Transport and intracellular movement - Protein translocation via dedicated secretion systems

This chapter discusses the methods that are used to elucidate the action of the Yop virulon, an archetype of type III secretion/translocation systems on eukaryotic target cells. This ‘weapon’ is encoded by the pYV plasmid (Yersinia virulence) and consists of three major parts: (1) type III secretion machinery (Ysc) that directs Yop proteins over the bacterial membranes, (2) translocator Yops that allow delivery of effector Yops in the cytosol of the eukaryotic cell, and (3) Yop effector proteins that interfere with the eukaryotic host cells. Y. enterocolitica strains are routinely grown in tryptic soy broth (TSB) and plated on tryptic soy agar (TSA) containing the required antibiotics. Prior to cell infection, Y. enterocolitica is grown in BHI under selective pressure for maintaining the relevant plasmids. Various methods are used to show translocation of bacterial proteins across the eukaryotic cell membrane. To prove translocation it must be demonstrated that the prokaryotic protein is found inside the eukaryotic cell compartment in the absence of phagocytosed bacteria