The development of a FACS-based strategy for the isolation of Shigella flexneri mutants that are deficient in intercellular spread

In the disease course of bacillary dysentery, pathogenic Shigella flexneri invade colonic epithelial cells and spread both within and between host cel ls. The ability to spread intercellularly allows the organism to infect an entire epithelial layer without significant contact with the extracellular milieu. Using fluorescence activated cell sorter (FACS)-based technology, w e developed a rapid and powerful selection strategy for the isolation of S. flexneri mutants that are unable to spread from cell to cell. The majority of mutants identified using this strategy harbour mutations that affect th e structure of their lipopolysaccharide or the ability of the bacteria to m ove intracellularly via actin-based motility; both factors have previously been shown to be essential for cell-to-cell spread. However, using a modifi ed strategy that eliminated both of these types of mutants, we identified s everal mutants that provide us with evidence that bacterial proteins of the type III secretion system, which are essential for bacterial entry into ho st cells, also play a role in cell-to-cell spread.