Comparative Proteomic Analysis of Toxoplasma gondii RH Wild-Type and Four SRS29B (SAG1) Knock-Out Clones Reveals Significant Differences between Individual Strains

Abstract

In T. gondii, as well as in other model organisms, gene knock-out using CRISPR-Cas9 is a suitable tool to identify the role of specific genes. The general consensus implies that only the gene of interest is affected by the knock-out. Is this really the case? In a previous study, we generated knock-out (KO) clones of TgRH88_077450 (SRS29B; SAG1) which differed in the numbers of the integrated dihydrofolate-reductase-thymidylate-synthase (MDHFR-TS) drug-selectable marker. Clones 18 and 33 had a single insertion of MDHFR-TS within SRS29B. Clone 6 was disrupted by the insertion of a short unrelated DNA-sequence, but the marker was integrated elsewhere. In clone 30, the marker was inserted into SRS29B, and several other MDHFR-TS copies were found in the genome. KO and wild-type (WT) tachyzoites had similar shapes, dimensions, and vitality. This prompted us to investigate the impact of genetic engineering on the overall proteome patterns of the four clones as compared to the respective WT. Comparative shotgun proteomics of the five strains was performed. Overall, 3236 proteins were identified. Principal component analysis of the proteomes revealed five distinct clusters corresponding to the five strains by both iTop3 and iLFQ algorithms. Detailed analysis of the differentially expressed proteins revealed that the target of the KO, srs29B, was lacking in all KO clones. In addition to this protein, 20 other proteins were differentially expressed between KO clones and WT or between different KO clones. The protein exhibiting the highest variation between the five strains was srs36D encoded by TgRH_016110. The deregulated expression of SRS36D was further validated by quantitative PCR. Moreover, the transcript levels of three other selected SRS genes, namely SRS36B, SRS46, and SRS57, exhibited significant differences between individual strains. These results indicate that knocking out a given gene may affect the expression of other genes. Therefore, care must be taken when specific phenotypes are regarded as a direct consequence of the KO of a given gene.

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

“…Now, taken into consideration in one hand the off target molecular effects (see e.g. [149]) and the untrustworthy concept of one-drug for one-target for onedisease approach in other hand, experimental validation of drug target using genetic tools may ultimately leave more questions than answers.…”

“…For instance, a Giardia line overexpressing E. coli glucuronidase A differs by nearly 10 % of the detected unique proteins from the corresponding wildtype with a major focus on altered patterns of surface proteins, referred to as "antigenic variation" [45]. Moreover, T. gondii knock-out clones of the surface antigen SAG1 created by gene editing show distinct strain-specific proteome patterns besides the intended knock-down [149].…”

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

“…Certainly, experimental validation based on knockdown, knock-out, or overexpression of the target gene is feasible. Now, taking into consideration, on the one hand, the off-target molecular effects (see, e.g., [157]) and, on the other hand, the untrustworthy concept of the one drug-for one target-for one disease approach, the experimental validation of drug targets using genetic tools may ultimately leave more questions than answers.…”

“…For instance, a Giardia line overexpressing E. coli glucuronidase A differs by nearly 10% of the detected unique proteins from the corresponding wildtype with a major focus on altered patterns of surface proteins, referred to as "antigenic variation" [50]. Moreover, T. gondii knock-out clones of the surface antigen SAG1 created by gene editing show distinct strain-specific proteome patterns besides the intended knock-down [157].…”

Proteomics Applications in Toxoplasma gondii: Unveiling the Host–Parasite Interactions and Therapeutic Target Discovery

“…Comparative proteomics involves comparing the proteomes of T. gondii and its infected host cells/tissues under different conditions to identify differentially expressed proteins and reveal molecular changes associated with infection. This approach allows researchers to identify and quantify parasite-specific proteins, host factors targeted by the parasite, and alterations in host protein expression induced by T. gondii infection [23][24][25][26][27][28][29]. During this stage, more than 6000 proteins can be identified and quantified using iTRAQ-labeling and strong cation exchange (SCX) fractionation techniques [16].…”