Detection of Type III Secretion System Needle Assembly in Yersinia pestis by Cross-Linking YscF

Abstract

Type III secretion systems (T3SSs) are finely tuned molecular machines that are required for the virulence of many Gram-negative bacteria. Both the transcription and secretion of the Yersinia pestis Ysc T3SS are highly activated at 37 °C when the bacteria are cultured in low-calcium medium in vitro (Cornelis GR: Nat Rev Microbiol 4(11):811–825, 2006). The Y. pestis Ysc T3SS has evolved a sophisticated regulatory mechanism to control the assembly of the secretion apparatus and the secretion of substrates. Once the transcription and secretion of the T3SS have been triggered, the secretion apparatus, called the injectisome, is assembled such that it extends from the bacterial surface. The needle of the Yersinia injectisome has a helical structure that consists of a few hundred copies of the YscF protein (Hoiczyk E, Blobel G: Proc Natl Acad Sci USA 98(8):4669–4674, 2001). In the presence of the cross-linker bis(sulfosuccinimidyl)suberate, bacterial surface-exposed YscF molecules that constitute the needle structure can be cross-linked via a covalent bond to form ladders of multimers, which can be evidenced after separation by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and immunoblotting detection using a specific anti-YscF antibody (Wood SE et al: J Bacteriol 190(12):4252–4262, 2008). Thus, the presence of the YscF multimers indicates that the tested bacterial strain can assemble the needle. This chapter describes an immunoblotting detection method for analyzing the formation of the YscF needle of Yersinia strains expressing the Ysc T3SS, which can be used to analyze the impacts of different T3SS components on YscF needle formation.