STRUCTURAL AND FUNCTIONAL STUDIES ON TYPE III AND TYPE VI SECRETION SYSTEM PROTEINS JOBICHEN CHACKO (M Sc Agriculture) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES, FACULTY OF SCIENCE, NATIONAL UNIVERSITY OF SINGAPORE 2008 To my dear parents ii ACKNOWLEDGEMENTS This thesis is the most significant scientific accomplishment in my life so far and it is my pleasure to thank all those who made this possible and supported me in one way or other. First and foremost I would like to thank Almighty God for blessing me with the opportunity to complete my PhD studies. I am grateful to my PhD supervisor, Assoc Prof Jayaraman Sivaraman. With his enthusiasm, inspiration and great efforts to explain things clearly and simply, he helped me understand what crystallography is. Without his patience and help, the structure of this thesis would not have been possible. I am also indebted to my co-supervisor A/P Leung Ka Yin for his enthusiasm, support, sincerity and hard work. I am extremely grateful to Prof Adrian Compoy for analysing the ITC data, A/P Markus Wenk and Dr Aaron Fernandis for mass spectrometry and lipid identification experiments, Prof Ilan Rosenshine and Dr Henry Mok for helping in manuscript preparation. I am also thankful to Dr Li Mo, Dr Zheng Jun, and Gal Yerushalmi for all their help. I would like to thank Dr Anand Saxena and Dr J Seetharaman who helped me during my data collection at National Synchrotron Light Source, USA. I acknowledge the services iii provided by Protein and Proteomics Centre (NUS) and thank Mr Sashikant Joshi for all his help. I thank Dr Liang Zhao-Xun (SBS, NTU) and Haiwei SONG (IMCB, Singapore) for allowing me to use the analytical ultra centrifugation facilities. I would like to thank all my lab mates for their help and support especially, Yvonne, Lissa, Sunita and Rajesh. My sincere thanks to A/P K Swaminathan and all members of Structural Biology Lab 4, especially Shiva for their support and help. I am indebted to my peers at NUS for providing a stimulating and fun environment in which I could learn and grow. I am grateful to all my friends especially Dileep Vasudevan, Dileep G R and Jinu Paul. I offer my special thanks to my parents, who constantly encouraged me throughout my life in all my endeavors. Without their support this work would not have been possible. I am also thankful to my sisters and their families for their support. I am also indebted to all my family friends and relatives in Singapore for their help and support during my stay. Finally a big THANK YOU to Rose, my wife. The NUS research scholarship, which supported my research and stay in Singapore, is greatly acknowledged. Thank you all. iv Table of contents Page No: Acknowledgements iii Table of contents v Summary ix List of tables xii List of Figures xiii List of abbreviations xvi Publications xx Chapter 1: General Introduction 1.1 Host Pathogen Interaction 1.2 Pathogenicity islands 1.3 Protein Secretion System 1.4 Sec system 1.5 Type I Secretion System 1.6 Type II Secretion System 10 1.7 Type III Secretion System 12 1.8 Type IV Secretion System 26 1.9 Type V Secretion System 27 1.10 Type VI Secretion System 29 1.11 Aim of this thesis 36 v Chapter 2: Structure of GrlR and the implication of its EDED motif in mediating the regulation of type III secretion system in enterohemorrhagic Escherichia coli (EHEC) 37 2.1 Introduction 38 2.2 Materials and Methods 41 2.2.1 Plasmid and strain construction 41 2.2.2 Purification and crystallization. 42 2.2.3 Data collection, structure solution and refinement 43 2.2.4 In vitro pull-down assay 44 2.2.5 Analytical ultra centrifugation 45 2.2.6 MALDI-TOF MS and MS/MS analysis 45 2.2.7 Circular dichroism spectrometry 46 2.2.8 Extracellular proteins isolation and assay 46 2.3 Results 47 2.3.1 Characterization of GrlR 47 2.3.2 Structure of GrlR 51 2.3.3 Dimers of GrlR 57 2.3.4 EDED Motif Is Essential for the Recognition of GrlA 60 2.3.5 Regulatory Function of GrlR Is Mediated by EDED Motif 63 2.4 Discussion 65 Chapter 3: Structural Basis for the Lipid Recognition of GrlR, a Locus of Enterocyte Effacement Regulator 67 3.1 Introduction 68 3.2 Materials and methods 69 3.2.1 Protein purification and Crystallization 69 vi 3.2.2 Data collection, structure solution and refinement 70 3.2.3 Isothermal titration calorimetry 71 3.2.4 Lipid extraction 72 3.2.5 Mass spectrometry analysis 72 3.3 Results 73 3.3.1 GrlR has a lipocalin like fold 73 3.3.2 Isothermal Titration Calorimetric studies 77 3.3.3 Structure of GrlR Lipid complex 80 3.3.4 Mass spectrometry analysis to identify the physiological lipid species bound to GrlR 86 3.4 Discussion 91 Chapter 4: Structural basis for the secretion of EvpC: a key type VI secretion system protein from Edwardsiella tarda 95 4.1 Introduction 96 4.2 Materials and Methods 98 4.2.1 Plasmid and strain construction 98 4.2.2 Purification and crystallization 98 4.2.3 Data collection, structure solution and refinement 99 4.2.4 In-vitro pull down assay 99 4.2.5 Analytical Ultra centrifugation 100 4.2.6 MALDI-TOF MS and MS-MS Analysis. 100 4.2.7 Secreted proteins isolation and assay 101 vii 4.3 Results 101 4.3.1 Characterization of EvpC 101 4.3.2 Overall structure 106 4.3.3 Sequence and structural homology 109 4.3.4 Oligomerization 111 4.3.5 Interaction of EvpC with other E tarda T6SS secreted proteins 116 4.3.6 Identification of key residues for EvpC function 119 4.4 Discussion. 122 Chapter 5. Conclusion and Future Directions. 123 References 128 Appendices 147 viii Summary Transport of proteins across the bacterial cell envelope is a basic function performed by bacteria. The secretion pathways used for the transport of proteins are known as secretion systems. Secreted proteins are involved in various functions such as biogenesis of the cell envelope, motility and intercellular communication. To date, six types of secretion systems have been identified in gram-negative pathogenic bacteria. A detailed introduction to bacterial secretion systems in gram-negative bacteria is given in chapter1. Enterohemorrhagic Escherichia coli (EHEC) is a common cause of severe hemorrhagic colitis. EHEC’s virulence is dependent upon a type III secretion system (T3SS) encoded by 41 genes. These genes are organized in several operons that are clustered in the locus of enterocyte effacement (LEE). Most of the LEE genes, including grlA and grlR, are positively regulated by Ler, and Ler expression is in turn positively and negatively modulated by the proteins GrlA and GrlR, respectively. However, the molecular basis for the GrlA and GrlR activity is still elusive. In chapter of this thesis, we report the crystal structure of GrlR at 1.9 Å resolution. It consists of a typical β-barrel fold with eight β-strands containing an internal hydrophobic cavity and a plug-like loop on one side of the barrel. Furthermore, a unique surface-exposed EDED (Glu-Asp-GluAsp) motif is identified to be critical for GrlA–GrlR interaction and for the repressive activity of GrlR. ix GrlR adopts the typical lipocalin fold, which comprises an eight stranded β-barrel followed by an α-helix at the C-terminus. Lipocalins are a broad family of proteins with diverse functions. They are present in eukaryotes as well as gram-negative bacteria. In chapter of this thesis we report the structure of lipid-GrlR complex and the details of the binding of a lipid to recombinant GrlR as verified by isothermal titration calorimetry. Based on these results we identified glycerophosphatidyl phosphatidic acids and glycerophosphatidylethanolamines as potential lipid ligands of GrlR. In addition, we identified an endogenously bound lipid species to GrlR by electrospray ionization mass spectrometry. Our studies demonstrate the hitherto unknown lipid binding property of GrlR. We speculate that this property of GrlR may help to anchor and orient GrlR on membrane to facilitate its interaction with the positive regulator GrlA. The type VI secretion system (T6SS) is a recently identified secretion system used by gram-negative bacteria to inject virulence proteins into host cells. The T6SS cluster in Edwardsiella tarda is named as Evp (Edwardsiella virulence protein) and it contains 16 different genes that are classified into intracellular apparatus proteins, secreted proteins and a group of proteins non-essential for T6SS. It has been shown that the secretion of the apparatus protein Hcp1 from Pseudomonas aeruginosa, a close homolog of EvpC, is an essential characteristic of a functional T6SS. Here we report the crystal structure of EvpC from E. tarda refined at 2.8 Å resolution. EvpC is comprised of a loose β-barrel domain with extended loops. We speculate that similar to its homolog Hcp1, EvpC can form a hexameric ring with a diameter of 40Ǻ that is capable of transporting small proteins and ligands. Analytical ultra centrifugation studies on the oligomerization state of EvpC showed that depending on concentration, EvpC can exist as both dimer and hexamer in x phagocytosis resistance, cytotoxicity and disruption of actin microfilaments. 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Nature. 358, 167-169 146 Appendices 147 148 149 [...]... while type I and type III are sec-independent secretion systems Even though these systems transport various proteins and substrates, the research is mainly focused on transport of virulence proteins which is important for the pathogenicity Various secretion systems in gram-negative bacteria are explained below with a special emphasis on the type III secretion system (T3SS) and type VI secretion system. .. extra-cellular protein secretion assay 120 xii List of Figures Page no: Figure: 1.1 General structure of a Pathogenicity Island 5 Figure: 1 2 Type I–V secretion systems in Gram-negative bacteria 7 Figure: 1.3 Model of pilus-mediated secretion via the type II secretion system 11 Figure: 1.4 The Type Three Secretion System 14 Figure: 1.5 Overall architecture of the Type III secretion apparatus (T3SA) 16... 11 1.7 Type III Secretion System (T3SS) T3SS is a complex secretion system, which consists of more than 20 proteins This secretion system has the capability to inject effector proteins directly into the host cell (Hueck, 1998) Many of the T3SS proteins are conserved in various pathogens Both plant and animal pathogens use T3SS for injecting their virulence proteins Although the T3SS apparatus proteins. .. from Schmidt and Hensel, 2004) 5 1.3 Protein Secretion Systems Transport of proteins across the bacterial cell envelope is a basic function performed by bacteria The secretion pathways used for the transport of proteins are known as secretion systems and they vary from simple to complex systems Recently, several three-dimensional structures of proteins associated with these secretion systems have been... colonization of host organisms are commonly secreted proteins (Gerlach and Hensel, 2007) To date six secretion systems have been identified in gramnegative pathogenic bacteria (Fig 1.2) They vary from simple systems that involve a few proteins to complex systems that contain more than 20 proteins Type II and type V are sec-dependent secretion systems that require a sec pathway for transport of proteins, ... which is involved in the secretion process A secreted protein also needs chaperones to prevent its premature interaction with other proteins T3SS-associated ATPases play a major role in protein secretion, substrate recognition and chaperone release from T3SS proteins as well as unfolding of type III secreted proteins (Akeda and Galan, 2005) The regulation of protein secretion by contact with host surface... We have studied the structure and function of GrlR, a regulator protein in T3SS and EvpC, which is a secreted protein in T6SS The details of our studies on T3SS are given in chapter 2 and chapter 3 and those of T6SS in chapter 4 6 Figure 1.2 Type I–V secretion systems in gram-negative bacteria Type I, type III and type IV SSs (left) are believed to transport proteins in one step from the bacterial cytosol... surface and external medium In the case of type III and type IV SSs, the proteins are transported from the bacterial cytoplasm to the target cell cytosol One exception for type IV is the pertussis toxin, which is secreted in two steps and released into the extracellular medium This exception is represented by the dotted arrow, which connects Sec and the type IV SS Type II and type V SSs transport proteins. .. the T3SS apparatus proteins are conserved among various pathogens, they inject different types of effector/virulence proteins into the hosts T3SS mainly consists of three groups of proteins: the first group comprise the secretion system apparatus and are known as structural proteins, the second group which helps in the translocation of proteins are known as translocators and the third group which are... Leung Ka Yin, and J Sivaraman 2009 Structural and functional studies on EvpC, a Type six secretion system protein (Manuscript submitted) xx Chapter I General Introduction 1.1 Host-Pathogen Interaction The interaction between bacterial pathogens and their host such as humans, animals and plants is an important area of study in microbiological research The findings that emerge from these studies will . 1.5 Type I Secretion System 9 1.6 Type II Secretion System 10 1.7 Type III Secretion System 12 1.8 Type IV Secretion System 26 1.9 Type V Secretion System 27 1.10 Type VI Secretion System. STRUCTURAL AND FUNCTIONAL STUDIES ON TYPE III AND TYPE VI SECRETION SYSTEM PROTEINS JOBICHEN CHACKO (M Sc Agriculture). virulence properties. Our studies broaden the understanding of the structure, function and assembly of protein secretion system, particularly the type III and VI secretion systems in gram- negative