BIOINFORMATIC ANALYSIS AND IN VITRO EXPRESSION OF MALARIA PARASITE TRANSLOCON AND RIBONUCLEASE BINDING–LIKE RHOPTRY GENES MOSES Z. TIMTA Bachelors of Science in Medicine and Surgery University of Maiduguri, Borno, Nigeria January 1991 United States Medical Licensing Examinations Educational Commission for Foreign Medical Graduates Philadelphia, PA October 2005 Submitted in partial fulfillment of the requirements for The degree of MASTER OF SCIENCE IN BIOLOGY at the CLEVELAND STATE UNIVERSITY May 2013 We hereby approve this thesis of MOSES Z. TIMTA Candidate for the Master of Science in Biology degree for the Department of Biological, Geological, and Environmental Sciences and the CLEVELAND STATE UNIVERSITY College of Graduate Studies _________________________________________________________________ Tobili Y. Sam-Yellowe, Ph.D., MPH / Date Thesis Committee Chairperson Department of Biological, Geological, and Environmental Sciences _________________________________________________________________ Girish C. Shukla, Ph.D. / Date Department of Biological, Geological, and Environmental Sciences _________________________________________________________________ Harry VanKeulen, Ph.D. / Date Department of Biological, Geological, and Environmental Sciences DEDICATION This work is dedicated to my daughter for her patience and understanding during the long hours of absence while trying to bring this work to a fruitful end. ACKNOWLEDGEMENT My most sincere and grateful appreciation to my adviser, Dr. Tobili Y. Sam-Yellowe, who let me use her lab and gave me the necessary advice and tools that I needed while carrying out this research. I also thank the members of my committee, Dr. Girish S. Shukla, and Dr. Harry Van Kuelen, who both offered objective and helpful criticisms. I also thank Raghav Yadavalli and Ruth Kebede and friends in the department who also offered their valuable help during the course of this research. BIOINFORMATIC ANALYSIS AND IN VITRO EXPRESSION OF MALARIA PARASITE TRANSLOCON AND RIBONUCLEASE BINDING-LIKE RHOPTRY GENES MOSES Z. TIMTA ABSTRACT Malaria caused by the parasite Plasmodium, still remains a significant public health problem worldwide, due to lack of a vaccine and emerging drug and insecticide resistance, among malaria parasites and mosquito vectors, respectively. Rhoptry proteins of Plasmodium enable merozoite invasion of host erythrocytes. However, only a few of these proteins have been characterized. Thirty-six P. yoelii merozoite rhoptry proteins were identified as putative rhoptry proteins by proteome analysis. Some of these proteins have been characterized while others still remain an intense area of active research. Molecular characterization and understanding of these novel proteins may assist in vaccine development, design of diagnostic assays and better control of malaria disease. This study was aimed at characterizing two Plasmodium rhoptry genes; Translocon and Ribonuclease binding-like (RNB-like) genes using bioinformatics analysis and in vitro cell free expression. Bioinformatics analysis was performed using the databases: PlasmoDB, ExPaSy, PSORTb, SWISSPROT-workspace, GeneDB, National Center for Biotechnology Information (NCBI) and COBALT: Multiple Alignment Tool. Both genes were characterized for features such as conservation profiles, domain architecture and alignment of sequences, both within Plasmodium species and among members of the v phylum apicomplexa. The RNB protein domains are generally conserved across Plasmodium species but protein identity across species is 30%. The amino acid identity is about 40% across species for the Translocon protein. This study revealed that these genes are expressed early upon merozoite invasion of the host erythrocytes. The expressed translocon protein that is annotated as hypothetical or putative has been shown to be part of a transport complex and the Ribonuclease binding-like (RNB) gene expresses a putative RNB-like protein found in all species of Plasmodium. The translocon of Plasmodium falciparum was successfully PCR amplified, cloned and a 23 kDa protein was expressed in vitro. Expression was confirmed with rhoptry specific antibodies. vi TABLE OF CONTENTS DEDICATION ACKNOWLEDGEMENT PAGE ABSTRACT……………………………………………………………………………v LISTS OF TABLES........................................................................................................xii LISTS OF FIGURES…………………………………………………………………..xiii ABBREVIATIONS………………………………………………………………….....xiv CHAPTER I INTRODUCTION……………………………………………………………………...1 CLASSIFICATION OF Plasmodium…………………….……………………….…...2 SPECIES INFECTING HUMANS ……………………..……………………………..2 SPECIES INFECTING RODENTS………………………………………………..…..3 BIOLOGY OF Plasmodium………………………………………………….………..3 LIFE CYCLE OF Plasmodium......................................................................................4 MOSQUITO STAGE…………………………………..…………………………..….5 vii EXO-ERYTHROCYTIC STAGE………………………………………………….…5 ERYTHROCYTIC STAGE…………………………………………………………..6 TROPHOZOITE AND SCHIZONT STAGES……………………..………………...6 GAMETOGENESIS……………….………………………………………………….8 MECHANISM OF ERYTHROCYTE INVASION BY Plasmodium PARASITE…..9 FORMATION OF TIGHT JUNCTION AND PARASITOPHOROUS VACUOLE.12 INVASION MOTOR AND MOVING JUNCTION…………………………………12 APICAL COMPLEX ORGANELLES……………………………………………….15 ROLE OF NECK PROTEINS IN HOST CELL INVASION………………………..16 RHOPTRY PROTEOME…………….………………………………………………17 PURPOSE…………………………………………………………………………….22 OBJECTIVES…………………………..………………………………………….…22 CHAPTER II………………………………………………………………………….23 EXPERIMENTAL APPROACH………………………….………………………..23 BIOINFORMATICS ANALYSIS………………...….……………………………..23 PARASITE CULTURING…….………..…..……………..………………………..23 viii DNA ISOLATION ……………………………………………………………………24 POLYMERASE CHAIN REACTION, CLONING AND PROTEIN EXPRESSION..24 EXPRESSION OF PROTEINS FROM GENES………………………………………25 IN VITRO HUMAN CELL-FREE EXPRESSION SYSTEM…………………………25 SDS-PAGE AND WESTERN BLOT………………………………………………….26 CHAPTER III………………………………………………………………………….28 RESULTS…………………………………………………………………….………..28 BIOINFORMATICS RESULT FOR TRANSLOCON GENE PTEX150 PF3D7_1436300 ..….…………………………..…………………………..28 MECHANISM OF TRANSLOCON FUNCTION…………………………………..30 TRANSLOCON PROTEIN EXPRESSION…………………………………………31 BIOINFORMATICS RESULT FOR RNB-LIKE GENE PF3D7_0906000 …........................................................................................................31 PROTEIN EXPRESSION DATA……………………………………………………..33 RESULTS OF CLONING AND EXPRESSION OF PF3D7_1436300……………..36 RNB-LIKE GENE AMPLIFICATION………………………………………………37 CHAPTER IV…………………………………………………………………………40 ix DISCUSSION………….……………………….…………………………………......40 SUMMARY…………………………………………..……………………….…........48 FUTURE STUDIES.……………………………………………………….…………48 REFERENCES.……………………………………………………………..…,,……49 APPENDICES.………………………………………………….…………..…..……59 Appendix 1. Table1A. GPI-anchored membrane surface proteins (MSP) secreted during RBC invasion ………………………………………………………60 Appendix 2. Multiple sequence alignment for PF3D7_1436300 (PTEX150) in Plasmodium species and Theileria ……………………………………………….65 Appendix 3. Predicted protein sequence of PF3D7_1436300 (PTEX150) translocon gene ………………………………………………………………………66 Appendix 4. PF3D7_1436300 (PTEX150) Multiple sequence alignment for Group OG5161974 ……………………………………………………………….67 Appendix 5. RNB-like protein segment showing amino acid alignment in Plasmodium species and some members of the phylum apicomplexa in the region of location of RNB-like residues 829-1322 (GeneDB) ……………….71 Appendix 6. Point of amino acid sequence homology in RNB-like protein in Plasmodium and E. coli (GeneDB) ………………………………………………72 Appendix 7. RNB-like peptide sequence found in E. coli (Ambler et al. 2006) between residues 85-156 …………………………………………………………...73 Appendix 8. RNB-like protein identity between P. yoelii (XP_723884), P. falciparum (XP_ 002808916) and E. coli (ACI73414) ………………………….73 x Appendix 9. Multiple Sequence Alignment of RNB-like (PF3D7_0906000) for Group 0G5 145111 (clusters of interest) …………………………………….74 xi LIST OF TABLES TABLE 1. SUMMARY OF INVASION-RELATED Plasmodium falciparum MEROZOITE PROTEINS………………………………………………………………………………18 TABLE 2. LIST OF PRIMERS ……………………………………………………… 26 xii LISTS OF FIGURES FIGURE 1. Plasmodium falciparum. Life cycle……………………………………..5 FIGURE 2. Illustration of host cell invasion by Plasmodium …………………………9 FIGURE 3. Illustrated guide to Ultrastructure of Plasmodium merozoite ………….. .16 FIGURE 4. Model for Translocon (PTEX150) function ……………………………..30 FIGURE 5. Summary of RNB domain architecture seen in Plasmodium and some members of the phylum, apicomplexa …………………………….…35 FIGURE 6. Genomic DNA isolation from three Plasmodium falciparum strains, Dd2, FCR3 and 3D7 ……………………….………………………………38 FIGURE 7. Western blot analysis of expressed 23kDa translocon protein …………..39 xiii ABBREVIATIONS AAA ------------------------- ATPases associated with various cellular activities AMA-1 ------------------------- Apical membrane antigen-1 BSA -------------------------- Bovine serum albumin CSD -------------------------- Cold shock domain CR1 -------------------------- Complement receptor-1 DARC --------------------------- Duffy antigen receptor for chemokines DBP ---------------------------- Duffy binding protein EBA --------------------------- Erythrocyte binding antigen EBL ---------------------------- Erythrocyte binding like proteins EDTA ---------------------------- Ethylenediamine tetra acetic acid EGF ----------------------------- Epithelium growth factor ER ----------------------------- Endoplasmic reticulum EXP2 ---------------------------- Exported