Characterization of the pre, pro and CTE domains of the cysteine proteinase 2 (Lpcys2) of Leishmania pifanoi: paper in lysosomal targeting and infection
Aim of the present work was to study characteristics of the pre, pro and CTE domains of the cysteine proteinase 2 (Lpcys2) of L. pifanoi and the role of these domains in lysosomal targeting and infection. One of our objectives was to identify proteins that might interact with the targeting domain of Lpcys2, and for that we initially used the yeast two hybrid system approach. We obtained no positive interactions. We interpreted this result as the possible recognition of the Leishmania targeting signal by yeast. To investigate this possibility we made various constructs with an expression vector of yeast expressing the pre, pro and prepro Lpcys2 domains, fused to GFP. Results obtained showed the various construct directing GFP to the vacuole, while the plasmid alone produced fluorescence in the cytoplasm. Fluorescence observed with GFP fused to the signal peptide indicates that the mere entrance into the secretory pathway of yeast was sufficient to take GFP to the vacuole. Vacuolar targeting observed in yeast transformed with the construction containing the pro-domain, probably occurred through autophagy or the cytoplasm-to-vacuole transport Cvt. The recognition of the lysosomal targeting signal of Leishmania was also tested in mammalian cells. Chinese Hamster Ovary (CHO) cells were transformed with an expression plasmid containing GFP fused to the pre-pro signal. Fluorescence was observed in the Golgi complex, as confirmed by colocalization using anti-GM-130 marker. These results demonstrate that Lpcys2 signal peptide is recognized by the translocation machinery of CHO cells. On the other hand, contrary to yeast and Leishmania, the pro signal was not recognized by mammalian cells, causing the accumulation of the reporter protein in the Golgi complex. To follow up on our studies of Lpcys2 targeting and on the role of the CTE in infection, recombinant peptides and antibodies were produced against the two domains. Using the anti-pro antibody, we saw that the zymogen of Lpcys2 has different isoforms, is preferentially expressed in amastigotes, as seen by immunofluorescence, electron-microscopy and western blot. Using antibodies against the three domains, we saw that Lpcys2 is processed. We also saw that L. amazonenses has a cysteine proteinase with high level of homology to Lpcys2. Since no results of interaction were obtained with the yeast two hybrid system, we used other approaches to detect proteins that might interact with the pro signal. Using ligand blot and cross-linking, we saw interaction with some proteins, both in promastigotes as amastigotes. Using immunoprecipitation, it was possible to identify some candidate proteins from amastigotes, as a P4-nuclease, Gliceraldehyde-3-dehydrogenase, histone H4, F1 subunit amp;#945;-ATPase, and Lpcys2. Using electron microscopy and immunolocalization, we observed a localization of CTE in megasomes and flagellar pocket of axenic amastigotes. In amastigotes infecting macrophages, the CTE localized in the flagellar pocket and parasite surface when in contact with the phagosome. We verified a role for the CTE in macrophage infection by Leishmania. In infection inhibition assays, amastigotes pre-incubated with the anti-CTE antibody were less infective than control parasites incubated with other sera.
Advisor:Yara Maria Traub-Cseko
School:Faculdades Oswaldo Cruz
Source Type:Master's Thesis
Keywords:Leishmania pifanoi Cisteína Proteinase Cysteine proteinasew C-terminal Domain Infection
Date of Publication:11/10/2006