Generation and Use of Site-Directed Chromosomal cyaA′ Translational Fusions in Salmonella enterica

CyaA from Bordetella pertussis is a calmodulin-dependent adenylate cyclase. Fusions to the catalytic domain of CyaA (CyaA′) are useful tools to detect translocation of type III secretion system effectors from gram-negative pathogens like Salmonella enterica. These fusions are usually generated using plasmids with strong promoters. Here, we describe a protocol to insert the CyaA′-encoding sequence in a specific site in the bacterial chromosome in order to get a monocopy fusion whose expression is driven by the native promoter. We also describe the procedure to detect translocation of a CyaA′ fusion into mammalian cells.
Source: Springer protocols feed by Infectious Diseases - Category: Infectious Diseases Source Type: news