Re-expression of SYNJ1 in SYNJ1 KO fibroblasts rescues uptake of bacteria

integrin-mediated uptake of S.aureus, SYNJ1 KO cells were transiently transfected with vectors encoding GFP-tagged wild type SYNJ1 or the phosphatase dead version of SYNJ1 (D730A). Meanwhile, control KO cells and SYNJ1 KO cells were transiently transfected with GFP alone. Forty-eight hours after transfection, cells were infected with pacific blue stained and biotin labelled S.aureus for 2h and then fixed samples were differentially stained for extra- and intracellular bacteria (Fig. 2.17). Enumeration of bacteria in these transfected cells by microscopy demonstrated that the number of cell-associated bacteria was similar in all the samples (Fig. 2.17). Consistent with prior observations, the internalization of S.aureus in SYNJ1 KO cells was clearly increased compared to control cells (Fig. 2.17). More importantly, the uptake of S.aureus was decreased in SYNJ1 KO cells re-expressing wild-type SYNJ1, demonstrating that recue of SYNJ1 expression establishes the wild-type phenotype. In contrast, the re-expression of the phosphatase dead mutant （D730A）did not result in a significant decrease in bacterial internalization (Fig. 2.17). These results demonstrate that the enzyme activity of SYNJ1 is important to interfere with the integrin mediated uptake of S.aureus.

Results

Fig.2.17. Re-expression of active SYNJ1 in SYNJ1 KO cells reduced bacterial uptake. SYNJ1 KO cells were transfected with plasmids encoding GFP, GFP-SYNJ1, or a phosphatase dead mutant of SYNJ1 (SYNJ1 D730A), respectively, control KO cells were transfected with GFP as control. Forty eight hours after transfection, cells were infected with pacific-blue stained and biotin-labeled S.aureus for 2h. After fixation, extracellular bacteria were stained with streptavidin-AlexaFluor647. The numbers of total cell associated (left panel) or intracellular bacteria (right panel) per cell were quantified (n = 3 samples; at least 30 cells/sample). Bars show the mean values from three independent experiments done in triplicate. Significance was evaluated by student’s t-test. *** p<0.001; ** p<0.01.

Cell-associated bacteria Invasive bacteria

General Discussion

General discussion 3.1 S.aureus uptake

Staphylococcus aureus, the gram positive bacterium, which normally acts as a bacterial commensal of the human nostrils and skin, can cause a variety of diseases ranging from superficial skin infections to life- threatening infections (Lowy, Franklin D. 1998).

Nowadays, due to the ever-increasing prevalence of antibiotic resistance, the therapeutic control of S.aureus is becoming more and more difficult (Lowy, Franklin D.

2003; Chambers, Henry F., 2009). Furthermore, the frequent recurrence of staphylococcal infections, even after the patients are treated with antibiotics and surgical intervention, demonstrates that the prior disease does not elicit protection against subsequent S.aureus infection (Thammavongsa, Vilasack, et al. 2015). All of these features make it urgent to investigate the mechanisms of host-microbe interactions in detail and find more efficient strategies to prevent and treat the staphylococcal diseases. In this study, we concentrated on the roles of PI-4,5-P2 in the infected host cells and the enzymes responsible for its synthesis and hydrolysis during the integrin-mediated internalization of S.aureus, trying to find out how this tiny phospholipid displays a giant impact on bacteria internalization.

Although for a long time primarily considered to be an extracellular pathogen, recent evidence suggests that S.aureus has the capacity to function as an intracellular microbe (Lowy, Franklin D. 2000; Fraunholz, Martin, 2012). It was already reported more than two decades ago that S.aureus does invade a variety of non-professional phagocytic cells, like fibroblasts (Fowler, Trent, et al. 2000; Agerer, Franziska, et al. 2005), osteoblasts (Hudson, Michael C., et al. 1995; Bost, Kenneth L., et al. 1999), epithelial cells (Deitch, Edwin A., et al. 1995; Bayles, Kenneth W., et al. 1998) and endothelial cells (Menzies, Barbara E., 1998; Massey, Ruth C., et al. 2001). Through the invasion into host cells, S.aureus can evade the clearance by immune defenses and can be protected from extracellular antibiotics. However, the detailed pathways by which bacteria invade these cells are still not known.

In the last two decades, there are a number of excellent published articles, which analysed the signaling pathways underlying the invasion of host cells by S.aureus

General Discussion

(Hauck, Christof R., 2006; 2012; Hirschhausen, Nina, et al. 2010; Alva-Murillo, Nayeli, 2014) (see Fig.3.1). One novel mechanism discovered recently is autolysin (Atl)/adhesins-mediated. Bacterial surface associated Atl directly binds to the heat shock cognate protein Hsc70 on host cell surface, and mediates S.aureus internalization (Hirschhausen, Nina, et al. 2010). The other adhesins involved in S.aureus internalization are: (1) Fibronectin binding proteins (FnBPs), which can directly interact with portion of host heat shock protein 60 (Hsp60) which is exposed on the cytoplasmic membrane of epithelial cells or bind to Fn and Hsp60 at the same time (Dziewanowska, Katarzyna, et al. 2000); (2) Iron-regulated surface determinant-B (IsdB) that is able to bind to integrins and promote the bacterial adhesion and invasion of host cells (Zapotoczna, Marta, et al. 2013), and (3) extracellular adherence protein (Eap) as a broad-spectrum adhesin contributing to internalization of S.aureus by binding to a number of host matrix and plasma proteins, such as Fn, fibrinogen, prothrombin, vitronectin, thrombospondin-1 and collagen (Haggar, Axana, et al. 2003;

Bur, Stephanie, et al. 2013). However, the main and classic pathway is considered to be integrin-mediated. S.aureus associates with the type I repeats of Fn through cell wall anchored FnBPs. Via this interaction, Fn is deposited on the pathogen surface and recognized by the cellular Fn receptor, in particular, integrin α5β1, which binds to the RGD motif of Fn. Bacteria-induced clustering of integrins further causes the local recruitment of structural proteins such as tensin, vinculin and zyxin, as well as signaling enzymes such as Src family PTKs and FAK, and finally leads to cytoskeleton rearrangements and bacterial uptake (Hauck, Christof R., 2006).

Fig. 3.1 Model of mechanisms involved in S.aureus internalization by non-professional phagocytes (Hirschhausen, Nina, et al. 2010).

General Discussion

The molecular mechanism of integrin-mediated bacterial uptake has been partially deciphered in the last years. A focal adhesion-like complex is involved in the uptake of S.aureus, and several well-characterized integrin-associated proteins, including vinculin, paxillin, zyxin, tensin, FAK, talin, c-Src and PIP5KIγ90 are recruited to the bacterial attachment sites (Agerer, F., J. 2003; Agerer, F., 2005; Schröder, Andreas, et al.2006). Interestingly, pharmacological or genetic inhibition of Src severely diminishes staphylococcal invasion into epithelial cells and fibroblasts, and Src kinase activity is enhanced upon S.aureus infection (Agerer, F., J. 2003; Fowler, Trent, et al. 2003). In addition, the phosphorylation levels of FAK are elevated during the bacterial infection and a lack of FAK significantly decreases bacterial internalization (Agerer, F., 2005).

Also, cortactin, one of the downstream effectors of the FAK-Src complex, and a regulator of actin cytoskeleton assembly, shows increased tyrosine phosphorylation upon S.aureus infection (Agerer, F., 2005; Kirkbride, Kellye C., et al. 2011).

Surprisingly, vinculin, one characteristic component of the focal adhesion complex, which mechanically links integrin cytoplasmic tails with the actin cytoskeleton, did not contribute to the FnBP-mediated uptake of S.aureus (Borisova, Marina, et al. 2013).

Together, these findingsprovide a mechanistic understanding of host cell invasion by fibronectin-binding S.aureus, but there are some questions which are still a matter of debate. For instance, it is unclear how these recruitment and phosphorylation events are spatiotemporally regulated and organized and how the focal adhesion-like complexes are converted into endocytic vesicles after the bacterial attachment on host cell surface.

Also, since S.aureus internalization is mainly mediated by integrins, especially integrin α5β1 and integrin αVβ3, the surface levels and activities of these integrins are critical for bacterial uptake. In vinculin-deficient murine embryonic fibroblasts, both the number of cell-associated and invasive bacteria are increased, compared to the control cells (Borisova, Marina, et al. 2013). Consistent with this observation, the surface level of integrin α5 in vinculin-deficient cells is elevated while the level of integrin β1 is unaltered (Borisova, Marina, et al. 2013). This indicates that in vinculin-deficient MEFs, the pairing pattern of integrins probably be altered, because as mentioned before, all integrin α5, α8 and αV subunit can assort with β1 subunit to form integrins which are

General Discussion

binding to proteins containing RGD motif. More integrin α5 on the surface of vinculin-deficient cells may cause more integrins α5β1 formation, thereby resulting in increased attachment of bacteria to host cells and bacteria invasion. Later, the data obtained with SYNJ1 KO cells had shown that the number of cell-associated bacteria is not altered, while the amount of invasive bacteria is markedly increased (see Fig.2.12 in Results section). Surprisingly, in SYNJ1 KO cells, the surface level of integrin α5 is clearly decreased and the levels of integrin β1 and integrin αVβ3 are only slightly diminished (see Fig.2.15 in Results section). These finding are puzzling, and the reasons causing this conflict could be the altered activity or recycling rate of integrins, which needs further investigations.

Based on current understanding, to better interpret the roles of PI-4,5-P2 and its producing/hydrolyzing enzymes in S.aureus uptake, we artificially divided the whole process into three stages, as shown in Fig.3.2. Establishment of attachment of bacteria to host cells is considered as the prerequisite for the following events of internalization.

Then, the successful attachment triggers the assembly of focal adhesion like complexes, which will be subsequently converted to endocytic vesicles. To achieve the final bacteria internalization, the endocytic vesicles are separated from plasma membrane.

The presence and absence of PI-4,5-P2 may function at certain stage.

Fig. 3.2 Artificially divided phases of S.aureus internalization process based on current understanding. The establishment of attachment is initiated by the binding of FnBPs on S.aureus surface to Fn, which subsequently interacts with integrins. These protein engagements result in the recruitment of components of the focal adhesion complex, however, the sequence of events that lead to assembly of this complex is not fully clear. Later, after the conversion of focal adhesion like complex to endocytic

General Discussion

vesicles and the fission of these vesicles from plasma membrane, bacteria internalization is completed.

3.2 PI-4,5-P2 in S.aureus internalization

Although phosphoinositides make up only a small proportion of the lipids present within cellular membranes, they sophisticatedly participate in a series of cellular processes via interactions with phosphoinositide-binding proteins, directing their local accumulation and activation (McLaughlin, Stuart, et al. 2002; Di Paolo, Gilbert, 2006;

Krauß, Michael, 2007; Falkenburger, BH., et al. 2010). Microbe-host interaction is one of the cellular processes that phosphoinositides are extensively involved in.

For different bacterial internalization, there are several morphologically distinct mechanisms classified and described with two particularly prominent examples: the so-called “trigger” mechanism and the “zipper” mechanism (Fig.3.3) (Cossart, Pascale, and Philippe J. Sansonetti. 2004; Ribet, David, 2015). These two mechanismshave in common that they both rely on the activation of signaling cascades which subsequently lead to the reorganization of actin cytoskeleton at the level of the host plasma membrane (Haglund, Cat M., 2011; Ham, Hyeilin, 2011). In the case of the zipper mechanism, which is utilized by for example Yersinia pseudotuberculosis, Listeria monocytogenes and Staphylococcus aureus, bacterial surface factors associate with host cell membrane proteins, such as integrins, cadherins or IgCAMs, either directly or indirectly (Cossart, Pascale, 2004; Hauck, Christof R., 2006). These initial interactions trigger a signaling cascade, including the recruitment of adaptors and effectors, protein phosphorylation and activation of cytoskeleton components that culminate in phagocytic cup closure and bacterial internalization (Cossart, Pascale, 2004). Alternatively, the trigger mechanism is used by Shigella and Salmonella to enter host cell. This mechanism is characterized by the formation of large membrane ruffles at the bacterial entry sites (dos Reis, Roberta Souza, 2010; Velge, Philippe, et al. 2012; Da Silva, Claudio Vieira, et al. 2012). Through a dedicated secretory system, for example the type III secretion system (T3SS) for Shigella flexneri, pathogens inject effectors into host cells to regulate the actin cytoskeleton dynamics (Schroeder, Gunnar N., 2008; Ham, Hyeilin, 2011;

Velge, Philippe, et al. 2012). These ruffle structures finally engulf the bacterium in a

General Discussion

sort of macropinocytosis and the internalization was completed (Pizarro-Cerda, Javier, 2006; Cossart, Pascale, 2010).

Fig.3.3 Mechanisms used by bacteria to enter cells. (A) The zipper mechanism used by Yersinia and Listeria. (B) The trigger mechanism used by Salmonella and Shigella (Cossart, Pascale, and Philippe J.

Sansonetti. 2004).

Over the last decades, numerous studies have demonstrated that in vitro activities of most of the actin-binding proteins can be regulated by the direct interactions with phosphoinositides, particularly PI-4,5-P2 (Yin, Helen L., 2003; Janmey, Paul A., 2004;

Tsujita, Kazuya, 2015). For instance, a protein modulated by PI-4,5-P2 is profilin, a small G-actin–binding protein, which prevents actin filament assembly. Once profilin binds PI-4,5-P2 it can no longer interact with G-actin and therefore does not interfere anymore with the formation of actin dimers or trimers nuclei (Bezanilla, Magdalena, et al. 2015). Additional examples include N-WASP (an actin nucleation promotion factor) (Papayannopoulos, V., 2005) and α-actinin (an actin cross-linking protein) (Sjöblom, Björn, 2008). Specifically, PI-4,5-P2 regulates N-WASP, an activator of the Arp2/3 complex, which subsequently affects actin reorganization (Rohatgi, Rajat, 2000;

Campellone, Kenneth G., 2010). The nucleating activity of Arp2/3 complex is constitutively low and needs to be activated by nucleation promoting factors, such as

General Discussion

WASP. In the N-terminal region of N-WASP, there is a G-protein binding domain that binds to the Rho GTPase Cdc42 and a basic region which interacts with PI-4,5-P2. Via these interactions, N-WASP is activated and the VCA domain in the C-terminus, which consists of a verprolin homology segment (V), a cofilin homology segment (C) and an acidic region (A), is released to bind the Arp2/3 complex, thereby stimulating Arp2/3 interaction with actin filaments and enhancing actin nucleation (Rohatgi, Rajat, 2000;

Campellone, Kenneth G., 2010). Based on the importance of actin cytoskeleton reorganization in bacterial uptake and effects of PI-4,5-P2 on regulation of actin cytoskeleton reorganization, it is not surprising that PI-4,5-P2 plays a role in the internalization of bacteria, such as S.aureus.However, the role of PI-4,5-P2 for the integrin-mediated internalization of S.aureus has not been investigated before.

In addition, some new findings were gained to provide a new perspective on the role of PI-4,5-P2 in integrin activation (Orłowski, Adam, et al. 2015). Earlier studies showed that talin binds to the cytoplasmic tail of integrin β subunit and induces the conformational changes in the transmembrane part of the integrin αβ complex, which are crucial for integrin inside-out activation. To facilitate talin-binding induced integrin activation, PI-4,5-P2 binds to the talin head domain and cause the conformational changes of talin, which contributes to the exposing of integrin β binding sites (Saltel, Frédéric, et al. 2009; Wang, Jia-huai. 2012). In Adam Orłowski’s study, integrin αIIbβ3 were used as an example and the results showed, that the negatively charged PI-4,5-P2

head groups can perturb a clasp at the cytoplasmic face of the integrin heterodimer, between Arg995 in αIIb and Asp723 in β3 integrin. The breakage of salt bridge subsequently facilitates the integrin activation (Orłowski, Adam, et al. 2015). This finding further implied that PI-4,5-P2 can regulate integrin-mediated S.aureus uptake.

From former studies, PI-4,5-P2 recruitment was observed during bacterial uptake. For example, during the uptake of Yersinia pseudotuberculosis, which is initiated with the binding of bacterial invasin protein to integrin β1, PIP5Kα and PI-4,5-P2 were recruited tonascent phagosomes(Wong, Ka-Wing, and Ralph R. Isberg.2003). In addition, during the uptake of Chlamydia, PI-4,5-P2 was recruited at the sites of bacterial entry (Balañá, María Eugenia, et al. 2005). In this study, we observed that PI-4,5-P2 is strongly

General Discussion

enriched at the bacterial attachment sites and FnBP-mediated contact is sufficient to trigger the increased PI-4,5-P2 levels (see Fig.2.1 and Fig.2.2 in Results section).

Together, these observations indicate the importance of PI-4,5-P2 in S.aureus uptake.

Since there is no perfect way to directly manipulate PI-4,5-P2 level in mammalian cells, to address the effect of PI-4,5-P2 deficiency on bacteria uptake, we measured bacteria uptake in murine fibroblasts lacking PIP5KIγ90 (PIP5KIγ90^-/-), the kinase locally produces PI-4,5-P2 in focal adhesions. In PIP5KIγ90^-/-cells, the integrin-mediated bacteria uptake is markedly reduced and the recruited PI-4,5-P2 level is also decreased (see Fig.2.5 and Fig.2.6), suggesting a positive contribution of locally formed PI-4,5-P2 in S.aureus internalization. To further investigate the possible reasons of the diminished S.aureus uptake, the tyrosine phosphorylation of FAK was measured and it is markedly reduced in PIP5KIγ90^-/- cells upon bacterial infection (see Fig.2.8), which is consistent with former finding that the presence and activity of FAK are required for S.aureus internalization (Agerer et al., 2005). In line with the ability of PI-4,5-P2 to activate talin, the accumulation of talin at bacterial attaching sites was decreased in PIP5KIγ90^-/-cells (see Fig.2.6B). Since talin is one critical component of focal adhesion complex, the inhibited talin enrichment could also contribute to the reduced bacteria uptake, even if the function of talin in S.aureus uptake has not been studied.

Lastly, from live cell imaging, we observed that the recruitment of PI-4,5-P2 to the bacterial attachment sites, normallyoccurred within 5 min after the bacterial adhesion to host cells, and then vanished over time (data not shown). This observation, on the one hand, presented the difficultyin the study of PI-4,5-P2 function and the initial events of bacterial infection, due to the short time course, but on the other hand, again, suggested the importance of PI-4,5-P2 in S.aureus uptake and implied the kinase responsible for synthesis and other enzymes executing hydrolysis are involved.

However, further studies are still needed tobroaden our detailed understanding of how PI-4,5-P2 is spatiotemporally regulated and which influence locally synthetized PI-4,5-P2 exert on the formation of focal adhesion-like complexes.

3.3 The role of PIP5KIγ90 in S.aureus uptake

General Discussion

It is well established that PI-4,5-P2 generated by type I PIP5K at the plasma membrane is a critical regulator of constitutive endocytosis (Padrón, David, et al. 2003; Nakano‐

Kobayashi, Akiko, et al. 2007; Van den Bout, Iman, 2009). In particular, recruitment and functionality of clathrin adaptor complex AP2 depends on the interaction of AP2 subunits with PIP5K and PI-4,5-P2 (Padrón, David, et al., 2003). PIP5KIα appears to be the major PIP5KI family member providing PI-4,5-P2 during clathrin-mediated endocytosis (Antonescu et al., 2011), whereas PIP5KIγ seems to contribute to clathrin-mediated endocytosis in specific cell types only. Indeed, previous studies have shown that specific ablation of the PIP5KIγ90 isoform in non-neuronal cell types, including murine fibroblasts, does not interfere with clathrin-mediated endocytosis of different plasma membrane receptors (Legate et al., 2011). These findings suggest that PIP5KIα and the remaining PIP5KIγ isoforms are sufficient to allow regular levels of endocytosis in PIP5KIγ90^-/- cells and point to other functions of PIP5KIγ90, which might compromise integrin-mediated uptake of S.aureus.

At focal adhesion sites, several integrin- and actin-associated proteins have PI- 4,5-P2

binding capability and assist to connect integrins to the actin cytoskeleton. More precisely, the integrin-associated proteins talin, kindlin, vinculin, FAK as well as α-actinin belong to the core focal adhesion proteins, which are responsive to increased PI-4,5-P2 levels (Janmey, 1994; Toker, A, 2002). However, the source of locally increased PI-4,5-P2 is not completely understood, one possibility is through the recruited PIP5KIγ90 in focal adhesions. In the C-terminus of PIP5KIγ90, there is a short extension (26aa in mouse or 28aa in human) which has the ability to bind to talin.

Via this interaction, PIP5KIγ90 is recruited to the focal adhesions (Legate, Kyle R., et al. 2011). In this regard, it has been demonstrated that recruitment of talin and vinculin, but not kindlin, is affected in PIP5KIγ90-deficient cells (Legate, Kyle R., et al. 2011).

As vinculin is not involved in the uptake of S.aureus (Borisova et al., 2013), a reduced integrin-association of talin in the absence of PIP5KIγ90 might contribute to a diminished internalization of these bacteria. Furthermore, PI-4,5-P2 binding to the talin head domain is required to release intramolecular inhibitory constraints from this ~180 kDa protein and to allow proper orientation of talin at the inner leaflet of the plasma

General Discussion

membrane (Goksoy et al., 2008; Elliott et al., 2010; Goult et al., 2013). As a consequence, the recruitment and the spatial orientation of talin at integrin-rich focal adhesion sites are impaired in PIP5KIγ90^-/-cells, resulting in slower incorporation of talin into new focal adhesion sites (Legate et al., 2011). Therefore, PI-4,5-P2 local production by PIP5KIγ90 could be critical to mobilize talin to newly formed integrin clusters beneath fibronectin-bound bacteria.

Im Dokument The role of phosphoinositol-4,5-bisphosphate in the cellular uptake of staphylococcus aureaus (Seite 69-0)