While it has been well established that the PI3K/Akt pathway is modulated by many viruses and plays an important role in the establishment of viral infection 
, the appropriation of Akt by pathogenic bacteria is less well understood 
, and other intracellular bacteria 
, use Akt activation to block or delay apoptosis in infected cells. Given the diverse cellular roles of Akt, it is likely to have additional functions during bacterial infection.
In this study, we first showed that the Salmonella
effector protein SopB is necessary and sufficient for Akt phosphorylation in HeLa cells. To gain a better understanding of the role of Akt in Salmonella
pathogenesis we then compared SopB-mediated Akt activation with the canonical EGF signaling pathway common to all epithelial cells. Using different approaches we assessed the two essential steps in Akt activation i.e. membrane translocation and phosphorylation. The most striking difference that our study revealed is that the irreversible PI3K inhibitor wortmannin is unable to inhibit either of these steps in Salmonella
-infected HeLa cells. An obvious interpretation of this is that SopB-dependent Akt activation is independent of class I PI3K, supported by the finding that depletion of the p85 regulatory subunit of class I PI3K had no effect on this pathway. Surprisingly, the more specific PI3K inhibitor LY294002 did inhibit both membrane translocation and phosphorylation of Akt in Salmonella
infected cells. However, LY294002 does have other intracellular targets 
, including: casein kinase-2, GSK3α and GSK3ß, as well as p97/VCP, a member of the type II AAA ATPase family 
. Several other potential targets, DNA-PK, PI4K and mTOR, can be excluded since they are equally sensitive to wortmannin 
. We also found that SopB-dependent Akt phosphorylation was less sensitive than EGF-induced phosphorylation to two small molecule inhibitors of AKT. SH-6 is a phosphatidylinositol analog that competes with PI3K for PtdIns(4,5)P2 
whereas TCN is a cell-permeable tricyclic nucleoside that inhibits Akt phosphorylation 
. One possibility is that the SopB pathway engages a mammalian PI3K other than the canonical class I PI3K, although this is unlikely since WTM does not show significant isoform specificity. A final alternative is PI3K-independent activation of Akt. This is not without precedent since both cAMP/protein kinase A and dopamine have been shown to elicit wortmannin-insensitive Akt activation 
. Despite the above differences between the SopB-mediated and EGF-mediated pathways of Akt activation our data suggest that the Akt kinases, PDK1 and mTORC2, are essential components in both cases.
To get a better understanding of the role of SopB in recruitment of signaling components we also investigated recruitment of proteins and phosphoinoside specific PH domains to membrane ruffles. This semi-quantitative method revealed that Akt enrichment is SopB dependent, whereas in a previous study where enrichment was simply assessed visually, we could not detect any requirement for SopB 
. Furthermore, the PH domain translocation experiments indicated that SopB induces a localized increase in PtdIns(3,4)P2
rather than PtdIns(3,4,5)P2
-induced ruffles. This suggests that Akt phosphorylation in the Salmonella
-induced ruffle is dependent on PtdIns(3,4)P2
rather than PtdIns(3,4,5)P2
. Further studies are required to determine the roles of these phosphoinositides in SopB-dependent Akt activation. Interestingly, studies on the S. flexneri
effector protein IpgD, a homolog of SopB, have shown that sustained Akt phosphorylation is mediated by IpgD-dependent generation of PtdIns(5)P 
and indeed SopB causes localized conversion of PI(4,5)P2
to PI(5)P in regions of Salmonella
-induced plasma membrane ruffles 
. One possible effect of increased PtdIns(5)P is to prevent the dephosphorylation of Akt by inhibiting the catalytic subunit of PP2A phosphatases 
. However, these studies also found that PI3K played an essential role in IpgD-dependent Akt-phosphorylation. Unfortunately, PtdIns(5)P is a rare phosphoinositide 
, making it very difficult to detect and it remains poorly understood.
In conclusion, we have shown that Salmonella induces Akt activation via a wortmannin insensitive mechanism that probably involves a novel class I PI3K-independent pathway. Why Salmonella have not simply tuned into the canonical pathway is unclear, but one possibility is that it could allow the targeting of different downstream proteins. The molecular mechanisms involved in this process remain unknown, however, the work presented here provides a foundation for future experiments that should lead to a better understanding of bacterial pathogenesis as well as the multi-faceted essential kinase Akt.