Immunogenicity of PvCyRPA, PvCelTOS and Pvs25 chimeric recombinant protein of Plasmodium vivax in murine model.

In the Americas, P. vivax is the predominant causative species of malaria, a debilitating and economically significant disease. Due to the complexity of the malaria parasite life cycle, a vaccine formulation with multiple antigens expressed in various parasite stages may represent an effective approach. Based on this, we previously designed and constructed a chimeric recombinant protein, PvRMC-1, composed by PvCyRPA, PvCelTOS, and Pvs25 epitopes. This chimeric protein was strongly recognized by naturally acquired antibodies from exposed population in the Brazilian Amazon. However, there was no investigation about the induced immune response of PvRMC-1. Therefore, in this work, we evaluated the immunogenicity of this chimeric antigen formulated in three distinct adjuvants: Stimune, AddaVax or Aluminum hydroxide (Al(OH)3) in BALB/c mice. Our results suggested that the chimeric protein PvRMC-1 were capable to generate humoral and cellular responses across all three formulations. Antibodies recognized full-length PvRMC-1 and linear B-cell epitopes from PvCyRPA, PvCelTOS, and Pvs25 individually. Moreover, mice's splenocytes were activated, producing IFN-γ in response to PvCelTOS and PvCyRPA peptide epitopes, affirming T-cell epitopes in the antigen. While aluminum hydroxide showed notable cellular response, Stimune and Addavax induced a more comprehensive immune response, encompassing both cellular and humoral components. Thus, our findings indicate that PvRMC-1 would be a promising multistage vaccine candidate that could advance to further preclinical studies.

Flp/FRT-mediated disruption of ptex150 and exp2 in Plasmodium falciparum sporozoites inhibits liver-stage development.

Plasmodium falciparum causes severe malaria and assembles a protein translocon (PTEX) complex at the parasitophorous vacuole membrane (PVM) of infected erythrocytes, through which several hundred proteins are exported to facilitate growth. The preceding liver stage of infection involves growth in a hepatocyte-derived PVM; however, the importance of protein export during P. falciparum liver infection remains unexplored. Here, we use the FlpL/FRT system to conditionally excise genes in P. falciparum sporozoites for functional liver-stage studies. Disruption of PTEX members ptex150 and exp2 did not affect sporozoite development in mosquitoes or infectivity for hepatocytes but attenuated liver-stage growth in humanized mice. While PTEX150 deficiency reduced fitness on day 6 postinfection by 40%, EXP2 deficiency caused 100% loss of liver parasites, demonstrating that PTEX components are required for growth in hepatocytes to differing degrees. To characterize PTEX loss-of-function mutations, we localized four liver-stage Plasmodium export element (PEXEL) proteins. P. falciparum liver specific protein 2 (LISP2), liver-stage antigen 3 (LSA3), circumsporozoite protein (CSP), and a Plasmodium berghei LISP2 reporter all localized to the periphery of P. falciparum liver stages but were not exported beyond the PVM. Expression of LISP2 and CSP but not LSA3 was reduced in ptex150-FRT and exp2-FRT liver stages, suggesting that expression of some PEXEL proteins is affected directly or indirectly by PTEX disruption. These results show that PTEX150 and EXP2 are important for P. falciparum development in hepatocytes and emphasize the emerging complexity of PEXEL protein trafficking.

Discovery and Quantitative Dissection of Cytokinesis Mechanisms Using Dictyostelium discoideum.

The social amoeba Dictyostelium discoideum is a versatile model for understanding many different cellular processes involving cell motility including chemotaxis, phagocytosis, and cytokinesis. Cytokinesis, in particular, is a model cell-shaped change process in which a cell separates into two daughter cells. D. discoideum has been used extensively to identify players in cytokinesis and understand how they comprise the mechanosensory and biochemical pathways of cytokinesis. In this chapter, we describe how we use cDNA library complementation with D. discoideum to discover potential regulators of cytokinesis. Once identified, these regulators are further analyzed through live cell imaging, immunofluorescence imaging, fluorescence correlation and cross-correlation spectroscopy, micropipette aspiration, and fluorescence recovery after photobleaching. Collectively, these methods aid in detailing the mechanisms and signaling pathways that comprise cell division.

Methods to Assess Autophagic Activity in Dictyostelium.

Autophagy is an intracellular clearance and recycling pathway that delivers different types of cargos to lysosomes for degradation. In recent years, autophagy has attracted considerable medical interest, and many different techniques are being developed to study this process in experimental models such as Dictyostelium. Here we describe the use of different autophagic markers in confocal microscopy, in vivo and also in fixed cells. In particular, we describe the use of the GFP-Atg8-RFP-Atg8ΔG marker and the optimization of the GFP-PgkA cleavage assay to detect small differences in autophagy flux.

APEX2-Mediated Proximity Protein Labeling in Dictyostelium.

Largely due to its simplicity, while being more like human cells compared to other experimental models, Dictyostelium continues to be of great use to discover basic molecular mechanisms and signaling pathways underlying evolutionarily conserved biological processes. However, the identification of new protein interactions implicated in signaling pathways can be particularly challenging in Dictyostelium due to its extremely fast signaling kinetics coupled with the dynamic nature of signaling protein interactions. Recently, the proximity labeling method using engineered ascorbic acid peroxidase 2 (APEX2) in mammalian cells was shown to allow the detection of weak and/or transient protein interactions and also to obtain spatial and temporal resolution. Here, we describe a protocol for successfully using the APEX2-proximity labeling method in Dictyostelium. Coupled with the identification of the labeled proteins by mass spectrometry, this method expands Dictyostelium's proteomics toolbox and should be widely useful for identifying interacting partners involved in a variety of biological processes in Dictyostelium.

Evaluating the Activity of Ras/Rap GTPases in Dictyostelium.

Ras and Rap small GTPases of the Ras superfamily act as molecular switches to control diverse cellular processes as part of different signaling pathways. Dictyostelium expresses several Ras and Rap proteins, and their study has and continues to greatly contribute to our understanding of their role in eukaryote biology. To study the activity of Ras and Rap proteins in Dictyostelium, several assays based on their interaction with the Ras binding domain of known eukaryotic Ras/Rap effectors have been developed and proved extremely useful to study their regulation and cellular roles. Here, we describe methods to assess Ras/Rap activity biochemically using a pull-down assay and through live-cell imaging using fluorescent reporters.

Analysis of cGMP Signaling in Dictyostelium.

Biochemical assays are described to analyze signal transduction by the second messenger cGMP in Dictyostelium. The methods include enzyme assays to measure the activity and regulation of cGMP synthesizing guanylyl cyclases and cGMP-degrading phosphodiesterases. In addition, several methods are described to quantify cGMP levels. The target of cGMP in Dictyostelium is the large protein GbpC that has multiple domains including a Roc domain, a kinase domain, and a cGMP-stimulated Ras-GEF domain. A cGMP-binding assay is described to detect and quantify GbpC.

Proteomic Analysis of Dictyostelium discoideum by Mass Spectrometry.

The large-scale proteomic analysis of Dictyostelium discoideum has contributed to our understanding of intracellular as well as secreted proteins in this versatile model eukaryote. Mass spectrometry-based proteomic analysis is a robust, sensitive, and rapid analytical method for identification and characterization of proteins extracted from tissues, cells, cell fractions, or pull-down assays. The availability of core facilities which make proteomics inexpensive and easy to do has facilitated a wide range of research projects. In this chapter, we present a simple standard methodology to extract proteins and prepare samples from D. discoideum for mass spectrometry and methods to analyze the identified proteins.

T. gondii excretory proteins promote the osteogenic differentiation of human bone mesenchymal stem cells via the BMP/Smad signaling pathway.

BACKGROUND: Bone defects, resulting from substantial bone loss that exceeds the natural self-healing capacity, pose significant challenges to current therapeutic approaches due to various limitations. In the quest for alternative therapeutic strategies, bone tissue engineering has emerged as a promising avenue. Notably, excretory proteins from Toxoplasma gondii (TgEP), recognized for their immunogenicity and broad spectrum of biological activities secreted or excreted during the parasite's lifecycle, have been identified as potential facilitators of osteogenic differentiation in human bone marrow mesenchymal stem cells (hBMSCs). Building on our previous findings that TgEP can enhance osteogenic differentiation, this study investigated the molecular mechanisms underlying this effect and assessed its therapeutic potential in vivo. METHODS: We determined the optimum concentration of TgEP through cell cytotoxicity and cell proliferation assays. Subsequently, hBMSCs were treated with the appropriate concentration of TgEP. We assessed osteogenic protein markers, including alkaline phosphatase (ALP), Runx2, and Osx, as well as components of the BMP/Smad signaling pathway using quantitative real-time PCR (qRT-PCR), siRNA interference of hBMSCs, Western blot analysis, and other methods. Furthermore, we created a bone defect model in Sprague-Dawley (SD) male rats and filled the defect areas with the GelMa hydrogel, with or without TgEP. Microcomputed tomography (micro-CT) was employed to analyze the bone parameters of defect sites. H&E, Masson and immunohistochemical staining were used to assess the repair conditions of the defect area. RESULTS: Our results indicate that TgEP promotes the expression of key osteogenic markers, including ALP, Runx2, and Osx, as well as the activation of Smad1, BMP2, and phosphorylated Smad1/5-crucial elements of the BMP/Smad signaling pathway. Furthermore, in vivo experiments using a bone defect model in rats demonstrated that TgEP markedly promoted bone defect repair. CONCLUSION: Our results provide compelling evidence that TgEP facilitates hBMSC osteogenic differentiation through the BMP/Smad signaling pathway, highlighting its potential as a therapeutic approach for bone tissue engineering for bone defect healing.

Chemoproteomics validates selective targeting of Plasmodium M1 alanyl aminopeptidase as an antimalarial strategy.

New antimalarial drug candidates that act via novel mechanisms are urgently needed to combat malaria drug resistance. Here, we describe the multi-omic chemical validation of Plasmodium M1 alanyl metalloaminopeptidase as an attractive drug target using the selective inhibitor, MIPS2673. MIPS2673 demonstrated potent inhibition of recombinant Plasmodium falciparum (PfA-M1) and Plasmodium vivax (PvA-M1) M1 metalloaminopeptidases, with selectivity over other Plasmodium and human aminopeptidases, and displayed excellent in vitro antimalarial activity with no significant host cytotoxicity. Orthogonal label-free chemoproteomic methods based on thermal stability and limited proteolysis of whole parasite lysates revealed that MIPS2673 solely targets PfA-M1 in parasites, with limited proteolysis also enabling estimation of the binding site on PfA-M1 to within ~5 Å of that determined by X-ray crystallography. Finally, functional investigation by untargeted metabolomics demonstrated that MIPS2673 inhibits the key role of PfA-M1 in haemoglobin digestion. Combined, our unbiased multi-omic target deconvolution methods confirmed the on-target activity of MIPS2673, and validated selective inhibition of M1 alanyl metalloaminopeptidase as a promising antimalarial strategy.

The Trypanosoma cruzi pleiotropic protein P21 orchestrates the intracellular retention and in-vivo parasitism control of virulent Y strain parasites.

P21 is a protein secreted by all forms of Trypanosoma cruzi (T. cruzi) with recognized biological activities determined in studies using the recombinant form of the protein. In our recent study, we found that the ablation of P21 gene decreased Y strain axenic epimastigotes multiplication and increased intracellular replication of amastigotes in HeLa cells infected with metacyclic trypomastigotes. In the present study, we investigated the effect of P21 in vitro using C2C12 cell lines infected with tissue culture-derived trypomastigotes (TCT) of wild-type and P21 knockout (TcP21-/-) Y strain, and in vivo using an experimental model of T. cruzi infection in BALB/c mice. Our in-vitro results showed a significant decrease in the host cell invasion rate by TcP21-/- parasites as measured by Giemsa staining and cell count in bright light microscope. Quantitative polymerase chain reaction (qPCR) analysis showed that TcP21-/- parasites multiplied intracellularly to a higher extent than the scrambled parasites at 72h post-infection. In addition, we observed a higher egress of TcP21-/- trypomastigotes from C2C12 cells at 144h and 168h post-infection. Mice infected with Y strain TcP21-/- trypomastigotes displayed higher systemic parasitemia, heart tissue parasite burden, and several histopathological alterations in heart tissues compared to control animals infected with scrambled parasites. Therewith, we propose that P21 is important in the host-pathogen interaction during invasion, cell multiplication, and egress, and may be part of the mechanism that controls parasitism and promotes chronic infection without patent systemic parasitemia.

Life stage-specific poly(A) site selection regulated by Trypanosoma brucei DRBD18.

The kinetoplastid parasite, Trypanosoma brucei, undergoes a complex life cycle entailing slender and stumpy bloodstream forms in mammals and procyclic and metacyclic forms (MFs) in tsetse fly hosts. The numerous gene regulatory events that underlie T. brucei differentiation between hosts, as well as between active and quiescent stages within each host, take place in the near absence of transcriptional control. Rather, differentiation is controlled by RNA-binding proteins (RBPs) that associate with mRNA 3' untranslated regions (3'UTRs) to impact RNA stability and translational efficiency. DRBD18 is a multifunctional T. brucei RBP, shown to impact mRNA stability, translation, export, and processing. Here, we use single-cell RNAseq to characterize transcriptomic changes in cell populations that arise upon DRBD18 depletion, as well as to visualize transcriptome-wide alterations to 3'UTR length. We show that in procyclic insect stages, DRBD18 represses expression of stumpy bloodstream form and MF transcripts. Additionally, DRBD18 regulates the 3'UTR lengths of over 1,500 transcripts, typically promoting the use of distal polyadenylation sites, and thus the inclusion of 3'UTR regulatory elements. Remarkably, comparison of polyadenylation patterns in DRBD18 knockdowns with polyadenylation patterns in stumpy bloodstream forms shows numerous similarities, revealing a role for poly(A) site selection in developmental gene regulation, and indicating that DRBD18 controls this process for a set of transcripts. RNA immunoprecipitation supports a direct role for DRBD18 in poly(A) site selection. This report highlights the importance of alternative polyadenylation in T. brucei developmental control and identifies a critical RBP in this process.

Two decades of molecular surveillance in Senegal reveal rapid changes in known drug resistance mutations over time.

BACKGROUND: Drug resistance in Plasmodium falciparum is a major threat to malaria control efforts. Pathogen genomic surveillance could be invaluable for monitoring current and emerging parasite drug resistance. METHODS: Data from two decades (2000-2020) of continuous molecular surveillance of P. falciparum parasites from Senegal were retrospectively examined to assess historical changes in malaria drug resistance mutations. Several known drug resistance markers and their surrounding haplotypes were profiled using a combination of single nucleotide polymorphism (SNP) molecular surveillance and whole genome sequence based population genomics. RESULTS: This dataset was used to track temporal changes in drug resistance markers whose timing correspond to historically significant events such as the withdrawal of chloroquine (CQ) and the introduction of sulfadoxine-pyrimethamine (SP) in 2003. Changes in the mutation frequency at Pfcrt K76T and Pfdhps A437G coinciding with the 2014 introduction of seasonal malaria chemoprevention (SMC) in Senegal were observed. In 2014, the frequency of Pfcrt K76T increased while the frequency of Pfdhps A437G declined. Haplotype-based analyses of Pfcrt K76T showed that this rapid increase was due to a recent selective sweep that started after 2014. DISCUSSION (CONCLUSION): The rapid increase in Pfcrt K76T is troubling and could be a sign of emerging amodiaquine (AQ) resistance in Senegal. Emerging AQ resistance may threaten the future clinical efficacy of artesunate-amodiaquine (ASAQ) and AQ-dependent SMC chemoprevention. These results highlight the potential of molecular surveillance for detecting rapid changes in parasite populations and stress the need to monitor the effectiveness of AQ as a partner drug for artemisinin-based combination therapy (ACT) and for chemoprevention.

[Biological threats to global malaria elimination II Deletion in the malaria rapid diagnostic test target Plasmodium falciparum histidine-rich protein 2/3 genes].

The global malaria epidemic is still severe. Because of simple procedures, rapid detection and accuracy results, rapid diagnostic test (RDT) has become the most important and the most widely used diagnostic tool for malaria prevention and control. However, deletions in the RDT target Plasmodium falciparum histidine-rich protein 2/3 (Pfhrp2/3) genes may cause false-negative results of RDT, which has been included as one of the four biological threats to global malaria elimination. This article reviews the applications of RDT in the global malaria diagnosis, analyzes the threats and challenges caused by Pfhrp2/3 gene deletion, proposes methods for monitoring Pfhrp2/3 gene deletion, and summarizes the causes and countermeasures of negative RDT detections, so as to provide insights into consolidation of malaria elimination achievements in China and contributions to global malaria elimination.

[Preparation and preliminary application of the polyclonal antibody against Toxoplasma gondii dense granule protein 24].

OBJECTIVE: To prepare and characterize the mouse polyclonal antibody against the dense granule protein 24 (GRA24) of Toxoplasma gondii, and explore its preliminary applications. METHODS: The GRA24 coding sequences of different T. gondii strains were aligned using the MEGA-X software, and the dominant peptide of the GRA24 protein was analyzed with the Protean software. The base sequence encoding this peptide was amplified using PCR assay and ligated into the pET-28a vector, and the generated GRA24 truncated protein was transformed into Escherichia coli BL21. After induction by isopropyl-beta-D-thiogalactopyranoside (IPTG), the expression and purification of the recombinant GRA24 protein was analyzed using sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE). BALB/c mice were immunized by subcutaneous injection with the purified recombinant GRA24 truncated protein to generate the polyclonal antibody, and the titer of the polyclonal antibody was measured using enzyme linked immunosorbent assay (ELISA). The specificity of the polyclonal antibody was tested using Western blotting, and the intracellular localization of the polyclonal antibody was investigated using immunofluorescence assay (IFA). RESULTS: SDS-PAGE showed successful construction of the recombinant expression plasmid, and Coomassie brilliant blue staining showed the generation of the high-purity recombinant GRA24 truncated protein. ELISA measured that the titer of the polyclonal antibody against the GRA24 truncated protein was higher than 1:208 400, and Western blotting showed that the polyclonal antibody was effective to recognize the endogenous GRA24 proteins of different T. gondii strains and specifically recognize the recombinant GRA24 truncated protein. Indirect IFA showed that the GRA24 protein secreted 16 hour following T. gondii invasion in host cells. CONCLUSIONS: The polyclonal antibody against the T. gondii GRA24 protein has been successfully prepared, which has a widespread applicability, high titers and a high specificity. This polyclonal antibody is available for Western blotting and IFA, which provides the basis for investigating the function of the GRA24 protein.

The Toxoplasma secreted effector TgWIP modulates dendritic cell motility by activating host tyrosine phosphatases Shp1 and Shp2.

The obligate intracellular parasite Toxoplasma gondii causes life-threatening toxoplasmosis to immunocompromised individuals. The pathogenesis of Toxoplasma relies on its swift dissemination to the central nervous system through a 'Trojan Horse' mechanism using infected leukocytes as carriers. Previous work found TgWIP, a protein secreted from Toxoplasma, played a role in altering the actin cytoskeleton and promoting cell migration in infected dendritic cells (DCs). However, the mechanism behind these changes was unknown. Here, we report that TgWIP harbors two SH2-binding motifs that interact with tyrosine phosphatases Shp1 and Shp2, leading to phosphatase activation. DCs infected with Toxoplasma exhibited hypermigration, accompanying enhanced F-actin stress fibers and increased membrane protrusions such as filopodia and pseudopodia. By contrast, these phenotypes were abrogated in DCs infected with Toxoplasma expressing a mutant TgWIP lacking the SH2-binding motifs. We further demonstrated that the Rho-associated kinase (Rock) is involved in the induction of these phenotypes, in a TgWIP-Shp1/2 dependent manner. Collectively, the data uncover a molecular mechanism by which TgWIP modulates the migration dynamics of infected DCs in vitro.

Drug resistance markers in Plasmodium vivax isolates from a Kanchanaburi province, Thailand between January to May 2023.

BACKGROUND: Plasmodium vivax has become the predominant species in the border regions of Thailand. The emergence and spread of antimalarial drug resistance in P. vivax is one of the significant challenges for malaria control. Continuous surveillance of drug resistance is therefore necessary for monitoring the development of drug resistance in the region. This study aims to investigate the prevalence of the mutation in the P. vivax multidrug resistant 1 (Pvmdr1), dihydrofolate reductase (Pvdhfr), and dihydropteroate synthetase (Pvdhps) genes conferred resistance to chloroquine (CQ), pyrimethamine (P) and sulfadoxine (S), respectively. METHOD: 100 P. vivax isolates were obtained between January to May 2023 from a Kanchanaburi province, western Thailand. Nucleotide sequences of Pvmdr1, Pvdhfr, and Pvdhps genes were amplified and sequenced. The frequency of single nucleotide polymorphisms (SNPs)-haplotypes of drug-resistant alleles was assessed. The linkage disequilibrium (LD) tests were also analyzed. RESULTS: In Pvmdr1, T958M, Y976F, and F1076L, mutations were detected in 100%, 21%, and 23% of the isolates, respectively. In Pvdhfr, the quadruple mutant allele (I57R58M61T117) prevailed in 84% of the samples, followed by (L57R58M61T117) in 11%. For Pvdhps, the double mutant allele (G383G553) was detected (48%), followed by the triple mutant allele (G383M512G553) (47%) of the isolates. The most prevalent combination of Pvdhfr (I57R58M61T117) and Pvdhps (G383G553) alleles was sextuple mutated haplotypes (48%). For LD analysis, the association in the SNPs pairs was found between the intragenic and intergenic regions of the Pvdhfr and Pvdhps genes. CONCLUSION: The study has recently updated the high prevalence of three gene mutations associated with CQ and SP resistance. Genetic monitoring is therefore important to intensify in the regions to further assess the spread of drug resistant. Our data also provide evidence on the distribution of drug resistance for the early warning system, thereby threatening P. vivax malaria treatment policy decisions at the national level.

Expression and biochemical characterization of the putative insulinase enzyme PF11_0189 found in the Plasmodium falciparum genome.

PF11_0189 is a putative insulin degrading enzyme present in Plasmodium falciparum genome. The catalytic domain of PF11_0189 is about 27 kDa. Substrate specificity study shows PF11_0189 acts upon different types of proteins. The substrate specificity is found to be highest when insulin is used as a substrate. Metal dependency study shows highest dependency of PF11_0189 towards zinc metal for its proteolytic activity. Chelation of zinc metal with EDTA shows complete absence of PF11_0189 activity. Peptide inhibitors, P-70 and P-121 from combinatorial peptide library prepared against PF11_0189 show inhibition with an IC50 value of 4.8 µM and 7.5 µM respectively. A proven natural anti-malarial peptide cyclosporin A shows complete inhibition against PF11_0189 with an IC50 value of 0.75 µM suggesting PF11_0189 as a potential target for peptide inhibitors. The study implicates that PF11_0189 is a zinc metalloprotease involved in catalysis of insulin. The study gives a preliminary insight into the mechanism of complications arising from glucose abnormalities during severe malaria.

Investigating Antiprotozoal Chemotherapies with Novel Proteomic Tools-Chances and Limitations: A Critical Review.

Identification of drug targets and biochemical investigations on mechanisms of action are major issues in modern drug development. The present article is a critical review of the classical "one drug"-"one target" paradigm. In fact, novel methods for target deconvolution and for investigation of resistant strains based on protein mass spectrometry have shown that multiple gene products and adaptation mechanisms are involved in the responses of pathogens to xenobiotics rather than one single gene or gene product. Resistance to drugs may be linked to differential expression of other proteins than those interacting with the drug in protein binding studies and result in complex cell physiological adaptation. Consequently, the unraveling of mechanisms of action needs approaches beyond proteomics. This review is focused on protozoan pathogens. The conclusions can, however, be extended to chemotherapies against other pathogens or cancer.

The Effects and Mechanism of ATM Kinase Inhibitors in Toxoplasma gondii.

Toxoplasma gondii, an important opportunistic pathogen, underscores the necessity of developing novel therapeutic drugs and identifying new drug targets. Our findings indicate that the half-maximal inhibitory concentrations (IC50) of KU60019 and CP466722 (abbreviated as KU and CP) against T. gondii are 0.522 µM and 0.702 µM, respectively, with selection indices (SI) of 68 and 10. Treatment with KU and CP affects the in vitro growth of T. gondii, inducing aberrant division in the daughter parasites. Transmission electron microscopy reveals that KU and CP prompt the anomalous division of T. gondii, accompanied by cellular enlargement, nuclear shrinkage, and an increased dense granule density, suggesting potential damage to parasite vesicle transport. Subsequent investigations unveil their ability to modulate the expression of certain secreted proteins and FAS II (type II fatty acid synthesis) in T. gondii, as well as including the dot-like aggregation of the autophagy-related protein ATG8 (autophagy-related protein 8), thereby expediting programmed death. Leveraging DARTS (drug affinity responsive target stability) in conjunction with 4D-Label-free quantitative proteomics technology, we identified seven target proteins binding to KU, implicated in pivotal biological processes such as the fatty acid metabolism, mitochondrial ATP transmission, microtubule formation, and Golgi proteins transport in T. gondii. Molecular docking predicts their good binding affinity. Furthermore, KU has a slight protective effect on mice infected with T. gondii. Elucidating the function of those target proteins and their mechanism of action with ATM kinase inhibitors may potentially enhance the treatment paradigm for toxoplasmosis.