Knockdown of Asparagine Synthetase A Renders Trypanosoma brucei Auxotrophic to Asparagine

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 Mg2+. 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. Author Summary The amino acid asparagine is important not only for protein biosynthesis, but also for nitrogen homeostasis. Asparagine synthetase catalyzes the synthesis of this amino acid. There are two forms of asparagine synthetase, A and B. The presence of type A in trypanosomes, and its absence in humans, makes this protein a potential drug target. Trypanosomes are responsible for serious parasitic diseases that rely on limited drug therapeutic options for control. In our study we present a functional characterization of trypanosomes asparagine synthetase A. We describe that Trypanosoma brucei and Trypanosoma cruzi type A enzymes are able to use either ammonia or glutamine as a nitrogen donor, within the conversion of aspartate into asparagine. Furthermore, we show that asparagine synthetase A knockdown renders Trypanosoma brucei auxotrophic to asparagine. Overall, this study demonstrates that interfering with asparagine metabolism represents a way to control parasite growth and infectivity.