The zebrafish as a novel model for the in vivo study of Toxoplasma gondii replication and interaction with macrophages

ABSTRACT Toxoplasma gondii is an obligate intracellular parasite capable of invading any nucleated cell. Three main clonal lineages (type I, II, III) exist and murine models have driven the understanding of general and strain-specific immune mechanisms underlying Toxoplasma infection. However, murine models are limited for studying parasite-leukocyte interactions in vivo, and discrepancies exist between cellular immune responses observed in mouse versus human cells. Here, we developed a zebrafish infection model to study the innate immune response to Toxoplasma in vivo. By infecting the zebrafish hindbrain ventricle, and using high-resolution microscopy techniques coupled with computer vision-driven automated image analysis, we reveal that Toxoplasma invades brain cells and replicates inside a parasitophorous vacuole to which type I and III parasites recruit host cell mitochondria. We also show that type II and III strains maintain a higher infectious burden than type I strains. To understand how parasites are cleared in vivo, we further analyzed Toxoplasma-macrophage interactions using time-lapse microscopy and three-dimensional correlative light and electron microscopy (3D CLEM). Time-lapse microscopy revealed that macrophages are recruited to the infection site and play a key role in Toxoplasma control. High-resolution 3D CLEM revealed parasitophorous vacuole breakage in brain cells and macrophages in vivo, suggesting that cell-intrinsic mechanisms may be used to destroy the intracellular niche of tachyzoites. Together, our results demonstrate in vivo control of Toxoplasma by macrophages, and highlight the possibility that zebrafish may be further exploited as a novel model system for discoveries within the field of parasite immunity. This article has an associated First Person interview with the first author of the paper. Summary: We have established a novel zebrafish infection model to investigate Toxoplasma replication in vivo, and demonstrate a key role for macrophages in parasite control.

[1]  Yannick Schwab,et al.  AMST: Alignment to Median Smoothed Template for Focused Ion Beam Scanning Electron Microscopy Image Stacks , 2020, Scientific Reports.

[2]  S. Mostowy,et al.  The Case for Modeling Human Infection in Zebrafish. , 2020, Trends in microbiology.

[3]  Jerzy Samolej,et al.  Mimicry embedding for advanced neural network training of 3D biomedical micrographs , 2019, bioRxiv.

[4]  L. Collinson,et al.  Differential spatiotemporal targeting of Toxoplasma and Salmonella by GBP1 assembles caspase signalling platforms , 2019, bioRxiv.

[5]  M. Carrington,et al.  Visualizing trypanosomes in a vertebrate host reveals novel swimming behaviours, adaptations and attachment mechanisms , 2019, eLife.

[6]  Michael R. Elliott,et al.  Alarmin S100A11 initiates a chemokine response to the human pathogen Toxoplasma gondii , 2018, Nature Immunology.

[7]  Nagisa Yoshida,et al.  Macrophage–Microbe Interactions: Lessons from the Zebrafish Model , 2017, Front. Immunol..

[8]  S. Mostowy,et al.  Zebrafish Infection: From Pathogenesis to Cell Biology , 2017, Trends in cell biology.

[9]  J. Saeij,et al.  Exposing Toxoplasma gondii hiding inside the vacuole: a role for GBPs, autophagy and host cell death , 2017, Current opinion in microbiology.

[10]  B. Clough,et al.  The Toxoplasma Parasitophorous Vacuole: An Evolving Host-Parasite Frontier. , 2017, Trends in parasitology.

[11]  S. Mostowy,et al.  Septins restrict inflammation and protect zebrafish larvae from Shigella infection , 2017, PLoS pathogens.

[12]  R. Swerdlow,et al.  Mitochondria-Derived Damage-Associated Molecular Patterns in Neurodegeneration , 2017, Front. Immunol..

[13]  Constance A. M. Finney,et al.  Toxoplasma gondii: One Organism, Multiple Models. , 2017, Trends in parasitology.

[14]  C. Secombes,et al.  Analysis of interferon gamma protein expression in zebrafish (Danio rerio). , 2016, Fish & shellfish immunology.

[15]  A. Sher,et al.  Innate recognition of Toxoplasma gondii in humans involves a mechanism distinct from that utilized by rodents , 2016, Cellular & Molecular Immunology.

[16]  A. Koshy,et al.  Neurons are the Primary Target Cell for the Brain-Tropic Intracellular Parasite Toxoplasma gondii , 2016, PLoS pathogens.

[17]  A. Sher,et al.  The IL-12 Response of Primary Human Dendritic Cells and Monocytes to Toxoplasma gondii Is Stimulated by Phagocytosis of Live Parasites Rather Than Host Cell Invasion , 2016, The Journal of Immunology.

[18]  Stephan Saalfeld,et al.  Robust registration of calcium images by learned contrast synthesis , 2015, 2016 IEEE 13th International Symposium on Biomedical Imaging (ISBI).

[19]  E. Feeley,et al.  Ubiquitin systems mark pathogen-containing vacuoles as targets for host defense by guanylate binding proteins , 2015, Proceedings of the National Academy of Sciences.

[20]  R. Xavier,et al.  A Noncanonical Autophagy Pathway Restricts Toxoplasma gondii Growth in a Strain-Specific Manner in IFN-γ-Activated Human Cells , 2015, mBio.

[21]  Kelly J Pittman,et al.  Long-Term Relationships: the Complicated Interplay between the Host and the Developmental Stages of Toxoplasma gondii during Acute and Chronic Infections , 2015, Microbiology and Molecular Reviews.

[22]  Martin Weigert,et al.  ClearVolume: open-source live 3D visualization for light-sheet microscopy , 2015, Nature Methods.

[23]  N. Ueno,et al.  Toxoplasma gondii dissemination: a parasite's journey through the infected host , 2015, Parasite immunology.

[24]  P. Deloukas,et al.  Supplementary Figure 2 , 2014 .

[25]  Philipp J. Keller,et al.  Light-sheet functional imaging in fictively behaving zebrafish , 2014, Nature Methods.

[26]  Sarah E. Ewald,et al.  Toxoplasma Effector MAF1 Mediates Recruitment of Host Mitochondria and Impacts the Host Response , 2014, PLoS biology.

[27]  A. Sher,et al.  Innate resistance against Toxoplasma gondii: an evolutionary tale of mice, cats, and men. , 2014, Cell host & microbe.

[28]  C. Nüsslein-Volhard,et al.  transparent, a gene affecting stripe formation in Zebrafish, encodes the mitochondrial protein Mpv17 that is required for iridophore survival , 2013, Open.

[29]  C. Nüsslein-Volhard,et al.  transparent, a gene affecting stripe formation in Zebrafish, encodes the mitochondrial protein Mpv17 that is required for iridophore survival , 2013, Biology Open.

[30]  Seong-Ji Han,et al.  Motile invaded neutrophils in the small intestine of Toxoplasma gondii-infected mice reveal a potential mechanism for parasite spread , 2013, Proceedings of the National Academy of Sciences.

[31]  D. Roos,et al.  Replication and Distribution of Toxoplasma gondii in the Small Intestine after Oral Infection with Tissue Cysts , 2013, Infection and Immunity.

[32]  K. Takeda,et al.  A cluster of interferon-γ-inducible p65 GTPases plays a critical role in host defense against Toxoplasma gondii. , 2012, Immunity.

[33]  B. Butcher,et al.  Phagocyte Responses to Protozoan Infection and How Toxoplasma gondii Meets the Challenge , 2012, PLoS pathogens.

[34]  A. Cardona,et al.  Fiji: an open-source platform for biological-image analysis , 2012, Nature Methods.

[35]  Johannes E. Schindelin,et al.  TrakEM2 Software for Neural Circuit Reconstruction , 2012, PloS one.

[36]  N. Trede,et al.  A model 450 million years in the making: zebrafish and vertebrate immunity , 2012, Disease Models & Mechanisms.

[37]  J. Meseguer,et al.  Evolutionary conserved pro-inflammatory and antigen presentation functions of zebrafish IFNγ revealed by transcriptomic and functional analysis. , 2011, Molecular immunology.

[38]  M. Leptin,et al.  The role of gamma interferon in innate immunity in the zebrafish embryo , 2009, Disease Models & Mechanisms.

[39]  Michael S. Behnke,et al.  Phenotypic and Gene Expression Changes among Clonal Type I Strains of Toxoplasma gondii , 2009, Eukaryotic Cell.

[40]  M. Falagas,et al.  Toxoplasmosis snapshots: global status of Toxoplasma gondii seroprevalence and implications for pregnancy and congenital toxoplasmosis. , 2009, International journal for parasitology.

[41]  F. Pratlong,et al.  Genotype of 88 Toxoplasma gondii isolates associated with toxoplasmosis in immunocompromised patients and correlation with clinical findings. , 2009, The Journal of infectious diseases.

[42]  J. Meseguer,et al.  New Insights into the Evolution of IFNs: Zebrafish Group II IFNs Induce a Rapid and Transient Expression of IFN-Dependent Genes and Display Powerful Antiviral Activities1 , 2009, The Journal of Immunology.

[43]  A. Sher,et al.  Recognition of Toxoplasma gondii by TLR11 Prevents Parasite-Induced Immunopathology1 , 2008, The Journal of Immunology.

[44]  L. Sibley,et al.  Autophagosome-Independent Essential Function for the Autophagy Protein Atg5 in Cellular Immunity to Intracellular Pathogens , 2008, Cell Host & Microbe.

[45]  L. Sibley,et al.  Gr1(+) inflammatory monocytes are required for mucosal resistance to the pathogen Toxoplasma gondii. , 2008, Immunity.

[46]  R. Savan,et al.  An unexpected discovery of two interferon gamma-like genes along with interleukin (IL)-22 and -26 from teleost: IL-22 and -26 genes have been described for the first time outside mammals. , 2006, Molecular immunology.

[47]  J. Zerrahn,et al.  Disruption of Toxoplasma gondii Parasitophorous Vacuoles by the Mouse p47-Resistance GTPases , 2005, PLoS pathogens.

[48]  J. Boothroyd,et al.  Differences among the three major strains of Toxoplasma gondii and their specific interactions with the infected host. , 2005, Trends in parasitology.

[49]  L. Sibley,et al.  Recruitment of Gr-1+ monocytes is essential for control of acute toxoplasmosis , 2005, The Journal of experimental medicine.

[50]  C. Subauste,et al.  CD40 Signaling in Macrophages Induces Activity against an Intracellular Pathogen Independently of Gamma Interferon and Reactive Nitrogen Intermediates , 2005, Infection and Immunity.

[51]  L. Sibley,et al.  A novel population of Gr‐1+‐activated macrophages induced during acute toxoplasmosis , 2003, Journal of leukocyte biology.

[52]  H. Pelloux,et al.  Genotype of 86 Toxoplasma gondii isolates associated with human congenital toxoplasmosis, and correlation with clinical findings. , 2002, The Journal of infectious diseases.

[53]  L. Sibley,et al.  Biogenesis of nanotubular network in Toxoplasma parasitophorous vacuole induced by parasite proteins. , 2002, Molecular biology of the cell.

[54]  Soumaya Bennouna,et al.  CXCR2 Deficiency Confers Impaired Neutrophil Recruitment and Increased Susceptibility During Toxoplasma gondii Infection1 , 2001, The Journal of Immunology.

[55]  Sung-Kook Hong,et al.  Analysis of upstream elements in the HuC promoter leads to the establishment of transgenic zebrafish with fluorescent neurons. , 2000, Developmental biology.

[56]  J. Boothroyd,et al.  Lytic Cycle of Toxoplasma gondii , 2000, Microbiology and Molecular Biology Reviews.

[57]  L. Kasper,et al.  Differential Infectivity and Division ofToxoplasma gondii in Human Peripheral Blood Leukocytes , 2000, Infection and Immunity.

[58]  L. Sibley,et al.  Toxoplasma gondii comprises three clonal lineages: correlation of parasite genotype with human disease. , 1995, The Journal of infectious diseases.

[59]  J. Berger,et al.  When is a cluster? , 1993, The Lancet.

[60]  L. Sibley,et al.  Tumor necrosis factor-alpha triggers antitoxoplasmal activity of IFN-gamma primed macrophages. , 1991, Journal of immunology.

[61]  C. Nathan,et al.  Macrophage oxygen-dependent antimicrobial activity. II. The role of oxygen intermediates , 1979, The Journal of experimental medicine.

[62]  H. Murray,et al.  Macrophage oxygen-dependent antimicrobial activity. I. Susceptibility of Toxoplasma gondii to oxygen intermediates , 1979, The Journal of experimental medicine.

[63]  Ryan M. Anderson,et al.  Nitroreductase-mediated cell/tissue ablation in zebrafish: a spatially and temporally controlled ablation method with applications in developmental and regeneration studies , 2008, Nature Protocols.

[64]  G. Milon,et al.  CD11c- and CD11b-expressing mouse leukocytes transport single Toxoplasma gondii tachyzoites to the brain. , 2006, Blood.

[65]  D. Ferguson,et al.  The host-parasite relationship ofToxoplasma gondii in the brains of chronically infected mice , 2004, Virchows Archiv A.

[66]  M. Westerfield The zebrafish book : a guide for the laboratory use of zebrafish (Danio rerio) , 1995 .