MeBOP Module 2 Introduction

MeBOP Module 2 Introduction

Karine Frenal: The expert of Toxo glideosome and IMC Damien Jacot: Expert in about everything

Sunil Kumar Dogga: TgAsp3 Budhaditya Mukherjee: Biochemistry

Apicomplexans are human and animal pathogens Piroplasmida Theileriidiae Theileria East coast fever Babesiidae Babesia Haemosporida Plasmodium spp Malaria Apicomplexa Coccidia Eimeriida Eimeriidae Eimeria Coccidiosis Neospora Sarcocystidae Toxoplasma Toxoplasmosis Sarcocystis Cryptosporidiidae Cryptosporidium spp cryptosporidiosis

Malaria Caused by - Plasmodium falciparum - P. vivax - P. malariae - P. ovale - P. knowlesi Malaria - Fever - Anaemia - Metabolic dysfunctions : acidosis, hypoglycemia At risk - More than 40% of the world population Deaths - Around 0.7 million per year

Plasmodium falciparum life cycle oocyste' ookinete'

Plasmodium falciparum erythrocytic stages Feeding phase Replication phase Egress & Invasion phase

Plasmodium falciparum erythrocytic stages 0!5h%Rings% 5!10h%%%%%%%%%%%%%%%%%%%%%%%%%%%%% 10!20h%%%%%%%%%%%%%%%%%%%%%%%%% Early%trophozoite% Mid!Late%Trophozoite% >40h%Schizont%

Apicomplexan invasive tachyzoite and merozoite Micronemes Rhoptries Inner Membrane Complex (IMC) Dense granules Adapted from Frenal K et al (2013) Traffic

Toxoplasma gondii Model organism Easy genetics good looking Easy to grow Plasmodium berghei Mouse malaria Easy genetics In vivo only Full life cycle accessible Plasmodium falciparum Human malaria Tricky genetics Relevant

Aspartic endopeptidases (ASP/PM)! Present in all eukaryotes! Broad range of roles : - protein degradation - enzyme maturation - signal transduction - virulence factors! Use Asp residues in the motifs DTG or DSG! Pro-region inactivates enzyme! Proteolytic maturation leads to activation

Mode of action Activation with water hydroxyl-anion formation Peptidic bond lysis

Apicomplexan ASPs follow 6 distinct groups Protein*export* PV/PVM* PMVIII' TgASP4' TgASP2' PMVI' PMVII' TgASP5' PMV' D' F' B' C' E' TgASP6' TgASP7' A' TgASP3' TgASP1' PM1,2,4,HAP' PMX' PMIX' Plasmodium*specific* Hemoglobin*degrada4on*

Plasmodium falciparum Plasmepsins 10 aspartic proteases: PfPMI-PfPMX 7 expressed during the erythrocytic stages Food vacuole Hemoglobin degradation ER Export? Expressed during sexual stages

Hemoglobin degradation Goldberg, PNAS 2006 - A massive catabolic process. - Consumes 75% of the infected cell Hb, which provides an important source of amino acids for the parasite growth and maturation - In an acidic food vacuole - Catalyzed by four aspartic proteases (plasmepsins), three cysteine proteases (falcipains) and one metalloprotease (falcilicin) " Drug target?

The way you cut your meat reflects the way you live Confucius endocytosis Hemoglobin Hemoglobin Oxyhemoglobin Globin Ferriprotoporphyrin IX Large peptides Small peptides Plasmepsins aspartic-proteases Falcipains cysteine-proteases Falcilysin metallopeptidase Aminopeptidase metallopeptidase Transporter? Amino acids

The way you cut your meat reflects the way you live Confucius endocytosis Hemoglobin Hemoglobin Oxyhemoglobin Toxic' Globin Ferriprotoporphyrin IX Polymerization Large peptides Small peptides Hemozoin Non'toxic' Transporter? Amino acids

Plasmodium falciparum Plasmepsins 10 aspartic proteases: PfPMI-PfPMX 7 expressed during the erythrocytic stages Food vacuole Hemoglobin degradation ER Export? Expressed during sexual stages

Aspartyl proteases implicated in protein export in P. falciparum Maurer s cleft Red Blood Cell cytosol PV PEXEL/HT motif R/KxLxE/Q/D PfPMV - Plasmepsin V Boddey et al, Nature, 2010 Russo et al, Nature, 2010 PMV Nucleus ER Parasite Homologue in T. gondii RxLxE/D TgASP5 - Aspartyl Protease 5 Hsiao et al, Traffic, 2013 Curt-Varesano et al, Cell microbial, 2015 Hammoudi et al, PLoS pathogens, 2015 Coffey et al, elife, 2015

Plasmepsins expression throughout the life cycle PMI PMVI PMII PMV PMVII PMIX 2400 2000 PMIII/HAP PMIV PMVIII PMX RPKM 400 200 0 Ring Early Trophozoite Late Trophozoite Schizont Gametocyte II Gametocyte V Ookinete

Plasmodium falciparum Plasmepsins KO viable ' ' ' ' Phenotype None None None None ' Asexual growth???? ' '? Oocyst None?

PMIX localizes at the apical end of merozoites PM9-Ty AMA1 PM9-Ty CyRPA

PfPMIX-Ty-Lox expression/excision 100 70 55 PfPMIX expected size 74 kda 40 35 PRF

PMIX is critical for blood stages development 40% 35% 30% PM9LoxP%+%Rapa% WT%+%Rapa% PM9LoxP% Parasitemia'%' 25% 20% 15% 10% +%Rapa% 5% 0% 0% 1% 2% 3% 4% 5% 6% 7% Days'

TgASP3' PMIX' Toxoplasma gondii ASPs PMX' 331!351% 167!187% 340!360% 131!151% 500!521% 155!175% 269!290%

Asp3 is a post-golgi resident protease Asp3%3Ty ( 5 UTR' Modified%locus% 3Ty'tag ' SAG1'3 UTR' HXGPRT' 3 UTR' TgASP3LTy' αlmlc1' Merge' TgASP3LTy' GRASPLGFP' Merge'

Tet-inducible knock-down of ASP3 ASP3L3Ty' +'ATc' ASP3L3Ty' 130' 100' 70' Pro' 55' 40' Mature' Asp3L3Ty' LATC' +ATC' αty' αcatalase' DTGS% FLAP' DSGT% 30% 298% 489% 3Ty% DTGS% FLAP' DSGT% 298% 489% 3Ty% GAP45LTy'

TgAsp3 is critical for Toxo lytic cycle -ATC +ATc Gliding' moulity' AYachment' Invasion' Egress' Intracellular' growth'

hydroxyethylamine scaffold-based drug 49c Low%IC50%at%72%hr% High%IC50%at%24hr%

hydroxy-ethyl-amine scaffold-based drug 49c IC 50 (24 hours) >500 nm, IC 50 (72 hours) 0.6 nm Ciana et al. 2013 - Peptidomimetic inhibitor of aspartic proteases - Designed to target Plasmodium food vacuole aspartyl proteases - slow acting drug and dropped

Compound 49c efficiently blocks Toxo lytic cycle IC 50 650 nm (4 days) CTL' 1000'nM' 500'nM' 250'nM' 125'nM' 63'nM' 31'nM' 16'nM' 8'nM' 4'nM' 2'nM' 1'µM'PYR'

What we want! - Functional characterization of PfPMIX - Functional characterization of TgAsp3 - Molecular targets of compound 49c