Modelling Toxoplasma gondii infection in human cerebral organoids

Abstract

Pluripotent stem cell-derived cerebral organoids have the potential to recapitulate the pathophysiology of in vivo human brain tissue, constituting a valuable resource for modelling brain disorders, including infectious diseases. Toxoplasma gondii, an intracellular protozoan parasite, infects most warm-blooded animals, including humans, causing toxoplasmosis. In immunodeficient patients and pregnant women, infection often results in severe central nervous system disease and fetal miscarriage. However, understanding the molecular pathophysiology of the disease has been challenging due to limited in vitro model systems. Here, we developed a new in vitro model system of T. gondii infection using human brain organoids. We observed that tachyzoites can infect human cerebral organoids and are transformed to bradyzoites and replicate in parasitophorous vacuoles to form cysts, indicating that the T. gondii asexual life cycle is efficiently simulated in the brain organoids. Transcriptomic analysis of T. gondii-infected organoids revealed the activation of the type I interferon immune response against infection. In addition, in brain organoids, T. gondii exhibited a changed transcriptome related to protozoan invasion and replication. This study shows cerebral organoids as physiologically relevant in vitro model systems useful for advancing the understanding of T. gondii infections and host interactions.

Keywords: Cerebral organoid; Toxoplasma gondii; disease modelling; pluripotent stem cells; toxoplasmosis.

Figure 1.

Generation of cerebral brain organoids. (A) Schematic showing the method for generating hESC-derived brain organoids. (B) Representative images of a developing cerebral organoid at specific time points. (C) Immunohistochemistry of markers for the detection of neurons (SOX2 and TUJ1), radial glial cells (PAX6), astrocytes (GFAP) and oligodendrocytes (O1 and O4). Scale bars, as indicated. (D) Schematic representation of cerebral organoids generated in this study.

Figure 2.

Distribution of Toxoplasma gondii in the human cerebral organoids. (A) Schematic representation of the life cycle of T. gondii. (B) 3D images of a cerebral organoid infected with T. gondii (green). (C – F) Representative fluorescence images of cerebral organoids infected with 2 strains of T. gondii: ME49 (top) and RH (bottom) infected cerebral organoids are shown stained for (C) TUJ1, a neuronal marker; (D) GFAP, an astrocyte marker; (E) O1, an oligodendrocyte marker; and (F) SOX2, a radial glial cell marker. Scale bars, as indicated.

Figure 3

. T. gondii cyst formation in human cerebral organoids. Representative fluorescence image of cyst-like structures in an organoid infected with (A) ME49 and (B) RH. Images of transmission electron microscopy of (C–D) ME49- and (E) RH-infected cerebral organoids. Scale as indicated with the bar. PVM, parasitophorous vacuole membrane; Nu, nucleus; Rh, rhoptry; Co, conoid; Mt, mitochondrion; Dg, dense granule; and Am, amylopectin.

Figure 4.

Virulence of T. gondii in the infected cerebral organoids. (A) Schematic representation of the experimental design: T. gondii isolated from infected cerebral organoids was injected into mice. The levels of T. gondii P30 protein were measured by ELISA (n = 5, biologically independent mice) 2 months postinfection. (B) ME49 and (C) RH antibody titre presented as the optical density of ELISA . *p < 0.05. Quantitative data are expressed as the mean ± S.E.M. of at least 3 independent experiments.

Figure 5.

Transcriptome analysis of ME49 post-infection. (A) Differentially expressed genes of ME49 post-infection. Noninfectious ME49 was used as a control (|fc| ≥ 2, raw p-value < 0.05). A heat map was generated using Cluster grammer (http://amp.pharm.mssm.edu/clustergrammer/). (B) Volume plot showing differential expression of ME49 genes between 0 and 72 h post-infection (|fc| ≥ 2, raw p-value < 0.05). The top 5 gene names are indicated. (C) KEGG pathway analysis was performed using the differentially expressed genes (DEGs) of ME49 (|fc| ≥ 2, raw p-value < 0.05). (D) Gene Ontology (GO) annotation of T. gondii postinfection of cerebral organoids; p-value < 0.01. MF, molecular function; BP, biological process; and CC, cellular component.

Figure 6

. Transcriptome analysis of T. gondii-infected cerebral organoids. Heat map showing differentially expressed genes (DEGs) of (A) ME49- and (B) RH-infected cerebral organoids 72 h post-infection (|fc| ≥ 2, raw p-value < 0.05). Noninfected cerebral organoids were used as controls (n = 3, biologically independent samples). A heat map was generated using Cluster grammer (http://amp.pharm.mssm.edu/clustergrammer/). Gene Ontology analysis of DEGs in the (C) ME49- and (D) RH-infected organoids. Top five Gene Ontology terms (p-value < 0.005). Volume plot showing the top five genes (marked as red dots) in the (E) ME49- and (F) RH-infected organoids. Significantly upregulated and downregulated genes (|fc| ≥ 2, raw p-value < 0.05) are marked in blue. Noninfected organoids were used as controls. (G) Transcriptome data were analyzed using ingenuity pathway analysis (IPA) software (p-value < 0.05).