Thesis Reference

Thesis

Functional investigation of rhomboid proteases and their substrates in Toxoplasma gondii

MENDONCA DOS SANTOS, Joana

Abstract

Reference

MENDONCA DOS SANTOS, Joana. Functional investigation of rhomboid proteases and their substrates in Toxoplasma gondii. Thèse de doctorat : Univ. Genève, 2010, no. Sc. 4240

URN : urn:nbn:ch:unige-108163 DOI : 10.13097/archive-ouverte/unige:10816

Available at: http://archive-ouverte.unige.ch/unige:10816

Disclaimer: layout of this document may differ from the published version.

1 / 1 UNIVERSITÉ DE GENÈVE

Département de Biologie Moléculaire FACULTÉ DES SCIENCES Prof. Ueli Schibler

Département de Microbiologie FACULTÉ DE MÉDECINE et Médecine Moléculaire Prof. Dominique Soldati-Favre

______

Functional Investigation of Rhomboid Proteases and Their Substrates in Toxoplasma gondii

THÈSE présentée à la Faculté des Sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologie

par Joana Mendonça dos Santos de Torres Vedras (Portugal)

Thèse nº 4240 Imprimée par Genève Uni Mail 2010

UNIVERSITÉ DE GENÈVE

Département de Biologie Moleculaire FACULTÉ DES SCIENCES Prof. Ueli Schibler

Département de Microbiologie FACULTÉ DE MÉDECINE et Médecine Moléculaire Prof. Dominique Soldati-Favre

______

Functional Investigation of Rhomboid Proteases and Their Substrates in Toxoplasma gondii

THÈSE présentée à la Faculté des Sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologie

par Joana Mendonça dos Santos de Torres Vedras (Portugal)

Thése nº 4240 Imprimée par Genève Uni Mail 2010

The present thesis resulted in the publication of the following peer-reviewed scientific articles:

Friedrich N, Santos JM, Liu Y, Palma AS, Leon E, Saouros S, Kiso M, Blackman MJ, Matthews S, Feizi T, Soldati-Favre D (2009) Members of a novel protein family containing MAR domains act as sialic acid-binding lectins during host cell invasion by apicomplexan parasites. J Bio Chem 285(3): 2064-2076

Santos JM, Sheiner L, Klages N, Parussini F, Jemmely N, Friedrich N, Ward G, Soldati-Favre D (2010) Toxoplasma gondii transmembrane microneme proteins and their modular design. Mol Micro, in press

Santos JM, Ferguson D, Blackman MJ, Soldati-Favre D (2010) ROM4-mediated cleavage of AMA1 switches Toxoplasma from an invasive to a replicative mode. Submitted

Santos JM, Lebrun M, Daher W, Soldati D, Dubremetez JF (2009) Apicomplexan cytoskeleton and motors: key regulators in morphogenesis, cell division, transport and motility. Int J Parasitol 39(2): 153-62 Acknowledgments

I would like to thank first of all my great supervisor, Dominique Soldati, who always found a way to motivate me and who has given me the opportunity to do so many things during my PhD. I could have not asked for a better supervisor.

I would also like to thank Mike Blackman for all his support throughout the years and especially at the conclusion of my PhD.

I would like to thank Photini Sinnis for having given me the opportunity to work in her lab and for supporting all my decisions.

I would like to say a big thanks to the girls - Andrea, Louise, Christina, Hanni, Lilach, Karine and Valerie – and boys – Alberto and Christian – in Geneva. Thanks for always being there, for forcing me out, taking care of me and for being so fun to hang out with.

I want to thank Luís, Sara and Sofia for being as good long-distance friends as they were short-distance, and my parents and Pims for always supporting and listening to me.

I want to thank all the members of the Soldati lab, past and present, who have always supported me and made the lab such a great place to work: Paco, Mike and Tobias thanks for all the beer o’clock; Nikolas thank you for letting me contribute to your project; Lilach and Tim thank you for being so welcoming when I started in the lab; Noelle and Julien L., thanks for showing me around in the beginning; Karine and Valerie, thanks for always being so helpful in the lab and being such good companion; Karine thank you also for spending time translating the Abstract and figuring out Adobe Acrobat; Arnault, Damien, Julien S. and Christina thanks for making the lab a fun place to work; Jean Baptiste and Natacha thanks for all the hours in cell culture splitting cells; Louise thanks for being such a good friend.

I would also like to thank everyone involved in the MalPar and AntiMal PhD programs. I feel very fortunate for having been part of such a good PhD program. A special thank you to Aga and Francesc for being not only good PhD mates but also good friends.

1 Abstract

Toxoplasma gondii is a member of the phylum Apicomplexa, which groups important human and animal pathogens, including Plasmodium, the causative agent of malaria. Apicomplexan parasites invade host cells in an active manner, which critically relies on an actomyosin system and the regulated secretion of proteins from specialized organelles, named micronemes (Carruthers and Sibley, 1997). These micronemal proteins (MICs) are released onto the parasite’s surface as complexes, containing both soluble and transmembrane proteins. In Toxoplasma gondii a large repertoire of functionally non-redundant MICs participates in gliding motility, host cell attachment, moving junction formation, rhoptry secretion and invasion. Some transmembrane microneme proteins (TM-MICs) also function as escorters, assuring trafficking of their complexes to the micronemes. The MICs present a modular design, possessing an ectodomain, capable of interacting with host cell receptors, and a short cytoplasmic tail, shown in micronemal protein -2 and -6 (TgMIC2 and TgMIC6) to connect to the actomyosin system via binding to aldolase (Jewett and Sibley, 2003). Within the ectodomain a variety of domains have been shown to contribute to host cell adhesion and recognition. Among these, a new structural module termed Microneme Adhesive Repeat (MAR) present on micronemal protein 1 (TgMIC1) was shown to be responsible for the recognition of sialyated oligosaccharides on the host cell surface (Blumenschein et al., 2007). Sialic acids serve as key determinant for invasion by the Apicomplexa, in general, and by T. gondii, in particular. During invasion the adhesive complexes are shed from the parasite’s surface by the action of the micronemal protein protease 1 (MPP1), which cleaves the TM-MICs in the transmembrane spanning domain. The MPP1 activity is presumably important during invasion and is likely mediated by a rhomboid serine protease constitutively active at the plasma membrane of the parasite. In T. gondii, the plasma membrane rhomboid proteases -4 and -5 (TgROM4 and TgROM5) are the primary candidates for the MPP1 activity (Brossier et al., 2005; Dowse et al., 2005). In this study we aimed to better understand the function of the micronemal proteins during host cell invasion and identify the rhomboid-like protease responsible for the MPP1 activity in Toxoplasma.

1 Apicomplexan parasites are obligatory intracellular parasites, which need to invade host cells in order to survive and propagate, and any knowledge regarding host cell invasion may provide new tools in the fight against this deadly pathogens.

1 Résumé Toxoplasma gondii est un parasite appartenant au phylum des Apicomplexes qui contient de nombreux pathogènes d’importance médicale et vétérinaire, tel que les espèces du genre Plasmodium responsables de la malaria. Les parasites de ce phylum ont la particularité d’envahir les cellules hôtes de façon active grâce un complexe moteur et à des protéines provenant d’organelles apicaux appelés micronèmes (Carruthers and Sibley, 1997). Ces protéines MIC sont sécrétées à la surface du parasite sous forme de complexes contenant à la fois des protéines solubles et transmembranaires (MIC-TM). Toxoplasma gondii possède un vaste répertoire de protéines MIC dont la fonction est non-redondante et qui sont impliquées dans la motilité du parasite, son attachement à la cellule hôte, la formation d’une jonction mobile et l’entrée du parasite dans la cellule. Certaines protéines MIC transmembranaires servent aussi d’escorteurs, assurant le trafic des complexes vers les micronèmes. Les MIC sont des protéines modulaires. Elles présentent un ectodomaine capable d’interagir avec les récepteurs de la cellule hôte et une courte extrémité C-terminale cytoplasmique. C’est par cette partie cytosolique que TgMIC6 et TgMIC2 interagissent avec le complexe moteur du parasite via leur liaison à l’aldolase (Jewett and Sibley, 2003). Il a été montré que plusieurs types de domaines structuraux, présents au sein de l’ectodomaine, contribuent à l’adhésion ainsi qu’à la reconnaissance de la cellule-hôte. Par exemple, le domaine MAR (Microneme Adhesive Repeat), un nouveau repliement découvert dans la protéine TgMIC1, est responsable de la reconnaissance spécifiques des oligosaccharides syaliques présent à la surface des cellules (Blumenschein et al., 2007). Les acides syaliques jouent un rôle majeur dans l’invasion par les Apicomplexes en général et par Toxoplasma gondii en particulier. Au cours de l’invasion, les complexes établis entre le parasite et la cellule hôte ont besoin d’être rompus pour permettre la progression du parasite. Cette activité protéolytique, appelée activité MPP1 (micronemal protein protéase 1), est assurée par une protéase des micronèmes clivant les protéines MIC-TM au sein de leur domaine trans-membranaire. L’activité MPP1, constitutivement active au niveau de la membrane plasmique du parasite, est probablement assurée par une sérine protéase de type rhomboïde. Toxoplasma gondii exprime deux rhomboïdes à sa surface, TgROM4

1 et TgROM5, qui sont par conséquent les meilleurs candidats pour être responsable de l’activité MPP1 (Brossier et al., 2005; Dowse et al., 2005). L’objectif de cette étude est de comprendre le rôle des protéines micronémales au cours du processus d’invasion de la cellule hôte et d’identifier la rhomboïde à l’origine de l’activité MPP1 chez Toxoplasma gondii.

1 Contents

Acknowledgements………..…….……………………………………………………1 Abstract……...……………………………………………………………...………...2 Résumé………………………………………...……………………………………...4 Contents………………………………..……………………………………………..6 List of Figures………..……………………………………….………………………9 List of Abbreviations…………………….………………………………...………..11 Chapter I: Introduction…………………………………………………………….14 1. Phylum Apicomplexa……………………………………………………………...14 1.1 Parasite ultrastructure…………………………………………………………….15 1.2 Life cycle…………………………………………………………………………16 1.2.1 Toxoplasma life cycle…………………………………………………………..16 1.2.2. Plasmodium life cycle…………………………………………………………18 1.3 Parasite cell division……………………………………………………………...19 2. Host Cell Invasion…………………………………………………………………21 2.1 Micronemal proteins…………………………………………………………..…23 2.1.2 Apical membrane antigen 1…………………….………………………………26 2.1.3 Parasite lectins………………………………………………………………….28 2.1.3.1 Plasmodium sialic acid-dependent and -independent pathways…………….29 3. Micronemal protein proteolysis during invasion………………………………….32 3.1 Toxoplasma micronemal proteins proteolysis……………………………………32 3.1.1 MPP1 activity…………………………………………………………………..33 3.2 Plasmodium micronemal proteins proteolysis…………………………………...34 3.2.1 Shaving: Merozoite surface sheddase (MESH activity)……………………….35 3.2.2 Rhomboid activity……………………………………………………………...36 4. Rhomboids and regulated intramembrane proteolysis…………………………….38 4.1 Rhomboids……………………………………………………………………….38 4.1.1 Rhomboids in the Apicomplexa………………………………………………..40 4.2. Regulated intramembrane proteolysis…………………………………………...45 5. Hypothesis and Aims of the Project……………………………………………….48 Chapter II: Materials and Methods…………………………………………….....50 1. Reagents and Suppliers……………………………………………………………50

1 1.1 Enzymes………………………………………………………………………….50 1.2 Kits……………………………………………………………………………….50 1.3 Antibodies………………………………………………………………………..50 2. Solutions………………………….………………………………………………..51 2.1 Culture media…………………………………………………………………….51 2.2 General solutions…………………………………………………………………51 3. Cell lines and microbiological strains……………………………………………..52 3.1 Bacteria…………………………………………………………………………...52 3.2 Mammalian cells…………………………………………………………………52 3.3 Toxoplasma gondii strains………………………………………………………..52 4. Culture conditions…………………………………………………………………52 4.1 Bacterial culture………………………………………………………………….52 4.2 Mammalian cell culture…………………………………………………………..53 4.3 Parasite propagation.……………………………………………………………..53 5. Transformations and transfections………………………………………………...53 5.1 Eschericia coli transformation…………………………………………………...53 5.2 Toxoplasma gondii transfection………………………………………………….53 6. Cloning of DNA constructs………………………………………………………..54 6.1 DNA constructs used in the in vitro cleavage assays…………………………….54 6.2 Agarose gel electrophoresis……………………………………………………...55 6.3 Preparation of nucleic acids……………………………………………………...55 6.4 Polymerase chain reaction (PCR)………………………………………………..56 6.5 Ligations……………………………………………………………………….…56 6.6 Site-directed mutagenesis…………………………………………………….…..56 7. Sodium Dodecyl Sulphate (SDS) Polyacrylamide gel (PAGE) Electrophoresis and Western blotting……………………………………………………………………...56 8. Immunofluorescence assays (IFA) and confocal microscopy……………………..57 9. Assays……………………………………………………………………………..58 9.1 In vitro cleavage assay…………………………………………………………...58 Chapter III: Results…………………………………………………………….…..59 1. Members of a novel protein family containing microneme adhesive repeat domains act as sialic acid-binding lectins during host cell invasion by apicomplexan parasites………………………………………………………………………………60

1 2. Toxoplasma gondii transmembrane microneme proteins and their modular design………………………………………………………………………………...74 3. Study of TgROM4 as a candidate to the MPP1 activity…………………………121 3.1 Insight into the role of the conserved C-terminus domain of the large apicomplexan rhomboids…………………………………………………...………121 3.2 ROM4-mediated cleavage of AMA1 switches Toxoplasma from an invasive to a replicative mode…………………………………………………………………….125 Chapter IV: Discussion and Concluding Remarks……………………………...174 1. Toxoplasma tachyzoites: multi target cells………………………………………174 2. Proteolytic shedding of Plasmodium surface proteins…………………………...177 3. MPP1 activity: TgROM4 or TgROM5?...... 178 4. AMA1: a multi-functional protein……………………………………………….180 4.1 AMA connection to the glideosome…………………………………………….181 4.2 AMA1 function during parasite division……………………………………….181 5. Rhomboid-mediated RIP: a conserved mechanism for regulation of signal transduction in the Apicomplexa?...... 186 6. Rhomboids as drug targets?...... 188 7. Concluding remarks……………………………………………………………...188 References…………………………………………………………………………..190 Appendix……………………………………………………………………………205

1 List of Figures

Figure 1.1 Conoid and subpellicular microtubules of T. gondii……………………...15 Figure 1.2 Ultrastructure of T. gondii tachyzoite and P. falciparum merozoite……..16 Figure 1.3 Life cycle of T. gondii…………………………………………………….17 Figure 1.4 Lytic cycle of T. gondii…………………………………………………...18 Figure 1.5 Life cycle of P. falciparum……………………………………………….19 Figure 1.6 Schematic of endodyogeny and schizogony……………………………...20 Figure 1.7 Schematic of Plasmodium merozoite invasion of an erythrocyte………...21 Figure 1.8 Formation of the moving junction during host cell invasion by Toxoplasma…………………………………………………………………………..22 Figure 1.9 The glideosome…………………………………………………………...23 Figure 1.10 Repertoire of micronemal proteins encoded in the genomes of Eimeria, Toxoplasma, Plasmodium, Cryptosporidium and Neospora parasites……………….24 Figure 1.11 Schematic of the four known Toxoplasma micronemal complexes and their functions………………………………………………………………………...25 Figure 1.13 Schematic showing export of the Toxoplasma RON proteins to the host cell membrane………………………………………………………………………..28 Figure 1.14 Capping of TgMIC2……………………………………………………..33 Figure 1.15 MPP1 activity on the Toxoplasma micronemal complexes……………..34 Figure 1.16 Alignment of the transmembrane domain of micronemal proteins from different apicomplexan parasites……………………………………………………..36 Figure 1.17 Schematic representation of a rhomboid protease and its substrate…….38 Figure 1.18 Schematic of the protein structure of a bacterial rhomboid……………..40 Figure 1.19 Phylogenetic tree of the apicomplexan rhomboids……………………...41 Figure 1.20 Schematic representation of the intracellular localization of the Toxoplasma rhomboids………………………………………………………………42 Figure 1.21 RIP mechanisms………………………………………………………...47 Figure 3.1 Alignment of the large rhomboids from Plasmodium and Toxoplasma...122 Figure 3.2 Schematic of the cell-based cleavage assay……………………………..123 Figure 3.3 Western-blot of a cell-based cleavage assay…………………………….125 Figure 4.1 Expression of “invasion proteins” at the surface of Plasmodium merozoites and Toxoplasma tachyzoites………………………………………………………...178

1 Figure 4.1 Schematic representation of the functions proposed for the MPP1 activity………………………………………………………………………………180 Figure 4.3 Schematic representation of the five functions proposed for the MPP1 activity………………………………………………………………………………182 Figure 4.4 Alignment of the conserved C-terminal domain of AMA1 from different apicomplexan species……………………………………………………………….183 Figure 4.5 Model for signaling of rhoptry secretion in Toxoplasma…………….....187

1 List of Abbreviations

AMA-1 Apical Membrane Antigen 1 APP Amyloid Precursor Protein APS Ammonium Persulphate Atc Anhydrous Tetracycline BLAST Basic Local Alignment Search Tool BSA Bovine Serum Albumin CBL Chitin-Binding-Like CDPK Calcium-Dependent Protein Kinase CHO Chinese Hamster Ovary CIP Calf Intestinal Alkaline Phosphatase COS CV-1 Origin SV40 carrying DAPI Diamidine-2’phenylindole Dihydrochloride DBP Duffy-binding Protein DCI 3, 4-Dichloroisocoumarin DD Destabilization Domain DHFR Dihydrofolate Reductase-thymidylate synthase gene EBA Erythrocyte-binding Antigen EBL Erythrocyte-binding Ligand EGF Epidermal Growth Factor EGFR EGF Receptor ER Endoplasmic Reticulum EST Expressed Sequence Tag F-actin Filamentous actin FCS Fetal Calf Serum GAP Gliding Associated Protein GAG Glycosaminoglycan GFP Green Fluorescent Protein GPI Glycosylphosphatidylinositol GST Gluthatione S-Transferase HEK Human Embryonic Kidney HFF Human Foreskin Fibroblasts

1 iCLiP Intramembrane-cleavage Protease IFA Immunofluorescence Assay IMC Inner Membrane Complex LB Luria-Bertani MAPK Mitogen-Activated Protein Kinase MAR Microneme Adhesive Repeat MESH Merozoite Surface sheddase MCP MAR-Containing Protein MHC Major Histocompatibility Complex MIC Microneme protein MJ Moving Junction MLC Myosin Light Chain MPA Mycophenolic Acid MPP1 Microneme Protein Protease 1 MPP2 Microneme Protein Protease 2 MPP3 Microneme Protein Protease 3 MSP Merozoite Surface Protein MTIP Myosin Tail Interacting Protein ORF Open Reading Frame PAF Paraformaldehyde PAGE Polyacrylamide Gel Electrophoresis PAN Plasminogen Apple Nematode PBS Phosphate Buffered Saline PCR Polymerase Chain Reaction PM Plasma Membrane PV Parasitophorous Vacuole PVM Parasitophorous Vacuole Membrane RBC Red Blood Cell ROM Rhomboid-like protease RON Rhoptry Neck protein ROP Rhoptry bulb protein RT-PCR Reverse-Transcriptase PCR RBC Red Blood Cells RBL Reticulocyte Binding-Like

1 RBP Reticulocyte-binding Protein Rh Reticulocyte-binding Protein Homologue RIP Regulated Intramembrane Proteolysis SAG Surface Antigen Glycoprotein SDS Sodium Dodecyl Sulphate S1P Site-1-Protease S2P Site-2-Protease Sia Sialic acid SREBP Sterol Regulatory Element Binding Protein SUB Subtilisin-like protease TAE Tris/Acetate/EDTA TE Tris/EDTA TEMED N, N, N’, N’-Tetramethyl-1-,2-Diaminomethane TGN Trans Golgi Network TMD Transmembrane Domain TM-MIC Transmembrane micronemal protein TPCK Tosyl Phenylalanine Chloromethyl Ketone TRAP Thrombospondin-related Anonymous Protein YFP Yellow Fluorescent Protein

1 Chapter I

Introduction

(Some sections of this introduction are adapted from a published review (Santos et al., 2009) written to summarize the current knowledge regarding the role of the cytoskeleton and motors to the different steps of the parasites lytic cycle. The manuscript in its published form can be found in the appendix)

1. Phylum Apicomplexa

The Apicomplexa is a phylum of unicellular eukaryotic organisms that includes numerous parasites responsible for several animal and human diseases. This thesis focused on the study of Toxoplasma, a member of the Coccidian (cyst-forming) group of parasites, and Plasmodium, the causative agent of malaria. Toxoplasma gondii is considered the most successful of all protozoan parasites because it can invade any nucleated cell and most warm blooded animals and it chronically affects a vast percentage of the adult human population (Su et al., 2003). Clinical toxoplasmosis is rare and infections are generally asymptomatic, but complications can take place when infection is transmitted congenitally or occurs on immunocompromised individuals. Toxoplasma is seen as the model organism of the phylum because it is easily genetically manipulated and cultivated in vitro. Four species of Plasmodium cause malaria in humans – P. falciparum, P. vivax, P. malariae and P. ovale – but P. falciparum is responsible for the highest number of deaths. Malaria is still an endemic disease in many tropical countries, having caused nearly one million deaths only in 2008 (WHO). Such high mortality is consequence of the inexistence of vaccines and the development of resistance by the parasite or the mosquito vector to the majority of the anti-malarial drugs or insecticides available, respectively. It is thus emergent to discover new drug targets and to better understand the mechanisms involved in the establishment of infection by these parasites.

1 1.1 Parasite ultrastructure Apicomplexa parasites are delimited by the pellicle, a tri-bilayer structure, comprising the plasma membrane and two tightly associated membranes named the inner membrane complex (IMC). The IMC extends throughout the body of the parasite and serves as support for the gliding machinery, which drives motility. Closely associated to the parasite pellicle is the subpellicular network, which acts as the parasite’s skeleton. Underneath the subpellicular network, at the apical tip, is found the apical complex and on the opposite end, is localized the basal complex (figure 1.1).

Figure 1.1 Conoid and subpellicular microtubules of T. gondii The cytoskeleton structures at the apical end of the parasite are shown in detail, including the polar rings and the apical (preconoidal) rings; scale 0.3µm. Taken from (Dubey et al., 1998).

The apical complex is characteristic of this group of parasites but only some members of the phylum possess the entire set of organelles that compose it. Permanent features are the presence of three specialized secretory organelles named micronemes, rhoptries and dense granules, and a cytoskeleton element named the apical polar ring. The conoid, another cytoskeleton element, is only present in the coccidians (figure 1.2). The parasites possess all the organelles characteristic of a eukaryotic cell (endoplasmic reticulum-ER, Golgi apparatus, nucleus, mitochondrion) but also a plastid called the apicoplast (Foth and McFadden, 2003; Kohler et al., 1997; Roos et al., 1999) (figure 1.2) and a plant-like vacuole at the tachyzoite stage of Toxoplasma (Miranda et al.; Parussini et al.) or a food vacuole at the erythrocytic stages of Plasmodium (Langreth et al., 1978).

Figure 1.2 Ultrastructure of T. gondii tachyzoite and P. falciparum merozoite On the left is a schematic of the T. gondii tachyzoite and P. falciparum merozoite with the intracellular structures labelled. Taken from (Baum et al., 2006a). On the right is an electron micrograph of an intracellular T. gondii tachyzoite showing the parasite’s conoid (C), micronemes (m), rhoptries (R), dense granules (G), apicoplast (A) and nucleus (N); scale bar 1µm. Taken from (Dubremetz, 2007).

1.2 Life cycle Apicomplexa parasites have a complex life cycle characterized by conversion into different morphological stages, which either undergo intense replication or migrate and invade other hosts or cell tissues. The invasive stages are called zoites. The life cycle always include a phase of asexual reproduction and one of sexual development that can take place in the same host (moxenous species) or into two distinct hosts (heteroxenous species), as in Toxoplasma and Plasmodium. The host where there’s formation of the sexual stages is named the definitive host, and the one where there’s asexual differentiation is termed the intermediate host.

1.2.1 Toxoplasma life cycle Toxoplasma parasites undergo an intestinal phase in the definitive host and a tissue phase within the intermediate host, and differentiate into three distinct forms - oocysts, tachyzoites, and bradyzoites (Dubey et al., 1998) (figure 1.3). While the definitive host is always a feline, the intermediate host can be virtually any warm-

1 blooded animal. The intestinal phase of the infection occurs when felines ingest animals infected with the tissue stage of the parasite. The parasites invade the intestinal epithelial cells and undergo merogony, producing merozoites that can then either undergo additional rounds of replication or undertake gametogony. Gametogony originates the sexual forms - the macrogametes (“female” gametes) and the microgametes (“male” gametes). The bi-flagellated microgametes are released into the lumen of the intestine and fertilize the macrogametes in the epithelial cells, generating oocysts that are excreted with the feces. In the outside environment, the immature oocysts undergo sporogony, differentiating into mature oocysts containing two sporocysts, each with four sporozoites. These oocysts remain infective for months and are very resistant to environmental conditions. Infection of the intermediate host occurs via ingestion of food contaminated with oocysts. Inside the host, the sporozoites are released and penetrate the intestinal epithelium. The intracellular parasites undergo merogony by endodyogeny and produce tachyzoites that can invade new host cells and repeat the replicative cycle. Infected macrophages and dendritic cells can then disseminate the tachyzoites throughout the host during acute infection (Lambert and Barragan). Development of the host immune response slows down the replication rate and the infected host cells become encapsulated, originating tissue cysts containing bradyzoites. This is the stage that causes chronic infection. The bradyzoites can be transmitted congenitally or to other intermediate hosts through carnivorism. A new cycle initiates when a feline consumes meat contaminated with cysts (Dubey et al., 1998).

Figure 1.3 T. gondii life cycle A feline like a cat functions as definitive host, while a warm-blood animal functions as intermediate host. Human infection can occur via congenital transmission, ingestion of animals infected with tissue cysts (right blue arrow) or contact with congenital material contaminated with oocysts transmission (left blue arrow). Adapted from dpd.cdc.gov.

In the laboratory it is cultured the tachyzoite stage, which can differentiate into bradyzoites when under stress conditions (Gross et al., 1996a; Gross et al., 1996b; Skariah et al.; Soete et al., 1994; Soete et al., 1993). Tachyzoites undergo a lytic cycle comprising host cell attachment, invasion, replication, egress from the infected cells and gliding (figure 1.4).

Figure 1.4 T. gondii lytic cycle Scheme of the parasite’s lytic cycle showing (clockwise) attachment to the host cell, invasion, replication within the parasitophorous vacuole inside the host cell, egress from the infected cells and gliding of the free parasites. Taken from (Soldati and Meissner, 2004).

1.2.2 Plasmodium life cycle The malaria parasite life cycle is split between an Anopheles mosquito that is simultaneously the vector and the definitive host, and a human that functions as intermediate host. Three invasive forms are produced - merozoites, sporozoites and ookinetes - that differ regarding the types of cells or tissues they invade and the ability to be motile (figure 1.5). Individuals are infected when they are bitten by the mosquito and hence receive the sporozoites stored in the salivary glands. These parasites can either start developing already in the skin, invade blood vessels or lymphatic vessels (Amino et al., 2006) but only the parasites that reach the blood circulation seem to lead to a productive infection (Amino et al., 2006). When sporozoites reach the liver, they migrate through several hepatocytes (Mota et al., 2001) until they establish themselves and ensure asexual replication (exoerythrocytic schizogony), leading to the production of merozoites. A proportion of the liver-stage Plasmodium vivax and Plasmodium ovale parasites go through a dormant period (hypnozoites) instead of immediately undergoing asexual replication. These hypnozoites can reactivate after several weeks or months after the primary infection and are responsible for relapses. The thousands of merozoites produced by schizogony are delivered into the blood stream inside specialized bags named merosomes (Sturm et al., 2006). Once there, they infect red blood cells (RBCs) and

1 multiply asexually, generating a large number of new merozoites that can invade new erythrocytes. This is the symptomatic stage of the disease. As an alternative to the asexual replicative cycle, the parasite can differentiate into macrogametocytes and microgametocytes that are transferred to the vector by a new mosquito bite. In the mosquito midgut, gametogenesis is induced and fertilization produces a zygote and later an ookinete. This zoite is motile and can thus migrate through the gut wall and divide, originating oocysts filled with sporozoites. The sporozoites migrate to the salivary glands and can re-initiate a new cycle.

Figure 1.5 P. falciparum life cycle On the left are represented the asexual stages of the life cycle within the intermediate host, a human, and on the right is shown the sexual development phase within the definitive host, the mosquito. Taken from (Menard, 2005).

1.3. Parasite cell division Different Apicomplexa, and even different life cycle stages of a same species, adopt distinct strategies to ensure the completion of their replicative cycle. Most intracellular stages are not infectious, and therefore, cell division has to be precisely timed in order to ensure that the new daughter zoites are fully formed and prepared to

1 invade at the time of host cell egress. The usual rule is schizogony, where several rounds of DNA synthesis and nuclear division occur prior to zoite genesis and cytokinesis and all daughter cells are produced concomitantly; but in some cases the parasites replicate via endodyogeny (a variant form of schizogony where DNA replication is immediately followed by nuclear division and cytokinesis), which leads to the production of only two new daughter cells per replication cycle (figure 1.6). Plasmodium undergoes schizogony while most Toxoplasma stages, including tachyzoites, undergo endodyogeny. Multiple studies have suggested that there is no fundamental distinction between the various modes of reproduction, apart from the number of nuclear divisions preceding zoite genesis. In all cases, the morphogenesis of Apicomplexan zoites has been described as being coordinated with mitosis. Regulation of endodyogeny seems to involve cell cycle checkpoints similar to those of other eukaryotes (reviewed in (Gubbels et al., 2008)). These master switches were recently suggested to be up/down-regulated according to each parasite specific program, i.e., parasites that execute several rounds of DNA synthesis before cytokinesis (i.e. schizogony) would down-regulate proteins involved in the checkpoint at the end of DNA replication (Striepen et al., 2007). Nothing is known regarding the signals that lead to initiation of the replication programme.

Figure 1.6 Schematic of endodyogeny and schizogony The top scheme depicts endodyogeny, as undergone by Toxoplasma tachyzoites, in which a mother cell gives raise to two new daughter cells at each round of multiplication and the mother cell and apical structures remain intact until the last steps of division. The bottom scheme depicts schizogony of Plasmodium merozoites, in which all daughter cell are formed simultaneously and the mother cell structures are broken down and many nuclear divisions occur. Adapted from (Striepen et al., 2007).

1 2. Host cell invasion

All members of the phylum Apicomplexa are obligatory intracellular parasites that therefore need to continuously invade host cells in order to survive and propagate. Host cell recognition and invasion has to be accomplished rapidly in order to evade the host immune system. In consequence, host cell invasion is a critical step in the establishment of infection. The invasion process is exceptionally fast, taking 10-30s, and involves a series of steps believed to follow the same scheme on both Plasmodium merozoites and Toxoplasma tachyzoites: initial low affinity binding of the parasite to the host cell is followed by tighter attachment to the host and parasite reorientation. Subsequently, an electron dense junction (moving junction-MJ) is formed between the parasite and the host cell membranes leading to penetration of the host cell. Migration of the MJ results in the formation of a specialized vacuole, the parasitophorous vacuole (PV), derived from both the host cell plasma membrane and parasite material originated from the rhoptries (Hakansson et al., 2001). Finally, there is sealing of the parasitophorous vacuolar membrane (PVM) (reviewed in (Carruthers and Boothroyd, 2007)). At the end of the penetration process, the parasite is completely secluded inside the PV surrounded by the PVM (figure 1.7).

Figure 1.7 Schematic of Plasmodium merozoite invasion of an erythrocyte Invasion is a multi-step process involving (clockwise) reversible attachment of the merozoite to the host cell, reorientation so that the apical end of the parasite faces the erythrocyte, formation of the moving junction, translocation of the moving junction from the apical to the posterior pole concomitant with penetration of the host and closure of the parasitophorous vacuole (adapted from (Cowman and Crabb, 2006)).

1 The MJ is a zone of intimate contact (Aikawa et al., 1978; Michel et al., 1980) between the parasite and host cell membranes created by interaction of parasite ligands with specific host cell receptors (Sibley, 2004) (figure 1.8). It begins as a cup covering the parasite apex and rapidly converts into a ring that moves across the parasite’s surface as it penetrates the host cell (Santos et al., 2009). The MJ serves as a molecular sieve and as an anchor for entry (Mordue et al., 1999a; Santos et al., 2009). The sieving process serves to push toward the rear or even completely remove the parasite transmembrane ligands that are engaged with the host receptors as well as the host cell integral membrane proteins and hence avoids fusion of the PVM with the host membrane (Brecht et al., 2001b; Jewett and Sibley, 2003; Mordue et al., 1999a; Mordue et al., 1999b).

Figure 1.8 Formation of the moving junction during host cell invasion by Toxoplasma Electron micrograph of a tachyzoite invading a host cell (HC). As the parasite invades the host cell, there is formation of an invagination in the host cell membrane called the moving junction (MJ). The MJ migrates towards the posterior end of the parasite, leading to the formation of the parasitophorous vacuole, surrounded by the parasitophorous vacuole membrane (PVM). The star indicates the position of the rhoptries near the apical cytoskeleton (AC); scale bar 0.5µm. Taken from (Boothroyd and Dubremetz, 2008).

The apicomplexan parasites differ from most other pathogens because they actively invade host cells, using their own energy and actomyosin machinery, the glideosome, anchored at the IMC and the plasma membrane (Opitz and Soldati, 2002) (figure 1.9). The glideosome is strictly conserved in all parasites of the phylum and in all motile stages (Kappe et al., 1999) and includes a myosin motor, MyoA (Baum et al., 2006a; Baum et al., 2006b; Jones et al., 2006; Schuler and Matuschewski, 2006; Wetzel et al., 2005); its associated myosin light chain - MLC1 in Toxoplasma (Herm- Gotz et al., 2002) and myosin tail interacting protein (MTIP) in Plasmodium (Baum et al., 2006b; Jones et al., 2006); and two gliding associated proteins, GAP45 and GAP50 (Gaskins et al., 2004; Johnson et al., 2007), that anchor the complex to the IMC. MyoA presumably pulls on short actin filaments (F-actin) (Dobrowolski et al., 1997; Dobrowolski and Sibley, 1996) that are linked, likely via aldolase (Bosch et al.,

1 2006; Buscaglia et al., 2003; Jewett and Sibley, 2003), to the parasite adhesins bound to host receptors. Consequently the adhesin-receptor complexes translocate towards the rear of the parasite, resulting in forward movement of the parasite into the host cell. Gliding motility not only assists invasion but also migration through biological barriers (reviewed in (Tardieux and Menard, 2008)).

Figure 1.9 The glideosome On the left is a schematic of the parasite’s pellicle and on the right is a schematic of the parasite’s glideosome sandwiched between the plasma membrane and the inner membrane complex. Taken from (Keeley and Soldati, 2004).

2.1 Micronemal proteins Invasion involves the sequential discharge of the micronemes and rhoptries (Carruthers and Sibley, 1999). The micronemal adhesins (MICs) are discharged onto the parasite surface upon the release of calcium from intracellular stores in the parasite (Carruthers and Sibley, 1999; Lovett et al., 2002; Lovett and Sibley, 2003) and form in many cases tight complexes with host cell receptors (Alexander et al., 2005). Formation of the complexes adhesins-receptors is essential for glideosome function and formation of the MJ but not for initial low-affinity attachment to the host cell. During invasion, the complexes MICs-receptors are rapidly re-distributed towards the posterior end of the parasite powered by the glideosome, and, as a result, the parasite is propelled into the host cell. Ultimately, these parasite adhesins are released from the parasite surface by proteolytic cleavage and, as a consequence, the parasite can disengage itself from the host cell membrane and complete penetration.

1 Polarized secretion, translocation and proteolytic processing must thus be tightly coupled for efficient invasion (Sibley, 2004).

Figure 1.10 Repertoire of micronemal proteins encoded in the genomes of Eimeria, Toxoplasma, Plasmodium, Cryptosporidium and Neospora parasites Taken from (Friedrich et al.).

Numerous MICs have been identified in Toxoplasma (figure 1.10) and shown to assemble in complexes, already in the ER prior to transit to the micronemes. Four complexes have been characterized so far (figure 1.11). The complexes comprising micronemal protein 2 and micronemal associated protein 2 (TgMIC2-M2AP), micronemal proteins -1, -4 and -6 (TgMIC1-MIC4-MIC6) or micronemal proteins -3 and -8 (TgMIC3-MIC8) are stored in the micronemes and re-localize to the parasite’s surface upon invasion. In contrast, the complex containing apical membrane antigen 1, and rhoptry neck proteins -2, -4, -5 and -8 (TgAMA1-RON2-RON4-RON5-RON8) involves the participation of proteins secreted from both the micronemes and the rhoptry necks and is assembled on the parasite’s surface immediately post-exocytosis (Alexander et al., 2005; Besteiro et al., 2009b; Straub et al., 2008); this complex is only found at the MJ. Only the latter complex is conserved in Plasmodium and other apicomplexan parasites (Cao et al., 2009; Collins et al., 2009; Straub et al., 2009). These complexes contain soluble (TgM2AP, TgMIC1 and TgMIC4) and transmembrane proteins (TgMIC2, TgMIC6, TgMIC8 and TgAMA1). The

1 transmembrane MICs portray a modular structure comprising a soluble ectodomain, a membrane-spanning domain and short cytoplasmic tail. Whilst the ectodomain establishes connections with host receptors and hence engages the parasite with the host cell surface, the C-terminal domain can, in some cases, escort the complex to the micronemes and associate with the glideosome via binding to aldolase. TgMIC1, TgMIC2 and TgMIC4 are all known to function as adhesins (Blumenschein et al., 2007; Brecht et al., 2001b; Fourmaux et al., 1996; Hehl et al., 2000; Huynh and Carruthers, 2006; Mital et al., 2005) but only TgMIC2 and TgMIC6 were shown to function as escorters (Di Cristina et al., 2000; Opitz et al., 2002; Reiss et al., 2001) and associate with aldolase (Jewett and Sibley, 2003; Zheng et al., 2009).

Invasion Invasion Invasion Invasion Gliding Virulence ROP secretion MJ formation RON secretion

Figure 1.11 Schematic of the four known Toxoplasma micronemal complexes and their functions

Only three micronemal proteins – TgMIC2, TgMIC8 and TgAMA1 – seem to play an essential function in Toxoplasma (Hehl et al., 2000; Huynh and Carruthers, 2006; Kessler et al., 2008; Mital et al., 2005), since for all the others disruption of the encoding genes does not produce a lethal phenotype. Parasites conditionally depleted for TgMIC2 (mic2iko) are deficient in host cell attachment, invasion and motility (Huynh and Carruthers, 2006). The TgMIC1-TgMIC4-TgMIC6 complex has been demonstrated to play an important role in invasion in vitro and to contribute to virulence in vivo (Blumenschein et al., 2007; Cerede et al., 2005; Sawmynaden et al., 2008). Genetic disruption of TgMIC8 interferes with rhoptries secretion, preventing formation of the MJ and completion of invasion (Kessler and Soldati, 2008). Parasites lacking TgAMA1 efficiently attach to host cells (Mital et al., 2005) but are defective

1 in secretion of the rhoptries necks (Alexander et al., 2005), fail to create a MJ and are unable to invade host cells (Mital et al., 2005). In Plasmodium, four families of transmembrane adhesins have been implicated in invasion (figure 1.10). PfAMA1 has been suggested to be involved in merozoites reorientation (reviewed in (Remarque et al., 2008)). The Duffy binding ligand- erythrocyte (DBL-EBP) and reticulocyte binding-like (RBL) families have been implicated in the establishment of high-affinity interactions with host cell receptors, at the time of host cell invasion (reviewed in (Iyer et al., 2007)). The thrombospondin- related anonymous protein (TRAP) family has been proposed to link the MJ to the parasite cytoskeleton (Bosch et al., 2007; Buscaglia et al., 2003; Moreira et al., 2008), as well as to act as adhesins in the sporozoite (PfTRAP, TRAP-related protein – PfTREP/S6 and TRAP-like protein - PfTLP), merozoite (merozoite TRAP - PfMTRAP and PFF0800w) and ookinete (circumsporozoite and TRAP-related protein - PfCTRP) stages (reviewed in (Morahan et al., 2009)). Both PfTRAP and PfTREP are also important for gliding (Combe et al., 2009; Sultan et al., 1997).

2.1.2 Apical membrane antigen 1 AMA1 was first identified in Plasmodium (Peterson et al., 1989; Waters et al., 1990) but it is now known to be conserved in all members of the phylum. It is encoded by a single copy gene refractory to genetic disruption that translates into a type I transmembrane protein, including a signal peptide, a pro-domain region, a long ectodomain divided into three functional regions called domain I, II and III, a transmembrane domain (TMD) and a short cytoplasmic tail (reviewed in (Remarque et al., 2008)). The structure of the ectodomain has been solved in both P. falciparum (Bai et al., 2005) and T. gondii (Crawford et al.) but little is known regarding the TMD and cytosolic regions, with the exception that the PfAMA1 C-terminal domain undergoes phosphorylation and is essential for invasion (Treeck et al., 2009; Lyekauf et al.). AMA1 is expressed by Toxoplasma tachyzoites (Donahue et al., 2000; Hehl et al., 2000) and Plasmodium merozoites (Peterson et al., 1989; Waters et al., 1990) and sporozoites (Silvie et al., 2004), and localizes to the micronemes. Targeting to the micronemes is mediated by the ectodomain in Plasmodium (Healer et al., 2002). Three functions have been attributed to this protein: parasite reorientation during invasion (Mital et al., 2005), formation of the MJ complex (Alexander et al., 2005) and regulation of rhoptries secretion (Mital et al., 2005).

1 AMA1 is one of the most studied apicomplexan proteins because several antibodies and peptides raised against its ectodomain confer protection against Plasmodium infection by inhibiting host cell invasion by both merozoites and sporozoites but its exact function during invasion remains however a mystery (reviewed in (Remarque et al., 2008)). AMA1 has been shown to associate with the RON proteins at the MJ in T. gondii tachyzoites (Alexander et al., 2005) and P. falciparum merozoites (Cao et al., 2009; Collins et al., 2009) but the complex is likely conserved across the phylum because AMA1 and RON proteins are present in the genomes of all Apicomplexa, with the exception of the Cryptosporidium genus, which invades the host in a distinct manner (Santos et al., 2009). In both Plasmodium and Toxoplasma only a minority of the surface expressed AMA1 associates with the RON proteins at the MJ (Alexander et al., 2005; Collins et al., 2009). The MJ complex includes at the moment four RON proteins in T. gondii and three in Plasmodium. While RON2, RON4 and RON5 are ubiquitous (Alexander et al., 2005; Besteiro et al., 2009a; Collins et al., 2009) RON8 seems to be restricted to the coccidians (Besteiro et al., 2009a; Straub et al., 2009). The Plasmodium RON proteins have been shown to form a pre-complex in the rhoptries and then be delivered onto the parasite’s surface (Collins et al., 2009) where they bind to AMA1 (Alexander et al., 2005; Collins et al., 2009). PfAMA1 association with the PfRONs occurs via a conserved Tyr residue at the hydrophobic trough (Collins et al., 2009) and TgAMA1 binds to TgRON2 in the absence of the other components of the complex (Besteiro et al., 2009a), suggesting that AMA1 associates directly with RON2 and indirectly with the rest of the complex. At the same time TgRON2 and TgRON4 bind strongly (Alexander et al., 2005), indicating that they are directly associated. All members of the complex are proteolytically processed (Besteiro et al., 2009a; Collins et al., 2009; Straub et al., 2009) but maturation is not required for complex formation (Besteiro et al., 2009a). Experiments in Plasmodium with an inhibitory peptide (Richard et al.) suggested that the MJ complex forms only after parasite reorientation and establishment of the initial tight junction, indicating that it only plays a role in the following steps of invasion. In Toxoplasma, it was shown that the RONs complex is targeted to the host cell membrane during invasion by association of TgRON4, TgRON5 (a membrane- anchored protein (Straub et al., 2009)) and TgRON8 with the host cell membrane (Besteiro et al., 2009a). TgRON2, which has three TMDs (Straub et al., 2009),

1 provides the association to TgAMA1 (Besteiro et al., 2009a) (figure 1.13). Based on these results, Besteiro et al. suggested that the Toxoplasma ability to invade a wide range of host cells relies on export of its own receptor on the surface of the host cells (Besteiro et al., 2009a). Alternatively, export of the complex is just important for the sieving function of the MJ. It is unclear if the same model can be applied to Plasmodium.

Figure 1.13 Schematic showing export of the Toxoplasma RON proteins to the host cell membrane On the left is a representation of the TgAMA1-RONs complex formed at the moving junction. On the right is a detailed view of the TgRONs anchored at the host cell plasma membrane potentially serving as a receptor for TgAMA1, which is anchored at the parasite’s plasma membrane and can function as a ligand. Taken from (Besteiro et al., 2009a).

2.1.3 Parasite lectins Glycans and in particular sialic acids are ubiquitously distributed on the surface of vertebrate cells (Anantharaman et al., 2007) and the apicomplexan parasites seem to have adapted to this situation by expressing adhesins (lectins) specialized in binding to sialic acid determinants at the host cell surface (Friedrich et al.). P. falciparum EBA-175 (Adams et al., 1992; Sim et al., 1994) and N. caninum MIC1 (Keller et al., 2002) bind to sialic acid or sulfated glycosaminoglycans, respectively, and recognition of sialic acid is responsible for 90% of all T. gondii host cell invasion events (Blumenschein et al., 2007). Two types of adhesive domains related to lectins have been identified in the Toxoplasma MICs – the chitin-binding like (CBL) domain, that mediates binding to N-acetyl glucosamine (Wright et al., 1991) and the Micronemal Adhesive Repeat

1 (MAR) domain, that confers adhesive properties against sialic acid (Blumenschein et al., 2007) (figure 1.10). The CBL domain has been shown to be present at the N-terminus of TgMIC3 (Garcia-Reguet et al., 2000), TgMIC8, TgMIC8.2 and TgMIC8.3 (Meissner et al., 2002), followed by several EGF-like domains. The CBL domain of MIC3 binds to host cells to an unknown receptor (Cerede et al., 2002; Cerede et al., 2005) but only upon dimerization mediated by the EGF-like domains (Cerede et al., 2002), and this binding is important for virulence (Cerede et al., 2005). TgMIC1 possesses two sialic-acid binding sites uniquely arranged in tandem repeated MAR domains (Blumenschein et al., 2007). The major oligosaccharide binding activity lays within the second MAR domain (Garnett et al., 2009) and binding targets notably gangliosides, which are abundantly expressed on neurons, suggesting that TgMIC1 might play an important role during establishment of a chronic infection (Blumenschein et al., 2007). MAR domains are also present in three other uncharacterized TgMIC1-like proteins in T. gondii (Blumenschein et al., 2007). TgMIC1 also possesses a galectin-like fold at its C-terminus. This domain does not confer adhesive properties but binds to the EGF-like domains 2 and 3 of TgMIC6 and is important for formation of the complex (Saouros et al., 2005). Association with TgMIC4 is mediated via the MAR domains (Saouros et al., 2005). Several MICs are predicted to harbour thrombospondin type 1 repeat (TSR-1) domains (Labaied et al., 2007; Tossavainen et al., 2006) and apple domains (Anantharaman et al., 2007) (figure 1.10) and some of these proteins have been demonstrated to carry lectin properties. Apple domains mediate association of the PfAMA1 N-terminus to the RONs complex (Bai et al., 2005; Collins et al., 2009; Crawford et al.; Richard et al.) and binding of TgMIC4 to carbohydrates (Brecht et al., 2001b).

2.1.3.1 Plasmodium sialic acid-dependent and -independent pathways Different strains of Plasmodium invade host cells in a sialic acid-dependent or - independent manner (Baum et al., 2003; Dolan et al., 1990; Persson et al., 2008; Stubbs et al., 2005) and the parasite switches between the different invasion pathways during infection, in order to escape the host’s immune response. Two protein families are involved in this mechanism: the Duffy-binding-like or erythrocyte-binding-protein (DBL-EBP) family and the reticulocyte-binding-like (RBL) family.

1 The DBL family is characterized by the presence of the Duffy binding ligand domain (DBL), which has different amino acid composition but similar structure in different proteins (Mayor et al., 2005; Singh et al., 2006; Tolia et al., 2005). Whereas some proteins of the family mediate interactions with sialyated oligosaccharides on glycoproteins, others recognize specific protein epitopes. P. vivax only encodes one DBL protein, the Duffy-binding protein (DBP) (Wertheimer and Barnwell, 1989). In P. falciparum the family is composed by the members functioning in erythrocyte invasion, the so-called erythrocyte-binding ligands (EBL) (Adams et al., 1992), and the ones functioning as variant surface antigens of the erythrocyte membrane protein 1 (PfEMP-1) family (Scherf et al., 2008). Six PfEBL proteins have been identified - erythrocyte-binding antigen 175 (PfEBA-175) (Orlandi et al., 1990; Sim et al., 1992), erythrocyte-binding antigen 140 (PfBAEBL/EBA-140) (Thompson et al., 2001), erythrocyte-binding antigen 181 (PfEBA-181/JESEBL) (Gilberger et al., 2003), erythrocyte-binding antigen 165 (PfEBA-165/PEBL) (Triglia et al., 2001b), AMA1- and EBL-related protein (PfMAEBL) (Ghai et al., 2002; Kariu et al., 2002) and erythrocyte-binding ligand 1 (PfEBL-1) (Taylor et al., 2001). All PfEBLs, with the exception of PfMAEBL (Blair et al., 2002), are micronemal type I transmembrane proteins containing a signal peptide, a duplicated DBL domain (F1 and F2) called together region II, a cysteine-rich domain (region VI), a membrane-spanning domain and a cytoplasmic tail (Adams et al., 2001). PfEBA-140, PfEBA-175, PfEBA-181 and PfEBL-1 bind to glycophorins (major sialoglycoproteins on the surface of the RBC). While the receptor for PfEBA-181 is unknown (Maier et al., 2009), PfEBA-175 preferentially binds to a cluster of O-linked sialyated oligosaccharide structures on glycophorin A (Orlandi et al., 1992), PfEBA-140 binds preferentially to a N-linked glycan on glycophorin C (Jiang et al., 2009; Maier et al., 2009) and PfEBL-1 binds to glycophorin B (Mayer et al., 2009) (summarized in table 1.1). PfEBA-175 is the most studied member of the EBL family because antibodies directed against it strongly inhibit RBC invasion (Sim et al., 1990). Moreover it is required for both sialic acid- dependent and -independent invasion pathways (Duraisingh et al., 2003a) and signals for rhoptries secretion upon engagement with the host receptor (Singh et al.). It is also implicated in the selection of hosts by the parasite (Martin et al., 2005). The RBL family is composed in P. yoelii by the Py235 group, containing 14 homologues suggested to play a similar role to PfEMP1 (Preiser et al., 1999); in P. vivax by the reticulocyte binding proteins -1 and -2 (RBP-1 and RBP-2) (Galinski et

1 al., 1992); and in P. falciparum by the family of RBL-homologues (Rh), containing PfRh1, PfRh2a, PfRh2b, PfRh3, PfRh4 and PfRh5 (Triglia et al., 2001a). All localize to the rhoptries necks in the merozoite and, with the exception of PfRh5, all are type I transmembrane proteins (Baum et al., 2009; Duraisingh et al., 2003b; Kaneko et al., 2002; Rayner et al., 2000). PfRh1, PfRh2a, PfRh2b, PfRh4 and PfRh5 function as adhesins during the sialic acid-independent pathway (Baum et al., 2009; Desimone et al., 2009; Gao et al., 2008; Gaur et al., 2007; Rayner et al., 2001; Stubbs et al., 2005) and PfRh4 is also essential for switching invasion pathways (Stubbs et al., 2005) (summarized in table 1.1).

Table 1.1 Summary of the P. falciparum ligands involved in the sialic acid - dependent (Sia-dep) and –independent (Sia-indep) pathways (adapted from (Friedrich et al.)) Protein RBC binding RBC receptor Invasion pathway PfEBA-175 Sia-dep (Orlandi et al., Glycophorin A (Orlandi et Sia-dep and Sia-indep 1992) al., 1992) (Duraisingh et al., 2003a) PfEBA-140 Sia-dep (Maier et al., Promiscuous, Glycophorin C Sia-dep (Maier et al., 2009) (Maier et al., 2009; Mayer et 2003) al., 2009; Mayer et al., 2006) PfEBA-181 Sia-dep (Maier et al., Unknown Sia-dep (Maier et al., 2009) 2009) PfEBL-1 Sia-dep (Mayer et al., Glycophorin B (Mayer et al., Sia-dep (Mayer et al., 2009) 2009) 2009) PfRh1 Sia-dep (Triglia et al., Unknown Sia-dep (Triglia et al., 2005) 2005) PfRh2a Not detected Unknown Sia-indep (Desimone et al., 2009) PfRh2b Not detected Unknown Sia-indep (Duraisingh et al., 2003a) PfRh4 Sia-indep (Gaur et al., Unknown Sia-indep (Stubbs et 2007) al., 2005) PfRh5 Sia-indep (Baum et al., Unknown Sia-indep (Baum et al., 2009) 2009)

1 3. Micronemal proteins proteolysis during invasion

Numerous proteases are encoded in the genomes of the Plasmodium and Toxoplasma parasites (Wu et al., 2003) and protease inhibitors indicate that distinct classes of proteases might be implicated in invasion (Conseil et al., 1999; Olaya and Wasserman, 1991) but only two families of serine proteases - subtilisins and rhomboids - have been functionally proven to be implicated in the removal of excess adhesins (surface shedding) during invasion of host cells by these parasites (reviewed in (Carruthers, 2006; Dowse et al., 2008)).

3.1 Toxoplasma micronemal proteins proteolysis TgMICs undergo a series of proteolytic processing events. There is a first cleavage event to remove the signal peptide and subsequently a series of cleavage events occur during trafficking along the secretory pathway (Carruthers, 2006; Dowse and Soldati, 2004). Some TgMICs also contain pro-peptides that are removed by cleavage by a cathepsin protease (Parussini et al.), so that there is activation of the adhesive properties of the protein, as for TgMIC3 (Cerede et al., 2002; El Hajj et al., 2008), or to assure assembly and efficient release of the complex onto the parasite surface, as for TgM2AP (Harper et al., 2006). The other proteolytic processing events occur post- micronemal exocytosis at the parasite’s surface. Three proteolytic activities have been shown to occur at the parasite’s surface - microneme protein protease 1 (MPP1), microneme protein protease 2 (MPP2) and microneme protein protease 3 (MPP3) (Carruthers et al., 2000; Zhou et al., 2004). MPP1 seems to be the only essential activity given that inhibition of the two others does not prejudice invasion (Carruthers et al., 2000). MPP2 and MPP3 perform the so-called surface trimming. The MPP2 activity is most likely mediated by a chymotrypsin-like serine protease or a calpain-like cysteine protease (Brydges et al., 2006). It is responsible for a series of cleavage events on TgMIC2 (Carruthers et al., 2000; Zhou et al., 2004) and its associated partner protein TgM2AP (Zhou et al., 2004) and also for the cleavage of TgMIC4 (Brecht et al., 2001a). Most likely it also mediates the proteolytically processing of subtilisin-like protease 1 (TgSUB1) (Zhou et al., 2004). Its function is unknown and may be specific to the substrate as processing of TgMIC2 enhances binding to host cells (Barragan et

1 al., 2005) but the same might not hold true for TgMIC4 (Brecht et al., 2001a). The MPP2 activity is regulated by micronemal protein 5 (TgMIC5) (Brydges et al., 2006), which acts an inhibitory pro-domain (S. Matthews, personal communication). TgM2AP is the only protein cleaved by MPP3 (Zhou et al., 2004) and it is not known if this cleavage event is a pre-requisite for the one mediated by MPP2.

3.1.1 MPP1 activity Most single membrane-anchored MICs on the surface are excluded from the forming PV at the level of the MJ and are redistributed, at least for TgMIC2 and TgMIC3, towards the posterior end of the parasite during invasion still as a membrane- associated proteins, on a process called capping dependent on the actomyosin machinery (Carruthers, 1999; Garcia-Reguet et al., 2000) (figure 1.14). Shedding of these MICs from the parasite’s surface has been suggested to occur by proteolytic shedding of the MICs by the MPP1 protease. Cleavage prevents surface accumulation of excess adhesins at the parasite’s surface and limits the vulnerability of the MICs as target for neutralizing antibodies (Carruthers and Boothroyd, 2007), and may be responsible for the disengagement of the parasite from the host cell, at the end of the invasion process.

αTgAMA1 αTgM2AP merge

Figure 1.14 Capping of TgMIC2 Immunofluorescence assay of a T. gondii tachyzoite invading a host cell. TgMIC2 and its associated soluble partner, TgM2AP, are excluded from the moving junction (MJ) and the invading parasite (red). TgAMA1 is not excluded from the MJ and can be detected on intracellular parasites (green).

MPP1 was shown by in vitro cleavage assays and studies in the parasite to cleave TgMIC2, TgMIC6, TgMIC12 and TgAMA1 (Brossier et al., 2003; Howell et al., 2005; Opitz et al., 2002; Urban and Freeman, 2003; Zhou et al., 2004) (figure 1.15). The site of cleavage was mapped to an Ala residue within the TMD of TgMIC2 (Zhou et al., 2004), TgMIC6 (Opitz et al., 2002) and TgAMA1 (Howell et al., 2005) and this

1 cleavage motif is conserved in other proteins (Dowse and Soldati, 2005). The process is essential at least for TgMIC2 function because over-expression of a cleavage mutant inhibits invasion (Brossier et al., 2003).

Figure 1.15 MPP1 activity on the Toxoplasma micronemal complexes proteins

Unlike MPP2, MPP1 is constitutively expressed and active at the surface of the parasites, even when intracellular (Opitz et al., 2002), and regulation of its activity is assured by compartmentalization of the substrates, which are stored at the micronemes, and the enzyme, which is expressed at the plasma membrane. The MPP1 activity is conserved in different species of the phylum Apicomplexa because expression of the Plasmodium berghei TRAP protein in Toxoplasma parasites leads to cleavage within the TMD (Opitz et al., 2002). Proteases of the rhomboid family are seen as the best candidates to the MPP1 activity because the enzyme cleaves the MICs within the TMD in a conserved consensus sequence, it shows a very restricted sensitivity to serine protease inhibitors (it is only inhibited by DCI (Carruthers et al., 2000)) and a ubiquitous activity (Opitz et al., 2002).

3.2 Plasmodium micronemal proteins proteolysis In Plasmodium, the surface adhesins are shed from the parasite surface by a process of shaving, mediated by a protease that cleaves the substrates in a juxtamembrane position, removing them from the parasite’s surface during penetration of the host cell; and by rhomboid-mediated cleavage performed by a protease that cleaves substrates within the TMD at the end of the invasion process, promoting the

1 disengagement between the parasite and the host cell (O'Donnell and Blackman, 2005). The characteristics of the shaving activity suggested that it is performed by a subtilisin-like protease. Three protein subtilases have so far been identified in Plasmodium - subtilisin 1 (PfSUB1), subtilisin 2 (PfSUB2) and subtilin 3 (PfSUB3). All of them are expressed at the RBC stages, and PfSUB2 is also expressed at the mosquito stages (Florens et al., 2002; Han et al., 2000). PfSUB1 and PfSUB2 are encoded by essential genes but PfSUB3 is the only subtilisin protease to which functional data is not yet available (reviewed in (Withers-Martinez et al., 2004)). While PfSUB1 is localized to new secretory organelles named exonemes (Yeoh et al., 2007) and functions primarily during merozoites egress (Koussis et al., 2009), PfSUB2 functions during invasion mediating the shaving activity (Harris et al., 2005).

3.2.1 Shaving: Merozoite surface sheddase (MESH activity) The SUBs are synthesized as pre-pro-proteins and the pro-domain functions as a natural inhibitor (Withers-Martinez et al., 2004). Taking advantage of this, Harris and colleagues showed that a synthetic PfSUB2 pro-peptide inhibits cleavage of merozoite surface protein 1 (PfMSP1) and PfPfAMA1 (Harris et al., 2005) and that PfSUB2 is thus the subtilisin responsible for the merozoite surface sheddase (MESH) activity. PfSUB2 is a type I membrane protein localized to the micronemes and secreted onto the parasite’s surface during invasion (Harris et al., 2005). It is responsible for the cleavage of PfMSP1 (Harris et al., 2005) and for the predominant form of PfAMA1 shedding during invasion (Harris et al., 2005; Howell et al., 2005; Howell et al., 2003). It also sheds PfTRAMP from the merozoites surface (Green et al., 2006) and putative PfSUB2 cleavage sites can be found in PfRON2, PfRON5 and PfRON8 (Besteiro et al., 2009a; Miller et al., 2003). PfMSP1 may be involved in initial low-affinity attachment to the RBC and is expressed as a protein precursor that is cleaved into four fragments that then assemble into a complex at the merozoite’s surface, together with PfMSP6 and PfMSP7 (reviewed in (Blackman, 2000)). This large complex is cleaved by both PfSUB1 and PfSUB2 in a sequential and tightly regulated manner (Koussis et al., 2009). Primary processing takes place upon release of PgSUB1 from the exonemes into the PV lumen just before egress and secondary processing, which releases the complex from the

1 merozoite’s surface, occurs after release of PfSUB2 from the micronemes following egress. It is unclear if primary processing by PfSUB1 is a pre-requisite for secondary processing. PfAMA1 has been shown by mass-spectrometry to be shed from the merozoite’s surface by PfSUB2 cleavage (Howell et al., 2003) at a juxtamembrane site (Howell et al., 2005). Interestingly, the site of cleavage has no similarities with that of PfMSP1. Cleavage of PfAMA1 by PfSUB2 only occurs at the surface but both proteins localize to the micronemes and it has been suggested that the PfSUB2 pro-domain remains associated to the enzyme until exocytosis from the micronemes (Dowse et al., 2008).

3.2.2 Rhomboid activity PfEBA-175 (O'Donnell et al., 2006), PfAMA1 (Howell et al., 2005), PfRh1, PfRh4 (Triglia et al., 2009) and PfTRAP (P. Sinnis, personal communication) are the only Plasmodium adhesins shown to be cleaved by a rhomboid in vivo but several proteins possess a recognizable rhomboid cleavage motif within their TMDs (figure 1.16) and in vitro studies suggest that a rhomboid-like protease potentially cleaves substrate adhesins expressed at all the invasive stages of the life cycle (Baker et al., 2006), indicating that this activity is ubiquitous throughout the parasite’s life cycle.

1 Figure 1.16 (previous page) Alignment of the transmembrane domain of micronemal proteins from different apicomplexan parasites Alignment of the transmembrane domains and some surrounding amino acids of various micronemal proteins from Toxoplasma gondii (Tg), Neospora caninum (Nc), Sarcocystis muris (Sm), Eimeria tenella (Et), Cryptosporidium parvum (Cp), Babesia bovis (Bb), Theileria annulata (Ta) and Plasmodium falciparum (Pf) and comparison of their rhomboid cleavage motif with the one of Drosophila melanogaster (Dm) Spitz. The similar amino acid residues are labeled in blue and the identical ones are labeled in pink. The arrowhead indicates the position of rhomboid cleavage. Taken from (Dowse and Soldati, 2005).

PfAMA1 is an intriguing case when it comes to shedding because it is cleaved in Toxoplasma by a rhomboid protease and in Plasmodium it is preferentially cleaved by SPfUB2 and rhomboid cleavage is only favored in conditions in which PgSUB2 activity is impaired (Howell et al., 2005). However, PfAMA1 can be recognized by a rhomboid protease when expressed in in vitro cleavage assays (Baker et al., 2006), supporting the idea that the protein is cleaved by a rhomboid protease. Studies in sporozoites are all the more intriguing because they suggest that at this stage of the life cycle, PfAMA1 is cleaved by the same enzyme as PfTRAP and that the cleavage activity is sensitive to inhibitors different from PfSUB2 (Silvie et al., 2004). The significance of PfEBA-175 cleavage by a surface expressed rhomboid in the parasite has been functionally dissected (O'Donnell et al., 2006). The protein is cleaved at an alanine residue three residues into the TMD and this residue is conserved across the DBL-EBP family (figure 1.16), indicating that further members of the family are most likely rhomboid substrates. Surprisingly, cleavage is essential for invasion regardless of whether the protein is used as a primary ligand during invasion (O'Donnell et al., 2006). PfRh1 and PfRh4 are processed to produce fragments consistent with cleavage within the TMD consistent with rhomboid cleavage (Baker et al., 2006; Triglia et al., 2009) and PfTRAP, PfCTREP, PfMTRAP and PfMAEBL are susceptible to rhomboi- mediated cleavage in vitro (Baker et al., 2006). PfRh1 (Triglia et al., 2009) and PfEBA-175 (O'Donnell et al., 2006) localize to the apical end of the merozoite and move with the MJ during invasion, and rhomboid cleavage is suggested to release the ectodomains from the merozoite’s surface at the end of the invasion process.

1 4. Rhomboids and regulated intramembrane proteolysis

Rhomboids are serine proteases that along with three other groups of proteases perform regulated intramembrane proteolysis (RIP) (Erez and Bibi, 2009).

4.1 Rhomboids Rhomboids are serine proteases, possessing six to seven TMDs and the two residues forming the catalytic dyad are positioned within two different TMDs located deep into the lipid bilayer of the membrane (figure 1.17). Catalytic activity is only sensitive to the serine protease inhibitors 3, 4-Dichloroisocoumarin (DCI) and N- tosyl-L-phenylalanine chloromethyl ketone (TPCK) (Urban et al., 2001).

Figure 1.17 Schematic representation of a rhomboid protease and its substrate In blue is depicted a rhomboid protease possessing seven transmembrane domains; the catalytic serine residue is labeled in pink. In green is depicted a type I transmembrane protein substrate. Cleavage occurs within the substrate membrane-spanning domain. Not drawn to scale.

Drosophila melanogaster rhomboid-1 was the first member of the class identified (Lee et al., 2001) but since then it was shown that rhomboid-like genes are present throughout evolution in archea, prokaryotes and eukaryotes (Koonin et al., 2003; Pascall and Brown, 1998; Wasserman et al., 2000), being perhaps the most widely conserved membrane protein family (Koonin et al., 2003; Lemberg and Freeman, 2007; Urban et al., 2001). The overall sequence identity between rhomboids from different organisms is nevertheless low (Koonin et al., 2003; Pascall and Brown, 1998; Wasserman et al., 2000). The most recent classification of the family in eukaryotes (Lemberg and Freeman, 2007) proposed a three groups-based organization into Rhomboid-like proteases (active rhomboids), iRhomboids (inactive rhomboids) and a group containing all the proteins that could not be clustered with the previous

1 groups. The Rhomboid-like proteases can be further classified according to their intracellular localization (Koonin et al., 2003) into rhomboids residing in the secretory pathway with a structure comprising 6 + 1 TMDs (secretase rhomboids or RHO) and proteins localized to the mitochondrion possessing only 6 TMD (PARL-like rhomboids after the presenilin-associated rhomboid like protein found in mammals (Pellegrini et al., 2001)). The iRhomboids and the PARL group are typically represented by a single member in each species (Koonin et al., 2003; Pellegrini et al., 2001). Rhomboids appear to require no co-factors (Lemberg et al., 2005; Maegawa et al., 2007; Urban and Wolfe, 2005) and activity is regulated most likely according to the timing of gene expression and localization of the substrates. RHBDL2, a human rhomboid, was shown to be translated as an inactive proenzyme, and to have its activity regulated by proteolysis (Lei and Li, 2009), but it is unclear if this kind of regulation can be extrapolated to other members of the family. The rhomboid substrates are type I or type II transmembrane proteins that are cleaved in conserved sites at the or in proximity to the TMD at helix-breaking residues (GG or GA). The proteases don’t appear to recognize a specific sequence within the substrate motif but rather a common conformation (Akiyama and Maegawa, 2007; Urban and Freeman, 2003). It also seems that the presence of these motifs is necessary but not sufficient for cleavage to occur (Akiyama and Maegawa, 2007; Lohi et al., 2004). Substrate specificity seems to exist as not all rhomboids cleave the same substrates, but different proteases can cleave synthetic model substrates or non-cognate substrates from different species, indicating that either there is minimal substrate specificity or shared structural features in the proteases, substrates or both (Pascall and Brown, 2004; Urban et al., 2002). Recently a sequence cleavage motif was identified that is recognized by rhomboids from evolutionary distant species (Strisovsky et al., 2009) but it is unknown if this motif is universal and sufficient for cleavage by all rhomboids. Several groups have published the crystal structure of a bacterial rhomboid (reviewed in (Lemberg and Freeman, 2007)). These studies revealed that the catalytic dyad is found below the surface and that the movement of a loop at the C-terminus may allow access of the substrate, between helices 2 and 5, to the active site. Nevertheless, no mechanism for substrate recognition has been proposed and because the eukaryotic rhomboids present one more TMD and show limited sequence conservation with the

1 prokaryotic ones, it is not possible to extrapolate and speculate about the structure and mode of action of the eukaryotic rhomboids (figure 1.18).

Figure 1.18 Schematic of the protein structure of a bacterial rhomboid On the left is represented the consensus structure of a bacterial rhomboid in which the residues are labeled from yellow to red according to percentage of conservation; the active site consensus GxSx is presented in helix 4 (H4). On the right is depicted a model for rhomboid function based on the known protein structure; it is still unclear what is the substrate’s point of entry. Taken from (Lemberg and Freeman, 2007).

Conservation of rhomboid proteins in all organisms suggests that these proteases are implicated in important biological processes. However the study of their role in many species is still at an early stage and there are therefore few studies linking rhomboid activity to a specific mechanism. Several examples will be discussed later in this introduction.

4.1.1 Rhomboids in the Apicomplexa Rhomboids are encoded in all apicomplexans genomes sequenced to date and each species possesses several genes, suggesting that they are implicated in multiple processes (Dowse and Soldati, 2005). Toxoplasma expresses six rhomboid-like genes, five of which are expressed at the tachyzoite stage, and Plasmodium possesses eight genes coding for putative rhomboids but not all of them exhibit the conserved catalytic dyad. ROM1, ROM3, ROM4 and ROM6 are present in both Toxoplasma and Plasmodium genomes, ROM2 is only encoded in Toxoplasma, ROM5 is present in both Toxoplasma and Eimeria, ROM7 was only identified in Plasmodium and Theileria and ROM8-10 are Plasmodium specific (Dowse and Soldati, 2005). The PfROMs and TgROMs form four distinct phylogenetic clusters with the rhomboids of

1 other Apicomplexa (figure 1.19) (Dowse and Soldati, 2005) and can be divided into three different groups based on size: the short ROMs, the large ROMs (TgROM4, TgROM5 and PfROM4) and the PARL-like ROMs (TgROM6 and PfROM6). The shorter and larger ROMs are all predicted to have seven TMDs but the large ROMs are characterized for possessing significantly longer N- and C-termini domains.

Figure 1.19 Phylogenetic tree of the apicomplexan rhomboids Phylogenetic relationships between the rhomboids encoded in the genome of T. gondii (Tg), P. falciparum (Pf), P. berghei (Pb), E. tenella (Et), T. annulata (Ta) and C. parvum (Cp). The human (RHBDL2) and fly rhomboids (DmRho1) were also included in the analysis. The tree is based on neighbor-joining/distance analysis. Taken from (Dowse and Soldati, 2005).

Localization of the PfROMs and TgROMs in the parasite by N-terminal epitope- tagging (figure 1.20) revealed that ROM1 is a micronemal protein in both Toxoplasma (Brossier et al., 2005; Dowse et al., 2005) and Plasmodium (O'Donnell et al., 2006), TgROM2 is localized to the Golgi (Dowse et al., 2005), TgROM6 is a mitochondrial rhomboid (Dowse, Sheiner and Soldati, unpublished) and TgROM4 (Brossier et al., 2005; Dowse et al., 2005) and PfROM4 (O'Donnell et al., 2006) are expressed at the parasite plasma membrane. The localization of TgROM4 at the surface was also validated with specific antibodies raised against a recombinant TgROM4 fragment (Sheiner et al., 2008). TgROM5 intracellular location is still a matter of debate. While our laboratory localized it to the parasite’s surface and to internal unknown structures, as well as residual bodies (Dowse et al., 2005), other group reported a surface localization with an accumulation at the posterior pole (Brossier et al., 2005). The different results could be explained by different gene annotations by the two groups (TgROM5 annotated by Brossier et al. starts at Met 257 of the second exon of TgROM5 annotated by Dowse et al.) and the use of different promoters for expression of the epitope-tagged copies (a strong tubulin promoter in

1 the case of Dowse et al. and the endogenous promoter in the case of Brossier et al.). Until specific antibodies or tagging of the endogenous gene is available, the true localization of TgROM5 cannot be clarified.

ROM5

Figure 1.20 Schematic representation of the intracellular localization of the Toxoplasma rhomboids The TgROM1, TgROM2 and TgROM4 localizations have been taken from (Sheiner et al., 2008). The TgROM5 localization on the left has been taken from (Brossier et al., 2005) and the one on the right has been taken from (Dowse et al., 2005).

Validation of the apicomplexan rhomboids as active proteases was performed by expression on an in vitro heterologous system previously validated for other rhomboid proteases (Lee et al., 2001) (summarized in table 1.2). While Toxoplasma TgROM1, TgROM2 and TgROM5 (Brossier et al., 2005; Dowse et al., 2005) and Plasmodium PfROM1 and PfROM4 were active in this context, TgROM4 could either not be expressed (Dowse et al., 2005) or was not active (Brossier et al., 2005). Dowse et al. reported that while TgROM1, TgROM2 and TgROM5 were all able to cleave Spitz, only TgROM2 was able to cleave the parasite adhesins TgMIC2 and TgMIC12, which were also cleaved by the fly Rhomboid-1 and the human RHBDL2 (Dowse et al., 2005). In another study (Brossier et al., 2005), different results were obtained. TgROM5 was found to be able to cleave Spitz but also TgMIC2, TgMIC6 and TgMIC12 (summarized in table 1.2). The different results can be consequence of the

1 use of different substrates in the assay. While in the study of Dowse et al., the expressed substrate was a chimera containing the Spitz N-terminus, 5 myc tags and the TMD and C-terminal domain of TgMIC2 and TgMIC12 (Dowse et al., 2005); in the study of Brossier et al., the substrate contained GFP, the MIC TMD and the cytoplasmic tail of Transforming Growth Factor Alpha (TGF-α) (Brossier et al., 2005). More recently, an in vitro study tested the substrate specificity of the apicomplexan rhomboids TgROM5, PfROM1 and PfROM4 against Spitz and Plasmodium adhesins (Baker et al., 2006). All the enzymes, with the exception of PfROM4, could cleave Spitz. TgROM5 was found to have a promiscuous specificity as it could cleave almost all of the substrates tested and PfROM1 and PfROM4 showed more limited specificity (summarized in table 1.2). The authors suggested that TgROM5 has a “dual specificity”, being able to cleave Spitz-like and Spitz-unlike substrates, and PfROM1 and PfROM4 share the two activities in Plasmodium. The Baker et al. study revealed the potential of PfROM4 to cleave substrate adhesins expressed at all invasive stages of the Plasmodium life cycle. Parasites depleted for T. gondii TgROM4 and T. gondii, P. falciparum and P. berghei ROM1 are available. The rom1 knock out does not abrogate any essential step of the lytic cycle of the parasite and it is thus assumed to be either a non-essential protein or to have its function redundantly fulfilled by another rhomboid on its absence (Brossier et al., 2008; Srinivasan et al., 2009). TgROM4 is an essential protein but unlike what was hypothesized its essentiality does not rely on invasion. The protease mediates cleavage of TgMIC2, TgAMA1 and most likely of TgMIC8 (Buguliskis et al.). Parasites conditionally depleted for the enzyme only present a four-fold defect in invasion, which is a rather mild phenotype, suggesting that TgROM4 mediates an essential function at another step of the lytic cycle (Buguliskis et al.). PfROM4 seems to be encoded by an essential gene since it cannot be disrupted (Pino and Soldati, unpublished) and modification of the gene at the 3’ is deleterious (O'Donnell et al., 2006). Further studies of this rhomboid protease await the development of a conditional knock out system for Plasmodium.

Table 1.2 Toxoplasma and Plasmodium adhesins cleavage by rhomboid proteases as demonstrated by in vitro cleavage assays and in vivo studies in the parasite Substrate TgROM1 TgROM2 TgROM5 PfROM1 PfROM4 In vivo DmSpitz     DmRho1 (Baker et al., (Dowse et al., (Baker et al., (Baker et al., (Lee et al., 2001) 2006; Dowse 2005) 2006; 2006) et al., 2005) Brossier et al., 2005; Dowse et al., 2005) TgMIC2   TgROM4 (Dowse et al., (Brossier et (Buguliskis et al.) 2005) al., 2005) TgMIC6  TgROM5? (Brossier et (Buguliskis et al.; al., 2005) Opitz et al., 2002) TgMIC12   ? (Dowse et al., (Brossier et (Opitz et al., 2002) 2005) al., 2005) TgAMA1 TgROM4 (Buguliskis et al.; Howell et al., 2005) PfAMA1   PfSUB2 (Baker et al., (Baker et al., PfROM? 2006) 2006) (Howell et al., 2005; Howell et al., 2003) PfEBA175   ? (Baker et al., (Baker et al., (O'Donnell et al., 2006) 2006; 2006) O'Donnell et al., 2006) PfJESEBL   (Baker et al., (Baker et al., 2006) 2006) PfBAEBL   (Baker et al., (Baker et al., 2006) 2006) PfRh1    ? (Baker et al., (Baker et al., (Baker et al., (Triglia et al., 2009) 2006) 2006) 2006) PfRh2A   (Baker et al., (Baker et al., 2006) 2006) PfRh2B    (Baker et al., (Baker et al., (Baker et al., 2006) 2006) 2006) PfRh4   ? (Baker et al., (Baker et al., (Triglia et al., 2009) 2006) 2006) PfTRAP   PfROM4? (Baker et al., (Baker et al., (Srinivasan et al., 2006) 2006) 2009) PfMTRAP   (Baker et al., (Baker et al., 2006) 2006) PFF0800c   (Baker et al., (Baker et al., 2006) 2006) PfMAEBL    (Baker et al., (Baker et al., (Baker et al., 2006) 2006) 2006) PfCTRP   (Baker et al., (Baker et al., 2006) 2006)

1 4.2. Regulated intramembrane proteolysis Regulated intramembrane proteolysis (RIP) is a simple, powerful, precise and irreversible strategy for signal transduction in which intramembrane proteolysis triggers intracellular or intercellular signaling processes by the release of sequestered protein domains (Brown, 2000; Erez and Bibi, 2009; Freeman, 2009; Urban et al., 2002). The substrates are membrane-anchored proteins that are inactive in their membrane-tethered form, and are activated upon cleavage within the TMD, through release of their cytoplasmic or luminal/extracellular domains. RIP is catalyzed by four different protease families collectively named intramembrane cleavage proteases (iCliPs) that can be grouped into three major groups (Erez and Bibi, 2009): aspartyl proteases, including presenilin and signal peptide peptidase (SPP); rhomboid serine proteases; and zinc metaloproteases site -1 and -2 proteases (S1P and S2P). All these proteases are multi-spanning membrane proteins that cleave their substrates within residues buried inside or in proximity to the TMD. Rhomboids are the only iCLiPs that do not require a previous cleavage event in order to recognize the substrate. With the exception of presenilin, which is part of the γ-secretase protease complex (De Strooper et al., 1998; Wolfe et al., 1999), all appear to work as monomers (reviewed in (Erez et al., 2009)). An additional family of metalloproteinases named ADAM (a desintegrin and metalloproteinase) has also been implicated in RIP. The family includes transmembrane and secreted proteins and are not iCLiPs but function as upstream regulators of many RIP mechanisms (reviewed in (Edwards et al., 2008; Reiss and Saftig, 2009)). Rhomboids-mediated RIP is most commonly associated to intercellular signal transduction pathways. The “original” rhomboid, D. melanogaster Rhomboid-1 regulates signaling of the epidermal growth factor receptor (EGFR) by mediating cleavage of Spitz, the EGFR ligand (Lee et al., 2001). Full-length Spitz is trapped in the ER until Star chaperones it to the Golgi apparatus (Tsruya et al., 2002), where it is activated by Rhomboid-1-mediated cleavage (figure 1.21). Rhomboid-1 also cleaves Star to regulate the levels of secreted Spitz (Tsruya et al., 2007). In Caenorhabditis elegans, an EGFR-like pathway also involves a rhomboid. C. elegans cells express an EGF-like signaling factor called LIN-3, which bears similarities to Spitz. When the LIN-3 signal is received by the EGFR-like receptor LIN-23 of neighboring cells, the RAS/mitogen-activated protein kinase (MAPK) pathway is activated (Sundaram,

1 2004). In this case, rhomboid cleavage only increases the range of the LIN-3 signal (Dutt et al., 2004). The human rhomboid, RHBDL2, is also involved in signaling (Pascall and Brown, 2004). The mechanism involves binding of the B-type ephrins ephrinB2 and ephrinB3 of one cell to the Eph tyrosine kinase receptor of another cell, upon cell-to-cell interaction. Binding not only activates the receptor but also regulates rhomboid cleavage and generates a reverse signal into the cells that express the ligands. A common response to Eph activation is repulsion of neighboring cells or cellular projections. In Pseudomonas stuartii, cleavage of the substrate TatA by the rhomboid AarA activates TatA function as a channel and initiates a program of quorum sensing (Gallio et al., 2002; Stevenson et al., 2007). S1P- and S2P-mediated cleavage has been shown to be involved in the control of expression of genes implicated in stress relief in several organisms, including humans (Erez and Bibi, 2009; Urban and Freeman, 2002). Mammalian sterol regulatory element-binding protein (SREBP) is tethered in the ER membrane by two TMDs. When cholesterol levels drop, SREBP traffics to the Golgi apparatus where it is cleaved by S1P (figure 1.21). This cleavage separates the two membrane-spanning segments but the N-terminus remains attached to the membrane by the first TMD and is only released upon cleavage by S2P. The released cytoplasmic domain travels to the nucleus and switches on the expression of genes required for cholesterol and fatty- acid synthesis (Ye et al., 2000a; Ye et al., 2000b). Activation of transcription factor 6 (ATF6) is also regulated by cleavage. Stress signals at the ER generate unfolded proteins and promote trafficking of ATF6 from the ER to the Golgi apparatus. In the Golgi, the ATF6 cytoplasmic domain is released by proteolytic processing by S1P and S2P and migrates to the nucleus where it induces the expression of factors that alleviate ER stress together with XBP1 (X-box binding protein 1) (Ye et al., 2000b). Response of some bacterium to cell-envelope insults (reviewed in (Heinrich and Wiegert, 2009; Makinoshima and Glickman, 2006)) and virulence of Vibrio cholerae and Mycobacterium tuberculosis (Matson and DiRita, 2005) also involves S2P- mediated cleavage. RIP mediated by presenilin has been widely studied in the recent years because it is known to be involved in the cleavage of APP (amyloid-β-precursor protein), which generates amyloid-β-peptide, the major component of the amyloid plaques formed in Alzheimer’s disease (Steiner, 2008; Wolfe et al., 1999) (figure 1.21). Presenilin also

1 regulates Notch signaling. In the Notch pathway, cells expressing the Delta, Jagged, or Serrate proteins at the cell membrane activate neighboring cells that contain the Notch protein at the surface. When associated to one of these ligands, Notch undergoes a conformational change that enables it to be cleaved first by ADAM-10 (van Tetering et al., 2009) and secondly by presenilins -1 and -2, and the released C- terminal tail enters the nucleus and induces expression of target genes (Bray, 2006). Gene transcription and cell proliferation by epithelial cell adhesion molecule (EpCAM) is also activated via RIP by ADAM and presenilin (Maetzel et al., 2009) upon cell-to-cell-contact (Denzel et al., 2009). ADAM-17 and presenilin sequentially cleave EpCAM and release an intracellular domain that shuttles to the cell nucleus and associates with transcription factors, inducing transcription of target genes (Maetzel et al., 2009). SPP is involved in generating the human lymphocyte antigen E (HLA-E) involved in antigen presentation to natural killer cells. During biosynthesis, the signal sequence of the major histocompatibility complex class I (MHC-I) is first cleaved by signal peptidase and then by SPP. The resultant fragment is released into the cytosol, where it is further processed, and transported back to the ER until it is loaded onto HLA-E and transported to the cell surface for antigen presentation (Narayanan et al., 2007) (figure 1.21).

1 Figure 1.21 (previous page) RIP mechanisms RIP mechanisms mediated by: top left - site-2 protease (S2P) on sterol regulatory element-binding protein (SREB); top right - rhomboid protease (GlpG, crystal structure of E. coli rhomboid) on epidermal growth factor ligand Spitz; bottom left – presenilin N-terminal fragment (NTF) and C- terminal fragment (CTF) on amyloid precursor protein (APP); bottom right – signal peptide peptidase (SPP) on the signal sequence of a major histocompatibiliy complex class I (MHC-I) molecule. Taken from (Erez et al., 2009).

5. Hypothesis and Aims of the Project

Toxoplasma, unlike Plasmodium, can invade a wide range of cells. The molecular mechanisms behind this phenomenon are unknown and one of the hypothesis is that it is consequence of expression of a different number of adhesin proteins that when discharged onto the surface can bind to different host cell receptors. Sialic acid is widely distributed on the host cell and is a major determinant of host cell invasion by T. gondii but the parasite sialic-acid binding ligands remain to be determined. TgMIC1 is a good candidate to this activity because it specifically recognizes sialyated oligosaccharides on the host cell (Blumenschein et al., 2007) and parasites depleted for TgMIC1 are highly deficient in invasion (Cerede et al., 2005). The first aim of this thesis was to determine the role of TgMIC1 for sialic acid-dependent parasite invasion and identify new sialic acid-binding lectins. Besides TgMIC1, other TgMICs perform essential functions during invasion. Four micronemal complexes, composed of both soluble and transmembrane proteins, have been identified in Toxoplasma. The membrane-anchored proteins have a modular structure that enables them to potentially bind to aldolase, mediate targeting of the complexes to the micronemes and be shed from the parasite surface at the end of the invasion process by rhomboid cleavage. These functions have only been attributed to a few transmembrane MICs (TM-MICs) and the study of the entire repertoire of TM- MICs has never been conducted. The second aim of this study was thus to determine the role of each of the previously identified TM-MICs for host cell invasion. MPP1-mediated cleavage seems to be a conserved, essential activity throughout the Apicomplexa phylum but the protease responsible for this proteolytic activity has yet to be identified. MPP1 is most certainly a rhomboid-like protease and we hypothesize

1 that in both Plasmodium and Toxoplasma it is mediated by ROM4 since this is the only rhomboid-like gene conserved in all apicomplexan parasite genomes (Dowse and Soldati, 2005) and it is expressed throughout the life cycle at the parasite’s plasma membrane (Brossier et al., 2005; Dowse et al., 2005; O'Donnell et al., 2006). The third aim of the study was to validate this hypothesis by studying the function of TgROM4.

1 CHAPTER II

MATERIALS AND METHODS

(For further details please report to the material and methods section of the articles presented in the Results section).

1. Reagents and Suppliers All chemicals were obtained from Sigma except where stated. The reagents used in parasite culture were purchased from GIBCO.

1.1 Enzymes Restriction enzymes were purchased from New England Biolabs T4 DNA ligase was purchased from Promega GoTaq Green Master Mix (Promega) TaKaRa LA Taq DNA polymerase from TaKaRa.

1.2 Kits Nucleobond maxi-prep kit for large scale DNA preparation (BD Biosciences) WizardMini-prep kit for small scale DNA preparation (Promega) Wizard SV gel and PCR Clean-Up kit (Promega) pGEM T-easy kit (Promega) Quikchange II kit (Stratagene)

1. 3 Antibodies

Table 2.1 Antibodies used in this study Antibody Source Mouse anti-myc hibridoma GE10 Invitrogen Mouse anti-Ty1 hibridoma BB2 (Bastin et al., 1996) Rabbit anti-PfAMA1 Kindly provided by M J Blackman Rabbit anti-TgMIC4 (Brecht et al., 2001b) Rabbit anti-TgGAP45 (Plattner et al., 2008)

1 Rabbit anti-TgProfilin (Plattner et al., 2008) Goat anti-mouse horse radish peroxidase (HRP) Molecular Probes conjugated Goat anti-rabbit HRP conjugated Molecular Probes Goat anti-mouse, Alexa 488 conjugated (green) Molecular Probes Goat anti-mouse, Alexa 594 conjugated (red) Molecular Probes Goat anti-rabbit, Alexa 488 conjugated (green) Molecular Probes Goat anti-rabbit, Alexa 594 conjugated (red) Molecular Probes Anti-TgROM4Nt (Sheiner et al., 2008)

All primary antibodies were used at a 1:1000 dilution for both immunofluorescence and western blot, with the exception of anti-TgGAP45, which was used at a 1:3000 dilution. All the secondary antibodies were used at a 1:3000 dilution.

2. Solutions

2.1 Culture media LB (Luria Bertani) liquid media: 1% w/v Bacto-Tryptone, 0.5% w/v yeast extract, 1% w/v NaCl LB Agar: LB liquid with 1.5% w/v Agar T. gondii culture media: Dulbecco´s Modified Eagle´s Medium (DMEM) + 4500mg/L Glucose + L-Glutamine + Pyruvate (GIBCO) supplemented with 5% (v/v) fetal calf serum (FCS), 2mM L-glutamine and 25 µg/ml gentamicin

2.2 General solutions

Table 2.2. Solutions used in this study Solution Composition Mini-prep Solution I 50mM glucose, 25mM Tris-Cl, 10mM EDTA, pH 8.8 Mini-prep Solution II 0.2M NaOH, 1% SDS Mini-prep Solution III 3M potassium acetate, 5M glacial acetic acid Phosphate buffer solution (PBS) 137mM NaCl, 10mM Phosphate, 2.7mM KCl, pH 7.4 SDS-PAGE gel-loading buffer 50mM Tris-Cl pH 6.8, 100mM Dithiothreitol, 2% SDS, 0.1% Bromophenol blue, 10% glycerol

1 Agarose gel-loading (6X) 0.02% Bromophenol blue, 0.02% Xylencyanol

FF, 30% Glycerol in H2O

Cytomix 120mM KCl, 0.15mM CaCl2, 10mM

K2HPO4/KH2PO4, 25mM Hepes, 5mM MgCl2, pH 7.6, 5mM ATP, 5mM Glutathione Tris/Acetate/EDTA (TAE) 0.04M Tris-Acetate, 0.001M EDTA, pH 8 SDS-PAGE running buffer 25mM Tris, 250mM Glycine, pH 8.3, 01% SDS Semi-dry transfer buffer 2.5mM Tris, 19.2mM Glycine, 20% Methanol Tris EDTA (TE) 10X 100mM Tris-HCl pH 7.5, 10mM EDTA pH 8

3. Cell lines and microbiological strains

3.1 Bacteria

Table 2.3 Bacteria strains used in this study Strain name Genotype XL1-Blue F’::Tn10 proA+B+laclqΔ(lacZ)M15/recA1 endA1 r - + gyrA96(Nal ) thi hsdR17 (rK mK ) glnV44 relA1 lac - - BL21 F- ompT gal [dcm] [lon] hsdSB (rB mB ) λ(DE3)

3.2 Mammalian cells Human foreskin fibroblasts (HFF), the African Green Monkey Kidney Cell line (Vero) and the human embryonic kidney 293T cell line (HEK293T) were obtained from the American Type Culture Collection (ATCC).

3.3 Toxoplasma gondii strains The RHΔhxgprt is a virulent strain in which the hxgprt gene has been disrupted (Donald et al., 1996).

4. Culture conditions

4.1 Bacterial culture Bacteria cultures were grown in LB Broth at 37ºC or on plates of LB Agar. Ampicillin was used at a concentration of 100µg/ml. Kanamycin was used at a

1 concentration of 30µg/ml.

4.2 Mammalian cell culture HFF, Vero and HEK293T cells were grown as monolayers in supplemented DMEM medium and were split using Trypsin (GIBCO).

4.3 Parasite propagation T. gondii tachyzoites were grown in Vero or HFF cell cultures. Selection and cloning of stably transfected parasites took place in HFF cell cultures, with medium supplemented with the appropriate drug (25µg/ml mycophenolic acid (MPA) and 50µg/ml Xanthine or 1µM Pyrimethamine).

5. Transformations and transfections

5.1 Eschericia coli transformation Competent E. coli was prepared using the protocol of Inoue et al. (Inue et al., 1990). XL1-Blue bacteria transformation was performed by incubating the DNA with the bacteria on ice for 20 minutes and performing heat-shock (1min, 42ºC). BL21 bacteria were transformed by electroporation. The transformed bacteria were plated on LB- Agar plates supplemented with the appropriated antibiotic.

5.2 Toxoplasma. gondii transfection 5×107 freshly egressed RHΔhxgprt parasites were centrifuged at 300g for 10min, resuspended in 700µl cytomix buffer and mixed with 80µg of linearized plasmid carrying the selectable marker gene and the expression cassette containing the DNA sequences. Restriction enzyme-mediated integration (REMI) was employed by adding 50-100 units of restriction enzyme to the transfection mix. The parasites/DNA mix was electroporated at 2 kV, 25 mF, 48 V (Soldati and Boothroyd, 1993) using a BTX electroporator (Harvard biosciences, Holliston, MA, USA) before being added to a monolayer of HFF cells in the presence of the appropriate drug. Cloning by limiting dilution on 96-well microtiter plates following one growth cycle.

1 6. Cloning of DNA constructs

6.1 DNA constructs used in the in vitro cleavage assays The construct based on pcDNA3.1 (Invitrogen) for transient expression of HA-tagged PfROM4 synthetic gene wild type (pcDNA-PfROM4) or mutated at the catalytic serine (pcDNA-PfROM4S-A), as well as the constructs based on pSectag2a (Invitrogen) for transient expression of the HA-tagged EBA175 minigene and the chimeric construct AMA1-EBA175tm-ct were provided by M J Blackman. A pcDNA3.1 based plasmid for transient expression of HA-tagged TgROM4 (pcDNA- TgROM4) was a gift of S. Urban and the pCAN constructs for expression of HA- tagged TgROM5 wild type (pCAN-TgROM5) were previously described (Dowse et al., 2005). Deletion constructs expressing PfROM4 and TgROM5 without the C-terminal domain were amplified from pcDNA-PfROM4 and pCAN-TgROM5 with primers 1795 and 1796 and 1175 and 2049, respectively. The PCR products were then digested with XbaI and EcoRI or BglII and EcoRI and cloned in pcDNA-PfROM4 or pCAN-TgROM5 digested with XbaI and EcoRI or BamHI and EcoRI, respectively, originating pcDNA-PfROM4ΔCt and pCAN-TgROM5ΔCt, respectively. The PfROM4 chimeric constructs were generated by amplifying the PbROM4, TgROM4 and TgROM5 tails with primers 2029 and 2030, 1797 and 1798 and 1799 and 1800, respectively, digesting with EcoRI and NotI and cloning in the same sites in the pcDNA-PfROM4ΔCt plasmid. The resultant plasmids were named pcDNA- PfROM4-CtPbROM4, pcDNA-PfROM4-CtTgROM4 and pcDNA-PfROM4- CtTgROM5. A plasmid expressing a PfROM4 carrying the last C-terminal cysteine replaced by an alanine residue was generated by amplifying PfROM4 with primers 1795 and 2171, digesting the PCR product with XbaI and EcoRI and cloning in the same sites in the pcDNA-PfROM4 plasmid. This way was generated pcDNA-PfROM4C-A.

Table 2.4 Primers used in construction of the plasmids used in the cell-based assays Primer Sequence Restriction Resulting name site plasmid 1795 GGTATTGAGGGTCGCTCTAGAATGTC XbaI pcDNA- AGGTTACCCCTATGACGTGC PfROM4ΔCt

1 1796 AGAGGAGAGTTAGAGCCGAATTCTTA NotI pcDNA- TGCGGCCGCGAGCAGGTAGATAAACA EcoRI PfROM4ΔCt GGACGATC 1797 CCGCTCGCGGCCGCAGATCCCTCACTG NotI pcDNA- TACAAGAGTTACTC PfROM4- CtTgROM4 1798 GGGTGGGAATTCTTACGGTTCAAGATAA EcoRI pcDNA- TACTGCGCATCC PfROM4- CtTgROM4 1799 CCGCTCGCGGCCGCACCGTCGTACTACGA NotI pcDNA- GTCTCTGAG PfROM4- CtTgROM5 1800 CCGGTGGAATTCTTATTGGCCTGCCCTCGT EcoRI pcDNA- CTGCTG PfROM4- CtTgROM5 1175 GGCATGCATAAAGATCTGTCGTCGAAAGGT BglII pCAN- GGATCTTCTC TgROM5ΔCt 2049 CCGGAATTCTTAAACCAGCAAATACAGCCA EcoRI pCAN- CAACACCG TgROM5ΔCt 2029 CCGCTCGCGGCCGCAGATGAAAGTGCTTAT NotI pcDNA- CGATCTTATACACCAATG PfROM4- CtPbROM4 2030 GGGTGGGAATTCTTATTCCTTGCAATAATAA EcoRI pcDNA- TCAAATGCTTCTTGATTGC PfROM4- CtPbROM4 2171 CGAATTCGCGGCCGCTTACTTGTTGGCGTAAT NotI pcDNA-

ACCGAGTGG EcoRI PfROM4C-A

6.2 Agarose gel electrophoresis To prepare 0.5-2% agarose gels agarose was dissolved (Sigma) in heated TAE and the mix was poured in a gel-casting tray (peqLab). SYBR Safe 0.4X (Invitrogen) was added to the gel to stain the nucleic acids. Electrophoresis was performed at 100- 200V in TAE buffer.

6.3 Preparation of nucleic acids Mini-preps of plasmid DNA from E. coli were performed using cell pellets from a 5ml culture and the WizardMini-prep kit for small scale DNA preparation (Promega). Large preparations of plasmid DNA were performed using the Nucleobond maxi-prep

1 kit (BD Biosciences). Both kits were used according to the manufacturer’s instructions.

6.4 Polymerase chain reaction (PCR) Standard analytical PCR reaction were performed with GoTaq Green Master Mix, using the supplied master mix and specific primers at 0.5 µM. Template DNA was used in the form of plasmid or genomic DNA (10-100ng per reaction) or a bacterial colony. The PCR reaction was set according to the manufacturer’s indications and to specificities of the primers used and the product to be amplified. PCR products for cloning were prepared using the high fidelity TaKaRa LA Taq polymerase, according to the manufacturer’s protocol.

6.5 Ligations TA cloning of PCR products was performed using pGEM T-easy cloning kit according to the manufacturer’s instructions and sequencing of selected clones was performed to verify the DNA sequence. Restriction digest of the correct clones excised the product of interest from pGEM. Other products were prepared by restriction digest of plasmids containing the sequence of interest. The cloning vectors were prepared by restriction digestion. Following digestion, gel purification of the appropriate bands was conducted with the Wizard SV gel and PCR Clean-Up kit, according to the manufacturer’s instructions. Ligation of the insert to the cloning vector was performed on a ratio of 7:1 with T4 DNA ligase in the supplied buffer, at 4ºC overnight. The ligation mixes were used for E. coli transformation.

6.6 Site-directed mutagenesis Site-directed mutagenesis by PCR was performed with Quikchange II kit, according to the manufacturer’s instructions, with specific sense and antisense primers containing the desired mutation(s). The presence of the desired mutation(s) was confirmed by DNA sequencing.

7. Sodium Dodecyl Sulphate (SDS) Polyacrylamide gel (PAGE) Electrophoresis and Western blotting SDS-PAGE was performed using standard methods (Dlouhy et al., 1989). Resolving gel (8-15% acrylamide, 0.1% SDS, 375mM Tris, pH 8) and stacking gel (5%

1 acrylamide, 0.1% SDS, 200mM Tris, pH 6.8) solutions were polymerized using 0.04- 0.1% N,N,N’,N’-Tetramethyl-1-2,-diaminomethane (TEMED) and 0.1% ammonium persulphate (APS). 2x107 freshly lysed parasites were harvested after complete lysis of the host cells and protein extracts were prepared in 1xPBS by five consecutive freeze/thaw cycles with intermediate homogenization. The protein suspension was mixed with SDS–PAGE-loading buffer and the proteins were separated by electrophoresis performed in SDS-PAGE running buffer. The separated proteins were transferred to a nitrocellulose membrane using a semidry electroblotter as described previously (Dlouhy et al., 1989). Western blots were performed by blotting the membranes in blocking solution (5% non-fat milk powder in 1X PBS-0.05% Tween), 1h at room temperature, followed by 1h incubation with specific primary antibodies diluted in blocking solution. Blots were washed with 1X PBS-0.05% Tween and incubated 1h with HRP-conjugated secondary antibodies also diluted in blocking solution. Bound antibodies were visualized using the ECL system (Amersham Corp).

8. Immunofluorescence assays (IFA) and confocal microscopy Intracellular parasites grown in glass coverslips with HFF attached were fixed with 4% paraformaldehyde (PAF) or 4% PAF-0.005% Glutaraldehyde (PAF/GA) in PBS for 10min at room temperature, followed by neutralization with 1X PBS-0.1M Glycine. Fixed parasites were then permeabilized 20min with permeabilization solution (1X PBS-0.2% Triton) and blocked 20min with blocking solution (1% BSA/1X PBS-0.2% Triton). Protein staining was performed by 1h incubation with specific primary antibodies diluted in blocking solution, followed by washes with permeabilization solution and 1h incubation with fluorochrome-conjugated secondary antibodies in the dark. The host cell nuclei were stained with 0.1mg-DAPI/mL diluted in 1XPBS. The treated coverslips were mounted into glass slides with Fluoromount G (Southern Biotech) and stored at 4°C in the dark. Micrographs were obtained on a Zeiss Axioskop 2 equipped with an Axiocam color CCD camera. Images where recorded and treated on computer through the AxioVision™ software. Confocal images were collected with a Leica laser scanning confocal microscope (TCS- NTDM/IRB) using a 63 Plan-Apo objective with NA 1.40. Optical sections were recorded at 250 nm per vertical step and four times averaging.

1 9. Assays

9.1 In vitro cleavage assay HEK-293T cells grown as a monolayer on 6-well chambers in supplemented DMEM medium were transiently transfected with 200ng (50ng in the case of TgROM5) of expression constructs encoding a rhomboid and substrate using the Fugene transfection solution (Roche), according to the manufacturer’s indications. 24 hours later, the medium and the cell extracts were harvested and analyzed by SDS and Western-blot. Upon cleavage, the substrate’s soluble shed form can be detected in the cell medium samples. Analysis of the cell extracts determines the levels of expression of both proteins.

1 CHAPTER III

RESULTS

1 1. Members of a novel protein family containing microneme adhesive repeat domains act as sialic acid-binding lectins during host cell invasion by apicomplexan parasites

It is known that while some parasites of the phylum Apicomplexa, such as Plasmodium, can invade a restricted set of host cells, others, such as Toxoplasma, can invade a wide range of cells and we hypothesize that this is consequence of expression of a different number of adhesin proteins that when discharged onto the surface can bind to different host cell receptors. TgMIC1 binds to host cell sialic acid through a new adhesive domain named Microneme Adhesive Repeat (MAR) but the contribution of this binding to the T. gondii ability to invade in a sialic acid-dependent manner is not known. By performing cell-binding assays with a strain depleted for TgMIC1, we could show that TgMIC1 is the major sialic acid-binding adhesin but that most likely the parasite encodes other sialic acid-binding lectins. The study of the three other MAR domain- containing proteins (MCPs) encoded in T. gondii genome revealed that TgMCP2 could function as a sialic acid-binding adhesin. The personal contribution to this work restricted to the analysis of the localization and cell binding capacity of the new proteins TgMCP2, TgMCP3 and TgMCP4 in the parasite and Eschericia coli. More specific contributions can be found at the authors contribution section.

1 Supplemental Material can be found at: http://www.jbc.org/content/suppl/2009/11/09/M109.060988.DC1.html

Members of a Novel Protein Family Containing Microneme Adhesive Repeat Domains Act as Sialic Acid-binding Lectins during Host Cell Invasion by Apicomplexan Parasites*□S Received for publication, August 29, 2009, and in revised form, November 5, 2009 Published, JBC Papers in Press, November 9, 2009, DOI 10.1074/jbc.M109.060988 Nikolas Friedrich‡, Joana M. Santos‡1,2, Yan Liu§1, Angelina S. Palma§3, Ester Leon¶, Savvas Saouros¶, Makoto Kisoʈ, Michael J. Blackman**, Stephen Matthews¶, Ten Feizi§4, and Dominique Soldati-Favre‡5 From the ‡Department of Microbiology and Molecular Medicine, Faculty of Medicine, University of Geneva Centre Medical Universitaire, 1 Rue Michel-Servet, 1211 Geneva 4, Switzerland, §Glycosciences Laboratory, Division of Medicine, Imperial College London, Northwick Park Campus, Harrow HA1 3UJ, United Kingdom, ¶Division of Molecular Biosciences, Imperial College London, South Kensington Campus, London SW7 2AZ, United Kingdom, ʈDepartment of Applied Bioorganic Chemistry, Gifu University, Gifu 501-11, Japan, and **Division of Parasitology, Medical Research Council National Institute for Medical Research, Mill Hill, London NW7 1AA, United Kingdom Downloaded from

Numerous intracellular pathogens exploit cell surface glyco- share a preference for ␣2–3- over ␣2–6-linked sialyl-N-acetyllac- conjugates for host cell recognition and entry. Unlike bacteria tosamine sequences. However, the three lectins also display differ- and viruses, Toxoplasma gondii and other parasites of the phy- ences in binding preferences. Intense binding of TgMIC13 to lum Apicomplexa actively invade host cells, and this process ␣2–9-linked disialyl sequence reported on embryonal cells and rel-

critically depends on adhesins (microneme proteins) released atively strong binding to 4-O-acetylated-Sia found on gut epithe- www.jbc.org onto the parasite surface from intracellular organelles called lium and binding of NcMIC1 to 6sulfo-sialyl Lewisx might have micronemes (MIC). The microneme adhesive repeat (MAR) implications for tissue tropism. domain of T. gondii MIC1 (TgMIC1) recognizes sialic acid (Sia), a key determinant on the host cell surface for invasion by this at SMAC Consortium - Geneve, on May 29, 2010 pathogen. By complementation and invasion assays, we demon- Sialic acids (Sias)6 occur abundantly in glycoproteins and gly- strate that TgMIC1 is one important player in Sia-dependent colipids on the cell surface and are exploited by many viruses invasion and that another novel Sia-binding lectin, designated and bacteria for attachment and host cell entry. Recognition of TgMIC13, is also involved. Using BLAST searches, we identify a carbohydrates and in particular sialylated glycoconjugates is family of MAR-containing proteins in enteroparasitic coccid- important also for host cell invasion by the Apicomplexa (1–4), ians, a subclass of apicomplexans, including T. gondii, suggest- a phylum that includes several thousand species of obligate ing that all these parasites exploit sialylated glycoconjugates on intracellular parasites, among them the Plasmodium spp. caus- host cells as determinants for enteric invasion. Furthermore, ing malaria. Enteroparasitic coccidians are a subclass of Api- this protein family might provide a basis for the broad host cell complexa comprising Eimeria spp. responsible for coccidiosis range observed for coccidians that form tissue cysts during in poultry, Neospora spp. causing neosporosis in cattle, and chronic infection. Carbohydrate microarray analyses, cor- Toxoplasma, the causative agent of toxoplasmosis in warm- roborated by structural considerations, show that TgMIC13, blooded animals and humans. TgMIC1, and its homologue Neospora caninum MIC1 (NcMIC1) The host range and cell type targeted by these parasites vary widely across the phylum. Whereas Plasmodium falciparum merozoites exclusively invade erythrocytes of humans and * This work was supported in part by the Swiss National Foundation (to D. S.), great apes (5), Toxoplasma gondii tachyzoites (the form of the the UK Medical Research Council, and UK Research Council Basic Technol- ogy Grant GR/S79268 and Translational Grant EP/G037604/1 (to T. F.). This parasite associated with acute infection) invade an extremely work is part of the activities of the BioMalPar European Network of Excel- broad range of cell types in humans and virtually all warm- lence supported by a European Grant LSHP-CT-2004-503578 from the Pri- blooded animals, enabling rapid establishment of infection in ority 1 “Life Sciences, Genomics, and Biotechnology for Health” in the 6th Framework Programme. the host and dissemination into deep tissues (6). Information is □S The on-line version of this article (available at http://www.jbc.org) contains emerging on the involvement of carbohydrate-protein interac- supplemental Materials and Methods, Figs. S1–S7, and Tables I and II. tions in this broad host cell recognition (1). 1 Both authors contributed equally to this work. 2 Recipient of the European Union-funded Marie Curie Action MalPar Training Many intracellular pathogens have evolved to manipulate the Grant MEST-CT-2005-020492 (The Challenge of Malaria in the Post- phagocytic pathways of host cells during invasion. This con- genomic Era). trasts with invasion by apicomplexans, which express their own 3 Fellow of the Fundac¸a˜o para a Cieˆncia e Tecnologia and supported by Grant SFRH/BPD/26515/2006, Portugal. Present address: REQUIMTE, Centro de Química Fina e Biotecnologia, Dept. de Química, Faculdade de Cieˆncias e 6 The abbreviations used are: Sia, sialic acid; EGF, epidermal growth factor; Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal. ESA, excreted secreted antigens fraction; HFF, human foreskin fibroblast; 4 To whom correspondence may be addressed. Tel.: 44-20-8869-3460; E-mail: MAR, microneme adhesive repeat; MCP, MAR-containing protein; MIC, [email protected]. microneme protein; NANA, N-acetylneuraminic acid; 3ЈSiaLacNAc, 3Јsialyl- 5 International Howard Hughes Medical Institute scholar. To whom corre- N-acetyllactosamine; PBS, phosphate-buffered saline; CHO, Chinese ham- spondence may be addressed. Tel.: 41-22-379-5672; Fax: 41-22-379-5702; ster ovary; IFA, immunofluorescence assay; WT, wild type; EST, expressed E-mail: [email protected]. sequence tag; SP, signal peptide. 1 2064 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285•NUMBER 3•JANUARY 15, 2010 Supplemental Material can be found at: http://www.jbc.org/content/suppl/2009/11/09/M109.060988.DC1.html Family of MAR Domain-containing Proteins in Apicomplexa machinery for active host cell entry. Invasion is a multistep complex is not the only molecular player involved in Sia-de- process requiring the tightly regulated discharge of parasite pendent invasion by T. gondii tachyzoites. By BLAST searches, organelles called micronemes and rhoptries (7). Micronemes we identify a family of MAR domain-containing proteins release adhesins (MICs) onto the parasite surface, which form (MCPs) in T. gondii and related apicomplexan parasites Sarco- multiprotein complexes with nonoverlapping roles in motility, cystis neurona, Neospora caninum, and Eimeria tenella. Among host cell attachment, secretion of rhoptry organelles, and cell these we characterize TgMIC13, and we compare the binding penetration (8). After attachment and reorientation of the par- specificities of recombinant TgMIC13 with TgMIC1 and its asite, invasion induces the formation of a nonfusogenic parasi- homologue from the closely related organism N. caninum, tophorous vacuole derived in large part from host cell plasma NcMIC1, by carbohydrate microarray and cell binding assays. membranes (9). The MICs share a limited number of adhesive Our results indicate that the MAR domain is unique to and domains arranged in various combinations and numbers (10). conserved among coccidians and that it acts as an important These domains are implicated in host cell recognition and determinant in host cell recognition by these parasites through attachment and are believed to contribute to host cell type selective binding to sialylated glycoconjugates. specificity and hence disease pathology. T. gondii microneme protein 1 (TgMIC1) forms a complex EXPERIMENTAL PROCEDURES with TgMIC4 and TgMIC6 (11, 12) and binds to sialylated gly- Generation of T. gondii Mutant Parasite Strains—All T. gon- coconjugates on the host cell surface (1). Previous studies based dii strains were grown in human foreskin fibroblasts (HFF) or Downloaded from on gene disruption have established a critical role for the com- Vero cells in Dulbecco’s modified Eagle’s medium (Invitrogen), plex in host cell invasion in tissue culture and its contribution to 10% fetal calf serum, 2 mM glutamine, 25 ␮g/ml gentamicin. virulence in vivo (13). The N-terminal region of TgMIC1 inter- The RH strain is referred to as “wild type.” acts with TgMIC4, a protein comprising six “apple” domains The mic1ko parasite strain, generated previously (12),

that has been shown to bind to host cells in the presence of was stably complemented with linearized plasmids www.jbc.org TgMIC1 (11). TgMIC6 contains three epidermal growth factor pM2MIC1myc, pM2MIC1T220Amyc, pM2MIC1T126A,T220Amyc, (EGF)-like domains and is a type I membrane protein, which and pROP1mycMIC1-GLD coding for the expression of serves as an escorter and anchors the TgMIC1-MIC4-MIC6 TgMIC1myc, TgMIC1T220Amyc, TgMIC1TTAAmyc, and

(TgMIC1-4-6) complex to the parasite surface during invasion mycTgMIC1-GLD (encompassing amino acids 299–456 of at SMAC Consortium - Geneve, on May 29, 2010 (12). The first EGF-like domain (TgMIC6-EGF1) is cleaved off TgMIC1), respectively, using a standard electroporation during secretory transport of the complex, probably in a post- transfection protocol with restriction enzyme-mediated Golgi compartment (14). Each of the remaining two EGF-like insertion. For selection, plasmids were cotransfected with domains is able to recruit one molecule of TgMIC1 via interac- p2854_DHFR or pTUB5-CAT (carrying the pyrimethamine tion with its C-terminal galectin-like domain (for a schematic and the chloramphenicol resistance marker, respectively) see Fig. 1A) (12, 15, 16). Correct trafficking of the complex to at a 5:1 ratio. pM2MIC1T220Amyc was generated from the micronemes depends not only on a sorting determinant in pM2MIC1myc using the QuikChange kit (Stratagene) and the C-terminal tail of TgMIC6 but also on the interaction primers TgMIC1-20_1731 and TgMIC1-21_1732. between the galectin-like domain of TgMIC1 (TgMIC1-GLD) pM2MIC1T126A,T220Amyc was obtained by replacing the with the third membrane-proximal EGF-like domain of fragment between restriction sites NdeI and EcoNI in TgMIC6 (TgMIC6-EGF3). This interaction is crucial for trans- pM2MIC1myc with the equivalent region in plasmid port of the entire complex through the early secretory pathway pPICZ␣-TgMIC1NTT126A,T220A (see Ref. 1). To obtain a as it assists proper folding of TgMIC6-EGF3, providing a qual- clonal line, parasites were cloned at least twice by limiting ity control checkpoint (12, 15, 17). dilution from the drug-resistant pool of parasites obtained Several studies have shown that recognition of carbohydrate from transfection and selection. structures on the host cell surface is critical for efficient inva- Cloning of T. gondii MCPs—Restriction enzymes were pur- sion by T. gondii (18–20). We have recently demonstrated that chased from New England Biolabs. All primers are listed in the N-terminal region of TgMIC1 contains two copies of a supplemental Table 2. Accession numbers are listed in Table 1. novel MAR domain and that this region termed TgMIC1- TgMIC13, TgMCP3, and TgMCP4 were amplified by PCR MARR binds specifically to sialylated oligosaccharides as from a T. gondii tachyzoite cDNA pool. TgMIC13 with or with- shown by cell binding assays and carbohydrate microarray out the signal peptide was amplified using primer pairs MIC1/ analyses (1). Also, we observed a 90% reduction of invasion 2–5/1047 and MIC1/2–6/1048 or MIC1/2–1/1147 and MIC1/ efficiency when N-acetylneuraminic acid (NANA) was used as 2–2/1148, respectively. TgMCP3 was cloned using primers a competitor or when host cells were treated with neuramini- MIC1/3-7_1981 and MIC1/3-7_1982. To obtain the coding dase (1). This suggested that Sia is a major determinant of host sequence for TgMCP4 with or without the signal peptide, cell invasion by T. gondii and that the effect observed in the primer pairs TgMIC1/4-7_1983 and TgMIC1/4-7_1984 as well invasion assays may be attributed, at least in part, to inhibition as TgMIC1/4-3_1847 and TgMIC1/4-4_1848 were used for of the interaction between TgMIC1 and its host cell receptor(s). amplification, respectively. The fragments were cloned into Here, we compare the invasion efficiency of several T. gondii pGEM and sequenced. The sequence encoding full-length knock-out strains and complemented mutants and demon- TgMIC13 was digested with EcoRI and SbfI and subcloned into strate that the TgMIC1-Sia interaction is indeed important for pTUB8TgMLCTy_HX between EcoRI and NsiI restriction efficient host cell invasion. We also show that the TgMIC1-4-6 sites. 1 JANUARY 15, 2010•VOLUME 285•NUMBER 3 JOURNAL OF BIOLOGICAL CHEMISTRY 2065 Supplemental Material can be found at: http://www.jbc.org/content/suppl/2009/11/09/M109.060988.DC1.html Family of MAR Domain-containing Proteins in Apicomplexa Downloaded from www.jbc.org at SMAC Consortium - Geneve, on May 29, 2010

FIGURE 1. Cooperative function for the Sia-binding TgMIC1-MAR domains and TgMIC4 in host cell invasion by T. gondii. A, schematic summarizing the domain organization of the components of the TgMIC1-4-6 complex in the WT and mic1ko/MIC1myc complemented strains. B, Western blot analysis of parasite lines expressing TgMIC1 mutant proteins on the mic1ko background. C–E, TgMIC1 mutant proteins expressed on the mic1ko background were assessed for their ability to substitute for TgMIC1 and target the components of the TgMIC1-4-6 complex to the micronemes. IFA was performed on intracellular parasites multiplying in their vacuole. TgAMA-1 is used as a micronemal marker independent of the TgMIC1-4-6 complex. Scale bars,1␮m. A schematic summarizes the association/dissociation of the components of the TgMIC1-4-6 complex in the different strains. An asterisk indicates a Thr to Ala substitution in the Sia-binding site of the MAR domain. F, comparison of host cell invasion efficiency by the various T. gondii mutant strains using an RH-2YFP strain as internal standard for parasite fitness. Error bars, standard deviation.

pTUB8TgMLCTy_HX was cut with EcoRI, blunt-ended with Full-length TgMCP4 was cloned into pTUB8TgMLCTy_HX endonuclease, and cut again with NsiI prior to insertion of full- using EcoRI and NsiI restriction sites. The sequence corre- length TgMCP3 coding sequence cut from pGEM-TgMCP3 sponding to TgMCP4 without SP was subcloned into with EcoRV and PstI. TgMCP3 without signal peptide (SP) was pROP1mycMIC1-GLD using NsiI and PacI restriction sites. cloned into pROP1mycMIC1-GLD in between NsiI and PacI Constructs were used in transient transfection experiments (blunt end cloning) resulting in pROP1mycTgMCP3(3430). in either RH⌬Hx, mic1ko,ormic6ko parasites. Stable parasite 1 2066 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285•NUMBER 3•JANUARY 15, 2010 Supplemental Material can be found at: http://www.jbc.org/content/suppl/2009/11/09/M109.060988.DC1.html Family of MAR Domain-containing Proteins in Apicomplexa lines were derived from transfection of the pTUB8TgMLCTy_ Invasion Assays—Comparison of different T. gondii strains HX-based vectors carrying the mycophenolic acid/xanthine for invasion efficiency was done using an RH-2YFP strain (21) resistance marker into the RH⌬Hx strain. as internal standard for parasite fitness. The details were Expression and Purification of MCPs in P. pastoris—Pichia described previously (16). Total number of parasite vacuoles pastoris transformation and expression were performed using and RH-2YFP parasite vacuoles were counted on 20 micro- pPICZ␣-based plasmids according to the Pichia expression kit scopic fields on each IFA slide with a minimum of 440 vacuoles protocols (Invitrogen). Transformation of the supplied host in total per slide. Only vacuoles containing at least two parasites strain GS115 was performed by electroporation following lin- were counted to be sure not to count extracellular parasites that earization of the vector with PmeI, SacI, or DraI. To inhibit resisted washing. Each experiment was repeated at least three glycosylation, expression was occasionally carried out in the times. Statistical analysis was performed using PRISM. presence of 10 ␮g/ml tunicamycin. Recombinant TgMIC13 Microarray Analyses of Recombinant MIC Proteins—The sia- (amino acids 23–468) was either used directly in the form of lyloligosaccharide microarrays were generated with 88 lipid- culture supernatant or purified on nickel-nitrilotriacetic acid linked oligosaccharide probes (supplemental Table 1), which

(Qiagen) and concentrated into 20 mM NaH2PO4/Na2HPO4, were arrayed in duplicate on nitrocellulose-coated glass slides pH 7.3. TgMCP3 (amino acids 54–565) and TgMCP4 (amino at 2 and 5 fmol per spot using a noncontact instrument (22). acids 526–1016) were not secreted, but soluble ␣-factor fusion Analysis of carbohydrate binding of the recombinant His- Downloaded from protein was obtained by lysis of cells in 50 mM NaH2PO4/ tagged MIC proteins was performed essentially as described (1). Na2HPO4, pH 7.4, 5% glycerol, 1 mM phenylmethylsulfonyl In brief, each His-tagged MIC protein was precomplexed with fluoride. mouse monoclonal anti-polyhistidine and biotinylated goat Preparation of Excreted Secreted Antigen (ESA) Fraction—T. anti-mouse IgG antibodies (Sigma) in a ratio of 1:2.5:2.5 (by gondii tachyzoites freshly lysed from their host cells were har- weight) and overlaid onto the arrays at 40 ␮g/ml for TgMIC1- ϫ ␮

vested by centrifugation at 240 g for 10 min and washed twice MARR and TgMIC13 and 20 g/ml for NcMIC1-MARR. Bind- www.jbc.org in IM (Dulbecco’s modified Eagle’s medium, 3% fetal bovine ing was detected using Alexa 647 fluor-conjugated streptavidin serum, 10 mM HEPES) prewarmed to 37 °C. A pellet of 2.0– (Molecular Probes). Microarray data analysis and presentation 4.88 ϫ 108 parasites was resuspended in 1 ml of IM, and an were carried out using dedicated software.7 The binding to aliquot of 50 ␮l was taken as reference standard to allow esti- oligosaccharide probes was dose-related, and results of 5 fmol at SMAC Consortium - Geneve, on May 29, 2010 mation of the degree of secretion, and microneme secretion per spot are shown. was stimulated by adding 10 ␮l of 100% EtOH to the remaining 950 ␮l. The sample was incubated for 10 min at room temper- RESULTS ature, then 40 min at 37 °C, and finally cooled down to 0 °C in an TgMIC1 Is an Important Player in Sia-dependent Host Cell ice-water bath for 5 min. Parasites were pelleted at 1000 ϫ g, Invasion by T. gondii—A previously generated TgMIC1 knock- 4 °C for 5 min. The supernatant was transferred to a new tube out strain (mic1ko) showed a 50% reduction in invasion effi- and centrifuged again for 5 min, 4 °C at 2000 ϫ g. The superna- ciency compared with the wild-type strain (13). As the tant (ESA) was collected and stored at Ϫ80 °C. TgMIC1-4-6 complex is disrupted in this strain, and the trans- Cell Binding Assays—These were performed as described port of TgMIC4 and TgMIC6 to the micronemes is ablated previously (1). Briefly, confluent monolayers of HFF cells were resulting in their retention in the early secretory pathway (12), blocked for1hat4°Cwith 1% bovine serum albumin in cold the contribution of the individual components to host cell

PBS, 1 mM CaCl2, 0.5 mM MgCl2 (CM-PBS). Excess bovine invasion has remained unresolved (for a schematic of the com- serum albumin was removed by two 5-min washes with ice- plex see Fig. 1A). To address this question, we complemented cold CM-PBS. The proteins to be assayed were then added in the mic1ko strain with three different TgMIC1 mutant con- the form of P. pastoris culture supernatant (ϳ0.25 ␮gin250␮l) structs and then examined whether expression of these pro- or parasite ESA, together or not with different concentrations teins is able to improve or restore invasion efficiency. A first of competitors (N-acetylneuraminic acid and glucuronic acid parasite mutant line named mic1ko/mycMIC1-GLD were purchased from Sigma; stock solutions in PBS were expresses the TgMIC1 galectin-like domain (TgMIC1-GLD) adjusted to pH 7.0), and diluted in cold CM-PBS to a total vol- on the mic1ko background. A second parasite line called ume of 500 ␮l. After incubation at 4 °C for 1 h, the supernatant mic1ko/MIC1TTAAmyc expresses full-length TgMIC1 carrying was removed, and the cells were washed four times for 5 min Thr to Ala substitutions at two positions (126 and 220) in the with ice-cold CM-PBS. The cell-bound fraction was collected binding site of each of the two MAR domains, previously shown by the direct addition of 50 ␮lof1ϫ SDS-PAGE loading buffer to be critical for the host cell binding activity of the TgMIC1- with 0.1 M dithiothreitol. In some cases, prior to blocking, HFF MAR region (TgMIC1-MARR) (1). A third complemented line cells were pretreated with 66 milliunits/ml ␣2–3, -6, -8 Vibrio called mic1ko/MIC1T220Amyc expresses TgMIC1 containing cholerae neuraminidase (Roche Applied Science) in RPMI 1640 just a single T220A substitution that also abolishes the host cell medium, 25 mM HEPES, L-glutamine (Invitrogen) for1hat binding activity of the protein (1). As a control, the mic1ko 37 °C in a total reaction volume of 1 ml. strain was complemented with full-length wild-type TgMIC1 CHO-lec2 cells were purchased from the ATCC. All CHO carrying a Myc tag epitope at the C terminus (parasite line cells were cultured according to ATCC indications. C6 rat gli- mic1ko/MIC1myc). Expression of the respective mutant pro- oma cells were propagated in RPMI 1640 medium (Invitrogen), 5% fetal calf serum, 2 mML-glutamine, 25 ␮g/ml gentamicin. 7 M. S. Stoll, unpublished data. 1 JANUARY 15, 2010•VOLUME 285•NUMBER 3 JOURNAL OF BIOLOGICAL CHEMISTRY 2067 Supplemental Material can be found at: http://www.jbc.org/content/suppl/2009/11/09/M109.060988.DC1.html Family of MAR Domain-containing Proteins in Apicomplexa teins was confirmed by Western blot (Fig. 1B). In addition, the previously generated mic4ko line (12) was included in the analyses. Fig. 1F shows a comparison of invasion efficiency of the parental wild-type and the mic1ko lines and confirms the 50% reduction in invasion phenotype previously reported for the mic1ko (13). Given that TgMIC4 and TgMIC6 are retained in the early secretory pathway in the mic1ko strain, assessment of the subcellular localization of the TgMIC1 mutant proteins, as well as of TgMIC4 and TgMIC6, in the complemented mic1ko parasite lines was important for interpretation of the invasion assay results. Expression of TgMIC1myc in the mic1ko strain rescued the targeting of TgMIC4 and TgMIC6 to the micronemes supplemental Fig. S1 (12). Invasion efficiency in this strain was FIGURE 2. Cell invasion assays using the T. gondii mic1ko strain demonstrate the existence of at least one more Sia-specific parasite lectin. restored to a level rather higher than the parental wild-type line A, invasion by mic1ko parasites in the absence and presence of 10 and 20 mM (Fig. 1F), showing that a complex composed of endogenous free NANA or galactose. B, assay comparing invasion of mic1ko parasites into TgMIC4 and TgMIC6 together with the epitope-tagged host cells (HFFs) pretreated or not with neuraminidase. Error bars, standard Downloaded from deviation. Note that, compared with the parental strain, the mic1ko shows a TgMIC1myc is functional in invasion. In agreement with pre- 50% reduced invasion phenotype (Fig. 1F), but its invasion efficiency was set vious studies (15), expression of TgMIC1-GLD in the mic1ko to 100% here. strain brought TgMIC6 to the micronemes but TgMIC4 remained in the endoplasmic reticulum (Fig. 1C). In the inva- function for TgMIC6 and TgMIC1-GLD. The invasion experi-

sion assay (Fig. 1F), when compared with mic1ko, mic1ko/ www.jbc.org ments establish that TgMIC1 function and its contribution to mycMIC1-GLD showed no rescue of phenotype, demonstrat- efficient invasion resides in TgMIC1-MARR binding to sialy- ing that the TgMIC6-TgMIC1-GLD mutant complex does not lated glycoconjugates. Furthermore, our data support the idea contribute to invasion, likely due to the absence of adhesive that TgMIC4 is an adhesin with an important function in host domains. at SMAC Consortium - Geneve, on May 29, 2010 In the mic1ko/MIC1T220Amyc line, TgMIC1T220A was tar- cell invasion. geted to the micronemes, restoring proper trafficking of T. gondii Possesses More Than One Sia-binding Factor TgMIC4 and TgMIC6 (Fig. 1D). This mutant invaded signifi- Involved in Host Cell Invasion—To address the question of cantly better than mic1ko/mycMIC1-GLD (Fig. 1F) despite the whether other parasite lectins bind to host sialylated glycocon- fact that the mutation in TgMIC1T220A abrogates the adhesive jugates during invasion, we tested the effect of free NANA on properties of the protein (1). This suggested that the invasion invasion by the mic1ko strain. Interestingly, this assay showed enhancement observed upon expression of this protein might that host cell invasion by the mic1ko strain was considerably be solely a result of its capacity to recruit TgMIC4 to the mem- impaired in the presence of free NANA (Fig. 2A), indicating brane-bound complex and/or residual weak binding of the first that TgMIC1 is not the only Sia-binding parasite lectin contrib- TgMIC1 MAR domain not carrying a Thr to Ala substitution. uting to invasion by T. gondii tachyzoites. This result was cor- Invasion efficiency of this line was similar to the wild type but roborated by performing an invasion assay with neuramini- lower than the control line mic1ko/MIC1myc, underlining the dase-treated host cells that resulted in a substantial reduction of importance of the adhesive properties of TgMIC1. In the invasion by the mic1ko strain (Fig. 2B). mic1ko/MIC1TTAAmyc line, TgMIC1TTAA was found mainly in Identification of a Novel Family of MCPs in Coccidia—In the the early secretory pathway (Fig. 1E) probably due to incorrect light of the above observations, we hypothesized that T. gondii folding of this mutant protein, precluding corroboration of the has at least one additional Sia-binding parasite lectin. A survey above results with the single T220A mutant. Therefore, it was of the T. gondii genome sequence revealed that it encodes a not surprising that there was no improvement of invasion effi- family of four MCPs, herein named TgMIC1, TgMCP2, ciency in this line compared with the mic1ko (Fig. 1F). TgMCP3, and TgMCP4 (see Table 1, Fig. 3A, and supplemental In the mic4ko strain, the partial TgMIC1-6 complex was Fig. S2). The presence in all cases of a predicted N-terminal SP delivered to the micronemes (12). This strain invaded more suggests that these proteins are delivered to the secretory efficiently than mic1ko and mic1ko/mycMIC1-GLD (t tests, p Ͻ pathway. In contrast to TgMIC1, which includes two MAR 0.05, respectively), confirming the critical role of TgMIC1- domains (TgMIC1-MARR) followed by a galectin-like MARR in invasion (Fig. 1F). In addition the mic4ko strain did domain (TgMIC1-GLD), the other three sequences contain not invade as well as the wild type (t test, p Ͻ 0.05), suggesting four consecutive MAR domains but lack a galectin-like a role for TgMIC4 in invasion in agreement with our observa- domain. In addition, TgMCP4 possesses a novel N-terminal tions with the mic1ko/MIC1T220Amyc line or, alternatively, that repeat region (16 repeats, 17–22 amino acids per repeat), the presence of TgMIC4 impacts on the proper function of the with the number of repeats varying between different strains MAR domains. of T. gondii. Examination of the EST data set indicate that all Collectively, the characterization of these mutants suggests a these MCP genes are transcribed both in tachyzoite and bra- cooperative function for TgMIC1-MARR and TgMIC4 in dyzoite stages, and a proteomic study (23) showed the receptor binding and confirms the absence of any adhesive expression of TgMCP2 in tachyzoites. 1 2068 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285•NUMBER 3•JANUARY 15, 2010 Supplemental Material can be found at: http://www.jbc.org/content/suppl/2009/11/09/M109.060988.DC1.html Family of MAR Domain-containing Proteins in Apicomplexa

Our earlier studies have established that TgMIC1-MARR has logues in N. caninum (Table 1 and supplemental Fig. S2). the potential to bind to two molecules of Sia through its two NcMIC1, the homologue of TgMIC1, has been described pre- binding sites, one in each MAR domain, characterized by the viously (24), whereas TgMCP2 is highly similar to genemodel presence of conserved His and Thr residues. In both binding NCLIV_026810 (73% identical and 86% similar), and genes cod- sites, the Thr residue (Thr-126 and Thr-220, respectively) ing for proteins highly similar to TgMCP3 (67% identical and makes principal contacts to the Sia moiety (1). Sequence com- 75% similar) and TgMCP4 (73% identical and 80% similar in the parison reveals that these important Thr residues are not pres- MAR region) are present on N. caninum chromosome 1b (sup- ent in either TgMCP3 or TgMCP4 but are conserved in three of plemental Figs. S3 and S4 show full amino acid sequence align- the four MAR domains in TgMCP2 (supplemental Fig. S2). ments). Like TgMCP4, NcMCP4 displays an N-terminal region Consequently, we hypothesized that TgMCP2 may be another with short repeats but including only 8 units. Interestingly, the Sia-binding parasite lectin. His and Thr signature residues of the MAR Sia-binding site are Genome-wide BLAST searches of related apicomplexan par- conserved when comparing the N. caninum and T. gondii asites allowed identification of a complete set of four homo- homologues, suggesting that these could be functionally equivalent and hence orthologues (supplemental Fig. 2). This view is TABLE 1 supported by a phylogenetic analysis comparing the first two Proposed orthologous relationships and GenBankTM (and predicted MAR domains of each member of the family (Fig. 3B). toxoDB/geneDB/EuPathDB) accession numbers for various MCPs Downloaded from NI means not identified. In this analysis the putative orthologues are indeed most closely T. gondii N. caninum S. neurona E. tenella related. TgMCP3, TgMCP4, NcMCP3, and NcMCP4 lacking MIC1 MIC1 NI NI the critical Thr residues form a separate cluster, consistent with CAA96466 AAL37729 an expected functional divergence. (80.m00012) (NCLIV_043270) MIC13/MCP2 MCP2 NI NI Unfortunately, there were insufficient genomic data avail- ABY81128 (NCLIV_026810) able to establish whether a similar gene family exists in www.jbc.org (55.m04865) another genus of coccidians, Sarcocystis spp., but EST data MCP3 MCP3 NI NI CAJ20583 (NCLIV_003260) (SnEST4a79g11.y1, SnEST4a34d02.y1, SnESTbab01e09.y1, (25.m00212) SnEST4a69h07.y1, and SnESTbab30h02.y1) indicate the pres- at SMAC Consortium - Geneve, on May 29, 2010 MCP4 MCP4 NI NI ence of several MAR-containing proteins, one of which (termed GQ290474 (NCLIV_003250) (25.m01822) SnMCP5) has a homologue in N. caninum (NCLIV_066750 on Not present MCP5 MCP5 NI chromosome12, named NcMCP5 hereafter, see Fig. 3A and (NCLIV_066750) EST4a79g11.y1 EST4a34d02.y1 supplemental Fig. S2 and Fig. S4). Despite a high level of syn- Not present MCP6 NI NI teny between the two parasites, the gene encoding NcMCP5 is (NCLIV_054450) absent at the corresponding locus in T. gondii. Two additional Not present MCP7 NI NI loci coding for putative MCPs were found in the N. caninum (NCLIV_054425) genome (NCLIV_054450 and NCLIV_054425, named Not present Not present NI MIC3 ACJ11219 NcMCP6 and NcMCP7, respectively), both composed of an SP Not present Not present NI MCP2 and two MAR domains (Fig. 3A and supplemental Fig. S2 and Contig 29262 Fig. S5). In E. tenella, the previously described EtMIC3 contains

FIGURE 3. Family of MAR domain-containing proteins in apicomplexans. A, schematic of the domain organization of various MCPs. MAR domain type I (light blue), MAR domain type II (light green), MAR domain type II extension the “␤-finger” (green), galectin-like domain (orange), and region of short repeats (blue stripes) are shown. The presence of a Thr in a MAR domain in an equivalent position to those critical for Sia binding in TgMIC1-MARR is indicated by a black T. The fourth MAR domain in TgMCP4 contains this Thr, but the sequence context does not fit with it being indicative of a potential Sia-binding site; therefore, the T is in parentheses. B, phylogenetic relationship of MCPs from T. gondii, N. caninum, and E. tenella. Because the domain structure of the different proteins varies, only the sequence corresponding to the first two predicted MAR domains of each protein was used for the analysis. All bootstrap values are Ͼ80. Sequences were aligned in ClustalX, and alignment positions containing gaps in Ͼ50% of the sequences were excluded from phylogenetic analyses. Phylogenetic analyses were carried out using POWER (neighbor-joining distance method, bootstrapping with 1000 replicates). Phylogenetic trees were generated using TREEVIEW. 1 JANUARY 15, 2010•VOLUME 285•NUMBER 3 JOURNAL OF BIOLOGICAL CHEMISTRY 2069 Supplemental Material can be found at: http://www.jbc.org/content/suppl/2009/11/09/M109.060988.DC1.html Family of MAR Domain-containing Proteins in Apicomplexa

adopts a ␤-hairpin (1). Curiously, in this regard all the MAR domains in EtMIC3 and in all other predicted MCPs in Eimeria resemble the first of the two tandemly arranged MAR domains, referred to as type I (Fig. 3A and supplemental Fig. S2). This suggests that these proteins have evolved differently in Eimeria compared with the MCPs found in Toxo- plasma, Neospora, and Sarcocystis. Therefore, despite the fact that some of the MCPs in Eimeria display the binding site His-Thr or a similar motif, these proteins might possess different properties and functions. Downloaded from TgMIC13, a Novel Sia-binding Lectin—A parasite line expressing a C-terminal ty-epitope-tagged copy of TgMCP2 was generated.

TgMCP2ty was co-localized with www.jbc.org TgMIC4 to the micronemes by IFA and was renamed TgMIC13 according to the current nomenclature

status. IFA on wild-type parasites at SMAC Consortium - Geneve, on May 29, 2010 using antibodies raised against recombinant TgMIC13 expressed in P. pastoris confirmed localization to the micronemes, although only FIGURE 4. Characterization of TgMIC13 (TgMCP2) in T. gondii. A, IFA shows co-localization of TgMIC13 with microneme proteins in wild-type (wt)(top, confocal images) and in WT/TgMIC13ty parasites (bottom). B, West- partial co-localization with TgMIC3 ern blot analysis of endogenous and epitope-tagged TgMIC13 in wild-type and WT/TgMIC13ty parasites. An was observed (Fig. 4A). Analysis of extract of HFF cells was also loaded as a control. C, assessment of TgMIC13 solubility by fractionation (top). Cell lysates from wild type and binding assays using ESA from WT/TgMIC13ty parasites (bottom). I, input; W, last of four washes; CB, cell-bound fraction. D, transient transfection of TgMIC13ty into the mic1ko and the mic6ko show correct localization of WT/TgMIC13ty parasites by West- TgMIC13ty to the micronemes. Scale bars, 1 ␮m. ern blot indicated that TgMIC13 migrates on SDS-PAGE as a single seven MAR domains, in which repeats 3–5 are identical (25). protein species with an apparent molecular mass (56 kDa) EtMCP2, lying downstream of EtMIC3 on contig 29262, was somewhat higher than expected (49.1 kDa calculated from the annotated in silico by comparison with EST data from sporu- amino acid sequence) (Fig. 4B). TgMIC13ty migrates slightly lated oocyst and sporozoite stages. EtMCP2 is composed of an faster than endogenous TgMIC13. A similar discrepancy SP and a single MAR domain (Fig. 3A and supplemental Fig. S2 between apparent molecular mass on SDS-PAGE and the and Fig. S5). In addition, this parasite possesses a number of expected mass was observed for recombinant TgMIC13 hypothetical MCPs (two gene models, respectively, on contigs expressed in P. pastoris (Fig. 5A, 59 kDa on SDS-PAGE versus 29652 and 14843). EST data support expression of at least one 51.9 kDa expected). No glycosylation sites are predicted for of them in first generation merozoites. Our BLAST searches TgMIC13, and mass spectrometric analysis of recombinant failed to identify any MCPs in Plasmodium, Theileria,orCryp- TgMIC13 confirmed that its true mass is close to that predicted tosporidium. Accession numbers and proposed orthologous (supplemental Fig. S7). Therefore, we conclude that the aber- relationships for all MCPs are depicted in Table 1. rant migration behavior is related to intrinsic properties of the A comparison of MAR domains from Toxoplasma, Neos- protein. The solubility profile of TgMIC13 is identical to pora, and Sarcocystis highlights differences in disulfide bond TgMIC4 and is consistent with it being a soluble protein within patterns between two tandemly arranged MAR domains in a the parasite organelles (Fig. 4C, upper panel). To assess whether given MCP (supplemental Fig. S2). As in the TgMIC1-MARR TgMIC13 displays cell binding activity, ESA prepared from prototype, the beginning of the second domain (referred to as WT/TgMIC13ty parasites was tested in a cell binding assay; the type II) lacks a stretch of amino acids containing two cysteine protein was found to bind to the cell surface (Fig. 4C, lower panel). residues shown to participate in the formation of a disulfide Furthermore, transient expression of TgMIC13ty in mic1ko and bond in the TgMIC1-MARR crystal structure, but it possesses mic6ko recipient strains indicated that TgMIC13 traffics to the an additional C-terminal extension (with the notable exception micronemes independently of the TgMIC1-4-6 complex and does of the second MAR domain in TgMCP2 and NcMCP2) that not associate with its components (Fig. 4D). These results establish 1 2070 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285•NUMBER 3•JANUARY 15, 2010 Supplemental Material can be found at: http://www.jbc.org/content/suppl/2009/11/09/M109.060988.DC1.html Family of MAR Domain-containing Proteins in Apicomplexa

tions of a control acidic monosaccharide, glucuronic acid (Fig. 5C). However, the binding properties of TgMIC13 differed from TgMIC1 in the following ways: 1) at equivalent concentrations, more TgMIC13 appeared to bind to HFFs) (Fig. 5A); 2) compared with TgMIC1, higher concentrations of NANA were required to fully inhibit receptor binding of TgMIC13 (10 mM for TgMIC1 (1) versus 50 mM for TgMIC13) (Fig. 5C); 3) only extensive treatment of cells with neuraminidase completely abolished the binding of TgMIC13 (data not shown); and 4) TgMIC1 did not bind to CHO lec2 cells that have greatly reduced surface expression of Sia (26), whereas TgMIC13 can weakly bind to them (Fig. 5D). No other differences were observed in cell binding assays with CHO mutants deficient in glycosaminoglycan synthesis (CHO-pgsA and CHO-pgsB) or with CHO cells lacking two glycosyltransferase activities (CHO-pgsD) (Fig. 5D) (27). Taken together, these experiments indicated that TgMIC13 is a Sia-binding lectin with the poten- Downloaded from tial to act during host cell invasion independent of the TgMIC1- 4-6 complex. Furthermore, our observations suggested that the cell-binding characteristics of TgMIC13 are different from TgMIC1, possibly binding to different sialylated receptors.

Binding Properties of Other T. gondii Proteins of the MAR www.jbc.org Domain Family—Given that the MAR domains on TgMIC1 and TgMIC13 are involved in carbohydrate recognition, we postulated that TgMCP3 and TgMCP4 could also be functional

lectins. In cell binding assays using ESA prepared from trans- at SMAC Consortium - Geneve, on May 29, 2010 genic parasites expressing TgMCP3ty and TgMCP4ty, neither of the two epitope-tagged proteins showed detectable binding activity (data not shown). Full-length TgMCP3 as well as the MAR region of TgMCP4 (TgMCP4-MARR) encompassing all four MAR domains without the N-terminal repeat region were expressed in both P. pastoris and Escherichia coli. No binding to HFFs could be observed with the recombinant proteins in cell- binding assays (Fig. 5A and data not shown). The lack of binding to Sia-containing moieties would be consistent with the absence of the critical Thr residues in the MAR domains of FIGURE 5. Binding characteristics of recombinant TgMIC13. A, cell binding TgMCP3 and TgMCP4 (supplemental Fig. S2). Homology assays comparing recombinant TgMIC1myc, TgMIC13myc, and ␣-factor- TgMCP3myc fusion protein produced in P. pastoris; ϩtu/Ϫtu, protein modeling and sequence alignments suggest that although expressed in the presence/absence of tunicamycin. Top panel, Western blot TgMCP3 and TgMCP4 adopt the MAR fold, they present a indicating the relative concentration of the proteins in supernatants used for largely hydrophobic surface in the equivalent position to the the assay. Middle panel, cell-bound fractions (CB) probed for bound protein. Bottom panel, control for use of equivalent amounts of cell material in each hydrophilic carbohydrate-binding site in TgMIC1 and experiment. B, pretreatment of cells with neuraminidase abolishes binding therefore would not be expected to bind sugars (data not by TgMIC1 and TgMIC13. S, supernatant; W, last of four washes; CB, cell-bound fraction; c, background control. C, binding of TgMIC13 is strongly reduced in shown). cell binding assays by competition with free NANA but not glucuronic acid. Carbohydrate Microarray Analyses Reveal Differing Binding D, binding of TgMIC1 and TgMIC13 to various CHO cell lines (K1, WT strain; 2, Characteristics for TgMIC13, TgMIC1, and NcMIC1—To lec2 mutant with strong reduction in Sia surface expression; A and B, pgsA745 and pgsB618 mutants deficient in glycosaminoglycan synthesis; D, pgsD677 examine the binding specificities of TgMIC13, we performed mutant deficient in two glycosyltransferase activities). Only the cell-bound cell-independent carbohydrate microarray studies. Preliminary fractions of the assay are shown. screening analyses with more than 300 lipid-linked oligosaccharide probes (1, 28) showed that recombinant TgMIC13 that expression of TgMIC13 cannot functionally rescue the expressed in P. pastoris bound exclusively to sialylated oligosac- mic1ko, strongly suggesting that TgMIC13 belongs to a distinct charide probes (supplemental Fig. S6). This is in accord with complex. results of the cell binding assays and shows that Sia is a require- The cell binding activity of TgMIC13 was reproduced with ment for TgMIC13 binding. recombinant protein expressed in P. pastoris (Fig. 5A). Similar Details of the binding specificity of TgMIC13 were compared to TgMIC1, the binding of TgMIC13 was abolished after pre- with those of TgMIC1 and NcMIC1 using an array of 88 oligo- treatment of cells with neuraminidase (Fig. 5B). Binding of saccharide probes. These included 82 diverse sialylated oligo- TgMIC13 to host cell receptor(s) could be competed out with saccharide probes with different sialyl linkages and backbone free NANA, but it was not affected by increasing concentra- sequences (Fig. 6; supplemental Table S1); 6 neutral oligosac- 1 JANUARY 15, 2010•VOLUME 285•NUMBER 3 JOURNAL OF BIOLOGICAL CHEMISTRY 2071 Supplemental Material can be found at: http://www.jbc.org/content/suppl/2009/11/09/M109.060988.DC1.html Family of MAR Domain-containing Proteins in Apicomplexa Downloaded from www.jbc.org at SMAC Consortium - Geneve, on May 29, 2010

FIGURE 6. Carbohydrate microarray analyses of recombinant TgMIC1-MARR expressed in E. coli (A), TgMIC13 expressed in P. pastoris (B), and NcMIC1- MARR expressed in E. coli (C) using 88 lipid-linked oligosaccharide probes. Numerical scores of the binding signals are means of duplicate spots at 5 fmol/spot (with error bars). The complete list of probes and their sequences and binding scores are in supplemental Table 1. The binding signal for probe 88 was saturated and could not be accurately quantified (asterisk in B). charide probes served as negative controls. In these experi- NcMIC1. The three proteins bound to various sialylated but not ments we used full-length TgMIC13 expressed in P. pastoris,as to neutral oligosaccharides in the microarrays. Binding was pre- well as TgMIC1-MARR (amino acids 17–262) and NcMIC1- dominantly observed to ␣2–3-linked sialyl probes with little or no MARR (amino acids 17–259), both expressed in E. coli. binding to the ␣2–6-linked sialyl probes. This is in agreement with TgMIC1 lacking its C-terminal galectin-like domain is known a recent study revealing the importance of a glutamic acid residue to reflect the binding specificities of the full-length protein (1, (Glu-222) in TgMIC1 for this preference (29), which is conserved 16), and we assumed that this is also the case for its homologue in TgMIC13 and NcMIC1 (supplemental Fig. S2). 1 2072 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285•NUMBER 3•JANUARY 15, 2010 Supplemental Material can be found at: http://www.jbc.org/content/suppl/2009/11/09/M109.060988.DC1.html Family of MAR Domain-containing Proteins in Apicomplexa

Interestingly, for each protein, a distinct binding profile was which might induce electrostatic repulsion of the negatively observed. Among the broad range of sialylated probes bound by charged carboxylate group and hinder any preference for 2–9- TgMIC1-MARR, the preferred ligands are ␣2–3-linked sialyl- disialyl sugars. In TgMIC13, Glu-221 and -223 are replaced by N-acetyllactosamine (3ЈSiaLacNAc) sequences with or without positively charged Arg and Lys residues, which would not only fucosylation/sulfation on the N-acetylglucosamine residue reduce any electrostatic repulsion but provide opportunities for (highlighted probes in Fig. 6A). This is in overall agreement the formation of specific charge-charge interaction with the with previous findings (1). Although the oligosaccharide second Sia. sequences of probes 18, 20, 21, and 22 are short tri- or tetrasac- The preference of NcMIC1-MARR for the two sialyl Lex- charides, their recognition demonstrates the effective presen- related probes sulfated in the 6-position of galactose could be tation of these as oxime-linked neoglycolipids on the array sur- explained by the presence of a positively charged Lys (Lys-176) face (30). in NcMIC1-MAR2, which is replaced by an Ala in TgMIC1 (Fig. TgMIC13 bound to a more restricted spectrum of probes 7C). By analogy to the complex of TgMIC1-MARR with Ј Ј compared with TgMIC1-MARR (Fig. 6B). Several 3 SiaLacNAc- 3 SiaLacNAc1–3, Lys-176 of NcMIC1 would be spatially closely based probes were bound, but there was little or no binding to positioned to the 6-position of galactose and could form a spe- sialyl sequences having long chain polyLacNAc or N-glycan cific charge-charge interaction with the negatively charged sul- backbones (probes 38–41) and to ␣2–8-polysialyl sequences fate. Lys-176 in NcMIC1 is not conserved in TgMIC13 but is (probes 76–85). Strikingly, the strongest binding of TgMIC13 present in NcMCP2. Downloaded from was to a disialyllactose probe having ␣2–9 linkage between the two Sia residues (probe 88) (31). The binding signal for this DISCUSSION probe was extremely high (saturated) at the protein concentra- Sia is abundant on glycoconjugates decorating the surface of tion tested. In addition, TgMIC13, but not TgMIC1, bound to a all vertebrate cells and is widely exploited by viruses and bacte- Ј ria for host cell entry. In this study, we have characterized key little studied 4-O-acetylated 3 sialyllactose sequence as in www.jbc.org probe 16. It is interesting that the binding to probe 16 was parasite molecular players involved in Sia-dependent host cell stronger than that to the non-O-acetylated analogue (probe 12). invasion by T. gondii. The salient findings are as follows: first, For NcMIC1-MARR, there was strikingly high binding to two the characterization of TgMIC1-4-6 complex as one important x sialyl Le -related probes (probes 33 and 37, Fig. 6C), which have player in Sia-dependent host cell invasion; second, the reliance at SMAC Consortium - Geneve, on May 29, 2010 in common a sulfate group at the 6-position of the galactose of the parasite on an additional Sia-binding lectin for invasion, residue. In contrast, if the sulfation is on the N-acetylglucos- identified as TgMIC13; and third, the determination of distinct amine residue as in probe 35, binding of NcMIC1-MARR was Sia-dependent binding specificities for three members of a much weaker. NcMIC1-MARR gave stronger binding signals novel family of MCPs conserved in coccidians. In T. gondii, all than TgMIC1 and TgMIC13 with polysialyl sequences. the four MCPs are expressed in tachyzoites. Among them, only TgMIC1-MARR Crystal Structure Provides Explanations for TgMIC1 and TgMIC13 possess the sequence characteristics of the Findings in Microarray Analyses—To better understand the a Sia-binding MAR domain, and only these specifically recog- basis of the binding preference revealed by the microarray stud- nize sialyloligosaccharides. ies, we compared the crystal structure of TgMIC1-MARR in Role of TgMIC13 in Host Cell Invasion by T. gondii—To con- Ј complex with 3 SiaLacNAc1–3 (Protein Data Bank code 3F5A) tribute to host cell invasion, TgMIC13 needs to be targeted (29) to the TgMIC13 MAR domains. Although the first three accurately to the micronemes and then delivered in a timely MAR domains of TgMIC13 possess the Sia-binding signature, fashion to become firmly anchored on the parasite surface. we have assumed that the type II domains (MAR2) make the Because TgMIC13 does not contain a predicted transmem- dominant contribution to Sia recognition as observed for brane domain or glycosylphosphatidylinositol-anchor motif, it TgMIC1-MARR (1); therefore, MAR2 of TgMIC1 and likely belongs to a yet undefined complex. Expression of TgMIC13, in particular, was considered. In TgMIC1-MAR2, TgMIC13 in the mic1ko and mic6ko strains ruled out any inter- Lys-216 and Phe-169 from loops ␤1-␤2 and ␤4-␤5 are posi- action with the TgMIC1-4-6 complex. TgMIC13 does not con- tioned in proximity to the hydroxyl group on C4 of the Sia ring tain a galectin-like domain like TgMIC1, which precludes a (Fig. 7A). In the equivalent MAR domain from TgMIC13, the similar architecture for the putative TgMIC13-containing bulky side chains of Lys-216 and Phe-169 are replaced by complex. Unraveling the mechanism of TgMIC13 sorting to the smaller Ser and Thr residues, respectively, which together with micronemes must await the identification of its escorter pro- a movement in backbone atoms could enable TgMIC13-MAR2 tein, as described for other soluble microneme proteins (12, 14). to accommodate the additional acetyl group at the C4 of Sia. To assess precisely the role of TgMIC13 in invasion, we In a 2–9-linked disialyloligosaccharide, the two Sia moieties repeatedly tried to knock-out the gene in wild-type (RH strain) are separated by three additional bonds compared with the sep- and in mic1ko parasites without success using a knock-out cas- aration between Sia and galactose in 3ЈSiaLacNAc resulting in sette with 5-kb flanking elements homologous to the TgMIC13 increased flexibility and making it difficult to predict which 5Ј- and 3Ј-UTRs.8 In addition, knock-out attempts8 failed in a amino acids will interact. Assuming a similar mode of recogni- ⌬-ku80 strain efficiently amenable to gene disruption by dra- tion for the outermost Sia moiety, one possibility is that the matically enhanced frequency of homologous recombination longer linkage would shift the second Sia to a position directly over the end of helix II in TgMIC1-MAR2 (Fig. 7B). In TgMIC1, three consecutive Glu residues (Glu-221–223) cap this helix, 8 N. Friedrich, J. Santos, and D. Soldati-Favre, unpublished data. 1 JANUARY 15, 2010•VOLUME 285•NUMBER 3 JOURNAL OF BIOLOGICAL CHEMISTRY 2073 Supplemental Material can be found at: http://www.jbc.org/content/suppl/2009/11/09/M109.060988.DC1.html Family of MAR Domain-containing Proteins in Apicomplexa Downloaded from www.jbc.org at SMAC Consortium - Geneve, on May 29, 2010

FIGURE 7. Three orientations of the crystal structure of TgMIC1-MARR in complex with 3 SiaLacNAc1–3 (Protein Data Bank code 3F5A) (29); 3 SiaLacNAc1–3 is shown in an orange stick representation. Key side chains are drawn as stick representations minus attached protons and labeled with sequence position. A and B, amino acid differences between TgMIC1 and TgMIC13 relevant to their binding specificities are labeled in cyan and violet, respectively. C, amino acid differences between TgMIC1 and NcMIC1 relevant to their binding specificities are labeled in cyan and violet, respectively.

(32, 33). These results suggest that this gene might play an There is so far limited information on the occurrence of 2–9- important role for parasite propagation. linked Sia in animals. It has been reported as a component on a Distinct Binding Specificities Might Determine Tissue Tro- murine neuroblastoma cell line (38) and a human embryonal pism of Parasitic Infections—With carbohydrate microarray carcinoma cell line (39). The distribution of ␣2–9-linked disia- technology, we examined details of carbohydrate-binding spec- lyllactose in HFFs, C6 rat glioma, and CHO cells has not been ificities of TgMIC13 in comparison with MAR regions of described; this and its susceptibility to neuraminidases will be TgMIC1 and NcMIC1. The preponderant binding activity of all the subject of future investigation. three proteins was to ␣2–3-linked sialyl probes. However, the A special feature of NcMIC1 as revealed by carbohydrate relative binding intensities to specific sialyl probes were quite microarray analyses is the strong binding to the two sulfated different for the three proteins. TgMIC13 bound to two sialyl- sialyl Lex-related probes. Sulfation was previously reported to oligosaccharide probes, namely the 4-O-acetylated sialyllactose play a role in cell surface binding by NcMIC1 (24); in that study, and the ␣2–9-linked disiayl sequences, which were not recog- glycosaminoglycans were proposed to be targeted by NcMIC1; nized by TgMIC1 or NcMIC1. The 4-O-acetyl-substituted Sia however, the influence of Sia was not investigated. The distinct has been described in various tissues in mice, especially in the binding specificities of the three MIC proteins might have gut; its presence has been documented in a number of other implications for tissue tropism, and the cellular targets for these animal species and in trace amounts in humans (34, 35). The proteins warrant further investigation. resistance of 4-O-acetyl-substituted Sia toward neuramini- In general, TgMIC1 and TgMIC13 bind with high affinity to dases from V. cholerae and Clostridium perfringens has previ- 3ЈSiaLacNAc sequences. Considering the potential spectrum of ously been described (36, 37), but the susceptibility of this Sia sialyloligosaccharides that can be bound by these two lectins, form to neuraminidases requires further detailed investigation. and the wide distribution of Sia in vertebrate tissues, it is likely 1 2074 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285•NUMBER 3•JANUARY 15, 2010 Supplemental Material can be found at: http://www.jbc.org/content/suppl/2009/11/09/M109.060988.DC1.html Family of MAR Domain-containing Proteins in Apicomplexa that the two proteins will bind to most cell types. This is in the apple domain, present in TgMIC4, is specific to coccidians marked contrast to the P. falciparum erythrocyte-binding anti- (45), although a divergent fold belonging to the plasminogen gen 175 (PfEBA175), which binds selectively to a unique sialy- apple nematode superfamily is present in apical membrane lated glycoprotein, glycophorin A (40, 41). Our results suggest antigen-1 homologues from all Apicomplexans (46, 47). Fur- cooperativity between TgMIC1 and TgMIC4 in receptor bind- thermore, our BLAST searches indicate that the chitin binding- ing by the TgMIC1-4-6 complex. It will be interesting to inves- like domain is restricted to coccidians. The chitin binding-like tigate if receptor recognition of TgMIC1 is influenced by its domain is present in TgMIC3 and TgMIC8, which form association with TgMIC4. another major complex involved in invasion. Dimerization of Is There a Molecular Basis for Invasion of a Broad Range of these chitin binding-like domains allows host cell binding and Cells?—The presence of the MAR domain-containing proteins has been associated with parasite virulence (13, 48). Intrigu- in Toxoplasma, Neospora, Sarcocystis, and Eimeria parasites ingly, a synergy effect has been demonstrated between this and its absence in Plasmodium, Theileria, and Cryptospo- complex and TgMIC1-4-6 (13). However, for most protein ridium suggest that this domain is exclusively conserved among complexes functioning in invasion, the timing and coordina- the Coccidia. We hypothesize that sialylated receptors may be tion of action are only beginning to be understood. In summary exploited as a common target by a subset of MCPs carrying the our work provides detailed insights into recognition of a broad appropriate binding motif in this group of enteroparasites. As host cell range by cyst-forming enteroparasites during early suggested in our study, not all MCPs would bind to Sia or even stages of invasion and raises the possibility that glycomimetic Downloaded from to host cells, which raises questions regarding their alternative drugs might be useful to reduce parasite burden in tissues dur- functions. We have identified a complete set of homologues of ing acute infection. T. gondii MCPs in N. caninum, a parasite causing significant economic loss by infecting cattle. T. gondii and N. caninum Acknowledgments—We thank C. Cherbuliez and N. Klages for excellent technical assistance; N. Jemmely for the generation of the anti- belong to a group of tissue-cyst-forming coccidians that also www.jbc.org include the genera Hammonida, Besnoita, Sarcocystis, and TgAMA-1 antibodies; W. Chai and Y. Zhang for mass spectrometric Frenkelia. Similar to T. gondii tachyzoites, N. caninum analyses of oligosaccharide probes; R. Childs for the microarrays; and tachyzoites and S. neurona merozoites are known to be promis- M. P. Stoll for the microarray analyses software. We thank Dr. C. Rodriguez (University of Oviedo), Dr. F. Tomley (Institute for Animal cuous pathogens that are able to infect a large variety of cells at SMAC Consortium - Geneve, on May 29, 2010 (42, 43). This is probably related to their common behavior of Health, Compton), and Dr. A. Hemphill (University of Bern) for kindly providing us with the C6 rat glioma cell line, CHO cells, and NcMIC1 spreading through tissues during acute infection prior to cyst cDNA, respectively. We also thank M. Messer and T. Urashima for the formation. Considering the fact that the MAR family is 4-O-acetylated sialyllactose used in the microarray analyses. We also restricted to enteroparasites, these adhesins might also be of acknowledge Dr. A. Pain and Dr. A. Reid (Sanger Institute, Hinxton, importance in the context of the natural route of infection via UK) for integrating our gene models into public data bases. the intestine. To our knowledge, no study has so far addressed the importance of receptor-ligand interactions for invasion of the midgut epithelium. REFERENCES It is tempting to speculate that the MAR family of lectins is 1. Blumenschein, T. M., Friedrich, N., Childs, R. A., Saouros, S., Carpenter, instrumental to the infection of such a broad range of cells. E. 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25. Labbe´, M., de Venevelles, P., Girard-Misguich, F., Bourdieu, C., Guil- (2009) Exp. Parasitol. 123, 111–117 www.jbc.org laume, A., and Pe´ry, P. (2005) Mol. Biochem. Parasitol. 140, 43–53 45. Chen, Z., Harb, O. S., and Roos, D. S. (2008) PLoS One 3, e3611 26. Deutscher, S. L., Nuwayhid, N., Stanley, P., Briles, E. I., and Hirschberg, 46. Chesne-Seck, M. L., Pizarro, J. C., Vulliez-Le Normand, B., Collins, C. R., C. B. (1984) Cell 39, 295–299 Blackman, M. J., Faber, B. W., Remarque, E. J., Kocken, C. H., Thomas, 27. Esko, J. D., Rostand, K. S., and Weinke, J. L. (1988) Science 241, 1092–1096 A. W., and Bentley, G. A. (2005) Mol. Biochem. Parasitol. 144, 55–67 at SMAC Consortium - Geneve, on May 29, 2010 28. Feizi, T., and Chai, W. (2004) Nat. Rev. Mol. Cell Biol. 5, 582–588 47. Pizarro, J. C., Vulliez-Le Normand, B., Chesne-Seck, M. L., Collins, C. R., 29. Garnett, J. A., Liu, Y., Leon, E., Allman, S. A., Friedrich, N., Saouros, S., Withers-Martinez, C., Hackett, F., Blackman, M. J., Faber, B. W., Re- Curry, S., Soldati-Favre, D., Davis, B. G., Feizi, T., and Matthews, S. (2009) marque, E. J., Kocken, C. H., Thomas, A. W., and Bentley, G. A. (2005) Protein Sci. 18, 1935–1947 Science 308, 408–411 30. Liu, Y., Feizi, T., Campanero-Rhodes, M. A., Childs, R. A., Zhang, Y., 48. Ce´re`de, O., Dubremetz, J. F., Bout, D., and Lebrun, M. (2002) EMBO J. 21, Mulloy, B., Evans, P. G., Osborn, H. M., Otto, D., Crocker, P. R., and Chai, 2526–2536

1 2076 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285•NUMBER 3•JANUARY 15, 2010 2. Toxoplasma gondii transmembrane microneme proteins and their modular design

Host cell invasion by Toxoplasma gondii is mediated by type I transmembrane proteins, which are secreted in a regulated manner from the micronemes onto the parasite’s surface, where they assemble into complexes. These proteins function as adhesins, binding to receptors on the host cell, and, in the case of TgMIC2 and TgMIC6, can also associate to the glideosome via binding to aldolase (Jewett and Sibley, 2003; Zheng et al., 2009). In some cases, they also escort the other members of the complex to the micronemes. Shedding from the parasite’s surface is thought to occur by cleavage within the transmembrane domain by a rhomboid. In order to gain insight into the ability of the different transmembrane micronemal proteins of T. gondii to function as escorters, associate with aldolase or function as rhomboid substrates, we tested these aspects for all the previously identified transmembrane microneme proteins, as well as for a newly identified micronemal protein. These analysis revealed that none of the proteins examined act as escorter but that several proteins can be cleaved by a rhomboid protease and bind to aldolase. The significance of aldolase binding for the in vivo function of TgMIC6 and TgAMA1 was also investigated and it was found that it is only essential for TgAMA1 function during invasion. The personal contribution to this study resumed to the study of the new TgMIC16 protein, analysis of the TgAMA1 cleavage and examination of the ability of TgMIC6 to bind to aldolase and its importance for gliding motility. The manuscript was written in collaboration with Lilach Sheiner.

1 Molecular Microbiology

Toxoplasma gondii transmembrane microneme proteins and their modular design

For Peer Review Journal: Molecular Microbiology

Manuscript ID: MMI-2010-10009.R1

Manuscript Type: Research Article

Date Submitted by the 31-May-2010 Author:

Complete List of Authors: Sheiner, Lilach; University of Georgia, Center for Tropical and Emerging Tropical Diseases; CMU-Universite de Geneve, Microbiology Santos, Joana; CMU-Universite de Geneve, Microbiology Klages, Natacha; CMU-Universite de Geneve, Microbiology Parussini, Fabiola; University of Vermont, Microbiology and Molecular Genetics Jemmely, Noelle; CMU-Universite de Geneve, Microbiology Friedrich, Nikolas; CMU-Universite de Geneve, Microbiology Ward, Gary; University of Vermont, Microbiology and Molecular Genetics Soldati-Favre, Dominique; CMU-Universite de Geneve, Microbiology; CMU University of Geneva, Microbiology and Molecular Medicine

Key Words: Toxoplasma gondii, trafficking, polytopic, rhomboid, microneme

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Toxoplasma gondii transmembrane microneme proteins and their modular design

Lilach Sheiner*1,3, Joana M. Santos§*1, Natacha Klages*1, Fabiola Parussini2, Noelle 1 Jemmely1, Nikolas Friedrich1, Gary E. Ward2 and Dominique Soldati-Favre

For Peer Review

§Corresponding author: [email protected] * These authors contributed equally to this work 1Department of Microbiology and Molecular Medicine, CMU, University of Geneva, 1 rue Michel-Servet, 1211 Geneva 4, Switzerland Phone: + 41 22 379 5656, Fax: + 41 22 379 5702 2Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, Vermont 05405, USA 3 Current address: Center for Tropical & Emerging Global Diseases, University of Georgia, USA

Running title: Transmembrane Microneme Proteins Modular Function Keywords: Toxoplasma gondii, trafficking, polytopic, rhomboid, protease, Golgi, microneme. Abbreviations: IFA, Indirect immunofluorescence assay; ROM, Rhomboid protease; MIC, microneme protein; TM-MIC, type-I single transmembrane MIC; ADL, aldolase; CTD, cytoplasmic C-terminal domain; TMD, transmembrane domain; DG, dense granules; RON, rhoptry neck protein; ROP, rhoptry bulb protein; TSR, thrombospondin repeat; TRAP, thrombospondin-related anonymous protein; TGN, trans-Golgi network

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Summary

Host cell invasion by the Apicomplexa critically relies on regulated secretion of transmembrane micronemal proteins (TM-MICs). Toxoplasma gondii possesses functionally non-redundant MICs complexes that participate in gliding motility, host cell attachment, moving junction formation, rhoptry secretion and invasion. The TM-MICs are released onto the parasite’s surface as complexes capable of interacting with host cell receptors. Additionally, TgMIC2 simultaneously connects to the actomyosin system via binding to aldolase.For During invasion Peer these adhesive Review complexes are shed from the surface notably via intramembrane cleavage of the TM-MICs by a rhomboid protease. Some TM- MICs act as escorters and assure trafficking of the complexes to the micronemes. We have investigated the properties of TgMIC6, TgMIC8, TgMIC8.2, TgAMA1 and the new micronemal protein TgMIC16 with respect to interaction with aldolase, susceptibility to rhomboid cleavage and presence of trafficking signals. We conclude that several TM- MICs lack targeting information within their C-terminal domains, indicating that trafficking depends on yet unidentified proteins interacting with their ectodomains. Most TM-MICs serve as substrates for a rhomboid protease and some of them are able to bind to aldolase. We also show that the residues responsible for binding to aldolase are essential for TgAMA1 but dispensable forTgMIC6 function during invasion.

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Introduction

Toxoplasma gondii is an obligate intracellular parasite of the phylum Apicomplexa, which also includes the deadly agent of malaria, Plasmodium falciparum. Host cell invasion by these parasites is a multi-step process (Carruthers & Boothroyd, 2007) propelled by the gliding motility machinery. It is initiated by apical attachment of the parasite to the host cell, followed by reorientation, formation of a junction between the parasite and host cell membranes, penetration and, finally, sealing of the parasitophorous vacuolar membrane.For Peer Review Some of the proteins implicated in invasion are sequentially released from two types of secretory organelles, named micronemes and rhoptries (Carruthers & Sibley, 1997). In T. gondii, four complexes composed of soluble and transmembrane microneme proteins or including rhoptry neck proteins (RONs) have been investigated and shown to perform non-overlapping functions during invasion (Figure 1A). More complexes are, however, likely to contribute to invasion since additional uncharacterized transmembrane microneme proteins (TM-MICs) are encoded in the genome. The selective participation of each of the four complexes in the invasion process has been uncovered by generating conventional or conditional knockouts of the genes encoding components of the complexes. The TM-MIC TgMIC2 forms a multimeric complex with the soluble partner TgM2AP (Rabenau et al., 2001, Jewett & Sibley, 2004). Parasites depleted in TgMIC2 are markedly deficient in host-cell attachment, motility and hence unable to invade host cells (Huynh & Carruthers, 2006). Another complex, composed of the transmembrane protein TgMIC6, is interacting with two soluble molecules TgMIC1 and TgMIC4. Genetic disruption of any of the three encoding genes is still compatible with parasite survival (Reiss et al., 2001) even if the complex has been demonstrated to play an important role in invasion in vitro and to contribute to virulence in vivo (Blumenschein et al., 2007, Cerede et al., 2005, Sawmynaden et al., 2008). A third complex is composed of the TM-MIC TgMIC8 and the soluble protein TgMIC3. Genetic disruption of TgMIC8 interferes with rhoptries secretion and, consequently, prevents formation of the moving junction (MJ) and completion of invasion (Kessler, 2008). A fourth complex, which uniquely localizes to the MJ (Alexander et al., 2005), contains the

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rhoptry proteins TgRON2, TgRON4, TgRON5 and TgRON8 (Alexander et al., 2005, Besteiro et al., 2009, Straub et al., 2008) and the TM-MIC TgAMA1, which anchors the complex to the parasite plasma membrane. Parasites lacking TgAMA1 efficiently attach to host cells but are defective in rhoptry secretion, fail to create a MJ and are consequently unable to invade host cells (Mital et al., 2005). Gliding motility is not significantly altered in the absence of TgAMA1 or TgMIC8 (Mital et al., 2005, Kessler, 2008). So far, TgMIC2 and TgMIC6 are the only TM-MIC shown to play a crucial role as force- transducer during For motility and Peer invasion. TgMIC2 Review binds to receptor(s) on the host cell surface and establishes simultaneously a connection, via its C-terminal cytoplasmic domain (CTD), with the parasite’s actomyosin system, hence powering parasite motility. The CTD of TgMIC2, TgMIC6 and other members of the TRAP family in Plasmodium (TRAP, CTRP and TLP) interact with aldolase, a glycolytic enzyme also capable of binding to filamentous-actin (F-actin) (Buscaglia et al., 2003, Jewett & Sibley, 2003, Heiss et al., 2008, Zheng et al., 2009). It is unknown whether other T. gondii TM-MICs, that are part of adhesive complexes and exhibiting crucial functions in invasion, can as well interact with aldolase and thus act as bridge molecules. At the end of the penetration process, the tight interactions formed between the different MIC complexes and the host cell receptors have to be disengaged to let the parasite freely replicate. This has been proposed to occur by proteolytic shedding of the MIC complexes from the parasite’s surface. Cell-based cleavage assays and studies on parasites have demonstrated that one of these critical cleavage events takes place at a conserved motif within the luminal part of the transmembrane domains of TgMIC2, TgMIC6, TgMIC12 and TgAMA1. The protease responsible for this intramembrane cleavage was named microneme protein protease 1 (MPP1) and likely corresponds to a plasma membrane rhomboid-like protease (Opitz et al., 2002, Brossier et al., 2003, Urban & Freeman, 2003, Zhou et al., 2004, Howell et al., 2005). Prime candidates for this shedding activity are TgROM4 and TgROM5, which are found at the plasma membrane of the parasite (Brossier et al., 2005, Dowse et al., 2005). More recently, parasites depleted in TgROM4 indicate that this protease acts as sheddase for TgMIC2 and TgAMA1 and hence critically contributes to the creation of an apical-posterior gradient of adhesins necessary

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for an apical orientation of the parasite during invasion (Buguliskis et al.). At the end of the penetration process, the tight interactions formed between the different MIC complexes and the host cell receptors have to be disengaged to let the parasite freely replicate. This has been proposed to occur by proteolytic shedding of the MIC complexes from the parasite’s surface by the TgROM5 activity. A prerequisite for successful invasion is the correct trafficking of the MIC complexes, from the endoplasmic reticulum (ER), where they are pre-assembled, to the micronemes, where they are stored prior to invasion. Similar to other eukaryotic sorting mechanisms, some TM-MICs areFor accurately Peer targeted to the Review micronemes via recognition of a tyrosine- based motif in the cytoplasmic CTDs (Sheiner & Soldati-Favre, 2008). TgMIC2 and TgMIC6 CTDs contain such a microneme targeting motif (EIEYE) and have been shown to serve as escorters for the soluble MICs that are part of the respective complexes (Di Cristina et al., 2000, Opitz et al., 2002, Reiss et al., 2001). Recent studies have also revealed an important contribution of some of the soluble MICs to trafficking. TgMIC1 was shown to promote folding of TgMIC6 by serving as a quality control mechanism (Saouros et al., 2005) and other soluble MICs contain pro-peptides that act as luminal forward targeting elements and are indispensable for correct trafficking of the entire complex (Brydges et al., 2008, El Hajj et al., 2008, Harper et al., 2006). To gain insight into the mechanistic contribution of each of the MIC complexes to invasion, we have undertaken a detailed analysis of the TM-MICs currently identified in T. gondii and included a new member, TgMIC16. We have searched for the presence of trafficking determinants, assessed their susceptibility to intramembrane cleavage and their ability to interact with aldolase. The results indicate that in contrast to TgMIC2, TgMIC6 and TgMIC12, the CTDs of TgMIC8, TgMIC8.2, TgAMA1 and TgMIC16 do not carry the information for proper trafficking to the micronemes and cannot therefore be considered as escorters. All these TM-MICs, apart from TgMIC8.2, appear to be susceptible to intramembrane cleavage and the CTDs of TgMIC6, TgMIC12 and TgAMA1 can bind to aldolase in pull down assays. Additionally, we have identified specific residues within the CTD of TgAMA1 that are required for both association with aldolase and host cell invasion. Collectively these data support a model describing the involvement of TM-MICs, as part of complexes with distinct and non-overlapping

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functions during invasion.

Results

TgMIC16 is a conserved Coccidia TM-MIC containing 6 TSR domains

A search in the T. gondii genome database for putative new microneme proteins containing TRAP-family-like transmembrane sequences led to the identification of a gene encoding a hypotheticalFor protein Peer (TGME49_089630) Review of 669 amino acids. This gene model (80.m00085) has also been identified by a recent in silico screen for secretory proteins and was proposed to reside in an apical compartment (Chen et al., 2008). The amino acid sequence of the protein includes an N-terminal predicted signal peptide, six putative TSR type 1 domains (Figure S1) and one TMD (transmembrane domain). This TMD is located close to the C-terminal end, contains a motif reminiscent of a rhomboid cleavage site and delimits a very short C-terminal tail (Figures 2A and 2B). Another TMD is also predicted at the N-terminal end of the protein (TMHMM prediction program), but with a low probability and therefore it is not depicted as such in the schemes. A search of the available apicomplexan genomes revealed that homologues of TGME49_089630 are present in the genomes of N. caninum and E. tenella but are absent in Hemosporidia, suggesting that this gene is restricted to the Coccidia. Alignment of the amino acid sequences of these genes (Figure 2A) uncovered a very similar domain structure. Transient expression of this new protein carrying a Ty epitope at the C-terminus revealed a micronemal localization in T. gondii tachyzoites (Figure 2B). Reflective of this localization and the nomenclature status, this protein has been named TgMIC16 (accession number EU791458). Given its predicted domain structure and the presence of a putative rhomboid cleavage site in the TMD, this protein was included in this study along with the TM-MICs that are part of the four major T. gondii MIC complexes (TgAMA1, TgMIC2, TgMIC6 and TgMIC8). We also included one of the homologues of TgMIC8, TgMIC8.2, previously known as MIC8-like 1 (Kessler, 2008). A chimera of the TgMIC8 ectodomain fused to the TM-CTD portion of TgMIC8.2 was able to functionally complement mic8ko parasites, indicating that the TM-CTDs of these proteins are

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functionally equivalent (Kessler, 2008). Finally, TgMIC12, the homologue of the Eimeria TM-MICs EtMIC4 and EmTFP250 (Witcombe et al., 2004, Periz et al., 2009), shown before to be susceptible to rhomboid cleavage (Opitz et al., 2002), was also included in the comparative analysis.

Multiple motifs and functions are conserved in the TM-CTDs of the TM-MICs

Several lines of evidence indicate that the TM-CTDs of the TM-MICs play an essential role in supporting Forthe functionality Peer of their respectiveReview complexes (information regarding the composition and susceptibility to proteolytic cleavage of these complexes is recapitulated in Figure 1A), and therefore an alignment of the amino acid sequences of these domains was performed and carefully examined (Figure 1B). Previous studies on the aldolase binding capacity of the CTDs of TgMIC2 and other TRAP-related TM-MICs have demonstrated the importance of both a stretch of acidic residues and a penultimate tryptophan residue in the extreme C-terminal sequence (Buscaglia et al., 2003, Starnes et al., 2006). TgMIC6 and TgMIC12 possess both the acidic stretch and a tryptophan residue near the C-terminus (Figure 1B). The CTDs of the two TgMIC8 homologues possess a penultimate tryptophan residue but are not of acidic nature. Conversely, TgAMA1 contains the C-terminal acidic residues, but the most C-terminal tryptophan is 21 residues in from the C-terminus (W520). This residue lies within a FW motif that is highly conserved in the AMA1 homologues of different apicomplexans (Hehl et al., 2000, Donahue et al., 2000) and is known to be essential for invasion in P. falciparum (Treeck et al., 2009). The short TgMIC16 CTD does not exhibit any feature of the aldolase-binding motifs. TgAMA1, TgMIC2, TgMIC6 and TgMIC12 possess a rhomboid cleavage site, IAGG or IAGL, at a conserved position within the TMD, and were previously shown to be cleaved in the parasite and in in vitro cleavage assays (Opitz et al., 2002, Urban & Freeman, 2003, Dowse et al., 2005, Brossier et al., 2005, Howell et al., 2005, Buguliskis et al.). A very similar motif is found at the corresponding position within the TMD of TgMIC16 and in a different position within the TMD of TgMIC8. No rhomboid cleavage motif could be identified in the TMD of TgMIC8.2.

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From the two motifs shown to be essential for TgMIC2 targeting to the micronemes (Di Cristina et al., 2000), the sequence SYHYY is not conserved in any of the CTDs of the TM-MICs analyzed, whereas the motif EIEYE is strictly conserved in the tail of TgMIC6. A sequence resembling EIEYE is similarly positioned in the TgMIC12 and TgAMA1 CTDs but the critical last glutamine residue is only present in TgMIC12. No sequence reminiscent of such a targeting motif could be identified in the CTDs of the TgMIC8 family members or TgMIC16.

Several TM-MICsFor lack trafficking Peer signals inReview their CTDs

The TM-CTDs of TgMIC2, TgMIC6 and TgMIC12 were shown to be able to target to the micronemes the surface antigen 1 protein (TgSAG1), lacking its GPI anchor signal (Di Cristina et al., 2000, Opitz et al., 2002, Reiss et al., 2001). From these studies it was concluded that these three TM-MICs act as escorters, bringing the soluble components of their respective complexes to the organelle. TgMIC8 was initially suspected to act as an escorter based on the ability of a GPI anchored TgMIC8 construct to bring TgMIC3 to the plasma membrane (Meissner et al., 2002). However this experiment only demonstrated that TgMIC3 and TgMIC8 were part of the same complex and the escorter hypothesis had to be revisited in the light of a recent study, which established that the soluble partner TgMIC3 was correctly targeted to the micronemes, even in the absence of TgMIC8 (Kessler, 2008). To assess if TgMIC8, TgMIC8.2 and TgMIC16 contain trafficking information, their TM-CTDs were C-terminally fused to the SAG1 coding sequence, lacking its GPI anchoring signal, and to a Ty-1 tag epitope, and expressed under the control of the TgMIC2 promoter (the constructs are depicted in Figure S2). Depending on the information carried by the CTDs, these chimeras were expected to travel through the secretory pathway and to be secreted onto the parasite surface, either via the micronemes by extracellular parasites at the time of invasion, or via the dense granules (DGs) in a constitutive fashion. In parasites expressing pMS1tyMIC16TM-CTD or pMS1tyMIC8TM-CTD, the fusion proteins accumulated in the DGs and were delivered to the parasitophorous vacuole (PV), as

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shown by co-localization with the DG marker GRA3 (Figure 3). An identical SAG1 fusion with the TM-CTD of TgMIC8.2 (pMS1tyMIC8.2TM-CTD) accumulated in the trans- Golgi, since it accumulated in a compartment in the proximity of cis-Golgi as shown by staining with the marker GRASP-YFP (Pelletier et al., 2002) (Figure 3). Due to the presence of a TMD, the chimeric proteins were expected to accumulate at the plasma membrane (PM). The absence of PM staining suggests that these proteins are cleaved once delivered to the plasma membrane, and what is being detected is the processed form. Insight into the traffickingFor ofPeer TgAMA1 was Review performed by expressing its TM-CTD C- terminally fused to the SAG1 and Ty-1 tag epitope, under the control of the endogenous promoter. The fusion pAS1tyAMA1TM-CTD localized to the DGs, and not to the PM, as previously shown for pMS1tyMIC16TM-CTD and pMS1tyMIC8TM-CTD, suggesting that this protein also undergoes proteolysis (Figure 4). A series of truncated variants of TgAMA1 fused N-terminally to a His tag, under the control of the endogenous promoter, were also expressed. Unlike pAS1tyAMA1TM-CTD, pAhisAMA1ΔTM-CTD, encoding the ectodomain only, or pAhisAMA1ΔCTD, encoding the ectodomain and TMD, were predominantly targeted to the micronemes, as shown by co-localization with TgMIC4. The same localization was obtained for the full-length protein, pAhisAMA1 (Figure 4). These data suggest that the ectodomain, but not the CTD, of TgAMA1 assures correct trafficking to the micronemes potentially via interaction with a yet unidentified protein. This is in accordance with the observations made on the Plasmodium AMA1 and other micronemal proteins (Treeck et al., 2009, Treeck et al., 2006, Healer et al., 2002). These results indicate that unlike TgMIC2, TgMIC6 and TgMIC12, none of the other TM-MICs analyzed here carry the necessary signal in their CTD to travel to the micronemes.

Several TM-MICs are susceptible to cleavage within the membrane-spanning domain

To determine if the SAG1-ty-TM-CTD chimeras were serving as substrates for intramembrane cleavage we generated parasites expressing SAG1 fusion constructs

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mutated in the predicted rhomboid cleavage sites (the mutated residues are boxed in the schemes depicted in Figures 5B-5E). Analysis by IFA of the corresponding transgenic parasite lines revealed that there was a dramatic change on the subcellular localization when compared to the wild type chimeras (Figure 3). The mutant fusion proteins accumulated at the PM and residually at the DGs (Figure 5A), suggesting that they were indeed subject to intramembrane proteolysis and introduction of the mutations conferred resistance to cleavage and accumulation at the parasite surface. To confirm that the changes in localization coincided with abrogation of cleavage, western blot analysesFor were Peer performed onReview total lysates from transgenic parasites expressing wild type or mutated SAG1-ty-TM-CTD chimeras (an additional blot can be seen on Figure S2). pMS1tyMIC16TM-CTD is detectable as a processed form that is no longer detected in the mutant chimera, pMS1tyMIC16mTM-CTD, when the putative rhomboid cleavage site AGGI was mutated to VVLV. The size difference between the processed and non-processed forms suggests that this cleavage is occurring downstream of the Ty-1 epitope within the TMD (Figure 5B). Similarly, when the motif IAGG in pMS1tyMIC8TM-CTD was mutated to IILV in pMS1tyMIC8mTM-CTD, there was a change in the migration pattern, compatible with the occurrence of a proteolytic cleavage downstream of the Ty-1 epitope, within the putative cleavage motif (Figure 5C). In sharp contrast to all the other rhomboid cleavage sites identified to date in apicomplexan substrates, this potential cleavage motif lies close to the cytoplasmic side of the TgMIC8 TMD (Figure 1A). Expression of pMS1tyMIC8.2TM-CTD led to the generation of two products suggesting that the protein undergoes proteolytic maturation (Figure 5D). The smaller product shows the same migration behaviour on SDS page as the intramembrane cleavage product observed for pMS1tyMIC16TM-CTD and other fusions (Figure S2), suggesting that the cleavage occurs within or close to the TMD but there is no recognizable rhomboid cleavage site in the TMD of TgMIC8.2 and therefore we could not test for rhomboid cleavage. pAS1ty1AMA1TM-CTD was mainly detected in the DGs and was subject to proteolysis at a site compatible with intramembrane cleavage as determined by western-blot (Figures 4 and 5E). In fact shedding of TgAMA1 from the parasite surface, during invasion, was previously reported to occur by proteolytic cleavage at a precise site within the TMD

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(Howell et al., 2005, Buguliskis et al.). These results indicate that pAS1tyAMA1TM-CTD, pMS1tyMIC8TM-CTD and pMS1tyMIC16TM-CTD, are cleaved likely by a rhomboid protease at the plasma membrane.

Several TM-MICs bind to aldolase

To determine whether other TM-MICs besides TgMIC2 can interact with aldolase, we examined the abilityFor of bacterially Peer expressed ReviewGST-CTD fusions to bind to aldolase by in vitro pull-down assays. Purified recombinant rabbit aldolase was used as source of aldolase and GST-MIC2CTD and GST alone served as positive and negative controls, respectively. The sequences of the TM-MICs used to generate the GST-fusions are listed in Figure S3. The experiment was repeated several times using independent purifications of each GST fusion and reproducibly showed that GST-MIC8.2CTD and GST-MIC16CTD were unable to bind to aldolase. In contrast, significant binding was monitored with GST- MIC6CTD, GST-MIC12CTD and GST-AMA1CTD (Figure 6A), confirming previous results with TgMIC6 (Zheng et al., 2009). In the case of GST-MIC8CTD, no conclusions could be taken regarding binding, due to aberrant migration of the protein on the gel, possibly result of protein un-stability. It is known that mutation of the conserved tryptophan residue at the C-terminus of PbTRAP, PfTRAP, PfTLP and TgMIC2 abrogates interaction with aldolase (Buscaglia et al., 2003, Heiss et al., 2008, Jewett & Sibley, 2003). TgMIC6 possesses a tryptophan residue in the same position as the one in TgMIC2 (Figure 1B), suggesting that this is the residue responsible for binding to aldolase. Indeed a GST-MIC6mCTD, in which F349 was replaced by an alanine residue (MIC6W/A) (Figure S3), showed a significant reduction in binding to aldolase (Figure 6B). Although there is not a tryptophan residue at the extreme C-terminus of TgAMA1, site-directed mutagenesis was performed to mutate F519W520, which is more distal to the C-terminus but represents a highly conserved motif in all apicomplexan AMA1 proteins and precedes a stretch of acidic residues in the TgAMA1 CTD (Figure S3). Intriguingly, the replacement of F519W520 by AA (AMA1FW/AA) led to a significant reduction in the binding of GST-AMA1mCTD to

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aldolase (Figure 6A).

Mutations in the CTD of TgAMA1 that block binding to aldolase inhibit invasion

The availability of mutant parasite strains in which the TgMIC6 and TgAMA1 genes have been disrupted by double homologous recombination offered the opportunity to examine the importance of the tryptophan residue in TgMIC6 and TgAMA1 for invasion (Reiss et al., 2001, Mital et al., 2005). A mutant of TgMIC6,For TgMIC6 PeerW/A-Ty, was generatedReview in which the residue W348, lying in a similar position as the tryptophan residue involved in TgMIC2 binding to aldolase, was converted to an alanine residue (Figure 1B). TgMIC6-Ty and TgMIC6W/A-Ty expressing vectors were used to complement the mic6ko strain and the resulting proteins were shown to localize to the micronemes (Figure 7A). Given that TgMIC6 is acting as escorter, in the absence of the protein, the soluble partners of its adhesive complex, TgMIC1 and TgMIC4, are mistargeted to the DGs and hence unable to participate in the invasion process (Reiss et al., 2001). In consequence, the mic6ko mutant is virtually comparable to a triple-knockout of TgMIC6, TgMIC1 and TgMIC4 (Reiss et al., 2001), in a situation parallel to the mic1ko strain, where TgMIC4 and TgMIC6 fail to traffic to the micronemes. Consistent with the invasiveness of mic1ko (Cerede et al., 2005), mic6ko shows about a 50% reduction of invasion efficiency compared to the RH-2YFP strain, which was used as an internal standard for parasite fitness (Figure 7B). Complementation of mic6ko with either MIC6Ty or MIC6W/ATy restored the invasion phenotype to a level comparable to wild type level. Gliding assays showed that mic6ko parasites are not defective in gliding and, as expected, the MIC6W/ATy complemented parasites also glide normally (Figure 7C). These results suggest that the residue W348, and therefore aldolase binding, is not critical for the function of the TgMIC4-MIC1- MIC6 complex during invasion. To study whether the residues F519W520 contribute to the function of the TgAMA1 during invasion we used a previously reported TgAMA1 conditional knockout parasite

(ama1koi; Mital et. al., 2005), in which the expression of wild type (myc-tagged) TgAMA1 can be controlled by the addition of anhydrotetracycline (ATc). In the absence

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of ATc, AMA1myc is expressed in these parasites and they are fully invasive; in the presence of ATc, AMA1myc expression is repressed and the parasites are severely

defective in invasion (Mital et al., 2005). The ama1koi parasites were transfected with plasmids encoding Flag-tagged wild type or mutant TgAMA1 (AMA1WTFlag and AMA1FW/AAFlag, respectively), and independent clones expressing similar levels of AMA1WTFlag and AMA1FW/AAFlag in the presence of ATc were isolated. Both the wild type and mutant proteins localized to the apical end of the parasite, as shown by co- localization with M2AP, indicating proper localization (Figure 8A and data not shown). While AMA1WTFlagFor was able Peer to complement Review the ATc-induced invasion defect in the FW/AA ama1koi parasites, AMA1 Flag was not (Figure 8B). These data demonstrate that the hydrophobic residues F519W520 within the CTD of TgAMA1 are essential for both aldolase binding and host cell invasion.

Discussion

MIC complexes serve essential roles during host cell invasion, by mediating parasite attachment, MJ formation and bridging of the host cell receptors to the actomyosin system, hence promoting gliding and invasion. The smooth transition through the various steps of the invasion process requires a high level of coordination not only between the different MIC complexes but also between each component of a given complex. The TM- MICs, in particular are multitasks and execute distinct functions that are specified by their modular design. The ectodomains, on one hand, recruit microneme or rhoptry proteins to the complex and, in several instances also interact directly with host cell receptors; and the TM-CTDs, on the other hand, contribute to targeting, proteolytic shedding and connection to the actomyosin system of the parasite. TgMIC2 and other members of the TRAP family are suited to carry out these multiple tasks (Morahan et al., 2009). In this study, we have investigated and compared to TgMIC2, the biological properties of the TM-MICs associated with the three other major MIC complexes known to be involved in invasion, as well as of TgMIC12, TgMIC8.2 and TgMIC16, whose functions

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remain to be established. Targeting to the micronemes, as demonstrated for the rhoptries (Ngo et al., 2003, Richard et al., 2009), resembles post-TGN targeting in other eukaryotes (Sheiner & Soldati-Favre, 2008). Complexes of soluble and TM-MICs are formed in the ER and travel through the secretory pathway until they are finally secreted (Huynh et al., 2003, Reiss et al., 2001). Some TM-MICs have been shown to act as escorters, implying that their CTDs are recognized by components of the vesicular sorting machinery (Meissner et al., 2002). Consistent with this idea, two micronemal targeting motifs, SYHYY and EIEYE, were identified in TgMIC2For (Di Cristina Peer et al., 2000) Review. The apparent absence of such motifs in the CTDs of TgAMA1, TgMIC16 and the TgMIC8 family members is in agreement with the findings here that the respective SAG1-ty-TM-CTD chimeras fail to traffic to the micronemes. Consequently, these TM-MICs do not function as escorters and are likely to interact, via their ectodomains, with other proteins that carry a determinant for micronemal targeting. Consistent with this hypothesis, the chimera the chimera MIC8 fused to the CTD of P. berghei TRAP localizes to the micronemes (Kessler, 2008) although the PbTRAP TM-CTD does not confer trafficking to micronemes in T. gondii (Di Cristina et al., 2000). Similarly, the refined analysis of TgAMA1 clearly established that it is the ectodomain of the protein that carries the necessary traffic information to the micronemes. Studies on AMA1 in P. falciparum led to the same conclusion (Treeck et al., 2009, Treeck et al., 2006, Healer et al., 2002). These observations imply that TgAMA1, TgMIC8, TgMIC8.2 and TgMIC16 may belong to complexes that are composed of more than one type of TM-MIC. These observations imply that TgAMA1, TgMIC8, TgMIC8.2 and TgMIC16 may belong to complexes that are composed of more than one type of TM-MIC. The SAG1-ty-TM-CTDs chimeras localized either to the DGs and PV (TgMIC8, TgMIC16 and TgAMA1), or were retained in the Golgi (TgMIC8.2). An alignment of the TM-CTDs of the selected TM-MICs predicted the presence of a rhomboid cleavage motif similar to IAGG in the TMDs of TgMIC2, TgMIC6, TgMIC12, TgAMA1 and TgMIC16. An IAGG motif is also present within the TMD of TgMIC8, but it is significantly shifted within the TMD, closer to the CTD. No apparent rhomboid cleavage site signature could be identified in the TMD of TgMIC8.2. Consistent with cleavage at the rhomboid

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cleavage motif, proteolytic processing at the expected position was observed for pMS1tyMIC8TM-CTD, pMS1tyMIC16TM-CTD and pAS1tyAMA1TM-CTD chimeric proteins. To provide further evidence for intramembrane processing by a rhomboid, point mutations were introduced in the identified cleavage motifs. The majority of the mutations introduced abrogated processing and thus provided strong evidence that the chimeras are cleaved within their TMD by a rhomboid-like protease. Interestingly, while TgMIC8 appears to be cleaved by a rhomboid, this proteolysis occurs much closer to the cytoplasmic region of the TMD than in all the other TM-MICs. This may allow the direct release of the CTDFor into the Peer cytoplasm, where Review it can initiate a signaling cascade, as previously proposed (Kessler, 2008). Given the absence of a recognizable rhomboid cleavage motif in the TMD of TgMIC8.2, we could not assess the nature of the processing event. However, the cleavage product runs at a size compatible with intramembrane processing and TgMIC8.2-CTD can replace that of TgMIC8 (Kessler, 2008), which is susceptible to intramembrane cleavage. It is consequently still plausible that the TgMIC8.2-chimera is as well processed within the TMD. Preventing the proteolytic cleavage by mutagenesis had an anticipated impact on the localization of the SAG1-ty-TM-CTD chimeras. All the mutant chimeras showed a dramatic change in subcellular localization, accumulating at the parasite’s surface, indicative of cleavage abrogation. In a prior study, a similar accumulation at the parasite plasma membrane was observed for the uncleaved SAG1TgMIC12TM-CTD mutant, which was mistargeted to the DGs (Opitz et al., 2002). This observation suggested that MPP1 is a constitutively active rhomboid-like protease at the plasma membrane of the parasite. We cannot discriminate between cleavage of the SAG1-ty-TM-CTD constructs at the plasma membrane or inside the parasite, but it is likely that TgMIC16 and TgMIC8 fusion constructs are cleaved by MPP1, because the corresponding non-cleaved mutants accumulate at the parasite’s surface. The unambiguous assignment of each of the TM- MIC substrates to a given protease awaits further investigations. Several Plasmodium TM-MIC proteins have been reported to interact with the F-actin binding protein aldolase and this way bridge the host cell surface with the actomyosin motor of the parasite. These proteins share the structural features characteristic of TRAP, namely an N-terminal secretion signal, a van Willebrand A-domain, one or more TSR

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domains, a TMD with a rhomboid-cleavage motif and an acidic CTD with a unique tryptophan residue close to the C-terminus (Morahan et al., 2009). In T. gondii, TgMIC6 and TgMIC2 are the only TM-MICs shown to bind to aldolase (Jewett & Sibley, 2003, Starnes et al., 2006, Zheng et al., 2009), in a model compatible TgMIC2 redistribution along the parasite’s surface upon invasion (Carruthers & Sibley, 1999) and demonstrated role in motility and invasion (Huynh & Carruthers, 2006). The patches of acidic amino acids constituting the aldolase binding site within the TgMIC2 CTD (Starnes et al., 2006) are not strictly conserved in the TgMIC6, TgMIC12 and TgAMA1 CTDs (Figure S4) but as shown in this study,For these proteinsPeer are able Review to bind to aldolase in an in vitro pull down assay. This suggests that the composition in acidic amino acids and their precise location within the CTD can accomodate a level of variation. The second prominent feature of aldolase binding is the presence of a conserved tryptophan residue at the extreme C- terminus of the CTD. All the TM-MICs studied here possess this residue except TgAMA1 and TgMIC16, and as shown by mutation of the residue in TgMIC6, the residue mediates binding to aldolase. Although the TgMIC8 family members possess a tryptophan residue at the extreme C-terminus, the acidic patch is absent and none of these CTDs bind to aldolase in the in vitro assay. This is in accordance with functional analysis showing no motility defect in TgMIC8 depleted parasites (Kessler, 2008). In contrast, TgAMA1 is able to bind to aldolase without an extreme C-terminal tryptophan, although TgAMA1 does contain a tryptophan just N-terminal to a patch of acidic residues (W520; Figure S4) within an FW motif that is well conserved among AMA1 homologues in other Apicomplexans (Hehl et al., 2000). Mutation of this FW motif in TgAMA1 disrupted both aldolase binding and invasion. This result suggests that TgAMA1 serves as a bridging protein that physically connects the glideosome (via its CTD) to other components of the MJ complex (via its ectodomain) and thus plays a critical role in the posterior translocation of the MJ complex during invasion. However, during invasion the majority of the TgAMA1 is not restricted to the MJ but is found over the parasite’s surface (Alexander et al., 2005, Howell et al., 2005). This suggests that there may be two pools of TgAMA1 at the parasite surface, one of which is bound to aldolase and is responsible for anchoring the MJ complex to the actomyosin motor. Whether the remaining fraction of TgAMA1 serves a distinct function or is simply available to be

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recruited to the MJ is unknown, but is it possible that the two pools of TgAMA1 are distinguished by different post-translational modifications of their CTDs, such as phosphorylation (Treeck et al., 2009). Disappointingly, our repeated efforts to monitor TgAMA1, or even TgMIC2, interaction with aldolase in the parasite by co- immunoprecipitation were unfruitful. The extreme C-terminus of TgMIC12 shares a nearly strictly conserved amino acid sequence with TgMIC2, and this reflects its comparable propensity to bind to aldolase in pull down experiments. Given the fact that no functional data are available to date on TgMIC12, the physiologicalFor relevancePeer of these Review observations is not known. TgMIC6 binds less efficiently to aldolase compared to the CTDs of TgMIC2 or TgMIC12, and this can simply reflect the fact that fewer acidic residues are present at its extreme C-terminus. TgMIC6 wild type or TgMIC6 carrying a W348/A mutation are both able to functionally complement the invasion defect in parasites depleted of TgMIC6, suggesting that the tryptophan residue may not be crucial for the function of the TgMIC1- MIC4-MIC6 complex. Consistent with these findings mic6ko showed not defect in gliding motility, suggesting that the function of TgMIC1-MIC4-MIC6 complex in invasion might be assisted via the formation of a macrocomplex by another TM-MIC that connects to the actomyosin system. It remains to be determined for TgMIC12, if aldolase plays a role in its functions in vivo and if it reflects a direct interaction with the actomyosin motor or another biological role. Taken together, there is an excellent correlation between the predictions made from sequence analysis and the three biological properties examined experimentally: presence of trafficking determinants, susceptibility to rhomboid protease cleavage and binding to aldolase. Moreover the assessed properties of the TM-MICs are in good accordance with their functional contribution to the individual steps of the invasion process (Table 1).

Material and Methods

Reagents and parasite culture Restriction enzymes were purchased from New England Biolabs and secondary antibodies for western blots and IFA from Molecular Probes. T. gondii tachyzoites (RH

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strain wild-type and RHhxgprt-) were grown in human foreskin fibroblasts (HFF) or Vero cells in Dulbecco´s Modified Eagle´s Medium (DMEM, GIBCO) supplemented with 5% fetal calf serum (FCS), 2mM glutamine and 25 µg/ml gentamicin. ama1koi parasites were cultured in DMEM supplemented with 1% fetal bovine serum, 25µg/ml mycophenolic acid, 50µg/ml xanthine, and 6.8µg/ml chloramphenicol (Mital et. al., 2005).

Downregulation of AMA1-myc expression in intracellular ama1koi parasites was achieved by incubation of infected cells for 36 hr in medium containing 1,5µg/ml anhydrotetracycline (ATc, Clontech). For Peer Review Cloning of DNA constructs For determination of MIC16 localization in T. gondii, the full-length gene was amplified from tachyzoite cDNA by PCR using the primers 1969 and 1971 (Supplementary table 1). The PCR product was purified, digested with EcoR1 and NsiI, and cloned into the corresponding sites in pTUB8Ty (Meissner et al., 2002). For expression in T. gondii as N-terminal SAG1-Ty fusions, DNA fragments coding for the TMD and CTD of MIC8, MIC8.2 and MIC16 were amplified from tachyzoite cDNA by PCR using the primers 1887 and 1888, 1889 and 1890, 1970 and 1972 (supplementary table 1). MIC8TMCTD, MIC8.2TMCTD and MIC16TMCTD were digested with SalI and PacI and each fragment was cloned into the corresponding sites in pMSAG1Ty vector, which drives expression under control of the TgMIC2 promoter, originating pMSAG1tyMIC8TMCTD, pMSAG1tyMIC8.2TMCTD and pMSAG1tyMIC16TMCTD (Di Cristina et al., 2000). For expression of the different AMA1 constructs under control of its endogenous promoter in RH strain T. gondii, DNA fragments coding for TMD and CTD, the full length protein, only the ectodomain (aminoacids 1-456) or the ectodomain and TMD (aminoacids 1-479) were amplified from tachyzoite cDNA by PCR using the primers 1079 and 1080, 2225 and 1080, 2247 and 2249 and 2247 and 2248, respectively (Supplementary table 1). The three last pair of primers added a 8His tag immediately after amino acid 25. AMA1TMCTD was digested with XhoI and PacI and cloned in pMSAG1Ty vector, originating pMSAG1tyAMA1TMCTD, and the other PCR products were digested with Nsi1 and Pac1 and cloned into the corresponding sites in pROP1

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vector, originating pROPhisAMA1, pROPhisAMA1ΔTM-CTD and pROPhisAMA1ΔCTD (Soldati et al., 1998). The AMA1 promoter was amplified with primers 2462 and 2463 and cloned between Kpn1 and Nsi sites in the pROP1 vectors, originating pAhisAMA1, pAhisAMA1ΔTM-CTD and pAhisAMA1ΔCTD, or between Kpn1 and Nsi1 sites in the pMSAG1Ty vector expressing AMA1 TM and CTD, originating pAS1tyAMA1TM-CTD. Generation of plasmid pSK+A/AMA1-Flag for expression of Flag-tagged TgAMA1 in

the ama1koi parasites has been described elsewhere (Parussini et al, submitted). For bacterial expression, DNA fragments corresponding to TM-CTDs of TgMIC2, TgMIC12, TgMIC8,For TgMIC8.2, Peer TgMIC6, TgMIC6ReviewW/A, TgAMA1 and TgMIC16 were amplified from tachyzoite cDNA by PCR using the primers 176 and 177, 1599 and 710, 325 and 326, 1832 and 1833, 211 and 212, 211 and 3100, 1948 and 1949 and 2001 and 2002, respectively (supplementary table 1). PCR products were purified using the Easy Pure-DNA Purification Kit (Biozym). MIC2TMCTD, MIC6TMCTD and MIC6W/ATMCTD were digested with EcoRI and SalI, MIC12 TMCTD and AMA1 TMCTD with EcoRI and XhoI, MIC8TMCTD with BamHI and MIC8.2TMCTD and MIC16TMCTD with BamHI and XhoI. Each fragment was cloned into the corresponding sites in the pGEX4T1 vector to generate N- terminal GST fusions.

Mutated constructs To mutate the motif FW to AA on the AMA1CTD of the plasmid pGEX4T1-AMA1TMCTD the primers 1830 and 1831 were used in a site-directed mutagenesis reaction using the commercial QuikChange II Site-Directed Mutagenesis Kit (Stratagen) according to the manufacturer’s instructions. Similarly, the residues AGG, YTG, AG or AGGI were mutated to ILV, VL or VVLV in the TMDs of MIC8, MIC8.2 and MIC16 in the plasmids pMSAG1tyMIC8TMCTD, pMSAG1tyMIC8.2TMCTD and pMSAG1tyMIC16TMCTD, respectively, using the primers 2003 and 2004, 2005 and 2006 or 2226 and 2227, respectively. To mutate F519W520 to AA on the AMA1 cytosolic tail in pSK+A/AMA1- Flag, primers TgAMA1FW/AA.f and TgAMA1FW/AA.r (Supplementary Table 1) were used for site directed mutagenesis as described above, generating the vector pSK+A/AMA1FW/AAFlag.

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Protein expression and purification pGEX-4T1 vectors encoding the MIC2, MIC12, MIC6, MIC8, MIC8.2, MIC16 or AMA1 TMCTD GST fusion proteins were transformed into the E. coli BL21 strain (Novagen, Madison, WI). Protein expression was induced using 1mM isopropyl-beta-d- thiogalactopyranoside (IPTG) for four hours at 37 ºC. Bacterial pellets were resuspended in 1X PBS supplemented with 1mg/ml of Lysozym, 10µg/ml DNAse, 20µg/ml RNAse and 1mM PMSF, and were allowed to homogenize for 30 minutes at 4 ºC, following which, cells were disrupted by 5 consecutive cycles freeze/thaw. After centrifugation (30 minutes, 30 000RPM),For the supernatant Peer containing Review the soluble GST-fusions was collected, and purified using GSH-beads (Glutathione Sepharose 4 Fast Flow, Amersham) according to the manufactures advice.

GST-Fusion protein pull-down experiment Glutathione-sepharose beads were incubated with 0.5mg of GST fusion proteins or GST for 1 hr at 4 ºC. Beads were washed twice with PBS and once with buffer XB (50mM

KCL, 20mM Hepes, 2mM MgCL2, 0.2mM EDTA, 0.2% Tween2, pH7.7), prior to the addition of approximately 400µg of recombinant aldolase (Sigma). Aldolase was incubated with the GST-fusion purified proteins for 4 hours at 4 ºC, washed five times with buffer XB, and bound proteins were eluted using SDS–PAGE-loading buffer supplemented with 100mM DTT.

Parasite transfection and selection of clonal stable lines Parasites transfection was performed by electroporation as previously described (Soldati & Boothroyd, 1993). The HXGPRT gene was used as a positive selectable marker in the presence of mycophenolic acid (25 mg/ml) and xanthine (50 mg/ml) as described previously (Donald et al., 1996). Briefly, freshly released parasites (5×107) of the RHhxgprt strain were resuspended in cytomix buffer in the presence of 50-80µg of linearized plasmid carrying the selectable marker gene and the expression cassette containing the DNA sequences. Transfection of ama1koi conditional knockout parasites was carried out by electroporation using 2.6x107 freshly released parasites, 100 µg of pSK+A/AMA1-Flag or pSK+A/AMA1FW/AAFlag, and 6 µg of pDHFR*.Tsc3ABP (Roos

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et. al., 1997) plasmids. Parasites were electroporated at 2 kV, 25 mF, 48 V using a BTX electroporator (Harvard biosciences, Holliston, MA, USA) before being added to a monolayer of HFF cells in the presence of mycophenolic acid/xanthine. Selection with 1µM pyrimethamine was initiated 24 hr later and continued for 7-10 days, after which resistant clones were isolated by limiting dilution.

Western blotting For AMA1 Western blots, 108 parasites were harvested after complete lysis of the host cell monolayer andFor extracted Peerfor 30 min at 4Review °C in 1 ml of RIPA buffer (50 mM Tris- HCl, pH 7.5; 1 % (vol/vol) Triton X-100; 0.5 % (vol/vol) sodium deoxycholate; 0.2 %

(wt/vol) SDS; 100 mM NaCl2; 5 mM EDTA) containing protease inhibitors (Sigma P8340), added directly to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer and boiled for 10 min. For all other samples, extracts from 2 x 107 parasites were prepared in 1x PBS by five consecutive freeze/thaw cycles with intermediate homogenization, following two consecutive sonications, and the suspension was boiled in SDS–PAGE loading buffer containing 100mM DTT. SDS-PAGE was performed using standard methods. Separated proteins were transferred to a nitrocellulose membrane using a semidry electroblotter. Western blots were performed using anti-Ty1 mAb (Bastin et al., 1996), anti-AMA1 mAb (B3.90, Donahue et. al., 2000), anti-myc mAb (Mital et al, 2005) and anti-Flag mAb (Sigma F3165) in 5% non-fat milk powder in 1X PBS. As secondary antibody, a peroxidase-conjugated goat anti-mouse or anti-rabbit antibody was used (Molecular Probes, Paisley, UK). Bound antibodies were visualized using either the ECL system (Amersham Corp) or with SuperSignalTM West Pico chemiluminescent substrates (Pierce).

IFA and confocal microscopy All manipulations were carried out at room temperature. Intracellular parasites grown in HFF seeded on glass coverslips were fixed with 4% paraformaldehyde (PFA) for 10 minutes or 4% PFA-0.002% Glutaraldehyde for 10 minutes. Following fixation, slides were quenched in 1X PBS-0.1M glycine. Cells were then permeabilized in 1X PBS-0.2 % Triton-X-100 (PBS/Triton) for 20 minutes and blocked in the same buffer

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supplemented with 2% FCS (PBS/Triton/FCS). Slides were incubated for 60 minutes with the primary antibodies anti-GAP45, anti-MIC4 (Brecht et al., 2001) anti-Myc, anti- GRA3 (kindly provided by JF Dubremetz) or anti-Ty1 diluted in PBS/Triton/FCS, washed and incubated for 40 minutes with Alexa488- or Alexa594-conjugated goat anti- mouse or goat anti-rabbit IgGs diluted in PBS/Triton/FCS. DAPI staining was performed with a concentration of 0.1mg-DAPI/mL 1XPBS for 5 minutes incubation before slides were mounted in Fluoromount G (Southern Biotech) and stored at 4°C in the dark. Micrographs were obtained on a Zeiss Axioskop 2 equiped with an Axiocam color CCD camera. Images whereFor recorded Peer and treated Review on computer through the AxioVision™ software. Confocal images were collected with a Leica laser scanning confocal microscope (TCS-NTDM/IRB) using a 63 Plan-Apo objective with NA 1.40. Optical sections were recorded at 250 nm per vertical step and four times averaging.

Cell invasion assays Comparison of different T. gondii strains for invasion efficiency was done using an RH- 2YFP strain as internal standard. A confluent 60mm-dish of human foreskin fibroblasts was heavily infected with a mixture of the strain of interest and RH-2YFP parasites. Some hours later the dish was washed to remove any non-invaded parasites. Two days later parasites egressed from their host cells and were collected by centrifugation at 240g, RT for 10 minutes and resuspended in 5ml culture medium (DMEM complemented with 2mM L-Glutamine, 5% FCS and 25µg/ml Gentamicin) preheated to 37°C. From this suspension a 1:10 dilution was made in preheated medium, the ratio of non-YFP to YFP parasites was determined in a Neubauer chamber (ratio between 0.8 and 7) and 500µl were inoculated into a well on a 24-well IFA plate. Invasion was allowed to take place for 1h at 37°C, then the wells were washed two times in CM-PBS (1 mM CaCl2, 0.5 mM

MgCl2 in PBS) and refilled with fresh medium. The plate was incubated for another 24- 32 hours in order for the parasites to devide. Afterwards cells were fixed with 4% paraformaldehyde for 20 min, followed by 3 min incubation with 0.1 M glycine in PBS to quench the reaction and subjected to an indirect immunofluorescence assay (IFA). Fixed cells were permeabilised with 0.2% Triton-X100 in PBS for 20 min and blocked in 2% bovine serum albumin, 0.2% Triton-X100 in PBS for 20 min. The cells were then

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stained with rabbit anti-TgMLC antibodies followed by Alexa 594 goat anti-rabbit antibodies (Molecular Probes). Total number of parasite vacuoles and RH-2YFP parasite vacuoles were counted on 20 microscopic fields on each IFA-slide with a minimum of 750 vacuoles in total per slide. Only vacuoles containing at least two parasites were counted. The ratio of YFP to non-YFP vacuoles was calculated and compared to the ratio obtained from live parasites at the beginning of the experiment. Each experiment has been repeated six times. Alternatively, host cell invasion was measured using a laser scanning cytometer-based assay (Mital et al.,For 2006). Briefly, Peer parasites grownReview for 36 hr in the presence of ATc were harvested, added to HFF monolayers and incubated at 37°C. One hr post-infection, the coverslips were fixed, blocked, and labeled with an anti-SAG1 antibody (mAb GII-9; Argene, North Massapequa USA) followed by an R-phycoerythrin-conjugated secondary antibody ("orange," DAKO, Carpenteria USA). Samples were then permeabilized, blocked, and labeled with anti-SAG1 followed by an Alexa647-conjugated secondary antibody ("red," Molecular Probes). Samples were analyzed on a CompuCyte Laser Scanning Cytometer equipped with a BX50 upright fluorescence microscope (Olympus America, Melville USA), 20X objective (N.A. 0.5), argon ion (488 nm) and helium/neon (633 nm) lasers, and three filter blocks/photomultiplier tubes (530-555 nm [green], 600- 640 nm [orange], and 650 nm [long-red]). Data were acquired and analyzed using Wincyte 3.4 Software (CompuCyte, Cambridge USA). Red parasites were counted to determine the total number of parasites per field. The number of orange, extracellular parasites was counted and subtracted from the total to calculate the number of invaded parasites per field. One-way ANOVA and Dunnett’s Multiple Comparison post-test were used to determine the significance of differences between groups. P values of less than 0.05 were considered significant.

Gliding motility assay Freshly egressed tachyzoites were filtered, pelleted, and resuspended in Calcium-Saline containing 1µM of ionomycin. The suspension was deposited on coverslips previously coated with Poly-L-Lysine (3 hr at RT). Parasites were fixed with PAF/GA and IFA using the α-SAG1 antibody was performed to visualize the trails.

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Acknowledgments

We thank Anne Kelsen for providing expert technical support. This work is part of the activities of the BioMalPar European Network of Excellence supported by a European grant (LSHP-CT-2004-503578) from the Priority 1 "Life Sciences, Genomics and Biotechnology for Health" in the 6th Framework Program, from supports to DS by the Swiss National FoundationFor and Peer the Howard HughesReview Medical Institute. Additional funding was provided by USPHS grant AI063276 (GEW). JMS is a recipient of the EU-funded Marie Curie Action MalParTraining (MEST-CT-2005-020492), “The challenge of malaria in the post-genomic Era”.

References

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Functional analysis of the leading malaria vaccine candidate AMA-1 reveals an essential role for the cytoplasmic domain in the invasion process. PLoS Pathog 5: e1000322. Urban, S. & M. Freeman, (2003) Substrate specificity of rhomboid intramembrane proteases is governed by helix-breaking residues in the substrate transmembrane domain. Mol Cell 11: 1425-1434. Witcombe, D. M., D. J. Ferguson, S. I. Belli, M. G. Wallach & N. C. Smith, (2004) Eimeria maxima TRAP family protein EmTFP250: subcellular localisation and induction of immune responses by immunisation with a recombinant C-terminal derivative. Int J Parasitol 34: 861-872. Zheng, B., A. He, M. Gan, Z. Li, H. He & X. Zhan, (2009) MIC6 associates with aldolase in host cell invasion by Toxoplasma gondii. Parasitol Res. (2):441-5. Zhou, X. W., M. J. Blackman, S. A. Howell & V. B. Carruthers, (2004) Proteomic analysis of Forcleavage events Peer reveals a dynamicReview two-step mechanism for proteolysis of a key parasite adhesive complex. Mol Cell Proteomics 3: 565-576.

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Figure Legends

Figure 1 A. Schematic representation of the four major microneme complexes in T. gondii, as well as of TgMIC12 and TgMIC16, when in the micronemes (top) and on the parasite’s surface (bottom). Represented is the currently known composition of the complexes, including the various proteolytic cleavage events as demonstrated in (Carruthers et al., 2000, Harper et al., 2006) for TgMIC2/M2AP, in (Opitz et al., 2002, Meissner et al., 2002, SawmynadenFor et al., 2008)Peer for TgMIC6/MIC1/MIC4, Review in (Meissner et al., 2002, Cerede et al., 2005) for TgMIC8/MIC3, in (Hehl et al., 2000, Alexander et al., 2005, Straub et al., 2008, Besteiro et al., 2009) for TgAMA1/RON2/RON4/RON5/RON8, in (Opitz et al., 2002) for TgMIC12 and in this study for TgMIC16. B. Amino acid sequence alignment of the TMDs and CTDs of TgMIC2, TgMIC6, TgMIC12, TgAMA1, TgMIC16, TgMIC8 and TgMIC8.2. Boxed in grey are the TMDs, in green the putative rhomboid cleavage sites and in pink the motifs for traffic to the micronemes. The tryptophan signature residue boxed in yellow and the acidic residues boxed in blue are both involved in binding to aldolase.

Figure 2 A. Amino acid sequences alignment of the T. gondii TgMIC16 (EU791458) and the homologous gene in E. tenella (SNAP00000003913). The predicted annotations of N. caninum and E. tenella genes await experimental confirmation. Boxed in light blue is the putative signal peptide, in grey the putative TMD and in light green the rhomboid cleavage motif. The strictly conserved residues are boxed in yellow. The six putative TSR domains are indicated. The TMD prediction was performed with the program TMHMM. B. Double immunofluorescence analysis by confocal microscopy of intracellular parasites transiently expressing MIC16-Ty carried out with anti-Ty-1 (green) and anti- MIC4 (red), a micronemal marker. Scale bars indicates 10µm.

Figure 3

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Localization of several stably transfected chimeras in the parasite using double immunofluorescence analysis and confocal microscopy. Anti-GRA3 or GRASP-YFP were used as dense granules and Golgi markers, respectively. Scale bars indicate 5µm. pMS1tyMIC16TM-CTD and pMS1tyMIC8TM-CTD expressing parasites were stained with anti-Ty-1 (in green) and anti-GRA3 (in red). pMS1tyMIC8.2TM-CTD was stained with anti- Ty-1 (in red) and co-localized with expression of GRASP-YFP (in green). The nucleus was stained with DAPI (in blue).

Figure 4 For Peer Review Localization of several stably transfected chimeras in the parasite using double immunofluorescence analysis and confocal microscopy and schemes of the different constructs. Anti-MIC4 was used as micronemal marker. Scale bars indicate 5µm. pAS1tyAMA1TM-CTD expressing parasites were stained with anti-Ty-1 (in green) and co- localized with anti-GRA3 (in red). pAhisAMA1ΔTM-CTD, pAhisAMA1ΔCTD and pAhisAMA1 expressing parasites were stained with anti-his (in green) and co-localized with anti-MIC4 (in red).

Figure 5 A Subcellular distribution of the SAG1-TM-CTD mutant chimeras by IFA and documented by confocal microscopy. Anti-GAP45 antibodies (in red), Anti-GRA3 (in red) and GRASP-YFP (in green) were used as IMC, DGs and Golgi markers, respectively. The nucleus was stained with DAPI (blue). pMS1tyMIC16mTM-CTD, pMS1tyMIC8mTM-CTD (in green) and pAS1tyAMA1mTM-CTD (in red) expressing parasites were stained with anti-Ty-1. Scale bars indicate 5µm. B-E Analysis of the cleavage events in the different chimeras. On the top, is shown the TMD of each TM-MIC in the original (wt) and mutated chimera (mut) and the residues mutated in the rhomboid cleavage motif are boxed. Below are western-blot analysis of lysates from parasites stably expressing the different pMS1tyMICTM-CTD fusion constructs. Indicated by arrows are the migration of the full (f) and shed (s) forms with indication of the molecular weight. B Migration of pMS1tyMIC16TM-CTD (wt) and pMS1tyMIC16mTM-CTD (mut). C Migration of pMS1tyMIC8TM-CTD (wt) and

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pMS1tyMIC8mTM-CTD (mut). D Migration of pMS1tyMIC8.2TM-CTD. E Migration of pAS1tyAMA1TM-CTD. The proteins were detected with anti-Ty-1. Molecular weight markers are indicated in kDa.

Figure 6 In vitro aldolase binding assay. A. GST-pull-down assays were performed with GST alone or with the GST fusion constructs GST-MIC2CTD, GST-MIC6CTD, GST-MIC8CTD, GST-MIC8.2CTD, GST- MIC12CTD, GST-AMA1ForCTD andPeer aldolase alone. Review GST-MIC16CTD and GST-MIC6W/ACTD were tested separately with GST-MIC2CTD. Note that GST-MIC8CTD migration is aberrant, as indicated by an asterisk. B. GST-pull-down assays were performed with GST alone or with the GST fusion constructs GST-MIC2CTD, GST-MIC6CTD, GST-MIC6W/ACTD and aldolase alone. SDS-page gel were stained with coomassie-blue. The migration of aldolase, GST and GST-MIC2CTD is indicated by arrows.

Figure 7 Invasion and gliding assays of mic6ko and complemented strains. The penultimate tryptophan residue in the CTD of TgMIC6 is not critical for productive invasion. A. IFA documented by confocal microscopy of intracellular parasites deficient in TgMIC6 (mic6ko) and complemented with TgMIC6-Ty (mic6ko/MIC6Ty) or MIC6W/ATy (mic6ko/MIC6W/ATy). Co-localization with the micronemal marker TgMIC4 was assessed using antibodies anti-TgMIC4 (red) and anti-Ty-1 (green). Scale bars indicate 5µm. B. Quantification of the relative invasion efficiency of the four parasite strains as determined by a cell invasion assay normalized to RH-YFP strain co-cultivated and used as internal standard for parasite fitness. Error bars indicate standard deviations. C. Gliding assays of mic6ko and mic6ko/MIC6W/ATy parasites. The trails were stained with anti-SAG1. The arrow indicates a trail.

Figure 8

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Invasion assay of TgAMA1 depleted parasites and complemented strains. Residues F519W520 within the CTD of TgAMA1 are critical for productive invasion. A. IFA analysis of AMA1FW/AAFlag expressed in the AMA1 conditional knockout

(ama1koi) parasites shows proper colocalization of the mutant protein with the microneme marker protein, M2AP. Two independent AMA1FW/AAFlag-expressing clones are shown (AMA1FW/AAFlag-1 and -2), as are the corresponding DIC images (left panels). Scale bars indicate 5 µm. B. Invasion assay. Host cell invasion by AMA1Flag, AMA1FW/AAFlag-1, FW/AA AMA1 Flag-2,For and ama1ko Peeri parasites, eachReview grown in the presence of ATc for 36 h, was measured 1 h postinfection using the laser scanning cytometer-based assay. The average number of intracellular parasites per scan area is presented (two independent experiments, two replicates within each experiment), with error bars representing the SD of the mean between experiments. The invasion levels for each parasite population (expressed relative to the AMA1Flag parasites) are shown. The total number of intracellular parasites counted is also listed below each sample. * p < 0.05, relative to AMA1Flag.

Supplementary material

Figure S1 Alignment of the six putative TSR domains of MIC16 with the six TSR domains of human F-spondin (HsF.spond.TSR.1441-499, HsF.spond.TSR.2500-555, HsF.spond.TSR.3556-

611, HsF.spond.TSR.4612-666, HsF.spond.TSR.5667-725, HsF.spond.TSR.6753-897), the six

TSR domains of T. gondii MIC2 (TgMIC2.TSR.1270-338, TgMIC2.TSR.2339-402,

TgMIC2.TSR.3403-469, TgMIC2.TSR.4470-530, TgMIC2.TSR.5531-597, TgMIC2.TSR.6598-

650)- The cysteine, arginine and tryptophan signature residues of a TSR domain are boxed in yellow. Most TSR type I domains fall into two groups, the TSR domains from human TSP-1 are representative for group I, the TSR domains of human F-spondin are representative of group II (Tan et al., 2008). From the six hypothetical TSR type I domains identified, four (TSR-1/-2/-4/-6) resemble the group I, and two (TSR-3/-5) the

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group II. Domains TSR-1 and TSR-3 are quite divergent from those from other organisms but it has been noticed previously that TSR domains in Apicomplexa can be of divergent nature (Tossavainen et al., 2006).

Figure S2 Analysis of the cleavage events in the different chimeras and comparison of the size of the shed product with that of pMS1tyMIC12TM-CTD (MIC12) (Opitz et al., 2002). On the top, scheme of the chimera constructs. Below western blot analysis of lysates from parasites stably expressingFor pMS1ty PeerMIC16TM Review-CTD (MIC16), pMS1tyMIC8TM-CTD (MIC8), pMS1tyMIC8.2TM-CTD (MIC8.2) and pAS1tyAMA1TM (AMA1). The proteins were detected with anti-Ty-1. Molecular weight markers are indicated in kDa.

Figure S3 Schematic representation of the GST fusion constructs used in aldolase pull down experiment. The residues mutated in GST-AMA1FW/AA-CTD and GST-AMA1W/A-CTD are boxed in blue.

Figure S4 Alignment of amino acid sequences of TM-CTDs of several T. gondii (upper alignment) and P. falciparum micronemal proteins. Boxed in grey are the TMDs, in green the putative rhomboid cleavage sites. The tryptophan residue is indicated in yellow and the acidic residues are boxed in blue. These residues are involved in binding to aldolase.

Table 1 TS – this study; ND - not determined.

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TM-MIC MIC2 MIC12 MIC6 MIC8 MIC8.2 AMA1 MIC16

Role in Motility, ND Host cell Rhoptry CTD MJ ND motility and Host cell binding secretion complement formation, invasion binding s MIC8 Rhoptry For Peer Review secretion Trafficking ++ + + - - - - determinant (Di Cristina et (Opitz et al., (Reiss et al., TS TS TS TS al., 2000) 2002) 2001) Binding to + + + - - + - aldolase (Buscaglia et al., TS TS TS TS TS TS 2003, Jewett & Sibley, 2003, Heiss et al., 2008) Substrate + + + + - + + for (Opitz et al., (Opitz et al., (Opitz et al., TS TS (Howell et al., TS rhomboid 2002, Brossier 2002, Brossier 2002, Brossier 2005) et al., 2003, et al., 2003, et al., 2003, cleavage Urban & Urban & Urban & Freeman, 2003, Freeman, Freeman, Brossier et al., 2003, Brossier 2003, Brossier 2005, Dowse et et al., 2005, et al., 2005, al., 2005) Dowse et al., Dowse et al., 2005) 2005)

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Supplementary Table 1 – primers and constructs used in this stud

Primer name No. Enz Sequence Resulting plasmid

TgMIC2 TMCTD 176 EcoRI CGGAATTCGCCAGTTACCACTACTATTTGA pGEX4T1-MIC2 TMCTD TgMIC2 TMCTD 177 SalI AACTGCAGGTCGACCCTACTCCATCCACATATCACT pGEX4T1-MIC2 TMCTD

TgMIC12TMCTD 1599 XhoI CCGCTCGAGTTAGTCCATGTCTGCCC pGEX4T1-MIC12 TMCTD TgMIC12TMCTD 710 EcoRI CGGAATTCGCCGTGTACGCATCCCAAGGTG pGEX4T1-MIC12 TMCTD

TgMIC6 TMCTD 211 EcoRI CGGAATTCGTTGCATACATGAGAAAGAGTGGGAGC pGEX4T1-MIC6 TMCTD TgMIC6 TMCTD 3100 SalI CTGCAGTCGACCTTAATCCGCTGTTTTGCTATCCAAAT pGEX4T1-MIC6 W/ATMCTD

TgMIC6 TMCTD 212 SalI CTGCAGTCGACCTTAATCCCATGTTTTGCTATCCAAAT pGEX4T1-MIC6 TMCTD

TgMIC8 TMCTD 325 BamHI GGCGGATCCGGAGGAATTTCTTACGCCAGAAACA pGEX4T1-MIC8 TMCTD TgMIC8 TMCTD 326 BamHI ForCGCGGATCCTTAGGACCAGATACCGCCCGA Peer Review pGEX4T1-MIC8 TMCTD TgMIC8 TMCTD 1887 SalI CCGGGTCGACAACAAAGGTCGATATTCGAAAG pMSAG1tyMIC8 TMCTD TgMIC8 TMCTD 1888 PacI GGCTTAATTAAGACCAGATACCGCCCGAAGG pMSAG1tyMIC8 TMCTD TgMIC8 TMCTD 2003 - GGTGTGTAGCCTTGTTGGGTATTATAATCCTAGTAATTTCTTACGCCAG pMSAG1tyMIC8m TMCTD AAACAGAGG TgMIC8 TMCTD 2004 - CCTCTGTTTCTGGCGTAAGAAATTACTAGGATTATAATACCCAACAAG pMSAG1tyMIC8m TMCTD GCTACACACC

TgMIC8.2 TMCTD 1832 BamHI CCGGGATCCTGGTTCTCGAATTCTCAAGAAGAACAAAC pGEX4T1-MIC8.2 TMCTD TgMIC8.2 TMCTD 1833 XhoI GGCCTCGAGTTATGACCACATTGAGCCTGACGGG pGEX4T1-MIC8.2 TMCTD TgMIC8.2 TMCTD 1889 SalI CCGGGTCGACGATAGCGGGAGCGACAACTCATC pMSAG1tyMIC8.2 TMCTD TgMIC8.2 TMCTD 1890 PacI GGCCTTAATTAAGACCACATTGAGCCTGACGGG pMSAG1tyMIC8.2 TMCTD TgMIC8.2 TMCTD 2005 - GGGAGCGACAACTCATCTATCCTTGTACTGGCAACGGGAGCCGTG pMSAG1tyMIC8.2m TMCTD TgMIC8.2 TMCTD 2006 - CACGGCTCCCGTTGCCAGTACAAGGATAGATGAGTTGTCGCTCCC pMSAG1tyMIC8.2m TMCTD

TgAMA1TMCTD 1948 EcoRI GCGAATTCGGCTGCTACTTCGCGAAGAG pGEX4T1-AMA1 TMCTD TgAMA1TMCTD 1949 XhoI CCGCTCGAGCTAGTAATCCCCCTCGACCATAAC pGEX4T1-AMA1 TMCTD TgAMA1 1830 - CATGCAAGAGGCTGAACCGTCGGCTGCGGATGAGGCAGAGGAGAAC pGEX4T1-AMA1 TMCTD(WF/AA) TgAMA1 1831 - GTTCTCCTCTGCCTCATCCGCAGCCGACGGTTCAGCCTCTTGCATG pGEX4T1-AMA1 TMCTD(FW/AA) TgAMA1 1079 XhoI CGGGATCCCCTCGAGACTGCGTTGATCGCTGGACTCGC pASAG1tyAMA1 TMCTD TgAMA1 1080 PacI CCGCAATTGTTAATTAACTAGTAATCCCCCTCGACCATAAC pASAG1tyAMA1 TMCTD TgAMA1 2225 NsiI CCGATGCATGAAGTTGATGGCACATTATACCGG pROPhisAMA1 TgAMA1 2247 NsiI GCCATGCATAGCTCAAGCACAAGGTCTCGCG pROP1hisAMA1ΔTM- CTD/ΔCTD TgAMA1 2248 PacI CCGTTAATTAAGCGAAGTAGCAGCCTCCTCCTAG pROPhisAMA1ΔTM-CTD TgAMA1 2249 PacI CCGTTAATTAAGCAGTGTTAGAGCCACATTCATTTTGTTCG pROPhisAMA1ΔCTD TgAMA1 2462 KpnI GCCGGTACCGCGCTACCACGATTCACTGGGTGC pA constructs TgAMA1 2463 NsiI CGGATGCATTGTGCTTGAGCTGAGTCCCGATGCG pA constructs TgAMA1FW/AA.f - - GCAAGAGGCTGAACCGTCGGCTGCGGATGAGGCAGAGGAGAAC pSK+A/AMA1FW/AAFlag TgAMA1FW/AA.r - - GTTCTCCTCTGCCTCATCCGCAGCCGACGGTTCAGCCTCTTGC pSK+A/AMA1FW/AAFlag

TgMIC16 1969 MfeI CAATTGCGTCGCGTTTTCTACAGGAGTTGATGG pTUB8MIC16Ty TgMIC16 TMCTD 1970 PacI GGCTTAATTAAAGGTAGTTGTCCCGTGTCCG pMSAG1tyMIC16TMCTD TgMIC16 1971 NsiI CCATGCATAGAGGTAGTTGTCCCGTGTCCG pTUB8MIC16Ty TgMIC16 TMCTD 1972 SalI CGGGTCGACAGCATTTTCTCCAATGGAGTCACTTACG pMSAG1tyMIC16TMCTD

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TgMIC16 TMCTD 2001 BamHI GGCGGATCCGGACGCAAGTTTTATCGAGCTCTG pGEX4T1-MIC16 TMCTD TgMIC16 TMCTD 2002 XhoI GGCCTCGAGTTAGAGGTAGTTGTCCCGTGTCC pGEX4T1-MIC16 TMCTD TgMIC16 2226 - GTCGTACTAGTTGGGCTGGTAGTTGTCATTGG pMSAG1tyMIC16mTMCTD TgMIC16 2227 - AACTAGTACGACAACTGCGTAAGTGACTCCATTG pMSAG1tyMIC16mTMCTD

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1 3. Study of TgROM4 as a candidate to the MPP1 activity

3.1 Insight into the role of the conserved C-terminus domain of the large apicomplexan rhomboids PfROM4 possesses the seven TMDs characteristic of most eukaryotic rhomboids but also atypical extended N- and C-terminal tails. While the N-terminal end is predicted to be cytoplasmic, the C-terminal end is extracellular and it is therefore exposed at the surface of the parasite, which, together with the fact that it is a highly conserved region in all Plasmodium orthologues (figure 3.1), suggests a role in substrate recognition, enzymatic activity and/or protein folding. Alignment of the C-terminal tail of PfROM4 with all the apicomplexans paralogues, reveals that a pattern of 5 cysteine residues is strictly conserved across the ROM4 of all species as well as in TgROM5, but not in EtROM4 or TgROM4, which have the third and last cysteine residues replaced by a glycine and a leucine residue, respectively (figure 3.1). The existence of another conserved set of cysteine residues in the extracellular loops connecting the several transmembrane domains suggests the potential formation of intramolecular disulfide bridges. If that is the case, the C- terminus could be critical for maintenance of the protein structure integrity and, indirectly, for its function.

M M

M D - - P K G - S N Q - - - - - G G A - G G D 0 0 0 0 0 0 I A A K - - V P - - E I M ------Y Y A K Y Y 4 2 6 0 4 8 T V K N - - - P - - H N R ------K R L L M F

1 4 5 7 8 2 H H G I - - - P - - L D E ------T A S S S A

L L R L - - - I - R I E ------E S G G G G * ------A A A R F - - - - R T R ------E P S S S S

D D G G S - - - - P N Q - -

- - - - - R L S S S S ------T N G S E - - - - R R P ------R D G G G G

S K K - - - A - S Y S - - - - S L Y F G

I I V V ------H - K - - - H K - S N S - - - - A A A L L T T T T

- E - - - V K - S D E - - - - S S L S A V V V I

0 0 0 0 0 0 K - D - G - K K N S V F - - - - E E E S V G G S T 3 1 5 9 3 7 N - G - K - N Q V S K E - - - - E D E G G - - - - 1 4 5 6 8 2 A - N - E - * A I E S P H - - - - F L L L L C C C C

I - I - I - L A E A S S - -

- - Q N N N N P P P P ------Q - S - I - Q Q R G L S - - - - R Y Y Y H D D D N

K - F - N - K G Q L S L - - - - T * E E E E C C A A

------K - S - P - P L S G K K - - - - Q

V V V V C C C C

R R

E - N G I A G D R R - - - - G

A A A A A A G A ------Y E K - E E A R K G F R -

- - - Y

S S S S G G G G

N E

K - P A V S K P N R - - - - V

L L L L Y Y Y Y 0 0 0 0 0 0 E R E - V N A S L T E I - -

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L L L L G G G G 2 0 4 8 2 6 G E D - E V V S K A G A - - - - A

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G P K G E K R P

K E R F F F W K N Q E 0 0 0 0 0 K T K E 0 E E R R Q R K A E E *

L L L L N K G L S K S 1 9 3 7 1 5 E G S P E R N S S R G I L L

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N - - A

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L - T E E * K

P P C C C C L L L L L L R F N - - K ------P T - A L A

A P I I I T R R R R K G G D F - - -

S - K D T S

S T

Q Q Q Q G G G G G R L N I ------R S R T S

- V

M I I I A N N N N N N N V N - - -

- P F A L A

L A F F F F Q Q Q Q K K V G I - - - 0 0 0 0 0 - A I H M 0 M

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- F F F C M L L L T T - - P - N ------L D - - T R

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- T T

- S G T S S A A A S S V V Q V S - A - G P I -

A A

- S T T T T W S A W W I I V I R S K - P A S ------* -

F F

- T T T T F F F V F F Q D R T K K K - T A N - 0 0 0 0 0 0

G G

- C C C L L L V G G S S S L G K T I - 7 5 9 3 7 A T K - *

1 Y F

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G G

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------

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A K I - 6 4 8 2 6

H H - N N N N H H F S N N N I I L K K K -

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A A - T T T T A A N G N N N Q Q Q F T V -

A D R A

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D D - R R R K G G G A D D P P P P - - - - -

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- - - -

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- - - - -

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A T L E A

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- - - -

K T N N

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- - - 0 -

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A K E S D 0 0 0 0 0 0 G G H N A A A G W Y Y L G T F F F

8 - - - -

V - - A A L V F R 4 2 6 0 4 8 F V E T A L K K K F Y Y P A L L L

8 - - -

D - - V P T R S L - 3 4 6 7 1 M M R P G G S M M A V G G A V I I

- - -

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- - - -

- Q - - S T D V K V ------I G K K G G G W I I M V T Q W W W

- - -

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F F R K L P P S I F D D A A M L Y

- - - -

- I - - Q N G G E C ------I Q W Q S S S I I T P P S R I I A

- - -

- E - - S G L R D S

G A A I G V S S N G N N G G I I F

- - -

- - L - - A L N V G A ------F V C V L E D N R F I F F F L L L

- - -

- D - - T V V R N P

I M S G E E P Q I M P P P G T I A

- - -

0 - S - - A A S G I A 0 0 0 0 0 0 V V H I V V V V V V N N N V S S A

- - -

7 - F - - - N L S S A 3 1 5 9 3 7 V L R - K L L V A L L A V K A A A

- - - 8 - I - - - Y G D F E - 3 4 5 7 1 L V R - D D D V N L A P P P A S C

- - - -

S - - - T D R S A

I S P - G K V S F A I A A A T T L

- - - - - S - - - K T L E A ------M V G - F M M P P P P R R R I I G

- - - -

R - S - K R E K E

F C G - F R F R R R W W W W R R R

- - - - - K - N - A R R K L ------I F D - G G G G V V G G G I V V I

- - - - K - I -

S N E E E

I L G - E N K D C V V V L L I I V

- - - - E - I - - K S T D P ------V F G - D D D D I A A V V W W W W

- - - - A G A - V

G R G L

C W N - S S S C G G G C E G E E E

- - - - N S K - L 0 A S N R 0 0 0 0 0 0 C A K - G R S S D C A T A A R R R

- - - - K R K - R 6 R T I K 2 0 4 8 2 6 P P T - P P P P P K Q S C E L P F

- - - - P P P - 8 Q S M S K - 3 4 5 7 1 R S L - S T T R R L L L R H K K S

- - - - N G - - W A K F E

D P A - G G G G P T T N P - M M M

- - - - F G - - V S K S Q ------G - I I I I F S H H H I I - R K K

- - - - G G - - S G L K R

S -

T H F F S A L L I T S C C C -

- - - - T R K - A H D E L ------R - - K D H H A C N E L K K P S P -

- - - - N A N - S P K K E

P -

W W R R R R E S P W W P P P -

- - - - S V T - E S S E G ------E - - Y Y N D P E D E D Y Y R R R -

- - - - G G E - G A G D A

S -

E E - - - T S N D E E G G G -

- - - - M M M - E M L K S 0 0 0 0 0 0 S - 0 V I - - - L T N D E E E I V -

------E V R N G 5 1 9 3 7 1 L - 5 Y S - - - P V V G Y C D D E -

------K V S I G 8 - 2 4 5 7 E - 1 Y F - - - G I L I Y Y N N Q -

------A A S L S

R -

A P - - - P I I A A I I L G P

------I S G F G ------R - - F V - - - T K R Q F I R R R R

------R T G S G

G -

L - - - W L I I L N N M F L L

------L W K E R ------T - - - - - R D E A A A K K K K A A

------P V S K G

E - - - - G N D E

G G M M V W G G

------A M S K S ------Q - - - - R I L E - I L V I A V I L

------E - M E A P - - - - P E R V

L L A S Y F L L

4 4 4 4 5 4 4 4 4 4 4 5 4 5 5 4 4 4 4 5 4 4 4 5 4 4 4 4 5

M M M M M M M M M M M M M M M M M M M M M M M M M M M M M

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

R R R R R R R R R R R R R R R R R R R R R R R R R R R R R

b g t f g f k b g b g g f g g b f b g g f b g g g f b g g

P P P T T P P T T P P T T P P T T P P T T P P T T P P T E T

1 Figure 3.1 (previous page) Alignment of the large rhomboids from Plasmodium and Toxoplasma Alignment of P. falciparum ROM4 (PfROM4), P. berghei ROM4 (PbROM4), T. gondii ROM4 (TgROM4) and T. gondii ROM5 (TgROM5). The TMpred predicted TMDs are highlighted in grey. The catalytic serine is represented in yellow and is underlined and the conserved cysteine residues are represented in green and are underlined; these residues are also marked with stars. Identical residues are labelled in red and 75% conserved residues are labelled in blue.

In order to study the role of the conserved C-terminal domain for PfROM4 function, a series of deletion and chimeric mutants were created and tested in an in vitro cleavage assay. This assay was fist used to analyze the activity of D. melanogaster Rhomboid- 1 against Spitz (Lee et al., 2001) and consists in the expression of the substrate and rhomboid proteins in mammalian cells. Upon cleavage, the substrate C-terminus is secreted into the medium. Western-blot analysis of the cell lysates probe for expression of the rhomboid and substrate (figure 3.2).

rhomboid substrate HEK293T cells

cell lysates medium samples

Figure 3.2 – Schematic of the cell-based cleavage assay A monolayer of HEK-293T cells is transiently transfected with plasmids encoding a rhomboid protein and a substrate. If the rhomboid recognizes the substrate, cleavage takes place within the cells. 24h post-transfection, the cell and medium extracts are harvested and analysed by western-blot to detect expression of the proteins and cleavage of the substrate.

1 The role of PfROM4 and TgROM5 C-terminus for catalytic activity was accessed by expressing constructs lacking the C-terminal tail (PfROM4ΔCt and TgROM5ΔCt). To determine if the C-terminus plays a role in substrate specificity, and taking advantage of the fact that PfROM4 and TgROM5 present different substrate specificity, we tested cleavage by chimeras in which the C-terminus from different Plasmodium species and from TgROM4 and TgROM5 were exchanged (PfROM4-CtPbROM4, PfROM4-CtTgROM4 and PfROM4-CtTgROM5). Finally, a PfROM4 mutant in which the last conserved C-terminal cysteine has been mutated to an alanine residue was also expressed to determine if the pattern of 5 conserved C-terminal cysteines plays a role in activity (PfROM4C-A). The enzymatic activity of the different constructs was accessed against a chimeric substrate construct comprising the ectodomain of PfEBA175 and the TMD and C- terminal tail of PfAMA1 (AMA1-EBA175tm-ct), or a substrate expressing PfAMA1 (AMA1). These two constructs allowed testing the substrate specificity of the different rhomboid constructs because PfROM4 can cleave PfEBA175 and therefore

AMA1-EBA175tm-ct but not PfAMA1, and TgROM5 can cleave both proteins. When testing for rhomboid activity in a cell-based cleavage assay, it is necessary to assure that the expressed rhomboid and substrate proteins localize to the same intracellular compartment and can thus meet for cleavage to occur. Determination of the location of the expressed proteins showed that both substrate constructs localized mainly to intracellular compartments - Golgi, endosomes and plasma membrane (data not shown) - and all the rhomboid constructs were localized mainly to the ER and in a minor amount to the Golgi apparatus (data not shown). Cleavage can consequently occur either in the Golgi or during transit of the substrate proteins in the secretory pathway in route to the plasma membrane. Analysis of the catalytic activity of the different rhomboid constructs suggested that the C-terminus of TgROM5 and PfROM4 play a role in activity (PfROM4ΔCt and TgROM5ΔCt are inactive) (figure 3.3 and data not shown) and that this function is conserved within the same species (PfROM4-CtPbROM4 is active) (figure 3.3) but not inter-species (PfROM4-CtTgROM4, PfROM4-CtTgROM5, TgROM4- CtPfROM4 and TgROM5-CtPfROM4 are inactive) (figure 3.3 and data not shown). The C-terminal tail does not seem to be involved in determining substrate recognition and specificity (PfROM4-CtTgROM5 is unable to cleave AMA1, unlike TgROM5)

1 (data not shown) but might be important for the proper folding of the protease as the pattern of conserved C-terminal cysteines is essential for activity (PfROM4C-A is inactive) (figure 3.3).

1 2 3 4 5 6 7 8

αHA

AMA1 α

← * αAMA1

Figure 3.3 Western-blot of a cell-based cleavage assay In the upper and middle panels is shown expression of the rhomboids and substrate in the cell extracts, as detected by with αHA and αAMA1, respectively. In the lower panel is shown expression of the shed form in the medium extract as detected with αAMA1. 1: TgROM5wt, 2: TgROM5mut, 3: PfROM4wt, 4: PfROM4mut, 5: PfROM4ΔCt, 6: PfROM4-CtPbROM4, 7: PfROM4-CtTgROM5, 8: PfROM4C-A. A shed form correspondent to rhomboid cleavage (arrow) can be detected upon expression of TgROM5wt, PfROM4wt and PfROM4-CtPbROM4. PfROM4mut carries the catalytic serine substituted by an alanine residue and was used as negative control. The star indicates background cleavage by the cell metalloproteases. In the assay it was used the chimeric substrate AMA1- EBA175tm-ct.

3.2 ROM4-mediated cleavage of AMA1 switches Toxoplasma from an invasive to a replicative mode The results presented in the previous section revealed that the C-terminus of both PfROM4 and TgROM5 is important for activity, possibly due to a role in protein folding. The cell-based cleavage assays are an artificial situation and the relevance of the results obtained for the in vivo situation is debatable. It was therefore important to analyze the function of TgROM4 in the parasite. When this study was initiated, the conditional knockout of TgROM4 had yet to be reported and all attempts in the laboratory to generate a parasite strain conditionally

1 depleted for the enzyme were unsuccessful (Dowse, Sheiner and Soldati, unpublished). Previous results had also indicated that expression of an inactive mutant of TgROM4 in the parasite caused a dominant negative effect (Dowse and Soldati, unpublished). Taking the previous informations into account, we decided to study the function of TgROM4 with an inducible dominant negative mutant carrying the catalytic serine substituted by an alanine residue. Analysis of the phenotype of parasites stably expressing this dominant negative form revealed that TgROM4 does not play an essential role during invasion but mediates instead an essential function during intracellular growth linked to the processing of TgAMA1. The results reported here constitute the major achievement of this PhD thesis.

1 Elsevier Editorial System(tm) for Cell Manuscript Draft

Manuscript Number:

Title: ROM4-mediated cleavage of AMA1 switches Toxoplasma from an invasive to a replicative mode

Article Type: Research Article

Keywords: apicomplexan parasite; invasion; replication; rhomboid protease; intramembrane proteolysis

Corresponding Author: Dr. Dominique Soldati-Favre, PhD

Corresponding Author's Institution: University of Geneva

First Author: Joana M Santos, PhD student

Order of Authors: Joana M Santos, PhD student; David J Ferguson, Dr.; Michael J Blackman, Dr.; Dominique Soldati-Favre, Dr.

Abstract: Apicomplexan parasites invade host cells and immediately initiate a program of cell division. The extracellular parasite discharges several type I transmembrane proteins onto the surface to participate in motility and invasion. These proteins are shed by intramembrane proteolysis, a process associated with invasion but otherwise poorly understood. Functional analysis of Toxoplasma gondii rhomboid 4 (ROM4), a surface intramembrane serine protease, by conditional over-expression of a catalytically inactive form, produced a profound block in parasite replication. This block was reversible, not linked to invasion and could be rescued by transgenic expression of the cleaved cytoplasmic tail of Toxoplasma or Plasmodium apical membrane antigen 1 (AMA1). These results reveal a function for AMA1 in parasite replication distinct1 from its role in invasion, and establish a new concept in apicomplexan biology in which invasion proteins are concomitantly implicated in a checkpoint signaling the parasite to switch from an invasive to a replicative mode.

Suggested Reviewers: Oliver Billker Dr. Head of Research unit, Sanger Institute [email protected] expert on Plasmodium biology and genetic manipulation.

Photini Sinnis Dr. Head of Research unit, New York University School of Medicine, [email protected] expert on proteases in Plasmodium

Volker Heussler Dr. Prof., – Bernhard Nocht Institute [email protected] expert on Plasmodium biology and proteases.

David Sibley Dr. Prof., University of Washington *Manuscript (required for ALL submissions) Click here to view linked References

ROM4-mediated cleavage of AMA1 switches Toxoplasma from an invasive to a replicative mode

Joana M. Santos1,3, David J. P. Ferguson2, Michael J. Blackman3, Dominique Soldati- Favre1,4

1 Department of Microbiology, Faculty of Medicine, University of Geneva, 1 rue- Michel Servet, 1211 Geneva 4, Switzerland 2 Nuffield Department of Clinical Laboratory Science, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, England, UK 3 Division of Parasitology, MRC National Institute for Medical Research, Mill Hill, London NW7 1AA, UK 4Correspondence should be addressed to D.S-F. (e-mail: Dominique.Soldati- [email protected])

Running title: TgROM4 and TgAMA1: a checkpoint in parasite replication

1 1 SUMMARY

Apicomplexan parasites invade host cells and immediately initiate a program of cell division. The extracellular parasite discharges several type I transmembrane proteins onto the surface to participate in motility and invasion. These proteins are shed by intramembrane proteolysis, a process associated with invasion but otherwise poorly understood. Functional analysis of Toxoplasma gondii rhomboid 4 (ROM4), a surface intramembrane serine protease, by conditional over-expression of a catalytically inactive form, produced a profound block in parasite replication. This block was reversible, not linked to invasion and could be rescued by transgenic expression of the cleaved cytoplasmic tail of Toxoplasma or Plasmodium apical membrane antigen 1 (AMA1). These results reveal a function for AMA1 in parasite replication distinct from its role in invasion, and establish a new concept in apicomplexan biology in which invasion proteins are concomitantly implicated in a checkpoint signaling the parasite to switch from an invasive to a replicative mode.

HIGHLIGHTS:

• ddROM4S-A expression leads to a blockage in parasite replication • Blockage in replication is reversible and invasion-independent • ROM4 mediated cleavage of AMA1 signals the switch to a replicative mode • The dual function of AMA1 is likely conserved in Plasmodium

1 2 INTRODUCTION

Toxoplasma gondii is a member of the phylum Apicomplexa, which encompasses important human and animal pathogens including Plasmodium, responsible for malaria. Apicomplexan parasites invade host cells by a process that is powered by the parasite actomyosin system and involves the discharge of proteins from secretory organelles called micronemes and rhoptries. The micronemal proteins, which usually exist as complexes of soluble and single-span integral membrane proteins fulfill several non-redundant functions during invasion (Soldati-Favre, 2008). Apical membrane antigen 1 (AMA1), a micronemal protein found in all apicomplexan species, is crucial for formation of the moving junction (MJ), a transient zone of contact between the parasite and the host cell plasma membranes during invasion (Mordue et al., 1999). At the MJ, AMA1 associates with four rhoptry neck (RON) proteins (Alexander et al., 2005; Besteiro et al., 2009; Straub et al., 2009). In Toxoplasma, AMA1 and the micronemal adhesins microneme protein -2 (MIC2), -6 (MIC6), -8 (MIC8) and -12 (MIC12) are all cleaved during invasion within their transmembrane domain (TMD) by a protease activity termed microneme protein protease 1 (MPP1) (Carruthers et al., 2000; Donahue et al., 2000; Howell et al., 2005; Opitz et al., 2002), which releases them from the parasite surface. This shedding is essential for MIC2 function during invasion (Brossier et al., 2003). Shedding of AMA1 during invasion also occurs in Plasmodium. In that parasite, the majority of the shedding is mediated through juxtamembrane cleavage by a subtilisin-like protease called SUB2 (Harris et al., 2005; Howell et al., 2005; Howell et al., 2003) but some shedding also occurs through cleavage at an intramembrane site by a rhomboid protease (Howell et al., 2005). The role of AMA1 shedding in any apicomplexan genus is unknown. AMA1 behaves differently from other MICs, as it is not only detected anterior to the MJ in invading parasites (Carruthers et al., 1999a), but its C-terminal released product is readily detectable in the newly-invaded intracellular parasite (Donahue et al., 2000). Similarly, the C-terminal product of Plasmodium AMA1 cleavage is also stably carried into the invaded erythrocyte (Howell et al., 2005; Howell et al., 2003; Howell et al., 2001). Previous studies have indicated that MPP1 is a member of the rhomboid family, a group of broad substrate-specificity serine proteases that recognize helix-destabilizing residues within the TMD of their substrates (Urban and Freeman, 2003). Among the

1 3 six rhomboid-like proteins encoded in the Toxoplasma genome (Dowse and Soldati, 2005), only ROM4 and ROM5 localize to the parasite plasma membrane (Brossier et al., 2005; Dowse et al., 2005). ROM1 is expressed in micronemes, but a crucial role in invasion was recently excluded in T. gondii and P. berghei (Brossier et al., 2008; Srinivasan et al., 2009). ROM4 was recently shown to be responsible for the cleavage of MIC2, AMA1 and most likely MIC8, and to have an important but not essential function during invasion (Buguliskis et al., 2010). The P. falciparum orthologue of ROM4, PfROM4, is expressed at the surface of merozoites and is responsible for the intramembrane cleavage and shedding of the micronemal erythrocyte-binding protein EBA175 (O'Donnell et al., 2006), and possibly several other transmembrane adhesins expressed throughout the life cycle of the parasite (Baker et al., 2006). Immediately following invasion, the parasite comes to rest within a parasitophorous vacuole and at once initiates a program of cell division that leads to the production of new daughter cells equipped to egress and repeat the lytic cycle. The Toxoplasma tachyzoite undergoes replication by endodyogeny, in which the intracellular tachyzoite gives rise to two invasion-competent daughter cells by internal budding. Several repetitions of this cycle within the same host cell eventually produces intracellular vacuoles containing numerous new parasites. In contrast, in Plasmodium species and most other apicomplexans, replication is by schizogony, in which repeated rounds of mitosis give rise to a multinucleated syncitium (schizont) that undergoes cytokinesis only after nuclear division is complete. Despite these well- described apicomplexan division strategies, in no case are the signals that govern initiation of the replicative phase known. In this study we have exploited the destabilization domain system (ddFKBP) (Herm- Gotz et al., 2007) to conditionally express ROM4 in a novel strategy to dissect its function. We reasoned that expression of a catalytically inactive mutant of ROM4, able to recognize and bind to the substrate but unable to cleave it, would sequester the substrate from the endogenous protease and hence behave as a dominant negative.

Surprisingly, expression of the ddROM4S-A mutant had little effect on invasion but caused instead a profound impairment in parasite replication. This effect was reversible and independent of invasion. Remarkably, expression of the cytoplasmic tail of AMA1 overcame the block in cell division. Our findings uncover a previously unsuspected signaling role for AMA1, mediated by ROM4 processing, that acts as a checkpoint between invasion and replication.

1 4

RESULTS

Conditional expression of an enzymatically inactive form of ROM4 blocks intracellular replication of Toxoplasma late in the cell cycle To study the function of T. gondii ROM4, we used the ddFKBP system (Herm-Gotz et al., 2007) to regulate transgenic expression of the protease in the parasite. We expressed a control construct in which wild type ROM4 was N-terminally fused to the FKBP destabilization domain (dd) and a myc-epitope tag (ddROM4), plus a mutant form of the same construct in which the predicted catalytic Ser at position 409 was

substituted with an Ala residue (ddROM4S-A). A similar mutation has previously been shown to ablate rhomboid hydrolytic activity (Baker et al., 2006; Brossier et al., 2005;

Dowse et al., 2005). To validate the system, an inactive mutant of ROM1 (ddROM1S-

A) was also generated to allow comparison with the previously reported conditional knock-down of the gene using a tetracycline-inducible system (Brossier et al., 2008). Analysis of the resulting stable parasite lines revealed that the presence of the dd-myc at the N-terminus of the transgenic proteins did not interfere with their localization to the plasma membrane (ddROM4 and ddROM4S-A) or micronemes (ddROM1) (Figure 1A), and that expression of the transgenes was tightly controlled by the presence of Shld-1 (Figure 1 A and B). Expression of ddROM4 could be detected by Western blot as early as 30 min after addition of Shld-1, reaching a level similar to that of the endogenous protein after 480 min of treatment (Figure 1C). By IFA, a signal corresponding to ddROM4 could be detected after just 5 min of Shld-1 treatment; however correct trafficking of the protein to the plasma membrane was only observed after ~180 min (Figure 1D). T. gondii tachyzoites undergo a lytic cycle comprising host cell invasion, intracellular growth, egress and spreading to neighboring host cells by gliding motility. Plaque assays recapitulate multiple lytic cycles over a period of 7 days. Parasites expressing ddROM4 or yellow fluorescent protein (RH-2YFP, used as control) formed plaques of similar size whether grown with or without Shld-1 (Figure 2A), indicating that stable over-expression of functional ROM4 had no detrimental effect on growth. The

ddROM1S-A parasites formed slightly smaller plaques in the presence of Shld-1,

indicating a modest effect on growth. In stark contrast, the ddROM4S-A parasites produced no plaques at all in the presence of Shld-1 (Figure 2A). This profound

1 5 defect compared to expression of ddROM4 suggested that expression of a catalytically inactive form of the protease exerts a dominant negative effect, and that ROM4 fulfils an important function during the lytic cycle. A closer examination of

the phenotype of the ddROM4S-A line revealed an unanticipated defect in intracellular replication (Figure 2B). Quantification of the number of parasites per vacuole 24h post-invasion indicated that the 2YFP, ddROM1S-A and ddROM4 expressing parasites replicated at a similar rate regardless of the presence of Shld-1. In contrast, the ddROM4S-A line grew normally in the absence of Shld-1 but was severely impaired in division in the presence of Shld-1, with more than 80% of vacuoles stalled at the two parasites stage (Figure 2B). This evaluation of the defect is likely to be an underestimate, as vacuoles containing only a single parasite were not scored in order to avoid counting dead parasites. Introduction of an epitope-tag at the extreme C-terminus of several rhomboids, including PfROM4, interferes with enzymatic activity (Brossier et al., 2005; Dowse et al., 2005; O'Donnell et al., 2006) likely due to misfolding of the protease or interference with substrate recognition. We postulated that if the observed effects of ddROM4S-A expression were genuinely due to a dominant negative activity, inclusion of a Ty-1 epitope tag at its C-terminus should abrogate the effect. Fusion of C- terminal tags to produce ddROM4Ty and ddROM4S-ATy did not interfere with regulation or trafficking of the transgene proteins, as both were correctly stabilized at the parasite plasma membrane upon addition of Shld-1 (Figures S1A and S1B). Importantly, parasites expressing either protein grew normally in the presence of Shld-1, forming large plaques (Figure S1C) and replicating equally well (Figure S1D). These results support our interpretation that the phenotype observed upon

stabilization of ddROM4S-A was the result of a dominant negative activity, probably mediated through sequestering of one or more ROM4 substrates important for parasite replication. With the exception of rhoptries and micronemes, which are synthesized de novo, all other Toxoplasma sub-cellular organelles are delivered from the mother into the new daughter cells in a coordinated and precise order during intracellular division. The centriole and Golgi are the first organelles to be delivered, then the apicoplast, followed by the nucleus, the endoplasmic reticulum and, finally, the mitochondrion (Nishi et al., 2008). An examination of the integrity and inheritance of the different organelles by IFA using well-characterized markers was undertaken to define the

1 6 point of cell cycle arrest following stabilization of ddROM4S-A. While the inner membrane complex and the apicoplast appeared normal in parasites treated with Shld- 1 (Figure S2A), the unique tubular mitochondrion accumulated abnormally in residual bodies at the posterior pole of the intracellular parasites (Figure 3A). The micronemes accumulated at the apical pole and were less numerous, whilst the rhoptries appeared elongated and disorganized (Figure S2A). A marker labeling the nascent apical cone present early in the formation of the two daughter cells can be used to estimate the stage of endodyogeny. Although the apical cone was properly formed in parasites

expressing ddROM4S-A, nascent dividing structures were very infrequently detected, indicating that few daughter cells were initiating a second round of division (Figure 3B). In the absence of Shld-1, 40% of the vacuoles showed staining of developing daughter cells, whereas only 10% of the vacuoles were positive in parasites treated with Shld-1 (Table S2). Staining with DAPI (Figure 3C) revealed that the nuclei were enlarged and uncondensed in the presence of Shld-1. Collectively, the observed aberrant morphologies of the nucleus and mitochondrion, but not the apicoplast, indicate that the block in cell division occurred late in the cell cycle.

To further characterize the phenotype, ddROM4S-A parasites grown in the presence or absence of Shld-1 for 18, 24 and 36h were examined by electron microscopy (EM). At 18h, Shld-1 treated and untreated samples were morphologically similar with most vacuoles containing two fully formed tachyzoites (Figure 3D panels A and B). The majority of parasites contained a normal compliment of organelles but in some cases the nuclei appeared elongated and lobated with no evidence of initiation of daughter formation (Figure 3D panel F). It was also possible to observe the mitochondrion protruding through the posterior pore and in the residual body (Figure 3D panel E). At 24h and 36h in the Shld-1 treated samples, the vast majority of vacuoles still contained only two parasites (Figure 3D panel C) in contrast to the untreated samples where at 24h many vacuoles contained 4 to 16 parasites (Figure 3D panel D) and by 36h the majority of host cells had ruptured with large numbers of free tachyzoites. The frequency of parasites undergoing endodyogeny was low in the Shld-1 treated samples (less than 5%) while the incidence was much higher in the untreated samples (>12% at 18h and >20% at 24h) (Table S2). In summary, both the IFA and the EM analysis indicate that, while repeated rounds of endodyogeny occur normally in

untreated ddROM4S-A parasites, stabilization of ddROM4S-A expression with Shld-1 results in arrest of growth following one cycle of endodyogeny.

1 7

The block in cell division induced by ddROM4S-A is reversible and independent of invasion At the inception of this work, we had anticipated that ROM4 activity likely plays a dominant role at the invasion step of the parasite life cycle, as others have shown that ROM4 plays an important, but non-essential role during invasion (Buguliskis et al.,

2010). We therefore investigated the effect of ddROM4S-A expression on invasion, gliding motility and egress. Addressing this experimentally was complicated by the replication phenotype, since prolonged treatment of intracellular ddROM4S-A parasites with Shld-1 prevented egress as a direct result of the block in replication. To circumvent this problem, we performed invasion assays with non-dividing tachyzoites treated extracellularly for 6h with Shld-1. Stabilization of ddROM4S-A expression produced a modest but statistically significant defect in invasion (Figure S3A).

Growth of intracellular ddROM4S-A parasites with Shld-1 for 6h had no effect on egress (Figures S3C and S3D) but extension of the treatment to 12h resulted in an inability to glide, most likely as consequence of the defect in cell division (Figure S3B). These results confirm that, although the dominant effect resulting from interfering with ROM4 activity is on intracellular parasite replication, ROM4 does indeed play a role in invasion. To determine whether the impairment in cell division was linked to host cell invasion, we performed pulse-chase experiments, treating with Shld-1 for different times and then assessing intracellular growth at 24h post-invasion. When Shld-1 was added to intracellular parasites 12h prior to egress, and maintained for only 1h post-invasion, the ddROM4S-A parasites were able to recover and undergo normal cell division (Figure 4A). Similar results were obtained when treatment with Shld-1 was prolonged to 6h post-invasion (Figure 4B). This indicates that the dominant negative effect

produced by inducing ddROM4S-A expression over the course of invasion and early in the intracellular growth phase, was fully reversible. Previous studies have established that MPP1 is constitutively active at the plasma membrane of extracellular and intracellular parasites (Opitz et al., 2002). One interpretation of the reversibility of the dominant negative effect is that it results from the constitutive secretion onto the parasite surface of one or more substrates responsible for a signaling event leading to parasite replication. To investigate if the block in parasite replication was independent of invasion, we performed assays in which parasites started being treated with Shld-1

1 8 only at 6h post-invasion. These parasites remained severely impaired in cell division (Figure 4C). We conclude that ROM4 activity signals the switch from an invasive to a replicative mode, and that a new stimulus is needed at each new replication cycle. ROM4 is expressed at the surface of tachyzoites (Brossier et al., 2005; Dowse et al., 2005; Sheiner et al., 2008) and this observation had suggested that it plays a role during invasion. Having shown that it is critical for intracellular growth, we wondered if it was also expressed in other replicative stages. To address this question, we analyzed its sub-cellular localization throughout the entire life cycle of T. gondii by IFA. In the intermediate host, ROM4 located not only to the surface of tachyzoites in the lungs of acutely infected mice (data not shown), but also to the surface of bradyzoites in the brain of a chronically infected mouse (Figures S4A and S4B). In the definitive host, the cat, mature schizonts showed the strongest staining of all developmental stages (Figures S4C and S4D). A more detailed examination revealed that the early intracellular stages were also surface labeled with ROM4 (Figure S4E) however as the parasite differentiated into schizonts, microgametocytes or macrogametocytes, levels of surface staining decreased (Figure S4F). Reduction in the signal was concordant with growth of the mother cell, which is associated with loss of the IMC. A dramatic increase in staining occurred with the final stage of daughter formation. The strong ROM4 staining at the surface of the fully formed merozoites was associated with an intact pellicle (Figure S4F). Developing microgametocytes and macrogametocytes showed only background staining (Figure S4D). Collectively these results show that ROM4 is only expressed at the surface of parasite stages with an invasive role, whether extracellular or briefly after invasion, or in the final stages of daughter formation when a new cycle of parasite replication is about to start.

The AMA1 tail specifically rescues the division defect induced by ddROM4S-A All T. gondii rhomboid substrates characterized to date are micronemal proteins. During invasion, MIC2 is excluded from the apical invading part of the parasite at the site of the MJ, accumulating at the posterior of the parasite until being shed by MPP1 activity (Howell et al., 2005). The residual cytosolic tail of MIC2 is undetectable following invasion, suggesting it is rapidly degraded following cleavage. In contrast the tail of AMA1 is readily detectable in the intracellular tachyzoites shortly after invasison (Donahue et al., 2000) and is also stably carried into invaded erythrocyte

1 9 and detectable at the ring stage (Howell et al., 2005; Howell et al., 2003; Howell et al., 2001). Although the AMA1 cytoplasmic domain does not contribute to trafficking to the micronemes (Healer et al., 2002; Treeck et al., 2009; Sheiner et al., 2010) or formation of the MJ (Treeck et al., 2009) it plays an essential role in invasion (Treeck et al., 2009; Sheiner et al., 2010). In Plasmodium the phosphorylation at the serine residue 610 in PfAMA1 cytoplasmic domain by the cAMP regulated protein kinase A (PfPKA) is critical for invasion (Leykauf et al., 2010; Treeck et al., 2009). In the light of these observations, we hypothesized that one explanation for the apparent ability of AMA1 to cross the MJ during invasion is that AMA1 is in fact constitutively discharged onto the parasite surface. If this were so, its C-terminal product, generated by rhomboid cleavage at or following invasion, would be a plausible candidate for the signal that governs transition to the replicative phase of growth. In a previous study using a tetracycline-inducible system, AMA1 was reported not to play a role in intracellular replication (Mital et al., 2005). However, in that work, examination of the phenotypic consequences of AMA1 depletion was performed with the conditional knockout strain of AMA1 (Δama1/AMA1-myc) only 30h after switching off expression of the inducible copy of AMA1, AMA1-myc, with Atc, and, under those conditions, AMA1-myc only becomes undetectable after 24h. In the light of our observations here, we reexamined the phenotype of the Δama1/AMA1-myc strain with parasites that had been treated with Atc for up to 48h. Growth assays (Figure 5A), revealed that, as previously shown (Mital et al., 2005), parasites Atc treated for only 24h post-invasion are not defective in replication. However, when the treatment was extended to 48h, a cell division defect became evident, suggesting that AMA1 plays a role in intracellular division of the parasite and so supporting our hypothesis. In the absence of Atc, the Δama1/AMA1-myc strain expresses AMA1-myc at a level ten times lower than endogenous AMA1 in wild type parasites (Mital et al.,

2005). Stabilization of ddROM4S-A in these parasites (Figures 5B and 5C) led to a profound defect in parasite division and a minor impact on invasion similar to that seen with wild type parasites (Figures 5D, 5E, 5F), confirming that ROM4 is unlikely to play an essential function during host cell entry. We reasoned that if our model is correct, expression of the AMA1 cytosolic tail in intracellular parasites should complement the block in division mediated by

expression of ddROM4S-A. In anticipation that constitutive expression of such a

1 10 construct might have a deleterious effect on parasite growth, we tested this hypothesis by expressing in the parasite a construct in which the predicted cytosolic portion of the AMA1 tail was fused to ddFKBP and a mycHis epitope-tag (ddAMA1). Cytosolic expression of ddAMA1 was tightly regulated by Shld-1 (Figure S5) and did not affect growth of wild type parasites or ddROM4 parasites, as shown by plaque and intracellular growth assays (Figure S6). As predicted, upon treatment with Shld-1 ddAMA1 expression in ddROM4S-A parasites efficiently rescued the cell division

phenotype; parasites simultaneously expressing both ddAMA1 and ddROM4S-A formed plaques of similar size (Figure 6A) and replicated at a similar rate (Figure 6B), whether Shld-1 treated or not. To assess the specificity of the results obtained with the AMA1 cytosolic tail, we tested the effect of expression of the cleavage

product of MIC2 in the ddROM4S-A strain. MIC2 plays an essential function during both motility and invasion (Huynh and Carruthers, 2006) and cleavage by ROM4 is important for both (Buguliskis et al. 2010). Expression of the predicted MIC2 cytosolic tail as a ddFKBPmycHis fusion (ddMIC2) was efficiently regulated in a Shld-1 dependent manner (Figure S5) and did not affect growth of the RHΔHX or the ddROM4 parasite strains, as evaluated by plaque and replication assays (Figure S6). In contrast to the effects observed with AMA1 tail, ddMIC2 did not rescue the

ddROM4S-A phenotype, and parasites expressing ddMIC2 were unable to form lysis plaques (Figure 6A) or to replicate at a normal rate (Figure 6B), when Shld-1 treated. Collectively, our results indicate that the cytoplasmic tail of AMA1 performs an essential function during cell division that cannot be performed by the corresponding region of MIC2.

The dual functions of AMA1 are mutually independent and conserved in the Apicomplexa A recent investigation of AMA1 function revealed that the cytosolic tail can interact with aldolase in vitro and that this binding requires conserved Phe and Trp (FW) residues (Sheiner et al., 2010). Expression of an AMA1 mutant in which the FW

motif had been replaced with Ala residues (AMA1FW-AA) in the Δama1/AMA1-myc strain (Mital et al., 2005) was unable to functionally complement the invasion defect, indicating that these two conserved residues are essential for AMA1 function during invasion (Sheiner et al.,2010). To determine whether the FW motif is also essential

1 11 for AMA1 function during replication, ddAMA1FW-AA was examined for its ability to complement the cell division phenotype induced by stabilization of ddROM4S-A.

Expression of ddAMA1FW-AA in a Shld-1 dependent manner (Figure S5) produced no deleterious effect in RHΔHX or ddROM4 strains (Figure S6). Importantly, it also

rescued the cell division defect of parasites expressing ddROM4S-A, since parasites expressing both constructs were able to form plaques of similar size (Figure 6A) and replicated equally well regardless of the presence of Shld-1 (Figure 6B). This result clearly differentiates the dual functions of AMA1 in replication and invasion, showing that they are independent. The C-terminal tail region of AMA1 is highly conserved in all apicomplexan parasites, suggesting functional conservation. To test whether the Plasmodium AMA1 tail also functions during cell division, we expressed the P. falciparum protein as a

ddFKBPmycHis fusion (ddPfAMA1) (Figure S5) in ddROM4S-A parasites and tested for trans-complementation. The protein was able to restore the cell division defect as

efficiently as the Toxoplasma ddAMA1 or ddAMA1FW-AA. The trans- complementation was best illustrated in a mixed pool of parasites expressing either ddROM4S-A alone or expressing ddROM4S-A plus ddPfAMA1 or ddAMA1FW-AA (Figure 6C). The double transgenic parasites could be identified by the expression of

ddPfAMA1 or ddAMA1FW-AA in the cytosol, and only these parasites showed a clear reversal of the intracellular replication defect caused by ddROM4S-A expression. Our results strongly suggest that the cytosolic tail region of AMA1 performs a conserved role in regulating initiation of intracellular growth in apicomplexan parasites.

DISCUSSION

This study has revealed a previously unsuspected function for AMA1, an essential molecule that is conserved in all known apicomplexan parasites. Furthermore, it has provided the first evidence in any system that rhomboid-like proteases are implicated in intracellular signaling events. Conditional expression of an inactive form of ROM4 in T. gondii led to a dominant negative effect, likely due to sequestration of substrate(s) from the endogenous protease (Figure 7A), in a manner analogous to that previously reported for other proteases (Bailey and O'Hare, 2004; Wolfe et al., 1999). Efforts to substantiate this

1 12 postulate by attempting to pull down the protease bound to its substrate(s) using His-

tagged ddROM4S-A have failed (data not shown). Nevertheless the absence of a defect

in ddROM4S-ATy parasites strongly supports the notion that the dominant negative

effect is dependent on substrate interaction. Expression of ddROM4S-A had only a minor effect on invasion but caused a severe cell cycle arrest at a late stage of the cell cycle in a reversible and invasion-independent manner. There is an apparent discrepancy between these observations and previous results of Buguliskis et al. (Buguliskis et al., 2010), who failed to observe a replication phenotype upon conditional knockdown of ROM4. There are several plausible explanations for this.

First, in our approach the block in parasite replication upon stabilization of ddROM4S-

A hampers an accurate assessment of the contribution of ROM4 to invasion, since if parasites are not properly formed, defects in egress, gliding motility and invasion are unsurprising. Second, given the abundance of transmembrane MICs delivered onto the parasite surface during invasion, we believe it is unlikely that the expression of

ddROM4S-A could sequester and neutralize enough molecules to impact severely on invasion. Prolonged treatment with Shld-1 did dramatically affect gliding and egress (data not shown) but the effect was modest if the dominant-negative protease was stabilized for just a short period of time. A conditional knockout of ROM4 (as used by Buguliskis et al., 2010) may be a better-suited system to address the role of ROM4 in invasion. In their hands knockdown of ROM4 led to only a four-fold reduction in host cell invasion suggesting that ROM4 functions during invasion but its essentiality lies at another step of the lytic cycle (Buguliskis et al., 2010). Finally, parasites conditionally depleted of ROM4 still produce 10-20% of ROM4 mRNA transcripts (Buguliskis et al., 2010). We suspect that the authors did not observe a cell replication defect because the role of ROM4 in parasite division is likely to depend on a very limited amount of substrate, and even trace expression of active ROM4 could be sufficient to release the substrate and initiate the signaling cascade required to trigger intracellular replication (Figure 7C). The fact that ddAMA1 but not ddMIC2 functionally rescued the dominant effect caused by expression of ddROM4S-A, establishes that AMA1 plays a specific role in parasite replication that is not shared by other single span transmembrane micronemal proteins. Supporting this idea we could show that parasites conditionally depleted of AMA1(Mital et al., 2005) are impaired in cell division. The lack of a profound defect on cell division was expected because there is never complete repression of

1 13 AMA1myc expression using that system (Figure 8C). This function is independent of

the role of AMA1 in invasion, since an AMA1FW-AA mutant can complement the cell division but not the invasion defect (Sheiner et al., 2010). Orthologues of ROM4 and AMA1 exist in all sequenced apicomplexan genomes (Dowse and Soldati, 2005). AMA1 is unique amongst known microneme proteins in its particularly high degree of conservation across the phylum, and in all cases AMA1 possesses a rhomboid cleavage motif within its predicted TMD. The fact that the Plasmodium AMA1 tail can trans-complement the cell division phenotype imposed

by expression of ddROM4S-A supports our proposal for its role as a key mediator of an intracellular signaling event conserved across the phylum. Toxoplasma tachyzoites divide by endodyogeny, involving multiple rounds of parasite replication, each time generating two new daughter cells within the mother parasite. We postulate that replication is initiated by AMA1 cleavage during invasion followed by repeated stimulation engendered by further cleavage of AMA1 or another ROM4 substrate at each round of endodyogeny. In contrast the erythrocytic cycle of the malaria parasite, in which large numbers of daughters are generated simultaneously by schizogony, would only require one signaling event per cycle, which could be generated by ROM4-mediated cleavage of AMA1 during invasion (Figure 7B). Our model is supported by three observations: the localization of ROM4 at the surface of parasites only before the initiation of a new round of replication; the intracellular growth data, which indicate that arrest occurs at the two parasites per vacuole stage, regardless of the length of the Shld-1 treatment; and our finding that the blockage in cell division is reversible, suggesting that each round of endodyogeny requires renewed cleavage.

According to our model, parasites expressing ddROM4S-A were able to invade and replicate once because the inactive protease could not sequester all the AMA1 discharged during invasion. The substrate then becomes limiting and parasites are unable to progress further in their lifecycle. Our model also predicts that AMA1 is constitutively secreted to the Toxoplasma surface whilst intracellular. Demonstrating this is technically challenging especially if only trace amounts of AMA1 were sufficient to support replication. However, the proposal is supported by previous secretion assays performed with AMA1 and MIC2 (Donahue et al., 2000) and microscopy analysis showing that AMA1 can be detected on invading parasites on both sides of the MJ (Donahue et al., 2000; Howell et al., 2005; Mital et al., 2005). It is equally difficult to demonstrate cleavage of AMA1 by ROM4 in intracellular

1 14 parasites because the majority of the AMA1 population is stored unprocessed at the micronemes at this stage. Regulated intramembrane proteolysis (RIP) is a powerful and precise strategy for signal transduction in which intramembrane proteolysis of substrate membrane- anchored proteins triggers a signaling cascade by release of sequestered protein domains (Erez et al., 2009). Whereas all previous studies examining rhomboid- mediated RIP have concluded that rhomboids produce signals that are released to the exterior of the cell, our work suggests that a ROM4-mediated RIP mechanism operating on AMA1 releases its tail into the parasite cytosol to trigger the parasite to switch to a replicative mode (Figure 7B). This work therefore represents the first evidence for a direct, intracellular signaling role for a rhomboid protease analogous to that performed by other classes of proteases that mediate RIP (Erez et al., 2009). Our study highlights a novel and striking dual role for one of the most important apicomplexan proteins and shows that this group of parasites has opted to use invasion molecules as signaling factors for replicative growth.

1 15 FIGURE LEGENDS

Figure 1 ddROM1 and ddROM4 expression is tightly regulated by Shld-1

(A) Stabilization of ddROM4, ddROM4S-A and ddROM1S-A expression following 12h incubation of intracellular parasites with Shld-1. Parasites were probed with α- GAP45 (a marker for the parasite intramembrane complex, the IMC, which lies just beneath the plasma membrane; red) and α-myc antibodies (green). Scale bar, 5 µm.

(B) Robustness of ddROM4, ddROM4S-A and ddROM1S-A regulation determined by western blot analysis of parasites treated with Shld-1 for 12h using α-myc antibody (top panel). In the middle panel, levels of expression of endogenous ROM4 are compared to that of the ddROM4 fusion using antibodies specific to the ROM4 N-

terminus (ROM4Nt). Bottom panel: T. gondii profilin (PRF) was used as loading control. (C) Stabilization of ddROM4 in intracellular parasites as a function of time, monitored by western-bot of parasites treated with Shld-1 from 30 to 720 min, as compared to the level of expression of the endogenous protein as detected with

antibodies specific to the ROM4 N-terminus (ROM4Nt). (D) Stabilization of ddROM4 in intracellular parasites as a function of time, monitored by IFA of parasites treated with Shld-1 from 5 to 360 min using α-myc (green) and α-GAP45 (red) antibodies.

Figure 2 Expression of ddROM4S-A but not ddROM4 or ddROM1S-A severely impairs intracellular growth

(A) Plaque assays of ddROM4, ddROM4S-A, ddROM1S-A and RH-YFP parasites grown ± Shld-1 for 7 days.

(B) Intracellular replication rate of ddROM4, ddROM4S-A, ddROM1S-A and RH-YFP parasites grown 12h ± Shld-1 before host cell egress and for further 24h during the assay in a total of 36h. Number of parasites per vacuole (x axis) was counted 24h after inoculation of host cells. * indicates statistically significant results (*: p<0.1 and **: p<0.01). Data are represented as ± SEM (view also Figure S1).

Figure 3 ddROM4S-A parasites are blocked late in the cell cycle

1 16 ddROM4S-A parasites have abnormal mitochondria (α-APT1, green) (A), are predominantly arrested after a single round of division as determined by staining of the apical cone (anti-ISP1, green) (B) and are defective in karyocytokinesis as observed by the diffused DAPI staining (C). The arrows indicate accumulation mitochondria in residual bodies (A), staining of the nascent daughter apical cones of the mother and daughter parasites (B) or abnormal nuclei (C). In the merge parasites are labeled in red with α-GAP45. Parasites were treated ± Shld-1 for 36h prior to fixation. Scale bar, 5µm. (D) Electron micrographs of parasites cultured ± Shld-1 for 18h or 36h. Longitudinal section through two daughters cells after 18h in the presence (a) or absence of Shld-1 (b), and after 36h in the presence of Shld-1 (c). Section though a typical vacuole at 36h minus Shld-1 showing a rosette of tachyzoites after three cycles of repeated endodyogeny (d). Detail of a vacuole at 18h plus Shld-1 (e) showing the connection of the posterior end of a daughter to the residual body and the mitochondrion running between the posterior pore and the residual body. Longitudinal section through one of two daughters at 18h plus Shld-1 (f) showing the elongated and lobed appearance of the nucleus observed in certain parasites. (N – nucleus, DG – dense granule, R – rhoptry, C - conoid, MN – micronemes, RB- residual body, Mi- mitochondrion, PP – posterior pole). Scale bars: a – d, 1 µm; e-f, 0.5 µm (view also Table S2 and Figure S2).

Figure 4 The dominant negative effect of ddROM4S-A is reversible and independent of invasion

Intracellular ddROM4 and ddROM4S-A parasites were treated 12 h with Shld-1 prior to host cell egress and the Shld-1 was then removed 1h (A) or 6h (B) post-invasion. In

(C), ddROM4 and ddROM4S-A parasites were allowed to invade and Shld-1 was added to the culture 6h post-invasion and maintained for the duration of the assay. The number of parasites per vacuole (x axis: mean ± SEM) was counted 24h after invasion. Note that Shld-1 treatment of ddROM4 parasites produced no defect, under any of the experimental conditions.

Figure 5 ΔAMA1-AMA1myc parasites are affected in cell division

1 17 (A) Intracellular growth assays of ΔAMA1-AMA1myc (left) and RH-2YFP parasites (right) not Atc treated (-Atc), Atc treated for only the time of the assay (+Atc 24h) or pre-treated 24h with Atc prior to egress and for the time of the assay in a total of 48h (+Atc 48h). The number of parasites per vacuole (x axis: mean ± SEM) was counted 24h after invasion. * indicates statistically significant results (*: p<0.1, **: p<0.01).

Stabilization and down-regulation of ddROM4S-A and AMA1myc on ΔAMA1-

AMA1myc parasites expressing ddROM4S-A following 24h treatment with Shld-1 or Atc, respectively as shown by immunofluorescence (B) and western-blot assays (C). Parasites were stained with α-GAP45 (red) and α-myc (green) antibodies. Scale bar, 5 µm. The proteins were detected with α-myc antibodies. Profilin was used as a loading control.

(D) Intracellular growth assays of ΔAMA1-AMA1myc parasites expressing ddROM4S-

A ± 24h Shld-1. The number of parasites per vacuole (x axis: mean ± SEM) was counted 24h after invasion. * indicates statistically significant results (***: p<0.001). (E) Invasion assays of RHΔHX (left) or ΔAMA1-AMA1myc parasites (right) expressing ddROM4S-A ± 6h Shld-1, when extracellular, as compared to the RH-2YFP strain. Data are represented as mean ± SEM. * indicates statistically significant results (*: p<0.1) (view also Figure S3).

Figure 6 Expression of ddAMA1, ddAMA1FW-AA and ddPfAMA1 trans-

complements the dominant negative effect of ddROM4S-A on parasite replication

(A) Plaque assays of ddROM4S-A parasites expressing ddAMA1, ddAMA1FW-AA or ddMIC2. Parasites were incubated 7 days ± Shld-1 prior to fixation and Giemsa staining (view also Figures S5 and S6).

(B) Intracellular replication assays performed on parasites expressing only ddROM4S-

A or ddROM4S-A and ddAMA1/ddAMA1FW-AA/ddMIC2. Parasites were treated 24h ± Shld-1 prior to fixation. Data are represented as mean ± SEM. (C) Immunofluorescence assays of a mixed population of parasites expressing only ddROM4S-A or ddROM4S-A and ddAMA1FW-AA (left) or ddROM4S-A and ddPfAMA1 (right) treated 24h with Shld-1 prior to fixation. Parasites were stained with α-GAP45

(red) and α-myc (green) antibodies. The star indicates parasites expressing ddROM4S-

A and ddAMA1FW-AA/ddPfAMA1 and the arrowheads show parasites expressing only ddROM4S-A.

1 18 Figure 7 Proposed model for the function of ROM4 and AMA1 during intracellular growth

(A) Expression of ddROM4S-A induces a dominant effect on cell division due to the sequestration of a limited substrate from the endogenous, active ROM4 copy. Ectopic

expression of ddAMA1 trans-complements the dominant effect of ddROM4S-A and reconstitutes the signaling cascade leading to the switch to the replication mode.

Endogenous ROM4 is depicted in blue, inactive ddROM4S-A is in yellow, AMA1 is red and the ddFKBP domain is in pink. (B) In T. gondii, the switch to a replicative mode is triggered by cleavage of the substrate during invasion but following the first cycle of division further cleavage events are required to stimulate additional rounds of replication. In Plasmodium, by contrast, since all daughter cell are generated simultaneously by schizogony, initiation of the replication phase only requires a single round of AMA1 cleavage during invasion. (C) In a wild type situation (RH), AMA1 and ROM4 are expressed at the surface of extracellular and intracellular parasites but AMA1 is expressed at a lower, basal level in intracellular parasites. Expression of ddROM4 increases the amount of ROM4 expressed at the surface of the parasites and leads to a blockage in intracellular

growth when there is mutation of the catalytic serine (ddROM4S-A) due to sequestration of the few AMA1 expressed in intracellular parasites. Parasites conditionally depleted of ROM4 (iROM4ko) express reduced levels of rhomboid at the surface and are blocked in invasion but not cell division because the level of ROM4 expression is still sufficient to mediate AMA1 cleavage in intracellular parasites. Parasites conditionally of AMA1 (iAMA1ko) express reduced levels of AMA1 at the surface and are blocked in invasion and mildly in cell division.

MATERIAL AND METHODS

Reagents and parasite culture Restriction enzymes were purchased from New England Biolabs and secondary antibodies for western blots and IFA from Molecular Probes. T. gondii tachyzoites (RHΔHX) were grown in human foreskin fibroblasts (HFF) in Dulbecco´s Modified Eagle´s Medium (DMEM, GIBCO) supplemented with 5% fetal calf serum (FCS), 2mM glutamine and 25 µg/ml gentamicin.

1 19

Cloning of DNA constructs For expression of ROM1 and ROM4 in T. gondii as N-terminal ddFKBPmyc-epitope tagged fusions, mycROM1 and mycROM4 from pT8mycROM1 and pT8mycROM4 plasmids (Dowse et al., 2005) were digested with NsiI and PacI and cloned into the pT8ddmycGFP vector, which drives expression of N-terminal ddFKBP-myc tagged fusions under control of tubulin promoter, originating pT8ddmycROM1 and pT8ddmycROM4, respectively. The pT8ddmycGFP plasmid was obtained by digesting the plasmid pT8ddYFP (Herm-Gotz et al., 2007) with EcoRI and NsiI and cloning the ddFKBP sequence in the same sites in the pT8mycGFPty vector (Meissner et al., 2002). The primers 1703 and 1704 and 1516 and 1517 were used in a site-directed mutagenesis reaction using the commercial QuikChange II Site-Directed Mutagenesis Kit (Stratagen) according to the manufacturer’s instructions, to mutate the catalytic serines into alanines in the pT8ddmycROM1 and pT8ddmycROM4

plasmids, respectively, originating pT8ddmycROM1S-A and pT8ddmycROM4S-A,

respectively. Digestion of pT8ddmycROM4 and pT8ddmycROM4S-A with NaeI and XbaI and cloning into the same sites in the p30/11 plasmid (Hettmann et al., 2000),

originated plasmids pT8ddmycROM4(DHFR) and pT8ddmycROMS-A(DHFR), respectively.

Parasites transfection and selection of clonal stable lines Parasites transfection was performed by electroporation as previously described (Soldati and Boothroyd, 1993). The HXGPRT gene was used as a positive selectable marker in the presence of mycophenolic acid and xanthine, as described before (Donald et al., 1996). Briefly, 5×107 freshly released RHΔHX parasites were resuspended in cytomix buffer in the presence of 80µg of linearized plasmid carrying the selectable marker gene and the expression cassette containing the DNA sequences. Parasites were electroporated at 2 kV, 25 mF, 48 V using a BTX electroporator (Harvard biosciences, Holliston, MA, USA) before being added to a monolayer of HFF cells in the presence of mycophenolic acid (25 mg/mL) and xanthine (50 mg/mL) and were then cloned by limiting dilution on 96-well microtiter plates following one growth cycle. The same procedure was followed to transfect the

ΔAMA1/AMA1-myc, the ddROM4 or the ddROM4S-A strains, but the DHFR gene was

1 20 used as positive selectable marker. Selection with 1µM pyrimethamine was initiated 24 hr after transfection and continued for 7-10 days, after which resistant clones were isolated by limiting dilution.

Transmission electron microscopy

ddROM4S-A parasites, which had undergone or not a 12h pre-treatment with 0.5µM Shld-1, were used to infect a host cell layer ± 0.5µM Shld-1. Samples were collected at 18, 24 and 36h post-infection and processed for electron microscopy using routine techniques. In summary, parasite pellets were fixed in 2.5% glutaraldehyde in 0.1M phosphate buffer, post-fixed in osmium tetroxide, dehydrated in ethanol and treated with propylene oxide prior to embedding in Spurr’s epoxy resin. Thin sections were stained with uranyl acetate and lead citrate prior to examining in a Jeol 1200EX

electron microscope.

Western blotting 2x107 freshly lysed parasites ±0.5µM Shld-1 treated for 24h were harvested after complete lysis of the host cells. Protein extracts were prepared in 1xPBS by five consecutive freeze/thaw cycles with intermediate homogenization. SDS- polyacrylamide gel electrophoresis (SDS-PAGE) was performed using standard methods: The suspension was mixed with SDS–PAGE-loading buffer and proteins were separated by electrophoresis in a 10-12% polyacrylamide gel. Separated proteins were transferred to a nitrocellulose membrane using a semidry electroblotter. Western blots were performed using anti-Myc mAb 9E10, anti-Ty mAb, anti-ROM4Nt pAb (Sheiner et al., 2008) or anti-Profilin pAb (Plattner et al., 2008) in 5% non-fat milk powder in 1X PBS-0.05% Tween. As secondary antibody, a peroxidase-conjugated goat anti-mouse or anti-rabbit antibody was used (Molecular Probes, Paisley, UK). Bound antibodies were visualized using the ECL system (Amersham Corp).

IFA and confocal microscopy Intracellular parasites grown in HFF were fixed with 4% paraformaldehyde (PAF) or 4% PAF-0.005% Glutaraldehyde (PAF/GA) in PBS depending on the antigen to be labeled and processed as previously described (Hettmann et al., 2000).

1 21 Plaque assays Monolayers of HFF grown in 6-well plates were infected with tachyzoites and incubated 6-7 days at 37ºC, after which they were fixed and stained with Giemsa stain for 10 minutes and washed with water.

Intracellular Growth assays New host cells seeded on 24-well IFA plates were inoculated with freshly egressed parasites ± 0.5µM Shld-1. Parasites were allowed to grow for 24h before fixation with 4% parafolmaldehyde. Double immunofluorescence assays (α-GAP45 and α -myc antibodies) were performed and the number of parasites on at least 100 vacuoles were counted for each condition. The pulse-chase intracellular growth assays were performed in the same way but the parasites were treated ± 0.5µM Shld-1 for the times indicated. For the assays with the ΔAMA1-AMA1myc and RH-2YFP strains, parasites were treated 24h ± 1µM Atc, prior to host cell egress. New host cells seeded on 24-well IFA plates were inoculated with freshly egressed parasites ± 1µM Atc. Parasites were allowed to grow 24h before fixation with 4% PAF. Immunofluorescence assays were performed and the number of parasites on at least 100 vacuoles were counted for each condition. The results are representative of at least three independent experiments.

Statistics P values were calculated in Excel using the Student’s t-test assuming equal variance, unpaired samples, and using 2-tailed distribution. Means and standard deviations (SEM) were also calculated in Excel.

ACKNOWLEDGMENTS The research leading to these results has received funding from MalParTraining, an FP6-funded Marie Curie Action under contract number MEST-CT-2005-020492. The challenge of Malaria in the Post-Genomic Era. This work is supported by the Swiss National Foundation (FN3100A0-116722) and the Medical Research Council, UK (U117532063). DS is an international scholar of the Howard Hughes Medical Institutes. We would like to thank P. Bradley for the monoclonal antibodies 7E8- ISP1, 11G8-Atrx1, 5F4 and anti-ROP7, M. Meissner for the ddFKBP constructs, and L. Sheiner and T. Dowse for all their efforts in the study of ROM4.

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1 25 Figure 1 Click here to download Figure: Figure 1_Santos.pdf

A B ddROM4 ddROM4 ddROM4S-A ddROM1S-A Shld - + - + - +

αmyc

ddROM4

ddROM4 S-A ddROM1

αmyc αGAP45 merge αmyc

ddROM4 αROM4 ROM4

αPRF PRF ddROM1S-A

αmyc αGAP45 merge ddROM4 72

1 Figure 2 Click here to download Figure: Figure 2_Santos.pdf

A RH-YFP ddROM4

ddROM4S-A ddROM1S-A

-Shld +Shld -Shld +Shld

parasites/vacuole

RH-YFP ddROM1S-A

parasites/vacuole

ddROM4 ddROM4S-A

1 Figure 3 Click here to download Figure: Figure 3_Santos.pdf A B C

αAPT1 merge α-ISP1 merge DAPI αmyc merge

1 Figure 4 Click here to download Figure: Figure 4_Santos.pdf

- Shld + Shld + Shld until 1h pi

parasites/vacuole

ddROM4 ddROM4S-A B

-12 0+1 +6 +24hours

- Shld + Shld + Shld until 6h pi

ddROM4 ddROM4S-A

** -12 0+1 +6 +24hours

- Shld + Shld + Shld 6h pi

ddROM4 ddROM4S-A

1 Figure 5 Click here to download Figure: Figure 5_Santos.pdf

A -Atc +Atc 24h

parasites/vacuole parasites/vacuole ΔAMA1-AMA1-myc RH-2YFP

B D

αmyc αGAP45 merge -Shld +Shld

*** *** -Shld-1 +Shld-1 C

55 F

-Shld-1 43 +Shld-1

αmyc

αPRF

ddROM4S-A ddROM4S-A RHΔHX ΔAMA1/AMA1myc

1 Figure 6 Click here to download Figure: Figure 6_Santos.pdf

ddMIC2 in ddROM4S-A

-Shld +Shld -Shld +Shld -Shld +Shld

parasites/vacuole parasites/vacuole

ddROM4S-A

parasites/vacuole parasites/vacuole ddROM4 + ddMIC2 ddROM4S-A+ ddAMA1 S-A

* *

1 Figure 7 ClickA here to download Figure: Figure 7_Santos.pdf

ROM4 ddROM4S-A

+ ddAMA1 tail

1 Inventory of Supplemental Information

INVENTORY SUPPLEMENTARY MATERIAL

Table S1. List of primers used in the study

Table S2. Number of parasites per vacuole and percentage of endodyogeny of

ddROM4S-A parasites at different time points as determined by IFA and EM

Figure S1. C-terminal epitope tagging of ddROM4S-A abrogates its dominant negative effect

Figure S2. Assessment of the arrest in replication in ddROM4S-A parasites by immunofluorescence assay

Figure S3. Shld-1 treatment of ddROM4S-A parasites affects gliding, host cell invasion and egress

Figure S4 ROM4 expression is confined to invasive stages of the Toxoplasma life cycle

Figure S5. Expression of ddAMA1, ddAMA1FW-AA and ddMIC2 in RHΔHX,

ddROM4 or ddROM4S-A parasites is efficiently regulated by Shld-1

Figure S6. Plaque and intracellular growth assays of RH and ddROM4 parasites

expressing ddAMA1, ddAMA1FW-AA or ddMIC2

1 FIGURE LEGENDS

Figure S1. C-terminal epitope tagging of ddROM4S-A abrogates its dominant negative effect

(A) Expression of both ddROM4Ty and ddROM4S-ATy is efficiently stabilized by treatment with Shld-1 for 12 h as determined by indirect immunofluorescence assay using α-Ty antibodies. α-GAP45 antibodies were used to stain the parasites. Scale bar, 5 µm. (B) Western blot probed with showing stable expression of ddROM4Ty and

ddROM4S-ATy (α-Ty antibody, top panel) as compared to the expression of endogenous ROM4 (α-ROM4 antibody, middle panel) upon treatment with Shld-1 for 12 h. Profilin was used as a loading control (bottom panel).

(D) Intracellular replication assays of ddROM4Ty or ddROM4S-ATy parasites. Parasites were grown 24 h ± Shld-1 prior to fixation. Data are represented as mean ± SEM.

Figure S2. Assessment of the arrest in replication in ddROM4S-A parasites by immunofluorescence assay

(A) Parasites expressing ddROM4S-A have normally developed apicoplast (α-F1- ATPase, green), but abnormal micronemes (α-MIC2, green) and rhoptries (α-ROP7, green). Parasites were treated ± Shld-1 for 36 h prior to fixation. Arrows indicate concentration of the micronemes at the apical tip of the parasite or elongation of the rhoptries. α-GAP45 antibodies were used to stain the parasites. Scale bar, 5 µm.

Figure S3. Shld-1 treatment of ddROM4S-A parasites affects gliding, host cell invasion and egress

(A) Stabilization of ddROM4S-A expression in extracellular parasites treated with Shld-1 for 6 h modestly affected host cell invasion, whereas expression of ddROM4

or ddROM1S-A had no effect. The y-axis represents the percentage of intracellular

1 parasites as the mean ± SEM compared with the RH-2YFP control line. * indicates a statistically significant reduction (p<0.1). (B) In vitro gliding motility assay based on the detection of trails using α-SAG1 antibodies by indirect immunofluorescence assay. A trail is indicated by the white arrow. Intracellular parasites were treated for 12 h ± Shld-1 prior to the assay. 2+ Ca -ionophore (A23187)-induced egress of extracellular ddROM4S-A (C) or ddROM4 (D) parasites following treatment for 6 h ± 0.5 µM Shld-1. The parasites surface was stained with α-SAG1 antibodies and α-GRA3 antibodies were used as a marker of the parasitophorous vacuole. Scale bar, 5 µm.

Figure S4 ROM4 expression is confined to invasive stages of the Toxoplasma life cycle Micrographs showing sections through tissue cysts stained with anti-ROM4 and ani- ENO1 showing the surface of the bradyzoites outlined in green (a, b). Low power image of a 1µm plastic section stained with Azure A showing the morphology of the various stages of coccidian development within the apical cytoplasm of the epithelial cells (c) and only four mature schizonts strongly stained for ROM4 (arrowheads d). Various developmental stages strongly stained with anti-ENO2 (d). Detail of early intra-cellular stages showing the surface labeling of ROM4 (arrowheads) with the apicoplast located in the parasite cytoplasm (e). Section showing mature schizonts with strong ROM4 surface labeling of the fully formed merozoites (f). No surface labeling could be detected in the early multi-nucleate schizonts (f). All sections were double labeled with anti-ROM4 visualized with FITC (green) and either anti-ENR (E), anti-ENO1 (A, B) or anti-ENO2 (E-F) visualized with Texas Red (red) and counter stained with DAPI (blue). Scale bars: a, b, e, f, 1 µm; c, d, 10 µm. (N – nucleus, Ma – macrogamete, A – apicoplast, Br – bradyzoites, S – mature schizont, ES – early multi-nucleated schizont)

Figure S5. Expression of ddAMA1, ddAMA1FW-AA and ddMIC2 in RHΔHX, ddROM4 or ddROM4S-A parasites is efficiently regulated by Shld-1

(A) Scheme of ddAMA1, ddAMA1FW-AA, ddPfAMA1 or ddMIC2 fusion proteins. The sequence of the cloned micronemal tails (MIC) is shown. The mutated residues

on ddAMA1FW-AA are boxed in grey.

1 (B) Western-blot of RHΔHX (left), ddROM4 (middle) or ddROM4S-A (right) parasites

stably expressing ddAMA1 or ddAMA1FW-AA as detected with α-myc. Profilin was used as loading control. Parasites were treated ± Shld-1 for 12h.

(C) Western-blot of RHΔHX, ddROM4 or ddROM4S-A parasites stably expressing ddMIC2 (top) or ddPfAMA1 (bottom) as detected with α-myc. Profilin was used as loading control. Parasites were treated ± Shld-1 for 12h.

(D) Immunofluorescence assay of RHΔHX, ddROM4 or ddROM4S-A parasites expressing ddAMA1, ddAMA1FW-AA or ddMIC2 treated ± Shld-1 for 12h. The parasites were stained in red with α-GAP45 antibodies and in green with α-myc. In the double transgenic parasites both ddROM4 or ddROM4S-A and ddAMA1 or

ddAMA1FW-AA are undetectable in the absence of Shld-1 (B, D, F). Profilin (PRF) was used as a loading control. The arrow indicates expression of ddAMA1 or ddROM4. Scale bar, 5 µm.

Figure S6. Plaque and intracellular growth assays of RH and ddROM4 parasites

expressing ddAMA1, ddAMA1FW-AA or ddMIC2 (A) Plaque assays of RHΔHX or ddROM4 parasites expressing ddAMA1 (left),

ddAMA1FW-AA (middle) or ddMIC2 (right) treated ± Shld-1 for 7 days. Intracellular growth assays of RHΔHX (B) or ddROM4 parasites (C) expressing ddAMA1 (left), ddAMA1FW-AA (left) or ddMIC2 (right). Parasites were treated 24 hrs ± Shld-1 prior to fixation. Data are represented as mean ± SEM.

1 MATERIAL AND METHODS

Cloning of DNA constructs

To express ROM4 Ty-epitope tagged, rom4 and rom4S-A were amplified from

pT8ddmycROM4wt or pT8ddmycROM4S-A, respectively, by PCR using the primers 1483 and 1484 (supplementary table 1), and cloned between EcoRI and NsiI in the

pT8mycGFPty vector. To generate ddFKBP fusions, rom4Ty and rom4S-ATy were cloned between MfeI and PacI in the pT8ddmycGFP vector, generating

pT8ddmycROM4Ty and pT8ddmycROM4S-ATy, respectively. To express the TgAMA1 and TgMIC2 C-terminus as a N-terminal myc-His tagged ddFKBP fusion, AMA1 and MIC2 were amplified from tachyzoites cDNA by PCR with primers 1080 and 2614, 2945 and 2946, respectively, and cloned between NsiI and PacI in pT8ddmycHisROM4 or pT8ddmycHisROM4(DHFR), originating pT8ddmycHisAMA1 or pT8ddmycHisAMA1(DHFR), pT8ddmycHisMIC2 or pT8ddmycHisMIC2(DHFR), respectively. The primers 1830 and 1831 were used in a site-directed mutagenesis reaction to mutate the FW into AA, generating pT8ddmycHisAMA1FW-AA and pT8ddmycHisAMA1FW-AA (DHFR). The plasmids pT8ddmycHisROM4 or pT8ddmycHisROM4(DHFR) were generated by amplification HisROM4 by PCR with primers 2577 and 1073, digestion with NsiI and PacI and cloning into the same sites in pT8ddmycROM4 or pT8ddmycROM4(DHFR), respectively. The PfAMA1 tail was expressed as a synthetic gene (CGGATGCATAAGCGCAAGGGCAACGCGGAGAAGTACGACA AGATGGACGAGCCGCAGGACTACGGCAAGTCGAACTCGCGCAACGACGA GATGCTCGACCCGGAGGCGTCGTTCTGGGGCGAGGAGAAGCGCGCGTCGC ACACGACGCCGGTCCTCATGGAGAAGCCGTACTACTGATTAATTAAGGC) and cloned between NsiI and PacI in pT8ddmycHisROM4 or pT8ddmycHisROM4(DHFR), originating pT8ddmycHisPfAMA1 or pT8ddmycHisPfAMA1(DHFR).

Cell invasion assays The assay was done using an RH-2YFP strain as internal standard, as previously described [33]. Briefly, a confluent 60mm-dish of HFF was infected with a mixture of the strain of interest and RH-2YFP parasites. Parasites were either treated ± Shld-1for 12h, when intracellular, or for only 6h, after lysing the host cells. The ratio of non-

1 YFP to YFP parasites was determined and the mix of parasites was inoculated into

IFA plates. The plate was incubated for 1h (in the case of the ddROM4S-A strain) or 24h at 37ºC and the cells were subjected to IFA. The total number of parasite vacuoles and the ratio of YFP to non-YFP vacuoles were counted on at least 100 vacuoles per slide. Each experiment was repeated at least 3 independent times.

Induced Egress assays Parasites were grown for 6 hr ± 0.5µM Shld-1. Freshly egressed tachyzoites were used in the inoculation of new host cell layers. After 30 hr of intracellular growth, the infected host cell layers were incubated for 5 min at 37ºC with DMEM containing DMSO or the calcium ionophore A23187 (from Streptomyces chartreusensis, Calbiochem). The host cells were fixed with PAF and IFAs with the α-GAP45 and α- GRA3 antibodies were performed.

Induced Gliding assays Parasites were grown for 12 hr ± 0.5µM Shld-1. Freshly egressed tachyzoites were filtered, pelleted, and resuspended in Calcium-Saline containing 1mM of ionomycin. The suspension was deposited on coverslips previously coated with Poly-L-Lysine. Parasites were fixed with PAF/GA and IFA using the α -SAG1 antibody was performed to visualize the trails.

Immunofluoresce assay (IFA)

For the in vivo study with the anti-ROM4Nt pAb, sections of cat small intestine containing the coccidian stages and mouse lung and brain containing the tachyzoite and bradyzoite stages of T. gondii were isolated as previously described (Ferguson et al., 1999).

1 Supplemental Figure 1 Click here to download Supplemental Figure: Figure S1_Santos.pdf

A B

αmyc αGAP45 merge ddROM4Ty 72

55 43

αmyc

ddROM4Ty

αROM4 ROM4

PRF αPRF

ddROM4Ty ddROM4S-ATy

-Shld +Shld -Shld +Shld

D -Shld-1 +Shld-1

parasites/vacuole

ddROM4Ty ddROM4S-ATy

1 Supplemental Figure 2 Click here to download Supplemental Figure: Figure S2_Santos.pdf

αF1-ATPase αGAP45 merge

αMIC2 αGAP45 merge

αROP7 αGAP45 merge

1 Supplemental Figure 3 Click here to download Supplemental Figure: Figure S3_Santos.pdf

B A ddROM4

ddROM4S-A

-Shld +Shld RHΔHX ddROM1S-A ddROM4 ddROM4S-A

C D

ddROM4S-A ddROM4

αSAG1 αGRA3 αSAG1 αGRA3

1 Supplemental Figure 4 Click here to download Supplemental Figure: FigureS4_Santos.pdf

1 Supplemental Figure 5 Click here to download Supplemental Figure: FigureS5_Santos.pdf A ddFKBP myc His MIC tail

ddAMA1 CYFAKRLDRNKGVQAAHHEHEFQSDRGARKKPSDLMQEAEPSFWDEAEENIEQDGETHVMVEGDY ddAMA1FW-AA CYFAKRLDRNKGVQAAHHEHEFQSDRGARKKPSDLMQEAEPSAADEAEENIEQDGETHVMVEGDY ddPfAMA1 KRKGNAEKYDKMDEPQDYGKSNSRNDEMLDPEASFWGEEKRASHTTPVLMEKPYY ddMIC2 AAGGFAYNFVLSSSVGSPSAEIEYEADDGEQKLISEEDLETLVPVDDDSDMWME

ddAMA1 ddAMA1FW-AA ddAMA1 ddAMA1FW-AA ddAMA1 ddAMA1FW-AA Shld − + − + − + − + − + − + 95 ddROM4S-A 74

26 ddAMA1 αmyc

PRF αPRF

RHΔHX ddROM4 ddROM4S-A C D

RH HX ddROM4 ddROM4 Δ S-A ddAMA1 ddAMA1FW-AA ddMIC2 Shld − + − + − +

ddROM4 αmyc αGAP45 αmyc αGAP45 αmyc αGAP45

ddMIC2 αmyc αmyc αGAP45 αmyc αGAP45 PRF αPRF

74 ddROM4

55 αmyc αGAP45 αmyc αGAP45 αmyc αGAP45 34

26 ddPfAMA1

αmyc αmyc αGAP45 αPRF PRF

1 Supplemental Figure 6 Click here to download Supplemental Figure: FigureS6_Santos.pdf A

ddAMA1 ddAMA1FW-AA ddMIC2

-Shld +Shld -Shld +Shld -Shld +Shld

-Shld-1 +Shld-1

parasites/vacuole

ddAMA1 ddAMA1FW-AA ddMIC2

-Shld-1 +Shld-1

parasites/vacuole parasites/vacuole parasites/vacuole

ddAMA1 ddAMA1FW-AA ddMIC2

1 Supplemental Table 1 Click here to download Supplemental Figure: Table S1_Santos.pdf

Primer No. Enz Sequence Resulting plasmid name

ROM4 1516 CAGTCGGATCGGCTGGTTCCATGTATG pT8ddmycROM4S-A

ROM4 1517 CATACATGGAACCAGCCGATCCGACTG pT8ddmycROM4S-A

ROM4 1483 EcoRI CCGGAATTCCCTTTTTCGACAAAATGGTGTGGACTT pT8ddmycROM4S-ATY CGGCCGTC ROM4 1484 NsiI GGCATGCATGCGGTTCAAGATAATACTGCGCATCC pT8ddmycROM4S-ATY ROM4 2577 NsiI ATGCATATGGTGATGGTGGTGATGGTGGTGGGCCA pT8ddmycHisROM4 TGGCCAGGTCCTCC ROM4 1073 PacI GGAATTCTTAATTAAGGTTCAAGATAATACTGCGCAT pT8ddmycHisROM4 CC

ROM1 1703 CTCTCAAAGTTGGAGCCGCTACGGCAGGCTTCGG pT8ddFmycROM1S-A

ROM1 1704 CCGAAGCCTGCCGTAGCGGCTCCAACTTTGAGAG pT8ddmycROM1S-A AMA1 2614 NsiI ATGCATGGAGGCTGCTACTTCGCGAAGAG pT8ddmycHisAMA1 AMA1 1080 PacI CCGCAATTGTTAATTAACTAGTAATCCCCCTCGACC pT8ddmycHisAMA1 ATAA

AMA1 1830 NsiI CATGCAAGAGGCTGAACCGTCGGCTGCGGATGAGG pT8ddmycHisAMA1FW- CAGAGGAGAAC

AA AMA1 1831 PacI GTTCTCCTCTGCCTCATCCGCAGCCGACGGTTCAG pT8ddmycHisAMA1FW- CCTCTTGCATG

AA MIC2 2945 NsiI ATGCATGCAGCTGGAGGATTTGCATATAATTTTG pT8ddmycHisMIC2

MIC2 2946 PacI TTAATTAACTACTCCATCCACATATCACTATCGTC pT8ddmycHisMIC2

1 Supplemental Table 2 Click here to download Supplemental Figure: Table S2_Santos.pdf

1 Chapter IV

DISCUSSION AND CONCLUDING REMARKS

1. Toxoplasma tachyzoites: multi target cells

The invasion molecular machinery is highly conserved in Plasmodium merozoites and Toxoplasma tachyzoites (Kappe et al., 1999) but the two cells are distinct in size and host cell niche - merozoites are small cells (1.5µm) restricted to invasion of erythrocytes while tachyzoites are 7.5µm and can invade a wide array of host cells - and have thus adopted different strategies regarding surface expression of adhesins. In Plasmodium there is phenotypic variation of protein ligands, i.e., within a same parasite population, different merozoites express a different pattern of surface molecules and favor different invasion pathways (reviewed in (Cowman and Crabb, 2006)) (figure 4.1). Silencing of the expression of invasion-related genes is reversible and epigenetically transmitted (Cortes et al., 2007) and can thus be easily and rapidly reverted. This type of expression system is highly advantageous for the merozoite because the RBC surface is highly polymorphic depending on the individual and lifespan and the number of parasite receptors available is therefore limited, restricting the routes of parasite entry and forcing the merozoite to express surface proteins that ensure a rapid adjustment to different conditions. Only disruption of some genes induce switching of invasion pathways, indicating that the invasion phenotype of a particular parasite line depends not only on the set of ligands expressed but also on a molecular hierarchy that determines which of the expressed ligands are used, possibly because of competition for space at the tip of the merozoite (Baum et al., 2005).

Figure 4.1 Expression of “invasion proteins” at the surface of Plasmodium merozoites and Toxoplasma tachyzoites On the top panel, is represented the mechanism of phenotypic variation of surface ligands in Plasmodium merozoites; a same parasite within two different hosts can express different proteins at the surface depending on the host cell immune response. On the bottom panel, is represented the expression of “invasion proteins” at the surface of Toxoplasma tachyzoites; these parasites do not control expression of protein ligands and therefore always express same pattern of proteins at the surface. Adapted from (Cortes, 2008).

The mechanism allowing Toxoplasma to invade almost any cell type remains a mystery. It has been hypothesized that it can be due to export of its own receptor into the host cell, expression of low specific ligands able to bind to many receptors, or expression of multiple high specificity ligands that can recognize receptors common to many cell types. The first hypothesis is supported by secretion of the TgRON proteins to the host cell (Besteiro et al., 2009b), suggesting that they may act as receptor for TgAMA1, which would function as ligand but, although export has only been validated for Toxoplasma, the AMA1-RONs complex is conserved in Plasmodium (Richard et al.), suggesting that alone this aspect cannot explain the variety of cells invaded by the Toxoplasma parasite. Sialic acid is widely distributed at the surface of many types of cells and several parasites use sialylated structures as receptors for invasion (reviewed in (Friedrich et al.)). T. gondii relies on sialic acid binding for invasion of certain cell types more than others (reviewed in (Hager and Carruthers, 2008)), suggesting that the parasite is able to select for the most appropriate invasion pathway for each cell type, depending on receptor availability. Unlike Plasmodium, a phenomenon of surface variant expression has not been reported in Toxoplasma, and since the tachyzoites surface is less space constrained than that of merozoites, choice of the most appropriate invasion

1 pathway is believed to rely on the simultaneous expression of multiple ligands targeting different receptors at the parasite surface (figure 4.1). Previous studies (Blumenschein et al., 2007) and the work reported on this thesis indicated that Toxoplasma relies on at least two MAR-domain containing proteins (MCPs) – TgMIC1 and TgMIC13 – for binding to the host cells sialic acid during invasion. Plasmodium does not encode MCPs and mice infected with a Tgmic1-3ko strain have lower parasite load and higher survival rate (Cerede et al., 2005; Moire et al., 2009) and it is therefore tempting to suggest that TgMIC1 and TgMIC13 can contribute to the broad host range of T. gondii. It should be noted, however, that micronemal proteins other than the MCPs contribute to the adhesion of T. gondii tachyzoites to host cells and Eimeria and Neospora parasites, which have a much more limited host range, also possess MCPs (Keller et al., 2002; Lai et al., 2009). The contribution of the MAR domain to this aspect must thus be mediated through recognition of an appropriately narrow or wide array of sialic acid structures in the context of particular glycoproteins. Supporting this idea, TgMIC1 and TgMIC13 were found to selectively bind to different carbohydrates, suggesting that they may be involved in the determination of tissue tropism. TgMIC1 binds with higher affinity to polyvalent carbohydrates possessing two or more sialic acid residues such as gangliosides found in neuronal tissues (Blumenschein et al., 2007) and TgMIC13 preferentially bound to sialic acid determinants most commonly found in the gut. This selective binding suggests that TgMIC1 may be an important player during the asexual stage of the parasite life cycle during bradyzoites invasion (Blumenschein et al., 2007), and TgMIC13 plays an important role during infection of the definitive host, the cat. This level of specificity is mediated by the different amino acid sequence, spacing and configuration of sialic acid binding sites on the MAR domains of the two parasite ligands as indicated by homology modeling of the TgMIC13 MAR domains, based on the crystal structure of TgMIC1. In the TgMIC1-MIC4-MIC6 complex, TgMIC4 also contributes to binding to the host cells (Brecht et al., 2001b), providing a further level of specificity. TgMIC13 was unable to restore micronemal targeting of TgMIC4 and TgMIC6 in the Tgmic1ko strain and it is thus not expected to associate with these proteins. Given the sequence similarity between TgMIC1 and TgMIC13 MAR domains and the fact that TgMIC1 associates with TgMIC4 via a MAR domain (Saouros et al., 2005), a strong premise is that TgMIC13 assembles in a complex with a PAN domain-containing protein with

1 similarity to TgMIC4. A search of the Toxoplasma genome revealed the presence of 12 PAN-domain containing proteins and study of their function will indicate if they are indeed partners of TgMIC13. Since TgMIC13 possesses neither a TMD nor motifs responsible for micronemal targeting (El Hajj et al., 2008), a protein functioning as escorter is also expected to assemble into this new micronemal complex. The specific contribution of TgMIC1 and TgMIC13 for binding of tachyzoites to sialic acid awaits the study of TgMIC13 function by disruption of the encoding gene but it remains possible that other adhesins also function as sialic acid-binding lectins. Two other MCPs - TgMCP3 and TgMCP4 – are encoded in the genome of T. gondii but the threonine residues shown to be crucial for binding to sialic acid (Blumenschein et al., 2007) are missing in their MAR domains and no carbohydrate- binding activity could be detected, indicating that these proteins are most likely not adhesins. Expression of C-terminal epitope-tagged versions of TgMCP3 and TgMCP4 in the parasite suggested that the proteins are stored in the dense granules and accumulate in the PV. It is possible that the epitope-tagged versions are mistargeted due to expression under the control of a strong promoter or interference of the tag but the same localization was obtained with expression under a weaker promoter or by inserting an epitope-tag just after signal peptide (data not shown), suggesting that they might be bona fide DG proteins. Conclusive information regarding the localization of TgMCP3 and TgMCP4 awaits epitope-tagging of the encoding genes by knock-in in the recently generated ku80ko parasite strain (Fox et al., 2009; Huynh and Carruthers, 2009) or the use of specific antibodies raised against the endogenous proteins.

2. Proteolytic shedding of Plasmodium surface proteins

Malaria merozoites employ both a subtilisin (PfSUB2) and a rhomboid activity (PfROM4) for shedding of surface proteins. PfSUB2 is responsible for the shedding of the complex PfMSP-1-6-7 from the parasite surface (Koussis et al., 2009). The exact function of PfMSP-1 is unknown but it has been suggested that it is involved in initial attachment to the host cell (reviewed in (Kadekoppala and Holder)). Merozoites perform invasion in the bloodstream, a turbulent environment, and the interactions with the host surface need to be stringer

1 than those between tachyzoites or sporozoites so that the parasite doesn’t accidentally disengage from the host cell surface. At the same time cleavage needs to occur quickly in order to allow passage of the parasite into the PV and invasion. PfSUB2 is ideal for this function because it is stored in the micronemes until contact with the host cell triggers secretion onto the surface (Harris et al., 2005). According to this model, one would expect that all Plasmodium proteins stored at the micronemes are shed from the parasite’s surface by rhomboid cleavage. Indeed most micronemal proteins are susceptible to cleavage by PfROM4 in vitro (Baker et al., 2006) but PfAMA1 is primarily cleaved in vivo by PfSUB2 (Howell et al., 2005). TgAMA1, the Toxoplasma homologue is exclusively cleaved by a rhomboid-like activity (Howell et al., 2005). Since the function of the protein is expected to be conserved in both parasites it is counter-intuitive to expect that shedding from the surface is mediated by two different proteases. Taking into account that PfAMA1 is susceptible to cleavage by a rhomboid protease in vitro (Baker et al., 2006) and in vivo (Howell et al., 2005), one hypothesis is that cleavage of the protein is mediated by both PfSUB2 and PfROM4 in merozoites. This is an unexpected situation but having shown that PfAMA1 like TgAMA1 might function during cell division, it can be envisioned that PfSUB2 mediates shedding of the protein from the merozoite’s surface and PfROM4 is responsible for releasing the cytosolic tail to initiate the signaling cascade leading to replication. Supporting this theory, the PfAMA1 stub carried into the ring stages of the erythrocytic cycle is generated by rhomboid- mediated cleavage (Howell et al., 2005).

3. MPP1 activity: TgROM4 or TgROM5?

Toxoplasma micronemal transmembrane proteins are exclusively shed from the surface by a rhomboid-like activity named MPP1. In order to determine if TgROM4 is responsible for the MPP1 activity, we expressed a dominant negative mutant of TgROM4 carrying a mutation at the catalytic serine

(ddROM4S-A). Expression of ddROM4S-A failed to inhibit invasion but blocked parasite replication at a late stage of the cell cycle, suggesting that the protease does not play an essential role during invasion. Our system revealed to be sub-optimal for

1 the study of TgROM4 function during invasion but the results obtained indicating that the MPP1 activity is most likely not performed by a single rhomboid protease are supported by the conditional knockout of TgROM4 (TgROM4iko), which only partially affected protein shedding (Buguliskis et al.). The new model for the MPP1 activity proposes that rhomboid activity is not restricted to the final steps of invasion, and that both TgROM4 and TgROM5 perform important non-overlapping functions (figure 4.2). TgROM4 is expressed throughout the parasite’s surface (Brossier et al., 2005; Dowse et al., 2005) and cleaves substrate proteins during the entire invasion process, removing adhesins that have not productively engaged in attachment and might thus be easily targeted by the host immune response and creating a gradient of adhesins from the anterior to the posterior end of the parasite that promotes parasite reorientation (Buguliskis et al.). TgROM4 activity is also essential for gliding since the inability to shed TgMIC2 results in a twirling movement in which the parasites are constantly attached to the host surface by their posterior end (Buguliskis et al.). At the end of invasion, TgROM5 might execute the final pinching activity that leads to closure of the PV and disengagement of the parasite from the host cell. Such scheme assumes that TgROM5 is exclusively localized to the posterior end of the parasite. In Plasmodium, only PfROM4 is expressed at the parasite’s surface (O'Donnell et al., 2006) and is therefore expected to perform both activities. Alternatively, TgROM4 and TgROM5 could play similar roles during invasion and just have different substrate specificity. Tgrom4iko parasites have enhanced surface staining of TgMIC2, TgAMA1 and TgMIC3 but not of TgMIC6 (Buguliskis et al.) despite the sequence similarity between the TgMIC2 and TgMIC6 rhomboid cleavage motifs. TgROM4 expression in Tgrom4iko parasites is not completely suppressed (Buguliskis et al.) and TgMIC6 might be less sensitive than the other TgMICs to changes in expression of the protease. Alternatively, inhibition of TgROM4 can be compensated by up-regulation of TgROM5 activity. If indeed TgROM4 does not mediate cleavage of TgMIC6, the apicomplexan rhomboids must rely on sequences beside the TMD for determination of substrate specificity. Previous studies had already suggested that this is the case; mutation of a lysine residue 11 residues upstream of the TMD abolishes cleavage of TgMIC2 by MPP1 (Brossier et al., 2003).

1 TgROM4 TgROM4 TgROM5? TgROM4

Figure 4.2 Schematic representation of the functions proposed for the MPP1 activity The MPP1 activity has been proposed to (from left to right): create a gradient of parasite adhesins from the anterior to the posterior end of the parasite; promote movement by disrupting the complexes formed between parasite adhesins and host cell receptors during invasion; be responsible for parasite disengagement from the host cell at the end of the invasion process; and promote gliding by breaking the interaction between TgMIC2 and the host cell surface.

Validation of TgROM5 as mediator of the MPP1 activity awaits clarification of its localization and insight into its function. Our attempts to study TgROM5 function with inducible dominant negative mutants were uninformative due to problems in localization of the expressed copy: expression of the gene annotated by Dowse et al. (Dowse et al., 2005) localized the protein to internal unknown structures; and expression of the alternative gene model (Brossier et al., 2005) resulted in a non- homogenous localization of the protein in the parasite population to either the plasma membrane or unknown intracellular compartments (data not shown). Interestingly, over-expression of TgROM5 did not produce a growth defect phenotype in contrast to what was previously reported by Dowse (Dowse and Soldati, unpublished) and Brossier et al. (Brossier et al., 2005).

4. AMA1: a multi-functional protein

AMA1 has been reported to act during parasite reorientation, participate in formation of the MJ and signal for rhoptries secretion (reviewed in (Remarque et al., 2008)). In this thesis, it is proposed that the protein may additionally anchor the MJ complex to

1 the parasite’s motor machinery and participate in a signaling mechanism leading to initiation of parasite division. Both activities are encoded in AMA1 tail but are mediated by different amino acid residues.

4.1. AMA1 connection to the glideosome In vitro pull down assays with GST-TgAMA1 revealed that the TgAMA1 C-terminus is capable of binding to aldolase. This result was surprising because previous data suggested that PfAMA1 surface distribution is insensitive to cytochalasin D (Howell et al., 2003; Silvie et al., 2004) and is therefore actin independent, and the immunoprecipitation experiments that led to identification of the MJ complex did not find any association between TgAMA1 and aldolase (Alexander et al., 2005). It is possible however that TgAMA1 association with aldolase may be less strong than the one reported for TgMIC2 because the speed of gliding and invasion are different. All our attempts to validate the in vitro data with pull down assays similar to those performed with TgMIC2 (Jewett and Sibley, 2003) were unsuccessful but this seems to be an experimental challenging assay as we were also unable to reproduce the results previously obtained with TgMIC2 (Jewett and Sibley, 2003). Supporting the importance of TgAMA1 association with aldolase, we could show that the FW motif implicated in aldolase binding is essential for invasion. In a parallel situation, mutation of the same residues in the C-terminus of PfAMA1 impairs invasion (Treeck et al., 2009). Although, because the tryptophan residue implicated in TgAMA1 binding to aldolase is in a different position than that present in the C- terminus of TgMIC2 (Jewett and Sibley, 2003), it remains to be confirmed if the essentiality of the FW motif for AMA1 function during invasion is associated to binding to aldolase or to another function. In fact the tryptophan residue present at the extreme C-terminus of TgMIC2 is strictly conserved in TgMIC6 and the protein is able to bind to aldolase in vitro but mutation of this amino acid residue does not interfere with function of the TgMIC1-MIC4-MIC6 complex during invasion.

4.2. AMA1 function during parasite division The study of TgROM4 function with a dominant negative mutant revealed that TgROM4-mediated cleavage of TgAMA1 is not essential for invasion but triggers the parasite to switch from an invasive to a replicative mode. These results were highly unexpected because rhomboids are not known to mediate intracellular signaling

1 pathways (reviewed in (Urban, 2006)) and such function had not been exposed by the study of parasites conditionally depleted for TgROM4 (Tgrom4iko) (Buguliskis et al.) or TgAMA1 (Tgama1iko) (Mital et al., 2005). However, Pprasites grown under repressive conditions still express background levels of TgROM4 and TgAMA1 mRNA transcripts (Buguliskis et al.; Mital et al., 2005) and it is expected that little TgAMA1 cytosolic tail is needed to participate in cell division. This idea is supported by an independent study showing that expression of a TgAMA1 construct mutated in the rhomboid cleavage motif in parasites expressing low levels of TgAMA1 (Tgama1iko) results in a cell division defect but does not interfere with invasion, and even undetectable levels of shed TgAMA1 can trigger parasite replication (G. Ward, personal communication). Furthermore, re-examination of the intracellular growth phenotype of Tgama1iko parasites revealed that they are impaired in cell division. Our results revealed then a fifth function for the MPP1 activity in Toxoplasma parasites mediated by the TgROM4 intramembrane proteolysis of TgAMA1 (figure 4.3).

Host cell invasion

Gliding motility Parasite replication

Figure 4.3 Schematic representation of the five functions proposed for the MPP1 activity in Toxoplasma The study of TgROM4 function with an inducible dominant negative mutant indicates that the MPP1 activity is essential not only during host cell invasion and gliding motility but also post-invasion during parasite replication inside the parasitophorous vacuole.

1 Pulse-chase intracellular growth experiments revealed that the cell division phenotype induced by expression of the TgROM4 dominant negative mutant is reversible and independent of invasion, suggesting that each cycle of replication requires a new boost of signaling, i.e., cleavage. Our results clearly pinpoint for the release of the cytosolic domain of TgAMA1 from the membrane as the signal switching on the replication mode of the parasite, but it remains unclear if TgAMA1 cleavage is responsible for triggering subsequent cycles of endodyogeny in Toxoplasma or this relies on cleavage of another rhomboid substrate because TgAMA1 is not expressed at the bradyzoite stage (Alexander et al., 2005) and it is believed to be secreted only during invasion, on extracellular parasites, upon a calcium signaling. It is experimentally challenging to demonstrate constitutive secretion of the protein onto the parasite’s surface and it remains possible that other molecules are involved in the switching mechanism post-invasion. Curiously the parasite encodes two TgAMA1 homologues of unknown function (figure 4.4). Requiring a new signaling boost at each replication round might be counter-intuitive but it is in fact a smart strategy from the parasite to tightly regulate its lytic cycle. Endodyogeny ensures that the new parasites produced are fully competent for egress from the host cell and invasion of new cells and therefore, at the end of each cycle of replication, it is necessary to decide if replication should be continued or arrested. The molecular aspects behind such decision are unknown for the moment but one can envision that, since rhomboids are constitutively active proteases, it can be either at the level of substrate secretion or protease expression. Study of TgROM4 expression throughout the Toxoplasma life cycle supports the first hypothesis since the protease is expressed throughout the cell cycle.

TgAMA1 YFAKRLDRNKGVQAAHHEHEF-QSDRGARKKRPS--DLMQEAE--PSFWDEA--EENIEQDGETHVMVE-GDY NcAMA1 YYSKRLNTNQGVPAVDHDHEF-QTQRNAQKKRPS--DLMQEAE--PSFWDEA--EENIDQSGETQVLVE-GDY EtAMA1 NPEKKKLLDEDEER-DEEFLKVQEKR---KHKQS--DLAQEAE--PSFWGETPQDHTNVVVDH--NAHD-AYY PfAMA1 YLYKRKGNAEKYDKMDE-----QH---YGK-SNSRNDEMLDPE--ASFWGE---EKRASHT--TPVLMEKPYY PbAMA1 YFFKSNKKGENYDRMG------QADI-YGK-ANSRKDEMLDPE--VSFWGE---DKRASHT--TPVLMQKPYY BbAMA1 IKAKKEPAPPSFDKYLSNYDYDTTLDA-DNETEQRLDSS--AY---S-WGEAVQRPSDV----TPVKLSKIN- TaAMA1 YS.KNHLKKHNSQIYEDDNVNNYYNEDFDD--EQDRDEYASNVRGDQIWSRHTPDRSEV----TPVRISRLNH NcAMA1.2 YYRKER-----DP------QAEQ------PT--VEAAGEGDREQETLL-GSRA---VEADY NcAMA1.3 YWIARRKKEEPEVGK-PNIVDETREHAVRARQNQA-DLLQEAE--PSFWGEAASQP--TNVILEPGAIDRDF-

Figure 4.4 (previous page) Alignment of the conserved C-terminal domain of AMA1 from different apicomplexan species Alignment of the C-terminal domain of AMA1 from T. gondii (TgAMA1), N. caninum (NcAMA1), E. tenella (EtAMA1), P. falciparum (PfAMA1), P. berghei (PbAMA1), B. bovis (BbAMA1) and T. anullata (TaAMA1) with the two AMA1 homologues encoded in N. caninum genome (NcAMA1.2 and NcAMA1.3). The AMA1 homologues are also encoded in T. gondii genome; their annotation is provisory and awaits experimental validation. Residues at least 50% conserved are labeled in blue. In red is highlighted the FW motif responsible for aldolase binding, in green is labeled the Y residue tested for function in cell division, in orange is labeled the serine residue phosphorylated in PfAMA1 (Leykauf et al.) and in pink are labeled the residues shown in (Treeck et al., 2009) to be essential for Plasmodium invasion.

It is unclear how cell division is triggered by the release of the TgAMA1 C-terminus from the plasma membrane. It is tempting to speculate that TgAMA1 tail could initiate a program of signal transduction that ultimately leads to expression of genes involved in parasite replication but this hypothesis has not been experimentally challenged. Moreover RIP mechanisms mediated by iCliPs are known to activate membrane-tethered transcription factors in response to various signals but the TgAMA1 tail seems too small to encode any DNA binding domains. Mapping of the residues involved in TgAMA1 tail function during cell division will aid clarifying this question. TgAMA1-mediated roles during invasion and replication are physically independent because mutation of the FW motif essential for function during invasion (figure 4.4) does not interfere with function during cell division. We also tested for a role of the extremely C-terminal Tyr residue, since it is strictly conserved throughout all apicomplexan AMA1 proteins (figure 4.4), but did not find impairment of function (data not shown). Although the PfAMA1 cytosolic tail can trans-complement the cell division deficiency imposed by expression of the dominant negative mutant of TgROM4, it is premature to conclude that a similar mechanism dictates initiation of the replication program of the malaria merozoites or any other Plasmodium replicative stages. Although PfROM4 and PfAMA1 are expressed at both stages of the life cycle, it is unclear what is the role of rhomboid cleavage for PfAMA1 function. Furthermore, Plasmodium merozoites do not replicate by endodyogeny but schizogony, in which all

1 daughter cells are generated simultaneously, and in that case a single PfAMA1 cleavage event during invasion would be sufficient to trigger the replication cycle. PfAMA1 is phosphorylated at its C-terminal domain at a serine residue (figure 4.4) just after schizont rupture by protein kinase A and this post-translational modification is important for function during invasion (Treeck et al., 2009; Leykauf et al.). However, mutation of the phosphorylated serine residue does not completely inhibit invasion and substitution by residues that mimic phosphorylation does not complement function (Leykauf et al.), suggesting that phosphorylation might not be essential. Intriguingly, it seems that the cleaved cytosolic tail of AMA1 that is carried into the newly invaded ring stages is not phosphorylated, raising the possibility that there is a de-phosphorylation event at this stage (Leykauf et al.). One possibility is that phosphorylation regulates AMA1 function: phosphorylation prompts for function during invasion and de-phosphorylation for function during replication. It should be noticed, though, that the modified serine residue is only conserved in Plasmodium species and in Toxoplasma, for instance, it is replaced by a glutamic acid (figure 4.4), suggesting that if phosphorylation plays a role during cell division, this phenomenon is not conserved throughout the phylum. AMA1 C-terminal domain is especially conserved and in fact mutation of several highly conserved residues susceptible or not to phosphorylation abrogates PfAMA1 function during invasion (figure 4.4) (Treeck et al., 2009). The strategy taken by Treeck et al. to gain insight into the role of the PfAMA1 tail during invasion does not allow one to investigate function during cell division but since TgAMA1 functions during invasion and cell division are physically displaced, it is expected that the residues mapped by Treeck et al. for PfAMA1 function during invasion will be dispensable for function during parasite division. Future experiments will address this hypothesis. TgMIC2, unlike TgAMA1 (Mital et al., 2005), is essential for gliding motility (Huynh and Carruthers, 2006) and TgROM4-mediated cleavage of TgMIC2 is essential for this function (Buguliskis et al.). The results excluding a function for TgMIC2 during parasite replication are thus of special relevance during parasite migration/traversal of tissues. Replication should only be triggered post-invasion and not in the context of parasite motility and participation of only TgAMA1 in a post-invasion event guarantees the necessary independence between migration and initiation of parasite division. In the same line of thought, it is expected that neither cleavage of PfTRAP

1 nor cleavage of any of the Plasmodium protein involved in sporozoite or ookinete migration (reviewed in (Morahan et al., 2009) will trigger replication.

5. Rhomboid-mediated RIP: a conserved mechanism for regulation of signal transduction in the Apicomplexa?

Rhomboids are common effectors of RIP but their function in the Apicomplexa has been limited to the surface shedding of parasite membrane-anchored proteins during invasion. As discussed in the previous section, it is now clear that TgROM4 is responsible for a mechanism resembling RIP acting on TgAMA1. TgROM4 and TgAMA1 are also important players during invasion and this sort of regulation of parasite division is thus a good strategy to assure a quick transition from the invasive to the replicative stage of the lytic cycle. The C-terminal domains of rhomboid substrates other than TgAMA1 have been proposed to participate in signaling pathways, raising the question of whether the apicomplexan rhomboids are involved in the regulation of other mechanisms. Rhoptries discharge relies on contact with the host cell (Alexander et al., 2005; Carruthers and Sibley, 1997) and, in Plasmodium, signaling is mediated by binding of the parasite micronemal ligands PfEBA-175 or PfEBA-140 to their host receptors (Singh et al.). The current model for rhoptry secretion in Plasmodium advocates that adhesin-receptor binding restores the basal calcium levels, which were elevated to induce microneme discharge, through a signal transmitted by the C-terminal tails of the PfEBA protein family members (Singh et al.). PfROM4 mediates cleavage of PfEBA-175 (O'Donnell et al., 2006), and possibly of PfEBA-140, and this event is essential for invasion independently of the use of PfEBA-175 as an adhesin during invasion (O'Donnell et al., 2006), raising the possibility that PfROM4-mediated cleavage is involved in the signaling mechanism. In Toxoplasma both TgAMA1 (Mital et al., 2005) and TgMIC8 (Kessler et al., 2008) are involved in rhoptries secretion. In the absence of TgAMA1 there is still secretion of the TgRONs (Alexander et al., 2005) but release of the rhoptry bulbs proteins (TgROPs) is impaired (Mital et al., 2005). In contrast, there is complete abrogation of rhoptry secretion in the absence of TgMIC8 (Friedrich et al.; Kessler et al., 2008). It is

1 then plausible to postulate that TgMIC8 association to an unknown host cell receptor signals for secretion of the TgRONs and, upon TgAMA1 association with the TgRONs complex, it is triggered secretion of the TgROPs. TgMIC8 is shown in this work to be susceptible to rhomboid cleavage and a previous study demonstrated that the C-terminal domain of TgMIC8 is functionally important and can only be complemented by that of an homologue, TgMIC8.2 (Kessler et al., 2008). Direct evidence for cleavage of TgMIC8 by TgROM4 is lacking but parasites conditionally depleted for the protease (Tgrom4iko) have enhanced surface staining of its associated soluble partner, TgMIC3, which is not a rhomboid substrate (Buguliskis et al.). It is unclear if Tgromi4ko parasites are defective in rhoptry secretion but most parasites are unable to form a MJ (Buguliskis et al.). This model of regulation would offer complete synchronization between parasite attachment to the host cell, microneme secretion, formation of adhesive complexes between parasite micronemal transmembrane proteins and host cell receptors, secretion of the rhoptry necks, formation of the MJ, secretion of the rhoptry bulbs, formation of the PV and initiation of the parasite’s replication program, once host cell penetration has been completed (figure 4.5). Nevertheless the importance of TgMIC8 rhomboid cleavage for rhoptries secretion is unclear because, as shown in this work, TgMIC8.2 is most likely not a rhomboid substrate.

Figure 4.5 Model for signaling of rhoptry 1 2 3 4 5 secretion in Toxoplasma Microneme proteins, including TgMIC8 and TgAMA1, are secreted onto the surface upon host cell contact by a calcium signal (1). Interaction of TgMIC8 with an unknown host receptor triggers secretion of the rhoptry neck proteins (RONs) (2). Formation of the AMA1-RONs complex at the moving junction (MJ) (3) triggers discharge of the rhoptry bulb contents (4). At the end of invasion, the parasite resides inside the PV (5) and can initiate replication. Adapted from (Alexander et al., 2005).

1 Rhomboid protein substrates of the Rh family in P. falciparum have also been implicated in signal transduction pathways. The PfRh2b encoding gene can be readily disrupted but it is impossible to generate tail-less PfRh2b parasites, suggesting that modification of the cytoplasmic tail results in a dominant negative effect (Desimone et al., 2009; Dvorin et al.).

6. Rhomboids as drug targets?

Several micronemal proteins in both Toxoplasma and Plasmodium have been shown to be non-essential and the parasites are able to switch invasion pathways in the absence of a determined adhesin or when under drug pressure. Targeting a specific adhesin is therefore not a good therapeutic strategy. Rhomboid activity, on the other hand, can been seen as an attractive drug target because it seems to be ubiquitous throughout the parasite’s life cycle, cleaving adhesins expressed at all zoite stages (Baker et al., 2006), and as shown in this thesis it also mediates other essential functions during the parasite’s life cycle. So far no specific drugs targeting rhomboid- like enzymes have been developed and there is a lack of knowledge regarding their mechanism of action and substrate specificity. On a positive note, some success has been obtained in the development of drugs specifically targeting presenilin activity (reviewed in (Bergmans and De Strooper)), also an iCLiP, suggesting that in the future it may be possible to specifically target rhomboid function.

7. Concluding Remarks

The study of the Toxoplasma MCP proteins indicated that TgMIC1 and TgMIC13 are sialic-acid binding lectins but it remains to be determined what is the identity of their host cell receptors and the function of TgMIC13. TgMIC13 and TgMIC16, a new micronemal protein identified in Toxoplasma, associate most likely in a complex with other micronemal proteins and future studies will possibly identify the composition of these new micronemal complexes.

1 The comprehensive study of the known Toxoplasma micronemal proteins revealed that TgMIC8 and TgMIC16 can function as rhomboid substrates and TgAMA1 and TgMIC6 may act as linkers between the host cell surface and the parasite’s actomyosin system. The functional analysis of TgROM4 indicated that the protease is not responsible alone for the MPP1 activity but that this role is most likely shared with TgROM5 and validation of this assumption requires now the functional study of TgROM5. Although we were unable to corroborate TgROM4 as the rhomboid responsible for the MPP1 activity, we could identify a new, unexpected role for the protease on a mechanism implicated in switching the parasite from an invasive to a replicative mode linked to the release of TgAMA1 cytoplasmic domain from the plasma membrane. This is the first mechanism of intracellular signaling mediated by a rhomboid protease and the first in apicomplexan parasites shown to be associated to the control of initiation of the parasite’s replication cycle. The work reported in this thesis inaugurates a new concept in Apicomplexa biology in which invasion and replication are linked by proteins with a dual function. Future experiments will dissect the details of the putative signal transduction pathway and indicate if PfROM4 performs a similar function in Plasmodium.

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1 Appendix

1 International Journal for Parasitology 39 (2009) 153–162

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International Journal for Parasitology

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Invited Review Apicomplexan cytoskeleton and motors: Key regulators in morphogenesis, cell division, transport and motility

Joana M. Santos a, Maryse Lebrun b, Wassim Daher a, Dominique Soldati a, Jean-Francois Dubremetz b,* a Department of Microbiology and Molecular Medicine, Faculty of Medicine–University of Geneva CMU, 1 rue Michel-Servet, 1211 Geneva 4, Switzerland b UMR CNRS 5235, Bt 24, CC 107 Université de Montpellier 2, Place Eugène Bataillon, 34095 Montpellier cedex 05, France article info abstract

Article history: Protozoan parasites of the phylum Apicomplexa undergo a lytic cycle whereby a single zoite produced by Received 30 July 2008 the previous cycle has to encounter a host cell, invade it, multiply to differentiate into a new zoite gen- Received in revised form 13 October 2008 eration and escape to resume a new cycle. At every step of this lytic cycle, the cytoskeleton and/or the Accepted 16 October 2008 gliding motility apparatus play a crucial role and recent results have elucidated aspects of these processes, especially in terms of the molecular characterization and interaction of the increasing number of partners involved, and the signalling mechanisms implicated. The present review aims to summarize Keywords: the most recent ﬁndings in the ﬁeld. Apicomplexa Ó 2008 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. Cytoskeleton Actin Myosin Mitosis Invasion Egress Glideosome

1. Introduction named the parasitophorous vacuole (PV). This distinct form of invasion, as well as migration, replication, invasion and egress (exit This review describes the most recent progress made in under- from host cells) takes advantage of the high flexibility of the cyto- standing the contribution of cytoskeletal elements and protein mo- skeleton that while, on one side, assures maintenance of the para- tors in governing the conserved mechanisms behind the site’s cell shape and structural integrity, on the other side, enables Apicomplexa lytic cycle events (Fig. 1). This phylum includes many adjustment of this same shape during migration and host cell inva- parasites, and while some aspects of their life cycle, such as the sion. Consequently, while some characteristics of the apicom- machinery that leads to gliding motility and the process of inva- plexan cytoskeleton are conserved with those of other sion, have been extensively dissected and demonstrated to be eukaryotes, others are unusual and specific to these organisms. shared among the members of the group, others, such as cell divi- Apicomplexan parasites are delimited by the pellicle (Fig. 2), a sion and morphogenesis, or host cell egress, are poorly understood. tri-bilayer structure, comprising the plasma membrane and two The most recent data suggests that both conserved and specific tightly associated membranes formed by endoplasmic reticulum mechanisms are involved in these processes. (ER)-derived flattened vesicles named the inner membrane complex (IMC). The IMC extends throughout the body of the parasite 2. Apicomplexan cytoskeleton – taking advantage of the best of and provides support for the gliding machinery, which drives two worlds: stability and flexibility motility. Closely associated to the parasite pellicle is the subpellicular network, which acts as the parasite’s skeleton and is consti- 2.1. The apical complex and cytoskeleton tuted by the intermediate filament-like TgIMC1 (Mann and Beckers, 2001). In contrast, an updated annotation of the TgIMC2 The invasive stages of the apicomplexan life cycles are named gene reveals that it codes for a phosphatase-like protein that car- zoites. These highly polarized cells attach apically to host cells ries a signal peptide and is the resident protein of the IMC (Frenal and invade them by building a unique membranous structure and Soldati, unpublished data). One of the IMC-associated proteins, photosensitized INA-labeled protein 1 (PhIL1), is thought to be responsible for the cytoskeleton-pellicle association (Gilk et al., * Corresponding author. Tel.: +33 467143455; fax: +33 467144286. E-mail address: [email protected] (J.-F. Dubremetz). 2006). Underneath the subpellicular network, at the apical tip, is

0020-7519/$34.00 Ó 2008 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2008.10.007 1 154 J.M. Santos et al. / International Journal for Parasitology 39 (2009) 153–162

Fig. 1. Apicomplexan life cycle illustrating all the lytic events: parasite division inside the invaded host cell, egress of the new daughter cells from the host cell, gliding motility, migration inside the host and recognition of the right target cell, and invasion of the new target host cell (clockwise).

Fig. 2. Scheme of a ‘‘model” apicomplexan parasite. Shown are the cytoskeleton elements (microtubules and centrocone), the apical complex (micronemes, rhoptries, conoid and apical polar ring), the pellicle (inner membrane complex (IMC) and plasma membrane), the secretory organelles (exonemes, dense granules, micronemes and rhoptries), the non-secretory intracellular organelles (mitochondrion, apicoplast, nucleus, endoplasmic reticulum (ER) and Golgi) and the basal complex. Note that not all members of the phylum contain the full repertoire shown in the ﬁgure.

the apical complex, after which the phylum is named, and the basal of the phylum; whilst the specialized secretory organelles, micro- complex is localized at the opposite end (Gubbels et al., 2006). nemes, rhoptries and dense granules, as well as the apical polar The apical complex is an exclusive structure of this group of ring, are present in all Apicomplexa, the full repertoire, which in- parasites but its composition can vary depending on the members 1 cludes the conoid, is only present in a set of parasites named coc- J.M. Santos et al. / International Journal for Parasitology 39 (2009) 153–162 155 cidians. The apical polar ring and the conoid are both elements of iti (Cortes et al., 2006), another coccidian parasite, was shown to the cytoskeleton but while the first one is the microtubule organiz- change shape and modify its surface when invading host cells, ing center (MTOC) of the subpellicular microtubules, the conoid is due to re-arrangements of its cytoskeleton, despite having the organized into a hollow cylinder composed of a polymer of alpha same subpellicular microtubule organization as the other and beta tubulins assembled into a new type of protofilament Coccidia. sheets (Hu et al., 2002) and can move up and down through the apical polar ring and protrude apically at the time of cell invasion 2.2. Building zoites: different ways of dividing; same mechanism? in a calcium-dependent fashion (Mondragon and Frixione, 1996; Monteiro et al., 2001). Three proteins likely to be involved in this Different Apicomplexa, and even different life cycle stages of motility are dynein light chain (TgDLC), which could be part of the same species, adopt distinct strategies to ensure the comple- the motor, and calcium-binding proteins 1 and 2 (TgCAM-1 and - tion of their replicative cycle. Most intracellular stages are not 2), which may regulate this kind of motion (Mondragon and Frixi- infectious and therefore cell division has to be precisely timed in one, 1996; Hu et al., 2006). order to ensure that the new daughter zoites are fully formed As mentioned above, the subpellicular microtubules, which are and prepared to invade at the time of host cell egress. The usual important for shape, apical polarity and organelle trafficking, are rule is schizogony, where several rounds of DNA synthesis and nu- organized from the apical polar ring, but two other sets of microtu- clear division occur prior to zoite genesis and cytokinesis, but in bules exist in these parasites. One set is found in the mitotic spin- some cases parasites replicate via endodyogeny (a variant form dle, where it coordinates chromosome segregation and originates of schizogony where DNA replication is immediately followed by from MTOCs organized by centrioles in Coccidia (Dubremetz, nuclear division and cytokinesis), which leads to the production 1973), or by rudimentary spindle pole bodies in other Apicomplexa of only two new daughter cells per replication cycle. Regulation such as Plasmodium (Schrevel et al., 1977), and the other set is of this process seems to involve cell cycle checkpoints similar to localized in the conoid. These different microtubules are uniquely those of other eukaryotes reviewed in Gubbels et al. (2008b),as specialized, in a phenomenon reflective of the apicomplexan para- a forward genetic screen of temperature-sensitive Toxoplasma gon- sites’ lifestyle. For instance, it was recently found that the subpel- dii cell cycle mutants led to the identification of multiple proteins, licular microtubules of Plasmodium berghei sporozoites (Cyrklaff several of which were shown to be orthologues of known cell cycle et al., 2007) are maintained in a state of ‘‘suspended depolymeriza- factors (Gubbels et al., 2008a). These master switches were re- tion” by an as yet unidentified molecule that allows them to bend cently suggested to be up/down-regulated according to each para- far beyond what is allowed by regular microtubules undergoing site-specific program, i.e. parasites that execute several rounds of treadmilling, an ability that is especially important during the DNA synthesis before cytokinesis (i.e. schizogony) would down- transmigration and invasion processes. regulate proteins involved in the checkpoint at the end of DNA rep- The number and organization of the microtubules can also lication (Striepen et al., 2007). Apicomplexan mitosis is known, differ between parasite species and even life cycle stages. Cryp- however, to also involve unique aspects. In T. gondii, for example, tosporidium parvum, for instance, was shown not to have subpel- the S phase is bipartite (Radke et al., 2001) and in Coccidia, the mi- licular microtubules but longitudinal ridges that might perform a totic spindle undergoes a complex cycle (Dubremetz, 1973), devel- similar function (Matsubayashi et al., 2008), and Besnoitia besno- oping first extranuclearly, then becoming intranuclear and, at last,

Fig. 3. Scheme of a mother parasite undergoing division by endodyogeny. Two identical new daughter cells are produced, completely protected within the mother cell until the end of the process. IMC, inner membrane complex. 1 156 J.M. Santos et al. / International Journal for Parasitology 39 (2009) 153–162 turning into a pair of centrocones, derived from both the nuclear of the daughter cytoskeletons as the new cells grow (Hu, 2008). envelope and the spindle poles, and characterized by the presence Two new conoids are constituted within the apical polar ring of the membrane occupation and recognition nexus motif contain- close to the TgMORN1 rings (Hu et al., 2006) and the growing ing protein TgMORN1 (Gubbels et al., 2006). subpellicular microtubules provide the scaffold for the construc- Multiple studies performed during the past 40 years have sug- tion of the new daughter IMC (Hu et al., 2002) and subpellicular gested that there is no fundamental distinction between the vari- network (Mann et al., 2002). An actin-like protein (TgALP-1) (Gor- ous modes of reproduction, apart from the number of nuclear don et al., 2008) appears to be connected to the formation of this divisions preceding zoite genesis. In all cases, the morphogenesis new IMC, either by escorting ER vesicles or by scaffolding mem- of apicomplexan zoites has been described as being coordinated branes. The newly formed IMCs continue to elongate and progres- with mitosis. The mitotic poles were clearly shown to be the pri- sively wrap the mitotic poles of the dividing nucleus together mary organizing centers of both the mitotic spindle and the apical with the apical organelles (Nishi et al., 2008). Concomitantly, cytoskeleton of the nascent zoites. The pattern of differentiation the new basal complexes gain polarity, upon recruitment of the has been described in many Apicomplexa and it was found to be centriolar marker, TgCentrin2, and the dynein light chain, TgDLC, conserved ((Dubremetz, 1975). What has been revealed more re- and move away from the apical complexes in a process thought cently are the molecular features of the structures previously de- to be microtubule-dependent (Gubbels et al., 2006). The new par- scribed. These findings essentially concern the process of asites continue to expand and fill the mother until they bud out, endodyogeny of T. gondii tachyzoites, thus we will only report on acquiring in the process their plasma membrane. At this time this process (Figs. 3 and 4). there is complete closure of the basal complex in a process pos- The first sign of cell division is the migration of the centrioles sibly driven by TgCentrin2 (Hu, 2008). This kind of replication en- to the basal pole of the nucleus and their replication. These rep- sures, that the new parasites are produced and protected within licated centrioles sandwich the spindle and the poles of the spin- an infectious mother parasite. dle give rise to the centrocones, on which the kinetochores of replicated chromosomes are attached. The primary structures which develop next to these centrioles are the two apical polar 3. Host cell egress: signalling and mechanism rings, from which the subpellicular microtubules extend, covered with the early IMC. The outer edges of the IMCs appear as ring The intracellular growth of Apicomplexa eventually causes structures decorated with TgMORN1 and are the precursors of lethal lysis of the host cells in a mechanism termed egress, which the daughter basal ring complexes, remaining at the basal ends results in the exit of infective parasites from their PVs. This pecu- liar process occurs after a highly variable number of parasite division cycles, the general rule being that a fixed number of mitosis occurs at each schizogonic stage of the cycle. Such a mechanism ensures that the daughter zoites are completely differentiated before the host cell is damaged by parasite development. In T. gondii, host cell lysis is a less critical issue since endodyogenic division allows production of infective parasites at the end of every round of replication. In T. gondii, egress is an active process relying on the parasites’ ability to sense that their host cell is dying or dead. Treatment with agents, such as the Ca2+ ionophore (A23187), which leads to an increase in the intra-parasitic level of calcium, is a potent artificial inducer of parasite egress (Endo et al., 1982) as early as 2 h p.i. (Caldas et al., 2007). During this process the micronemes discharge their contents, the conoid extends and the parasites become motile. Changes in host cell membrane permeability and host cell io- nic homeostasis seem to be involved but the exact origin and nature of such a signal remains elusive. It is known that potassium (K+) signalling is involved as decreasing the potassium concentration leads to premature egress (Black et al., 2000; Moudy et al., 2001) and the same phenomenon occurs following treatment with the K+ ionophore nigericin (Fruth and Arrizabalaga, 2007). This mechanism is thought to involve a yet unidentified parasite sensor that detects a decrease in the concentration of K+ inside the host cell and PV. The ability of the parasite to sense changes in the environment might also allow a prompt exit from compro- mised cells such as those targeted by the immune defence system. In such a model, molecules described to be crucial for Ca2+ signalling, such as phospholipase C, calmodulin and a Ca2+-dependent calmodulin domain protein kinase have been proposed to be required for egress and invasion (Moudy et al., 2001; Caldas et al., Fig. 4. Late stage of Toxoplasma gondii endodyogeny showing several of the 2007). A more recent study revealed that T. gondii tachyzoites cytoskeletal molecules involved in the process; Cc, centrocone; Ce, centriole; Co, can produce the hormone abscisic acid that induces the production conoid; DBR, daughter basal ring; DIMC, daughter inner membrane complex; G, of cyclic ADP ribose (cADPR) and which in turn stimulates calcium- golgi; HC, host cell; M, mitochondrion; N, nucleus; MBR, mother basal ring; MIMC&P, mother inner membrane complex and plasmalemma; PV, parasitophor- dependent protein secretion and leads to egress. The critical role ous vacuole; R, rhoptry. Bar = 1lm. 1 played by this hormone is moreover supported by the fact that J.M. Santos et al. / International Journal for Parasitology 39 (2009) 153–162 157 the same effect is obtained with the addition of exogenous abscisic state (Schmitz et al., 2005; Baum et al., 2006a; Sahoo et al., 2006; acid and that the selective disruption of its synthesis by the inhib- Schuler and Matuschewski, 2006) that can be rapidly polymerized itor fluridone leads to a delay in egress and prompts parasite differ- into microfilaments at a concentration three to fourfold lower entiation into bradyzoites (Nagamune et al., 2008). The notion that than mammalian muscle actin, in a process dependent on the egress depends on the parasite actin-dependent motility was re- presence of salt, magnesium and ATP (Sahoo et al., 2006). This al- cently challenged by the finding that treatment with actin-disrupt- lows a rapid treadmilling process that facilitates directional ing drugs does not delay parasite egress. A new model for egress migration, the fast regeneration of new actin subunits for future was then proposed in which the disruption of host cell actin would rounds of assembly, and avoids unwanted locomotion (Baum lead to internal pressure and mechanical rupture of the host cell et al., 2006a; Sahoo et al., 2006; Schuler and Matuschewski, membrane, which in turn would activate parasite motion due to 2006). the loss of ions from the host cell (Lavine and Arrizabalaga, Both these actin dynamics and microfilament turnover are 2008). suggested to arise from a sophisticated interaction with a vast ar- In the case of Plasmodium falciparum, the egress of merozoites ray of actin binding proteins (Schmitz et al., 2005). However, api- from red blood cells (RBCs) is very tightly regulated and involves complexan genomes contain relatively few conventional actin- the timely breakdown of the PV membrane followed by vesicula- binding proteins. Among this limited repertoire are actin depoly- tion of the RBC membrane (Glushakova et al., 2005). A new class merizing factor (ADF1) (Schuler et al., 2005), capping protein al- of secretory organelles named exonemes have recently been pha and beta (Gordon and Sibley, 2005), profilin (Plattner et al., identified and shown to control egress via release of the serine 2008), toxofilin (cofilin) (Poupel et al., 2000) and coronin (Tardi- protease PfSUB1 (Yeoh et al., 2007). Indeed, PfSUB1 is discharged eux et al., 1998; Figueroa et al., 2004). Unexpectedly, Apicom- from exonemes into the PV space before host cell rupture and in- plexa lack a canonical actin regulator Arp2/3 complex, which is duce a proteolytic maturation of the vacuolar marker PfSERA5. otherwise widespread among eukaryotes where it drives actin This last hypothetical protease is known to be essential for the assembly by nucleating filaments from the pointed end. Instead, efficient release of parasites from host RBCs (Delplace et al., the Apicomplexa possess formins (Gordon and Sibley, 2005; 1988; Yeoh et al., 2007; Arastu-Kapur et al., 2008). Similar find- Baum et al., 2006a) that are known, along with profilin, to drive ings have been reported for sporozoite release from mosquito actin polymerization in a mechanism alternative to that of the midgut oocysts, which is completely prevented by the disruption Arps (Higgs and Peterson, 2005). In T. gondii, profilin was recently of another SERA family member, SERA8 (ECP1), (Aly and Matus- shown to play a vital role in parasite motility and invasion in a chewski, 2005). process conserved across the phylum, since the Plasmodium profilin fully complements a T. gondii profilin knockout strain. Further- more, purified recombinant profilins from three different 4. Gliding machinery in Apicomplexa: the motor that drives Apicomplexa are able to control actin polymerization (Plattner infection et al., 2008). Apicomplexan genomes also encode two or more large formins that feature a typical forming homology domain 2 Migration across biological barriers and active penetration of (FH2) and a recent study highlighted Plasmodium formin 1 host cells and egress rely on the parasite’s ability to glide. Gliding (PfFRM1) as a potential effector in actin nucleation during inva- motility is critically dependent on actin polymerization and is sion, based on its localization at the moving junction and its abil- powered by a myosin motor (MyoA) ubiquitoulsy conserved ity to act as a potent actin nucleator of chicken actin in vitro across the phylum (Wetzel et al., 2005; Baum et al., 2006a,b; (Baum et al., 2008). Jones et al., 2006; Schuler and Matuschewski, 2006). Toxoplasma gondii MyoA (TgMyoA) was originally shown to belong to a motor complex including the myosin light chain (TgMLC1) (Herm-Gotz 5. Migration and host cell recognition: how to get there and et al., 2002) that is firmly anchored in the plane of the IMC by sense where you are the integral membrane glycoprotein GAP50 and the lipid modified GAP45 (Johnson et al., 2007). This organization extends to 5.1. Migration: getting there Plasmodium, where the orthologues of PfMTIP, PfGAP45 and PfGAP50 have been identified in P. falciparum merozoites (Baum Plasmodium sporozoites are only able to invade a restricted set et al., 2006b; Jones et al., 2006), and to all other members of of cell-types and have to endure a long journey in order to reach the phylum. their final destination, making migration undeniably fundamental It has been established that aldolase offers a bridge between the for the establishment of a malaria infection. Progress in investi- actomyosin system and the host receptor-parasite ligand com- gating this phenomenon has vastly benefited from the sophisti- plexes (Jewett and Sibley, 2003; Bosch et al., 2006). This glycolitic cated improvements in bioimaging (Amino et al., 2005, 2007; enzyme is unexpectedly able to bind to both the C-terminal do- Frevert et al., 2005; Tarun et al., 2006; Thiberge et al., 2007), main of an adhesin (TgMIC2 in Toxoplasma and TRAP, MTRAP and and this review will only focus on migration of the Plasmodium TLP in Plasmodium) and the parasite’s actin filaments (Buscaglia sporozoites from the site of injection to the liver. It is now known et al., 2003; Jewett and Sibley, 2003; Baum et al., 2006b; Heiss that once deposited in the skin, sporozoites do not leave immedi- et al., 2008). This interaction involving aldolase, and potentially ately but remain at their site of inoculation for 1–3 h after the other proteins, is important for parasite survival (Starnes et al., mosquito bite (Yamauchi et al., 2007) before entering blood or 2006). In such a model, motility is presumably generated by the lymph vessels (Amino et al., 2006). If invasion of a blood vessel posterior translocation of F-actin-aldolase bound to the adhesin occurs, the sporozoites are carried in the bloodstream and readily proteins driven by the myosin tracks firmly anchored and immobi- reach the liver. Once in the liver sinusoids, the next barrier that lized in the IMC (Johnson et al., 2007). sporozoites need to overcome is the endothelial barrier. It has Despite a clear role of F-actin dynamics in gliding, formal dem- been suggested that to access hepatocytes, sporozoites pass onstration of the presence of F-actin in Apicomplexa has been dif- through the resident liver macrophages (Kupffer cells) (Baer ficult due to the short size and inherent instability of these et al., 2007). At this point the parasite circumsporozoite protein filaments. Indeed, apicomplexan actin exhibits unusual proper- (CSP) binds to the liver surface LRP-1 and proteoglycans and pre- ties. The majority of actin molecules are maintained in a globular 1 vents activation of the respiratory burst, hence contributing to 158 J.M. Santos et al. / International Journal for Parasitology 39 (2009) 153–162 parasite survival (Usynin et al., 2007). As previously shown for A set of new data concerning recognition of liver cells by Plas- Eimeria sp. interacting with cells in vitro (Roberts et al., 1971), modium sporozoites revealed that the level of sulfation of surface when encountering the hepatic cells the sporozoites do not hepatocyte glycoproteins named HSPGs serves as a local position- immediately establish infection but first traverse several hepato- ing system (Coppi et al., 2007). The sporozoites seem to be ‘‘acti- cytes (Mota et al., 2001). It was initially hypothesized that this vated” for invasion when they contact the highly sulfated HSPGs would occur so that the host cells would be activated and become of the hepatocytes due the induction of the proteolytic processing more receptive to infection (Carrolo et al., 2003), but this theory of CSP that occurs just prior to invasion (Coppi et al., 2005). Pbs36p was revised when transgenic parasites lacking sporozoite micro- and Pbs36 are two members of the 6-cys domain-containing pro- neme proteins essential for cell traversal (SPECT-1 and -2/PPLP1) teins family that participate in this process of commitment for were shown to be unable to migrate through host cells but none- invasion as disruption of these genes leads to continuous traversal theless were able to productively invade hepatocytes (Ishino of hepatocytes and failure to find suitable host cells (Ishino et al., et al., 2004, 2005b). Given that SPECT mutant parasites are less 2005a). infective than wild type ones in vivo using the rodent malaria Despite intense studies on the malaria liver stage, a receptor for model, it appears that host cell traversal is not essential but sporozoites on hepatocytes has yet to be identified. CD81, a tetra- might help the parasite to encounter the optimal host cells (Ami- spanin family member, is involved in the permissiveness of hepa- no et al., 2008). In contrast, crossing hepatocytes also causes the tocytes to infection but this role seems to be indirect since CD81 release of several host cell factors such as NF-jB, which can alert appears to act as a modulator of an unidentified sporozoite protein the immune system and limit the extent of malaria infection in receptor (Silvie et al., 2003a,b; 2006; Yalaoui et al., 2008). New the liver (Torgler et al., 2008). clues regarding the answer to this question may be provided by In addition to SPECT-1 and SPECT-2 (Ishino et al., 2004, 2005b), the analysis of the belr1 locus of chromosome 17, which encodes two new members of the TRAP family TLP (Moreira et al., 2008) several host cell genes involved in susceptibility of mice to a liver and TRSP (Labaied et al., 2007), CelTOS (Kariu et al., 2006) and a infection (Goncalves et al., 2008). A productive infection requires phospholipase PbPL (Bhanot et al., 2005) have been reported to more, however, than a successful invasion of hepatocytes. It was be involved in cell traversal, however little is known about their recently shown that parasites lacking the sporozoite low complex- mechanistic contribution to the process. ity asparagine-rich protein (SAP1) were able to migrate and invade Several homologues of these proteins have been identified in but failed to develop of a productive infection due to the repression ookinetes. MAOP is a SPECT-2 homologue (Kadota et al., 2004) of several genes required for efficient development in the liver (Aly and CelTOS is expressed at both sporozoite and ookinete stages et al., 2008). (Kariu et al., 2006) suggesting that a common mechanism might explain membrane rupture of the hepatocyte and of mosquito midgut cells. 6. Invasion: how to go in? New steps were also made towards elucidation of the signals that induce this type of migration. Calcium-dependent protein ki- 6.1. Moving junction formation nase 3 (PbCDPK-3) was shown to regulate ookinete invasion of the midgut wall (Ishino et al., 2006) and PbCDPK-6 was demon- Invasion is a unique process tightly coupled to the sequential strated to be involved in the switch between migration and inva- secretion of two types of apical organelles named micronemes sion (Coppi et al., 2007). This calcium signalling is conserved and rhoptries. The micronemes are first discharging proteins across the Apicomplexa phylum, as T. gondii CDPK-1 (TgCDPK-1) thought to participate in gliding motility and host cell recognition appears to regulate motility and host cell invasion (Kieschnick (reviewed in Carruthers and Tomley, 2008) followed by the release et al., 2001). Potassium signalling also contributes to migration gi- from the rhoptries, club-shaped organelles with an anterior part ven that exposure of parasites to high concentrations of potassium called rhoptry ‘neck’ extending in the apical end. leads to a decrease in migration (Kumar et al., 2007), and activation In T. gondii, successful subcellular fractionation resulted in an of a potassium channel stimulates apical exocytosis, which causes enrichment in rhoptries, allowing the identification of more than a decrease in cell traversal (Ono et al., 2008). 30 rhoptry proteins, some sequestered in the bulb (ROPs) and others located in the duct part of the rhoptry (RONs) (Bradley 5.2. Host cell recognition: how to know it is time to stop migrating and et al., 2005). Lipids are also known to be contents of these start invading organelles (Foussard et al., 1991; Besteiro et al., 2008). Since many of the identified proteins are conserved across much of Apicomplexa exhibit very diverse preferences in terms of host the phylum and are secreted during invasion, the rhoptries have and host cell-type specificities with some parasites being able to long been suspected as playing a key role in the intracellular invade a wide repertoire of host cells while others are extremely lifestyle of the Apicomplexa. However, it was only recently restricted. shown that their contribution to invasion is not restricted to A generally common feature of host cell recognition seems to providing building material for the developing PV; they are also involve the binding to sialic acids on receptors at the surface of involved in modifying of the host cell following invasion (Brad- host cells. In Plasmodium, the erythrocyte surface protein 175 ley et al., 2005; Bradley and Sibley, 2007; Boothroyd and Dubr- (EBA-175) binds to the heavily sialyated receptor glycophorin A emetz, 2008). of RBCs (Tolia et al., 2005). A similar type of interaction might gov- Host cell invasion is exceptionally fast, taking about 10s, and it ern Babesia bovis binding to erythrocytes as the presence of a sialy- is intimately linked with gliding motility (see above). During this ated receptor similar to glycophorin A was shown to contribute to process host cell plasma membrane transmembrane (TM) proteins host cell invasion (Takabatake et al., 2007). Recognition of sialic but not glycosylphosphatidylinositol (GPI)–anchored proteins are acid was also previously reported to be critical for T. gondii inva- largely excluded from the newly formed PV, as shown for invasion sion (Monteiro et al., 1998). More recently, the adhesive domain of cells by T. gondii (Mordue et al., 1999) and of RBCs by Plasmo- called microneme adhesive repeat (MAR), present on TgMIC1, dium merozoites (Aikawa et al., 1978). This remarkable vacuole was demonstrated to bind selectively to sialic acid (Blumenschein remodelling remains a conundrum, but it is known to take place et al., 2007) however the nature of the receptor(s) on the host cell at the site of close attachment between the parasite and the host surface awaits further investigations. 1 cell membrane, named the moving junction (MJ), suggesting that J.M. Santos et al. / International Journal for Parasitology 39 (2009) 153–162 159 molecules that build the MJ are involved in this molecular sieving. RONs complex inserted into the host plasma membrane. Since The term Moving Junction, coined by Aikawa et al. (1978) to de- RON4 and RON5 contain no predicted TM domains, RON2, which scribe ‘‘a zone of attachment between the erythrocyte and merozo- is predicted to have two or three TM domains, may be responsi- ite that moves along the confronted membranes to maintain its ble for such a function. This model is supported by electron position at the orifice of the invagination” is a region of tight mem- microscopy of the MJ showing a thickening of the host cell membrane contact (less than 6 nm) between the parasite and the host brane and a specific substructure (Aikawa et al., 1978), which cell membranes, with the latter being markedly thickened. It be- suggests recruitment of proteins at this level. In this exciting sce- gins as a cup covering the parasite apex and rapidly turns into a nario, the insertion of the parasite’s own invasion apparatus into ring encircling the parasite, moving backwards relative to the par- the host cell membrane would not only act as a grip and contrib- asite, and when entry is completed, fusion occurs at the posterior ute to exclusion of cytoskeletal and TM proteins from the vacu- end of the parasite. The movement that propels the parasite into ole, but would also explain the large diversity of cells invaded the nascent PV is possible because the proteins forming the MJ by most Apicomplexa. are probably connected to yet unidentified components of the Interestingly, AMA1 and RONs proteins are present in all the actomyosin motor. genomes of Apicomplexa sequenced to date, with the exception The molecular components of the MJ remained a mystery for a of the Cryptosporidium genus, which displays a markedly distinct long time but recent reports highlighted an association between mode of zoite-host cell interaction. In the case of Theileria,noMJ microneme and rhoptry proteins (Alexander et al., 2005). Some is visualized during leucocyte invasion by sporozoites or merozo- of these are hypothetical proteins restricted to Apicomplexa, sug- ites but this process is considered as a zippering interaction driven gesting that these parasites have developed a specific machinery by the host cell (Fawcett et al., 1984), and these sporozoites and for host cell invasion that has no counterpart in other cells. The merozoites are known to differ from classical apicomplexan zoites cooperation between proteins of the micronemes and the rhoptries in many significant aspects (they have no clear apical complex or at the MJ is supported by several pieces of data published through- IMC and are non-motile). Nevertheless, in the tick vector the Thei- out the years. It was first shown that antibodies against the micro- leria kinete possesses an apical complex and is motile, suggesting neme protein PfAMA1 inhibit the committed attachment between formation of a MJ. Plasmodium merozoites and RBCs – the initial random surface Further studies are obviously necessary to dissect the functional attachment of merozoites to RBCs was not affected but the close significance of RONs at the MJ, since almost all data reported to junctional contact was absent (Mitchell et al., 2004). Then Mital date are derived from studies on only one stage of one species, T. et al. (2005), using an engineered T. gondii strain expressing less gondii. than 0.5% of TgAMA1, showed that this protein is not involved in gliding motility, or in the initial step of attachment, or in micro- 6.2. Secretion and post-secretory fate of MICs and ROPs: signalling neme release, but it is needed for an intimate attachment to the issues host cells. Finally, it was demonstrated that the rhoptry neck protein TgRON4 co-localizes with the MJ (Lebrun et al., 2005) where it One of the most sophisticated features of invasion by Apicom- associates with TgAMA1 (Alexander et al., 2005), and that TgAMA1 plexa is the coordinated secretion of micronemes and rhoptries. deletion has no effect on RON4 secretion but abolishes its recruit- As discussed above, microneme secretion is regulated by an in- ment at the MJ and blocks invasion. Moreover the presence of crease in the cytoplasmic calcium that is released from intracellu- PfRON4 at the MJ of merozoites invading RBCs (Baum et al., lar stores in the parasite. Host cell calcium is dispensable, as 2008) and the association of PfAMA1 with PfRON4 in P. falciparum, microneme discharge can be artificially triggered by ionophores suggest that the collaboration between micronemal AMA1 and (Carruthers and Sibley, 1999) and occurs before the interaction rhoptry RON proteins is a conserved feature (Alexander et al., with a host cell since it is required for gliding motility (Lovett 2006). and Sibley, 2003). On the other hand, secretion of rhoptries cannot The MJ complex is now known to contain other rhoptry proteins be mimicked in the absence of host cells, suggesting that it is (RON2, RON4, RON5). They were identified in T gondii by pull- dependent on intimate contact of the parasite with the host cell down experiments with anti-RON4 or anti-AMA1 antibodies, and membrane. cross-linking experiments during host cell invasion by this same As the parasite penetrates the host cell, most MIC proteins (ex- parasite have demonstrated that this RON complex is probably cept AMA1) are excluded from entering the vacuole and are pro- pre-formed inside the parasite but only associates with AMA1, gressively capped behind the MJ, remaining confined to the which is secreted first, upon discharge of the rhoptries onto the portion of the parasite that still protrudes from the host cell. In parasite’s surface (Alexander et al., 2005). It is still unclear, how- contrast, rhoptry proteins reach at least four destinations: (i) sev- ever, how the complex is organized at the MJ. eral RONs remain associated with the MJ; (ii) some ROPs end up As mentioned above, the MJ complex represents a stable in the PV, (iii) others associate with the PVM, and (iv) another frame at the cell surface onto which the parasite grabs to propel group of ROPs are found beyond the PVM, in the host cell nucleus itself inside the cell using its gliding motion, implying that it is (Boothroyd and Dubremetz, 2008). linked to the subpellicular motor of the parasite. In such a model How the different proteins reach their final destination is un- AMA1, which shares homologies with TgMIC2 in its C-terminal known. It may be due to the association with lipids that are some- domain, should interact with the glideosome to ensure transloca- times visualized using electron microscopy as membrane whorls tion of the MJ during invasion. However, this protein does not inside the rhoptries (Nichols et al., 1983). These lipid vesicles possess the critical tryptophan in its C-terminus that appears (termed e-vacuoles) are secreted in the host cell cytoplasm and necessary for its connection to the sub-membranous motor (Jew- then fuse with the parasite-containing vacuole, in a mechanism ett and Sibley, 2003) and therefore an indirect interaction of blocked by cytochalasin D (Hakansson et al., 2001). While the sig- TgAMA1 with other transmembrane MIC proteins such as nalling leading to microneme exocytosis and its direct conse- TgMIC2 at the MJ seems more likely. This model also implies that quence, i.e. gliding motility, is rather well known (discussed the MJ is held at the host cell surface by interacting with a stable above), the trigger for rhoptry exocytosis is entirely distinct and cytoskeletal structure, indirectly via association with integral has not been elucidated. It is therefore unlikely that microneme host proteins linked to the cytoskeleton, or directly by interac- and rhoptry neck fuse before exocytosis, as is sometimes suggested tion of the host cell subplasmalemmal cytoskeleton with the 1 in the literature. 160 J.M. Santos et al. / International Journal for Parasitology 39 (2009) 153–162

7. Conclusion Bosch, J., Turley, S., Daly, T.M., Bogh, S.M., Villasmil, M.L., Roach, C., Zhou, N., Morrisey, J.M., Vaidya, A.B., Bergman, L.W., Hol, W.G., 2006. Structure of the MTIP-MyoA complex, a key component of the malaria parasite invasion motor. This short review aims to highlight the questions that still re- Proc. Natl. Acad. Sci. USA 103, 4852–4857. main concerning every step of the apicomplexan lytic cycle and Bradley, P.J., Sibley, L.D., 2007. Rhoptries: an arsenal of secreted virulence factors. the most recent findings regarding the proteins and mechanisms Curr. Opin. Microbiol. 10, 582–587. Bradley, P.J., Ward, C., Cheng, S.J., Alexander, D.L., Coller, S., Coombs, G.H., Dunn, J.D., involved (check Supplementary Table S1 for a summary). It is still Ferguson, D.J., Sanderson, S.J., Wastling, J.M., Boothroyd, J.C., 2005. 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