Genetic and chemical analyses reveal that trypanothione synthetase but not glutathionylspermidine synthetase is essential for Leishmania infantum.
Trypanothione is a unique and essential redox metabolite of trypanosomatid parasites, the biosynthetic pathway of which is regarded as a promising target for antiparasitic drugs. Synthesis of trypanothione occurs by the consecutive conjugation of two glutathione molecules to spermidine. Both reaction steps are catalyzed by trypanothione synthetase (TRYS), a molecule known to be essential in Trypanosoma brucei. However, other trypanosomatids (including some Leishmania species and Trypanosoma cruzi) potentially express one additional enzyme, glutathionylspermidine synthetase (GSPS), capable of driving the first step of trypanothione synthesis yielding glutathionylspermidine. Because this monothiol can substitute for trypanothione in some reactions, the possibility existed that TRYS was redundant in parasites harboring GSPS. To clarify this issue, the functional relevance of both GSPS and TRYS was investigated in Leishmania infantum (Li). Employing a gene-targeting approach, we generated a gsps(-/-) knockout line, which was viable and capable of replicating in both life cycle stages of the parasite, thus demonstrating the superfluous role of LiGSPS. In contrast, elimination of both LiTRYS alleles was not possible unless parasites were previously complemented with an episomal copy of the gene. Retention of extrachromosomal LiTRYS in the trys(-/-)/+TRYS line after several passages in culture further supported the essentiality of this gene for survival of L. infantum (including its clinically relevant stage), hence ruling out the hypothesis of functional complementation by LiGSPS. Chemical targeting of LiTRYS with a drug-like compound was shown to also lead to parasite death. Overall, this study disqualifies GSPS as a target for drug development campaigns and, by genetic and chemical evidence, validates TRYS as a chemotherapeutic target in a parasite endowed with GSPS and, thus, probably along the entire trypanosomatid lineage.
IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, 4150-180 Porto, Portugal.
André F
AF
Sousa
IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, 4150-180 Porto, Portugal.
Ana G
AG
Gomes-Alves
Laboratory of Redox Biology of Trypanosomes, Institut Pasteur de Montevideo, CP 11400 Montevideo, Uruguay.
Diego
D
Benítez
Laboratory of Redox Biology of Trypanosomes, Institut Pasteur de Montevideo, CP 11400 Montevideo, Uruguay.
Marcelo A
MA
Comini
Departamento de Bioquímica, Universidad de la República, CP 11800 Montevideo, Uruguay, and Department of Molecular Medicine, Università degli Studi di Padova, 35131 Padova, Italy.
Leopold
L
Flohé
German Center for Infection Research, 38124 Braunschweig, Germany.
Timo
T
Jaeger
IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, 4150-180 Porto, Portugal.
Joana
J
Passos
Schirm GmbH, D-39218 Schönebeck, Germany.
Friedrich
F
Stuhlmann
IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, 4150-180 Porto, Portugal; ICBAS - Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, 4099-003 Porto, Portugal.
Ana M
AM
Tomás
IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, 4150-180 Porto, Portugal. Electronic address: hcastro@ibmc.up.pt.
Helena
H
Castro
D019732:Amide Synthases / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics*; D000818:Animals; D000981:Antiprotozoal Agents / Q000494:pharmacology*; D055786:Gene Knockout Techniques; D005978:Glutathione / Q000031:analogs & derivatives / Q000096:biosynthesis / Q000737:chemistry; D018314:Leishmania infantum / Q000201:enzymology* / Q000235:genetics; D007898:Leishmaniasis, Visceral / Q000188:drug therapy; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D013095:Spermidine / Q000031:analogs & derivatives / Q000096:biosynthesis / Q000737:chemistry
D000981:Antiprotozoal Agents; C012333:glutathionylspermidine; C044809:trypanothione; D019732:Amide Synthases; C081536:glutathionylspermidine synthetase; C064407:trypanothione synthetase; D005978:Glutathione; D013095:Spermidine
Free radicals; Glutathionylspermidine; Leishmania; Target validation; Trypanosoma; Trypanothione