Activation of Akt/Protein Kinase B in epithelial cells by the Salmonella typhimurium effector SigD

is a proto-oncogene involved in the of cell and Activation of is initiated by binding to the phospholipid products of phosphoinositide 3-kinase at the inner leaflet of the plasma membranes followed by phosphorylation at Ser473 and Thr308. We have found that Akt is activated by Salmonella enterica serovar Typhimurium in epithelial cells. A bacterial effector protein, SigD, which is translocated into host cells via the specialized type III secretion system is essential for Akt activation. In HeLa cells, wild type S. typhimurium induced translocation of Akt to membrane ruffles, phosphorylation at residues Thr308 and Ser473 and increased kinase activity. In contrast, infection with a SigD deletion mutant did not induce phosphorylation or activity although Akt was translocated to membrane ruffles. Complementation of the SigD deletion strain with a mutant containing a single Cys to Ser mutation (Cys462Ser), did not restore the Akt activation phenotype. This residue has previously been shown to be essential for inositol phosphatase activity of the SigD homologue, SopB. Our data indicate a novel mechanism of Akt activation in which the endogenous cellular pathway does not convert membrane associated Akt into its active form. SigD is also the first bacterial effector to be identified as an activator of Akt. localization of Akt in infected epithelial using typhimurium . Localized translocation of Akt vicinity of both S. typhimurium wild type and D sigD mutant was clearly evident within 2 min of adding bacteria 5A and B). At this time point extensive membrane ruffling, characteristic of Salmonella invasion, is evident where the bacteria are in contact with the host cell (29). Bacterial-induced translocation of Akt is still evident 30 min post-infection, which is similar to the localization pattern induced by cell-cell or cell-matrix contact in epithelial cells (22). These results show that Akt is translocated to membrane ruffles induced by Salmonella and, furthermore, that this translocation is independent of SigD.


Introduction
The serine/threonine kinase Akt (also known as PKBα) is a key regulator of cellular survival which is activated by a variety of extracellular signals including mitogens {for review (1)}. In the current model for Akt activation Akt is first recruited from the cytoplasm to cellular membranes by interaction of its PH (Pleckstrin Homology) domain with the phosphatidylinositol 3-kinase (PI3-K 3 ) lipid products phosphatidylinositol-3,4,5-triphosphate (PIP 3 ) and phosphatidylinositol-3,4-biphosphate {PI(3,4)P 2 } (2). Akt activity is not stimulated by translocation itself, but is dependent upon phosphorylation at two residues (Ser 473 and Thr 308 ).
The facultative intracellular pathogen Salmonella enterica serovar Typhimurium (S. typhimurium) is an important causative agent of food-borne gastroenteritis in humans.
Penetration of the intestinal epithelium and survival within a variety of mammalian cell types is essential for pathogenicity and is dependent on a number of virulence factors. The genes encoding these factors are located within five discrete Salmonella pathogenicity islands (SPIs) on the bacterial chromosome {reviewed in (8)}. Encoded in SPI1 is a type III secretion system (TTSS), which translocates effectors directly into the cytoplasm of host cells. and is required for bacterial invasion of nonphagocytic cells (9). This remarkable process is instigated by interactions between bacterial effectors, components of eukaryotic signaling pathways and the actin cytoskeleton, resulting in membrane ruffling on the cell surface, and subsequent bacterial internalization (10)(11)(12)(13). Two GTPases, Cdc42 and Rac-1, which are key regulators of eukaryotic actin dynamics, are directly targeted by Salmonella. For example, the SPI1 effector protein SopE, when microinjected into eukaryotic cells, catalyzes the exchange of GDP for GTP on Cdc42 and Rac-1 thus activating them, to induce membrane ruffling (11).
Although Salmonella-induced membrane ruffling is growth-factor receptor independent (14), and invasion is not blocked by inhibitors of PI3-K (15), we show that S. typhimurium rapidly activates Akt in epithelial cells. One translocated bacterial effector protein, SigD, was found to be absolutely required for both phosphorylation and activation of Akt. A single point mutation in a conserved inositol phosphatase catalytic domain of SigD (16) abolished both Akt phosphorylation and activation. SigD was not required for Salmonella-induced membrane translocation of Akt, indicating that in this novel pathway the membrane translocation of Akt does not lead to its activation unless an exogenous bacterial factor is present.

Cell culture and bacterial strains
HeLa cells, Salmonella typhimurium 1344 wild type and the SB111/pRI203, A1A1 and E12A2 mutants were as described previously (17,18)

Plasmid construction and generation of S. typhimurium sigD mutant
To construct the sigD mutant (missing amino acids 34-465), we used the primers D1 (5'-GCG   AAT TCT ATC TGT TCA AGC ATG-3)  were confirmed by sequencing. These two PCR products were fused at a SalI site (underlined in D2 and D3) and ligated into pCRTOPO2.1 (Invitrogen). The resulting deletion cassette was released by digestion with SacI and XbaI, ligated into the corresponding sites of the positive selection suicide vector pRE112 (Cm R ) and transformed into SY327 λpir (19). The SL1344 sigD deletion mutant (sigD) was then constructed by allelic exchange as described (20). The resulting plasmid contains part of the sigD ORF from the EcoRV site to the StuI site in sigE.
The corresponding fragment of pMWDE was removed and replaced with the mutated fragment, producing pMWDE*.

Bacterial infection of eukaryotic cells
Bacteria were grown in Luria-Bertani (LB) broth overnight at 37°C with shaking. Overnight cultures were subcultured at a dilution of 1:33 in fresh LB and incubated at 37°C with shaking for a further 3 h. The culture was centrifuged at 1,000 g for 2 min at room temperature and then directly resuspended in phosphate buffered saline (PBS). Invasion was initiated by the addition of these bacteria directly to cultured cells. Cells were then incubated at 37°C in 5% CO 2 for 5 to

Immunofluorescence and confocal microscopy
HeLa cells grown on glass coverslips were transfected with GFP-Akt (22)

Salmonella typhimurium induces phosphorylation of Akt in cultured epithelial cells
The activation state of Akt in infected epithelial cells was initially investigated by determining its phosphorylation state. HeLa cells were serum starved for 3 h and then infected with S.
typhimurium for 2-10 min. After infection, the cells were washed to remove extracellular bacteria and incubated for varying times. Western blots were probed with phospho-specific antibodies recognizing either Thr 308 or Ser 473 phosphorylation of Akt. Phosphorylation on both residues was induced within 10 min of the addition of bacteria and peaked at approximately 60 min ( Figure 1A and B). In comparison, activation of Akt by epidermal growth factor (EGF) produces a much more rapid and short lived phosphorylation response ( Figure 1C).

Akt phosphorylation is PI3-K dependent and requires the SPI1 type III secretion system
To determine whether the observed Akt phosphorylation was PI3-K-dependent we used the integrin receptors (25). Under the conditions employed here, the mutant was internalized at approximately half the efficiency of the wild type bacteria. However, the mutant did not induce Akt phosphorylation even when extremely high inocula (320 cfu/cell) were used ( Figure 2D).
Thus Akt phosphorylation is dependent on one or more effectors secreted by the SPI1 TTSS.

The SPI5 encoded type III effector SigD is required for Akt phosphorylation
To identify effectors involved in Akt phosphorylation we screened several S. typhimurium mutants. These mutants were constructed by the random insertion of a promoterless luciferase gene cassette into the S. typhimurium chromosome, and had been selected in a previous screen for increased intracellular expression (18). One of these mutants, A1A1, was unable to induce Akt phosphorylation in HeLa cells ( Figure 3A). In this mutant, the transposon is inserted into a previously described gene, sigD located in SPI5 the gene product of which, SigD, is translocated  Figure 3A and B).
A non-polar sigD deletion mutant, S. typhimurium sigD, was constructed by removing 430 codons from the sigD open reading frame. Like the A1A1 strain, S. typhimurium sigD did not induce Akt phosphorylation ( Figure 3C). Under the conditions used in this study, the invasion efficiency of the SigD mutants did not differ significantly from the wild type S. typhimurium (not shown). Transcomplementation of the A1A1 and sigD mutants with a plasmid (pMWDE) encoding the sigD and sigE genes under the control of the lacZ promotor, rescued the ability to phosphorylate Akt ("DE" in Figure 3C). The sigE (pipC) gene was included in the pMWDE plasmid as it encodes a putative SigD chaperone which is essential for secretion of the effector (26).

Akt kinase activity is increased in S. typhimurium infected cells in a SigD-dependent manner
Having defined the phosphorylation status of Akt, we next investigated whether the enzymatic activity of Akt was increased by S. typhimurium.. HeLa cells were infected with bacteria and then solubilized in lysis buffer after which total Akt was immunoprecipitated using a polyclonal antibody that recognizes both phosphorylated and nonphosphorylated forms. Immunoprecipitates were incubated with a synthetic peptide (Crosstide), containing the consensus sequence surrounding Ser 9 of GSK-3B, a known target of Akt, and 32 P-γ-ATP. Infection with wild type S. typhimurium for 10 min increased Akt kinase activity by approximately 12-fold compared to uninfected cells. Increasing the infection time to 20 min lead to a further increase (40-fold) in Akt activity (Compare Figures 4A and B). These results correlate well with the phosphorylation kinetics shown in Figure 1. Stimulation of kinase activity was almost completely abolished by the PI3-K inhibitor LY29 and was comparable to the level of stimulation obtained with EGF.
Furthermore, no significant Akt activity was detected when HeLa cells were infected with either Steele-Mortimer et al 11 the SPI1 TTSS mutant (SB111/pRI203) or with the sigD strain. Complementation of the sigD strain with pMWDE restored the bacterial stimulation of Akt activity ( Figure 4A). These results demonstrate that S. typhimurium activates Akt and that this activation is SigD dependent.
SigD, and the S. dublin and Shigella flexneri homologues, SopB and IpgD respectively, all contain two inositol 4 phosphatase domains and a conserved cysteine residue in one of these domains is essential for the activity of mammalian inositol 4-phosphatases as well as in vitro phosphatase activity of SopB (16,27). To investigate whether this residue is also required for SigD-induced Akt phosphorylation/activation, we constructed the equivalent mutant (C462S), designated pMWDE*. When this plasmid was used to complement the sigD strain, no Akt activation was detected ( Figure 4B) and neither Ser 473 or Thr 308 phosphorylation was induced ( Figure 4C). Thus, a motif common to 4-phosphatases is required for Akt phosphorylation and activation by SigD.

PI3-K activation and rapid translocation of Akt to the plasma membrane are independent of SigD
Since LY29 inhibited Akt activation, the association of PI3-K with tyrosine phosphorylated proteins was examined. Such interactions lead to the activation of PI3-K by localization with its substrates at the plasma membrane and through allosteric modifications (28).
Immunoprecipitation with anti-phosphotyrosine antibody showed that the p85 subunit of class 1α-PI3-K was recruited to tyrosine-phosphorylated proteins during infection with either wild type or sigD strains ( Figure 4D). This demonstrates that while Salmonella invasion does activate PI3-K, this activation is independent of SigD. activity and/or substrate specificity especially as translocated SopB/SigD appears to associate with cellular membranes (30). Identification of the substrates of SopB/SigD in vivo will help define the roles of PIP 3 and PI(3,4)P 2 in Akt activation, which to date are unclear (28,(31)(32)(33).
It is well documented that Akt is a general mediator of survival signals and that Akt is both necessary and sufficient for survival of eukaryotic cells {for review see (34)). Thus it is possible that Salmonella-induced activation of Akt in epithelial cells would increase host cell survival, perhaps allowing the pathogen a greater intracellular time frame within which to replicate. Antiapoptotic mechanisms are employed by at least two intracellular bacterial pathogens Chlamydia trachomatis (35) and Rickettsia ricketsii (36), although the bacterial factors mediating these processes have not been identified. In macrophages, Salmonella and other bacterial pathogens including Shigella, specifically induce apoptosis (12,37). The S. typhimurium TTSS effector, SipB, cleaves caspase-1 leading to rapid induction of the apoptotic cascade in macrophages but not in non phagocytic cells (12). This dichotomy may reflect the specific roles played by different cell types in host cell defense, giving those bacteria which can induce apoptosis in macrophages a distinct advantage in the establishment of infection (38).
While SigD does not appear to be required for virulence in a mouse model for typhoid fever (18,26), it is required for both increased fluid secretion and recruitment of subepithelial neutrophils, which correlates with an increase in intracellular Ins(1,3,4,5)P 4 , in a ligated bovine intestinal loop model of diarrhea (16,30,39). The transcription factor NF-κB, which is activated by S. typhimurium, is an important mediator of inflammation in the intestinal epithelium, and also transmits both pro-and anti-apoptotic signals (40)(41)(42). Since Akt can regulate the transcriptional activity of NF-κB (43)(44)(45), SigD may also regulate NF-κB induction in Steele-Mortimer et al 15 epithelial cells.
Although several bacterial effectors have shown to interact with eukaryotic signalling molecules this is the first time that one has been shown to activate the Akt pro-survival pathway.
While the effects of this activation in pathogenesis remain undefined it is certain that, by