TgTKL1 Is a Unique Plant-Like Nuclear Kinase That Plays an Essential Role in Acute Toxoplasmosis

Abstract

In the protozoan parasite Toxoplasma gondii, protein kinases have been shown to play key roles in regulating parasite motility, invasion, replication, egress, and survival within the host. The tyrosine kinase-like (TKL) family of proteins are an unexplored set of kinases in Toxoplasma Of the eight annotated TKLs in the Toxoplasma genome, a recent genome-wide loss-of-function screen showed that six are important for tachyzoite fitness. By utilizing an endogenous tagging approach, we showed that these six T. gondii TKLs (TgTKLs) localize to various subcellular compartments, including the nucleus, the cytosol, the inner membrane complex, and the Golgi apparatus. To gain insight into the function of TKLs in Toxoplasma, we first characterized TgTKL1, which contains the plant-like enhanced disease resistance 1 (EDR1) domain and localizes to the nucleus. TgTKL1 knockout parasites displayed significant defects in progression through the lytic cycle; we show that the defects were due to specific impairment of host cell attachment. Transcriptomics analysis identified over 200 genes of diverse functions that were differentially expressed in TgTKL1 knockout parasites. Importantly, numerous genes implicated in host cell attachment and invasion were among those most significantly downregulated, resulting in defects in microneme secretion and processing. Significantly, all of the mice inoculated intraperitoneally with TgTKL1 knockout parasites survived the infection, suggesting that TgTKL1 plays an essential role in acute toxoplasmosis. Together, these findings suggest that TgTKL1 mediates a signaling pathway that regulates the expression of multiple factors required for parasite virulence, underscoring the potential of this kinase as a novel therapeutic target.IMPORTANCEToxoplasma gondii is a protozoan parasite that can cause chronic and life-threatening disease in mammals; new drugs are greatly needed for treatment. One attractive group of drug targets consists of parasite kinases containing unique features that distinguish them from host proteins. In this report, we identify and characterize a previously unstudied kinase, TgTKL1, that localizes to the nucleus and contains a domain architecture unique to plants and protozoa. By disrupting TgTKL1, we showed that this kinase is required for the proper expression of hundreds of genes, including many that are required for the parasite to gain entry into the host cell. Specifically, parasites lacking TgTKL1 have defects in host cell attachment, resulting in impaired growth in vitro and a complete loss of virulence in mice. This report provides insight into the importance of the parasite tyrosine kinase-like kinases and establishes TgTKL1 as a novel and essential virulence factor in Toxoplasma.

Keywords: Toxoplasma gondii; apicomplexan parasites; host cell invasion; kinase.

FIG 1

Toxoplasma TKL kinases localize to multiple subcellular regions. (A) Schematic representation of TgTKL proteins, highlighting locations of conserved kinase (blue) and EDR1 (yellow) domains. Putative NLS sequences are shown above TgTKL1 and TgTKL2 data. (B) Immunofluorescence analysis showing localization of six endogenously tagged Toxoplasma TKL proteins. TgTKLs were visualized by staining with anti-HA antibody. Myosin light chain (MLC1) was used as a marker for the IMC, and TgSortilin was used as a marker for the Golgi apparatus. TgTKL1 to TgTKL6 were all found to have negative relative fitness scores in a genome-wide CRISPR screen (37), suggesting that they have an important function in T. gondii viability. Transcript levels for each gene throughout the cell cycle as reported in reference are shown at the right. Scale bar, 2 μm. RMA, robust multiarray average.

FIG 2

TgTKL1 is a nuclear kinase. (A) The localization of TgTKL1 in extracellular and intracellular parasites was visualized using IFA and staining with an anti-HA antibody. Myosin light chain 1 (MLC1), which localizes to the pellicle, is used as a marker in extracellular parasites. IMC3, which localizes to the inner membrane complex (IMC), is used as a marker in intracellular parasites to visualize progression through different cell cycle stages. Scale bar, 2 μm. (B) Immunoelectron microscopy of intracellular TgTKL1-HA using anti-HA antibody conjugated to gold particles showed that the protein localizes to the nuclei (n) of the parasites in the parasitophorous vacuole (PV) (panel 1), and higher magnification showed that the protein is largely localized to the euchromatin region (panel 2). (C) The amino acid sequence of the TgTKL1 kinase domain (residues 2620 to 2885) was aligned with the kinase domain of Homo sapiens protein kinase A (HsPKA) (residues 44 to 298), using ClustalOmega in the JalView v.2 software package. Regions of sequence conservation based on percent identity are illustrated by blue shading, and the locations of structural domains (I to XI) based on the sequence of PKA are shown at the top. (D) Predicted structure of TgTKL1 kinase domain, highlighting the ATP binding site and the N-lobe and C-lobe. (E) Visual representation of the conserved structural domains in the predicted TgTKL1 kinase domain (top) and overlay of predicted TgTKL1 kinase domain (dark gray) with the PKA (light gray) crystal structure (bottom) (PDB ID 4WB5) (root mean square deviation [RMSD] = 1.055 Å).

FIG 3

Generation of TgTKL1 knockout and complemented strains. (A and B) Semiquantitative RT-PCR and Western blot (WB) analysis (A) and immunofluorescence analysis (B) of wild-type (TgTKL1-HA), knockout (TgTKL1-KO), and complemented (TgTKL1-CO) parasites. For RT-PCR, primers that amplify parasite GAPDH (glyceraldehyde-3-phosphate dehydrogenase) were used as controls. For Western blotting, SAG1 was used as the loading control. In IFAs, TgTKL1 (red) was visualized by staining with anti-HA antibody, while TgGAP45 (green) was used as a marker for the IMC. Scale bar, 2 μm. (C) Plaque assay examining the growth of wild-type (TgTKL1-HA), knockout (TgTKL1-KO), and complemented (TgTKL1-CO) parasites in HFF cells. Plaques are visible as clear zones on the background of a crystal violet-stained HFF monolayer. (D) Quantification of plaque area sizes of TgTKL1-HA, TgTKL1-KO, and TgTKL1-CO strains. ***, P < 0.0001; n.s., not significant.

FIG 4

TgTKL1 is required for efficient microneme processing and host cell attachment. (A) Red-green invasion assay of Toxoplasma tachyzoites after 30 min of incubation with HFF cells. Data represent averages of results from three independent experiments each performed with technical triplicates ± standard errors of the means (SEM). *, P < 0.05. HCN, host cell nucleus. (B) Attachment of mycalolide B-treated tachyzoites to HFF cells. Parasites were treated for 10 min with mycalolide B, washed, and then added onto HFF cells. At 30 min postincubation at 37°C, slides were washed, fixed, and stained and the numbers of attached parasites were quantified. Data represent averages of results from three independent experiments each performed with technical triplicates ± SEM. *, P < 0.05. (C) Parasite lysate (cellular fraction) and ESA fractions from wild-type (TgTKL1-HA), knockout (TgTKL1-KO), and complemented (TgTKL1-CO) strains. ESA fractions were collected from culture supernatants after parasites were treated for 2 min at 37°C with 1% ethanol to stimulate microneme secretion. GRA1 was immunoblotted as the loading control for each strain. pM2AP, Pro M2AP; mM2AP, mature M2AP; M2AP-1, M2AP processed 1.

FIG 5

Loss of TgTKL1 alters the expression of multiple invasion-related factors. (A) Representative Western blot image of TgTKL1-HA, TgTKL1-KO, and TgTKL1-CO parasite lysates with anti-TgSUB1 antibody. SAG1 is used as the loading control. (B) Quantification of TgSUB1 protein levels in TgTKL1-HA, TgTKL1-KO, and TgTKL1-CO strains. The quantification of protein signal was determined using a FluorChem E (ProteinSimple) system. Data represent results from three independent experiments, and error bars represent SEM. *, P < 0.05. (C) Quantification of TgSUB1 transcript levels in TgTKL1-HA, TgTKL1-KO, and TgTKL1-CO strains by qRT-PCR. Data represent averages of results from three independent experiments each performed with technical triplicates ± SEM. *, P < 0.05. (D) Volcano plot of statistical significance (log odds ratio, B) versus fold change (log₂), highlighting genes identified as differentially expressed (|log₂ fold change| ≥ 1, FDR ≤ 1%) in TgTKL1-KO parasites compared to TgTKL1-HA parasites. Downregulated genes (n = 167) are highlighted in red, and upregulated genes (n = 63) are highlighted in blue. (E) Functional classification of the 167 genes downregulated and 63 genes upregulated in TgTKL1-KO parasites. Classifications were manually assigned according to known gene functions or putative functions based on conserved domains.

FIG 6

TgTKL1 is required for virulence in mice. (A) Mice were injected intraperitoneally with 500 tachyzoites of wild-type (TgTKL1-HA), knockout (TgTKL1-KO), or complemented (TgTKL1-CO) strains (five mice for each strain). Concomitant plaque assays were performed to corroborate the numbers of viable tachyzoites injected. (B) Naive mice and mice that survived TgTKL1 knockout parasite infection were rechallenged with 500 wild-type tachyzoites with 5 mice in each group.