Using such an inducible complementation system, deletion of the T3SS translocases as well as structural components of the T3SS, or transcriptional regulators of the T3SS, has been shown by electron microscopy to lead to an accumulation of bacteria in secondary vacuoles. These results have established that the T3SS is not only necessary for invasion of the primary infected cell and escape of the primary vacuole, but is also involved in escape of S. flexneri from the secondary vacuole. During host cell invasion, pre-translated effectors are secreted upon entry when the T3SS tip complex composed of the effector proteins IpaB and IpaD senses the plasma membrane leading first to secretion of the pore-forming translocases IpaB and IpaC, followed by other early effectors. Once IpaB and IpaC are secreted, their freed chaperone, IpgC, acts as a co-activator of transcription with MxiE, which leads to expression of a second subset of effectors with promoters that contain a MxiE-box motif. MxiE transcriptional regulation is further de-repressed by the secretion of OspD1, which inhibits MxiE activation until OspD1 is secreted with the first round of pre-translated effectors upon T3SS activation. The tight control of MxiE activation and MxiE-regulated gene expression make MxiE-regulated genes a good reporter system for activation of the S. flexneri T3SS.

Using lacZ-fusions to MxiE-regulated genes virA and ipaH7.8, and using a GFP-fusion to ipaH7.8, Demers et al. and CampbellValois et al. respectively demonstrated an BAY-60-7550 initial increase in MxiEregulated gene expression in cytosolic S. flexneri followed by a decrease in gene expression. The work by Campbell-Valois et al. proceeded further to demonstrate the re-initiation of the S. flexneri T3SS by detecting ipaH7.8-GFP expressing bacteria in protrusions prior to secondary vacuole formation. Much of the previous work studying defects in S. flexneri dissemination as an indicator of virulence has been done in the non-intestinal HeLa cell line. HeLa cells allow for S. flexneri invasion, cytosolic motility, and protrusion formation, but have a very low frequency of protrusion resolution into secondary vacuoles as compared to the colonic epithelial cell line HT-29. This low frequency of resolution is partly due to a defect in tyrosine kinase signaling in protrusions, essential to vacuole formation in HT-29 cells. In this work, we compared the cell-to-cell spread of wild-type 2457T S. flexneri serotype 2a in HT-29 cell monolayers to the cell-to-cell spread of a DmxiG mutant, which lacks a central component of the T3SS needle apparatus. By tracking the dissemination of individual bacteria, we uncovered multiple roles for the T3SS in the cell-to-cell spread of S. flexneri in intestinal cells, including the escape from secondary vacuoles, as previously reported in non-intestinal cells, and the tyrosine kinase signalingdependent resolution of protrusions into vacuoles.

The human pathogen S. flexneri displays the ability to invade epithelial cells of the human colon, followed by replication in the cytosolic compartment and dissemination to adjacent cells. Numerous studies conducted in various intestinal as well as nonintestinal cell lines have demonstrated an essential role for the T3SS in the invasive properties of S. flexneri.