Characterization of plasma myosin heavy chain in zebrafish as an important factor for ompa mediated anti phagocytic function

CHARACTERIZATION OF PLASMA MYOSIN HEAVY CHAIN IN ZEBRAFISH AS AN IMPORTANT FACTOR FOR OmpA-MEDIATED ANTI-PHAGOCYTIC FUNCTION PENG BO NATIONAL UNIVERSITY OF SINGAPORE 2008 CHARACTERIZATION OF PLASMA MYOSIN HEAVY CHAIN IN ZEBRAFISH AS AN IMPORTANT FACTOR FOR OmpA-MEDIATED ANTI-PHAGOCYTIC FUNCTION By PENG BO (M.Sc, B.Sc) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2008 ACKNOWLEDGEMENTS I would like to thank many people who have helped me over the years. First of all, I would like to express my heartfelt gratitude to Dr. Leung Ka Yin, my supervisor for keeping me on tract with his guidance, support discussion and suggestions, and Dr. Hew Choy Leong for the encouragement and financial support. I would also like to thank Mr. Yan Tie for technical help in fluorescence microscope and providing me aquarium space for culturing fish. I am also most appreciative to the fellow members of Dr. Leung’s lab, Zheng Jun, Yu Hong Bing, Smarajit Chakraborty, Xie Haixia, Li Mo and Tung Siew Lai for making my time there educational, and enjoyable. And also other lab members, Wang Xiaowei, Li Peng, Li Yue, Jiang Naxin and Chen Liming for sharing experiences, ideas and reagents. And finally, I am deeply indebted to my parents and my wife for their love, understanding and support over the years. i TABLE OF CONTENTS PAGE ACKNOWLEDGEMENTS i TABLE OF CONTENT ii LIST OF FIGURES vi LIST OF TABLES vii LIST OF ABBREVIATIONS viii SUMMARY x 1 CHAPTER 1 INTRODUCTION 1.1 Host-pathogen interaction 1 1.1.1 Host’s defense strategies 1 1.1.2 Pathogen’s (Gram-negative bacteria's) survival strategies 6 1.1.3 The roles of outer membrane proteins in host-pathogen interaction and vaccine development 1.2 1.3 Myosin heavy chain (MHC) and its Clinical significance 1.2.1 Overall review of myosin and MHC 1.2.2 Clinical Significance of plasma MHC and serum MHC The role of Outer membrane protein A (OmpA) in host-pathogen interaction 8 9 9 11 12 1.3.1 Basic structure of OmpA in E. coli 12 1.3.2 Physiological function of OmpA in E. coli 13 ii 1.4 1.3.3 The role of OmpA in virulence 17 1.3.4 The host’s immune system targets OmpA 19 E. coli and Zebrafish interaction model 23 1.4.1 E. coli as a model organism in prokaryotic cells 23 1.4.2 Zebrafish as a model organism in vertebrates 24 1.5 Objectives 26 28 CHAPTER 2 MATERIALS AND METHODS 2.1 Bacterial strains, media and bacterial culture 2.1.1 Bacterial strains 29 29 2.1.2 Bacterial culture media 29 2.1.3 Preparation of E. coli cultures 31 2.2 Cell culture medium and cell culture 31 2.3 Molecular Biology techniques 31 2.3.1 Genomic DNA isolation 31 2.3.2 Cloning and transformation into E. coli cells 32 2.3.3 Analysis of plasmid DNA 32 2.3.4 Purification of plasmid DNA 33 2.3.5 DNA sequencing 33 2.3.6 DNA sequence analysis 34 2.3.7 Construction of deletion mutants and plasmids 35 iii 2.4 Protein techniques 35 2.4.1 One-dimensional polyacrlamide gel electrophoresis (1D-PAGE) 2.4.2 Silver staining of protein gels 2.4.3 Western blot 2.4.4 Molecular cloning, expression and purification of OmpA in pET32a 35 37 38 39 2.4.5 Purification of outer membrane proteins from E. coli 40 2.4.6 Co-immuneprecipitation 41 2.5 Whole bacteria pull-down assay 41 2.5.1 Body fluid isolation from Zebrafish 41 42 2.5.2 Bacteria preparation 2.5.2.1 Paraformaldehyde fixed bacteria 42 2.5.2.2 Heat inactivated bacteria 42 2.5.2.3 Gentamycin-treated bacteria 42 2.5.2.4 Proteinase K-treated bacteria 42 2.5.3 Bacteria pull-down assay 43 2.6 Immunofluorescence microscopy examination of E. coli surface localization 43 2.7 Tissue lysis and cell lysis 44 2.7.1 Preparation of fish tissue lysis 44 2.7.2 Preparation of red blood cell lysis 44 2.7.3 Preparation of hemolysin and hemolysin-induced red blood cell lysate 45 2.8 Fluorescence labeling of bacteria 45 iv 2.9 Phagocytosis assay 46 2.10 Statistical analysis 47 48 CHAPTER 3 Results 3.1 Interactomics study between Zebrafish body fluid proteins and E. coli reveals MHC can bind to E. coli K12 50 3.2 Characterization of bacteria-interacting MHC 59 3.3 Characterization of the interaction between bacteria and MHC 64 3.4 Outer membrane protein A in E. coli can bind to MHC 68 3.5 Bacteria-interacting MHC involved in OmpA-mediated anti-phagocytic function 71 65 CHAPTER 4 Discussion 4.1 Interactomics is a powerful tool to study host-pathogen interaction 76 4.2 E. coli binds to plasma MHC and smooth muscle MHC (SM-MHC) 78 4.3 E. coli can actively bind to plasma MHC 80 4.4 OmpA-plasma MHC interaction may involve in anti-phagocytic function 82 References 87 v LIST OF FIGURES PAGE Title Fig. 1. A two-dimensional model of OmpA in the outer membrane of E. coli. 14 Fig. 2. Interaction profile between heat-inactivated E. coli and Zebrafish body fluid 51 Fig. 3. The Peptide Mass Fingerprinting (PMF) results of the identified proteins as reported in Table 3 54 Fig. 4. Localization of bacteria-interacting MHC 60 Fig. 5. Quantitiation of bacteria-interacting MHC in collected Zebrafish body fluids 62 Fig. 6. Characterization of interaction between MHC and E. coli 66 Fig. 7. Characterization of OmpA as the MHC binding protein in E. coli surface 69 Fig. 8. The interaction between OmpA and MHC involved in anti-phagocytic function 73 vi LIST OF TABLES Title PAGE Table 1 Bacteria strains and plasmids used for this study 27 Table 2 Oligonuclotides used in this study 33 Table 3 Summary of MS results of the identified proteins 49 vii LIST OF ABBREVIATIONS aa amino acid r Amp Ampicillin-resistant bp base pairs BSA Bovine serum albumin CFU Colony forming umits cm centimeter(s) Chlr Chloramphenicol-resistant Colr Colistin-resistant Da Daltons DMEM Dulbecco's Modified Eagle Medium DNA Deoxyribonucleic acid EDTA Ethylene diamine tetra acetic acid g gram g gravitational force HBSS Hank’s balanced salts solution IPTG Isopropyl-thiogalactoside Kanr Kanamycin-resistant kb kilo base l litre(s) LB Luria-Bertani broth LBA Luria-Bertani agar M molarity, moles/dm3 mg milligram(s) min minute(s) ml milliliter(s) mM milli moles/dm3 ºC Degree Celsius OD Optical density % percentage PAGE Polyacrylamide gel electrophoresis PBS Phosphate buffered saline PCR Polymerase chain reaction viii PVDF Polyvinylidene difluoride s second SDS Sodium dodocyl sulfate Tetr Tetracycline-resistant TE Tris-EDTA PBST Phosphate buffered saline with 0.05% Tween 20 U Unit(s) µg microgram(s) µl microlitre(s) v/v volume per volume w/v weight per volume X-gal 5- bromo-4-chloro-3-indolyl-β-D-galactopyranoside ix Summary Understanding the host-pathogen interaction is an important issue for the development of effective vaccines against pathogens. Many vaccine candidates were screened and developed based on the antigenic proteins located on pathogen’s surface. To gain more information about host-pathogen interaction from a systematic level, in this study, we chose the Zebrafish, Denio rerio, and Escherichia coli K12 as research models to investigate the host-pathogen interactions. By combining whole bacteria pull-down assay and proteomics tools, we first set up the interaction profile between E. coli surface and Zebrafish body fluid. Nineteen proteins were shown on the gel and finally only four proteins were identified: complement component 1, q subcomponent-like protein 1 (C1q1-like protein), vitellogenin, myosin heavy chain (MHC) and nucleoside diphosphate kinase-Z2. Among these four proteins, we were particularly interested in the fact that MHC can bind to E. coli. We first examined the distributions of bacteria-interacting MHC in plasma, serum and erythrocyte lysates. And we also studied the distributions in different tissues. Western-blot results showed that E. coli can bind to plasma MHC and smooth muscle MHC. This result also implied that E. coli might specifically bind to a subset of MHCs in Zebrafish. In addition, the interaction between MHC and E. coli surface was further confirmed by the treatment of E. coli with proteinase K and immunofluorescence microscopy study. The treatment of proteinase K of bacteria surface prevented the interaction of MHC to E. coli. And the immunofluorescence microscopy examination provided direct visualization of this x interaction. Furthermore, the interaction between E. coli and MHC was hydrophobic in nature as it could not be completely abolished upon the treatment of high NaCl concentrations. To further explore the interaction between MHC and E. coli, we used co-immunoprecipitation to identify the possible proteins that could interact with MHC in E. coli surface. Results showed that MHC can interact with outer membrane protein A (OmpA) of E. coli. This interaction was confirmed by using recombinant OmpA to do co-immunoprecipitaion of Zebrafish body fluid. Both the electrophoresis results and Western-blot results showed that OmpA can interact with MHC in Zebrafish body fluid. The above studies implied that this interaction may have biological functions. The phagocytic ability of J774 macrophages towards E. coli alone and E. coli that were preincubated with Zebrafish body fluid showed significant difference. The phagocytosis of E. coli preincubated with Zebrafish body fluid was greatly reduced when comparing to E. coli alone. In the contrary, the phagocytosis of E. coli ΔompA and E. coli ΔompA that preincuated with Zebrafish body fluid showed only minor difference. Furthermore, the bacteria-concentration dependent experiment showed that the increasing volumes of the bacteria suspension could increase the phagocytosis ratio. And the increase volumes of Zebrafish body fluids could decrease the phagocytosis ratio. We thus proposed that plasma MHC may work as a shield to protect the OmpA from being recognized by the J774 macrophages. xi CHAPTER 1 INTRODUCTION 1 1.1 Host-pathogen interaction The study of host-pathogen interaction is an old but prominent field in modern biology. The battle between host and pathogen has never stopped throughout the evolution. The pathogens have evolved different strategies to avoid being detected and killed by the host, which help them find niches inside hosts for survival and replication. Therefore, the host also developed effective mechanisms to fight against dangerous invaders and to clear them out. 1.1.1 Host’s defense strategies Human’s immune system is the most extensively studied defense system in the hosts. Human’s immune system consists of three important parts: fluid systems, innate immunity and adaptive immunity (Rotti et al., 2001). The fluid systems can be further divided into two systems, the blood system and the lymph system (Parham, 2001). These two systems are intertwined throughout the body and they are responsible for the transport of the agents of the immune system. The blood system provides an optimal environment for immune cells, leucocytes and platelets (Paul, 1999). The lymph system contains several important immune-related organs, such as thymus gland, spleen, lymph nodes, Peyer's patches and the appendix (Rotti et al., 2001). The innate immunity system is born with the humans and thus it is germ-line encoded and can be passed on to the offspring. One of the most important characteristics is that 2 innate immunity-mediated defense is non-specific, which means that they respond to infections in a generic method. And they can’t produce long lasting immunity to the pathogens (Alberts et al, 2002). Mucosal immunity belongs to innate immune system and is the first defense line in our human body (Ogra, 1998). Skin is the most important barrier to the invaders as most of the organisms cannot penetrate the skin unless it is broken. The hair of the lungs can expel pathogens by ciliary action, which leads to coughing and sneezing abruptly to eject the noncomparable substances from the respiratory tract. The low acidic pH of skin’s secretion will inhibit bacteria growth. In addition, saliva, tears, nasal secretions, and perspiration contain lysozyme, an enzyme that destroys Gram-positive bacterial cell walls and cause cell lysis. The stomach is a formidable obstacle as its mucosa secretes hydrochloric acid (pH < 3.0,) protein-digesting enzymes that kill many pathogens (Bos, 2005). Another important component of innate immune system is the normal flora. Normal flora is defined as a population of bacteria that live inside or on the human body under normal conditions. These microbes play pivotal roles in training immune tolerance after the birth of human beings to useful bacterial population, help digesting foods, keep proper environment and even kill other invading bacteria. Unfortunately, the normal flora can cause adverse effect to the populations that are out of control. An example is the stomach ulcer caused by Helicobacter pylori (O'Hara, 2006). 3 Phagocytes are a general name for the cells that can adhere to, engulf and ingest foreign substances in innate immune system. Macrophages, dendritic cells, natural killer cells and neutrophils are important cell types that are responsible for the “eating” of invaders (Rotti et al., 2001). The ability of the macrophages to phagocytose the pathogens is largely relied on their large number of receptors in their cell surface, such as mannose receptor, CD14 and Toll like receptors, complement receptors, Fc receptors and G-protein-coupled receptors (Martinez-Pomares & Gordon, 1999; Gordon & Mcknight, 2000; Linehan et al, 2000; Tunheim et al, 2007; van Lookeren et al, 2007). Toll-like recpetors, for example, are one of the most efficient pathogen detection systems. These receptors can specifically recognize the unique structures from pathogens, such as the LPS, CpG motif, lipoprotein, flagellin and viral RNAs (Akira, 2006; Kawai & Akira, 2006; Meylan & Tschopp, 2006). And this recognition is crucial for the activation of the downstream signaling, which mediate the activation of immune-specific genes including proinflammatory cytokines and chemokines (West et al, 2006). If the innate immunity is insufficient to clear pathogens, adaptive immunity will be activated. The adaptive immune system can be subdivided into two kinds of immune response, cell-mediated immunity and humoral immunity (Rotti et al., 2001). 4 Cell-mediated immune response does not involve antibodies but rather involves the activation of innate immune cells, such as macrophages, natural killer cells, antigen-specific cytotoxic T-lymphocytes and the release of cytokines. After the engulfment or phagocytosis of invading microbes, these cells can present the digested fragments from pathogens on their surface to the antigen-specific cytotoxic T-lymphocytes via major hiscompatibility complex I (MHC I) (Pamer & Cresswell, 1998). Subsequently, these cytotoxic T-lymphocytes will induce the apoptosis of the cell displaying epitopes of foreign antigens (Berke, 1994). Three mechanisms have been suggested on how it functions. It can clear pathogens by activating macrophages to destroy intracellular pathogens or by stimulating cells to produce a variety of cytokines to trigger adaptive immunity (Parham, 2001). Cell-mediated immunity is not only effective at removing virus-infected cells, but is also useful to clear fungi, protozoan and intracellular bacteria (Hahn & Kaufmann, 1981) The main participant of humoral immunity is B cell. The maturation of B cell is triggered by the interaction of B cell surface receptor and the antigen with the help of helper T cells (Slifka et al, 1998). The maturation of B cell will produce antigen-specific antibodies by gene recombination (Allison & Eugui, 1983). The antibody can inactivate the antigen by complement fixation, neutralization, agglutination and precipitation. Humoral immunity is effective in inducing the formation of memory B cell which can produce strong immune response at the second 5 infection, which is the basis for vaccine development (Allison & Eugui, 1983; Rotti et al., 2001; Kathryn et al, 2003) 1.1.2 Pathogen’s (Gram-negative) survival strategies Gram-negative bacteria are defined by its inability to retain the crystal violet dye in Gram staining protocol (Samuel, 1996). The characteristics of Gram-negative bacteria, which can be distinguished with other bacteria are: cell walls contain only a few peptidoglycan while Gram-positive bacteria contain a lot; the cell membrane has two layers: outer membrane and inner membrane while Gram-positive bacteria only has one membrane; there is space between inner membrane and outer membrane; porins are present in outer membrane and act as pores for particular molecules; lipoproteins are attached to lipopolysacchride backbone whereas in Gram-positive bacteria no lipoproteins are present. Many Gram-negative bacteria species are pathogenic, which means that they can cause disease in the hosts where they reside (Michael & John, 2005). They commonly utilized two methods to cause disease: toxins and virulence factors (Samuel, 1996). The toxins produced by Gram-negative bacteria can be subdivided into two classes: exotoxin and endotoxin. Exotoxins are soluble proteins excreted by pathogens. An exotoxin can cause damage to the host by interfering normal cells or tissues. The most known exotoxin is the hemolysin. Pathogenic E. coli, for example, can produce 6 α-hemolysin. The hemolysin precusors are secreted as monomers, which localize to host’s cell membrane and form ring-like polymers. This pore will cause the lysis of the target cell, erythrocytes (Weber & Osborn, 1969; Pavlovskis & Gordon, 1972; Chung & Colliur, 1977; Vasil et al, 1977; Snell et al, 1978) Almost all of the Gram-negative bacteria have endotoxins, which are not secreted in soluble forms by the bacteria but are a structural component of the bacteria. The most known endotoxin is lipopolysaccharide (LPS), which can cause “septic shock” in humans with the symptoms of low blood pressure and low blood flow (Glauser et al, 1991; Parrillo, 1993). In the serum of human body, LPS firstly bind to lipid binding protein (LBP). Working together with CD14 on the cell membrane, LBP transfer LPS to another protein, MD2, which has been associated with Toll like receptor 4 (TLR4) (Poltorak et al, 1998). However, TLR4 and CD14 are most present in immune system cells. The activation of TLR4 will trigger the activation of signaling pathways to secrete pro-inflammatory cytokines and nitric oxide that lead to “septic shock” (Wright et al, 1990; Shimazu et al, 1999) Gram-negative bacteria can also use type III secretion system (T3SS) to inject virulence factors directly into host cells. The T3SS is encoded as a gene cluster in Pathogenicity Island in bacteria genome or plasmid (Hacker et al, 1997; Wong et al, 1998). The T3SS is present only in Gram-negative bacteria. The secretion system 7 apparatus form a needle-like complex in cell membrane and the tip can penetrate host cell membrane and thus inject virulence factors into the host cells. The virulence factors, called effectors, can subvert normal cell functions, such as triggering apoptosis of immune cells or activating the phagocytosis of non-phagocytic cell to find niche inside the host cell for replication (Galan, 2001; Galan & Wolf-Watz, 2006) 1.1.3 The roles of outer membrane proteins in host-pathogen interaction and vaccine development As the outer membrane of Gram-negative bacteria is the exposed structure to the environment, the proteins anchored on the membrane thus are crucial for important functions of the bacteria. On one hand, the outer membrane proteins can play diverse roles in bacterial pathogenesis. They can work like adhesion molecules to aid colonization themselves in the hosts such as OmpU of Vibrio cholerae (Sperandio et al, 1995). The protease of Pla of Yersinia pestis can digest host proteins as a strategy to cause pathogenesis (Sodeinde et al, 1992). The bacterial membrane proteins can also bind to host proteins to inhibit their functions such as OmpA of E. coli K1 (Prasadarao, 2002b). They can work as sensors for the dangerous signals from the host, such as OprF of Pseudomonas aeruginosa to trigger activation of virulence-associated genes (Wu et al, 2005). Pathogens can also use the outer membrane proteins to interact with host cells for survival or to transverse barrier (Prasadarao, 2002a). On the other hand, some outer membrane proteins have antigenic properties which are good candidates 8 for the development of vaccines. By combining proteomics and immuno-blot technologies, researchers have identified the antigenic outer membrane proteins from A. hydrophila, Shigella flexneri 2a and Burkholderia pseudomallei. (Chen et al, 2004; Peng, et al, 2004; Xu et al, 2005; Harding et al, 2007). In A. hydrophila, for example, 3 out of 7 identified antigenic outer membrane proteins could effectively prevent the killing of fish by bacteria challenge followed by immunization of these proteins. Thus, these antigens, called protective antigens, can be further investigated for vaccine development (Chen et al, 2004). 1.2 Myosin heavy chain (MHC) and its Clinical significance 1.2.1 Overall review of myosin and MHC Myosins are a large superfamily of motor proteins found in almost all eukaryotic cells (Alberts et al, 2001). The functions of this protein family can be generally classified as intracellular molecules for transport and muscle contraction (Alberts et al, 2001). As motor proteins inside the cells, their cellular functions are including targeted organelle transport, endocytosis, chemotaxis, cytokinesis, modulation of sensory systems, and signal transduction. More broadly, they also play roles in developmental and functional disorders of the nervous, pigmentation, and immune systems (Dantzig et al, 2006). The function of myosin in muscle contraction is based on its ability to hydrolyze ATP, which provides the energy for the contraction of the muscle (Brooks et al, 2006). Several reports have demonstrated the mutations of myosin are associated 9 with human diseases, such as May-hegglin anomly / Fechtner syndrome and glomerulonephritis (Deutsch et al, 2003; Ghiggeri et al, 2003; Kunishima, et al, 2003) Typically, the myosin molecules are composed of two domains: a head domain and a tail domain. The head domain is responsible for the binding of filamentous actin and “walking” along the filament with the force generated from ATP hydrolysis (Tonomura & Oosawa, 1972). This was demonstrated by an experiment that myosin heads, which can be detached from myosin tails by protease treatment and fixed to a glass surface, promote the gliding of actin filaments labeled with fluorescent rhodamine-phalloidin and this process is ATP-dependent (Alberts et al, 2001). The tail domain is involved in the interaction with cargo molecules or / and other myosin subunits (Alberts et al, 2001). Thus based on the amino acid sequences of their ATP-hydrolyzing motor domains, the myosin protein family members can be divided into 20 classes. Different classes can be distinguished from their tail domains (Alberts et al, 2001). Each myosin protein was composed with one or two MHCs and myosin light chains. Myosin II, a subclass of myosin, for example, contains two heavy chains with each about 2000 amino acids in length (~200 kDa), which constitute the head and tail domains. Each of these heavy chains contains a N-terminal head domain, while the C-terminal tails contain heptad repeat sequence, which promote dimerization. 10 Furthermore, the C-terminal domain takes on rod-like α-helical coiled coil morphology and this structure can hold the two heavy chains together. Therefore, myosin II has two heads. It also contains 4 light chains (2 per head), which bind the heavy chains in the "neck" region between the head and tail (Tonomura & Oosawa, 1972 ; Korn et al, 1988). Being phosphorylated by myosin light chain kinases or Rho kinases, the myosin light chain can regulate the function of myosin by changing the conformation of myosin heads to detach from actin, increasing population placed close to thin filaments, potentiating actin-myosin interaction at low Ca2+ level, regulating ATPase activity of myosin and myosin assembly into filament (Wilson et al, 1992; Trybus, 1994; Stull et al, 1998; Depina & Langford, 1999; Nakamura & Kohama, 1999). 1.2.2 Clinical Significance of plasma MHC and serum MHC Although MHC is a structurally bound contractile protein of the thick filaments, this protein was reported that it can be released into circulation as the consequence of loss of cell membrane integrity. Thus, it has been proposed as an important indicator of muscle injury in clinical diagnosis (Onuoha et al, 2001). The concentration of MHC together with the concentrations of creatine kinase, myoglobin and cardiac troponin I in human plasma were used to assess the myoskeletal muscle damage. The results from 25 patients showed that after injury the concentration of MHC in human plasma increased when comparing to control groups (Onuoha et al, 2001). A similar study was conducted to examine the amounts of four proteins: MHC, creatine kinase, myoglobin 11 and cardiac troponin I in human plasma to see the mycoskeletal injuries after surgerical treatments when comparing to the people who did not receive treatments. This study also indicated that after surgerical treatment, the plasma MHC concentration increased almost 2 folds (Onuoha, et al, 1999). Meanwhile, after exercise, the concentration of MHC in human plasma was also found to be elevated (Mair et al, 1992). Furthermore, the MHC has been implicated to be present in human serum. The clinical significance of this serum protein was also reported. Two research groups have found that serum MHC can be the indicators for acute aortic dissection, the diagnosis of acute aortic emergency and acute aortic dissection (Hori et al, 1999; Suzuki et al, 2000). The serum MHC can also be the biomarker of rhabdomyolysis and ectopic pregnancy (Lofberg et al, 1995; Birkhahn et al, 2000). It can be used to predict restenosis after percutaneous transluminal coronary angioplasty (PTCA) and suspected appendicitis (Tsuchio et al, 2000; Birkhahn et al, 2002). Taken together, in clinical diagnosis, the change of the concentration of MHC in human serum and plasma is an important factor to examine the muscle injury and myosin-related diseases. 12 1.3 The role of Outer membrane protein A (OmpA) in host-pathogen interaction 1.3.1 Basic structure of OmpA in E. coli OmpA is one of the most extensively studied outer membrane proteins in Gram-negative bacteria. This protein in E. coli K12 contains 325 residues and is heat modifiable (Pautsch & Schulz, 1998). It contains two domains: N-terminal domain and C-terminal domain. Structural analysis and topological analysis showed that the classic N-terminal domain is 171 amino acids in length and span the outer membrane eight times in antiparalle-strands (Koebnik, 1995; Fig.1.). Four relatively large and hydrophobic surfaces-exposed loops and short periplamsic turns were found (Pautsch & Schulz, 2000). While the C-terminal domain is mainly located in the periplasm, a space between outer membrane and inner membrane, and binds to peptidoglycan, which connects it to the outer membrane (Vogel & Jahnig, 1986; Arora et al, 2001) The OmpA or OmpA-like proteins are present in almost all Gram-negative bacteria tested so far, which include 17 genera (Beher, 1980). The comparison of OmpA from five close related genera indicated that the β-sheet amino acid residues of OmpA N-terminal are highly conserved, while the extracellular loops are largely variated between different genera (Pautsch & Schulz, 1998; Wang, 2002). 1.3.2 Physiological function of OmpA in E. coli 13 Fig. 1. A two-dimensional model of OmpA in the outer membrane of E. coli. Predicted TM β-strands are boxed and residues whose side-chains are predicted to point to the lipid bilayer are shown in italics. The surface-exposed loops and periplasmic turns have been labeled L1 to L4 and T1 to T3, respectively. (Adopted from Membrane topology and assembly of the outer membrane protein OmpA of Escherichia coli K12 Ried et al., 1994) 14 15 The physiological function of OmpA in E. coli K12 is thought to contribute to the maintenance of the integrity of outer membrane along with murine lipoprotein (Braun & Bosch, 1972) and peptidoglycan-associated lipoprotein (Lazzaroni & Portalier, 1992). Both of these studies showed that the ompA deficient E. coli strains are highly susceptible to drugs such as cholic acid. The treatment of this drug could cause the release of periplasmic proteins into media. In addition, a recent study showed that the ompA deficient E. coli mutant is sensitive to detergents such as SDS, cholate acid, osmotic shock and serum-mediated killing (Wang, 2002). However, the introducing of a plasmid containing the full length ompA can restore these functions as the wide type E. coli. In addition, besides its role in keeping the proper structure of the outer membrane, the OmpA also has been shown to be required for the F-conjugation. The mutation of ompA will cause the Con phenotype, in which a number of addition outer membrane proteins were missing or decrease and are defective in mating tropic (Skurray et al, 1974). And the isolated OmpA protein can work together with LPS to inhibit the conjugation of the receptor cell, which confirmed that OmpA plays crucial role in conjugation (Schweizer & Henning, 1977). The fact that OmpA can serve as a bacteriophage receptor has long been determined. As early as 1973, Foulds and colleagues grouped the mutants that were independently 16 tolerant to bacteriocin into four classes (Foulds & Banett, 1973). Later, OmpA was found to be one of them. The research group led by Henning screened a series of ompA mutants that were able to either irreversibly or reversibly bind to the phage. Furthermore, the DNA sequence analysis revealed that the four surface-exposed loops are involved in recognition of different phage protein as well as involving in conjugation and in binding of a phage and a bacteriocin (Morona et al, 1984). 1.3.3 The role of OmpA in virulence The role of OmpA in virulence is mainly documented with the pathogenic E. coli K1. The sequence of OmpA in E. coli K1 is identical to that in E. coli K12. Several important functions have been reported. The evasion of serum-mediated killing was an important strategy utilized by E. coli K1 for the pathogenesis of meningitis in neonates. Earlier studies showed that the wide type E. coli K1 was much more virulent than the ompA deficient strain when they were inoculated simultaneously into the neonate’s rats. And the restoration of the ompA in ompA deficient strain could cause the same percentage death as the wide type. In addition, the ompA deficient strain was sensitive to classical complement pathway attack (Weiser& Gotschlich, 1991) Combining previous study that E. coli K1 can avoid the complement attack in human serum, Prasadarao and colleagues found that the OmpA in E. coli K1 surface can 17 specifically bind to human complement component C4 binding protein (C4bp), a complement fluid phase regulator. The interaction between OmpA and C4bp could not be interfered with the addition of C4b and heparin and is not salt sensitive, which implied that this interaction is naturally hydrophobic and with high binding affinity. Furthermore, they also demonstrated that C4bp binds to the N-terminal domain of OmpA (Prasadarao et al, 2002). The underling mechanism of OmpA-C4bp mediated survival within blood stream was deciphered recently. Prasadarao and colleagues found an interesting phenomenon that the log phase E. coli K1 can be more effective at avoiding complement attack than that the stationary E. coli K1, while the ompA mutant E. coli K1 cannot survive in the serum. The reason for the survival effectiveness of log phase E. coli K1 is due to the increasing binding of C4bp. The OmpA-C4bp complex acts as a co-factor for the factor I in the cleavage of C3b and C4b, which prevents the formation of membrane attack complex (Selvaraj, et al, 2007). The other aspects of the functions of OmpA during E. coli K1 pathogenesis have also been reported. OmpA can interact with a receptor on human brain microvascular endothelial cells, which causes the up-regulation of intracellular adhesion molecule 1 (ICAM-1). The upregulation of ICAM-1 is crucial for the pathogenesis and is depended on PKC-alpha and PI3-kinase signaling and NF-κB activation (Prasadarao, 2002; Selvaraj, et al, 2007). In addition, the OmpA in E. coli K1 is also a crucial important factor for the inhibition of proinflammatory response. Studies showed that 18 the incubation of the wide type E. coli K1 would significantly suppress the production of cytokines and chemokines, such as TNFα, IL-1beta and IL-8. However, if the monocytes were treated with ompA deficient E. coli K1, they will produce a robust production of cytokines and chemokines. Further investigation of the underlying mechanism showed that the wide type E. coli K1 can inhibit the phosphorylation of NF-κB thereby prevents the translocation of NF-κB to the nucleus. The mechanism of inhibiting the proinflammatory response can help the pathogenesis of E. coli K1 at the onset stage (Selvaraj et al, 2005). 1.3.4 The host’s immune system targets OmpA The importance of OmpA in the activation of immune system was first reported in 2000. The research group led by Jeannin from France found that the OmpA of Klebsiella pneumoniae (KpOmpA) could specifically bind to professional antigen-presenting cells, such as dendritic cells (Jeannin et al, 2000). The extended incubation caused immature dendritic cells to phagocytose this protein via a receptor-dependent manner, which triggered the activation of immune response. The dendritic cells produced IL-12 to induce the maturation of dendritic cells. Furthermore, if the whole antigen was coupled with OmpA, it could be taken up by dendritic cells and delivered to the conventional MHC-I presentation pathway. The KpOmpA, on the other hand, can prime antigen-specific CD8+ CTLs in the absence of CD4+ T cell or adjuvant. In addition, this research group also investigated whether KpOmpA played a 19 similar role toward macrophage, which is another important innate immune system cells. They smartly designed the experiment that the KpOmpA was firstly labeled with fluorescence and they examined the interaction between OmpA and macrophage. Similarly, the KpOmpA can adhere to macrophage and can be phagocytosed after a longer incubation, which will produce inflammatory cytokines (Soulas et al, 2000). Later, they showed that the immune activation by KpOmpA was dependent on Toll-like receptor 2 (TLR2). However, KpOmpA cannot bind to TLR2 directly. Instead, KpOmpA specifically bind to two scavenge receptors: LOX-1 and SREC-I rather than other family members. The LOX-1 can colocalize and cooperate with TLR2 to trigger the cellular response, which will produce a soluble pattern recognition receptor, PTX3. Thus, the OmpA-elicited immune response could be abolished in TLR2 knock-out mice and will be reduced in PTX knock-out mice (Jeannin et al, 2005). The characteristics of KpOmpA that can elicit host’s immune activation in the absence of adjuvant make it a potential candidate carrier for vaccines. Researchers found that immunization of mice with the antigen conjugated with KpOmpA could induce immune response effectively. For example, the polysaccharides derived from Streptococcus pneumoniae can induce only a minor immune response if it was injected alone: no production of high affinity antibody and no generation of memory B-cells. However, if the polysaccharides are conjugated with KpOmpA before immunization, 20 the anti-polysaccharides antibody can be detected and furthermore, the induced humoral response can protect the mice against a subsequent bacterial challenge (Libon et al, 2002). Another example is that the fusion of respiratory syncytial virus subgroup A (RSV-A) G protein with KpOmpA can induce both mucosal and systematic antibody response in a mice model. The immunization of this fusion protein in the absence of adjuvant still bolstered the protection of both upper and lower respiratory tracts against RSV-A infection (Goetsch et al, 2001). The OmpA of E. coli was also reported to play roles in immune activation. One study showed that OmpA was an important target of host’s immune system (Shafer et al, 1999). Neutrophil elastase (NE) is always regarded as an anti-bacteria protein. It is known for its nonoxidative bacteria killing. The molecular mechanism for its ability to kill bacteria was published earlier (Belaaouaj et al, 2000). It was found that the NE can specifically degrade the OmpA in E. coli surface, which could lead to the clearance of invading E. coli. However, the in vitro study showed that purified NE could not kill ompA deficient E. coli. Furthermore, the in vivo study of NE (-/-) mice showed that they had impaired survival rate to bacterial sepsis when comparing to the wide type mice. According to the results of this study, they then tested whether other neutrophil-derived defense systems can kill bacteria via OmpA of E. coli. Studies showed that the ompA deficient E. coli can induce neutrophils to produce intracellular oxygen radicals. This activation required an intact neurtrophil cytoskeleton but was not 21 related to bacterial phagocytosis. In addition, they also found the ompA deficiency will cause the bacteria more susceptible to membrane-acting bactericidal peptides when comparing to the wide type strain. This work highlights the importance of OmpA in the battle between host and pathogens (Fu et al, 2003). Besides these two works, the pathogenic E. coli O157:H7 (EHEC) OmpA can induce the activation of dendritic cells just as the protein KpOmpA. In this study, the researchers found that the OmpA of EHEC can induce the dendritic cells to produce cytokines, interleukin-1, interleukin-10, and interleukin -12 in a dose-dependent manner (Torres et al, 2006). Furthermore, a recent finding opens a new perspective on how the host can target OmpA in Gram-negative bacteria in order to clear them up. Serum amyloid A (SAA) has been determined as an acute phase protein during inflammation. This low molecular weight protein was conserved throughout the evolution from fish to mammals (Uhlar, 1999). The synthesis can be induced upon lipopolysacchride treatment (Santiago-Cardona et al, 2003). Now, this study showed that SAA can bind rapidly to almost all of the Gram-negative bacteria via OmpA with high binding affinity (Hari-Dass et al, 2005). More importantly, functional study of the interaction between SAA and OmpA revealed that SAA acts as an oposinin for phagocytosis by macrophage and neutrophil. Under lab conditions, the phagocytosis of the opsonined bacteria with SAA was greatly increased compared to the control group. In parallel 22 with increased phagocytosis, the production of cytokines is also elevated (Shah et al, 2006). 1.4 E. coli and Zebrafish interaction model In the modern life sciences study, model organism is an important tool to help us advance our knowledge in studying human disease. The so-called “model organisms” are the organisms that are cost less in purchasing and feeding, and have less ethical constraints when using them. Most importantly, they have long been examined and useful data sets have been gathered to describe basic biological processes. In other words, they must be simple in structure and features, which make them ameable to answer important biological questions (Bolker, 1995). 1.4.1 E. coli as a model organism in prokaryotic cells. E. coli, a prokaryotic microorganism without nuclear membrane, is one of the most popular model organisms in current life sciences study (Flannery, 1997). E. coli can reproduce very quickly under laboratory conditions, producing one generation per 20 min, which enable a number of experiments to be conducted in a short time. In addition, E. coli is easy to take up exogenous genetic materials under the procedure known as DNA-mediated cell transformation which also made it a popular model for studies using recombinant DNA technology (Moss, 1991). Most importantly, it shares fundamental characteristics, such as DNA and messenger RNA, with all other 23 organisms (Botstein & Fink, 1988). The value of E. coli in recombinant DNA makes it a good model organism for students to study the genetic material. 1.4.2 Zebrafish as a model organism in vertebrates Zebrafish is another important vertebrate model organism. Zebrafish is a small fresh water fish which are originated in rivers in India and is a common aquarium fish throughout the world (Josephine, 2002). This organism was first considered as a useful model for the study of developmental biology and genetic functions (Haffter et al, 1996; Mayden et al, 2007). The advantages to choose this organism for developmental study are that for each mating, the fish can give birth to a large numbers of eggs in a short time and more important, the fertilization is occurred in external space, thus all stages of development are accessible to the scientists (Streisinger et al, 1981). In addition, the embryonic development of Zebrafish also provides advantages over other vertebrate model organisms. Zebrafish embryos can develop rapidly from eggs to larvae in three days. The embryos are robust, large and more important, transparent. All of these characteristics facilitate the experimental manipulation and is ideal for dynamic observations. Furthermore, the morpholino antisense technology has been widely used in Zebrafish to study their early development. This morpholino is synthetic oligonucleotides containing the same bases as RNA or DNA. The injected morpholino bind to complementary RNA sequence and thus reduce specific gene expression (Ekker & Larson, 2001). 24 Recently, it is proposed that Zebrafish is also an ideal model for the study of host-pathogen interaction. By comparing with other invetebrate and vertebrate research models, there are several advantages for using the Zebrafish as a host model. Although the immune system of Zebrafish is still under study, we have already known this organism has innate immunity and adaptive immunity, which is similar to the mice and human. Thus, comparing to neomates and fruit flies, Zebrafish has a fully developed immune system. Evidence from teleost, which Zebrafish belongs to, shows that Zebrafish has active complement system and can be activated via three different pathways: the classic pathway, the alternative pathway and the lectin pathway, which are similar to mammals (Holland & Lambris, 2002). The homologous of toll like receptors found in mammals are also present in Zebrafish genome and are involved in pathogen detection (Jault et al, 2004; Meijer et al, 2004). For example, after the infection with Mycobacterium marinum, TLR1 and TLR2 can be induced in Zebrafish and the same genes were activated in mice when infected with the same bacteria (Jault et al, 2004). The adaptive immune systems also consist of T cells and B cells. Like mammalian immune development, the immunoglobulin and T-cell receptors also undergo recombinase-activating gene-dependent recombination during their developments (Kasahara et al, 2004; Lam et al, 2004). The most prominent advantages of Zebrafish over other vertebrates are the genetic screens and real-time visualization (van der Sar et al, 2004). Not only the reverse 25 genetic methods can be used to study the specific gene function of Zebrafish, forward genetic screening is also possible. This kind of screening can enable the researchers to isolate the mutant fish whose susceptibility of certain pathogen has been altered (van der Sar et al, 2004). While real-time visualization enabled the researchers to follow a single infected animal or monitor the injection concentration to mimic the natural infections using fluorescence-labeled bacteria. This approach has been successfully utilized to study M. marinum and Salmonella typhimurium infection in Zebrafish (Davis et al, 2002; van der Sar, 2003) 1.5 Objectives Although a lot of work has been done on host-pathogen interaction at molecular level, there are still lacking data to see the whole picture of how the pathogens interact with the host. In order to get a more detailed picture of the interaction between pathogen and host, we chose E. coli as the pathogen model and Zebrafish as the host model to investigate the interaction between E. coli surface and Zebrafish body fluid. The objectives of this study are as follows. i. To set up an interaction profile between E. coli surface and Zebrafish body fluid by using whole-bacteria pull down assay, MALDI-TOF-TOF protein identification and bioinformatic analysis. ii. To choose one identified protein and examine its distribution in Zebrafish. iii. To characterize the interaction between this Zebrafish host protein and E. coli 26 iv. To identify the possible bacterial protein that interacts with the Zebrafish host protein v. To explore the possible biological function induced by the interaction. All of these studies served to provide clues on how the Zebrafish body fluid proteins interact with E. coli surface proteins, which may advance our understanding between host-pathogen interactions. 27 CHAPTER 2 MATERIALS AND METHODS 28 2.1 Bacterial strains, media and bacterial culture 2.1.1 Bacterial strains The bacterial strains used in this study and sources are given in Table1. Cultures of E. coli were incubated at 37 ºC. Stock cultures of E. coli were maintained in a suspension of LB with 25% (v/v) glycerol at -80 ºC. When required, the media were supplemented with antibotics (Sigma, USA) at the following final concentrations unless otherwise stated: ampicillin (Amp, 100 μg / ml), chloramphenicol (Cm, 30 μg / ml), colistin (Col, 12.5 μg / ml), kanamycin (Km, 100 μg / ml) and tetracycline (Tc, 12.5 μg / ml). 2.1.2 Bacterial culture media E. coli were grown in Luria Bertani broth (LB) (BD biosciences) or LB agar (LBA) (BD Biosciences, USA) or brain-heart infusion agar (BHI) (Difco Laboratories, USA). These media were prepared according to manufacturer’s instructions. To make LB, 25 g of LB were dissolved in 1,000 ml of deionized water and then autoclaved at 121 ºC for 20 min. Preparation of TSA required dissolving 25 g of LB, 15 g of agar for every 1 L of LB needed. After autoclaving, LBA was cooled to 53 ºC and poured into sterile Petri dishes. Brain heart infusion-skim milk agar (BHISMA) was prepared by mixing 53 g BHIA and 0.5% of NaCl in 850 ml deionized water. 29 Table 1. Bacteria strains and plasmids used for this study Strain or plasmid Description Reference or source E. coli JM109 Kms, Cols, Cms Promega MC1061 (λpir) thi thr1 leu6 proA2 his4 argE2 lacY1 galK2 ara14 xyl5 supE44 pir Rubirés et al., 1997 SM10 (λpir) r thi thr leu tonA lacY supE recA-RP4-2-Tc-Mu Km pir Rubirés et al., 1997 BW25113 q lacI rrnB3ΔlacZ4787 hsdR514 DE(araBAD)567 DE(rhaBAD)568 Datsenko, et rph-1 Pro+ with P1kc on BW24321 al. 2000 ΔompA BW25113, in-frame deletion of ompA (missing amino acid 0 – 371) This study BL21(DE3)/pLysS F-, OmpT, hsdSβ(rβ-mβ-), dcm, gal, (DE3)tonA, pLysS(CmR) Stratagene pGP704 suicide plasmid, pir dependent, Chlr, oriT, oriV, sacB Edwards et al., Plasmid pRE112 1998 r pGEM-T Easy Amp Promega pET32a Expression vector Novagen 30 After autoclaving and cooling to 53 ºC, 150 ml of a sterile 10% (w/v) solution of skim milk powder (Difco) was added and plates were prepared. 2.1.3 Preparation of E. coli cultures A single colony of each E. coli strain was first picked up from LB agar plate and inoculated into 5 ml fresh LB. The culture was incubated at 37 ºC overnight. In the next day, another fresh culture was prepared by transferring overnight culture to fresh media with the ration of 1:200. The sub-cultured bacteria were then incubated for 3-4 h at 37 ºC until an optical density (OD) at 600 nm approximately reached 0.6-1.0. The cells were harvested by centrifugation at 4, 000 × g for 10 min at 4 ºC. Supernatant was discarded and the bacteria were washed three times with PBS. 2.2 Cell culture medium and cell culture Mouse BALB/c monocyte macrophage, J774, were grown in DMEM, and supplemented with 10% heat inactivated FBS and 2 mM glutamine. For the culturing of J774, cells are subcultured by harvesting the attached and suspended cells separately. Cells in suspensions were centrifuged and the adherent cells were collected using a cell scraper. Then, the suspended cells and adherent cells are combined and resuspended in fresh medium and seeded into a new flask at 1:3 to 1:4 31 ratio at 37 ºC in 5% CO2 incubator. All tissue culture reagents were obtained from Gibco Laboratories. (Grand Island, USA). 2.3 Molecular Biology techniques 2.3.1 Genomic DNA isolation E. coli strains were grown in 10 ml LB at 37 ºC overnight. Bacteria genomic DNA was extracted as described in the manuals of the genomic DNA isolation/purification kits (Promega, USA). The purified genomic DNA obtained was dissolved in 50 μl of TE buffer (10 mM Tris-HCl and 1 mM EDTA, pH 7.5) 2.3.2 Cloning and transformation into E. coli cells PCR products were cloned into pGEM-T Easy vector system (Promega, USA) according to manufacturer’s instructions and transformed into E. coli JM109. E. coli complement cells were prepared and transformed according to the procedure provided by Sambrook and co-workers (1989). Transformants were plated on LBA containing ampicillin, isopropylthiogalactoside (IPTG, 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside blue-white colony selection. 32 Bio-Rad), (X-gal, Bio-Rad), and for 2.3.3 Analysis of plasmid DNA The boiling lysis procedure described by Holmes and Quingley (1981) was used for the mini-preparation of plasmid DNA. Briefly, 30 μl of overnight bacteria culture was obtained and spun at 13,000 × g for 1min. The bacteria pellet was resuspended in 11 μl of STET solution [0.1 M NaCl, 10 mM Tris-HCl (pH 8.0), 1 mM EDTA (pH 8.0), 5% Triton X-100, 0.8 μg / μl lysozyme and 10 μg / μl RNase A]. Subsequently, the tubes were placed on a boiling water bath for 30 s and cooled down to room temperature. One μl of 10 × buffer of appropriate restriction enzyme and 0.1 - 0.2 units of restriction enzyme was added and the tubes were incubated at appropriate temperature for 30 min. The digested samples were analyzed by gel electrophoresis using 1% (w/v) agarose gel (Seakem®, BioWhittaker Molecular Applications, USA), followed by staining in ethidium bromide. Clones containing the right insert in the plasmid were selected for the purification. 2.3.4 Purification of plasmid DNA QIAGEN plasmid mini (for high copy plasmid) or midi (for low copy plasmid) (QIAGEN GmbH, Germany) were used for the plasmid DNA isolation. Bacteria strains containing plasmids were cultured in LB broth (with appropriate antibiotics) and incubated in a shaker (Forma scientific, USA) with 225 rpm shaking at 37 ºC. After the bacteria were cultured for 16 to 18 h, the plasmid DNA was extracted according to manufacturer’s protocol. The quality and concentration of DNA was 33 determined using the ration of A260/A280 in a spectrophotometer (Shimadzu, UV-1601, Japan). 2.3.5 DNA sequencing DNA sequencing was carried out on an ABI PRISM 3100 genetic analyzer with BigDye Terminator version 3.1 Cycle Sequencing Kit. Sequencing PCR reaction was set up as following: 1 μl of autoclaved water, 100 to 200 ng of the plasmid DNA, 0.2 μl of 5 μM primer and 1 μl of BigDye. PCR was carried out in an ABI PCR system 2400 or 2700 using the conditions: 1 min at 96 ºC, followed by 25 cycles of denaturation 96 ºC, 10 s, annealing 50 ºC, 5 s, extension 60 ºC, 1 min 30 s. The final holding temperature was 16 ºC. The PCR product was purified by ethanol and sodium acetate precipitation prior to automate sequencing. In brief, the PCR product was transferred to a 1.5 ml microcentrifuge tube, and 20 μl of 99% ethanol and 0.5 μl of 3 M sodium acetate (pH 4.6) were added. The mixture was vortexed for about 10 s and centrifuged for 15 min at 13,000 × g in a conventional benchtop centrifuge (Eppendorf, Germany). The supernatant was carefully removed and the pellet was washed with 500 μl 70% ethanol followed by centrifugation for 5 min at 13, 000 × g. The washing step was repeated once and the supernatant was carefully decanted. Residual ethanol was removed after pulse-spinning. The pellet was air-dried at room temperature and stored at -20 ºC for DNA sequencing. 34 Thirteen μl of Hi-DiTM formamide (ABI) was added to dissolve the pellet prior to loading to the 96-well plate (ABI) for sequencing. 2.3.6 DNA sequence analysis Vector NTI DNA analysis software (InforMax, USA) was used for the sequence assembly and DNA editing. DNA and protein sequences were submitted to the National Center for biological Information (NCBI) (http://www.ncbi.nih.nih.gov/Blast) for analysis using the basic local Alignment Search Tool (BLAST) network service (Altschul et al., 1990). DNA and protein homology were compared against a nucleotide and a protein sequence databases, respectively, using the corresponding BLASTN and BLASTP or PSI-BLAST program. 2.3.7 Construction of deletion mutants and plasmids Overlap extension PCR (Ho et al, 1989) was used to generate in-frame deletion of ompA on the E. coli BW25113 chromosome. For the construction of ΔompA, two PCR fragments were generated from BW25113 genomic DNA with the primer pairs of up-for plus up-rev, and down-rev plus down-for. The resulting products generated two 1000-bp fragments containing the upstream of ompA and downstream of the ompA, respectively. A 16-bp overlap in the sequences (underlined) permitted amplification of a 2 kb product during a second PCR with the primers up-for and 35 Table 2. Oligonuclotides used in this study Primer name Primer sequence upfor 5’-ATGTACCCGTGACGTAAGCGGATGG-3' uprev 5'-GCGCAAAAAGTTCTCGTCTGGTAGAAA-3' dnfor 5'-GACGAGAACTTTTTGCGCCTCGTTATC3' dnrev 5'-GGGTACCCCGTCACCAACGACAAAA-3' A2-for 5'-CGGAATTCAAAAAGACAGCTATCGATTG-3' A2-rev 5'-TGCGGCCGCAGCCTGCGGCTGAGTTAC-3' 36 down-rev, both of which were introduced into a KpnI restriction site, respectively. The resulting PCR product contained a deletion of the full length of OmpA. The PCR product was cloned into pGEMT-Easy vector, and DNA sequencing was performed to confirm that the construct was correct. The ΔompA fragment was excised with KpnI, ligated into suicide vector pRE112 (Cmr) (Edwards et al, 1998) and the resulting plasmid was then transformed into E. coli SM10 λ pir. The single-crossover mutants were obtained by conjugal transfer into E. coli BW25113. Double-crossover mutants were obtained by plating onto 10% sucrose-LB agar plates. The deletion mutants were confirmed by PCR and DNA sequencing. 2.4 Protein techniques 2.4.1 One-dimensional polyacrylamide gel electrophoresis (1D-PAGE) 1D-PAGE was performed according to a standard protocol (Sambrook et al., 1989) and 8%, 10% or 12% polyacrylamide gels were used for protein separation. Briefly, the resolving gel solution was poured into the gap between two glass plates and isopropanol was layered on top. The isopropanol overlay was poured off and the gel was washed several times with milli-Q water after the resolving gel was polymerized completely. The 4% stacking gel was poured on top of the resolving gel and a clean Teflon comb was immediately inserted. The Teflon comb was carefully removed after the stacking gel polymerized completely and the wells were washed with milli-Q water to remove any trace of unpolymerized acrylamide 37 Prior to loading, protein samples were mixed with the SDS gel-loading buffer [50 mM Tris-HCl, 100 mM DTT (Bio-Rad, USA), 2% (w/v) SDS, 0.1% (w/v) bromophenol blue (Bio-Rad, USA), 10% glycerol] and were boiled for 5 min. The samples were loaded to the gel wells, and a constant current 5 mA per gel was applied. After the dye front has moved into the resolving gel, the current was increased to 15 mA per gel. Tris-glycine electrophoresis buffer [25 mM Tris, 250 mM glycine (pH 8.3), 0.1% (w/v) SDS] was used for the electrophoresis. 2.4.2 Silver staining of protein gels Silver staining (Blum et al., 1987) was used for more sensitive detection of proteins in the gel. The gel was first fixed in 50% (v/v) methanol and 10% (v/v) acetic acid for at least 30 min, followed by 15 min in 50% (v/v) methanol. The gel was then washed 5 times (5 min each) with milli-Q water, followed by fresh 0.02% (w/v) sodium thiosulfate (Sigma, USA) for 1 to 2 min, and washed twice (1 min each) with milli-Q water. Freshly prepared, 0.2% (w/v) silver nitrate (Merck, Germany) solution was then added and the gel was stained for 25 min. The gel was then washed twice with milli-Q water and developed [3% (w/v) sodium carbonate, 0.025% formaldehyde (Sigma, USA)] until appropriate time. The staining was stopped by adding 1.4% (w/v) EDTA for 10 min. 2.4.3 Western blot 38 Protein samples were subjected to 1D-PAGE and transferred onto an Immun-BlotTM PVDF membrane [0.2 μm] (Bio-Rad, USA) with a Semi-Dry transfer system (Bio-Rad, USA) using transfer buffer consisting of 100 mM Tris (pH 7.4), 200 mM glycine, 20% (v/v) methanol. The membrane was then blocked using 5% (w/v) skim milk in phosphate buffered saline with 0.05% Tween-20 (Bio-Rad, USA) (PBST) for overnight at room temperature. The next day, the membrane was incubated with a primary antibody in 1% skim milk in PBST for 1 h and 30 min. The membrane was washed three times in PBST and incubated with HRP-conjugated secondary antibody in 1% skim milk in PBST for another 1 h, followed by three wash in PBST. Signal detection was performed using the SuperSignal WestPico Chemiluminescent substrate (Pierce, USA) and the Lumi-Film Chemiluminescent Detection film (Roche, USA). 2.4.4 Molecular cloning, expression and purification of OmpA in pET32a The full length of ompA was cloned from E. coli strain BW25115 with the primers, A-for and A-rev (Table 2). The PCR product was ligated into pGEMT-easy vector for blue-white screening and the positive clones were sequenced to confirm the correct sequence. The correct clones were then excised with EcoRI and NotI and the fragments was purified and ligated to pET32a which was predigested with the same enzymes. The ligated product was transformed into BL21 and transformants was confirmed by PCR. 39 For the expression, the fresh colony was inoculated into LB containing Amp and subcultured into fresh medium with a ratio of 1:100. The culture was grown at 37 ºC for 2-3 h until the OD600 is around 0.6. The IPTG was added to a final concentration of 1 mM. And the culture was further incubated for another 4 h. The bacteria were collected by centrifuge at 12,000 × g for 20 min and the pellet was stored at -80 ºC for up to one month. The bacterial pellet was suspended in sonication buffer [10 mM Tris-HCl, pH 7.5, 5 mM MgCl2] and sonicated until the cell suspension become clear. The inclusion body was collected by centrifuge at 20, 000 × g for 15 min at 4 ºC of the sonicated cell suspension. After that, the supernatant was removed and the pellet was washed with cell lysis buffer [10 mM Tris-HCl, 1 mM EDTA, 1% Triton X-100 and 1 × protease inhibitor cocktail] for 30 min at room temperature. Then, the solution was centrifuged at 20,000 × g to collect the pellet, which is the inclusion body. Thus, The purified inclusion body was dissolved in Inclusion Body Lysis Reagents (Pierce, USA) for another 30 min in room temperature and the solution was centrifuged at 20,000 × g for another 30 min. According to the manual of Inclusion Body Lysis Reagents, the supernatant was subjected to dialysis to exclude the urea. Briefly, the supernatant was firstly dialyzed in 6 M urea dissolved in 25 mM Tris-HCl for 6-12 h at cold room. Adding 250 ml 40 Tris-HCl, pH 7.4 for every 6-12 h until the final volume was 3 L. After that, the supernatant was further dialyzed for another 6-12 h in 2 L 25 mM Tris-HCl, pH 7.4. Finally, the sample was concentrated with Centricon (Millipore, USA). 2.4.5 Purification of outer membrane proteins from E. coli E. coli BW25113 was grown overnight in LB broth and collected by centrifuging at 12, 000 × g for 20 min. The cell pellet was resuspended in sonicatoin buffer [10 mM Tris-HCl, pH 7.4, 5 mM MgCl2] and sonicated until the suspension become clear, which was centrifuged at 4,000 × g at 4 ºC. The supernatant was centrifuged at a speed of 100, 000 × g for 40 min at 4 ºC. The pellet was resuspended in 2% Sodium lauroyl sarcosine (w/v) (Sigma, USA) dissolved in sonication buffer and loaded for ultracentrifugation at the same speed for another 40 min at 4 ºC. The final product was the outer membrane proteins and was dissolved in water or Tris-HCl. 2.4.6 Co-immunoprecipitation The co-immunoprecipitation was performed as previously described (Sambrook et al, 1989). Briefly, the purified outer membrane proteins or purified OmpA was mixed with Zebrafish body fluid and was incubated at 4 ºC with end-to-end roation for 16 h. After that, anti-myosin heavy chain monoclonal antibody or anti-his taq monoclonal antibody was added and incubated at 4 ºC for 1 h. Then, the protein A sepharose 41 slurry (Sigma, USA) was added and incubated at 4 ºC for 30 min. This mixture was thus centrifuged at 500 × g and the pellet was washed with NETN [20 mM Tris-HCl, pH 7.5, 1% NP-40, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 × protease inhibitor cocktail] supplemented with 900 mM NaCl for 5 times and washed with NETN for the last time. After decanting the supernatant, the pellet was added with 25 μl 1 × loading buffer and boiled for 5 min, which was used for SDS-PAGE analysis. 2.5 Whole bacteria pull-down assay 2.5.1 Body fluid isolation from Zebrafish The adult zebrafish was anesthetized in 0.5% 2-proxyehtnol (Sigma, USA) in water. The fish body was cut into several part and centrifuged at 4000 × g at 4 ºC for 30 min for 3 times. The collected supernatant was then centrifuged further at 15,000 × g for 10 min to exclude any insoluble substance and stored at -80 ºC until use. 2.5.2 Bacteria preparation The bacteria used in whole bacteria pull-down assay are all grown in LB for 3-4 h until the value of OD600 reached to 0.8 after subculturing. 2.5.2.1 Paraformaldehyde fixed bacteria 42 The bacteria was washed three times with PBS and incubated with 3.7% paraformaldehyde at 4 ºC for 1 h. The bacteria were washed three times and stored at -20 ºC until use. 2.5.2.2 Heat inactivated bacteria The bacteria was washed three times with PBS and incubated at 65 ºC for 2 h in water bath. The bacteria were washed three times and stored at -20 ºC until use. 2.5.2.3 Gentamycin-treated bacteria The bacteria were washed three times with PBS and added gentamycin to a final concentration of 50 μg / μl. The bacteria were washed three times and stored at -20 ºC until use. 2.5.2.4 Proteinase K-treated bacteria 1 × 108 bacteria were incubated with 5 mg / ml proteinase K solution at 37 ºC for 30 min. Subsequently, PMSF was added to a final concentration of 1mM and incubated at room temperature for 15 min and washed three times by PBS. The death of bacteria was confirmed by plating. 43 2.5.3 Bacteria pull-down assay 1 × 109 bacteria was mixed with 500 μl Zebrafish body fluid at 4 ºC for 2 h. Centrifuged at 8000 × g at 4 ºC for 5 min, the bacteria were washed with 1% NP-40 buffer [20 mM Tris-HCl, pH 8.0, 10% glycerol, 1% NP-40, 150 mM NaCl, 20 mM NaF, 3 mM Na3VO4, 1x Complete Protease Inhibitor cocktail]. This step was repeated 3 times. To elute the bacteria, 1% NP-40 buffer containing another 1M NaCl was used to elute the bound protein, which was incubated at 4 ºC for 1 h and collected at 12,000 × g for 10 min. Finally, the sample was concentrated by acetone. 2.6 Immunofluorescence microscopy examination of E. coli surface localization Overnight cultures of E. coli were washed with PBS and fixed in 3.7% paraformaldehyde for 30 min at 4 °C with rotation. In order to inhibit autofluorescence of washed E. coli, bacterial suspensions were blocked with 50 mM NH4Cl for 30 min. Non-specific binding sites of washed E. coli bacterial suspensions were blocked by 1% BSA for 1 h. Suspensions of 106 E. coli were then incubated with and without 200 μl isolated Zebrafish body fluid at 4°C for 2 h. Then the bacteria were washed three times with PBST. The anti-myosin heavy chain monoclonal antibody (Millipore, USA) was added at a dilution ratio of 1:100 and incubated at room temperature for 1 h. After extensive washing in PBST, the suspensions of E. coli were incubated with Alexa448 labeled anti-mouse sencondary antibody (Invitrogen, USA) for another 1 h. After washing 3 times, the bacteria were stained with DAPI 44 (Sigma, USA). The immunofluorescence stained bacteria were fixed to slides and examined for positive staining using fluorescence microscopy at oil immersion (× 1000). 2.7 Tissue lysis and cell lysate 2.7.1 Preparation of fish tissue lysate The Zebrafish was anesthetized with 0.5% 2-proxyehtnol in water. The cardiac muscle was collect mainly from fish heart, smooth muscle was collected from internal organs except heart and the skeletal muscle was collected from fish body. All of the tissues were washed three times in physiological solution [0.72% NaCl, 0.038% KCl, 0.0162% CaCl2, 0.023% MgSO4•7H2O, 0.1% NaHCO3, 0.041% NaH3PO4, and 0.1% glucose] (Wolf & Jackson, 1963). The tissues were homogenized in lysis buffer [150 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1% Triton X-100, 2 mM PMSF, 2 mM Na3VO4, 1 × protease inhibitors cocktail]. The tissue lysis was subsequently centrifuged at 15, 000 × g for 15 min at 4 ºC. Supernatant was immediately used for the whole bacteria pull-down assay or stored at -80 ºC as aliquots. 2.7.2 Preparation of red blood cell lysate 45 The red blood cell lysate was prepared by using 1% NP-40 lysis buffer as described previously (Martinez et al, 2005). Briefly, the red blood cell was washed three times with 0.6% NaCl solution and resuspended in the 1% NP-40 lysis buffer on ice for 15 min. Then the cell was centrifuged at 15, 000 × g for 15 min at 4 ºC. Supernatant was immediately used for bacteria pull-down assay or stored at -80 ºC until use. 2.7.3 Preparation of hemolysin and hemolysin-induced red blood cell lysate The hemolysin was isolated as described previously (Honda, et al, 1988). Briefly, the Vibrio parahaemolyticus was inoculated in BHI broth and was subcultured to fresh broth for another 16 h. The culture supernatant was obtained by centrifuge at 16,000 × g for 20 min and passed through 0.22 μm filter and concentrated with Centricon (10,000 KDa cut off). Solid ammonium sulfate (351 g / L) was added to the concentrated culture supernatant, and the resulting precipitate was dissolved in a small amount of 0.01 M phosphate buffer (Na2HPO4, pH 7.0), dialyzed overnight against the same buffer, and used as crude hemolysin. The lysis of the red blood cells was done by mixing 100 μl toxins diluted with 10 mM Tris-HCl (pH 7.0) and an equal volume of 2% fish red blood cells in the same buffer, which was incubated at 37 ºC for 30 min and centrifuged at 1,800 × g for 2 min. The supernantant was used for the whole bacteria pull-down assay. Meanwhile, the 46 supernatant was transferred to the cuvette and measured at A540 to confirm the release of hemoglobin from cell lysis. 2.8 Fluorescence labeling of bacteria Heat-killed E. coli were washed in PBS, then labeled with FITC by incubation with 0.1 mg / ml FITC isomer 1 (Sigma Chemical, USA) in 0.1 M NaHCO3, pH 9.0 at 25°C for 60 min (Gelfand et al., 1980). Bacteria were pelleted at 12,500 ×g for 5 min, then washed free of unbound fluorochrome with PBS and stored frozen at -20°C until used. 2.9 Phagocytosis assay The phagocytosis assay was performed using a standardized protocol as described previously (Czuprynski et al, 1984; Drevets & Campbell, 1991). Briefly, 5 x 106 J774 macrophages and 5 x 107 FITC-labeled E. coli or E. coli ΔompA were mixed with 10% FBS DMEM and diluted to 1 ml final volume in 12 x 75 mm polypropylene snap cap tubes (Falcon, Becton-Dickenson Labware, Lincoln Park, USA). The tubes were then rotated end-over-end for 30 min at 37 °C. After that, the tubes were centrifuged at 300 × g for 10 min at 4 °C. The bacteria that did not bind to macrophage were removed by washing the cells with 2 ml iced HBSS for three times. The cells were resuspended in 1.0 ml PBS with 5% fetal calf serum and 5 mM glucose, supplemented 47 with 0.5 μg / ml cytochalasin D (Sigma, USA). To quantify internal vs external bacteria, after the incubation, 100 μl aliquots of macrophages in suspension were mixed with ethidium bromide (EB) (Sigma, USA) to a final EB concentration of 50 μg / ml. Then a 10 μl of the mixture was immediately placed on a glass slide and overlaid with a coverslip. Phagocytosis of FITC-labeled bacteria was visualized with a fluorescence microscope (Olympus) using a 520 nm FITC filter under oil immersion (1000 x) and quantified by counting 60 consecutive individual macrophages. The intracellular bacteria appeared as green and the extracellular bacteria, which was quenched with ethidium bromide appeared as red-orange. 2.10 Statistical analysis All data from phagocytosis assay were analyzed using one-way ANOVA and a Duncan multiple range test (SAS software, SAS Insitute Inc., Cary, NC.). Values of p< 0.05 were considered to be significant. 48 CHAPTER 3 RESULTS 49 3.1 Interactomics study between Zebrafish body fluid proteins and E. coli reveals MHC can bind to E. coli K12. In order to identify protein molecules in Zebrafish body fluid that can interact with E. coli surface, we utilized a whole bacteria pull-down assay coupled with proteomics to set up an interaction profile. In this system, the heat-inactivated E. coli was used as the “beads” to pull-down the host proteins in Zebrafish body fluid. The eluted proteins from E. coli surface was resolved in a 4-20% NuPage gradient gel and protein bands were subsequently excised and subjected to MALDI-TOF-TOF analysis (Fig. 2). The proteins sent to mass spectrometry (MS) were indicated as arrows in Fig. 2 and the identified proteins and their descriptions were summarized in Table 3 and Fig. 3. Proteins that did not match to the Zebrafish protein database were not included in the list. Among the 12 samples, only 4 of them could be matched and they are complement component 1 q -like protein (C1q-like protein), vitellogenin, nucleoside diphosphate kinase-Z2 and MHC. The C1q-like protein has been extensively studied both in human and mouse. It can bind to pathogen surface directly or to antigen-antibody complex to trigger the activation of downstream complement components, which leads to the activation of classical complement pathway to form a membrane attack complex and to clean pathogens (Gasque , 2004). 50 Fig. 2. Interaction profile between heat-inactivated E. coli and Zebrafish body fluid. Bacteria alone were used as a control to exclude the bacterial proteins that might be eluted under high salt concentration. The eluted proteins from the bacteria surface were resolved in a 4-12% NuPage gradient gel, which was silver stained. 51 52 Table 3. Summary of MS results of the identified Zebrafish proteins. No. 3 Accession gi|5620786 Mass 21327 Score 83 Description complement component 1, q subcomponent-like 1 (C1QL1) [Danio rerio] 7 gi|94733731 149923 242 novel protein similar to vitellogenin 1 (vg1) [Danio rerio] 8 gi|113678366 149836 306 hypothetical protein LOC559475 [D. rerio] 9 gi|21391472 128476 294 vitellogenin 1 [D. rerio] 10 gi|66773050 228486 320 myosin, heavy chain polypeptide 11, smooth muscle [D. rerio] 11 gi|41053595 17229 231 Nucloside diphosphate kinase-Z2 protein [D. rerio] 53 Fig. 3. The Peptide Mass Fingerprinting (PMF) results of the identified proteins as reported in Table 3. Figs A, B, C, D, E and F indicated below the PMF profiles are corresponding to #3, #7, #8, #9, #10 and #11, respectively in the Table 3. 54 1276.6304 4700 Reflector Spec #1 MC[BP = 1276.6, 433] 100 432.9 90 80 % Intensity 70 60 50 40 0 799.0 1441.8 2059.1597 1642.9703 1778.9641 1515.7682 1160.5311 1235.7368 10 1003.5677 20 842.5249 30 2084.6 2727.4 3370.2 4013.0 Mass (m/z) Fig. 3A. 1115.7015 4700 Reflector Spec #1 MC[BP = 1115.7, 172] 100 171.8 90 80 1463.9004 60 50 10 0 799.0 1441.8 1699.9467 1622.8662 20 1149.6820 30 1477.9193 40 842.5112 % Intensity 70 2084.6 2727.4 Mass (m/z) Fig. 3B. 55 3370.2 4013.0 1496.7024 4700 Reflector Spec #1 MC[BP = 1496.7, 1108] 100 1108.2 90 1115.7000 80 1515.7039 60 50 1622.8635 1441.8 2174.2212 0 799.0 1391.7515 1173.6316 842.5090 888.5323 10 1097.6809 20 1699.9460 1723.9397 30 1477.9167 1463.9056 40 1983.0452 1814.8427 % Intensity 70 2084.6 2727.4 3370.2 4013.0 Mass (m/z) Fig. 3C. 100 975.4611 4700 Reflector Spec #1 MC[BP = 975.5, 547] 547.1 80 938.5112 90 60 50 40 0 799.0 1441.8 2467.2036 1616.8143 1635.8367 10 1398.7024 20 1203.6429 30 1007.4522 % Intensity 70 2084.6 2727.4 Mass (m/z) Fig. 3D. 56 3370.2 4013.0 20 10 0 799.0 1441.8 2212.1438 1330.7578 1441.8 2259.9988 2035.0786 1818.9618 1743.8364 1627.8026 1335.6996 0 799.0 1473.7253 1349.7162 1276.6097 90 1032.5980 1983.0441 1826.9905 1699.9409 1115.6987 1642.9403 1463.9042 1622.8630 1287.6750 1203.6389 842.5057 30 1137.5999 1187.6619 30 1052.4875 10 961.4849 1010.5358 % Intensity 938.5107 100 1017.5775 100 944.5341 20 863.4762 % Intensity 4700 Reflector Spec #1 MC[BP = 938.5, 242] 241.6 90 80 70 60 50 40 2084.6 Mass (m/z) 2727.4 2084.6 Mass (m/z) 2727.4 Fig. 3F. 57 3370.2 3370.2 4013.0 Fig. 3E. 4700 Reflector Spec #1 MC[BP = 1330.8, 1135] 1135.3 80 70 60 50 40 4013.0 The functions of other three proteins identified in our study were poorly understood in their interactions with bacteria. Vitellogenin, an egg yolk protein precursor, plays a crucial role for the further development of oocyte in the form of nutrient (Wahli, 1988). The synthesis of this protein is only expressed in female fish but dormant in male fish under normal conditions. However, in the presence of estrogenic endocrine disrupting chemicals (EDCs), males can express the vitellogenin gene in a dose dependent manner. Besides the functions of a nutrient source and chemical exposure indicators, there is no literature describing vitellogenin interacts with microorganisms. Nucleoside diphosphate kinase-Z2 (NDK-Z2) functions intracellularly. The presence of this protein in our bacteria-pull down assay may be due to the contamination of intracellular component as a limitation of our sample collection method. MHC is the major component of myosin. It functions together with myosin light chain to promote the movement of proteins along actin filament inside cells or provide energy for the muscle contraction by hydrolyzing ATPs. The presence of this protein in our whole cell pull-down assay is interesting after we check that no myosin light chain (~20 kDa) was found in our MS data. Thus we further characterized the MHC and its association with E. coli in this study. 58 3.2 Characterization of bacteria-interacting MHC Although MHC is largely found inside cells, it also has been reported to be present in human plasma and serum, which is an important factor for the evaluation of muscle injuries. In addition, the MHC also exists in erythrocytes, which can undergo lysis and contaminate the body fluid during sample preparation (Fowler et al, 1985). Thus, there are three possible sources of MHC in Zebrafish body fluids: they are erythrocytes, plasma and serum. To investigate the presence of MHC in fish plasma or serum, another fish, tilapia, a bigger fish than Zebrafish, was used for the isolation of plasma and serum. The blood was collected with or without sodium citrate, a commonly used anticoagulant, to generate plasma and serum fractions, respectively. In addition, care was taken to make sure the plasma and serum were not contaminated with the tissue fluids in the collection protocol. The supernatant was subsequently used for the whole-bacteria pull-down assay and the eluted proteins were subjected to Western-blot analysis with anti-MHC monoclonal antibody. As shown in Fig. 4A, only the plasma fraction contained the MHC could bind to the E. coli surface. However, fish serum or erythrocytes lysate, did not show any MHC bands after the bacterial pull-down assay. 59 Fig. 4. Localization of bacteria-interacting MHC. (A) Western-blot results of the distribution of bacteria-interacting MHC in plasma, serum and erythrocytes after bacteria pull-down assays. (B) Western-blot results of the distribution of bacteria-interacting MHC in different tissues. The tissue lysates were used for bacteria pull-down assay and also subjected to SDS-PAGE analysis directly (supernatant). Anti-myosin heavy chain monoclonal antibody (anti-MHC) was used as the primary antibody and anti-mouse IgG antibody was used as secondary antibody. 60 A. B. 61 Fig. 5. Quantitiation of bacteria-interacting MHC in collected Zebrafish body fluids. (A) Bacteria pull-down assay of MHCs from 20 Zebrafish body fluid. (B) Bacteria pull-down assay of MHCs from 25 Zebrafish body fluid. The eluted proteins were resolved on 4-12% NuPage gradient gels and stained with Commassie brilliant blue R-250. The relative intensity of MHC in the gel was quantified by Quantity One software. The arrows indicated the position of MHC, which was confirmed by MS and Western-blot. 62 63 In addition, the plasma MHC fragments were generated from tissue injury and thus it was possible that the bacteria could interact with the myosin heavy from tissues. We also divided the tissue types to three muscle types: cardiac muscle, smooth muscle and skeletal muscle. Interestingly, the smooth MHC from internal organs except heart could bind to bacteria but not the samples from heart or skeletal muscles (Fig 4B). These data showed that the bacteria do not only bind to plasma MHC but also can bind to SM-MHC. This specificity implied that E. coli-MHC interaction may be a novel host-pathogen interaction mechanism. As only a subset of MHC in Zebrafish body fluid was able to bind to E. coli, we thus wanted to quantitate the relative amount of MHC in our collected samples. Relative quantitation in the Zebrafish body fluid sample was measured by a bovine serum albumin (BSA) serial dilution-based method. Two independent experiments with the Zebrafish sample were derived from 20 and 25 fish, respectively. This result showed that the relative concentration of bacteria-interacting MHC was about 0.33 - 0.42 ng / μl in Zebrafish body fluids (Fig. 5). 3.3 Characterization of the interaction between bacteria and MHC 64 In the bacteria-pull-down assay, the heat-inactivated E. coli bacteria were used to absorb the Zebrafish host proteins rather than live bacteria. Heat-inactivated bacteria will not secrete proteins during the experiment and thus will not introduce contaminated proteins in the pull-down assay. We also investigated whether live bacteria as well as chemical-treated bacteria could bind to MHC as well. As expected, live bacteria, gentamycin-treated and paraformaldehyde-treated bacteria showed similar results and they all interacted with the MHC (Fig. 6A). However, if the bacteria were pretreated with proteinase K, a non-specific serine protease, they did not interact with MHC as detected by the Western-blot analysis. This result indicated that the MHC may bind to E. coli via surface proteins. In addition, the immunefluorescence-based microscopy examination provided direct evidence that MHC could bind to the E. coli surface. As shown in Fig. 4B, MHC was found to adhere to E. coli surface. However, E. coli alone could not produce any fluorescence except a dim background. Furthermore, there were several regions that showed much stronger signals (Fig. 6B). This data suggested that MHC may interact with surface proteins of E. coli. To further characterize the interaction between bacteria and MHC, we tested whether the increasing of salt concentration would influence the MHC-bacteria interaction. In this experiment, after the incubation with Zebrafish body fluids at 4 65 Fig. 6. Characterization of interaction between MHC and E. coli. (A) The effect of different treatments of bacteria surface in E. coli-MHC interaction. (B) Localization of MHC on bacteria surface by immunofluorescence microscopy. (C) The influence of increasing NaCl concentrations on the interaction between E. coli and MHC. The MHC was detected by an anti-MHC antibody. 66 A . B. C. 67 ℃ for 2 h, the bacteria-MHC complex was treated with different concentrations of NaCl ranging from 100 mM to 500 mM. As shown in Fig. 6C, the binding of MHC to bacteria was not reduced that much even in high salt concentrations, i.e. 500 mM. Most of the MHC was still retained in the bacterial pellet. Thus, MHC may bind to bacteria using hydrophobic contact and only a minor contribution is from electrostatic force (Prasadarao et al, 2002). 3.4 Outer membrane protein A of E. coli can bind to MHC Our results showed that the MHC could not bind to bacteria after the proteinase K treatment. This indicated that the MHC might interact with the surface protein of E. coli. We thus interested in characterizing this possible MHC interacting protein from the outer membrane fraction of E. coli. The purified outer membrane proteins were first incubated with Zebrafish body fluids and this mixture was used for co-immunoprecipitation. Only one band around 37 kDa was shown in one-dimensional SDS-PAGE gel when comparing to the control group in which there were Zebrafish body fluid proteins only (Fig. 7A). The band was cut for MALDI-TOF-TOF identification. MS result showed that it was the outer membrane protein A (OmpA) from E. coli. To confirm this interaction, recombinant OmpA tagged with 6×His at C-terminal domain was generated in an 68 Fig. 7. Characterization of OmpA as the MHC binding protein on E. coli surface. (A) Co-immunoprecipitation of MHC with purified outer membrane fractions from E. coli by using an anti-MHC antibody. The proteins were resolved on a 10% SDS-PAGE gel and silver stained. The arrow indicated the position of OmpA. (B) Co-immunoprecipitation of recombinant OmpA with Zebrafish body fluid by using anti-his tag monoclonal antibody. The proteins were run on a 10% SDS-PAGE gel and silver stained. The arrow indicated the position of MHC. (C) Western-blot results of the Co-immunoprecipitation of recombinant OmpA with an Zebrafish body fluid using an anti-his tag antibody. The presence of MHC was detected by anti-MHC antibody. 69 A. B. C. 70 overexpression plasmid. After refolding (See Materials and methods), the OmpA was pre-incubated with Zebrafish body fluid and an anti-his tag monoclonal antibody was used for co-immunoprecipitation. The anti-his tag monoclonal antibody alone was used as a negative control to exclude the possibility of unspecific interaction between anti-his tag antibody and MHC. Both the silver staining and Western-blot results showed that recombinant OmpA could bind to MHC although some other proteins were also present (Fig. 7B and 7C). We thus conclude that Zebrafish MHC interacts with E. coli through OmpA. 3.5 E. coli -interacting MHC involved in OmpA-mediated anti-phagocytic function. For the biological function of the interaction between OmpA and MHC, we speculate that OmpA is a target for the host immune system and E. coli utilizes OmpA to interact with host proteins as an escape mechanism. In addition, phgocytosis of the invaders is an important defense mechanism used by the host. Thus, the interaction between OmpA and MHC may involve in phagocytosis. To test this possibility, fluorescence microscope-based phagocytosis assay in J774 macrophage cell line was used. The bacteria were divided into four groups: E. coli (WT), E. coli preincubated with Zebrafish body fluids (WTi), E. coli ΔompA (ΔompA) and E. coli ΔompA preincubated with Zebrafish body fluids (ΔompAi). 71 Fig. 8. The interaction between OmpA and MHC involved in anti-phagocytic function. (A) Difference phagocytic ability of J774 macrophages toward 4 different groups of E. coli. The values represent the mean ± SD from one representative experiment performed with triplicate samples. Similar results were obtained at least in triplicates. (B) Zebrafish body fluids volume-dependent phagocytosis assay. (C) Bacteria-concentration dependent phagocytosis assay. In the figures of B and C, the series 1, series 2 and series 3 demonstrate three independent experiments. 72 A. B. C. 73 For the first two groups, it was shown that the phagocytosis ratio of E. coli pre-incubated with Zebrafish body fluid was significantly reduced when comparing with bacteria alone. This data indicated that the Zebrafish body fluid contained certain proteins that can prevent E. coli from being phagocytosed by J774 macrophages. To test whether this phenomena was relevant to MHC, the full length OmpA was deleted in the E. coli background. The comparison of the phagocytosis ratio between the group of ΔompA and the group of ΔompAi showed MHC might involve in the phagocytosis assay as there was not much difference between these two groups (Fig. 8A.). Further more, based on these results, the body fluid-dependent phagocytosis and bacteria concentration-dependent phagocytosis were performed. As shown in Fig. 8B and Fig. 8C, when the Zebrafish body fluid volume increased, the phagocytosis ratio, however, decreased in a dose-dependent manner. This suggests that the more MHC interacted with bacteria, the less chance the bacteria would be phagocytosed. In the contrary, in the bacteria-concentration-dependent experiment, the phagocytosis ratio increased along with the increasing concentration of bacteria. This suggests that bacteria were more susceptible by the J774 macrophages without the “cover” of the MHC. Taken together, these results showed that the interaction between MHC and OmpA was involved in phagocytosis. 74 CHAPTER 4 DISCUSSION 75 4.1 Interactomics is a powerful tool to study host-pathogen interaction. Interactomics is defined as a kind of system biology dealing with the study of interactome, which is the interaction among proteins and molecules within a cell (Kitano, 2001; Bock & Goode, 2002). The cellular life is organized as an interaction network, in which no one protein can be functioned alone. They must interact with others to deliver the signal and affect the gene activation, which will ultimately change the cellular function. Thus, interactomics is another form of proteomics, whose purpose is to study the protein-protein interaction in a system level by using proteomics tools such as mass spectrometry-based protein identification and bioinformatic analysis (Coulombe et al, 2004; Lee & Lee, 2004; Gingras , et al, 2005; Singh et al, 2006). The pathogen surface is an important place for the interaction between hosts and pathogens. On one hand, the bacterial surface contains proteins and chemical structures that enable them to survival, replicate and invade the host. Smith proposed that microbe’s surface characteristics can be the determinants of the virulence of microbes (Smith, 1913; Smith, 1934; Smith, 1977). The pathogenic and nonpathogenic bacteria can be distinguished by their surface chemical structure (Casadevall & Pirofski, 1999). On the other hand, the surface characteristics of the pathogens can also be potential targets by the host’s immune system. A lot of work has been done on the interaction between pathogen’s surface and host’s immune 76 system. However, the concept of interactomics was not well studied and examined in the host-pathogen interaction. For example, only two reports used whole bacteria pull-down assay coupled with proteomics tools to identify the proteins in the hosts that can bind to pathogen surfaces (Martinez et al, 2005; Zhu, et al, 2005). In this study, we chose E. coli K12 as the prototype of pathogen and Zebrafish as the host to investigate the host-pathogen interaction. The reason that bacteria was heatinactivated before the whole-bacteria pull-down assay is that the live bacteria will secrete a lot of proteins during sample preparation and thus will contaminate the Zebrafish host proteins (Zhu, et al, 2005). Among the four identified proteins, only C1q1-like protein has been reported to bind bacteria while the other three proteins have not. Although there is no study showing that the vitellogenin is associated with bacteria infection, indirect evidence from other insects, such as mosquitoes, bees, crayfish and beetle, however, implied that this proteins may be a potential target used by the invaders. Several research groups found that upon the infection with parasites, the amount of vitellogenin mRNA was decreased (Hall et al, 1999; Ahmed et al, 2001; Fievet et al, 2006; Warr et al, 2006). The detection of vitellogenin in our pull-down assay is interesting and this may indicate that E. coli may use this protein to reduce the nutrient source inside the fish host to cause systematic infection. Nucloside diphosphate kinase (NDK) is a metabolic enzyme both in prokaryotic cell and eukaryotic cells. Its physiological function is to transfer a phosphate group from a 77 nucleoside triphosphate to a nucleoside diphosphate (Lazarowski et al, 2000). And thus, the NDKs serve to maintain the balance between the concentrations of different nucleotriphosphates (Lazarowski et al, 1997). Although the NDK is typically an intracellular enzyme, it was also detected on the mammalian cell surface, and it can take part as secreted forms in pathogens and parasites (Zaborina et al, 1999a; Zaborina et al, 1999b; Kamath et al, 2000; Gounaris et al, 2001). In addition, in plant, the mRNA expression level of nucleoside diphosphate kinase 1 (NDK1) was greatly elevated after the Xanthomonas oryzae pv. oryzae infection, although the relationship between NDK1 and bacteria infection should be further addressed (Cho et al, 2004). In our study, the NDK from Zebrafish body fluid can bind to E. coli is an interesting finding. E. coli may inhibit the normal function of NDK, while the NDK may also be a non-immune related molecule to help the host to clear the pathogens. These issues should be further addressed by examining the in vivo expression level upon bacterial infection. 4.2 E. coli binds to plasma MHC and smooth muscle MHC (SM-MHC). The presence of MHC in the plasma may be due to the muscle injury which causes the loss of cell membrane integrity and thus leads to the release of MHC from the cells (Onuoha et al, 2001). The fact that E. coli can interact with plasma MHC in Zebrafish body fluid intrigues us to do further investigation about the possible biological function of MHC. During our sample preparation, the samples may be contaminated 78 with the intracellular contents from erythrocytes. To rule out the possibility, we used two methods to treat the isolated erythrocytes: lysed with lysis buffer or hemolysin-induced cell lysis. Using lysis buffer was a mild way to break the cell membrane and will not affect the stability of the proteins, while using hemolysin-induced lysis was to mimic the actual infection process. However, neither of them released E. coli-interacting MHC. The MHC of erythrocytes belongs to the family of non-muscle MHC. Studies from fruit flies showed that the non-muscle MHCs were encoded by another myosin gene families (Kiehart et al, 1989). This may result in the loss of the domains that can interact with OmpA in E. coli. And the interaction between MHC and E. coli may be a specific binding. Furthermore, our data showed that the MHC from smooth muscle binds to E. coli (Fig. 2B), whereas the MHCs of cardiac muscle and skeletal muscle could not. Actually, all of the SM-MHC fragments, cardiac muscle MHC fragments and skeletal muscle MHC fragments have been found in human plasma (Mair et al, 1992; Suzuki et al, 2000; Onuoha et al, 2001). This tissue-specific interaction thus raised another interesting question about whether the plasma MHC is from smooth muscle injury or the plasma MHC that binds to E. coli is distinct from the SM-MHC. We favored the possibility that the bacteria-interacting plasma MHC is from SM-MHC. Non-pathogenic E. coli is living in the lower intestine in the host and serves as waste processing, vitamin production and food absorption (Feng et al, 2002). While the 79 pathogenic E. coli, such as E. coli strain O157:H7, causes sever damage to the epithelial cells of intestine, disrupting cell-cell conjugation and causing lesions in the intestine smooth muscle (Finlay & Abe, 1998). The interaction between pathogenic E. coli and host cells may cause the loss of cell membrane integrity and this may lead to the release of the MHC. In addition, the OmpA on E. coli surface is responsible for the interaction to plasma MHC. OmpA is quite conserved, sequence between the pathogenic and non-pathogenic E. coli is very similar. The difference of migration distance between skeletal muscle myosin heavy chain and cardiac myosin heavy chain might be due to different molecular weight of MHC in different tissue. Since the genomic sequence of Zebrafish is incomplete, it is difficult to address the migration variation at the moment. Alternatively, it is possible that the skeletal muscle myosin heavy chain might bind some cellular components that affect its migration rates. Further studies should be carried out to clarify all the issues. Taken together, we proposed that E. coli can specifically bind to a subset of plasma MHC and SM-MHC, and can have important biological function. Whether the pathogenic E. coli can cause the release of MHC from intestine can be confirmed later. 4.3 E. coli can actively bind to plasma MHC 80 Although we identified the plasma MHC by using heat-inactivated bacteria, which may change the surface structure of the bacteria during the heat inactivation. Our further analysis, however, confirmed that the interaction is similar with live bacteria. The treatment of bacteria with proteinase K has been widely used to study the protein-protein interactions in cell membrane. In this study, after the treatment of this protease, the plasma MHC is no longer bind to E. coli (Fig. 4A). These results showed us that the plasma MHC may interact with the surface proteins of E. coli and moreover, the plasma MHC is not associated with non-protein structure of E. coli, such as LPS and peptidoglycan. In addition, both the heat-inactivated bacteria and the live bacteria can bind to plasma MHC indicated that the bacteria are not required to actively change the surface structure or specific gene sequence to trigger the interaction to MHC. The immunofluorescence-based surface localization showed the plasma MHC was indeed adhered to E. coli surface and more importantly, it seems localized at specific areas of the bacteria surface (Fig. 4B). There are three possible reasons. First, the MHC might aggregate upon the binding of OmpA on E. coli surface. To rule out this possibility, we need to do co-localization of OmpA with MHC. The second possible reason is that not only OmpA in E. coli surface can bind to MHC, but also other polar proteins can also bind to MHC. This may cause the accumulation of fluorescence 81 signal at the sites. Another possible reason is that different bacteria generated by different growth status may lead to the different binding capacity to MHC. Protein-protein interactions involve complex mechanisms and are predominantly dictated by van der Waals contacts, hydrogen bonds and electrostatic forces (Kollman et al, 1994). The binding between E. coli and plasma MHC is hydrophobic in nature as their interaction is insensitive to high ionic strength. The NaCl concentration ranging from 0.1 – 1 M was generally accepted to evaluate the contribution of long-range electrostatic force to the protein-protein interaction (Blom et al, 2000). As the NaCl concentrations within this range can reduce the long-range electrostatic interaction and limit the formation of salt bridges (Dill et al, 1990). Thus, to evaluate the interaction property between plasma MHC and potential membrane surface proteins, we chose the range of salt concentrations from 0.1 M to 0.5 M. Interestingly, the interaction was not disrupted. Taken together, E. coli can actively bind to plasma MHC via certain membrane proteins and this interaction may be due to hydrophobic interaction. 4.4 OmpA-plasma MHC interaction may involve in anti-phagocytic function 82 By using co-immunoprecipitaion, we identified that OmpA is the bacterial component that is responsible for the binding of plasma MHC. Actually, OmpA / OprF protein family consists one of the largest outer membrane proteins family in Enterobacteriaceae bacteria and its related members are found in almost all of the Gram-negative bacteria. The OmpA has been shown to be involved in a broad range of bacteria functions including the maintenance of outer membrane integrity, porin-like activity, bacteriaphage receptor and biogenesis of LPS. OmpA typically transverses the outer membrane 8 times and 4 hydrophobic loops are exposed outside. The most conserved domains are the transmembrane domains while the extracellular loops are greatly variated among different bacteria, which may lead to different functions of this protein. OmpA is also a target by the host’s immune system. Besides lipoprotein in bacteria surface that can activate innate immunity through Toll-like receptors (TLRs) (Kirschning et al, 1998), the KpOmpA can also activate Toll-like receptor 2 (TLR 2)-meidated immune activation by specifically binding to scavenge receptors. In addition, the tumor-specific antigen coupled with KpOmpA can be taken up by antigen-presenting cells (APCs) and are processed through the conventional MHC class I pathway. Furthermore, the protective anti-tumor cytotoxic response can be initiated without the help of CD4 T cell and adjuvant. Thus, OmpA was proposed as a 83 new type of pathogen-associated molecular pattern, which is considered as a potential antigen carrier (Jeannin et al, 2000; Soulas et al, 2000; Goetsch et al, 2001). Our choice of J774 as the cellular model for phagocytosis assay is based on three reasons. First, OmpA is targeted by TLR2, which is a functionally conserved protein from mammal to fish (Hilary et al, 2005). Therefore, the investigation of the role of OmpA-MHC interaction on J774 is valid. Second, J774 is a well-characterized cell line for phagocytosis assay (Czuprynski et al, 1984; Drevets & Campbell, 1991). And more importantly, the J774 has been extensively used in the study of signaling pathway. Therefore, this cell line offers an advantage for further functional study about the interaction between macrophage and MHC-coated bacteria. Considering the importance of the OmpA in host’s immune activation and its essential functions in bacterial life, we thus seek to understand the biological function of their interaction based on the assumptions that plasma MHC may serve as a shield to protect OmpA from being recognized by the host. Indeed, phagocytosis showed that the OmpA-plasma MHC interaction greatly altered the phagocytosis. In other word, the PM-MHC coated E. coli has less chance to be phagcytosed by the macrophages comparing to the E. coli alone. The quantitive studies further confirmed the role of plasma MHC in phagocytosis. 84 However, the molecular mechanism of the anti-phagocytic function should be followed up. Further studies are needed to determine whether recombinant OmpA can be recognized and phagocytosed by macrophages such as KpOmpA and whether the plasma MHC can cover the epitopes that are required for the phagocytosis. It will be informative to investigate whether in vivo OmpA can bind to plasma MHC, which has biological function. In conclusion, our studies reveal a new perspective on host-pathogen interaction. When the bacteria invades into the host, they will selectively bind to host proteins to help their survival. Otherwise, they will be targeted by host surveillance system and will then be destroyed. In our case, we utilized the powerful proteomics tools to study the interaction between E. coli and Zebrafish body fluid, which lead to the identification of plasma MHC as a bacteria binding component. Later, we showed that this host protein was selectively bound by E. coli via OmpA. This interaction helps the bacteria to escape from being phagocytosed. 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Zhu, Y., Thangamani, S., Ho, B. & Ding, J. L. (2005) The ancient origin of the complement system. EMBO J. 24:382-94. 97 [...]... also contains 4 light chains (2 per head), which bind the heavy chains in the "neck" region between the head and tail (Tonomura & Oosawa, 1972 ; Korn et al, 1988) Being phosphorylated by myosin light chain kinases or Rho kinases, the myosin light chain can regulate the function of myosin by changing the conformation of myosin heads to detach from actin, increasing population placed close to thin filaments,... together, in clinical diagnosis, the change of the concentration of MHC in human serum and plasma is an important factor to examine the muscle injury and myosin- related diseases 12 1.3 The role of Outer membrane protein A (OmpA) in host-pathogen interaction 1.3.1 Basic structure of OmpA in E coli OmpA is one of the most extensively studied outer membrane proteins in Gram-negative bacteria This protein in. .. Thus based on the amino acid sequences of their ATP-hydrolyzing motor domains, the myosin protein family members can be divided into 20 classes Different classes can be distinguished from their tail domains (Alberts et al, 2001) Each myosin protein was composed with one or two MHCs and myosin light chains Myosin II, a subclass of myosin, for example, contains two heavy chains with each about 2000 amino... phage protein as well as involving in conjugation and in binding of a phage and a bacteriocin (Morona et al, 1984) 1.3.3 The role of OmpA in virulence The role of OmpA in virulence is mainly documented with the pathogenic E coli K1 The sequence of OmpA in E coli K1 is identical to that in E coli K12 Several important functions have been reported The evasion of serum -mediated killing was an important strategy... protein was reported that it can be released into circulation as the consequence of loss of cell membrane integrity Thus, it has been proposed as an important indicator of muscle injury in clinical diagnosis (Onuoha et al, 2001) The concentration of MHC together with the concentrations of creatine kinase, myoglobin and cardiac troponin I in human plasma were used to assess the myoskeletal muscle damage... potentiating actin -myosin interaction at low Ca2+ level, regulating ATPase activity of myosin and myosin assembly into filament (Wilson et al, 1992; Trybus, 1994; Stull et al, 1998; Depina & Langford, 1999; Nakamura & Kohama, 1999) 1.2.2 Clinical Significance of plasma MHC and serum MHC Although MHC is a structurally bound contractile protein of the thick filaments, this protein was reported that it can... than that the stationary E coli K1, while the ompA mutant E coli K1 cannot survive in the serum The reason for the survival effectiveness of log phase E coli K1 is due to the increasing binding of C4bp The OmpA- C4bp complex acts as a co -factor for the factor I in the cleavage of C3b and C4b, which prevents the formation of membrane attack complex (Selvaraj, et al, 2007) The other aspects of the functions... after injury the concentration of MHC in human plasma increased when comparing to control groups (Onuoha et al, 2001) A similar study was conducted to examine the amounts of four proteins: MHC, creatine kinase, myoglobin 11 and cardiac troponin I in human plasma to see the mycoskeletal injuries after surgerical treatments when comparing to the people who did not receive treatments This study also indicated... 2007) In A hydrophila, for example, 3 out of 7 identified antigenic outer membrane proteins could effectively prevent the killing of fish by bacteria challenge followed by immunization of these proteins Thus, these antigens, called protective antigens, can be further investigated for vaccine development (Chen et al, 2004) 1.2 Myosin heavy chain (MHC) and its Clinical significance 1.2.1 Overall review of. .. membrane proteins can also bind to host proteins to inhibit their functions such as OmpA of E coli K1 (Prasadarao, 2002b) They can work as sensors for the dangerous signals from the host, such as OprF of Pseudomonas aeruginosa to trigger activation of virulence-associated genes (Wu et al, 2005) Pathogens can also use the outer membrane proteins to interact with host cells for survival or to transverse .. .CHARACTERIZATION OF PLASMA MYOSIN HEAVY CHAIN IN ZEBRAFISH AS AN IMPORTANT FACTOR FOR OmpA-MEDIATED ANTI-PHAGOCYTIC FUNCTION By PENG BO (M.Sc, B.Sc) A THESIS SUBMITTED FOR THE DEGREE OF MASTER... light chain can regulate the function of myosin by changing the conformation of myosin heads to detach from actin, increasing population placed close to thin filaments, potentiating actin -myosin interaction... change of the concentration of MHC in human serum and plasma is an important factor to examine the muscle injury and myosin- related diseases 12 1.3 The role of Outer membrane protein A (OmpA) in