates ofS aureus are encapsulated, eighty percent of which are capsular polysaccharide (CP) type 5 or 8. CPs are thought to shield S aureus from agglutination by antibodies to cell wall structures. For this reason, encapsulated strains are poorly phagocytosed by polymorphonuclear leukocytes. However, antibodies specific for the CP have been shown to be efficient in inducing phagocytosis of encapsulated strains. S aureus remains a problem for patients with decreased immunologic function because these individuals are often unable to form immune responses to carbohydrate molecules. Preformed antibodies against the CP ofS aureus would aid these patients in eliminating the bacterium. Presented in this study is the first step to the production oftherapeutic antibodies, the isolation and purification of S aureus CP. The CP was removed from the bacterium by autoclaving the bacterial cell suspension. The CP was then purified by enzymatic digestion and anion exchange chromatography. In this study, several methods are tested for their ability to detect the CP in the anion exchange column eluate. The CP, a reducing sugar, was ultimately detected by its ability to reduce Red Tetrazolium to an intensely colored diformazan whose production was measured spectrophotometrically. The CP, which was determined to be free ofteichoic acid contamination, will be used in further studies to produce preformed antibodies against S aureus CPs.