To hydrolyze PI-4,5-P2, there are three main ways found in mammalian cells, via the action of 5’-phosphatase to remove phosphate from the 5’-position, or through the kinase activity of phosphoinositide 3-kinase(PI3K) to phosphorylate at 3’-position, or by phospholipase C to generate two important second messengers, diacyl-glycerol (DAG)and inositol trisphosphate(IP3), as shown in Fig.3.4. In this study, we focused on the effect of phosphatase-mediated hydrolysis of PI-4,5-P2 on S.aureus uptake.
Similar to the comprehension of the roles of phosphoinositide kinases in bacterial uptake, the current understanding of the implication of phosphatases in bacterial internalization is also not satisfactory.
In the case of Yersinia pseudotuberculosis infection, the bacteria entry into host cells is mediated by the engagement of integrin β1 with bacteria outer membrane protein YadA.
During the invasion, Yersinia pseudotuberculosis first reside in the so called
“prevacuoles”, which are PI-4,5-P2-rich and membrane-bound structures. Later, these
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prevacuoles are separated from the plasma membrane, which finally results in the accomplishment of bacterial invasion. To achieve the successful membrane scission, PI-4,5-P2 hydrolysis is required, and this hydrolysis is carried out by the recruited inositol 5-phosphatases OCRL and INPP5B (Sarantis, Helen, et al. 2012). In the same year, while investigating the host cell invasion by Listeria monocytogenes, which is mediated through the interaction of the bacterial surface protein InlB with the cellular receptor Met, Andreas Kühbacher and colleagues discovered that OCRL is recruited to the L. monocytogenes invasion foci (Kühbacher, Andreas, et al. 2012). The siRNA mediated knockdown of OCRL increases the bacterial internalization, but surprisingly decreases the surface expression of the receptor Met (Kühbacher, Andreas, et al.2012).
These findings highlight the importance of inositol 5-phosphatases mediated PI-4,5-P2
hydrolysis in bacterial invasion and make the functions of PI-4,5-P2 in bacterial uptake more comprehensive; however, the detailed mechanisms are still missing.
Fig.3.4 The main hydrolyzing ways of PI-4,5-P2 in mammalian cells. Cleavage of PI-4,5-P2 by phospholipase C (PLC) produces diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3),both act as second messengers.IP3 stimulates the release of calcium ions from the smooth endoplasmic reticulum, while DAG activates protein kinase C (PKC). PI-4,5-P2 also can be dephosphorylated by 5-phosphatses to generate PI-4-P or be phosphorylated by PI3K to generate PI-3,4,5-P3.
Beyond the host cell phosphatases that are required during bacterial invasion, it has also been reported that some pathogens inject bacterial effectors acting as phosphoinositide phosphatase into host cells to promote bacteria internalization. For instance, Salmonella enterica, the main causative agent of food-borne gastroenteritis, expresses inositol phosphatase SopB (also known as SigD) and delivers it into host cells by the type III
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secretion system. Inside of host cells, SigD promotes the disappearance of PI-4,5-P2, which in turn reduces the rigidity of the membrane skeleton and induces plasma membrane invagination and fission (Terebiznik, Mauricio R., et al. 2002). Recently, the role of SopB in Salmonella invasion was further studied. SopB was found to simultaneously contain the inositol 4-phosphatase and 5-phosphatase activities.
Consequently, in SopB-deficient strain, neither the expression of 4-phosphatase nor the expression of 5-phosphatase is sufficient to rescue the impaired bacterial invasion, which indicates both of these phosphatase activities are required to induce membrane ruffling and promote bacterial invasion (Piscatelli, Heather L., 2015). In a similar way, Shigella flexneri, a facultative intracellular pathogen that causes bacillary dysentery in humans, use type III secretion system to translocate IpgD directly into eukaryotic cells.
Inside of the host cells, IpgD acts as a potent inositol 4-phosphatase and specifically dephosphorylates PI-4,5-P2 into PI5P. This lipids conversion triggers the dramatic morphological changes of host cell, leading to a decrease in membrane tether force that is associated with membrane blebbing and actin filament remodeling, which subsequently contribute the bacterial invasion (Niebuhr, Kirsten, et al. 2002).
As reported before, SYNJ1 is a multi-domain protein and has two main splice variants, named SYNJ1-145 and SYNJ1-170 based on their molecular sizes (Perera, Rushika M., et al. PNAS, 2006). In clathrin-mediated endocytosis, through its C-terminal region, SYNJ1-145 associates with the BAR domain containing protein endophilin. Via this interaction, SYNJ1-145 is recruited to the curved membranes and locally removes PI-4,5-P2. The elimination of PI-4,5-P2 facilitates membrane fission, which results in the final endocytosis (Chang-Ileto, Belle, et al. 2011). It is also found during clathrin mediated endocytosis, the two main splice variants, SYNJ1-145 and SYNJ1-170, are recruited to the clathrin coated pits (CCPs) in temporally different ways (Perera, Rushika M., et al. 2006). In detail, SYNJ1-145 is observed to be rapidly recruited at the late stage of CCP formation in an endophilin-dependent way, while SYNJ1-170 is present at all stages of CCP formation. Together, these observations indicate the contribution of SYNJ1 to CCP fission and uncoating (Perera, Rushika M., et al. 2006).
It is known that via the C-terminal part, SYNJ1-170 interacts with clathrin, endophilin,
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Ap2, and Eps15, indicating that SYNJ1-170 plays a role in endocytosis. In this study, the recruitment of SYNJ1-170 to the internalized S.aureus, as well as the phosphatase dead mutant (D730A), was observed. With the expression of myr motif tagged phosphoinositide phosphatase domain (from human INPP5J) in HEK 293T cells, the uptake of S.aureus was significantly decreased, while the cell-associated bacteria was not altered. In accordance with this result, S.aureus invasion is dramatically enhanced in SYNJ1-170 genetically depleted NIH 3T3 cells (see Fig.2.12 in Results section).
This observation was further confirmed by a flow cytometry-based bacterial invasion assay (Pils, Stefan, et al. 2006). In line with the 5-phosphatase activity of SYNJ1-170, the level of recruited PI-4,5-P2 around the internalized bacteria was obviously elevated in SYNJ1-170 KO cells (Fig.2.13in Results section), which pointed to the consequence of phosphatase deficiency at internalized vesicles containing bacteria. However, the surface level of integrins, especially integrin α5, is decreased in SYNJ1-170 KO cells, compared to the control KO cells. This seems to be contradictory to the increased bacterial internalization which is integrin dependent.
In addition, the FAK phosphorylation level, which is critical for S.aureus uptake, is not obviously changed in SYNJ1-170 KO cells, demonstrating that the synthesis, but not the hydrolysis of PI-4,5-P2, is essential for FAK activation, which may reflect the order of signaling pathway. Again, even the role of talin in S.aureus uptake is not investigated, talin recruitment around S.aureus in SYNJ1-170KO cells is enhanced, compared to the control cells.
One plausible explanations for the seemingly contradictory results between integrin surface level and bacteria uptake in SYNJ1 KO cells, could be the accelerated integrin recycling. Sorting nexins (SNX), the proteins that preferentially reside on early endosomes, are involved in diverse sorting processes which determine the fate of membrane protein during endocytosis, including integrins(Steinberg, Florian, et al.
2012; Böttcher, Ralph Thomas, et al. 2012; Tseng, Hui-Yuan, et al. 2014.). Interestingly, these SNX can bind to phosphoinositides, like PI3P, PI-4,5-P2 and PI-3,4,5-P3, via the PX domain or FERM domain (Worby, C. A., and J. E. Dixon. 2002; Cullen, Peter J.
2008). Ralph Thomas Böttcher et al. proved that SNX17 binds to the cytoplasmic tail
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of β1-integrin in early endosomes, to prevent β1-integrin degradation, resulting in their recycling to the cell surface (Böttcher, Ralph Thomas, et al. 2012). Interestingly, SNX17 contains the FERM domain which is supposed to bind to PI-4,5-P2, even if the evidence for direct interaction between SNX17 and PI-4,5-P2 is still missing. Similar effect was found with SNX31, which binds several β integrin tails in early endosomes and also has FERM domain (Tseng, Hui-Yuan, et al. 2014). Together, these results can give us some hints to interpret the decreased surface level of integrins in SYNJ1 KO cells.