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Trypanosoma brucei (UMP synthase null mutants) are avirulent in mice, but recover virulence upon prolonged culture in vitro while retaining pyrimidine auxotrophy.
Mol Microbiol. 2013 Oct; 90(2):443-55.MM

Abstract

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.

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Authors+Show Affiliations

Ong HB
Divisional of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee, DD1 5EH, UK.
Sienkiewicz N
No affiliation info available
Wyllie S
No affiliation info available
Patterson S
No affiliation info available
Fairlamb AH
No affiliation info available

MeSH

AmidesAnimalsBiological TransportCell LineDeoxyuridineFluorouracilGene Knockout TechniquesMiceMultienzyme ComplexesOrotate PhosphoribosyltransferaseOrotic AcidOrotidine-5'-Phosphate DecarboxylasePyrazolesPyrimidinesRibonucleosidesRiboseTransfectionTrypanocidal AgentsTrypanosoma brucei bruceiUracilUridineUridine MonophosphateVirulence

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

23980694

Citation

Ong, Han B., et al. "Trypanosoma Brucei (UMP Synthase Null Mutants) Are Avirulent in Mice, but Recover Virulence Upon Prolonged Culture in Vitro While Retaining Pyrimidine Auxotrophy." Molecular Microbiology, vol. 90, no. 2, 2013, pp. 443-55.
Ong HB, Sienkiewicz N, Wyllie S, et al. Trypanosoma brucei (UMP synthase null mutants) are avirulent in mice, but recover virulence upon prolonged culture in vitro while retaining pyrimidine auxotrophy. Mol Microbiol. 2013;90(2):443-55.
Ong, H. B., Sienkiewicz, N., Wyllie, S., Patterson, S., & Fairlamb, A. H. (2013). Trypanosoma brucei (UMP synthase null mutants) are avirulent in mice, but recover virulence upon prolonged culture in vitro while retaining pyrimidine auxotrophy. Molecular Microbiology, 90(2), 443-55. https://doi.org/10.1111/mmi.12376
Ong HB, et al. Trypanosoma Brucei (UMP Synthase Null Mutants) Are Avirulent in Mice, but Recover Virulence Upon Prolonged Culture in Vitro While Retaining Pyrimidine Auxotrophy. Mol Microbiol. 2013;90(2):443-55. PubMed PMID: 23980694.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Trypanosoma brucei (UMP synthase null mutants) are avirulent in mice, but recover virulence upon prolonged culture in vitro while retaining pyrimidine auxotrophy. AU - Ong,Han B, AU - Sienkiewicz,Natasha, AU - Wyllie,Susan, AU - Patterson,Stephen, AU - Fairlamb,Alan H, Y1 - 2013/09/09/ PY - 2013/08/22/accepted PY - 2013/8/29/entrez PY - 2013/8/29/pubmed PY - 2014/5/20/medline SP - 443 EP - 55 JF - Molecular microbiology JO - Mol Microbiol VL - 90 IS - 2 N2 - 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. SN - 1365-2958 UR - https://www.unboundmedicine.com/medline/citation/23980694/Trypanosoma_brucei__UMP_synthase_null_mutants__are_avirulent_in_mice_but_recover_virulence_upon_prolonged_culture_in_vitro_while_retaining_pyrimidine_auxotrophy_ L2 - https://doi.org/10.1111/mmi.12376 DB - PRIME DP - Unbound Medicine ER -