The spread of intracellular pathogens between host cells involves three steps: the formation of pathogen-containing protrusions from the donor cell, engulfment of these protrusions by the recipient cells and, finally, vacuolar escape and entry of the pathogens into the recipient cells. To identify differences in these steps between L. monocytogenes and R. parkeri, the authors used live imaging of bacteria that expressed fluorescent markers in host cell monolayers. R. parkeri formed shorter protrusions but they were resolved and engulfed faster than protrusions formed by L. monocytogenes. Interestingly, whereas R. parkeri had actin tails in the cytosol of host cells, it lost these tails when it neared the membrane and entered protrusions, which suggests that it uses a different mechanism from the one used by L. monocytogenes to spread.
To identify bacterial factors that are involved in spread, the authors used transposon mutagenesis and tested the ability of the mutants to form infectious plaques. One such mutant had a transposon insertion in Sca4 that caused the production of a weakly expressed, truncated version of the protein. Indeed, this mutant generated smaller infectious foci, while retaining normal growth kinetics, cell invasion and cytosolic actin-based motility, which confirmed that Sca4 is important for intercellular spread but does not affect any other steps that might decrease plaque formation. Importantly, the mutant showed substantially delayed resolution of protrusions in live-imaging experiments. This delay and the defect in spread could be rescued when Sca4 was expressed on a plasmid in the mutant.
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