What are the signals being sensed during systemic infection and how are they integrated to express virulence factors appropriately? 
There have been several studies to identify environmental factors that regulate expression of the type III secretion system encoded within SPI-2.
Carbon limitation, low concentrations of Mg2+ or Ca2+ [73], and acidic pH [47],[61] induce SPI-2 expression in vitro.
Inside professional phagocytic cells divalent cation concentrations and the presence of defensin-like molecules signal through phoP/phoQ [74],[75], and acidic pH and osmolarity signal through ompR/envZ [76].
OmpR has been shown to respond to acidic pH via cadC [77].
The signal(s) received by SsrA, the sensor of the SsrA/SsrB system, is not yet established.
It is surprising that over-expression of ssrB can compensate for a deletion of ssrA or ssrA/ssrB.
Previous results have shown that a conservative replacement of the amino acid that is the essential phosphate acceptor eliminated expression of SPI-2 genes suggesting that SsrB requires phosphorylation for activity.
Yet, expression of ssrB without ssrA results in expression of all 7 SPI-2 genes examined (Figure 7A).
It is possible that SsrB can be phosphorylated from other sources as noted by Walthers et al. [52] or that the over-expression results in dimerization and self activation similar to what has been observed for PhoP [78].
Mutations in rpoE, smpB, rpoS, and hfq showed only small decreases in SPI-2 gene transcription during growth in minimal acidic media but some of them showed large defects in survival assays performed in murine macrophages.
For rpoE and rpoS it is possible that the environmental conditions that distinguish growth in minimal media from those in the SCV inside hosts are sensed via these two alternative sigma factors.
Figure 6B shows that there is a close relationship between these two regulators and that they can both affect each other through the ompR/envZ two-component regulator.
Post-transcriptional SPI-2 regulation is a likely explanation for the large intracellular growth defects observed in the mutant smpB and hfq derivatives despite relative small transcriptional effects.
Strains containing mutations in spvR and hnr showed comparable survival levels to wild-type in macrophages from BALB/c mice despite the fact that they are attenuated in the same mouse strain.
Perhaps these regulators are selected during growth in other cell types or within phagocytic cells that are activated as a consequence of the inflammatory response generated by the bacteria.
