GIARDIA ADAPTOR PROTEIN 2 POSSESSES A CRITICAL ROLE DURING CYST WALL FORMATION

RIVERO R.; VRANYCH C; MIRAS S; ROPOLO A; TOUZ C

Santa Fe, Santa Fe, Argentina

Congreso; XXIII Reunión Científica Anual Sociedad Argentina de Protozoología; 2009

Sociedad Argentina de Protozoología

The differentiation of Giardia lamblia into the cyst form allows the parasite to survive in an infavorable environment, thereby increasing the likelihood of the infection of a new host. During this process, cyst wall proteins (CWPs) are synthesized and transported into encystations-specific secretory vesicles (ESVs) that migrate towards the periphery of the cell releasing their content to form the rigid cyst wall. How this discharge ocurred is still unclear or controversial. We found that at the cell periphery, the ESV establishes contact the peripheral vesicles (PVs), acidic organelles that correspond to an endosome lysosome system, and observed that an exchange of material migth occur between both organelles. Previously, we showed that the heterotetrameric clathrin-adaptor protein AP2 complex localizes in the PVs, cytoplasm, and plasma membrane in growing trophozoites. However, during encystations, we observed that the PVs containing gAP2 enfold the ESVs. To better detail the participation of AP2 during this process, we analyzed the localization of the medium subunit of gAP2 (gu2) together with the ESVs formation and CWP1 release by IFA, confocal miscroscopy, and inmunoelectro-microscopy of encysting trophozoites. We found that during encystation gu2 was decorating the ESVs membrane in patches with the subsequently dispersal of ESV into small secretory vesicles containing CWP1 and gu2 before secretion. At the end of this stage, gu2 was present at the plasma membrane remaining on the inner side of it, just where the CWPs were released to form the filamentous cyst wall. Conversely, formation of these small vesicles as well as the development of the cyst wall was inhibited when gu2 was knocked down. In this sense, quantitative analysis of cyst by FACS showed that the silencing of gu2 drastically reduced the number of cyst. Even though gu2 expression does not increase during encystation, our findings support the hypothesis where CWPs leave sorting PVs in gu2 compartments by segregation into discrete domains that can separate in small vesicles and then fuse with the plasma membrane in an atypical exocytic event. Protein trafficking during encystation needs to be explored in detail for a better understanding of the molecular bases involved in surviving and dissemination of this important human parasite.