Alpha-2-macroglobulin binds to the surface of Trypanosoma cruzi.
Trypanosoma cruzi, the causative agent of Chagas disease, infects vertebrate cells after an initial step of parasite/host-cell recognition. Alpha-2-macroglobulin (A2M), an important type of physiological proteinase inhibitor found in tissues and in the plasma of mammals, inhibits cell invasion by T. cruzi and accumulates in sites of the inflamed myocardium associated with parasite antigens. To study whether A2M would bind to T. cruzi, an indirect immunofluorescence reaction was performed using two different anti-mouse A2M sera. Intense labeling was observed in the membrane lining the cell body and the flagellum of bloodstream trypomastigotes obtained from experimentally infected mice in the peak of parasitemia, suggesting that the antisera recognize plasma A2M associated with the parasite surface. Metacyclic trypomastigotes obtained in a serum-free defined medium reacted with anti-A2M only after previous incubation with purified human A2M. Enzyme-linked immunosorbent assay (ELISA) studies were applied to characterize better the binding of native (N-A2M) and of proteinase-complexed (P-A2M) forms of A2M. The "in vitro" binding of N-A2M to trypomastigotes was better at pH 5.0, followed by pH 10.0 and pH 7.4. Cysteinly and serine proteinase inhibitors, E-64 and STI, respectively, inhibited the reaction. P-A2M also bound to T. cruzi in a dose-dependent way. Flow-cytometry studies showed that about 80% of the parasites stained with fluorescein isothiocyanate (FITC)-labeled P-A2M (50 micrograms/ml) with high affinity at pH 7.4 (but also at pH 10.0) in a process that was reverted by the addition of unlabeled P-A2M or the calcium-chelator agent EDTA and also by incubation at an acid pH (4.0). These results suggest that (a) native-A2M binds to T. cruzi proteinase(s) and (b) T. cruzi expresses a receptor(s) that binds proteinase-complexed A2M.
Coutinho
C M
CM
Depto. Ultra-estrutura e Biologia Celular, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Brasil.
Cavalcanti
G H
GH
van Leuven
F
F
Araújo-Jorge
T C
TC
D000818:Animals; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate; D019084:Fluorescent Antibody Technique, Indirect; D006790:Host-Parasite Interactions; D006801:Humans; D007106:Immune Sera; D051379:Mice; D011485:Protein Binding; D014349:Trypanosoma cruzi / Q000502:physiology*; D000511:alpha-Macroglobulins / Q000378:metabolism*
D007106:Immune Sera; D000511:alpha-Macroglobulins; D016650:Fluorescein-5-isothiocyanate