Infection of Epithelial Cells With Salmonella enterica - Bacterial Pathogenesis: Methods and Protocols - page 191

or SCV (3) . In contrast, T3SS2 effectors are translocated across the vacuole
membrane and mediate SCV biogenesis and intracellular survival (4) . Recent
evidence suggests that there is overlap in the temporal expression and function
of T3SS1 and T3SS2 although this remains largely uncharacterized (5,6,7,8) .
The T3SS1 translocon or injectosome, as well as the regulatory components and
some effector proteins are encoded on Salmonella pathogenicity island 1 (SPI1).
Significantly, other T3SS1 effectors, encoded elsewhere on the chromosome,
are also included in the SPI1 regulon (9,10) . As invasion is mediated by
T3SS1, the SPI1 regulon must be induced extracellularly; however, the cues
involved in this process remain incompletely characterized. We have found that
SPI1 induction is strongly growth phase dependent such that bacteria grown to
late log/early stationary phase are “hyper-invasive” ( see Fig. 1 ). Therefore, to
synchronize invasion of cultured epithelial cells, we use SPI1-induced bacteria
at a high multiplicity of invasion (MOI) for a short infection time (2-10 min)
followed by removal of the extracellular bacteria. Gentamicin is routinely
added to kill any remaining extracellular bacteria, as this antibiotic does not
cross the plasma membrane and should not kill intracellular bacteria. However,
gentamicin can be taken up by fluid phase endocytosis and could be delivered
to the SCV; adding antibiotic 15-20 min after removing the inoculum should
minimize the chance of killing intracellular bacteria.
Fig. 1. Salmonella enterica serovar Typhimurium invasiveness is growth phase
dependent. Salmonella grown to late log/early stationary phase (dotted line) are “hyper-
invasive” (solid line). The gray bar illustrates the subculture time required to obtain
“hyper-invasive bacteria.