To confirm the contribution of PIP5KIγ90 to integrin-mediated internalization of S.aureus, PIP5KIγ90-/- cells were transiently transfected with GFP-tagged PIP5KIγ90 or the inactive version of PIP5KIγ90 (PIP5KIγ90 D253A). As a negative control, PIP5KIγ90-/- cells were transiently transfected with GFP alone. The expression of GFP and fusion proteins was measured by western blot (data not shown). Forty-eight hours after transfection, cells were infected with S.aureus for 2h and fixed samples were differentially stained for extra- and intracellular bacteria (Fig. 2.7 A). Enumeration of bacteria in these transfected cells by microscopy demonstrated that the number of cell-associated bacteria was similar in all the samples (Fig. 2.7 B). However, the internalization of S.aureus by PIP5KIγ90-/- cells was clearly diminished compared to wild-type cells (Fig. 2.7 B). Importantly, the uptake of S.aureus was rescued in PIP5KIγ90-/- cells expressing the wild-type PIP5KIγ90 enzyme, whereas re-expression of the kinase-inactive enzyme did not result in increased numbers of
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intracellular S.aureus (Fig. 2.7 A and B). These results demonstrate that both the talin-binding capacity and the enzyme activity of PIP5KIγ90 are important for the integrin-mediated uptake of S.aureus.
Fig.2.7. Re-expression of active PIP5KIγ90 in PIP5KIγ90-/- fibroblasts rescues bacterial uptake (A) PIP5KIγ90-/- cells were transfected with plasmids encoding GFP, GFP-PIP5KIγ90, or a kinase inactive mutant of PIP5KIγ90 (PIP5KIγ90 D253A), respectively. PIP5KIγ90fl/fl cells were transfected with GFP, then infected with Pacific-blue stained and biotin-labeled S.aureus for 2h. After fixation, extracellular bacteria were further stained with streptavidin-AlexaFluor647. Arrows point to intracellular bacteria stained in blue only. Bars, 10 µm. (B) Experiments were performed as in (A) and the number of total cell associated (upper panel) or intracellular bacteria per cell (lower panel) was quantified (n = 3 samples; at least 30 cells/sample). Bars show the mean values from three independent experiments performed in triplicate. Significance was evaluated by student’s t-test. *** p<0.001; ** p<0.01; * p<0.05.
2.8 PIP5KIγ90 is critical for maximal FAK activity in response to integrin stimulation
Several signaling processes, including activation of the protein tyrosine kinase FAK as
A B
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well as phosphorylation of FAK binding partners, such as paxillin or cortactin, occur upon integrin engagement by S.aureus (Agerer et al., 2005). To monitor such downstream signaling events, PIP5KIγ90fl/fl or PIP5KIγ90-/- cells were either infected with S.aureus for different time periods or left uninfected. Western blot analysis of whole cell lysates using phospho-tyrosine-specific antibodies revealed that overall tyrosine phosphorylation was reduced in uninfected PIP5KIγ90-/- compared to the PIP5KIγ90 expressing cells (Fig. 2.8 A). Moreover, PIP5KIγ90 expressing cells showed a strong increase in tyrosine phosphorylation, and in particular in tyrosine phosporylation of FAK at the auto-phosphorylation site Y397 during the course of 2h upon S.aureus infection (Fig. 2.8 A and B). The increase in tyrosine phosphorylation was completely absent in cells lacking PIP5KIγ90 (PIP5KIγ90-/- cells) (Fig.2.8 A and B). These results indicate that maximal activation of tyrosine phosphorylation of FAK downstream of integrin engagement by S.aureus requires PIP5KIγ90 activity. To investigate, if FAK Y397 phosphorylation depends on PIP5KIγ90 activity during physiological stimulation of integrins during cell adhesion, PIP5KIγ90fl/fl or PIP5KIγ90-/- cells were plated on dishes coated with fibronectin or were kept in suspension. Clearly, cell adhesion to fibronectin stimulated a strong increase in tyrosine phosphorylation in both, PIP5KIγ90fl/fl and PIP5KIγ90-/- cells (Fig.2.8 C). Nevertheless, overall tyrosine phosphorylation was slightly elevated in PIP5KIγ90fl/fl compared to PIP5KIγ90-/- cells and in particular FAK Y397 phosphorylation, as an indicator of FAK activity, was more pronounced in wild-type cells within 90 minutes of adhesion to fibronectin (Fig. 2.8 C). These findings suggest that the local generation of PI-4,5-P2
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by PIP5KIγ90 is critical for maximal activation of FAK both in response to fibronectin-binding bacteria as well as during cell adhesion to a fibronectin matrix(Fig. 2.8 D).
Therefore, the reduced activation of FAK in the absence of the PIP5KIγ90 isoform can help to explain the impairment in the integrin-mediated internalisation of S.aureus.
Fig.2.8. PIP5KIγ90 is critical for maximal FAK activation in response to integrin stimulation. (A) PIP5KIγ90fl/fl and PIP5KIγ90-/- cells were serum starved overnight, then incubated in suspension medium for 45 min. 1.5 x 106 cells were seeded on poly-L-lysine coated 6-well plates and incubated for 1h. Then the cells were infected with fibronectin-coated S.aureus at MOI 100. At different time points, cells were lysed and tyrosine phosphorylation was monitored in whole cell lysates (WCL) by Western blot with phospho-tyrosine antibody, a phospho-specific antibody recognizing the phosphoY397 (pY397) form of FAK, a polyclonal rabbit anti-FAK antibody recognizing total FAK, a monoclonal antibody against
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C D
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vinculin, or a monoclonal antibody against tubulin as indicated. (B) Signals obtained from two independent blot experiments as described in (A) were analyzed by densitometry and the intensity ratio at different time points is plotted over the observed time frame. (C) Cells were serum-starved overnight and incubated in suspension medium as in (A), before seeding on fibronectin (15 µg/ml) coated 6-cm dishes (1.5 x 106 cells /dish) for the indicated periods. Furthermore, some cells were kept in suspension.
Whole cell lysates (WCL) prepared from adherent cells and suspended cells were analyzed by Western blotting with phospho-tyrosine antibody, a phospho-specific antibody recognizing the phosphoY397 (pY397) form of FAK, a polyclonal rabbit anti-FAK antibody recognizing total FAK, or a monoclonal antibody against tubulin as indicated. (D) Schematic summary of the role of PIP5KIγ90 in S.aureus internalization. The binding of PIP5KIγ90 to talin targets PIP5KIγ90 to the focal adhesion like complex, where it locally synthesizes PI-4,5-P2. The locally produced PI-4,5-P2 subsequently facilitates the full activation of FAK and promotes the final uptake of bacteria.
Collectively, current data have revealed that the local synthesis of PI-4,5-P2 via the action of PIP5KIγ90, which locates in focal adhesion like complexes through the interaction with talin, is crucial for S.aureus internalization. Since PI-4,5-P2 also can be hydrolyzed by phosphoinositide phosphatases, it is worth to address the effect of phosphatase during bacterial uptake. We already observed the expression of myr-tagged, an active 5’ directed phosphoinositide phosphatase substantially decreases S.aureus uptake, but which phosphatase is the main regulator during bacteria uptake is not known. In mammalian cells, there are more than 10 different phosphatases, and the function of most of these phosphatases is poorly understood. Fron now on, we started to further address the roles of PI-4,5-P2 via the manipulation of phosphoinositide phosphatases in cells.
2.9 SYNJ1 is the major phosphoinositide phosphatase to regulate S.aureus uptake To further understand, which cellular phosphoinositide phosphatase mainly regulates integrin-mediated bacterial uptake, we designed shRNAs targeting individual phosphoinositide phosphatases to generate corresponding knockdown cells. These
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stable knock-down cells were then used to carry out gentamicin protection assays upon infection with S.aureus. In the phosphatase knockdown cells, the number of cell associated bacteria was not altered, but the amount of internalized bacteria was consistently increased (Fig.2.9 A and B), even if the depletion of the different phosphatases had varying influence on bacterial uptake. Especially, in INPP5B, INPP5K, SYNJ-1, SYNJ-2, Fig4 and SHIP1 knockdown cells, bacteria internalization was increased, compared to the other phosphatase knockdown or control cells. Then bacteria uptake in INPP5B, INPP5K, SYNJ-1, SYNJ-2, Fig4 and SHIP1 knockdown cells was further measured via FACS-based internalization assay (Fig.2.9 C). Analysis of the mean fluorescence intensity in these cells revealed that depletion of SYNJ1 had the strongest and most consistent influence on bacterial uptake compared to depletion of the other candidate phosphatases, therefore SYNJ1 was selected for detailed further studies.
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Fig.2.9. SYNJ1 is the major phosphoinositide phosphatase to regulate S.aureus uptake. (A and B) HEK 293T cells with their individual phosphatase knockdown were infected for 2h with S.aureus. Total cell-associated and intracellular bacteria were quantified by gentamicin protection assays. (A) The number of invasive bacteria in phosphatase knockdown cells was normalized by the number in control knockdown cells, and the ratio is presented (%). (B) The number of invasive bacteria was divided by the number of cell associated bacteria to generate the uptake index, then the individual uptake index in phosphatase knockdown cells was normalized by the uptake index in control knockdown cells, the ratio is presented (%). (C) The mean fluorescence intensity in phosphatase knockdown cells, which was measured by flow cytometry, was normalized by the intensity gained from control knockdown cells.