Purification and detection of Shigella type III secretion needle complex

Abstract

Publisher Summary This chapter describes the method for purifying the type III secretion complex, tentatively named the needle complex, from S. flexneri 2a strain VirG- mutan (M94). Because M94 contains the intact type III secretion system required for invasion but is defective in actin-based motility in mammalian cells because of a Tn5 insertion in the vir G ( ics A) gene, attenuated strain have been used for extracting the type III secretion complex for the sake of laboratory biosafety. In brief, bacteria are suspended in sucrose solution supplemented with ethylenediaminetetraacetic acid, (EDTA)-2Na and lysozyme to allow spheroplast formation. Subsequently, bacteria are lysed by a nonionic detergent such as Triton X-100, which is followed by elimination of DNA by DNase digestion, and then the lysate is subjected to a low-speed centrifugation to remove cell debris. The intact needle complexes contained in the lysate are enriched by ultracentrifugation. The pellet is fractionated by caesium chloride, density gradient centrifugation, and the fractions are further ultracentrifuged to sediment the extensively purified needle complex.