Table 1.
Essentiality of Toxoplasma gondii metabolic mutants based on nutrient availability and media contenta
| Inhibited gene of interest | Methodology described in | Culturing conditions | Derived parasite strain | Host cell line | Culture media | Serum type and concentration | Incubation temperature | Incubation CO2 concentration | Antibiotics and concentration | Supplement and concentration | Essentiality |
|---|---|---|---|---|---|---|---|---|---|---|---|
| TgPYK1 and TgPYK2 | Xia et al. (2019) [10] Shen et al. (2017) [82] Roos et al. (1995) [83] |
Control | RH | HFF | MEM | 1% heat-inactivated FBS | 37°C | 5% | – | – | PYK2 becomes essential where PYK1 is inhibited |
| TgPYK1 and TgPYK2 | Xia et al. (2019) [10] Shen et al. (2017) [82] Roos et al. (1995) [83] |
High-nutrient conditions | RH | HFF | MEM | 1% heat-inactivated FBS | 37°C | 5% | – | 8 mM lactate + 8 mM glutamine, or 4500 mg/l glucose + 8 mM alanine | Not essential |
| TgBCKDH-E1a | Oppenheim et al. (2014) [19] | Propagation | RH | HFF | DMEM | 5% FCS | 37°C | 5% | 25 μg/l gentamicin | 2 mM glutamine | – |
| TgBCKDH-E1a | Oppenheim et al. (2014) [19] | Control | RH | HFF | DMEM 41966 | 5% FCS | 37°C | 5% | 25 μg/ml gentamicin | 6 mM glutamine | Not essential. Reduced growth rate and smaller plaques observed |
| TgBCKDH-E1a | Oppenheim et al. (2014) [19] | Glucose depleted | RH | HFF | DMEM 11960 | 5% FCS | 37°C | 5% | 25 μg/ml gentamicin | 6 mM glutamine | Not essential. Severely reduced growth rate |
| TgBCKDH-E1a | Oppenheim et al. (2014) [19] | Glutamine depleted | RH | HFF | DMEM 11966 | 5% FCS | 37°C | 5% | 25 μg/ml gentamicin | – | Not essential. Reduced growth rate |
| TgBCKDH-E1a | Oppenheim et al. (2014) [19] | Glucose depleted and high acetate | RH | HFF | DMEM 11960 | 5% FCS | 37°C | 5% | 25 μg/l gentamicin | 6 mM glutamine and 5 mM acetate | Not essential. Partially restored growth rate |
| TgGAD | MacRae et al. (2012) [20] | Control | RH | HFF or hTERT-BJ1 | DMEM | 5% FBS | 37°C | 5% | – | – | Not essential |
| TgGAD | MacRae et al. (2012) [20] | Control | PRU | HFF or hTERT-BJ1 | DMEM | 10% FBS | 37°C | 5% | – | – | Not essential |
| TgELO-A, B, or C | Ramakrishnan et al. (2012) [28] Striepen and Soldati (2007) [84] |
Control | RH | HFF | DMEM | 1% FCS | 37°C | 5% | 1:200 of 10 000 units/l penicillin/ streptomycin | 1:100 of 200 mM stock glutamine | Not essential. Effect of variable FBS not determined |
| TgDEH | Ramakrishnan et al. (2015) [32] Striepen and Soldati (2007) [84] |
Control | RH | HFF | DMEM | 1% FCS | 37°C | 5% | 1:200 of 10 000 units/l penicillin/ streptomycin | 1:100 of 200 mM stock glutamine | Essential. Complete growth arrest after 5 days. Effect of variable FBS not determined |
| TgDEH | Ramakrishnan, et al. (2015) [32] Striepen and Soldati (2007) [84] |
High-nutrient conditions | RH | HFF | DMEM | 1% FCS | 37°C | 5% | 1:200 of 10 000 units/l penicillin/ streptomycin | 1:100 of 200 mM stock glutamine and 250 μM mixture of [C18:1, C20:1, C22:1, and C24:1], or 250 μM of C20:1, or 250 μM of C22:1 | Not essential when supplemented with unsaturated fatty acid mix |
| TgLIPIN | Dass et al. (2021) [38] | Control | RH | HFF | DMEM | 0%/1%/10% FBS (escalating lipotoxicity) | 37°C | 5% | 25 μg/ml gentamicin | 2 mM glutamine | Essential in 10% FBS, but more tachyzoite growth in reduced FBS |
| TgNPT1 (TgApiAT1) | Rajendran et al. (2017) [8] | Control | RH | HFF | RPMI 1640 | 1% FBS | 37°C | 5% | Unspecified antibiotics | – | Essential in DMEM but not RPMI due to lack of amino acid availability |
| TgNPT1 (TgApiAT1) | Parker et al. (2019) [68] | Nutrient deprived | RH | HFF | MAAM | 1% FBS | 37°C | 5% | Unspecified antibiotics | – | Not essential. Reduced growth rate |
| TgApiAT5-3 | Parker et al. (2019) [68] | Control | RH | HFF | DMEM | 1% FCS | 37°C | 5% | Unspecified antibiotics | – | Not essential. Reduced growth rate and smaller plaques observed |
| TgApiAT5-3 | Parker et al. (2019) [68] | Nutrient deprived | RH | HFF | MAAM | 1% FCS | 37°C | 5% | Unspecified antibiotics | – | Essential |
| TgApiAT5-3 | Parker et al. (2019) [68] | High-nutrient conditions | RH | HFF | MAAM | 1% FCS | 37°C | 5% | Unspecified antibiotics | 2.5 mM tyrosine | Not essential |
| TgApiAT6-1 | Fairweather et al. (2021) [9] | Control | RH | HFF | RPMI 1640 | 1% FCS | 37°C | 5% | 50 U/ml penicillin, 50 μg/ml streptomycin, 10 μg/ml gentamicin, and 0.25 μg/ml amphotericin B | 2 mM glutamine | Essential. No rescue possible with arginine or lysine supplementation |
| TgFabD | Liang et al. (2020) [43] Shen et al. (2017) [82] Roos et al. (1995) [83] |
Control | RH | HFF | MEM | 1% heat-inactivated FBS | 37°C | 5% | – | – | Not essential. Reduced growth rate and smaller plaques observed |
| TgFabD | Liang et al. (2020) [43] | Isotope labelling | RH | HFF | Glucose free DMEM | 1% heat-inactivated FBS | 37°C | 5% | – | 8 mM glucose | Not essential in 10% FBS. Growth in variable FBS not determined |
| TgATS1 | Amiar et al. (2020) [6] | High glucose | RH | HFF | DMEM 41965-062 |
1% FBS/10% FBS (complement) | 37°C | 5% | – | 1 mM glutamine | Increased viability with high FBS |
| TgATS2 | Amiar et al. (2020) [6] | High glucose | RH | HFF | DMEM 41965-062 |
1% FBS/10% FBS (complement) | 37°C | 5% | – | 1 mM glutamine | Slightly reduced growth in low FBS |
| TgACP | Ramakrishnan et al. (2012) [28] | Control | RH | HFF | DMEM | 1% FCS | 37°C | 5% | – | – | Essential. Growth in variable FBS not determined |
| TgASPV | Amiar et al. (2020) [6] | High glucose | RH | HFF | DMEM 41965-062 |
1%/10% FBS (complement) | 37°C | 5% | – | 1 mM glutamine | Reduced plaques in 0% FBS, growth defect is exacerbated in reduced FBS |
| TgMYR1 | Amiar et al. (2020) [6] | High glucose | RH | HFF | DMEM 41965-062 |
1%/10% FBS (complement) | 37°C | 5% | – | 1 mM glutamine | Reduced growth but consistent between 0, 1, and 10% FBS. Likely lack of host rewiring leads to less nutritious host cell |
| TgACBP1 and TgSCP2 | Amiar et al. (2020) [6] | High glucose | RH | HFF | DMEM 41965-062 | 1%/10% FBS (complement) | 37°C | 5% | – | 1 mM glutamine | ACBP1 and SCP2 not essential in any FBS condition. Normal growth |
| TgACBP1 and TgSCP2 | Fu et al. (2019) [85] | Control | RH | HFF | DMEM | 1% FBS | 37°C | 5% | 10 units/ml penicillin, and 100 mg/ml streptomycin | – | ACBP1 and SCP2 not essential in 1% FBS. Moderately reduced growth rate |
| TgACBP2 | Amiar et al. (2020) [6] | High glucose | RH and PRU | HFF | DMEM 41965-062 | 1%/10% FBS (complement) | 37°C | 5% | – | 1 mM glutamine | Not essential in 0, 1, or 10% FBS |
| TgACBP2 | Fu et al. (2019) [85] | Control | RH and PRU | HFF | DMEM | 1% FBS | 37°C | 5% | 10 units/ml penicillin, and 100 mg/ml streptomycin | – | Not essential in 1% FBS in RH Type I tachyzoites. Important for growth in Type II. Difference is apparently strain specific |
| TgACS | Amiar et al. (2020) [6] | High glucose | RH | HFF | DMEM 41965-062 | 1%/10% FBS (complement) | 37°C | 5% | – | 1 mM glutamine | Not essential in 0, 1, or 10% FBS |
| TgACL and TgACS | Kloehn et al. (2020) [16] | Control | RH | HFF | DMEM | 5% FCS | 37°C | 5% | 25 μg/ml gentamicin | 2 mM glutamine | Individually dispensable, essential together. Effect of high-nutrient conditions not determined |
a
Abbreviations: ACBP, acyl-coenzyme A binding protein; ACL, ATP citrate lyase; ACP, acyl carrier protein; ACS, acetyl-coenzyme A synthetase; ApiAT, apicomplexan amino acid transporter; ASP, aspartyl protease; ATS, apicoplast glycerol-3phosphate acyltransferase; BCKDH-E1a, branched-chain ketoacid dehydrogenase E1a subunit; DEH, hydroxyacyl-coenzyme A dehydratase; ELO, elongase; DMEM, Dulbecco’s Modified Eagle’s Medium; FBS, fetal bovine serum; FCS, fetal calf serum; GAD, glutamate decarboxylase; HFF, human foreskin fibroblast; hTERT-BJ1, human telomerase reverse transcriptase-immortalized BJ fibroblasts; MAAM, minimal amino acid medium; MEM, modified Eagles medium; MYR, Myc regulation; NPT, novel putative transporter; PYK, pyruvate kinase; RPMI, Roswell Park Memorial Institute; SCP, sterol carrier protein.