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Secretion of Malaria Transmission-Blocking Proteins from Paratransgenic Bacteria Nicholas Bongio

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Secretion of Malaria Transmission-Blocking Proteins from Paratransgenic Bacteria Nicholas Bongio Duquesne University Duquesne Scholarship Collection Electronic Theses and Dissertations Spring 2015 Secretion of Malaria Transmission-Blocking Proteins from Paratransgenic Bacteria Nicholas Bongio Follow this and additional works at: https://dsc.duq.edu/etd Recommended Citation Bongio, N. (2015). Secretion of Malaria Transmission-Blocking Proteins from Paratransgenic Bacteria (Doctoral dissertation, Duquesne University). Retrieved from https://dsc.duq.edu/etd/335 This Immediate Access is brought to you for free and open access by Duquesne Scholarship Collection. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of Duquesne Scholarship Collection. For more information, please contact [email protected]. SECRETION OF MALARIA TRANSMISSION-BLOCKING PROTEINS FROM PARATRANSGENIC BACTERIA A Dissertation Submitted to the Bayer School of Natural and Environmental Sciences Duquesne University In partial fulfillment of the requirements for the degree of Doctor of Philosophy By Nicholas James Bongio May 2015 Copyright by Nicholas James Bongio 2015 SECRETION OF MALARIA TRANSMISSION-BLOCKING PROTEINS FROM PARATRANSGENIC BACTERIA By Nicholas James Bongio Approved May 19, 2014 ________________________________ ________________________________ Dr. David Lampe Dr. Nancy Trun Associate Professor of Biology Associate Professor of Biology (Committee Chair) (Committee Member) ________________________________ ________________________________ Dr. Marcelo Jacobs-Lorena Dr. Jana Patton-Vogt Professor of Molecular Biology and Associate Professor of Biology Immunology (Committee Member) (Committee Member) ________________________________ ________________________________ Dr. Philip Reeder Dr. Joseph McCormick Dean, Bayer School Chair, Biological Sciences Professor of Environmental Science Associate Professor of Biology iii ABSTRACT SECRETION OF MALARIA TRANSMISSION-BLOCKING PROTEINS FROM PARATRANSGENIC BACTERIA By Nicholas James Bongio May 2015 Dissertation supervised by Dr. David Lampe Malaria is a debilitating and deadly disease that afflicts over 200 million people and kills over 600 thousand each year. Due to quickly evolving drug resistance and lack of an affordable vaccine, novel interventions are needed to fight the Plasmodium parasites that cause malaria. Targeting Plasmodium inside their mosquito hosts is one approach that could complement other preventative and medicinal interventions by reducing the ability of the mosquitoes to transmit the disease to humans. The research presented here uses paratransgenesis, the genetic modification of symbiotic bacteria within the mosquito midgut, to provide antimalarial protein to the mosquito and to interfere with the life cycle of Plasmodium within the insect host. This research has produced three new antimalarial paratransgenic tools. The first tool is a set of new antimalarial effector proteins that were constructed by converting iv anti-Plasmodium mouse antibodies into single-chain variable fragment (scFv) versions for expression by bacteria. These antibodies bind to Plasmodium surface proteins and interfere with critical steps in the parasite life cycle. The second tool is a modified bacterial species, Pantoea agglomerans, which was engineered to secrete diverse antimalarial proteins via the hemolysin secretion pathway. Modified P. agglomerans were fed to mosquitoes and were capable of inhibiting the invasion of Plasmodium within the midgut. The third tool is another modified bacterial species, Asaia sp. SF2.1. Native Type II secretion signals were discovered that enable the creation of paratransgenic strains of these bacteria. Modified strains of Asaia sp. SF2.1 were also demonstrated to interfere with the invasion of Plasmodium within the mosquito. These tools have laid the groundwork for the future use of paratransgenic bacteria to combat malaria in the wild. Asaia sp. SF2.1 bacteria, in particular, are capable of spreading throughout mosquito populations, so they provide their own drive mechanism to establish themselves within the mosquito vectors of malaria. While further modifications will be required to make these bacteria ready for field use, the findings of this research provide proof of concept that the bacteria are suitable for eventual use in malaria transmission-blocking interventions. v DEDICATION This work is dedicated to my wonderful fiancée, Candice Kruth. She has inspired me to achieve all that I have, and my life would not be complete without her. She has helped me in countless ways to follow the path I have chosen and to accomplish the goals I set forth. I would like to thank her for the weeks she spent editing my writing into the legible form presented in this volume. Without her love and support, I would not be where I am today. vi ACKNOWLEDGEMENTS I would like to thank my committee members who were always helpful and generous with their time. A special thanks to my Ph.D. advisor, Dr. David Lampe, who has been a great friend and mentor over the years. Working in your lab has been a great experience, and I would not be the scientist I am today without having had the opportunity to study with you. Thank you Dr. Marcelo Jacobs-Lorena, Dr. Jana Patton- Vogt, and Dr. Nancy Trun for agreeing to serve on my committee. The time you have spent giving advice and feedback on my work has been integral to my growth and success, and all of your hard work is appreciated. I would also like to thank the Bayer School faculty, staff, and students for all the assistance they have provided to me in my time at Duquesne and for the friendship we shared. The community in the Department of Biological Sciences has made this place a great environment to work in. I will fondly remember my time at Duquesne. I’d like to thank both the Bongio and Kruth families for their love and support. My parents have always supported me and encouraged me to achieve great things. The Kruth family has been just as loving as my own, and they have always been there when I needed them. I am forever grateful to you all for your helping hands and open hearts over these years. vii ATTRIBUTIONS In Chapter 1, 2, 4, and 5, the design and execution of experiments were fully conducted by Nicholas Bongio. In Chapter 3, Nicholas Bongio was responsible for designing, cloning, and testing the bacterial expression of two of the antimalarial genetic constructs and overseeing the work of an undergraduate researcher on similar constructs. All other work in this chapter was conducted by the lab of Marcelo Jacobs-Lorena, including the writing of the manuscript. The appendix publication “a Draft Genome Sequence of Asaia sp. SF2.1, an Important Member of the Microbiome of Anopheles sp. Mosquitoes” was written by Dr Lavid Lampe, and edited by Nicholas Bongio and Jackie Shane. Jackie Shane prepared the DNA sample that was used for sequencing. The appendix publication “Paratransgenesis in Mosquitoes and Other Insects: Microbial Ecology and Bacterial Genetic Considerations” was written by Dr. David Lampe, and edited by Nicholas Bongio. Figures were created by Nicholas Bongio. viii TABLE OF CONTENTS Page Abstract .............................................................................................................................. iv Dedication .......................................................................................................................... vi Acknowledgement ............................................................................................................ vii Attributions ...................................................................................................................... viii List of Tables .................................................................................................................... xii List of Figures .................................................................................................................. xiii Chapter 1: The problem of malaria .............................................................1 MALARIA: THE DISEASE................................................................................................1 ANTIMALARIAL EFFORTS .............................................................................................3 THE BIOLOGY AND LIFE CYCLE OF PLASMODIUM .................................................4 TRANSMISSION-BLOCKING EFFECTOR PROTEINS .................................................7 THE MOSQUITO VECTORS OF HUMAN MALARIA.................................................10 MOSQUITO MICROBIOTA ............................................................................................11 TRANSGENESIS AND PARATRANSGENESIS ...........................................................13 PROTEIN SECRETION FROM GRAM-NEGATIVE BACTERIA ................................17 Chapter 2: Production of single-chain variable fragment antibodies and secretion from bacteria ....................................................25 ABSTRACT .......................................................................................................................25 BACKGROUND ...............................................................................................................26 MATERIALS AND METHODS .......................................................................................34 ix RESULTS ..........................................................................................................................48 DISCUSSION ....................................................................................................................52
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