The role of phosphoinositol-4,5-bisphosphate in the cellular uptake of staphylococcus aureaus

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The role of phosphoinositol-4,5-bisphosphate in the cellular uptake of staphylococcus aureus

Dissertation

Zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.)

vorgelegt von Yong Shi

im Fachbereich Biologie

Konstanz, 2016

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Table of Contents

S.aureus can invade non-professional phagocytic cells in an integrin dependent manner to escape from the clearance of the human immune system. Among the different virulence factors produced by S.aureus, Fibronectin-binding proteins (FnBPs) are the major ones responsible for the bacterial internalization. FnBPs first bind to Fibronectin (Fn), a member of extracellular matrix which acts as a molecular bridge in this case, the RGD motif of Fn interacts with integrins presenting on host cell surface, especially integrin α5β1 and αVβ3. Via these molecular interactions, the adhesion of bacteria to host cells is established, and this adhesion leads to the recruitment of a focal adhesion like complex and the re-arrangement of the actin cytoskeleton, which finally results in the internalization of bacteria.

Phosphoinositol 4,5 bisphosphate, in short, PI-4,5-P2, participates in a variety of cellular processes via the interactions with other proteins containing PI-4,5-P2 binding motif. It is well known that PI-4,5-P2 could regulate localization and activation of several members of the focal adhesion complex. In this study, we investigated the roles of PI-4,5-P2 and enzymes producing or hydrolyzing PI-4,5-P2 during cellular invasion of S.aureus. First of all, we observed strong accumulation of PI-4,5-P2 at the bacteria attachment sites, and FnBPs were sufficient to trigger this enrichment. Furthermore, knockdown of individual type I phosphatidylinositol-4-phosphate 5-kinase revealed

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Abstract

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that PIP5KIγ plays a role in bacterial internalization, as PIP5KIα does. In mammalian cells, there are two main splice variants of PIP5KIγ, based on their molecular size, one is named PIP5KIγ87 and the other is named PIP5KIγ90. PIP5KIγ90 is known to associate with talin FERM domain at integrin-rich focal adhesion complexes and is strongly recruited to sites of bacterial attachment. Selective genetic deletion of this isoform significantly reduced bacterial invasion, which could be rescued by the re- expression of active PIP5KIγ90, but not by its kinase inactive form. In PIP5KIγ90- deficient cells, overall PI-4,5-P2 levels in the plasma membrane were unaltered, but local accumulation of PI-4,5-P2 at bacterial attachment sites and FAK tyrosine phosphorylation were markedly reduced. These results highlight the importance of local synthesis of PI-4,5-P2 by a focal adhesion-associated lipid kinase to promote integrin-mediated internalization of S. aureus by non-phagocytic cells.

With the over-expression of myr-tagged phosphoinositide phosphatase domain in cells, which is assumed to catalyze the de-phosphorylation of PI-4,5-P2, bacterial invasion was significantly reduced. To find which phosphoinositide 5-phosphatase mainly performs this function in cells, we generated shRNA-mediated knockdown cells. Via gentamicin protection and FACS assays, synaptojanin1 (SYNJ1) was found to have more influence on bacterial uptake, compared to the other phosphatases. The increased bacterial uptake could also be confirmed in SYNJ1 knockout cells which were generated via CRISPR-Cas9 method. As expected, PI-4,5-P2 recruitment, as well as talin enrichment in SYNJ1 KO cells was much stronger, compared to control KO cells.

Interestingly, FAK tyrosine397 phosphorylation level was not altered in SYNJ1 KO

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cells, compared to control KO cells. And surprisingly, the surface level of integrins was decreased in SYNJ1 KO cells, which seems to be in conflict with the increased bacterial uptake. In the end, the enhanced bacterial uptake in SYNJ1 KO cells was rescued by the re-expression of wild-type SYNJ1, but not the phosphatase dead mutant. All these data demonstrated the essential role of PI-4,5-P2 in S.aureus uptake. However, the effect of precisely regulated synthesis and hydrolysis of PI-4,5-P2 in this process is not completely understood. In future, it is worth to put some effort into the investigation of how PI-4,5-P2 presence and absence is spatiotemporally controlled by the kinase and phosphatases in S.aureus uptake.

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Zusammenfassung

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Zusammenfassung

Staphylococcus aureus, ein kommensales Bakterium das besonders in der Nasenschleimhaut sowie auf der Haut zu finden ist, entwickelt sich mehr und mehr zu einem der gefährlichsten humanen Pathogene. Durch die steigende Verbreitung von Antibiotikaresistenzen wird die Behandlung von Infektionen mit S.aureus immer schwieriger. Das Bakterium ist in der Lage in nicht-professionelle Phagocyten integrin- abhängig aufgenommen zu werden und so dem Immunsystem zu entkommen. Unter den vielen verschieden Virulenzfaktoren die von S.aureus produziert werden, sind die Fibronectinbindeproteine (FnBPs) hauptverantwortlich für die Internalisierung der Bakterien. FnBPs binden an Fibronektin (Fn) welches als molekulare Brücke agiert und dem RGD Motiv des Fibronektins ermöglicht mit Integrinen zu interagieren. Diese Interaktion ist besonders ausgeprägt im Falle von α5β1 und αVβ3 Integrinen welche auf der Zelloberfläche als Rezeptoren agieren und so die Internalisierung initialisieren.

Phosphatidylinositol-4,5-bisphosphat, im Folgenden mit PI-4,5-P2 abgekürzt, spielt eine wichtige Rolle in einer Vielzahl von zellulären Prozessen, welche durch die Interaktion mit Proteinen vermittelt werden, die ein PI-4,5-P2 Bindemotif besitzen. Es ist seit langem bekannt, dass PI-4,5-P2 die Lokalisierung und die Aktivität mehrerer Mitglieder des fokalen Adhäsionskomplexes reguliert. Im Rahmen dieser Arbeit wurde die Rolle von PI-4,5-P2 und verschiedenen PI-4,5-P2 regulierenden Enzymen während der Internalisierung von S.aureus untersucht. Es konnte eine starke Akkumulierung von PI-4,5-P2 an der bakteriellen Adhäsionsstelle festgestellt werden die durch FnBPs ausgelöst wurde.

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Zusammenfassung

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Des Weiteren konnte der knockdown von einzelnen Phosphatidylinositol-4-phosphate 5 Kinasen (PIP5KI) zeigen, dass die γ-Untereinheit PIP5KIγ, ebenso wie PIP5KIα eine Rolle bei der Internalisierung von Bakterien spielt. In Säugetieren gibt es zwei Spleiß- Varianten von PIP5KIγ die anhand ihrer Molekülgröße PIP5KIγ87 und PIP5KIγ90 benannt wurden. Es ist bekannt, dass sich PIP5KIγ90 mit der Talin-FERM Domäne an integrinreichen Fokaladhäsionskomplexen assoziiert und stark an bakterielle Anlagerungsstellen rekrutiert wird. Die selektive Deletion dieser Isoform führte zu einer reduzierten Aufnahme von Bakterien. Durch Re-Expression der aktiven Form derselben, konnte der Effekt negiert werden. Im Gegensatz dazu zeigte die Re- Expression eines Konstrukts mit inaktiver Kinasedomäne keine Erhöhung der bakteriellen Aufnahme. In PIP5KIγ90 defizienten Zellen blieb das Plasmalevel von PI- 4,5-P2 unverändert, die lokale Akkumulierung an der bakteriellen Adhäsionsstelle sowie die Tyrosinphosphorylierung von FAK waren jedoch eindeutig vermindert. Diese Ergebnisse heben die Bedeutung von lokaler PI-4,5-P2 Synthese durch Lipidkinasen, die an fokalen Adhäsionen assoziiert sind, für die integrinabhängige Internalisierung von S.aureus durch nicht-professionelle phagozytierende Zellen hervor.

Durch die Überexpression von Phosphoinositolphosphatasedomänen, von welchen angenommen wird, dass sie die Dephosphorylierung von PI-4,5-P2 katalysieren, und das Lokalisieren dieser an die Plasmamembran mit Hilfe eines myr-tags, wurde die Aufnahme von Bakterien signifikant reduziert. Um zu identifizieren welche der Phosphoinositol- 5-phosphatasen diese Funktion in der Zelle ausübt, generierten wir shRNA basierte knockdown Zellen und überprüften deren Aufnahme von Bakterien

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mittels In vitro basierten Internalisierungsversuchen. Es konnte gezeigt werden, dass der Knockdown von Synaptojanin 1 (SYNJ1) einen größeren Anstieg der Internalisierungsrate verursacht als der anderer Phosphatasen. Die erhöhte Aufnahme von Bakterien konnte auch in SYNJ1 defizienten Zellen gezeigt werden welche mittels CRISPR-Cas erzeugt wurden. Wie erwartet war die Rekrutierung von PI-4,5-P2 in SYNJ1 knockout-Zellen deutlich stärker im Vergleich zu Kontroll-knockout-Zellen.

Interessanterweise war die Phosphorylierung der FAK am Tyrosin397 in SYNJ1 knockout-Zellen vergleichbar mit der Phosphorylierung in Kontrollzellen. Die Oberflächenkonzentration von Integrinen in SYNJ1 knockout-Zellen war jedoch vermindert, was im Gegensatz zu der erhöhten Aufnahme von Bakterien steht.

Schlussendlich konnte die erhöhte bakterielle Aufnahme in SYNJ1 knockout-Zellen durch die Re-Expression des SYNJ1 Wildtyps aber nicht durch SYNJ1 mit inaktiver Phosphatasedomäne wiederhergestellt werden.

Zusammen zeigen diese Ergebnisse, die wichtige Rolle von PI-4,5-P2 in der Aufnahme von S.aureus. Die Wirkung der präzise regulierten Synthese und Hydrolyse von PI-4,5- P2 während dieses Prozesses ist noch nicht vollständig aufgeklärt. Der Einfluss der in die Aufnahme von S.aureus involvierten Kinasen und Phosphatasen auf die Regulation von PI-4,5-P2 stellt ein vielversprechendes Gebiet für zukünftige Forschung dar.

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General Introduction

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General introduction

1 .1. Staphylococcus aureus 1.1.1. General information

Staphylococcus, the Gram-positive bacterium, is a member of the Staphylococcaceae family, which also contains the other three less studied genera Gemella, Macrococcus and Salinicoccus. Staphylococcus was first discovered in 1880 in Aberdeen, Scotland by Alexander Ogston, in the pus from surgical abscesses. About four years later, two Staphylococcus strains were described by German surgeon Anton J. Rosenbach according to the color of the colonies: S.aureus (from the Latin aurum for gold) and S.

albus (from the Latin albus for white). S. albus later was renamed as S. epidermidis because of its ubiquity on human skin (Orenstein A, 2011). Nowadays, there are 36 species and several subspecies recognized in the genus Staphylococcus (Götz, Friedrich, 2006), but only S.aureus, S. epidermidis and S. intermedius are able to colonize and infect humans (Pottumarthy, Sudha, et al. 2004). In contrast to the other species, S.aureus is the only coagulase positive strain found in humans (Hennekinne, Jacques- Antoine, et al. 2010), which could convert the soluble fibrinogen into insoluble fibrin and form the blood clot. This characteristic feature is used to distinguish S.aureus from the other members of Staphylococcus. Staphylococci are encapsulated, non-sporulating, non-motile cocci. When observed under the microscope, they are round or slightly oval with the diameter 0.5 to 1.5 μm and occur singly, in pairs, short chains or grape-like clusters (Ogston A, 1984). S.aureus have different doubling times under different conditions, for instance, in vivo or in vitro, iron poor or iron rich, however generally it is about 60 min in vivo and 24 min at laboratory conditions(Domingue, Gil, 1996).

Staphylococci are ubiquitous in the environment and can be detected in the air, dust, sewage, water, environmental surfaces and even in humans and animals (Hennekinne, Jacques-Antoine, et al. 2010). S.aureus can grow in a wide range of temperatures, ranging from 7°C to 48.5 °C with an optimum of 30 to 37 °C (Schmitt, Margrit, 1990);

pH between 4.0 and 10.0, with an optimum of 6.0- 7.0; and sodium chloride concentrations up to 25% (Valero, A., et al. 2009). These characteristics enable S.aureus

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to grow and survive in different environmental conditions as well as to persist in stressful environments (e.g. dry surfaces) for long periods.

1.1.2. Diseases

Normally, S.aureus acts as a commensal in the human body, with the anterior nares of the nose being the most frequent carriage site. At any moment in time, about 20% of healthy adults (range 12-30%) are continuously colonized with S.aureus, around 30%

are intermittent carriers (range 16- 70%), and approximately 50% (range 16-69%) are non-carriers (Wertheim, Heiman FL, et al., 2005). In addition to humans, cattle, sheep, and goat are also the natural hosts of S.aureus. Due to the colonization, the risk for subsequent infections is significantly increased, because this colonization provides a reservoir of the pathogen, from which bacteria are introduced when the host immune system is impaired or weakened (Immunocompromised). From former study, it was already demonstrated that individuals are usually infected by their own carriage isolate (Peacock, Sharon J., 2001). During the nasal colonization, wall teichoic acid (WTA), a surface-exposed staphylococcal polymer, is an essential factor. The ability of WTA- deficient strains to adhere to nasal cells is impaired, and therefore they are completely unable to colonize cotton rat nares (Weidenmaier, Christopher, et al. 2004).

Due to the weakened immune systems of patients, the balanced state between S.aureus virulence determinants and host defense mechanisms is disturbed and the bacteria spread over the host organism leading to infections. Diseases caused by S.aureus range from minor skin infections, such as pimples, impetigo, boils, cellulitis, folliculitis, carbuncles, scalded skin syndrome, and abscesses, to the life-threatening infections, for example, bacteremia, endocarditis, sepsis, toxic shock syndrome, pneumonia, and so on(Lowy, Franklin D. 1998).

Take bacteremia as an example, from the investigatory results of Sentry Antimicrobial Surveillance Program, S.aureus cause the most cases of nosocomial bacteremia in North America (prevalence, 26.0%) and Latin American (prevalence, 21.6%), and was the second most common cause in Europe (prevalence, 19.5%) (Naber, Christoph K.

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2009). In addition, S.aureus bacteremia (SAB), is frequently associated with a high mortality rate ranging from 11% to 43% and represents a significant economic burden on public health systems (Lowy, Franklin D. 1998; Lodise, Thomas P., 2007). To efficiently treat SAB, several management strategies are accepted as standard of care, including (a) performing a thorough history and physical examination; (b) obtaining follow-up blood cultures to document resolution of bacteremia; and (c) draining abscesses and removing infected prosthetic material (Holland, Thomas L., 2014).

Concerning treatment, the choice of antibiotic is especially important, while both delaying anitbiotic treatment and inappropriate antibiotic use could increase the risk of infection-related mortality.

1.1.3. Virulence factors

To accomplish survival within the human host, S.aureus has an extraordinary repertoire of virulence factors and maintains fine control of their expression. Now it is well known that S.aureus expresses a broad variety of virulence factors during growth, which are responsible for the bacterial internalization into host cells and the following infectious symptoms. According to different production times under the in vitro cultivation, these virulence factors are divided into two main groups (Fig.1.1 and Table1.1). One group is the surface-associated virulence factors that are covalently attached to peptidoglycan, which are also known as cell wall anchored (CWA) proteins and preferentially expressed in the exponential growth phase, the other group is the secreted proteins released in the post-logarithmic (or stationary) phase, most of which are toxins and some enzymes (Fig.1.1, Lowy, Franklin D. 1998). Furthermore, in the group of cell wall anchored proteins, several members have been termed as microbial surface components recognizing adhesive matrix molecules, in short, MSCRAMM (Hauck, CR., 2006). It is assumed that this biphasic expression of virulence factors directs the infection process. Initially, the surface-bound adhesins are recognized by host surface receptors, like integrins, establishing bacterial colonization, which is then followed by further growth of the microbes and secretion of toxins and enzymes, to cause the

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symptoms (Hauck, CR., 2006).

Fig.1.1 Structure of S.aureus. A shows the surface and secreted proteins. The synthesis of many of these proteins is dependent on the growth phase, as shown by the graph, and is controlled by regulatory networks such as the quorum-sensing agr system. B and C show cross sections of the cell envelope.

Many of the surface proteins have a structural organization similar to that of clumping factor, including repeated segments of amino acids (C). TSST-1 denotes toxic shock syndrome toxin 1(Lowy, Franklin D.

1998)

In the last years, numerous investigations were focused on the molecular events directing the uptake of S.aureus and there were several remarkable reviews published to summarize the functions of these virulence factors during bacterial uptake. These covered topics such as how these factors promote adhesion to the extracellular matrix (ECM) and to host cells, how these factors contribute to the invasion of host cells, the evasion of innate immune responses, how these factors perturb the adaptive immune response, are partly understood (Hauck, CR., 2006; Zecconi, Alfonso, and Federico Scali. 2013; Foster, Timothy J., et al. 2014). However, due to the functional redundancy, the contribution of individual virulence factors to the infection process in vivo, as analyzed by experimental models of infection, is often not entirely clear.

Table1.1 Virulence factors of S.aureus (Modified from Hauck, CR., 2006; Zecconi, Alfonso, and Federico Scali. 2013)

Full Name Abbreviation Functions

Fibronectin binding protein A FnBP A Adhesins of fibrinogen, fibronectin

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and elastin

Fibronectin binding protein B FnBP B Adhesins of fibronectin and elastin Clumping factor A ClfA Adhesin of Fibrinogen γ-chain and

Fibrin

Clumping factor B ClfB Adhesin of Fibrinogen α- and β-

chain and Type I cytokeratin 10 Collagen binding protein Cna Adhesin of collagen (type I and IV) Elastin binding protein EbpS Adhesin of Elastin

Protein A Spa Binds the Fcγ domains of IgG,

Inhibition of opsonophagocytosis;

B cell superantigen; inflammation Bone sialoprotein binding

protein

Bbp Adhesin of bone sialoprotein (SdrE allelic variant), binds fibrinogen Extracellular matrix binding

protein

EbhAB Adhesin of Fibronectin

Serin-aspartate repeat proteins C/D/E

Sdrs Adhesins

Polysaccharide intercellular adhesin

PIA Adhesin for aggregation; involved in biofilm formation

Von Willebrand factor binding protein

vWbp Binds and activates prothrombin;

binds fibrinogen and vW factor Serine-rich surface protein SraP Binds platelets

α toxin Hla Cytolytic pore-forming toxin

β toxin Hlb Sphingomyelinase with cytolytic

activity

Leukocidins D/E/M LukD/E/M Kill leukocytes; bi-component pore-forming leukotoxins

PSM peptides PSMs Pore-forming toxins or detergent

activity

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Exfoliative toxins A/B/D ETA/B/D Exotoxins with superantigen activity; gluamate-specific serine proteases that digest desmoglein 1

Enterotoxins SEs Gastroenteric toxicity;

immunomodulation via

superantigen activity

SE-like proteins SEls Unknown/No gastroenteric

toxicity; immunomodulation via superantigen activity

Toxic shock syndrome toxin-1 TSST1 Endothelial toxicity (direct and cytokine-mediated); superantigen activity

Formylpeptides fMLPs Ligands for formyl peptide receptor

Coagulase Coa Binds and activates prothrombin;

promotes conversion of fibrinogen to fibrin

V8 protease Serine protease

Glycerol ester hydrolases lip, geh, beh, Triacylglycerols degradation Fatty acid-modifying enzyme FAME Fatty acids modificatio O-acetyl transferase OatA Peptidoglycan O-acetylation PtdIns-phospholipase C Plc Phosphotidylinositol-specific lipase

activity

1.1.4. Antibiotic resistance

With the discovery of antibiotics, humans began to gain an upper hand in the battle against antibiotic resistant bacteria, leading to their critical importance today as therapeutic agents (Chambers, Henry F., 2009). However, the rate at which bacteria continue to gain antibiotic-resistance genes far exceeds our expectation, this makes it urgent to improve strategies and better antibiotics to prevent and treat S.aureus

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infections. On the other hand, for bacteria to survive under antibiotic treatment it is essential to evolve and acquire the resistance gene(s). So the competition between the development of antibiotics and the ability of bacteria in obtaining resistanceis always occurring.

In the early 1940s, when penicillin was introduced to treat staphylococcal infections, it dramatically decreased the mortality of patients. However, as early as 1942, S.aureus strains containing penicillin resistant genes were detected in hospitals. By the late 1960s, about 80% of both hospital- and community-acquired S.aureus isolates were penicillin resistant. These strains produced penicillinase, which is also called as β-lactamase, to hydrolyze the β-lactam ring of penicillin that is essential for its antimicrobial activity (Chambers, Henry F., 2009). The gene encoding β-lactamase is part of a transposable element located on a large plasmid, often with additional antimicrobial resistance genes (e.g., gentamicin and erythromycin).

In 1959, the semi-synthetic penicillinase-resistant penicillin, named as methicillin, was introduced to treat the infections caused by penicillin-resistant S.aureus. Unfortunately, in 1961, only two years later, S.aureus isolates which had acquired methicillin resistance (MRSA) were identified. In the chromosome of MRSA there is a large stretch of foreign DNA, approximately 30 to 50 kb, referred to as the mec element. In mec, the mecA gene encodes a 76-kDa protein, names PBP2a, which is a member of PBPs. PBPs are membrane bound DD-peptidases that catalyze the transpeptidation reaction that cross-links the peptidoglycan of the bacterial cell wall. Beta-lactam antibiotics can covalently bind to the PBP and therefor inactivate the enzyme. In methicillin-resistant bacteria, PBP2a has reduced affinity for all available β-lactam agents and confers resistance to most currently available β-lactam antibiotics (Chambers, Henry F. 1997;

Stryjewski, Martin E., 2014).

Vancomycin, a glycopeptide antibiotic, which was introduced in the mid-1950s, exerts antimicrobial effects by inhibiting the cell-wall synthesis of S.aureus, in the same way how β-lactam antibiotics work. Vancomycin is clinically used to treat MRSA infections, however, in 1996 the first MRSA to acquire resistance to vancomycin (VRSA) was isolated from a Japanese patient (Hiramatsu, Keiichi. 2001). In VRSA strains, the

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normal dipeptide D-Ala-D-Ala, which acts as building block in peptidoglycansynthesis, is replaced by D-Ala-D-Lacaltering cell wall structure and metabolism. (Fig.1.2). In addition, in VRSA strains, a significantly thickened cell wall is commonly observed, compared to the vancomycin susceptible S.aureus (VSSA) strains. (Hughes, Diarmaid.

2003）.

Fig.1.2. A two-component regulatory system VanR–VanS regulates vancomycin resistance in vancomycin-resistant Enterococci (VRE) and vancomycin-resistant Staphylococcus aureus (VRSA) (Hughes, Diarmaid. 2003).

Last year, a new antibiotic termed teixobactin was discovered from a screen of uncultured bacteria. This antibiotic has outstanding activity against Gram-positive pathogens but no toxicity against mammalian cells (like NIH/3T3 and HepG2 cells) at the highest testing dose. By binding to a highly conserved motif of lipid II (precursor of peptidoglycan) and lipid III (precursor of cell wall teichoic acid), teixobactin inhibits cell wall synthesis. More importantly, there was no any mutants of S.aureus resistant to teixobactin obtained in laboratory conditions so far (Ling, Losee L., et al. 2015). Based on these properties, the drug Teixobactin could be considered as the last resort for staphylococcal infections, which is a role that up until now has been Vancomycin.

1.1.5 Pathogen-host interaction

For bacterial infection, establishment of the attachment between bacteria and host cells is prerequisite. Though S.aureus was considered as an extracellular pathogen for a long

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time, now it is well recognized that S.aureus also can invade a variety of non- professional phagocytic cells, like human endothelial cells (Ogawa et al., 1985), a bovine mammary epithelial cell line (Bayles et al., 1998) and murine fibroblasts (Usui et al., 1992). In the past decades, significant advances have been made in the S.aureus internalization field to better understand the underlying mechanisms. In 2010, there was a novel staphylococcal internalization pathway discovered, in which the autolysin Atl was exploited as the bacterial adhesin and heat shock cognate protein Hsc70 was recognized as the host cell receptor (Hirschhausen, Nina, et al. 2010). But the fibronectin-binding proteins A and B (FnBP-A and FnBP-B) were well identified as major factors in initiating the uptake of S.aureus, in the integrin dependent manner.

Most clinical isolates of S.aureus harbor two closely related fnb genes, named fnbA and fnbB. The proteins encoded by these two genes, named FnBP-A and FnBP-B (Fig.1.3), are anchored by an LPXTG motif to the cell wall of S.aureus. With Cowan strain lacking FnBPA, the bacterial invasiveness is diminished by 80-85%, and when both FnBPA and FnBPB are deficient, the invasiveness is decreased by~95%. In addition, exogenous expression of either FnBPA or FnBPB in the non-invasive staphylococcal strains such as S. carnosus, is sufficient to confer an invasive phenotype (Sinha, Bhanu, et al. 1999). These observations strongly supported the function of FnBPs in S.aureus uptake.

Fig.1.3. Schematic representation of FnBPs and FN. Upper: structure of FnBPs, the position of the signal sequence (S), the fibrinogen and elastin binding A domain (N1, N2, N3), and the fibronectin binding motifs (numbered) are indicated. The wall/membrane-spanning region (W and M) and LPETG motif at the C terminus are required for covalent attachment to cell wall peptidoglycan (Geoghegan, Joan A., et al. 2013). Lower: structure of FN, N consists of type I (rectangles), type II (ovals) and type III (circles) repeats. Sets of repeats constitute binding domains for fibrin, FN, collagen, cells and heparin,

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as indicated. The three alternatively spliced segments, EIIIA, EIIIB and V (or IIICS), are in yellow. The assembly domain and FN-binding sites are highlighted in orange. SSindicates the C-terminal cysteines that form the dimer (Wierzbicka-Patynowski, Iwona, 2003).

FnBPA and FnBPB, both of them have a pronounced modular architecture. They are composed of a short signal sequence (S) which is responsible for targeting the proteins to a defined location in the cell envelope, followed by a long N-terminal domain (N1, N2 and N3) that binds to fibrinogen, then the fibronectin binding motifs (numbered), and the wall and membrane-spanning region (W and M) and LPETG motif at the C- terminus which are required for covalent attachment to cell wall peptidoglycan.

Between FnBPA and FnBPB, the sequences of fibronectin-binding motifs are highly conserved (95% sequence identity). By contrast, the sequences of fibrinogen and Elastin binding domains are more divergent (about 45% sequence identity) (Burke, Fiona M., 2010).

To initiate bacteria and host cell attachment, cell wall anchored FnBPs bind to the extracellular matrix, fibronectin (Fn). As with the structure of FnBPs, FN is also a modular protein composed of types I, II, and III repeating units (indicated in different shapes in Fig1.3). The Fn-binding motifs of FnBPs specifically bind to the 29 kD N- terminal region of Fn (F1 region). As shown in the figure, the F1 region contains five sequential repeats^1–5F1. The binding of FnBPs to the F1 region subsequently mediates the adherence of S.aureus to host cells, because in the centrally located 9th and 10th type III repeats of Fn, there is a well-characterized RGD (Arg–Gly–Asp) motif, which could recognize and associate with integrin α5β1 presenting on the host cell surface. In these protein-protein interactions, Fn acts as a molecular bridge, providing a tight connection between bacteria and host cells. The attachment of Fn-coated bacteria to host cells subsequently triggers the engagement and clustering of integrin α5β1, which in turn leads to characteristic signaling pathways in the host cell. A crucial outcome of these signaling events is the re-arrangement of the actin cytoskeleton, which is essential for final integrin-mediated uptake (Fig.1.4, Hauck, Christof R., 2006).

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Fig.1.4 Schematic summary of host cell signaling events induced by S.aureus engagement of integrin α5β1.S.aureus associates with host-derived Fn through FnBP. Fn deposited on the pathogen surface is recognized by the cellular Fn receptor, integrin α5β1. Bacteria-induced clustering of integrins leads to the local recruitment of structural proteins such as tensin, vinculin and zyxin, as well as signaling enzymes such as Src family PTKs and FAK, to the sites of bacterial attachment. The combined activity of FAK and Src results in tyrosine phosphorylation of multiple downstream effectors including cortactin.

Cortactin is functionally involved in bacterial internalization most likely by its influence on cytoskeleton rearrangements by the Arp2/3 complex or the regulation of endocytosis by dynamin. (Hauck, Christof R., 2006).

1.2. Phosphoinositides

1.2.1. General information of Phosphoinositides

A few decades ago, the structure of the cell membrane was deciphered and four different types of molecules, phospholipids, cholesterol, proteins and carbohydrates were classified. Phosphoinositides, since they only present a tiny portion of the total phospholipids, have not been interesting for many researchers for a long time, until they became known as the source of second messengers in transducing signals from cell surface receptors (Lemmon, Mark A. 2008; Balla, Tamas, 2013). For example, the cleavage of PI-4,5-P2 via Phospholipase C generates diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3), both of which function as second messengers, leading to calcium release and protein kinase C (PKC) activation (Brose, Nils, 2004; Kadamur, Ganesh, 2013). Afterwards, with the discovery of membrane proteins interacting with phosphoinositides, these lipids are becoming one of the most universal signaling

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entities in eukaryotic cells and have gained tremendous research interest. In mammalian cells, all phosphoinositides are derived from the same precursor, phosphatidylinositol, in short, PtdIns (see Fig.1.5)(Di Paolo, Gilbert, 2006), which is synthesized in the endoplasmic reticulum from CDP-DAG and myo-inositol by a PtdIns synthase (PIS) enzyme (Agranoff BW, 1958). In its inositol ring, there are five hydroxyl moieties, but only three of them (position -3, -4, and -5) are actually phosphorylated by the corresponding phosphoinositide kinases according to current information. Via these phosphorylation reactions, seven phosphoinositides are produced, including three phosphatidylinositol monophosphates (phosphatidylinositol 3-phosphate, PI3P;

phosphatidylinositol 4-phosphate, PI4P and phosphatidylinositol 5-phosphate, PI5P), three phosphatidylinositol bisphosphates (phosphatidylinositol 3.4-bisphosphate, PI- 3,4-P2; phosphatidylinositol 4.5-bisphosphate, PI-4,5-P2 and phosphatidylinositol 3.5- bisphosphate, PI-3,5-P2) and one phosphatidylinositol trisphosphate (phosphatidylinositol 3,4,5-trisphosphate, PI-3,4,5-P3)(see Fig.1.5)(Lemmon, Mark A.

2008; Sasaki, Takehiko, 2009). In cells, the phosphoinositides are much less abundant, compared to the three main acidic phospholipids, phosphatidylserine, phosphatidic acid and phosphatidylinositol, which make up approximately 8.5%, 1.5% and 1.0% of total lipid (by weight) in erythrocytes, respectively. Stephens et al. had estimated the approximate relative levels of phosphoinositides, for the monophosphates, PI3P, PI4P and PI5P, they represent to be around 0.002%, 0.05% and 0.002% of total lipid, respectively. About the phosphatidylinositol bisphosphates, PI-3,4-P2,PI-3,5-P2, PI- 4,5-P2, their relative levels are 0.0001%, 0.0001% and 0.05% (Lemmon, Mark A. 2008).

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Fig.1.5 Structures of the precursor phosphatidylinositol and its derivatives, the phosphorylated products, phosphoinositides. Phosphatidylinositol consists of a glycerol backbone that is esterified at its sn-1 and sn-2 positions to two fatty acids, and linked via phosphate at its sn-3 position to the D1 position of a D-myo-inositol head group. In mammalian cells, the hydroxyl groups at the D3, 4 and 5 positions of the myo-inositol head group can be phosphorylated by the phosphoinositide kinases to yield the indicated mono-phosphate, bis-phosphate and tris-phosphate phosphoinositide derivatives. The phosphorylation of the 2- and 6-hydroxyl groups has yet to be documented. (Sasaki, Takehiko, 2009).

Each phosphoinositide has different subcellular location in which it performs its distinct pivotal functions (Di Paolo, Gilbert, 2006; Van Meer, Gerrit, 2008). PtdIns is mainly enriched in the endoplasmic reticulum and PI3P is found in early endosomes, multivesicular bodies and phagosomes (Kutateladze, Tatiana G. 2006), PI4P is in the Golgi complex, however, the distribution of PI5P is not well understood, although it is reported that PI5P is found in several distinct subcellular locations, like nuclear (Jones, David R., et al. 2006) and plasma membrane (Sarkes, Deborah, 2010). PI-3,5-P2 is detected in late endosomes, whereas PI-3,4-P2, PI-4,5-P2 and PI-3,4,5-P3 are predominantly present in the plasma membrane (Stahelin, Robert V., 2014; Posor, York, et al. 2013) Among these phosphoinositides, PI-4,5-P2 has been particularly well studied, from its metabolism to its functions.

1.2.2. Functions of phosphoinositides

Although in the plasma membrane, the concentration of phosphoinositides is very low, they regulate a variety of cellular processes by the interactions with proteins containing

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inositide-binding modules with various degrees of selectivity (Cullen, Peter J., 2001;

Di Paolo, Gilbert, 2006; Lemmon, Mark A. 2008; Saarikangas, Juha, 2010; Shewan, Annette, 2011; Balla, Tamas, 2013). Among these inositide-binding motifs, Pleckstrin homology domains or PH domains are well studied and extensively used (Rameh, Lucia E., 1997; Lemmon, Mark A. 2003; Lemmon, Mark A. 2007). They are regions of approximately 120 amino acids that share sequence similarity with two such regions in pleckstrin, a major substrate of protein kinase C in platelets (Tyers, Michael, et al. 1989).

It is worth to note, these PH domains from different proteins bind to different phosphoinositides with distinct affinity and avidity (Lemmon, Mark A. 2008). Now the influence of PI-4,5-P2 on the actin cytoskeleton is well studied (Sechi, Antonio S., 2000;

Czech, Michael P. 2000; Yin, Helen L., 2003; Saarikangas, Juha, 2010). For example, ezrin, a membrane-cytoskeleton linker protein that can bind F-actin in its active conformation, has a FERM domain which binds PI-4,5-P2, inducing the relief of the autoinhibited state. Another example, Wiskott-Aldrich syndrome protein (WASP) family proteins, which promote actin assembly by activating the nucleating Arp2/3 complex, contain an NH2-terminal polybasic motif, that binds PI-4,5-P2 to get activated (Higgs, Henry N., 2000; Murray, Diana, 2005; Papayannopoulos, Venizelos, et al. 2005).

Integrin dependent cellular processes such as adhesion, migration, proliferation and survival rely on the dynamic interaction of integrin cytoplasmic tails with intracellular integrin binding proteins, for instance, cytoskeletal proteins (Brakebusch, Cord, 2003;

Delon, Isabelle, 2007; Ziegler, WolfgangH, et al. 2008; Huttenlocher, Anna, 2011).

Talin, vinculin and α-actinin, the well investigated members of cytoskeletal proteins, have emerged as critical players in linkage to the actin cytoskeleton and integrin activation (Otey, Carol A., 1990; Wang, Jia-huai 2012; Ziegler, WolfgangH, et al. 2008).

Interestingly, all these proteins contain PI-4,5-P2 binding domains(Fukami, Kiyoko, et al. 1994; Sjöblom, Björn, 2008; Yang, Jun, et al. 2014;). PI-4,5-P2 binds to the FERM motif in the talin head domain and disrupts the auto-inhibition between the talin head domain and rod domain and further promotes conformational unmasking of talin(Yang, Jun, et al. 2014). Vinculin, a modular protein composed of a 95kDa N-terminal, globular head domain followed by a short proline rich region to a 30-kDa tail domain,

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functions as a central regulator of adherens junctions and focal adhesions. The PI-4,5- P2 binding of vinculin via its tail domain promotes oligomerization and focal adhesion activity of vinculin (Chinthalapudi, Krishna, et al. 2014).

Moreover, PI-4,5-P2 also has been implicated in endocytosis(Boucrot, Emmanuel, et al.

2006; McMahon, Harvey T., 2011; Posor, York, 2015). In clathrin-mediated endocytosis, PIP5KIγ90 associates with the μ-subunit of the AP-2 adaptor complex via the C-terminal 26aa (or 28aa in human) extension and locally produces PI-4,5-P2, the synthesis of PI-4,5-P2 facilitates the initiation of clathrin-coated pit (CCP) formation.

During the later stages of this process, de-phosphorylation of PI-4,5-P2 regulates CCP maturation and vesicle uncoating (Posor, York, 2015).

1.2.3 Phosphoinositide kinase

As mentioned above, through the combined phosphorylation catalyzed by certain phosphoinositide kinases, the seven known phosphoinositides are synthesized.

Theoretically, PI-4,5-P2 could be produced through the activity of two distinct phosphoinositide kinases, one is the type I phosphatidylinositol-4-phosphate 5-kinase family, in short, PIP5K, which could catalyze phosphorylation of PI4P; the other is the type II phosphatidylinositol-5-phosphate 4-kinases, PIP4K, which use PI5P as the substrate. Since in mammalian cells, the PI4P level is much higher than PI5P, it is generally acknowledged that the majority of cellular PI-4,5-P2 is produced by PIP5K (Sasaki, Takehiko, 2009; Van den Bout, Iman, 2009). So far, three isozymes of PIP5K have been identified and are known as PIP5KIα, PIP5KIβ and PIP5KIγ. In addition, there are at least two splicing variants of PIP5KIγ. Based on their molecular sizes, they are named PIP5KIγ87 and PIP5KIγ90, respectively (Legate, Kyle R., et al. 2011;

Legate, Kyle R., et al. 2012).

For each isoform, the subcellular localization and function is different (Di Paolo, Gilbert, 2006; Sasaki, Takehiko, 2009). PIP5KIα is normally enriched in the plasma membrane and the Golgi complex, and has also been observed at sites of membrane ruffling induced by the Rho GTPase Rac and in the nucleus (Boronenkov, Igor V., et al.

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1998; Orth, James D., et al. 2002). PIP5KIβ generally localizes to the plasma membrane but is also found on vesicles in the perinuclear region of the cell. PIP5KIγ is present at the plasma membrane, with at least one isoform (PIP5KIγ90) enriched at focal adhesions and adherence junctions. PIP5KIα^-/-mice are born at the expected Mendelian ratio, are healthy and fertile, and show no developmental abnormalities, but also display enhanced passive cutaneous and systemic anaphylaxis (Sasaki, Junko, et al. 2005). For PIP5KIβ, the knockout mice appear grossly normal and survive to adulthood, but they are born at less than the expected ratio, breed poorly and produce few offspring (Wang, Yanfeng, et al. 2008). The role of PIP5KIγ in embryonic development was also clarified.

PIP5KIγ^-/-mice die a few hours after birth with an apparent inability to feed, but the PIPKI90^-/-mice are born at normal Mendelian ratios and are viable and fertile, which means at least one PIP5KIγ specific function is essential for life and PIP5KIγ90- specific functions are dispensable (Legate, Kyle R., et al. 2012).

PIP5KIγ90, the research target in this study, has a 26 (or 28aa in human) amino acids extension at its carboxyl-terminus compared to the other isoform PIP5KIγ87, and this extension binds to the FERM domain of talin, one important component of focal adhesions. Via the association with talin, PIP5KIγ90 is targeted to the focal adhesions and gets activated. Interestingly, PIP5KIγ90 is also critical for talin targeting to focal adhesions, and the kinase activity is required for this targeting.

1.2.4. Phosphoinositide phosphatases

Phosphoinositide phosphatases, together with phosphoinositide kinases, manage the conversion of PIs between distinctive phosphorylation states. About phosphoinositide 5-phosphatases, which hydrolyze the D5 phosphate of the inositol ring, there are more than 10 members discovered (Fig.1.6)(Sasaki, Takehiko, 2009; Hsu, Fo Sheng, 2015).

The first 5-phosphatase gene cloned in the early 90s is named INPP5A (Speed, Caroline J., et al. 1996). It has a molecular weight of 43-kD and hydrolyses the soluble inositol polyphosphates Ins(1,4,5)P3 (or IP3, a secondary messenger molecule from the PLC mediated PI-4,5-P2 hydrolysis) and Ins(1,3,4,5)P4. Currently, only SYNJ1, OCRL and

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SHIPs are comparably well investigated.

Fig.1.6. Schematic domain organization of 5-phosphatases. Abbreviations: 5-ptase, 5-phosphatase catalytic domain; PH, pleckstrin homology; ASH, ASPM-SPD2-Hydin; CB, clathrin binding; RBD, RNA binding domain; SH2, Src homology 2; SKICH, SKIP carboxyl homology (Hsu, Fo Sheng, 2015)

SYNJ1 protein contains an N-terminal suppressor of actin1 (Sac1) domain that predominantly dephosphorylates PI(3)P and PI(4)P, a central 5-Ptase domain, and a C- terminal proline-rich region (PRD). In mammalian cells, there are two major splice variants, SYNJ1–145 and SYNJ1–170, based on their molecular sizes (Fig.1.7) (Perera, Rushika M., et al. 2006; Drouet, Valérie, 2014.). SYNJ1–145 is present at high levels in the brain, while SYNJ1–170 is ubiquitously expressed in the non-neuronal cells. The PRD in both SYNJ1–145 and SYNJ1–170 binds to the SH3 domains of endocytic proteins such as endophilin, amphiphysin, syndapin/pacsin and intersectin/Dap160.

Compared to SYNJ1–145, SYNJ1–170 protein has an additional C-terminal 30 kDa extension that has three asparagine-proline-phenylalanine (NPF) repeats. The NPF repeat region is essential for the recognition of SYNJ1–170 by Eps15 homology (EH) domains, which are essential components of accessory proteins that are involved in endocytosis, cytoskeletal reorganization, neuronal development and growth factor signaling. SYNJ1–170 also bears a C-terminal tail that contains binding sites for

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clathrin and clathrin adaptor protein complex 2 (AP2) (Haffner, Christof, et al. 2000).

All these protein-protein interactions indicate SYNJ1–170 participates in endocytosis.

Interestingly, the 5-phosphatase activity and interactions with partner proteins of SYNJ1 is affected by its phosphorylation status. The phosphorylation of tyrosines in the SYNJ1-145 PRD by EphB2 inhibits the interaction with endophilin, but not the interaction with amphiphysin and suppresses the 5′-phosphatase catalytic activity (Irie, Fumitoshi, et al. 2005; Hopper, Neil A., and Vincent O'Connor. 2005). In addition, the phosphorylation of SYNJ1-145 on Ser1144 in PRD by cyclin-dependent kinase 5(cdk5) inhibits both SYNJ1 catalytic activity and the interactions with endophilin-1 and amphiphysin (Lee, Sang Yoon, et al. 2004). However, the effect of protein phosphorylation is not investigated in SYNJ1-170 yet.

Fig.1.7: Functional and interaction domains of the two major isoforms of SYNJ1. The 145 kDa (top) and the 170 kDa (bottom) SYNJ1 isoforms harbor two functional inositol phosphatase domains, an Nterminal Sac1 domain and a more central 5�-phosphatase domain. Several protein-protein interaction domains are found in the Cterminal part of the proteins: one or two PRD domains, AP2 binding motifs (WxxF, FxDxF, and DxF, in pink), and Eps15 binding motifs (NPF: asparagine-proline-phenylalanine, in blue). The homozygous mutation Arg258Gln, found in Parkinson’s disease patients, is indicated in red.

Numbers indicate the amino acid positions along the proteins. Sac1: suppressor of actin1; PRD: proline- rich domain; AP2: adaptor protein complex 2; Eps15: epidermal growth factor receptor pathway substrate 15. (Drouet, Valérie, 2014).

OCRL was originally identified as the product of the gene responsible for the oculocerebrorenal syndrome of Lowe (Attree, Olivier, et al. 1992). It is a multi-domain protein containing a conserved central inositol 5-phosphatase domain, followed by an

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ASH (ASPM, SPD2, hydin) domain and the RhoGAP-like domain. OCRL primarily performs the de-phosphorylation of the 5-phosphates of PtdIns(4,5)P2, PtdIns(3,4,5)P3, Ins(1,4,5)P3 and Ins(1,3,4,5)P4. Due to a critical amino acid change (arginine to glutamine) in the RhoGAP-like domain, the RhoGAP-like domain of OCRL is catalytically inactive (Peck, Jeremy, et al. 2002). Via the RhoGAP-like domain, OCRL interacts with the Rho family small GTPases Rac1, Cdc42, ARF1 and ARF6 (Lichter- Konecki, U., et al. 2006; Sasaki, Takehiko, et al. 2009; Pirruccello, Michelle, 2012).A loop of the RhoGAP-like domain of OCRL contains a classical clathrin-box motif (LIDID) that binds the clathrin heavy chain (Erdmann, Kai S., et al. 2007). Currently, there are two reported alternative splice variants of OCRL, termed OCRL1a and OCRL1b (Johnson, Jason M., et al. 2003). OCRL1a and OCRL1b are both expressed in almost all tissues but only OCRL1a is expressed in brain. OCRL KO mice have no major obvious defects, but OCRL/INPP5B double KO mice die embryonically, suggesting that the absence of abnormality in OCRL KO mice may be due to partial functional redundancy with INPP5B(Jänne, Pasi A., et al. 1998).

1.3. Integrins

1.3.1. Integrin family and structure

Integrins are a superfamily of type I transmembrane and heterodimeric proteins that are used by cells to transmit information from the extracellular environment to activate many intracellular signaling pathways, such as cell survival, cell proliferation, cell shape, and angiogenesis and so on(Giancotti, Filippo G., 1999; Harburger, David S., 2009). In vertebrates, there are 18α subunits and 8β subunits which are non-covalently associated to assemble into 24 different receptors (Hynes, Richard O. 2002; Humphries, Jonathan D., 2006). The molecular size varies, but typically the α- and β-subunits contain around 1000 and 750 amino acids, respectively. It is worth noting that Integrin α and β subunits are totally different, without detectable homology between them; even among α subunits, sequence identity is only about 30% and among β subunits 45%

(Takada, Yoshikazu, 2007). In addition, these receptors have different binding

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properties and different tissue distribution, and hence are grouped into several subsets.

For example, only 8 integrin heterodimers have been shown to recognize the RGD motif in the native ligands, fibronectin (Fig.1.8).

Both α and β subunits consist of a large extracellular domain that binds to a variety of ligands, like ECM proteins collagen, laminin and fibronectin (Fig.1.9) (Humphries, Jonathan D., 2006; Barczyk Malgorzata, 2010). Fibronectin functions as a molecular bridge in Integrin-dependent S.aureus uptake (Hauck, Christof R., 2006). In contrast to the large extracellular domain which is followed by a single-spanning transmembrane helix, integrins have only a short cytoplasmic domain (with the exception of β4) of about 20-60 amino acids in average (Luo, Bing-Hao, 2007). Coming to the detailed structure of each subunit, the α-subunit is composed of a seven-bladed β-propeller, which is connected to a thigh, a calf-1, and a calf-2 domain, together forming the extracellular region (Fig.1.9)(Barczyk Malgorzata, 2010). Nine of the α-subunits contain an I domain, also called the A domain, which is approximately 200 amino acids long, and is inserted between the second and third β-propeller (Larson, Richard S., et al. 1989; Tuckwell, Danny, et al. 1995). The extracellular domain of the β subunits contains a plexin-sempahorin-integrin (PSI) domain, a hybrid domain, a βI domain, and four cysteine-rich epidermal growth factor (EGF) repeats (Fig.1.9) (Barczyk Malgorzata, 2010).

Via the stimulation by various integrin ligands, including proteins and ions, for example, fibronectin, talin and Mn²⁺,the conformation of integrins is strictly regulated (Banno, Asoka, 2008; Campbell, Iain D., 2011). With electron microscopy, Takagi et al.

Fig.1.8. The Integrin Receptor Family. The figure depicts the mammalian subunits and their αβ associations; 8 β subunits can assort with 18α subunits to form 24 distinct integrins. These can be considered in several subfamilies on evolutionary relationships (coloring of α subunits), ligand specificity and, in the case of β2 and β7 integrins, restricted expression on white blood cells. (Hynes Richard O. 2002)

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investigated the different conformations of integrin αVβ3. In the presence of Ca²⁺, a cation that stabilizes integrins in the inactive or low affinity conformation, predominantly bent structures were observed. In bent integrins, the ligand-binding pocket may be oriented towards the plasma membrane, thereby impeding ligand engagement, but flexibility at the juxtamembrane domain could enable a “breathing”

movement for the conversion of bent to extended integrin. In the presence of Mn²⁺, integrins present an extended shape with a closed headpiece. With the addition of Ca²⁺

and RGD peptide, the third extended shape with open headpiece was observed, which stands for the active, high affinity conformation (Fig1.9) (Takagi, Junichi, et al. 2002;

Campbell, Iain D., 2011). The regulation of integrins activity is a fundamental process occurring in a variety of physiological events including embryogenesis, maintenance of tissue integrity, angiogenesis and immune response.

Fig.1.9. Representation of a prototypical αI-domain-containing integrin heterodimer. Nine out of the 18 integrin α chains contains an αI domain, as shown, but all integrins contain a βI domain in the βsubunit. Left-upper, Representation of the domains in αI domain-containing integrin (stars divalent cation-binding sites) (Barczyk Malgorzata, 2010); Left-lower, Electron micrographs of negatively stained αVβ3 integrin (Takagi, Junichi, et al. 2002); Right, Cartoon representation of bent and upright conformations showing approximate dimensions (Campbell, Iain D., and Martin J. Humphries. 2011).

To perform their functions properly, the activity state of integrins must be spatially and temporally controlled, in an accurate way. Now it is well known that integrins can be activated by proteins or other factors binding to their extracellular or their intracellular domain, which are termed as outside–in and inside–out signaling, respectively (Shattil,

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Sanford J., 2010; Bouvard, Daniel, et al. 2013). As a result, integrins possess the unique ability to signal bidirectionally. As shown in Fig.1.10, outside–in signaling is initiated by binding of the integrin extracellular domain to the ECM, like fibronectin. The engagement of integrin with ECM triggers the formation of a protein complex that binds to the actin cytoskeleton and activates several subsequent signaling pathways (Giancotti, Filippo G., 1999). The inside-out signaling is triggered by the binding of the integrin cytoplasmic tail to their ligands, such as talin and kindlin. Talin is activated by PI-4,5- P2 that is locally produced by recruited PIP5KIγ90. Active talin binds the Integrin β subunit, therefore disrupting the salt bridge between the integrin α and β subunits, changing the tilt angle of the β‑integrin transmembrane domain and in turn releasing the interactions at the interface between the transmembrane domains of the α- and β‑subunits (Bouvard, Daniel, et al. 2013).

Fig.1.10. Integrin activation. The Outside-in and Inside-out activation is dependent on the binding of integrins to their ligands (Bouvard, Daniel, et al. 2013).

1.3.2. Integrin in pathogen invasion

For bacterial colonization, a firm binding to host determinants is often a pre-requisite.

Via the interactions between cell adhesion molecules and bacterial invasins, the intimate contact with host cells and tissues is established. The cell adhesion molecules, including integrin, cadherin and immunoglobulin-related cell adhesion molecule

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(IgCAM) families are common targets for exploitation by distinct bacterial invasins (Table.1.2) (Hauck, Christof R. 2002; Scibelli, Antonio, et al. 2007; Hoffmann, Christine, 2011; Hauck, Christof R., 2012). Among them, integrins are well studied during bacterial internalization. Interestingly, bacteria utilize two distinct modes of integrin engagement: either, the microbial proteins directly bind to integrins (e.g.

invasion of Yersinia enterocolitica or Y. pseudotuberculosis, the Ipa proteins of Shigella flexneri, and the CagL protein of Helicobacter pylori) or the microbes indirectly associate with integrins via the recruitment of ECM proteins as found in the uptake of Staphylococcus aureus, Streptococcus pyogenes, Porphyromonas gingivalis, Neisseria gonorrhoeae, N.meningiditis, Bartonella henselae, or Mycobacterium leprae (Hoffmann, Christine, 2011). These two possibilities are not mutually exclusive, because some bacteria use both direct engagement of integrins as well as indirect binding via ECM proteins. This is best exemplified by Y. enterocolitica, which executes invasion for direct integrin β1 binding as well as YadA for binding via ECM proteins such as collagen and laminin (Roggenkamp, Andreas, et al. 1995; Isberg, Ralph R., 2000; Hoffmann, Christine, 2011).

Table.1.2 Pathogens targeting cell adhesion molecules (Hauck, Christof R., et al. 2006)

Species ECM protein/receptor

Integrins

Borrelia burgdorferi FN/β1 integrins

Mycobacterium leprae FN, LN/β1 and β4 integrins

Mycobacterium bovis BCG FN/β1 integrins

Neisseria gonorrhoeae and N. meningitidis FN, VN/β1and β3 integrins

Porphyromonas gingivalis β1 integrins

Shigella flexneri β1 integrins

Staphylococcus aureus FN, LN, Col/β1 integrins

Streptococcus pyogenes and S.dysgalactiae FN/β1 integrins Yersinia pseudotuberculosis and Y. enterocolitica β1 integrins Immunglobulin-related cell adhesionmolecules (IgCAMs)

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Haemophilus influenzae CEACAMs

Moraxella catarrhalis CEACAMs

Neisseria gonorrhoeae and N.meningitidis CEACAMs Cadherins

Listeria monocytogenes E-cadherin

Take S.aureus as an example, the role of integrins in bacterial uptake had been investigated. After the preincubation of cells with function-blocking anti-α5β1 antibodies, the internalization of S.aureus was markedly reduced, more than 50%.

(Sinha, Bhanu, et al. 1999). In summary, the association of fibronectin-binding protein on S.aureus surface with ECM component Fn allows bacteria to use Fn as molecular bridge to interact with host cell β1 integrin, which leads to clustering and activation of integrins. In turn, integrin-initiated signaling events, such as recruitment of a focal adhesion-like protein complex, and the rearrangement of the actin cytoskeleton result in the uptake of the ECM-coated bacteria. Several well-characterized integrin- associated proteins, e.g. vinculin, paxillin, zyxin, tensin, FAK and c-Src have been shown to be recruited in the vicinity of attached S.aureus (Agerer, Franziska, et al. 2005;

Schröder, Andreas, et al. 2006).

1.4. Focal adhesion complex

Focal adhesions, which are also called cell-matrix adhesions, are formed at the plasma membrane at the sites where cells touch their substrates. They are large and dynamic protein complexes through which integrins and scaffold proteins link to the actin cytoskeleton to the extracellular matrix, therefore regulating cellular processes including survival, gene expression, differentiation, migration and cell division (Wozniak, Michele A., et al. 2004; Geiger, Benjamin, 2009; Ivaska, Johanna. 2012).

There are four distinct types of cell adhesions with characteristic morphology and molecular composition: (1) classical focal adhesions, typically generated by interaction with a flat, rigid surface; such adhesions are usually several square micrometers in size, located at the ends of actin stress fibers, and stimulated by the small GTPase RhoA, (2)