title: Knockdown of asparagine synthetase A renders Trypanosoma brucei auxotrophic to asparagine.
abstract: Asparagine synthetase (AS) catalyzes the ATP-dependent conversion of aspartate into asparagine using ammonia or glutamine as nitrogen source. There are two distinct types of AS, asparagine synthetase A (AS-A), known as strictly ammonia-dependent, and asparagine synthetase B (AS-B), which can use either ammonia or glutamine. The absence of AS-A in humans, and its presence in trypanosomes, suggested AS-A as a potential drug target that deserved further investigation. We report the presence of functional AS-A in Trypanosoma cruzi (TcAS-A) and Trypanosoma brucei (TbAS-A): the purified enzymes convert L-aspartate into L-asparagine in the presence of ATP, ammonia and Mg(2+). TcAS-A and TbAS-A use preferentially ammonia as a nitrogen donor, but surprisingly, can also use glutamine, a characteristic so far never described for any AS-A. TbAS-A knockdown by RNAi didn't affect in vitro growth of bloodstream forms of the parasite. However, growth was significantly impaired when TbAS-A knockdown parasites were cultured in medium with reduced levels of asparagine. As expected, mice infections with induced and non-induced T. brucei RNAi clones were similar to those from wild-type parasites. However, when induced T. brucei RNAi clones were injected in mice undergoing asparaginase treatment, which depletes blood asparagine, the mice exhibited lower parasitemia and a prolonged survival in comparison to similarly-treated mice infected with control parasites. Our results show that TbAS-A can be important under in vivo conditions when asparagine is limiting, but is unlikely to be suitable as a drug target.
authors: Parasite Disease Group, Instituto de Biologia Molecular e Celular da Universidade do Porto, Porto, Portugal.
authors: Inês
authors: I
authors: Loureiro
authors: Joana
authors: J
authors: Faria
authors: Christine
authors: C
authors: Clayton
authors: Sandra Macedo
authors: SM
authors: Ribeiro
authors: Nilanjan
authors: N
authors: Roy
authors: Nuno
authors: N
authors: Santarém
authors: Joana
authors: J
authors: Tavares
authors: Anabela
authors: A
authors: Cordeiro-da-Silva
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000641:Ammonia / Q000378:metabolism; D000818:Animals; D001215:Asparaginase / Q000008:administration & dosage / Q000378:metabolism; D001216:Asparagine / Q000378:metabolism*; D001217:Aspartate-Ammonia Ligase / Q000037:antagonists & inhibitors* / Q000235:genetics; D001224:Aspartic Acid / Q000378:metabolism; D004195:Disease Models, Animal; D055785:Gene Knockdown Techniques; D008274:Magnesium / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D018512:Parasitemia / Q000469:parasitology; D016019:Survival Analysis; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D014349:Trypanosoma cruzi / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D014353:Trypanosomiasis, African / Q000469:parasitology
chemical_list: D001224:Aspartic Acid; D001216:Asparagine; D000641:Ammonia; D000255:Adenosine Triphosphate; D001215:Asparaginase; D001217:Aspartate-Ammonia Ligase; D008274:Magnesium