Background

Shigella infects about 160 million people leading to about 600,000 deaths every year (Reference 8). The clinical manifestations of Shigella infection, or shigellosis, arise only after the bacterium enters the epithelium, where it multiplies and spreads to adjacent cells causing cell death and tissue necrosis. Thus, the entry into the epithelial cells is a critical step in the infectious life cycle of Shigella (Ashida et al., 2015). Most of the determinants mediating this step have been ascribed to a large virulence plasmid that encodes a T3SS responsible for injecting bacterial proteins into the host cell via a needle-like projection (Puhar and Sansonetti, 2014).

The gentamicin protection assay is a classical method that is used to assess the invasiveness of Shigella and has led to the identification of a number of mediators of invasion by mutational analysis and comparisons with the wild-type bacteria. In a typical experiment, the bacteria are allowed to infect the intestinal cells in a synchronized manner followed by removal of external bacteria by gentamicin treatment. The invasiveness is assessed by determining the number of surviving bacteria (protected from gentamicin being intracellular) from the lysates of infected cells. A lower number of surviving bacteria with respect to the wild-type indicates a defect in invasion or intracellular survival. If short infection times are used after the gentamicin treatment (typically 1h), the gentamicin protection assay allows to compare the bacterial capacity to enter host cells only. However, if longer infection times are used after gentamicin treatment (2 h or more), the assay will also reflect the bacterial capacity to survive and multiply within the target cell besides the capacity to invade. Hence, if the gentamicin protection assay is employed to study the capacity to survive and multiply by allowing long infection times, these experiments should always be complemented with tests carried out at short infection times to ensure correct interpretation of the results. The gentamicin protection assay, however, is not suitable to assess the extent of intercellular spread of bacteria throughout the monolayer, which is conveniently determined by a plaque formation assay. Although the invasiveness of bacterial pathogens can also be studied by fluorescence microscopy-based techniques such as immunofluorescence and FACS; it requires the use of costly reagents like labeled probes, special reporter strains and sophisticated equipment. The gentamicin protection assay is not only relatively easy to perform and cheap, it is fairly rapid and less labor intensive making it amenable to higher throughput.

The protocol presented here reprises the classical gentamicin protection assay, which has been optimized for use with Shigella flexneri and TC7 intestinal epithelial cells but can be modified to be used with other intracellular pathogens and other host cell types.