Abstract
Trans-sialidase (TS), a virulence factor from Trypanosoma cruzi, is an enzyme playing key roles in the biology of this protozoan parasite. Absent from the mammalian host, it constitutes a potential target for the development of novel chemotherapeutic drugs, an urgent need to combat Chagas' disease. TS is involved in host cell invasion and parasite survival in the bloodstream. However, TS is also actively shed by the parasite to the bloodstream, inducing systemic effects readily detected during the acute phase of the disease, in particular, hematological alterations and triggering of immune cells apoptosis, until specific neutralizing antibodies are elicited. These antibodies constitute the only known submicromolar inhibitor of TS's catalytic activity. We now report the identification and detailed characterization of a neutralizing mouse monoclonal antibody (mAb 13G9), recognizing T. cruzi TS with high specificity and subnanomolar affinity. This mAb displays undetectable association with the T. cruzi superfamily of TS-like proteins or yet with the TS-related enzymes from Trypanosoma brucei or Trypanosoma rangeli. In immunofluorescence assays, mAb 13G9 labeled 100% of the parasites from the infective trypomastigote stage. This mAb also reduces parasite invasion of cultured cells and strongly inhibits parasite surface sialylation. The crystal structure of the mAb 13G9 antigen-binding fragment in complex with the globular region of T. cruzi TS was determined, revealing detailed molecular insights of the inhibition mechanism. Not occluding the enzyme's catalytic site, the antibody performs a subtle action by inhibiting the movement of an assisting tyrosine (Y119), whose mobility is known to play a key role in the trans-glycosidase mechanism. As an example of enzymatic inhibition involving non-catalytic residues that occupy sites distal from the substrate-binding pocket, this first near atomic characterization of a high affinity inhibitory molecule for TS provides a rational framework for novel strategies in the design of chemotherapeutic compounds.
Highlights
Chagas’ disease, the American trypanosomiasis, is a chronic disabling parasitic disease caused by the flagellate protozoon Trypanosoma cruzi
We report a complete characterization of this neutralizing monoclonal antibody, at the functional and molecular levels
Hybridomas were screened by TS-inhibition assay [30] and the 13G9 clone secreting a TSneutralizing mAb (IgG2ak) was obtained. The specificity of this mAb was confirmed by the absence of reactivity against the closely related sialidase from Trypanosoma rangeli and the TS from Trypanosoma brucei
Summary
Chagas’ disease, the American trypanosomiasis, is a chronic disabling parasitic disease caused by the flagellate protozoon Trypanosoma cruzi. With an estimated global burden of 100 million people at risk, 8 million already infected, and approximately 40,000 new cases/year, Chagas’ disease represents a major health and economic problem in Latin America [1]. Patients can transmit the disease either by in utero infection leading to the congenitally acquired disease or by accidental transmission through contaminated blood. The acute infection is characterized by patent parasite burden. During this initial stage, T. cruzi induces several alterations in the infected mammal including intense polyclonal activation of lymphocytes [2], transient thymic aplasia [3,4] and other clinical hematological findings [5,6]. Up to 20 years after the infection, ,35% of patients develop different pathologies, such as cardiomyopathy, peripheral nervous system damage, and/or dysfunction of the digestive tract [1]
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