Functional analysis of the apical polar ring and its role in secretion and motility of Toxoplasma parasites Submitted in total fulfilment of the requirements of the degree of Doctor of Philosophy (PhD) 2016 School of BioSciences, Faculty of Science The University of Melbourne Nicholas Jeremy Katris Student no. 327539 December 2016, Revised July 2017 i Abstract Human parasites Toxoplasma and Plasmodium species belong to the phylum Apicomplexa and are some of the most successful groups of human parasites on the planet. Part of this success can be attributed to the cytoskeletal components that afford them structural stability and flexibility required to efficiently attach to and invade host cells. As members of the superfamily Alveolata, they possess a pellicle comprised of a set of flattened vacuoles pressed up against the plasma membrane, with proteinaceous support network and actin actin-based motility system. In addition to this, Toxoplasma also possesses an apical complex which is a tubulin based structure comprised of a set of apical polar rings and a conoid, which is a tight-knit tubulin based structure that is evolutionarily derived from ancestral flagella components. The apical complex is biologically significant because it is the entry point the parasite uses to enter a host cell in order to parasitize it, and this process is conserved in Plasmodium species. However, unlike other organelles, the proteins of the apical complex have no known conserved targeting signals so identification of proteins that target here has been slow to progress. A Toxoplasma protein homologous to a predicted cytoskeletal Tetrahymena thermophila protein was identified and localized to the apical complex, which we call RNG2. RNG2 was functionally characterized by inducible knock down and found that RNG2 played a role in the cGMP signalling pathway upstream of calcium dependent activation of CDPKs, which severely impacted microneme secretion, conoid extrusion and even other downstream processes, particularly internal calcium release. In addition to this, I used various calcium and cyclic di- nucleotide signalling agonists and inhibitors to investigate novel regulation patterns of micronemes and dense granules. RNG2 and other cGMP and calcium signalling proteins, PKG, CDPK1 and CDPK3 all show altered secretion of dense granules showing for the first time a regulatory mechanism of dense granules based on calcium. ii Declaration This is to certify that: i) The thesis comprises only my original work towards the PhD except where indicated in the Preface, ii) Due acknowledgement has been made in the text to all other material used, iii) The thesis is fewer than 100 000 words in length, exclusive of tables, maps, bibliographies and appendices. ______________________ Nicholas J Katris iii Preface The majority of work presented here is my own and was undertaken during the period of my PhD candidature. Certain analyses and experiments presented in Chapter two and three of this thesis were extended from preliminary findings made during my undergraduate research project. These findings include rough localizations of the RNG2 protein, the generation of the inducible RNG2 mutant cell line and the preliminary observation of an invasion defect in this cell line. I acknowledge the following people for assistance during the course of my PhD as follows; Establishment of the GCaMP6 technology in Toxoplasma, introduction of the construct into the iΔHA-RNG2 mutant cell line, and subsequent FACS analysis which was performed by Dr. Rebecca Stewart, and Dr. Chris Tonkin at WEHI as part of a collaborative effort. My contribution was to assist in optimization of drug dosage of BIPPO for FACS analysis of the RNG2 mutant. I would like to thank Dr. Paul McMIlllan and Dr. Eric Hanssen for assistance with the OMX 3D-SIM microscopy from Bio21 Institute, Melbourne, Australia, and Dr. Nicola Lawrence for her assistance with the OMX 3D-SIM in Gurdon Institute, Cambridge, United Kingdom. I have published much of the work presented in Chapter 2 and Chapter 3 regarding localization and functional analysis of RNG2 in the journal PLoS pathogens (Katris, N.J., van Dooren, G.G., McMillan, P.J., Hanssen, E., Tilley, L., and Waller, R.F. (2014). The apical complex provides a regulated gateway for secretion of invasion factors in Toxoplasma. PLoS pathogens 10, e1004074.). The PLoS Pathogens publication was written by myself, together with my supervisor Dr. Ross F. Waller and Dr. Giel van Dooren. iv Acknowledgements I would like to thank Dr. Ross Waller for ongoing supervisory support. Prof. Geoff McFadden for use of shared laboratory consumables and lab space. Dr. Giel van Dooren for Toxoplasma training and provision of the base plasmids to generate these cell lines. Dr. Chris Tonkin and Dr. Sebastian Lourido for gifting mutant Toxoplasma cell lines. Also thanks to Dr. Oliver Billker for provision of the Compound 2 drug, and Dr. Phillip Campbell for providing the BIPPO drug. A big thanks to everyone in the McFadden lab for hosting me during the transition from Melbourne. Thanks to everyone in the Carrington Lab at Cambridge for their patience and support during the move to Cambridge, and thanks to everyone in the Waller lab for creating such a cool atmosphere that made it fun to go to work. I will miss you guys the most. Lastly thanks to everyone in Melbourne, Cambridge, France and everywhere in between for being there during this very chaotic, turbulent, and thrilling adventure that became my PhD. v List of Abbreviations: ATC: Anhydrotetracycline AKMT: Toxoplasma apical complex lysine methyl transferase A23187: Calcium Ionophore A23187 BLE: Phleomycin resistance gene BIPPO: Abbreviated from 5-benzyl-3-isopropyl-1H-pyrazolo[4,3-d]-pyrimidin-7(6H)-one. (See Howard et al. 2016) CAT: Chloramphenicol acyltransferase resistance gene. CDPK1: Calcium Dependent Protein Kinase 1 CDPK3: Calcium Dependent Protein Kinase 3 CytD: Cytochalasin D actin inhibitor DHFR: Dihydrofolate Reductase resistance gene in Toxoplasma GA: Glutaraldehyde used for fixing cells for microscopy. GFP: green fluorescent protein. GRA1: Dense Granule Protein 1 in Toxoplasma, no homologue in Plasmodium species. HA: haemaglutinin epitope tag HX: Mycophenolic acid resistance gene in Toxoplasma IFA: Immunofluorescence assay IMC: Inner Membrane Complex iΔ: inducible knockdown Δ: knock-out Ku80: DNA repair enzyme preventing homologous recombination. MIC2: Microneme protein 2, homologue of TRAP protein in Plasmodium species. Myc: c-Myc epitope tag. MyoH: Acronym for describing Myosins, in this case Myosin H protein. PFA: Paraformeldehyde fixative used for fixing cells for microscopy. PKG: Protein Kinase G pPR2: plasmid containing T7S4 promoter for promoter replacement of target genes. pPR2-HA3: plasmid containing T7S4 promoter for promoter replacement of target genes and addition of an N-terminal Haemaglutinin tag RNG1: Toxoplasma Apical Polar Ring protein 1 vi RNG2: Toxoplasma Apical Polar Ring protein 2 TEM: Transmission Electron Microscopy ToxoDB: Toxoplasma genome online database vii Table of Contents Abstract pg iii Declaration pg iv Preface pg v Acknowledgements pg vi List of abbreviations pg vii List of Figures and tables pg. xii Chapter 1, Literature review: The Toxoplasma cytoskeleton and its role in invasion. 1.1.1 The life cycle of Toxoplasma pg. 2 1.1.2 Origin from algal ancestors: not just the apicoplast. pg. 3 1.1.3 Introduction to the Apicomplexan pellicle structures. pg. 4 1.1.4 The apical complex in the cell division process of Toxoplasma tachyzoites. pg. 6 1.1.5 Cell replication in Toxoplasma; Mitotic assembly, nuclear division and formation of nascent daughter buds. pg. 6 1.2.1 Invasion components of Toxoplasma; the molecular composition of the apical complex. pg. 8 1.2.2 Functional studies of apical complex proteins. pg. 9 1.2.3 The Apical Complex in Plasmodium. pg. 12 1.2.4 The IMC. pg. 16 1.2.5 The glideosome. pg. 17 1.2.6 Cytoskeletal Morphology and TgPhil1/TgSIP. pg. 19 1.2.7 Sub-pellicular Microtubules (SPMs). pg. 20 1.3.1 Secretion factors in Toxoplasma and the tight junction. pg. 20 1.3.2 Micronemes. pg. 21 1.3.3 Rhoptries. pg. 22 1.3.4 Dense Granule Proteins. pg. 23 1.4.1 Signalling in Toxoplasma invasion. pg. 25 1.4.2 Calcium and Calcium-dependent protein kinases (CDPKs). pg. 26 1.4.3 The role of cyclic-guanosine-monophosphate (cGMP) and Protein Kinase G. pg. 28 1.4.4 Cyclic Adenosine monophosphate (cAMP) signalling. pg. 29 1.4.5 Conclusion and perspectives pg. 31 viii Chapter 2- Identification and localization of Apical Complex Proteins: 2.1 Introduction pg. 33 2.2 Results pg. 37 2.2.1 Bioinformatic analysis of the RNG2 protein. pg. 35 2.2.2 RNG2 localization in daughter cell formation. pg. 35 2.2.3 Constructing a 3D model of the apical complex: pg. 39 2.3 Discussion pg. 43 2.3.1 RNG2 is the earliest known protein to appear at the apical complex. pg. 43 2.3.2 RNG2 and the centrosome. pg. 43 2.3.3 RNG2 connects the apical polar ring and the conoid at the apical complex. pg. 44 2.3.4 The apical complex as a tube of rings? pg. 45 2.3.5 Apical complex and conoid loss in Plasmodium. pg. 45 Chapter 3-Functional Characterization of apical polar ring proteins 3.1 Introduction pg. 48 3.2 Results pg. 50 3.2.1 Generation of RNG2 mutant. pg. 50 3.2.2 RNG2 knockdown has a severe growth defect. pg. 50 3.2.3 Toxoplasma tachyzoites are morphologically intact following RNG2 knockdown. pg. 52 3.2.4 RNG2 knockdown cells have an invasion defect. pg. 55 3.2.5 RNG2 knockdown cells are impaired in motility and tight junction formation. pg. 55 3.2.6 RNG2 has a role in regulated secretion of micronemes. pg. 58 3.2.7 RNG2 has an additional defect in conoid extrusion.