Kai Hänggeli (Bern / CH), Prof. Dr. Andrew Hemphill (Bern / CH), Norbert Müller (Bern / CH), Dr Joachim Müller (Bern / CH), Prof. Manfred Heller (Bern / CH), Dr. Ghalia Boubaker (Bern / CH)
Abstract text
The role of the major T. gondii tachyzoite surface antigen 1 (SAG1), has remained elusive. We have previously generated four T. gondii RH SAG1 knock-out (KO) strains by CRISPR-Cas9 and characterized these KOs regarding the number of integrated dihydrofolate reductase-thymidylate synthase (mdhfr-ts) drug selectable marker. Accordingly, two clones have a single integration of the mdhfr-ts into the SAG1 gene. In one clone, sag1 was disrupted by insertion of a short DNA sequence unrelated to the resistance marker, but mdhfr-ts was integrated elsewhere in the genome. Another clone exhibited an insertion of mdhfr-ts into sag1, but several other mdhfr-ts copies were found in the genome.
In this study, we determined the overall change(s) in protein expression patterns in all four clones by applying a comparative shotgun proteomics approach in relation to wild-type (WT) parasites using biological triplicates of each KO and WT clone.
In SAG1 KO strains, the expression levels of 53 proteins were significantly altered. Of these, 12 were upregulated (fold change (FC) ≥ 1.5) and 41 were downregulated (FC ≤ 0.66) as compared to the WT parasites. In KO parasites, the expression of peptidase family M3 protein and putative transmembrane protein were 8- and 7-fold upregulated, respectively, and the expression of CBS domain-containing protein and TBC domain-containing protein was reduced by 90%, compared to WT tachyzoites. 39 SAG1 related sequence (SRS) proteins were found to be expressed in WT tachyzoites, and among those five were upregulated and three were downregulated in SAG1 KO parasites. In addition, depletion of SAG1 expression also impacted dense granule (GRA) protein expression; GRA 9 and GRA2 were upregulated. With regard to the phenotype, scanning electron micrographs did not reveal notable differences in the shape or dimensions of tachyzoites, but the surface of the mutants lacking SAG1 exhibited a higher density of particles compared to the smoother surface of WT tachyzoites. Plaque assays revealed reduced growth of KO tachyzoites compared to WT parasites.
In conclusion, this comprehensive analysis shows, for the first time, that the lack of SAG1 expression has a profound impact on the tachyzoite proteome, including the tachyzoite surface composition, and affects the tachyzoite growth characteristics in vitro. This highlights the need for further in vivo studies to elucidate the functional role of SAG1 during infection in the mammalian host.