The basic extension of cyst wall protein 2 and sintaxin I are essential for secretory granule biogenesis and release during Giardia encystation

NATALIA GOTTIG; MARIA JIMENA NORES; MARIA CAROLINA TOUZ; HUGO DANIEL LUJÁN

Mérida - Mexico

Congreso; Meeting of International Research Scholars; 2005

Giardia is an intestinal parasitic protozoan of humans. Trophozoites undergo fundamental changes to survive outside the intestine by differentiating into cysts. Encystation entails the synthesis, processing, transport, secretion and assembly of cyst wall components. Among those molecules are two closely related proteins that localize within encystation-specific secretory vesicles (ESVs) in encysting trophozoites and in the cyst wall of mature cysts. These CWP genes predict proteins of 26 (CWP1), 39 (CWP2), and 27 (CWP3) kDa, having 61% identity in a 26 kDa overlapping region. CWP2 differs from CWP1 and CWP3 by a 121-residue carboxy-terminal extension rich in basic amino acids. We previously demonstrated that this tail is cleaved before cyst wall assembly by a cysteine protease induced during encystation. In this work we determined the role of the alkaline extension of CWP2 and identify and characterize SNARE proteins involved in Giardia encystation. CWP constructs with or without the CWP2 basic tail were transfected into trophozoites and two types mammalian cells, those with and without endogenous secretory granules. Our results indicate that the CWP2 basic tail is necessary but not sufficient to induce the biogenesis of ESVs in non-encysting trophozoites and that there must exist a receptor for this tail in Giardia since no granules were generated in mammalian cells. By expression of HA-tagged molecules, antisense silencing, and yeast two-hybrid analysis of seven Giardia SNAREs, we found that these proteins localize to different subcellular compartments, are essential for parasite viability, and show specific SNARE-SNARE interactions. From these SNAREs, only Syntaxin 1 is involved in encystation. These results provide new insights into the minimal machinery for protein trafficking utilized by Giardia during differentiation into cyst.