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«Dissertation zur Erlangung des akademischen Grades des Doktors der Naturwissenschaften (Dr. rer. nat.) eingereicht im Fachbereich Biologie,Chemie, ...»

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Adherence to and invasion of epithelial cells by

Neisseria gonorrhoeae

Dissertation zur Erlangung des akademischen Grades des Doktors der

Naturwissenschaften (Dr. rer. nat.)

eingereicht im Fachbereich Biologie,Chemie, Pharmazie

der Freien Universität Berlin

vorgelegt von

Daniel de Graaf

aus Berlin

Januar 2009

1. Gutachter: Prof Dr. Thomas F. Meyer

2. Gutachter: Prof. Dr. Petra Knaus

Disputation am 23. November 2009

II

This work was prepared at the Max Planck Institute for Infection Biology in the department of Molecular Biology in Berlin under the supervision of Prof. Dr. Thomas F. Meyer

Parts of this work will be published under the following title:

de Graaf D, Kirchner M, Churin Y, Meyer TF Role of cAMP-dependent protein kinase in Neisseria gonorrhoeae invasion of epithelial cells (In preparation) III Dedicated to my family IV The greatest obstacle to discovery is not ignorance – it is the illusion of knowledge.

Daniel J. Boorstin V Table of contents Table of contents

1.1

Abstract

1.2 Zusammenfassung

Introduction

2.1 Bacteria – host cell interactions

2.1.1 Bacterial adherence to and invasion of target cells

2.2 Neisseria spp

2.2.1 Meningococcal pathogenesis

2.2.2 Gonococcal pathogenesis and clinical manifestations

2.2.3 N. gonorrhoeae adhesion to and invasion of epithelial cells.................. 8 2.2.3.1 Type IV pili

2.2.3.2 Opa proteins

2.2.3.3 Lipooligosaccharides (LOS)

2.2.3.4 Porin

2.2.3.5 Models for N. gonorrhoeae invasion

2.3 Caveolae and lipid rafts

2.3.1 Biochemical properties

2.3.2 Structure of caveolae and caveolins

2.3.3 Function of caveolae

2.3.3.1 Caveolae involvement in cellular signaling events

2.3.3.2 Caveolae and endocytosis

2.3.4 Role of lipid rafts and caveolae in bacterial infections

Objectives

Results

4.1 Microarray analysis of infected AGS vs infected caveolin-1 expressing AGS cells

4.2 Confirmation of microarray results by quantitative real-time PCR (qRT-PCR)

4.3 Impact of stimulation and inhibition of PKA activity on N. gonorrhoeae internalization

VI

4.4 N. gonorrhoeae activates the PKA pathway of epithelial cells

4.5 Recruitment of VASP to infecting N. gonorrhoeae

4.6 VASP knockdown affects N. gonorrhoeae internalization

4.7 AC/PKA pathway influences N. gonorrhoeae association with caveolin and lysosomal compartments

4.8 ErbB2 and Src in N. gonorrhoeae infection

4.9 N. gonorrhoeae triggered actin and VASP dynamics

Discussion

5.1 AC/PKA pathway in pilus mediated N. gonorrhoeae invasion

5.2 VASP and N. gonorrhoeae infection

5.3 Actin recruitment, dynamics and actin-caveolin interplay

5.4 Signalling through caveolae/lipid rafts relevant to N. gonorrhoeae internalization

Conclusion

Materials

7.1 Bacteria

7.1.1 E. coli

7.1.2 N. gonorrhoeae

7.2 Cell culture

7.3 Cell culture media and supplements

7.4 Media for bacterial culture

7.5 Plasmid vectors

7.6 Oligonucleotides

7.7 Antibodies

7.8 Buffers and solutions

7.9 Chemical reagents

7.10 Kits

7.11 Appliances and consumable materials

7.12 Software

Methods

8.1 Cell culture methods

VII 8.1.2 Transfection of cells

8.1.2.1 Transfection of plasmid DNA

8.1.3 Infection of cells with N. gonorrhoeae

8.1.4 Gentamicin protection assay

8.2 Growth and manipulation of bacteria

8.2.1 Growth of N. gonorrhoeae

8.2.2 Growth of E. coli

8.2.3 Preparation of DNA competent E. coli

8.2.4 Transformation of E. coli

8.3 Nucleic acid methods

8.3.1 Preparative isolation of plasmid DNA

8.3.2 RNA purification from adherent cells

8.3.3 Quantitative real-time polymerase chain reaction (qRT-PCR)............. 78 8.3.4 RNA interference (RNAi)

8.4 Protein biochemical methods

8.4.1 Discontinuous SDS polyacrylamide gel electrophoresis (SDS-PAGE) 80 Coomassie® stain of protein gels

8.4.2 8.4.3 Immuno blot (Western blot)

8.4.4 Membrane stripping

8.4.5 Protein co-immuno precipitation

8.4.6 Purification of PilC

8.5 Microscopy

8.5.1 Confocal laser scanning microscopy

8.5.1.1 Indirect immunofluorescence staining

8.5.1.2 Differential indirect immunofluorescence staining of bacteria...... 84 8.5.1.3 Confocal laser scanning microscopy

8.5.2 Life cell imaging microscopy

References

Index

10.1 Figure Index

10.2 Abbreviations

10.3 Curriculum vitae

10.4 Acknowledgements

VIII 1.1 Abstract

1.1 Abstract

The type VI pili (Tfp) of the Gram-negative bacterium Neisseria gonorrhoeae (Ngo) are crucial virulence factors for the colonization of its sole natural host, Homo sapiens. They do not only mediate the first step in infection by adhering to the host cell surface, but also contribute to other adhesion and invasion processes in concert with other virulence factors like the colony opacity associated (Opa) proteins and lipooligosaccharides (LOS). Furthermore, they are able to promote invasion into some cell lines.





Recently, it was shown that piliated gonococci elicit clustering of the membraneassociated protein caveolin-1 beneath attachment sites, and that downregulation of caveolin by RNA interference (RNAi) increased gonococcal invasion.

Complementarily, expression of caveolin-1 in caveolin-deficient epithelial cells blocked internalization. Here, it is demonstrated in a microarray experiment comparing gene regulation of infected epithelial cells with and without caveolin, that the regulatory subunit of the protein kinase A (PKA-RIβ) was highly upregulated in both cell lines. Pharmacological inhibition of PKA resulted in a strong increase of invasion in AGS and ME180 epithelial cells, whereas PKA agonists had the opposite effect. Adenylyl cyclase (AC) and PKA activity were increased during the first two hours of Ngo infection, starting around 10 minutes after the addition of bacteria and coinciding with pilus-mediated attachment. The PKA substrate vasodilator stimulated phosphoprotein (VASP) was phosphorylated in response to Ngo infection and enriched at the sites of attaching bacteria. Moreover, alteration of PKA activity had strong impact on caveolin-1 recruitment to gonococcal microcolonies. These findings suggest a role for PKA and VASP in the invasion process of N. gonorrhoeae by contributing to the assembly of an actin-caveolin network that blocks internalization during localized adherence.

Using life cell imaging microscopy, actin and VASP distribution and dynamics could be monitored, providing insights into the formation of actin clusters beneath gonococcal microcolonies. In addition, actin- and VASP rocketing was also observed in epithelial cells, and infection influenced actin-based comet tails. Whether internalized gonococci, like other microorganisms, have the capacity to induce or hijack actin comet tails to move intracellularly could not ultimately be proven.

Zusammenfassung

1.2 Zusammenfassung Die Typ IV Pili (Tfp) des Gram-negativen Bacteriums Neisseria gonorrhoeae sind wichtige Virulenzfaktoren für die Besiedelung ihres einzigen natürlichen Wirtes Homo sapiens. Diese vermitteln den ersten Infektionsschritt durch Adherenz an die Wirtszelloberfläche und tragen, gemeinsam mit anderen Faktoren wie Opa Proteinen und Lipooligosacchariden (LOS), zusätzlich zu anderen Adherenzvorgängen und zu Invasionsprozessen bei. Darüber hinaus verstärken Pili die bakterielle Invasion in bestimmte Zelllinien.

Vor kurzem konnte gezeigt werden, dass pilierte Gonokokken das membranassozierte Protein Caveolin unterhalb der Bindungsstelle auf Epithelzellen akkumulieren und das Ausschalten der Caveolinexpression mittels RNA-Interferenz (RNAi) die Gonokokkeninvasion erhöht. Komplementär hierzu wurde die Invasion durch Caveolinexpression in Zellen, welche normalerweise kein Caveolin synthetisieren, blockiert. In dieser Arbeit konnte bei dem Vergleich von infizierten Zellen mit und ohne Caveolin mit Hilfe eines Microarray-Experimentes eine starke Überexpression der regulatorischen Untereinheit der Proteinkinase A (PKA-RI) in beiden Zelllinien festgestellt werden. Pharmakologische Untersuchungen zeigten einen starken Anstieg der Invasion durch PKA-Inhibierung, eine Behandlung der Zellen mit PKA-Agonisten hingegen resultierte in verminderter Invasion. Die enzymatische Aktivität der Adenylylzyklase (AC) und der PKA war während der ersten beiden Stunden einer Gonokokkeninfektion erhöht. Dieser Effekt trat bereits zehn Minuten nach Bakterienzugabe auf und damit zeitgleich mit dem Beginn der Pilus-vermittelten Adherenz.

Das PKA-Substrat „vasodilator stimulated phosphoprotein“ (VASP) wurde durch Neisserieninfektion phosphoryliert und zu den Adherenzstellen der Bakterien rekrutiert. Des Weiteren wurde die Rekrutierung von Caveolin-1 zu bakteriellen Mikrokolonien durch die Beeinflussung der PKA-Aktivität stark verändert. Dies zeigt, dass PKA und VASP in Invasionsprozessen von N. gonorrhoeae beteiligt sind, indem diese zum Aufbau eines Caveolin-Actin-Geflechts beitragen, welche die Aufnahme der Bakterien in die Zelle hemmt.

Mittels „life cell imaging“ Mikroskopie konnte die Verteilung und Dynamik von Aktinund VASP-GFP Molekülen beobachtet werden, welche Einblicke in die Entstehung von Aktin-Clustern unterhalb von bakteriellen Mikrokolonien ergaben. Darüber hinaus

Zusammenfassung

wurden dynamische Strukturen, welche als Aktin- bzw. VASP-Kometen bezeichnet werden, in den verwendeten Epithelzellen entdeckt. Diese auf Aktinpolymerisation basierenden Strukturen wurden durch Neisserieninfektion beeinflusst. Ob intrazelluläre Gonokokken diese als Fortbewegungsmittel nutzen oder selbst induzieren, konnte nicht zweifelsfrei geklärt werden.

–  –  –

2 Introduction

2.1 Bacteria – host cell interactions In the long history of relations between bacteria and their human host, different bacterial species have evolved multiple strategies to survive in their differing biological niches. The different consequences of these strategies, such as host specificity, tropism and pathogenicity, have a basis at molecular level. That is humanmicrobe relations are determined by interactions of protein, carbohydrate and lipid macromolecules on both sides. Therefore, it is the major goal in infection biology to decipher these molecular interactions occurring between the host cell and the pathogen.

2.1.1 Bacterial adherence to and invasion of target cells In general, the first step of an infection process is the attachment of the microbe to the host cell. Binding to target cells is either followed by extracellular colonization of the specific tissue or by invasion of cells to establish intracellular accommodation.

Chlamydia spp for example are obligate intracellular organisms that multiply via a complex life cycle which can only be accomplished inside the cell in a special compartment termed inclusion (AbdelRahman and Belland, 2005). Although adhesion and invasion processes are crucial for Chlamydia infection, as they are for the infection of other bacteria, the underlying mechanisms for both events are still poorly understood (Ward and Murray, 1984; Allan and Pearce, 1987).

Pathogens eliciting enteric diseases exhibit elaborate mechanisms to invade host cells. Salmonella enterica which causes diarrhoea and typhoid fever, is able to adhere to and invade different cell types such as intestinal epithelial cells, microfold (M) cells and macrophages to overcome the epithelial barrier of the intestine (Haraga et al., 2008). Invasion of epithelial cells is preceded by the delivery of effector proteins by a secretion machinery designated type three secretion system (T3SS) into the cytosol, which leads to the rearrangement of the actin cytoskeleton and, in turn, to the engulfment and uptake of the microbe (Hardt et al., 1998; Unsworth et al., 2004). Shigella flexneri, another pathogen that infects the intestinal epithelium (LABREC et al., 1964), lacks, unlike Salmonella, adherence factors or flagella.

Despite this, Shigella efficiently invades M-cells, Macrophages and epithelial cells.

Introduction

The latter is stimulated by the interaction of the T3SS-effector IpaB to the cellular CD44 receptor, which is present in membrane areas rich in lipid rafts (Skoudy et al., 2000).

Escherichia coli, which normally constitute the gut flora in humans maintains a symbiosis with its host, but can also cause diarrhea. So called enteropathogenic (EPEC) and enterohaemorrhagic E. coli (EHEC) colonize the gut mucosa by establishing attaching and effacing (A/E) lesions. These lesions are characterized by loss of microvilli and close association of the bacteria with the enterocyte plasma membrane (Chen and Frankel, 2005). The association of EPEC and EHEC with the host cell is mediated via the adhesin intimin which binds to the translocated intimin receptor (Tir) that is introduced into the host cell membrane via T3SS (Kenny et al., 1997). Actin polymerization, which gives rise to pedestal formation underneath bacteria, is then triggered via the direct binding of the host adaptor protein Nck to Tir or indirectly through the bacterial T3SS injected effector protein TccP/EspFU (Campellone et al., 2004). Although actin polymerization is not thought to be involved in A/E lesion formation, it plays an as yet unidentified role in colonization (Bai et al., 2008).



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