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Cell invasion by Trypanosoma cruzi amastigotes of distinct infectivities: studies on signaling pathways
Adriana B Fernandes1, Ivan Neira, Alice T Ferreira
1Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de São Paulo-UNIFESP, Escola Paulista de Medicina, Rua Botucatu, 862 6th Floor, 04023-062, São Paulo, Brazil.
Abstract:
Trypanosoma cruzi metacyclic trypomastigotes of the major phylogenetic lineages use specific signaling pathways to invade host cells. Using a panel of drugs, we studied if the differences in the ability of extracellular amastigotes (EA) from G (T. cruzi I) and CL (T. cruzi II) strains to invade host cells could be associated to activation of specific signaling routes. Sonicated extracts from G or CL strain EA induced transient raises in HeLa cell intracellular Ca(2+) levels in a dose-dependent manner. Treatment of EA with drugs that affect Ca(2+) release from inositol-1,4,5-triphosphate-sensitive stores did not significantly affect the infectivity of either strain, whereas EA of both strains treated with ionomycin plus NH(4)Cl or nigericin that release Ca(2+) from acidocalcisomes had their infectivity reduced. Treatment of parasites with adenylate cyclase activator forskolin increased the infectivity of both strains towards HeLa cells. These data, taken together, suggest that, for host cell invasion, G and CL strain EA engage signaling pathways that lead to an increase of cyclic adenosine monophosphate and Ca(2+) mobilization from acidocalcisomes. Moreover, treatment of EA with genistein reduced by approximately 45% the invasion of HeLa cells by G but not by CL strain, implicating a protein tyrosine kinase in the process. In line with this, HeLa cell extracts contained a protein tyrosine kinase activity that mediated the phosphorylation of 87- and 175-kDa polypeptides of EA from G but not from CL strain. Regarding the target cell response, the activation of host PI3 kinase appears to be required for invasion by either strain as treatment of HeLa cells with wortmannin reduced EA infectivity. These data overall reinforce the concept that cell invasion by T. cruzi EA markedly differs from the process involving metacyclic trypomastigotes.
Insights
Extracellular amastigotes (EA) of Trypanosoma cruzi strains G and CL invade host cells via distinct signaling pathways. Invasion involves cyclic adenosine monophosphate, calcium mobilization from acidocalcisomes, and host PI3 kinase activation.
Area of Science:
- * Parasitology
- * Cell Biology
- * Molecular Biology
Background:
- * Trypanosoma cruzi, the causative agent of Chagas disease, exhibits distinct phylogenetic lineages (e.g., T. cruzi I and T. cruzi II).
- * Metacyclic trypomastigotes utilize specific signaling pathways for host cell invasion.
- * Extracellular amastigotes (EA) represent another infective stage with potentially different invasion mechanisms.
Purpose of the Study:
- * To investigate the signaling pathways utilized by extracellular amastigotes (EA) from different T. cruzi strains (G - T. cruzi I, CL - T. cruzi II) for host cell invasion.
- * To compare the invasion mechanisms of EA with those of metacyclic trypomastigotes.
- * To identify key molecular players involved in EA-mediated host cell invasion.
Main Methods:
- * Treatment of EA with various drugs targeting calcium signaling (inositol-1,4,5-triphosphate-sensitive stores, acidocalcisomes), cyclic adenosine monophosphate (forskolin), and protein tyrosine kinases (genistein).
- * Assessment of parasite infectivity using HeLa cells as host cells.
- * Analysis of host cell signaling pathways, including PI3 kinase activation (wortmannin treatment).
- * Detection of protein tyrosine kinase activity in host cell extracts and its effect on parasite proteins.
Main Results:
- * Sonicated EA extracts from both G and CL strains induced dose-dependent increases in HeLa cell intracellular calcium (Ca2+).
- * Infectivity of EA was reduced by drugs releasing Ca2+ from acidocalcisomes (ionomycin, NH4Cl, nigericin), but not by drugs affecting inositol-1,4,5-triphosphate stores.
- * Forskolin (adenylate cyclase activator) increased the infectivity of both strains.
- * Genistein (protein tyrosine kinase inhibitor) reduced invasion by G strain EA but not CL strain EA, suggesting strain-specific tyrosine kinase involvement.
- * Host cell PI3 kinase activation was essential for invasion by both strains, as shown by wortmannin treatment.
- * Host cell extracts exhibited tyrosine kinase activity phosphorylating specific parasite proteins in the G strain EA.
Conclusions:
- * Extracellular amastigotes (EA) of T. cruzi G and CL strains utilize distinct signaling pathways for host cell invasion, involving increased cyclic adenosine monophosphate and Ca2+ mobilization from acidocalcisomes.
- * Protein tyrosine kinase activity plays a role in the invasion process, potentially differing between T. cruzi strains.
- * Host cell PI3 kinase activation is a conserved requirement for invasion by both EA strains.
- * The invasion mechanism of T. cruzi EA differs significantly from that of metacyclic trypomastigotes.
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