In-solution behavior and protective potential of asparagine synthetase A from Trypanosoma cruzi

Highlights

• Recombinant Tc-AS-A is dimeric and stable in acidic environments.

• Our structural inspection showed that Tc-AS-A has the same fold and quaternary arrangement as prokaryotic AS-A.

• Immunization with Tc-AS-A confers protection to infective forms of T. cruzi.

• Tc-AS-A immunization hampered red blood cell lysis in early infection.

• Tc-AS-A immunization reduced the number of platelets and leukocytes.

Abstract

l-Asparagine synthetase (AS) acts in asparagine formation and can be classified into two families: AS-A or AS-B. AS-A is mainly found in prokaryotes and can synthetize asparagine from ammonia. Distinct from other eukaryotes, Trypanosoma cruzi produces an AS-A. AS-A from Trypanosoma cruzi (Tc-AS-A) differs from prokaryotic AS-A due to its ability to catalyze asparagine synthesis using both glutamine and ammonia as nitrogen sources. Regarding these peculiarities, this work uses several biophysical techniques to provide data concerning the Tc-AS-A in-solution behavior. Tc-AS-A was produced as a recombinant and purified by three chromatography steps. Circular dichroism, dynamic light scattering, and analytical size exclusion chromatography showed that Tc-AS-A has the same fold and quaternary arrangement of prokaryotic AS-A. Despite the tendency of protein to aggregate, stable dimers were obtained when solubilization occurred at pH ≤ 7.0. We also demonstrate the protective efficacy against T. cruzi infection in mice immunized with Tc-AS-A. Our results indicate that immunization with Tc-AS-A might confer partial protection to infective forms of T. cruzi in this particular model.

Introduction

l-Asparagine synthetase (EC 6.3.1.1) catalyzes the synthesis of l-asparagine from l-aspartate and ammonia upon ATP hydrolysis to AMP and PPi [1,2]. Enzymatic classification divides these enzymes into two families: asparagine synthetase A (AS-A) or asparagine synthetase B (AS-B), with AS-A being found in prokaryotes [[3], [4], [5], [6]] and AS-B being found in eukaryotes [[7], [8], [9], [10]]. Distinct from the majority of eukaryotes, Leishmania and Trypanosoma possess both AS-A and AS-B [11]. Moreover, while AS-A of other organisms are described as strictly ammonia dependent, the AS-A of Trypanosoma cruzi (Tc-AS-A, GenBank XP_812804), a causative agent of Chagas disease, was reported to use glutamine as a nitrogen donor [11]. The ability of using both glutamine and ammonia as nitrogen donors was only previously demonstrated for AS-B enzymes [[12], [13], [14], [15], [16]].

In Latin America, the infection of approximately 8 million people with the neglected Chagas disease placed this region among the endemic areas, where the mortality rate is approximately 10,000 people/year. In addition, more than 25 million people are at risk of infection [17]. Population movement spreads the disease from Latin America to other continents. In the United States of America, it is estimated that approximately 300,000 resident immigrants are infected by T. cruzi [18].

Currently, treatment is most effective in the acute phase of the disease, chronic infections of children under 12 years, congenital infections and laboratory accidents [19]. In addition to not being effective for all patients, drugs also have many side effects, such as hypersensitivity reactions and neuropathies. The treatment is not suitable for pregnant women; patients with systemic infections or heart, respiratory, renal or hepatic failure; untreatable homeopathies and neoplasms; or elderly or very debilitated people [19,20]. Thus, advances in Chagas treatment have been expected for a while.

Tc-AS-A has features that define a good target such as its presence in the parasite but not in the host and its unique biochemical behavior. However, it is indispensable to understand the enzyme functioning to define a better strategy to affect parasite viability through Tc-AS-A. This is especially due to the apparent parasite’s ability to grow using asparagine from the extracellular environment when the levels of AS-A are minimum [11]. Recent work with Trypanosoma brucei AS-A (Tb-AS-A) showed that only a combined therapy using both a Tb-AS-A inhibitor and an extracellular asparagine depletor (e.g., l-asparaginase) or an asparagine transport blocker could inhibit parasite growth [11]. However, a combined therapy could be too expensive and logistically inappropriate for the treatment of African trypanosomiasis [11]. However, whether the same profile would be necessary for the treatment of Chagas disease remains to be established.

In this work, Tc-AS-A was produced as recombinant and purified, and its structure was evaluated by circular dichroism, dynamic light scattering and analytical size exclusion chromatography. Our results indicate that α-β contents were preserved in recombinant expression and that the overall charge of the neutralizing enzyme might be important for its stability and quaternary arrangement. Moreover, Tc-AS-A is a dimer in solution similar to other AS-As. We also analyzed the protective effect of Tc-AS-A immunization. BALB/c mice were immunized with formulations containing Tc-AS-A and later infected with T. cruzi trypomastigotes. Our results indicate that immunization with Tc-AS-A might confer partial protection to infective forms of T. cruzi in this particular model.