Thesis
Reference
Functional investigation of rhomboid proteases and their substrates in Toxoplasma gondii
MENDONCA DOS SANTOS, Joana
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). [...]
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.
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.
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.
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.
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
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.
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 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
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
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
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
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
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
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
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 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).
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 1.1 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).
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-
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 material contaminated with oocysts (left blue arrow). Adapted from dpd.cdc.gov.
congenital transmission
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).
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
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).
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
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).
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)).
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).
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.,
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).
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.
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
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).
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
Invasion Invasion Invasion Invasion Gliding Virulence ROP secretion MJ formation RON secretion
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).
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),
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
(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 un- characterized 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.
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
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.,
1992)
Glycophorin A (Orlandi et al., 1992)
Sia-dep and Sia-indep (Duraisingh et al.,
2003a) PfEBA-140 Sia-dep (Maier et al.,
2009)
Promiscuous, Glycophorin C (Maier et al., 2009; Mayer et al., 2009; Mayer et al., 2006)
Sia-dep (Maier et al., 2003)
PfEBA-181 Sia-dep (Maier et al., 2009)
Unknown Sia-dep (Maier et al., 2009) PfEBL-1 Sia-dep (Mayer et al.,
2009)
Glycophorin B (Mayer et al., 2009)
Sia-dep (Mayer et al., 2009) PfRh1 Sia-dep (Triglia et al.,
2005)
Unknown Sia-dep (Triglia et al., 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.,
2007)
Unknown Sia-indep (Stubbs et al., 2005) PfRh5 Sia-indep (Baum et al.,
2009)
Unknown Sia-indep (Baum et al., 2009)
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
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.
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
αTgAMA1 αTgM2AP
MJ
merge
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
