Proteomic characterization of Toxoplasma gondii ME49 derived strains resistant to the artemisinin derivatives artemiside and artemisone implies potential mode of action independent of ROS formation

Müller, Joachim; Schlange, Carling; Heller, Manfred; Uldry, Anne-Christine; Braga-Lagache, Sophie; Haynes, Richard K.; Hemphill, Andrew

doi:10.1016/j.ijpddr.2022.11.005

Cited by 8 publications

(4 citation statements)

References 52 publications

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“…As in previously published studies [ 8 , 39 ], to enhance belief in our data, only proteins which were found differential between the strains by both LFQ and Top3 intensities were regarded as differentially expressed. Briefly [ 36 ], peptide forms were normalized by variance stabilization and imputed at this level to form the reported iTop3 values, whereas LFQ values were imputed at the protein group level to form the reported iLFQ values.…”

Section: Methodsmentioning

confidence: 84%

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

Hänggeli

Hemphill

Müller

et al. 2023

IJMS

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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.

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Section: Methodsmentioning

confidence: 84%

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

Hänggeli

Hemphill

Müller

et al. 2023

IJMS

Self Cite

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“…If only the IC50 is increased, but not the MIC, the corresponding strains tolerate the compound or have adapted to it, but are not resistant (see e.g. [130] or [131] for examples). Moreover, to allow robust investigation of the resistant strains including the preparation of single clones, the resistance must not be lost in the absence of drug pressure.…”

Section: General Considerationsmentioning

confidence: 99%

“…215 proteins downregulated in the artemisone resistant strain, 8 proteins in the artemiside resistant strain. [166] As in the case of the target deconvolution studies, the overview of studies of differentially expressed proteins in resistant vs. susceptible strains presented in Table 6 reveals a large number of differentials thus of candidate proteins involved in the mode of action of the respective drugs.…”

Section: Differential Analysis Of the Proteomes Of Susceptible Vs Res...mentioning

confidence: 99%

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

Müller,

Boubaker,

Müller

et al. 2024

Preprint

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Identification of drug targets and biochemical investigations on mechanisms of action is a are major issues in modern drug development. The present article reviews the classical “one drug” – “one target” paradigm. Novel methods for target deconvolution and for investigation of resistant strains based on protein mass spectrometry have shown that multiple gene products and adapta-tion mechanisms are involved in responses of pathogens to xenobiotics. This review is focused on protozoan pathogens. The conclusions can, however, be extended to chemotherapies against other pathogens or cancer. For this review we have searched Pubmed (pubmed.ncbi.nlm.nih.gov) and Web of Science (webofscience.com) using the key words listed below (status May 2024)

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“…Resistance to a given compound can now be defined by higher IC50s and higher MICs in resistant as compared to susceptible strains [137]. If only the IC50 is increased, but not the MIC, the corresponding strains tolerate the compound or have adapted to it but are not resistant (see, e.g., [138] or [139] for examples). Moreover, to allow robust investigation of the resistant strains, including the preparation of single clones, the resistance must not be lost in the absence of drug pressure.…”

Section: General Considerationsmentioning

confidence: 99%

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

Müller,

Boubaker,

Müller

et al. 2024

IJMS

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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.

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Proteomic characterization of Toxoplasma gondii ME49 derived strains resistant to the artemisinin derivatives artemiside and artemisone implies potential mode of action independent of ROS formation

Cited by 8 publications

References 52 publications

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

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

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

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

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