RT Journal Article
T1 Pleiotropic Effects on Tachyzoite and Host Cell Proteomes in Knock-Out Clones of the Open Reading Frames 297720 and 319730 Constitutively Expressed in T. gondii ShSp1 Tachyzoites
A1 Hänggeli, Kai Pascal Alexander
A1 Müller, Joachim
A1 Heller, Manfred
A1 Uldry, Anne-Christine
A1 Braga-Lagache, Sophie
A1 Arranz Solís, David
A1 Ortega Mora, Luis Miguel
A1 Hemphill, Andrew
AB Toxoplasma gondii, the causative agent of toxoplasmosis widespread in animals and humans, is an intracellular apicomplexan protozoan parasite infecting a variety of host cells. Gene editing using CRISPR-Cas9 has become a standard tool to investigate the molecular genetics of this interaction. With respect to gene knock-out (KO) studies, the general paradigm implies that the gene of interest is expressed in the wildtype and that only the gene of interest is affected by the knock-out. Consequently, the observed phenotype depends on the presence or absence of genes of interest. To challenge this paradigm, we knocked out two open reading frames (ORFs) constitutively expressed in T. gondii ShSp1 tachyzoites, but not essential, namely ORF 297720 encoding a trehalose-6-phosphatase homolog and ORF 319730 encoding a You2 C2C2 zinc finger homolog. We analyzed the proteomes of tachyzoites isolated at a late stage of infection, as well as intracellular tachyzoites and host cells at an early stage of infection. The intended KO proteins were present in the T. gondii Sp1 wildtype but absent in the KO clones. Moreover, besides differentially expressed (DE) proteins specific to each KO, 17 DE proteins common to both KOs were identified in isolated tachyzoites and 39 in intracellular tachyzoites. Moreover, 76 common DE proteins were identified in host cells. Network and enrichment analyses showed that these proteins were functionally related to antiviral defense mechanisms. These results indicate that the KO of a gene of interest may not only affect the expression of other genes of the target organism, which in our case is T. gondii, but also the gene expression of its host cells. Therefore, phenotypes of KO strains may not be causally related to the KO of a given gene. Overall, this study highlights that genetic manipulation in T. gondii can lead to system-wide proteomic shifts in both parasite and host, emphasizing the need for cautious interpretation of knock-out-based functional analyses
PB MDPI
SN 1661-6596
YR 2025
FD 2025
LK https://hdl.handle.net/20.500.14352/127322
UL https://hdl.handle.net/20.500.14352/127322
LA eng
NO Hänggeli, K. P. A., Müller, J., Heller, M., Uldry, A. C., Braga-Lagache, S., Arranz-Solís, D., Ortega-Mora, L. M., & Hemphill, A. (2025). Pleiotropic Effects on Tachyzoite and Host Cell Proteomes in Knock-Out Clones of the Open Reading Frames 297720 and 319730 Constitutively Expressed in T. gondii ShSp1 Tachyzoites. International journal of molecular sciences, 26(21), 10433. https://doi.org/10.3390/ijms262110433
NO Author Contributions: Data curation, J.M., M.H., A.-C.U. and S.B.-L.; formal analysis, J.M.; funding acquisition, A.H.; investigation, K.P.A.H. and J.M.; methodology, K.P.A.H., J.M., S.B.-L. and D.A.-S.; project administration, A.H.; resources, L.-M.O.-M. and A.H.; software, M.H., A.-C.U. and S.B.-L.; validation, J.M., M.H. and A.H.; visualization, K.P.A.H. and J.M.; writing—original draft, K.P.A.H. and J.M.; writing—review and editing, K.P.A.H., J.M., M.H., A.-C.U., S.B.-L., D.A.-S., L.-M.O.-M. and A.H. All authors have read and agreed to the published version of the manuscript
NO Swiss National Science Foundation
DS Docta Complutense
RD 20 ene 2026