ATOMIC FORCE MICROSCOPY STUDY OF MALARIA INFECTED RED BLOOD CELLS LI ANG NATIONAL UNIVERSITY OF SINGAPORE 2008 ATOMIC FORCE MICROSCOPY STUDY OF MALARIA INFECTED RED BLOOD CELLS LI ANG (B. S., FUDAN UNIVERSITY, CHINA) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF MACHANICAL ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2008 Acknowledgements Acknowledgements First of all, I would like to express my utmost gratitude and deepest appreciation to my supervisor, Associate Professor. Lim Chwee Teck for his dedicated support, invaluable insight and guidance, and continuous encouragement in the duration of the study. His influence on me is far beyond this thesis and will benefit me in my future research work. I am much grateful to my co-supervisor, Dr. Tan Shyong Wei, Kevin, for his inspirational help and valuable guidance in my research work. I would also like to thank Dr. Bruce Russel and Associate Professor. Brian Cooke, my outstanding research collaborators, for their constant support, helpful discussion, suggestion, recommendations and valuable perspectives. I really feel fortunate and enjoy working with them. I could not have made it through the four years in NUS without the support and encouragement from my friends, colleagues and current and former members in the Nano Biomechanics Lab. To Ms. Tan Phay Shing, Eunice, Mr. Hairul Nizam Bin Ramli, Ms. Nai Mui Hoon, Brenda, Mr. Lee Yew Yong, Gregory, Dr. Zhou En-Hua, Dr Xu Xiao-Jing for their helpful and friendly guidance during my early time. To Dr. Fu Hong-Xia, Mr. Vedula Sri Ram Krishna, Mr. Lim Tong Seng, Ms. Shi Hui, Ms. Tan Lee Ping, Mr. Chong Ee Jay, Ms. Qie Lan, Mr. Liu Ying, Ms. Ng Sin Yee, Dr. Lee Yew Hoe, Gabriel, Dr. Zhang Ji-Xuan, Ms. Low Yuen Hing, Mr. Li Qing-Sen, Ms. Jiao Gu-Yue, Mr. Tan Swee-Jin, Ms. Yow Soh Zeom, Dr. Zhang You-Sheng, Ms. Sun Wei, Mr. Yuan Jian, Ms. Zhang Rou, I would like to thank i Acknowledgements them for their assistance and contribution in one way or another to the success of this project. Thanks also go to my colleagues in the Laboratory of Molecular and Cellular Parasitology, especially to Ms. Yin Jing, Mr. Ammar Mansoor Hassanbhai, Ms. Ng Geok Choo, Mr. NP Ramachandran, for their assistance and time devoted to the culture of malaria parasite. In addition, I extend my gratitude to Dr. Monica A. Diez Silva, Associate Professor. Usa Lek-Uthai, Dr. Rossarin Suwanarusk, Ms. Kate Fernandez, Dr. John Mills, Professor. Ding Jeak Ling, Associate Professor. Ho Bow, Associate Professor. Sow Chorng Haur, for their helpful academic interactions. I would also like to acknowledge the following people for their friendship: Dr. Zhang Gui-Yong, Ms. Zhang Ying-Yan, Mr. Liu Zhuo, Mr. Li Zi-Rui, Ms. Li Mi, Mr. Luo Rong-Mo, Mr. Huang Liang, Mr. Pan Hai-Ning, Ms. Jiang Hai-Yan, Mr. Zheng Ye. To my family, I appreciate their love, encouragement and support in the duration of this thesis. Especially to my dear wife, Dr. Cheng Yuan, it is impossible for me to finish this work without her support and encouragement. Last but not the least, I am grateful to the National University of Singapore and NUSNNI for granting me the research scholarship which makes my study in NUS possible. Many thanks are conveyed to Department of Mechanical Engineering and Division of Bioengineering, for their material support to every aspect of this work. ii Table of contents Table of Contents ACKNOWLEDGEMENTS I TABLE OF CONTENTS III SUMMARY .VI LIST OF ABBREVIATIONS .VIII LIST OF SYMBOLS . X LIST OF TABLES XI LIST OF FIGURES XII CHAPTER INTRODUCTION . 1.1 Malaria Pathology . 1.1.1 Life Cycle of the Malaria Parasite 1.1.2 Pathogenesis of P. falciparum malaria . 1.2 Current Studies on Surface Morphology and Cytoadherence of IRBCs . 1.2.1 Imaging Techniques for Surface Morphological Studies . 1.2.2 Surface Morphological Studies on the other Plasmodium spp. IRBCs 10 1.2.3 Surface Morphological Studies on Babesia bovis IRBCs . 12 1.2.4 Molecular Mechanism of Cytoadherence of P. falciparum IRBCs 13 1.3 Objective and Scope of This Thesis . 24 1.3.1 Objectives . 24 1.3.2 Scope of Project 25 CHAPTER ATOMIC FORCE MICROSCOPY AND SINGLE-MOLECULE FORCE SPECTROSCOPIC TECHNIQUES 27 2.1 Instrumentation . 28 2.1.1 Working principle . 28 2.1.2 Components 29 2.2 Methods and applications for topographical imaging . 33 iii Table of contents 2.2.1 2.2.2 2.2.3 Operation modes . 33 Sample preparation for cell imaging . 38 Applications in imaging RBC surface 41 2.3 Methods and application in force spectroscopy . 43 2.3.1 Sample preparation . 44 2.3.2 Force mode 46 2.3.3 Interpretation of force curves 50 CHAPTER AFM IMAGING OF SURFACE MORPHOLOGY OF INFECTED CELLS 61 3.1 In-fluid Imaging and Limitations 62 3.1.1 Sample Preparation . 62 3.1.2 Results and Discussion . 64 3.2 Development of a Novel Imaging Method for Simultaneous Monitoring of Surface Morphology and Intracellular Development 68 3.2.1 Sample Preparation . 69 3.2.2 Results and Discussion . 71 3.3 Comparison of Different Laboratory Strains of P. falciparum . 77 3.3.1 Sample Preparation . 77 3.3.2 Results . 78 3.3.3 Discussion . 80 3.4 Changes to the surface of four Plasmodium spp. infected human erythrocytes from clinical isolates . 81 3.4.1 Sample Preparation . 83 3.4.2 Results . 85 3.4.3 Discussion . 95 3.5 Surface Morphology of Babesia-infected Red Cells . 100 3.5.1 Sample Preparation . 102 3.5.2 Results . 104 3.5.3 Discussion . 110 3.6 Conclusions 112 CHAPTER SINGLE MOLECULAR FORCE SPECTROSCOPY STUDY OF LIGAND-RECEPTOR INTERACTIONS INVOLVED IN CYTOADHERENCE . 113 4.1 Methods 114 4.1.1 Sample Preparation: Tip Functionalization 114 iv Table of contents 4.1.2 4.1.3 4.1.4 Sample Preparation: Enrichment of Infected Cells and Substrate Coating 118 AFM Data Collection 122 AFM Data Analysis 123 4.2 Results and Discussion 126 4.2.1 Results . 126 4.2.2 Discussion . 137 4.2.3 Limitations 143 CHAPTER CONCLUSIONS & RECOMMENDATIONS . 145 5.1 Conclusions 145 5.2 Recommendations . 149 PUBLICATIONS ARISING FROM THESIS . 150 REFERENCE 152 v Summary Summary Nano structural changes on the surface of the Plasmodium (P.) spp. infected red blood cells (IRBCs) have a profound importance on the pathobiology of human malaria. Knob-like protrusions have been reported on the surface of P. falciparum and P. malariae IRBCs whereas caveolae are found to be associated with P. vivax and P. ovale IRBCs. In particular, knobs of falciparum IRBCs are the focal adhesion sites mediating the cytoadherence of IRBCs to endothelium aligning the capillary, which is thought to be one of the key mechanisms involved in malaria pathology. However, due to tedious sample preparation and technical limitations associated with previous studies, surface ultrastructural changes of the clinical IRBCs remains unclear. Moreover, as cytoadherence occurs in a highly hydrodynamic environment, the intrinsic kinetic properties of different ligand-receptor interactions are critical in determining their pathological functions, which remain poorly understood. In this thesis, we developed a novel sample preparation protocol for combining Atomic Force Microscopy (AFM) scanning technique and different optical imaging techniques. Here, we comprehensively investigated the surface morphology of different strains as well as different species of human Plasmodium IRBCs and compared that with an animal model, Babesia bovis infection. Significant phenotypic differences between laboratory clones and clinical isolates of P. falciparum infection were found. Inparticular, knobs were not always associated with the surface of infected cells from clinical isolates, and the density of knobs was significantly higher in clinical samples than that in laboratory clones. In addision, distinct surface features of the vi Summary other species of Plasmodium as well as Babesia bovis infected cells were also revealed. A constant number of caveolae was found on the surface of all stages of P. vivax IRBCs whereas P. malariae IRBCs surfaces were covered by numerous ‘knob-like’ structures. The number of ‘ridges’ on the surface of B. bovis IRBCs correlated positively with the strain virulence, suggesting a ‘surface structure’ dependant mechanism determining the severity of the disease. We also applied the single molecular force spectroscopy technique to quantify the dynamic force spectra and characterize the intrinsic kinetic parameters for specific ligand-receptor interactions involved in cytoadherence of P. falciparum infection. Temperature was found to play an important role in affecting the dissociation rates as well as free energy barrier width of different ligands binding to IRBCs. Results from the comparison of CD36 with TSP (both being endothelial receptors that have binding affinities for the malaria exported proteins expressed at the knob) at physiological temperature showed that CD36 mediated interaction was much more stable than that mediated by TSP, although TSP-IRBCs interaction was stronger than CD36-IRBCs interaction in the fast pulling rate regime. This suggests that TSP may initiate the cell adhesion process by catching the fast flowing IRBCs whereas CD36 functions as the major ‘holder’ for providing stable binding. Our study should provide valuable information on the structure-property-function relationship and the biophysical and pathological functions of host receptors and parasite ligands, which will help to identify therapeutic targets and to develop novel drug candidates to reduce the morbidity and mortality burden caused by malaria. vii List of Abbreviations LIST OF ABBREVIATIONS aa AFM ANB-NOS APTES B. BFP BMM BS3 BSA CD36 CIDR CSA CVC EDC EC ECM EM eq. Fc GAGs GPIV GST HA His HOPG HS ICAM-1 LFA-1 Ig IL-1 IMPs IRBCs KAHRP KO. 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Journal of the American Chemical Society 127, 11230-11231. 176 [...]... constitute less than 2% of the total blood volume Whereas P malariae only infects older red cells with the number even more limited 2 The unique sequestration or cytoadherence property of P falciparum infected red cells may cause ‘mechanical’ vascular obstructions It has been observed for over a century (Bignami and Bastianelli, 1889) that only early ring stage falciparum infected red cells appear in peripheral... (account for 95% of total infection) and P falciparum being responsible for more than 90% of malaria related deaths The following sections of this chapter will discuss the general aspects of malaria parasite biology with a focus on P falciparum 1.1.1 Life Cycle of the Malaria Parasite The life cycle of the malaria parasite is shown in Figure 1.1 Infection in humans is initiated when an infected Anopheles... the number of ridges on the surface of B bovis -infected RBCs in relation to parasite maturity and level of virulence Typical AFM images showing the variation in the number of ridges present on the surface of bovine RBCs infected with either single (B and D) or paired (A and C) forms of B bovis (Anderson or K-strain) 107 Figure 3.20 Statistical quantification of density (A) and height (B) of ridges... scanning electron microscopy (SEM), which provides more detailed surface images of the sample than TEM A comprehensive study on the surface morphology of falciparum IRBCs was performed by Gruenberg et al in 1983 (Gruenberg et al., 1983) They compared the SEM images of different stages of infected cells and found the number and density of knob structures changed This is the first study focusing on quantitative... morphology of B bovis of different strains at different stages still remains unclear 1.2.4 Molecular Mechanism of Cytoadherence of P falciparum IRBCs Direct evidence of cytoadherence originates from histological examinations of the microcirculation in blood vessels from cerebral malaria patients in which large amounts of late stage parasitized cells accumulate and sequentially perturb or fully obstruct the blood. .. Parasite invasion of red blood cells (RBCs) involves specific interactions between merozoite surface antigens and erythrocyte membrane proteins and unfolds in four steps: initial contact; reorientation and junction formation; deformation of host cell membrane and entry of merozoite; and resealing of erythrocyte membrane (Aikawa et al., 1978; Dvorak et al., 1975) Once within the host red blood cells, the merozoites... knobby microgametocyte of P malariae (B) 94 Figure 3.18 Surface architecture of PRBCs imaged by atomic force microscopy (Hutchings et al., 2007) (A) Bovine RBC infected with B bovis showing ridge protrusions visible on the cell surface (B) Higher magnification of the RBC in (A) showing ridge morphology in greater detail (C) Bovine RBC infected with B bigemina Note the absence of ridges on the RBC... have been observed over the surface of malaria infected RBCs: the ‘knobs’ of P falciparum and P malariae (some evidence suggests their presence on P ovlae) and ‘caveolae’ of P vivax and P ovale (with one study suggesting caveole is also found on P malariae IRBCs) (Atkinson and Aikawa, 1990) Our current understanding of these structures is derived mainly from electron microscopy (EM) observations and there... Organization ix List of Symbols LIST OF SYMBOLS ∆G ∆G* ∆H ∆S F kB KD Keq koff kon NB P R r T t voff von xβ τ free energy barrier reduced free energy barrier at the presence of external force thermodynamic enthalpy thermodynamic entropy the force acting on the bond Boltzmann's constant dissociation constant equilibrium constant dissociation rate or off rate association rate or on rate probability of bond survival... probability density function of unbinding force the gas constant loading rate temperature time the natural vibration frequency factor of the dissociation of the bond in vacuum the natural vibration frequency factor of the association of the bond in vacuum Free energy barrier width or mechanical reaction coordinate life time of molecular bond x List of Tables List of Tables Table 2-1 Summary of different surface . ATOMIC FORCE MICROSCOPY STUDY OF MALARIA INFECTED RED BLOOD CELLS LI ANG NATIONAL UNIVERSITY OF SINGAPORE 2008 ATOMIC FORCE MICROSCOPY STUDY OF MALARIA INFECTED. surface of the Plasmodium (P.) spp. infected red blood cells (IRBCs) have a profound importance on the pathobiology of human malaria. Knob-like protrusions have been reported on the surface of P of Cytoadherence of P. falciparum IRBCs 13 1.3 Objective and Scope of This Thesis 24 1.3.1 Objectives 24 1.3.2 Scope of Project 25 CHAPTER 2 ATOMIC FORCE MICROSCOPY AND SINGLE-MOLECULE FORCE