Georgia State University ScholarWorks @ Georgia State University Biology Dissertations Department of Biology 4-30-2009 Endothelial Cell Factors Involved in Bartonella Bacilliformis Pathogenesis Tanushree Soni Follow this and additional works at: https://scholarworks.gsu.edu/biology_diss Part of the Biology Commons Recommended Citation Soni, Tanushree, "Endothelial Cell Factors Involved in Bartonella Bacilliformis Pathogenesis." Dissertation, Georgia State University, 2009. https://scholarworks.gsu.edu/biology_diss/39 This Dissertation is brought to you for free and open access by the Department of Biology at ScholarWorks @ Georgia State University. It has been accepted for inclusion in Biology Dissertations by an authorized administrator of ScholarWorks @ Georgia State University. For more information, please contact [email protected]. ENDOTHELIAL CELL FACTORS INVOLVED IN BARTONELLA BACILLIFORMIS PATHOGENESIS by TANUSHREE SONI Under the Direction of Dr. Barbara Baumstark, Ph.D. ABSTRACT The genus Bartonella comprises emerging pathogens that are causative agents of a wide range of clinical manifestations such as cat scratch disease, bacillary angiomatosis, and Carrion’s disease. All species are transmitted by blood-sucking arthropods and infect erythrocytes and endothelial cells of hosts. Carrion’s disease is a bi-phasic infection caused by Bartonella bacilliformis which is characterized by hemolysis of infected erythrocytes followed by invasion of the vascular endothelium. This provokes pronounced cellular proliferation, angiogenesis and skin eruptions called verruga peruana. Endothelial cells are thought to be the primary niche wherein bacteria reside between inoculation and erythrocyte infection. This study aims to elucidate some of the endothelial factors involved during the verruga peruana phase of Carrion’s disease. In order to adhere to and invade human microvascular endothelial cells (HMEC-1), B. bacilliformis engages a family of cell receptors called integrins. We used anti-integrin antibodies to show that the primary integrin involved is the fibronectin receptor α5β1, although the vitronectin receptor αVβ3 also plays a minor role. We show B. bacilliformis invasion is also dependent on integrin ligands, fibronectin and vitronectin as antibodies against these proteins decreased invasion and attachment, whereas pre-treatment of the bacteria with these molecules enhanced infection of endothelial cells. Bacterial uptake requires various host cytoplasmic signaling pathways to work in tandem, and our study identified three mitogen activated protein kinases involved. Apart from MAPKs, phosphotidylinositol 3 kinase plays a role during invasion and cell survival. PI3K inhibitors blocked bacterial internalization and B. bacilliformis infected cells showed accelerated apoptosis. Lastly, microarray analysis was performed to study the gene expression profile of B. bacilliformis infected HMEC-1 cells. Numerous molecules of the integrin signaling pathways are involved, suggesting integrins as the major receptor recruited for the successful infection by B. bacilliformis. In summary this is the first study to demonstrate the role of integrins as B. bacilliformis receptors and integrin ligands as facilitators of infection. Gene expression analysis suggests the possibility that integrin mediated signaling pathways are the key modulators of cellular alterations during B. bacilliformis infection. This hypothesis is supported by the identification of some members of the integrin signaling pathway necessary for B. bacilliformis entry into endothelial cells. INDEX WORDS: Bartonella, Endothelial cells, Integrins, Invasion, Extracellular Matrix Proteins, Kinases, Microarray Analysis ENDOTHELIAL CELL FACTORS INVOLVED IN BARTONELLA BACILLIFORMIS PATHOGENESIS by TANUSHREE SONI A Dissertation Presented in Partial Fulfillment of Requirements for the Degree of Doctor of Philosophy in the College of Arts and Sciences Georgia State University 2008 Copyright by Tanushree Soni 2008 ENDOTHELIAL CELL FACTORS INVOLVED IN BARTONELLA BACILLIFORMIS PATHOGENESIS by TANUSHREE SONI Committee Chair: Barbara Baumstark Committee: PC Tai Zehava Eichenbaum Julia Hilliard Electronic Version Approved: Office of Graduate Studies College of Arts and Sciences Georgia State University May 2008 iv Acknowledgements This dissertation would not have been completed without the good wishes and love of some wonderful people. I would like to express my gratitude to them all. I would like to thank Dr Barbara Baumstark for providing me an environment that developed my scientific skills and ability to think independently. I am grateful to Dr P.C. Tai and Dr Zehava Eichenbaum for their patience and guidance during the completion of my dissertation. I am pleased to thank Dr Julia Hilliard who gave me much needed confidence, encouragement and valuable insights that helped shape this project. I am blessed to have the most loving parents, sister and brother in law. You all have been pillars of support and inspiration thorough out my life. Thank you, mummy, papa, Sonu and Dhiraj. I am also very fortunate to receive endless support and encouragement from mummyji, papaji, bhaiya and bhabhi. I owe a huge debt of gratitude to my friends: Kavita, Pradeep, Goutam and Madhavi for giving me the strength, love and their invaluable friendship. I would like to thank Griselle, Feda, and Drew who laughed and cried with me and made this journey so memorable. This testament of gratitude is incomplete without mentioning my husband, Samit. I am so grateful to God for having you in my life. You have given me unconditional love and confidence that not only helped me complete my dissertation but also enriched my life and made me a better person. Thank you for loving me. v TABLE OF CONTENTS Acknowledgements...................................................................................................... iv List of Tables................................................................................................................. vii List of Figures..............................................................................................................viii List of Abbreviations..................................................................................................... x General Introduction ................................................................................................... 12 History of Carrion’s disease ............................................................................14 Bacteriology ....................................................................................................16 Clinical manifestations of Bartonella infections ...............................................17 Oroya Fever and verruga peruana...............................................................17 Trench Fever ...............................................................................................18 Cat Scratch Disease (CSD) .........................................................................19 Bacillary angiomatosis and endocarditis......................................................19 Virulent factors involved in adhesion and invasion..........................................20 Erythrocyte infection by B. bacilliformis........................................................21 Interactions of B. bacilliformis with endothelial cells ....................................22 Interactions of B. henselae and B. quintana with endothelial cells...............24 Concluding remarks ........................................................................................28 Chapter I: Role of Integrins in B. bacilliformis infection of endothelial cells ........................................................................................................................................... 30 Introduction .....................................................................................................30 Integrins involved in bacterial pathogenesis ................................................31 Fibronectin ...................................................................................................34 Vitronectin....................................................................................................36 Bacterial cell adhesion molecules that engage integrins .............................37 Materials and Methods....................................................................................39 Results ............................................................................................................54 Discussion.......................................................................................................89 Chapter II: Role of cell signal molecules during B. bacilliformis infection.. 98 Introduction .....................................................................................................98 Involvement of Mitogen Activated Protein Kinases in bacterial pathogenesis .....................................................................................................................99 Involvement of Phosphoinsotidine 3 Kinase in bacterial pathogenesis......100 Materials and Methods..................................................................................102 Results ..........................................................................................................107 Discussion.....................................................................................................134 Chapter III: Expression patterns