Toxoplasma Gondii: The Model Apicomplexan - Perspectives and Methods

Description

Toxoplasma gondii: The Model Apicomplexan - Perspectives and Methods, Third Edition, reflects significant advances in the field in the last five years, including new information on the genomics, epigenomics and proteomics of T. gondii, along with a new understanding of the population biology and genetic diversity of this organism. This edition expands information on the effects of T. gondii on human psychiatric disease and new molecular techniques, such as CAS9/CSPR. T gondii remains the best model system for studying the entire Apicomplexa group of protozoans, which includes Malaria, making this new edition essential for a broad group of researchers and scientists. Presents a complete review of molecular and cellar biology and immunology of Toxoplasma gondii combined with methods and resources for working with this pathogenProvides a single source reference for a wide range of scientists and physicians working with this pathogen, including parasitologists, cell and molecular biologists, veterinarians, neuroscientists, physicians and food scientistsCovers recent advances in the genomics, related bioinformatics analysis, epigenomics, gene regulation, genetic manipulation and proteomics of T. gondiiDetails advances in the molecular and cellular biology and immunology of Toxoplasma, and in the epidemiology, diagnosis, treatment and prevention of toxoplasmosis Cover Toxoplasma Gondii: The Model Apicomplexan—Perspectives and Methods Copyright Dedication Contents List of contributors Preface to the third edition 1 The history and life cycle of Toxoplasma gondii 1.1 Introduction 1.2 The etiological agent 1.3 Parasite morphology and life cycle 1.3.1 Tachyzoites 1.3.2 Bradyzoite and tissue cysts 1.3.3 Enteroepithelial asexual and sexual stages 1.4 Transmission 1.4.1 Congenital 1.4.2 Carnivorism 1.4.3 Fecal–oral 1.5 Toxoplasmosis in humans 1.5.1 Congenital toxoplasmosis 1.5.2 Acquired toxoplasmosis 1.5.2.1 Children 1.5.2.2 Toxoplasmosis in adults 1.5.2.2.1 Lymphadenopathy 1.5.2.2.2 Ocular disease 1.5.2.2.3 Acquired immunodeficiency syndrome epidemic 1.6 Toxoplasmosis in other animals 1.7 Diagnosis 1.7.1 Sabin–Feldman dye test 1.7.2 Detection of IgM antibodies 1.7.3 Direct agglutination test 1.7.4 Detection of Toxoplasma gondii DNA 1.8 Treatment 1.9 Prevention and control 1.9.1 Serologic screening during pregnancy 1.9.2 Hygiene measures 1.9.3 Animal production practices 1.9.4 Vaccination References Further reading 2 The ultrastructure of Toxoplasma gondii 2.1 Invasive stage ultrastructure and genesis 2.1.1 Basic ultrastructural morphology 2.1.2 Comparison of the invasive stages 2.1.3 Host cell invasion 2.1.4 Parasitophorous vacuole, intracellular development 2.1.5 Endodyogeny 2.1.5.1 Mitosis 2.1.5.2 Zoite biogenesis 2.2 Coccidian development in the definitive host 2.2.1 Host–parasite relationship 2.2.2 Asexual development 2.2.3 Sexual development 2.2.3.1 Microgametogony and the microgamete 2.2.3.2 Macrogametogony and the macrogamete 2.2.4 Oocyst wall formation 2.2.5 Fertilization 2.2.6 Oocyst and extracellular sporulation 2.2.7 Excystation 2.3 Development in the intermediate host 2.3.1 Tachyzoite development 2.3.2 Stage conversion: tachyzoite to bradyzoite 2.3.3 Structure of the tissue cyst and bradyzoite 2.3.4 Inflammatory changes in the brains of infected mice 2.3.5 Cyst rupture in immune competent hosts 2.3.6 Development in vitro 2.3.6.1 Tachyzoite development in vitro 2.3.6.2 Bradyzoite development in vitro References 3 Molecular epidemiology and population structure of Toxoplasma gondii 3.1 Introduction 3.2 Genetic markers 3.2.1 Microsatellites 3.2.2 Polymerase chain reaction restriction fragment length polymorphism 3.2.3 Multilocus DNA sequence typing 3.2.4 Serotyping 3.2.5 Whole-genome sequencing 3.2.6 Correspondence between haplogroups, polymerase chain reaction restriction fragment length polymorphism, and microsate... 3.3 Evolutionary history 3.4 Global diversity and population structure 3.4.1 Geographical distribution 3.4.1.1 Europe 3.4.1.2 Africa 3.4.1.3 Asia 3.4.1.4 Australia 3.4.1.5 North America 3.4.1.6 Central and South America 3.4.2 Factors affecting transmission and genetic exchange 3.4.2.1 Biological factors 3.4.2.2 Dynamics of transmission between different environments or hosts 3.4.2.3 Environmental and human factors 3.5 Outbreak investigations 3.6 Toxoplasma genotype and biological characteristics 3.7 Toxoplasma gondii genotype and human disease 3.7.1 Circumstances of isolation and genetic typing 3.7.2 Congenital toxoplasmosis 3.7.3 Postnatally acquired toxoplasmosis in immunocompetent patients 3.7.3.1 Ocular toxoplasmosis 3.7.3.2 Disseminated toxoplasmosis 3.7.4 Postnatally acquired toxoplasmosis in immunocompromised patients 3.8 Conclusion and perspective on Toxoplasma genotype and human disease References 4 Human Toxoplasma infection 4.1 Clinical manifestations and course 4.1.1 Introduction and history 4.1.2 Postnatally acquired infection in children and adults 4.1.2.1 Adults and older children with primary, acute acquired Toxoplasma gondii infection 4.1.2.2 The special problem of primary infection during gestation 4.1.2.3 Postnatally acquired infection in older children and adults—the chronic infection 4.1.3 Congenital infection 4.1.3.1 The fetus, infant, and older child 4.1.3.2 Congenital toxoplasmosis in different countries 4.1.3.2.1 France and Belgium 4.1.3.2.2 Austria, Germany, The Netherlands, and Italy 4.1.3.2.3 United States 4.1.3.2.4 Brazil 4.1.4 The special problem of ocular disease 4.1.5 Immune-compromised patients 4.1.5.1 HIV-infected patients 4.1.5.2 Persons with cardiac and renal transplants 4.1.5.3 Bone marrow and hematopoietic stem cell transplantation 4.2 Diagnosis of infection with Toxoplasma gondii 4.2.1 Toxoplasma antigens and diagnostic assays 4.2.2 The development of diagnostic assays 4.2.3 Diagnosis of Toxoplasma gondii infection in pregnant women 4.2.3.1 IgG avidity index 4.2.3.2 Combined, two-test strategies 4.2.4 Improvement of enzyme immunoassay tests for Toxoplasma-specific IgG and IgM antibodies 4.2.5 Recombinant IgG assays—adults 4.2.6 Recombinant IgM and IgG assays—newborns 4.2.7 The Toxoplasma-specific IgG avidity index 4.2.8 Molecular and other diagnostic techniques 4.2.9 Diagnosis of Toxoplasma gondii infection in newborn infants 4.2.10 Prompt diagnosis during gestation to facilitate treatment with unique spillover benefits 4.2.11 Immune-compromised patients 4.3 Treatment 4.3.1 Asymptomatic infection or latent infection 4.3.2 Acute/acquired toxoplasmosis 4.3.3 Acute/acquired toxoplasmosis during pregnancy 4.3.4 Congenital toxoplasmosis 4.3.5 Ocular toxoplasmosis 4.3.6 Toxoplasma infection in immune-compromised persons 4.3.7 Future development of newer improved anti–T. gondii agents 4.4 Prevention 4.5 Other considerations of pathogenesis in human infections 4.5.1 Recent studies of clinically identified associations of human brain or other diseases and presence of Toxoplasma infe... 4.5.2 Structural and functional neuroimaging in uninfected versus infected persons without recognized clinical symptoms 4.5.3 Genetic analyses: candidate human genes in cohort and transmission disequilibrium testing studies 4.5.3.1 National Collaborative Chicago-Based, Congenital Toxoplasmosis Study (sometimes EMSCOT) gestational and congenital ... 4.5.3.2 Case report and literature review concerning mutations and susceptibility to severe disease when infected with Toxo... 4.5.3.3 Brazil 4.5.3.4 Colombia 4.5.3.5 Poland 4.5.4 Signature pathways in neuronal stem cells, peripheral blood monocytic cells, and retinal cells modified by Toxoplasma... 4.6 Conclusion, unifying concepts, and toward the future References Further reading 5 Ocular disease due to Toxoplasma gondii 5.1 Introduction 5.2 Historical landmarks in ocular toxoplasmosis 5.3 Epidemiology 5.4 Pathophysiology: lessons from animal models and clinical studies 5.5 Host factors 5.6 Parasite factors 5.7 Animal models 5.8 Clinical characteristics 5.8.1 Recurrence 5.8.2 Congenital ocular toxoplasmosis 5.8.3 Ocular presentation in the elderly 5.8.4 Atypical presentations of ocular toxoplasmosis 5.8.4.1 Immunocompromised patients 5.8.4.2 Acute retinal necrosis 5.8.4.3 Punctate outer retinal toxoplasmosis 5.8.4.4 Other atypical clinical presentations 5.8.5 Classification systems for uveitis and retinochoroiditis 5.8.5.1 Anterior uveitis 5.8.5.2 Vitritis 5.8.5.3 Retinochoroiditis 5.8.6 Optic nerve involvement in ocular toxoplasmosis 5.8.7 Toxoplasma and glaucoma 5.9 Diagnostic tests 5.9.1 Histopathology 5.9.2 Ocular biopsies 5.9.3 Serology 5.9.4 Immunoblotting 5.9.5 Polymerase chain reaction 5.9.6 Clinical tissue culture systems 5.9.7 Ocular imaging 5.9.7.1 Fundus color photographs 5.9.7.2 Fluorescein angiography and indocyanine green angiography 5.9.7.3 Confocal scanning laser ophthalmoscopy 5.9.7.4 Fundus autofluorescence 5.9.7.5 Optical coherent tomography 5.9.7.6 Ultrasonography 5.10 Differential diagnosis 5.11 The treatment and management of ocular toxoplasmosis 5.11.1 Drug treatment of ocular toxoplasmosis 5.11.2 Corticosteroids 5.11.3 Laser treatment 5.11.4 Subconjunctival therapy 5.11.5 Surgical therapy 5.11.6 Intravitreal therapy 5.11.7 Prophylactic therapy 5.12 Conclusion References 6 Toxoplasmosis in wild and domestic animals 6.1 Introduction 6.2 Toxoplasmosis in wildlife 6.2.1 Felids 6.2.2 Canids 6.2.3 Bears 6.2.4 Raccoons 6.2.5 Squirrels 6.2.6 Rabbits and hares 6.2.7 Skunks and fisher 6.2.8 Beavers 6.2.9 Woodchuck and other large rodents 6.2.10 Insectivores 6.2.11 Bats 6.2.12 White-tailed and mule deer 6.2.13 Other deer 6.2.14 Other wild ruminants 6.2.15 Sea otters and other marine mammals 6.2.16 New world monkeys 6.2.17 Old world monkeys 6.2.18 American marsupials 6.2.19 Australian marsupials 6.2.20 African wildlife 6.2.21 Wild rodents 6.2.22 Wild birds 6.3 Toxoplasmosis in zoos 6.4 Toxoplasma gondii and endangered species 6.5 Toxoplasmosis in pets 6.5.1 Cats 6.5.2 Dogs 6.5.3 Ferrets 6.6 Domestic farm animals 6.6.1 Mink 6.6.2 Horses 6.6.3 Swine 6.6.4 Cattle 6.6.5 Sheep 6.6.6 Goats 6.6.7 Buffalos 6.6.8 Camels 6.6.9 Llamas, alpaca, and vicunas 6.6.10 Chickens 6.6.11 Turkeys 6.6.12 Ducks and geese 6.7 Fish, reptiles, and amphibians References Further reading 7 Toxoplasma animal models and therapeutics 7.1 Introduction 7.2 Congenital toxoplasmosis 7.2.1 Mouse 7.2.2 Rat 7.2.3 Calomys callosus 7.2.4 Hamster 7.2.5 Guinea pig 7.2.6 Primate 7.2.7 Rabbit 7.2.8 Other animals 7.3 Ocular toxoplasmosis 7.3.1 Models based on local eye infection 7.3.2 Models based on infection via the carotid artery 7.3.3 Models based on systemic infection 7.4 Cerebral toxoplasmosis 7.4.1 Acute infection models 7.4.2 Localized brain infection models 7.4.3 Progressive Toxoplasma encephalitis models 7.4.4 Chronic relapsing infection models (reactivated toxoplasmosis) 7.4.5 Latent infection models References 8 Biochemistry and metabolism of Toxoplasma gondii: lipid synthesis and uptake 8.1 Introduction 8.2 Fatty acids 8.2.1 Fatty acid biosynthetic pathways—generalities 8.2.2 Fatty acid synthesis in Toxoplasma 8.2.3 Fatty acid salvage by Toxoplasma 8.2.4 Fatty acid fluxes in Toxoplasma 8.3 Glycerophospholipids 8.3.1 Phospholipid biosynthetic pathways—generalities 8.3.2 Phospholipid composition and physiological relevance in Toxoplasma 8.3.3 Phospholipid synthesis in Toxoplasma 8.3.4 Phospholipid salvage by Toxoplasma 8.4 Acylglycerols 8.4.1 Acylglycerol biosynthetic pathways—generalities 8.5 Acylglycerol synthesis and storage in Toxoplasma 8.6 Sterols and steryl esters 8.6.1 Sterol lipid biosynthetic pathways—generalities 8.6.2 Sterol salvage and transport in Toxoplasma 8.6.3 Sterol storage in Toxoplasma 8.7 Sphingolipids 8.7.1 Sphingolipid biosynthetic pathways—generalities 8.7.2 Sphingolipid synthesis in Toxoplasma 8.7.3 Sphingolipid salvage by Toxoplasma 8.8 Isoprenoid derivatives 8.8.1 Isoprenoid biosynthetic pathways—generalities 8.8.2 Isoprenoid synthesis in Toxoplasma 8.8.3 Isoprenoid salvage by Toxoplasma 8.9 Concluding remarks References Further reading 9 Biochemistry and metabolism of Toxoplasma gondii: purine and pyrimidine acquisition in Toxoplasma gondii and other Apicom... 9.1 Introduction 9.2 Purines 9.2.1 Capture and transport 9.2.1.1 Genome analysis of purine transporters in Apicomplexa 9.2.1.2 Model of purine acquisition in Toxoplasma gondii 9.2.1.3 Properties of purine transporters in Apicomplexa 9.2.2 Purine transport in the parasitized host cell 9.2.3 Purine interconversion and salvage pathways in Apicomplexa 9.2.3.1 Purine salvage pathways in Toxoplasma gondii 9.2.3.2 Purine salvage pathways in Cryptosporidium parvum 9.2.3.3 Purine salvage pathways in Plasmodium falciparum 9.2.3.4 Alternative purine pathways in Apicomplexa 9.2.3.5 Polyamines in Apicomplexa 9.3 Pyrimidines 9.3.1 De novo pyrimidine synthesis in Apicomplexa 9.3.1.1 Organization and regulation of carbamoyl phosphate synthetase II in Apicomplexa 9.3.1.2 Pyrimidine biosynthetic pathways in Apicomplexa 9.3.1.3 Indirect inhibition of pyrimidine biosynthesis 9.3.2 Pyrimidine salvage in Apicomplexa 9.3.2.1 Salvage of pyrimidines in Cryptosporidium parvum 9.3.2.2 Salvage of pyrimidines in Plasmodium falciparum 9.3.2.3 Salvage of pyrimidines in Toxoplasma gondii 9.3.3 Pyrimidine synthesis and salvage pathways related to parasite niche 9.3.3.1 Plasmodium falciparum and Cryptosporidium parvum 9.3.3.2 Toxoplasma gondii 9.3.4 Folate pathways and synthesis of thymine nucleotides 9.3.4.1 Biosynthesis of folates in Apicomplexa 9.3.4.2 Antifolate chemotherapy and antifolate resistance 9.3.5 Toxoplasma gondii pyrimidine genetic selection strategies 9.3.6 Uracil auxotrophy, vaccination, and immunity References Further reading 10 Metabolic networks and metabolomics 10.1 Introduction 10.2 Genome-scale metabolic modeling 10.2.1 Systems biology approaches for understanding metabolism 10.2.2 Metabolic modeling and analysis of T. gondii 10.2.3 Harmonization of metabolic models with experimental data 10.2.4 Future perspectives 10.3 Central carbon metabolism 10.3.1 Glycolysis 10.3.2 Gluconeogenesis 10.3.3 Pentose phosphate pathway 10.3.4 Tricarboxylic acid cycle, 2-MCC, and the γ-aminobutyric acid shunt 10.3.5 Oxidative phosphorylation 10.3.6 Fatty-acid biosynthesis 10.3.7 Beta-oxidation 10.4 Carbohydrate metabolism 10.4.1 Sugar nucleotide synthesis 10.4.2 Glycosylation pathways in the secretory pathway 10.4.2.1 N-Glycans 10.4.2.2 Glycosylphosphatidylinositol glycolipids 10.4.2.3 O-Glycosylation 10.4.2.4 Nucleocytoplasmic glycosylation 10.4.3 Amylopectin 10.4.3.1 Synthesis and turnover of amylopectin 10.4.3.2 Regulation of amylopectin turnover 10.4.3.3 Amylopectin function 10.5 Vitamins and cofactor metabolism 10.5.1 Overview of vitamins and cofactors 10.5.2 Vitamins: thiamine B1, flavins B2, niacin B3, pantothenate B5, pyridoxal B6, biotin B7, Myo-inositol B8, and folates B9 10.5.2.1 Thiamine biosynthesis 10.5.2.2 Flavins biosynthesis 10.5.2.3 Niacin metabolism 10.5.2.4 Pantothenate biosynthesis for CoA production 10.5.2.5 Pyridoxal-phosphate metabolism 10.5.2.6 Folate and biopterins biosynthesis 10.5.2.7 Myo-inositol and biotin uptake and utilization 10.5.3 Cofactors: shikimate and chorismate, ubiquinone, heme, lipoic-acid, S-adenosyl-methionine, and glutathione 10.5.3.1 Shikimate and chorismate biosynthesis 10.5.3.2 Ubiquinone biosynthesis 10.5.3.3 Heme biosynthesis 10.5.3.4 Lipoic acid metabolism 10.5.3.5 S-Adenosyl-methionine biosynthesis 10.5.3.6 Glutathione biosynthesis and redox metabolism 10.6 Metabolomics approaches 10.6.1 Intracellular metabolite levels 10.6.2 Metabolic foot-printing 10.6.3 Stable isotope labeling approaches 10.6.4 Metabolomic analysis of host tissues 10.7 Discussion and outlook 10.7.1 Computational modeling 10.7.2 Molecular biology 10.7.3 Metabolomics References 11 The apicoplast and mitochondrion of Toxoplasma gondii 11.1 Introduction 11.2 The apicoplast 11.2.1 History 11.2.2 Evolution 11.2.3 The apicoplast genome 11.2.4 Expression and translation of the apicoplast genome 11.2.5 Apicoplast genome replication 11.2.6 Apicoplast division 11.2.7 Protein trafficking to the apicoplast 11.2.7.1 Targeting sequences 11.2.7.2 Trafficking mechanisms 11.2.8 Drug sensitivities and the phenomenon of “delayed death” 11.2.9 Apicoplast metabolism 11.3 The mitochondrion 11.3.1 Appearance and ultrastructure 11.3.2 Evolution 11.3.3 Replication and expression of the mitochondrial genome 11.3.4 Protein trafficking to the mitochondrion 11.3.5 Oxidative phosphorylation and energy metabolism 11.3.6 Biosynthetic pathways in the mitochondrion 11.3.7 The mitochondrion as a drug target 11.4 Conclusion References 12 Calcium storage and homeostasis in Toxoplasma gondii 12.1 Introduction 12.2 Fluorescent methods to study calcium in Toxoplasma 12.2.1 Probes for measuring calcium in Toxoplasma gondii 12.2.2 Ca2+ buffers 12.2.3 Genetic indicators 12.3 Regulation of [Ca2+]i in Toxoplasma gondii 12.3.1 Ca2+ transport across the plasma membrane 12.3.2 Calcium storage 12.3.2.1 Endoplasmic reticulum 12.3.2.2 Mitochondria 12.3.2.3 Acidocalcisomes 12.3.2.4 Plant-like vacuole/vacuolar compartment 12.4 Transducing Ca2+ signals 12.4.1 Calcium-binding proteins 12.4.2 Calcium-dependent protein kinases and their function 12.5 Conclusion References 13 Calcium and cyclic nucleotide signaling networks in Toxoplasma gondii 13.1 Introduction 13.2 Motility 13.3 Regulated secretion of micronemes 13.4 Release of intracellular calcium as a regulatory cascade 13.5 Calcium-dependent protein kinases 13.6 Nucleotide cyclases and cyclic nucleotide phosphodiesterases 13.6.1 Adenylate cyclases 13.6.2 Phosphodiesterases 13.6.3 Cyclic GMP-dependent protein kinase(PKG) 13.6.4 Cyclic AMP-dependent protein kinase (PKA) 13.7 Conclusion and future directions References 14 Toxoplasma secretory proteins and their roles in parasite cell cycle and infection 14.1 Introduction 14.2 Motility and invasion 14.2.1 Rapid and active processes unique to apicomplexan parasites 14.2.1.1 Motility 14.2.1.2 Invasion 14.2.1.3 Kinematic analysis of invasion process 14.2.1.4 Alternative routes of invasion 14.2.2 Motility and invasion: central role of micronemes 14.2.3 Moving junction formation: cooperative role between micronemes and rhoptries 14.3 Parasitophorous vacuole formation and maturation 14.3.1 Parasitophorous vacuole formation: role of rhoptries 14.3.2 Maturation of the vacuole: a prominent role of dense granules 14.3.2.1 A complex network of tubules and vesicles 14.3.2.2 Pore inside the parasitophorous vacuole membrane 14.3.2.3 Attraction of host organelles and structures to the parasitophorous vacuole membrane 14.3.2.4 Targeting ROPs and GRAs to the PVM and host cell to neutralize host defense 14.4 Egress 14.5 Micronemes 14.5.1 Trafficking of MICs and the biogenesis of microneme subpopulations 14.5.2 Microneme subpopulations 14.5.3 Microneme proteins 14.5.3.1 MICs sharing homologies with structural domains of eukaryotic proteins involved in protein–protein or protein–carb... 14.5.3.1.1 I- or A-domain 14.5.3.1.2 Thrombospondin type 1 (TSR) repeat domain 14.5.3.1.3 Epidermal growth factor-like domain 14.5.3.1.4 Plasminogen, apple, nematode/apple module 14.5.3.1.5 The chitin-binding-like domain 14.5.3.1.6 Galectin-like domain 14.5.3.1.7 Microneme adhesive repeat domain 14.5.3.2 Other MICs 14.5.3.3 MICs assemble in complexes 14.5.3.4 Cytosolic domain of transmembrane MICs 14.5.4 Microneme secretion 14.5.5 Postsecretory traffic of MICs 14.5.5.1 Parasite surface exposition and posterior capping of MICs 14.5.5.2 Proteolytic cleavages during invasion 14.5.6 Why does Toxoplasma gondii exhibit this patchwork of MICs? 14.5.6.1 MIC2: role in attachment and motility 14.5.6.2 MIC8 and claudin-like apicomplexan microneme protein: potential role in triggering rhoptry secretion 14.5.6.3 AMA1 and AMA1 homologs: role in moving junction formation 14.6 Rhoptries 14.6.1 Biogenesis of rhoptries—clustering and tethering to the apical end 14.6.1.1 Rhoptry: a complex organelle with subcompartments 14.6.1.2 Reshaping of the endosomal pathway for rhoptry biogenesis 14.6.1.3 Rhoptry-targeting signals 14.6.1.4 Rhoptry morphogenesis and clustering to apical end 14.6.2 ROPs and RONs processing 14.6.3 Secretion of rhoptries 14.6.4 Rhoptry proteins and functions 14.6.4.1 Rhoptry proteins associated with the cytosolic face of the rhoptry 14.6.4.1.1 Rab11a 14.6.4.1.2 ARO and its partners AIP and ACβ: apical targeting of rhoptries 14.6.4.1.3 Carbonic anhydrase–related protein 14.6.4.2 Integral membrane proteins 14.6.4.2.1 Acyltransferase DHHC7 14.6.4.2.2 Transporters Na+/H+ exchanger Transporter facilitator proteins: TFP2 and TFP3 RON11 14.6.4.3 Luminal rhoptry proteins 14.6.4.3.1 Rhoptry neck complex RON2/RON4/RON5/RON8/RON4L1: role in moving junction formation and invasion 14.6.4.3.2 The rhoptry kinase family (ROPKs): effectors to disarm the host immune response Parasitophorous vacuole membrane–associated ROPKs ROPK targeted to the host nucleus 14.6.4.3.3 Toxofilin: control of host cell actin polymerization 14.6.4.3.4 Other RONs/ROPs with less characterized functions 14.6.5 Stage-specific expression of ROPs/RONs 14.6.6 Rhoptry lipids 14.7 Dense granules 14.7.1 The dense granule organelles 14.7.2 The dense granule proteins: GRAs and others 14.7.3 Biogenesis of dense granules: features of both constitutive and regulated secretory pathways 14.7.4 Exocytosis of dense granules 14.7.5 Postsecretory trafficking of GRAs 14.7.6 Dense granule protein function 14.7.6.1 GRAs 14.7.6.2 Other dense granule proteins 14.7.7 Stage-specific expression of dense granule proteins 14.7.7.1 Bradyzoite tissue cyst and GRA proteins 14.7.7.2 Merozoite GRA proteins 14.7.7.3 Sporozoite GRA proteins 14.8 Conclusion References 15 Endomembrane trafficking pathways in Toxoplasma 15.1 Introduction 15.2 Sorting signals of secretory proteins 15.2.1 Trafficking of rhoptry proteins 15.2.2 Trafficking of micronemal proteins 15.2.3 The role of proteolytic maturation of secretory proteins for their transport 15.2.4 Recycling of maternal organelles during replication 15.3 Coding complement of the Toxoplasma gondii membrane-trafficking system 15.3.1 Overview of trafficking in the apicomplexa 15.3.2 Ras-related protein from brain (Rab) GTPases 15.3.3 Other GTPases 15.3.4 Tethers 15.3.5 Soluble N-ethylmaleimide-sensitive factor attachment protein receptors 15.3.6 Endosomal sorting complexes required for transport complexes 15.3.7 Coats 15.3.8 Adaptor proteins and cargo adapters 15.4 Organization of the Toxoplasma gondii membrane trafficking system 15.4.1 Overview 15.4.2 The endoplasmic reticulum 15.4.3 The Golgi 15.4.4 The dense granules 15.4.5 The endosomal system (micronemes, rhoptries, and the vacuolar compartment/plant-like vacuole) 15.5 An integrated model of exocytic trafficking through the membrane trafficking system 15.6 Dynamics of the endolysosomal system 15.6.1 Overview 15.6.2 Fragmentation and reformation of the vacuolar compartment/plant-like vacuole 15.6.3 Interactions between the vacuolar compartment/plant-like vacuole and endosomal-like compartments 15.7 Endocytosis and endocytic trafficking 15.7.1 Overview 15.7.2 Endocytosis of sulfated glycans 15.7.3 Endocytosis of lipids and surface proteins 15.7.4 Endocytosis of host-derived protein 15.8 Comparison of Toxoplasma gondii endosomal trafficking to model systems 15.8.1 Overview of yeast, mammalian, and plant systems 15.8.2 Similarities and distinctions of Toxoplasma gondii versus model systems 15.9 Autophagy 15.9.1 Coding capacity of the core Toxoplasma gondii autophagy machinery 15.9.2 Autophagy in Toxoplasma gondii 15.9.2.1 Canonical degradative autophagy 15.9.2.2 Evidence for stress-activated canonical degradative autophagy 15.9.2.3 Autophagy as part of an integrated stress response 15.9.2.4 A role for canonical autophagy in parasite virulence 15.9.3 Autophagy and differentiation 15.9.4 Noncanonical function of autophagy-related proteins at the apicoplast 15.10 Final remarks Glossary References 16 The Toxoplasma cytoskeleton: structures, proteins, and processes 16.1 Morphology 16.1.1 Life cycle and parasite appearance 16.1.2 Inner membrane complex and pellicle-associated structures 16.1.3 Apical structures 16.1.4 Basal structures 16.1.5 The nucleus 16.1.6 Centrioles, centrosomes, and basal bodies 16.2 Cytoskeletal elements 16.2.1 Tubulin, microtubules, microtubule-associated proteins, motors, and MTOC 16.2.2 Alveolins, glideosome-associated proteins with multiple membrane spans, and other inner membrane complex proteins 16.2.3 Actin, actin-like and actin-related proteins, and actin-binding proteins 16.2.4 Myosin motors, the glideosome, and other associated factors 16.3 Putting it all together: processes 16.3.1 Replication 16.3.1.1 Endodyogeny and endopolygeny 16.3.1.2 Nuclear division 16.3.1.3 Assembly of daughter cytoskeleton buds 16.3.1.4 Emergence of daughter parasites 16.3.1.5 The mature basal complex 16.3.2 Motility, invasion, and egress 16.3.2.1 Glideosome assembly, activation, and regulation 16.3.2.2 Actin polymerization for gliding motility in particular 16.3.2.3 Mechanism of conoid extrusion 16.3.2.4 The role of the host cell in invasion and egress 16.3.3 Other critical roles for Toxoplasma actin 16.4 Summary: a story of adaptation, loss, and novel components References 17 Effectors produced by rhoptries and dense granules: an intense conversation between parasite and host in many languages 17.1 Background 17.2 Rhoptry effectors—a potent class of host manipulators 17.3 Dense granule effectors—a second wave of manipulation 17.4 Conclusion Acknowledgments References 18 Bradyzoite and sexual stage development 18.1 Introduction 18.2 Bradyzoite and tissue cyst morphology and biology 18.3 The development of tissue cysts and bradyzoites in vitro 18.4 The cell cycle and bradyzoite development 18.5 The stress response and signaling pathways for bradyzoite formation 18.6 Heat shock proteins 18.7 Transcriptional control of bradyzoite genes 18.8 Cyst wall and matrix antigens 18.9 Surface antigens 18.10 Metabolic differences between bradyzoites and tachyzoites 18.11 Genetic studies on bradyzoite biology 18.12 Sexual stage morphology, biology, and antigens 18.13 Sexual stage development in cell culture 18.14 Sexual stage development in a mouse model 18.15 Summary References Further reading 19 Development and application of classical genetics in Toxoplasma gondii 19.1 Summary 19.2 Biology of Toxoplasma 19.2.1 Life cycle 19.2.2 Defining the sexual phase 19.2.3 Population structure and major strain types 19.3 Establishment of transmission genetics 19.3.1 Intra-strain crosses and meiosis 19.3.2 Genetic crosses between different lineages 19.3.3 Implications of selfing versus outcrossing for population structure 19.4 Development of genetic mapping 19.4.1 Advances in molecular genetic tools 19.4.2 Development of linkage maps for forward genetic analysis 19.4.3 Limitation of the current linkage maps 19.5 Mapping phenotypic traits by classical genetics 19.5.1 Mapping drug resistance 19.5.2 Mapping quantitative traits 19.5.3 Genetic approaches for defining virulence genes 19.5.3.1 Mapping differences in the type 1×3 cross 19.5.3.2 Mapping differences in the type 2×3 cross 19.5.3.3 Mapping differences in the type 1×2 cross 19.5.3.4 Mapping differences in crosses to “exotic” lineages 19.5.4 Expression quantitative trait locus mapping 19.5.4.1 Using eQTL mapping to characterize mechanisms of strain-specific gene regulation in Toxoplasma 19.5.4.2 Cross-species eQTL mapping: identifying Toxoplasma loci that affect host gene expression 19.5.5 Summary of differences between lineages 19.5.6 Relevance of the mouse model to other species 19.6 Future challenges 19.6.1 Overcoming current limitations 19.6.2 Expanding phenotypic analyses References 20 Genetic manipulation of Toxoplasma gondii 20.1 Introduction 20.2 The mechanics of making transgenic parasites 20.2.1 Transient transfection 20.2.2 Stable transformation and positive and negative selectable markers 20.2.3 Homologous recombination and random integration 20.2.4 Enhanced genetic manipulation through CRISPR/Cas9 20.3 Using transgenic parasites to study the function of parasite genes 20.3.1 Tagging subcellular compartments 20.3.2 Tagging of parasite proteins 20.3.3 Genetic analysis of essential genes 20.3.3.1 Tetracycline inducible systems 20.3.3.2 Regulation of protein stability 20.3.3.2.1 Destabilization domain (ddFKBP) 20.3.3.2.2 Auxin-based degron system 20.3.3.3 Site-specific recombination 20.3.3.3.1 Excision of LoxP flanked genes 20.3.3.3.2 U1 small nuclear ribonucleic particles–mediated gene silencing 20.3.4 Insertional mutagenesis and promoter trapping as tools of functional genetic analysis 20.3.5 Forward genetic analysis using chemical mutagenesis and complementation cloning 20.4 Perspectives 20.5 A selection of detailed protocols for parasite culture, genetic manipulation, and phenotypic characterization 20.5.1 Propagation of Toxoplasma tachyzoites in tissue culture 20.5.1.1 Maintenance of human foreskin fibroblast cells 20.5.1.2 Maintenance of tachyzoites 20.5.1.3 Cryopreservation of host cells and parasites 20.5.1.4 Mycoplasma detection and removal 20.5.1.5 Passaging Toxoplasma tachyzoites/bradyzoite cysts in animal 20.5.2 Transfection and stable transformation protocols 20.5.2.1 Transient transfection 20.5.2.2 Selection of stable transformants 20.5.2.3 Restriction enzyme-mediated integration 20.5.2.4 Cloning of transgenic lines by limiting dilution in 96 well plates 20.5.3 Measuring parasite survival and growth 20.5.3.1 Plaque assay 20.5.3.2 Fluorescence assay 20.5.3.3 β-Galactosidase (LacZ) assay 20.5.3.4 Uracil incorporation assay 20.5.4 Live-cell and indirect immunofluorescence microscopy 20.5.5 Cytometry of parasites and infected cells 20.5.6 Disruption of nonessential genes 20.5.6.1 Disruption of nonessential genes using a CAT/YFP positive/negative selection 20.5.6.2 Disruption of nonessential genes using CRISPR/Cas9 20.5.7 Disruption of essential genes 20.5.7.1 Tetracycline inducible systems 20.5.7.1.1 Two-step strategy 20.5.7.1.2 Single-step approach 20.5.7.2 Regulation of protein stability 20.5.7.2.1 Destabilization domain (ddFKBP) 20.5.7.2.2 Auxin-based degron system 20.5.8 Insertional mutagenesis and tag rescue 20.5.9 Chemical mutagenesis 20.5.10 Complementation cloning using Toxoplasma gondii genomic libraries 20.5.11 Recombinering cosmids of Toxoplasma gondii genomic libraries 20.5.12 Safety concerns working with Toxoplasma gondii References 21 Regulation of gene expression in Toxoplasma gondii 21.1 Introduction 21.2 Transcription in Toxoplasma 21.2.1 The parasite transcriptome and transcriptional regulation 21.2.2 Gene-specific cis-elements 21.2.3 The evolution of APETALA2-related proteins 21.2.4 ApiAP2 structure determination and DNA binding 21.2.5 The function of ApiAP2 proteins 21.2.6 Other factors that regulate gene expression 21.3 Epigenetics in Toxoplasma 21.3.1 Chromatin and chromatin remodeling 21.3.2 Mapping the Toxoplasma epigenome 21.3.2.1 Chromatin signatures in Toxoplasma biology 21.3.3 Histone-modifying enzymes 21.3.3.1 Histone acetylation 21.3.3.2 Histone methylation 21.3.3.3 Other histone covalent modifications 21.3.3.4 SWI2/SNF2 ATPases 21.3.4 Epigenetic mechanisms as drug targets 21.4 Posttranscriptional mechanisms in Toxoplasma 21.4.1 Translational control 21.4.2 Noncoding and small RNA 21.4.3 Other posttranscriptional mechanisms 21.5 Conclusion and future directions References 22 Proteomics and posttranslational protein modifications in Toxoplasma gondii 22.1 Introduction to Toxoplasma gondii proteomics 22.2 Toxoplasma gondii global proteomics 22.3 Toxoplasma gondii subproteomes 22.4 Toxoplasma gondii posttranslational modifications 22.4.1 Phosphorylation 22.4.2 Ubiquitination 22.4.3 Palmitoylation 22.4.4 Glycosylation 22.4.5 Methylation 22.4.6 Acetylation 22.4.7 Succinyllysine 22.4.8 SUMOylation 22.5 Studies on the function of posttranslational modifications in Toxoplasma. gondii biology 22.5.1 Posttranslational modifications in motility, invasion, and egress 22.5.2 Posttranslational modifications of the inner membrane complex 22.5.3 Posttranslational modifications in transcriptional and posttranscriptional regulation 22.5.4 Posttranslational modifications as regulators of parasite differentiation 22.5.5 Host–parasite interactions 22.6 Interactions of Toxoplasma gondii posttranslational modifications 22.7 Conclusion References 23 ToxoDB: the functional genomic resource for Toxoplasma and related organisms* 23.1 Introduction 23.2 Data content 23.3 Genome in ToxoDB 23.4 Functional data in ToxoDB 23.5 The ToxoDB home page 23.6 The search strategy system 23.6.1 Running your first search 23.6.2 Understanding and configuring the results page 23.6.3 Building a multistep search strategy 23.6.4 Defining genes based on their phylogenetic profile 23.7 Genomic colocation 23.8 The genome browser 23.9 Data analysis and integration into ToxoDB 23.9.1 Gene list analysis 23.9.2 Analyze my experiment 23.9.3 Galaxy result integration (my datasets) 23.10 Future directions References 24 Cerebral toxoplasmosis 24.1 Introduction 24.2 Models for understanding cerebral toxoplasmosis 24.3 Mouse and parasite genotype affect central nervous system outcomes 24.4 Overview of the central nervous system 24.5 Parasite entry into the central nervous system 24.5.1 Toxoplasma gondii dissemination to the central nervous system 24.5.2 Unique features of the blood–brain barrier 24.5.3 Breaching the blood–brain barrier 24.6 Brain regions and host cells infected in the brain 24.6.1 Human toxoplasmosis 24.6.2 Rodent cerebral toxoplasmosis 24.7 Control of cerebral toxoplasmosis 24.7.1 Parenchymal central nervous system cells 24.7.1.1 Neurons 24.7.1.2 Astrocytes 24.7.1.3 Microglia 24.7.2 Systemic immune cells 24.7.2.1 Immune cell infiltration into the central nervous system 24.7.2.2 Innate immune cells 24.7.2.2.1 Monocyte-derived macrophages and dendritic cells 24.7.2.2.2 Neutrophils and other granulocytes 24.7.2.3 Adaptive immune cells 24.7.2.3.1 T cells 24.7.2.3.2 Regulatory T cells 24.7.2.3.3 B cells 24.8 Physiologic effects of Toxoplasma gondii on the central nervous system 24.8.1 Effects on animal behavior 24.8.2 Effects on rodent neurophysiology and structure 24.8.3 Effects on human behavior 24.9 Conclusion References 25 Innate immunity to Toxoplasma gondii 25.1 Introduction 25.2 The intimate relationship between Toxoplasma gondii and its host cells 25.3 Establishment of infection and mucosal immunity 25.4 The role of IL-12-dependent IFN-γ production for innate resistance 25.5 Antigen processing and presentation 25.6 Molecular basis for innate recognition of Toxoplasma gondii 25.6.1 Toll-like receptor and MyD88 25.6.2 Inflammasome-mediated caspase activation 25.7 IFN-γ-dependent cell autonomous immunity 25.7.1 IFN-γ-induced nitrosative and oxidative defense 25.7.2 IFN-γ-induced restriction of nutrients 25.7.3 IFN-γ-inducible GTPases 25.7.3.1 Immunity-related GTPases (IRGs) 25.7.3.2 Guanylate-binding protein IFN-γ-inducible p65 GTPases 25.7.4 Autophagic processes 25.7.5 Cofactors for IFN-γ-dependent effector mechanisms 25.8 Additional immune pathways altered by Toxoplasma gondii 25.8.1 Parasite utilization of host cell pathways 25.8.2 Modulation of signal transducer and activator of transcription pathways 25.8.3 GRA proteins 25.9 Conclusion and perspectives References 26 Adaptive immunity 26.1 Introduction 26.1.1 αβ T cells 26.1.2 Other adaptive cell types 26.1.3 Dendritic cells: innate sentinels that initiate and shape adaptive immunity 26.2 How is Toxoplasma gondii “seen” by the adaptive immune system? 26.2.1 Antigen presentation by major histocompatibility complex molecules to T cells 26.2.2 Major histocompatibility complex class I presentation 26.2.2.1 The classical major histocompatibility complex I presentation pathway 26.2.2.2 The major histocompatibility complex I cross-presentation (or exogenous) pathways 26.2.3 Major histocompatibility complex I presentation of Toxoplasma gondii antigens 26.2.3.1 The role of secretion 26.2.3.2 The role of actively infected cells 26.2.3.3 The impact of antigen biochemical properties and trafficking 26.2.4 Modulation of the major histocompatibility complex I presentation pathway by Toxoplasma gondii 26.3 Initiation (priming) of T cell responses by dendritic cells 26.4 Major histocompatibility complex class II presentation 26.4.1 Major histocompatibility complex II presentation of Toxoplasma gondii antigens 26.4.2 Modulation of the major histocompatibility complex II presentation pathway by Toxoplasma gondii 26.5 Adaptive immune responses in the intestinal mucosa and associated lymphoid tissues 26.5.1 Early dissemination in the small intestine 26.5.2 Intestinal humoral responses to Toxoplasma gondii 26.5.3 Toxoplasma gondii acute ileitis: a T cell–mediated immune pathology 26.5.4 Th1/Th17 CD4+ T cells are main effectors of intestinal pathology 26.5.5 Treg and intraepithelial lymphocytes protect the host from gut pathology 26.5.6 Intestinal adaptive immunity in chronic phase 26.6 Lymphoid system 26.6.1 The pivotal role of the IL-12/IFN-γ axis 26.6.2 Immunoregulation during Toxoplasma gondii infection 26.6.3 IL-27 26.6.4 IL-10 26.6.5 Glucocorticoids and anti-inflammatory lipids 26.6.6 CD8+ T cells 26.6.6.1 CD8+ T cells play a prominent role in controlling Toxoplasma gondii 26.6.6.2 CD8+ T cell response in susceptible and resistant mouse strains 26.6.6.3 T cell dynamics during infection in vivo 26.6.7 CD4+ T cells 26.6.7.1 Help for CD8+ T cells 26.6.7.2 Immunosuppression and regulatory CD4+ T cells (Treg) 26.6.7.3 Antibody production and T follicular helper cells 26.7 Adaptive immunity in the brain 26.7.1 T cell entry and behavior in the Toxoplasma gondii–infected brain 26.7.1.1 Three ways to enter the brain 26.7.1.2 T cell entry in the Toxoplasma gondii–infected brain 26.7.1.3 Dynamics of Toxoplasma gondii–specific T cells in brain 26.7.1.4 T cell recirculation in the chronically infected brain 26.7.2 Th1 cytokines and cytotoxicity are essential for parasite control in the central nervous system 26.7.3 Roles of CD4+ and CD8+ T cells in infected brain 26.7.4 Resistance to encephalitis is mediated by CD8+ T cells that efficiently recognize tachyzoite-infected neurons 26.7.5 T cell exhaustion 26.7.6 Tissue-resident memory T cells 26.8 Adaptive immunity in the muscle 26.9 Conclusion References Appendix A The effect of murine gene deficiencies on the outcome of Toxoplasma gondii infection References Epilogue Index Back Cover