Transient Adaptation of Toxoplasma gondii to Exposure by Thiosemicarbazone Drugs That Target Ribosomal Proteins Is Associated with the Upregulated Expression of Tachyzoite Transmembrane Proteins and Transporters
Abstract
:1. Introduction
2. Results
2.1. In Vitro Activities of Three Gold Complexes and Their Salicyl-TSC Ligand (C4) against T. gondii-tachyzoites
2.2. C3 and C4 Induce Only Minor and Transient Ultrastructural Alterations in and Do Not Affect the Mitochondrial Membrane Potential T. gondii ME49 Tachyzoites
2.3. Identification of C3- and C4-Binding Proteins in Extracts of T. gondii ME49 tachyzoites
2.4. Long-Term Treatments and Generation of Clones of Drug-Adapted T. gondii Tachyzoites
2.5. Proteomic Analysis of T. gondii ME49 Clones Recovered after In Vitro Adaptation to C3 and C4
3. Discussion
4. Materials and Methods
4.1. Equipment and Reagents Used in Cell Culture
4.2. Compounds
4.3. Host Cells and Parasites
4.4. In Vitro Assessment of Toxicity and Efficacy of Compounds in HFF and T. gondii tachyzoites
4.5. Transmission Electron Microscopy (TEM)
4.6. Evaluation of the MMP by Tetramethyl Rhodamine, Ethyl Ester (TMRE) Uptake Assay
4.7. Drug Treatments for Extended Periods of Time and Generation of C3 and C4 Adapted T. gondii ME49 Clonal Strains
4.8. Differential Affinity Chromatography (DAC)
4.9. Proteomics
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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T. gondii β-Gal | HFF | ||
---|---|---|---|
EC50 When Added Concomitantly to Infection (nM) | EC50 When Added 3 h Post-Infection (nM) | No Viability Impairment at (µM) | |
Compound | |||
C3 | 28 (27–30) | 23 (20–26) | 25 |
C4 | 30 (40–20) | 20 (15–25) | 25 |
T. gondii ME49 | |||
C3 | 31 (16–62) | ||
C4 | 13 (6–27) |
ToxoDB ORF | Annotation | rAbu C3 |
---|---|---|
TGME49_262690 | ribosomal protein RPL27 | 741 |
TGME49_244110 | nucleosome assembly protein (nap) protein | 324 |
TGME49_309740 | LSM domain-containing protein | 100 |
TGME49_321520 | hypothetical protein | 91 |
TGME49_279390 | proliferation-associated protein 2G4, putative | 80 |
TGME49_319870 | ubiquitin-conjugating enzyme subfamily protein | 77 |
TGME49_313560 | 60S ribosomal protein L7a, putative | 71 |
TGME49_248810 | nuclear factor NF7 | 54 |
TGME49_231850 | serine-threonine phosophatase 2C (PP2C) | 51 |
TGME49_209170 | hypothetical protein | 43 |
TGME49_272400 | casein kinase ii regulatory subunit protein | 43 |
TGME49_227810 | rhoptry kinase family protein ROP11 (incomplete catalytic triad) | 35 |
TGME49_256050 | signal recognition particle 14kd protein | 28 |
TGME49_305340 | corepressor complex CRC230 | 9 |
ToxoDB ORF | Annotation | rAbu C4 |
---|---|---|
TGME49_213280 | SAG-related sequence SRS25 | 22,200 |
TGME49_253690 | hypothetical protein (putative transmembrane protein) | 15,804 |
TGME49_316710 | hypothetical protein | 4878 |
TGME49_266060 | ribosomal protein RPSA | 3977 |
TGME49_288245 | hypothetical protein | 2980 |
TGME49_231160 | hypothetical protein | 2724 |
TGME49_226570 | hypothetical protein | 1943 |
TGME49_285870 | SAG-related sequence SRS20A | 1652 |
TGME49_319560 | microneme protein MIC3 | 1600 |
TGME49_289680 | Ras-related protein Rab11 | 1461 |
TGME49_263630 | hypothetical protein | 1426 |
TGME49_214410 | hypothetical protein | 1316 |
TGME49_214770 | small GTP binding protein rab1a, putative | 1305 |
TGME49_225555 | hypothetical protein | 1284 |
TGME49_308020 | SAG-related sequence SRS57 | 1218 |
TGME49_249900 | adenine nucleotide translocator, putative | 1171 |
TGME49_233450 | SAG-related sequence SRS29A | 1156 |
TGME49_308840 | SAG-related sequence SRS51 | 1153 |
TGME49_245490 | microneme protein MIC8 | 1126 |
TGME49_227920 | hypothetical protein | 1043 |
ToxoDB ORF | Annotation | rAbu C3 | rAbu C4 |
---|---|---|---|
TGME49_309810 | ribosomal protein RPP2 | 3431 | 3473 |
TGME49_249250 | ribosomal protein RPL35A | 3406 | 214 |
TGME49_262670 | ribosomal protein RPL18A | 2651 | 522 |
TGME49_313390 | ribosomal protein RPL6 | 2390 | 482 |
TGME49_261570 | ribosomal protein RPL7A | 2348 | 372 |
TGME49_205340 | ribosomal protein RPS12 | 2026 | 493 |
TGME49_292130 | ribosomal protein RPL13A | 1678 | 447 |
TGME49_238070 | glutaredoxin domain-containing protein | 1666 | 332 |
TGME49_267060 | ribosomal protein RPL14 | 1540 | 317 |
TGME49_320050 | ribosomal protein RPL5 | 1492 | 223 |
TGME49_204020 | ribosomal protein RPL8 | 1487 | 114 |
TGME49_238250 | ribosomal protein RPL36 | 1422 | 517 |
TGME49_245680 | ribosomal protein RPL21 | 1362 | 219 |
TGME49_260260 | ribosomal protein RPP1 | 1354 | 2505 |
TGME49_314810 | ribosomal protein RPL7 | 1325 | 666 |
TGME49_270380 | ribosomal protein RPS13 | 1265 | 1846 |
TGME49_210690 | ribosomal protein RPS6 | 1242 | 759 |
TGME49_215470 | ribosomal protein RPL10A | 1090 | 400 |
TGME49_218820 | alba 2 | 1046 | 753 |
TGME49_232230 | ribosomal protein RPL30 | 986 | 50 |
EC50 (nM) | MIC (µM) | |
---|---|---|
C3 | ||
T. gondii Me49 wildtype (WT) | 31 (16–62) | 25 |
P1F3 | 45 (28–70) | 25 |
P1E5 | 90 (54–150) | 25 |
P2F5 | 51 (31–84) | 25 |
C4 | ||
T. gondii Me49 wildtype (WT) | 13 (6–27) | 25 |
P2F3 | 11 (6–18) | 25 |
P1G10 | 10 (4–23) | 25 |
P3E2 | 6 (4–9) | 25 |
ToxoDB ORF | Product Description | C3_Clone | Fold Change (FC) |
---|---|---|---|
Upregulated ↑ | |||
TGME49_258700 | transporter, major facilitator family protein | P1F3 | 6.5 |
TGME49_225480 | hypothetical protein | P1E5 | 6.4 |
TGME49_248140 | hypothetical protein | P1E5 | 5.6 |
TGME49_262120 | IQ calmodulin-binding motif domain-containing protein | P1E5 | 3.3 |
TGME49_253360 | SNARE protein STX20 | P1E5 | 3.2 |
TGME49_306300 | hypothetical protein | P1E5 | 3.2 |
TGME49_257160 | hypothetical protein | P1E5 | 3.1 |
TGME49_261740 | rhoptry protein ROP47 | P1E5 | 2.8 |
TGME49_293480 | MoeA N-terminal region (domain I and II) domain-containing protein | P1F3 | 2.6 |
TGME49_250950 | KRUF family protein | P1E5 | 2.5 |
TGME49_203630 | ribosomal protein RPL44 | P1E5 | 2.4 |
TGME49_315290 | hypothetical protein | P1E5 | 2.4 |
TGME49_214410 | hypothetical protein | P1E5 | 2.3 |
TGME49_268730 | glutaredoxin 1 | P1E5 | 2.3 |
TGME49_268740 | hypothetical protein | P1E5 | 2.2 |
TGME49_234180 | hypothetical protein | P1E5 | 2.1 |
TGME49_238200 | alpha/beta hydrolase fold domain-containing protein | P2F5/P1E5 | 2/1.6 |
TGME49_219690 | hypothetical protein | P1E5 | 2.0 |
TGME49_202940 | hypothetical protein | P1E5 | 2.0 |
TGME49_251180 | KRUF family protein | P1E5/P1F3/P2F5 | 2/1.7/1.5 |
Downregulated ↓ | |||
TGME49_248200 | ribosomal RNA (adenine(1779)-N(6)/adenine(1780)-N(6))-dimethyltransferase, putative | P1E5 | 0.1 |
TGME49_209755 | cyst matrix protein MAG2 | P1E5 | 0.1 |
TGME49_253350 | hypothetical protein | P1E5 | 0.3 |
TGME49_297110 | kinesin motor domain-containing protein | P1E5 | 0.3 |
TGME49_273780 | SWI2/SNF2-containing protein | P1E5 | 0.4 |
TGME49_280570 | SAG-related sequence SRS35A | P1E5 | 0.4 |
TGME49_272900 | DNA repair protein RAD51 | P1E5 | 0.4 |
TGME49_281900 | SET domain containing lysine methyltransferase KMTox | P1E5 | 0.4 |
TGME49_247390 | ATPase, AAA family protein | P1E5 | 0.4 |
TGME49_280390 | HEAT repeat-containing protein | P1E5 | 0.5 |
TGME49_320190 | SAG-related sequence SRS16B | P1E5 | 0.5 |
TGME49_318470 | AP2 domain transcription factor AP2IV-4 | P1E5 | 0.5 |
TGME49_297870 | inner membrane complex protein IMC36 | P1E5 | 0.5 |
TGME49_214970 | DNA replication licensing factor, putative | P1E5 | 0.5 |
TGME49_301170 | SAG-related sequence SRS19D | P1E5 | 0.5 |
TGME49_244500 | Tubulin-tyrosine ligase family protein | P1E5 | 0.5 |
TGME49_290970 | serine palmitoyltransferase SPT2 | P1E5 | 0.5 |
TGME49_219700 | DNA replication licensing factor MCM4, putative | P1E5 | 0.5 |
TGME49_237220 | DNA replication licensing factor Mcm7, putative | P1E5 | 0.5 |
TGME49_262990 | hypothetical protein | P1E5 | 0.5 |
ToxoDB ORF | Product Description | C4_Clone | Fold Change (FC) |
---|---|---|---|
Upregulated ↑ | |||
TGME49_306300 | hypothetical protein | P1G10 | 7.2 |
TGME49_258700 | transporter, major facilitator family protein | P2F3 | 4.2 |
TGME49_248140 | hypothetical protein (putative transmembrane protein) | P3E2 | 4.1 |
TGME49_234980 | hypothetical protein (putative transmembrane protein) | P2F3 | 3.8 |
TGME49_277740 | zinc finger, C3HC4 type (RING finger) domain-containing protein | P2F3 | 2.8 |
TGME49_268860 | enolase 1 | P2F3 | 2.8 |
TGME49_265190 | Ulp1 protease family, C-terminal catalytic domain-containing protein | P2F3 | 2.7 |
TGME49_276120 | histone lysine methyltransferase, SET, putative | P2F3 | 2.3 |
TGME49_220170 | hypothetical protein | P2F3 | 2.2 |
TGME49_200440 | hypothetical protein | P2F3 | 2.2 |
TGME49_273060 | ribosomal protein S17, putative | P2F3 | 2.1 |
TGME49_273550 | hypothetical protein | P2F3 | 2.1 |
TGME49_293480 | MoeA N-terminal region (domain I and II) domain-containing protein | P3E2 | 2 |
TGME49_238200 | alpha/beta hydrolase fold domain-containing protein | P2F3/P1G10 | 2.01/1.9 |
TGME49_284580 | ribose-phosphate diphosphokinase subfamily protein | P2F3 | 2 |
TGME49_211470 | Fcf2 pre-rRNA processing protein | P2F3 | 2 |
TGME49_229260 | basal complex component BCC4 | P2F3 | 2 |
TGME49_226640 | zinc binding protein, putative | P2F3 | 2 |
TGME49_224932 | hypothetical protein | P2F3 | 1.9 |
TGME49_297220 | AMP-binding enzyme domain-containing protein | P2F3 | 1.9 |
Downregulated ↓ | |||
TGME49_309580 | transporter, major facilitator family protein | P2F3 | 0.1 |
TGME49_268220 | hypothetical protein | P2F3 | 0.2 |
TGME49_305870 | DAD family protein | P1G10 | 0.4 |
TGME49_305610 | hypothetical protein | P2F3 | 0.5 |
TGME49_229140 | MaoC family domain-containing protein | P1G10 | 0.6 |
TGME49_320588 | glycosyl hydrolases family 35 protein | P2F3 | 0.6 |
Annotation | C3_Clone | FC | C4_Clone | FC | |
---|---|---|---|---|---|
Upregulated C3↑ C4 ↑ | |||||
TGME49_306300 | hypothetical protein | P1E5 | 3.2 | P1G10 | 7.2 |
TGME49_258700 | transporter, major facilitator family protein | P1F3 | 6.5 | P2F3 | 4.2 |
TGME49_248140 | hypothetical protein (putative transmembrane protein) | P1E5 | 5.6 | P3E2 | 4.1 |
TGME49_293480 | MoeA N-terminal region (domain I and II) domain-containing protein | P1F3 | 2.6 | P3E2 | 2.0 |
TGME49_238200 | alpha/beta hydrolase fold domain-containing protein | P1E5/P2F5 | 1.62/2.03 | P2F3/P1G10 | 2.0/1.9 |
TGME49_211470 | Fcf2 pre-rRNA processing protein | P2F5 | 1.8 | P2F3 | 2.0 |
TGME49_297220 | AMP-binding enzyme domain-containing protein | P1E5 | 2.0 | P2F3 | 1.9 |
TGME49_277920 | hypothetical protein (putative transmembrane protein) | P1E5 | 2.0 | P2F3 | 1.5 |
TGME49_224932 | hypothetical protein (putative jmjC domain protein) | P1E5 | 1.6 | P2F3 | 1.9 |
TGME49_269660 | TFIIH basal transcription factor complex helicase XPB subunit | P1E5 | 1.6 | P2F3 | 1.5 |
TGME49_274100 | hypothetical protein | P2F5 | 1.5 | P2F3 | 1.6 |
Downregulated C3↓ C4 ↓ | |||||
TGME49_229140 | MaoC family domain-containing protein | P1F3 | 0.5 | P1G10 | 0.6 |
Divergent C3↓ C4 ↑ | |||||
TGME49_312500 | hypothetical protein | P1E5 | 0.6 | P2F3 | 1.5 |
TGME49_315590 | macro domain-containing protein | P1E5 | 0.6 | P2F3 | 1.9 |
TGME49_242720 | aspartyl protease ASP5 | P1E5 | 0.7 | P2F3 | 1.5 |
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Semeraro, M.; Boubaker, G.; Scaccaglia, M.; Müller, J.; Vigneswaran, A.; Hänggeli, K.P.A.; Amdouni, Y.; Kramer, L.H.; Vismarra, A.; Genchi, M.; et al. Transient Adaptation of Toxoplasma gondii to Exposure by Thiosemicarbazone Drugs That Target Ribosomal Proteins Is Associated with the Upregulated Expression of Tachyzoite Transmembrane Proteins and Transporters. Int. J. Mol. Sci. 2024, 25, 9067. https://doi.org/10.3390/ijms25169067
Semeraro M, Boubaker G, Scaccaglia M, Müller J, Vigneswaran A, Hänggeli KPA, Amdouni Y, Kramer LH, Vismarra A, Genchi M, et al. Transient Adaptation of Toxoplasma gondii to Exposure by Thiosemicarbazone Drugs That Target Ribosomal Proteins Is Associated with the Upregulated Expression of Tachyzoite Transmembrane Proteins and Transporters. International Journal of Molecular Sciences. 2024; 25(16):9067. https://doi.org/10.3390/ijms25169067
Chicago/Turabian StyleSemeraro, Manuela, Ghalia Boubaker, Mirco Scaccaglia, Joachim Müller, Anitha Vigneswaran, Kai Pascal Alexander Hänggeli, Yosra Amdouni, Laura Helen Kramer, Alice Vismarra, Marco Genchi, and et al. 2024. "Transient Adaptation of Toxoplasma gondii to Exposure by Thiosemicarbazone Drugs That Target Ribosomal Proteins Is Associated with the Upregulated Expression of Tachyzoite Transmembrane Proteins and Transporters" International Journal of Molecular Sciences 25, no. 16: 9067. https://doi.org/10.3390/ijms25169067