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. © 2013 loureiro et al.