The myo -inositol-1-phosphate synthase gene is essential in Trypanosoma brucei

et al. 2013

African trypanosomes are capable of both de novo synthesis and salvage of pyrimidines. The last two steps in de novo synthesis are catalysed by UMP synthase (UMPS) – a bifunctional enzyme comprising orotate phosphoribosyl transferase (OPRT) and orotidine monophosphate decarboxylase (OMPDC). To investigate the essentiality of pyrimidine biosynthesis in Trypanosoma brucei, we generated a umps double knockout (DKO) line by gene replacement. The DKO was unable to grow in pyrimidine-depleted medium in vitro, unless supplemented with uracil, uridine, deoxyuridine or UMP. DKO parasites were completely resistant to 5-fluoroorotate and hypersensitive to 5-fluorouracil, consistent with loss of UMPS, but remained sensitive to pyrazofurin indicating that, unlike mammalian cells, the primary target of pyrazofurin is not OMPDC. The null mutant was unable to infect mice indicating that salvage of host pyrimidines is insufficient to support growth. However, following prolonged culture in vitro, parasites regained virulence in mice despite retaining pyrimidine auxotrophy. Unlike the wild-type, both pyrimidine auxotrophs secreted substantial quantities of orotate, significantly higher in the virulent DKO line. We propose that this may be responsible for the recovery of virulence in mice, due to host metabolism converting orotate to uridine, thereby bypassing the loss of UMPS in the parasite.

Section: Methodsmentioning

confidence: 99%

Trypanosoma brucei (UMP synthase null mutants) are avirulent in mice, but recover virulence upon prolonged culture in vitro while retaining pyrimidine auxotrophy

Ong

Sienkiewicz

et al. 2013

“…TRYS gene replacement cassettes were generated by amplifying a region of DNA encompassing the 5′‐UTR, open reading frame (ORF) and 3′‐UTR of T. brucei TRYS from genomic DNA with primers 5′UTR‐NotI_s and 3′UTR‐NotI_as, using Pfu polymerase. This sequence was then used as a template for the amplification of the individual regions used in the assembly of replacement cassettes containing the selectable drug‐resistance genes puromycin N ‐acetyl transferase ( PAC ) and hygromycin phosphotransferase ( HYG ), exactly as previously described (Martin and Smith, 2005).…”

Section: Methodsmentioning

confidence: 99%

Dissecting the essentiality of the bifunctional trypanothione synthetase‐amidase in Trypanosoma brucei using chemical and genetic methods

Oza

Patterson

et al. 2009

The bifunctional trypanothione synthetase-amidase (TRYS) comprises two structurally distinct catalytic domains for synthesis and hydrolysis of trypanothione (N1,N8-bis(glutathionyl)spermidine). This unique dithiol plays a pivotal role in thiol-redox homeostasis and in defence against chemical and oxidative stress in trypanosomatids. A tetracycline-dependent conditional double knockout of TRYS (cDKO) was generated in bloodstream Trypanosoma brucei. Culture of cDKO parasites without tetracycline induction resulted in loss of trypanothione and accumulation of glutathione, followed by growth inhibition and cell lysis after 6 days. In the absence of inducer, cDKO cells were unable to infect mice, confirming that this enzyme is essential for virulence in vivo as well as in vitro. To establish whether both enzymatic functions were essential, an amidase-dead mutant cDKO line was generated. In the presence of inducer, this line showed decreased growth in vitro and decreased virulence in vivo, indicating that the amidase function is not absolutely required for viability. The druggability of TRYS was assessed using a potent small molecule inhibitor developed in our laboratory. Growth inhibition correlated in rank order cDKO, single KO, wild-type and overexpressing lines and produced the predicted biochemical phenotype. The synthetase function of TRYS is thus unequivocally validated as a drug target by both chemical and genetic methods.

“…6A). Using INO1 [1-D-myo-inositol-3-phosphate synthase (Martin and Smith, 2005)] as a loading control ( Fig. 6C), the corrected ratios are 1:0.6:0.…”

Section: Can Endogenous Ptr1 Compensate For Lack Of Dhfr?mentioning

confidence: 99%

Chemical and genetic validation of dihydrofolate reductase–thymidylate synthase as a drug target in African trypanosomes

Sienkiewicz

Jaroslawski

et al. 2008

103

SummaryThe phenotypes of single-(SKO) and double-knockout (DKO) lines of dihydrofolate reductase-thymidylate synthase (DHFR-TS) of bloodstream Trypanosoma brucei were evaluated in vitro and in vivo. Growth of SKO in vitro is identical to wild-type (WT) cells, whereas DKO has an absolute requirement for thymidine. Removal of thymidine from the medium triggers growth arrest in S phase, associated with gross morphological changes, followed by cell death after 60 h. DKO is unable to infect mice, whereas the virulence of SKO is similar to WT. Normal growth and virulence could be restored by transfection of DKO with T. brucei DHFR-TS, but not with Escherichia coli TS. As pteridine reductase (PTR1) levels are unchanged in SKO and DKO cells, PTR1 is not able to compensate for loss of DHFR activity. Drugs such as raltitrexed or methotrexate with structural similarity to folic acid are up to 300-fold more potent inhibitors of WT cultured in a novel low-folate medium, unlike hydrophobic antifols such as trimetrexate or pyrimethamine. DKO trypanosomes show reduced sensitivity to these inhibitors ranging from twofold for trimetrexate to >10 000-fold for raltitrexed. These data demonstrate that DHFR-TS is essential for parasite survival and represents a promising target for drug discovery.