Giardia adaptor protein 2 possesses a critical role during cyst wall formation.

RIVERO MR; VRANYCH CV; MIRAS S; ROPOLO AS; TOUZ MC

Santa Fe.

Jornada; XXIII Reunión Científica Anual de la Sociedad Argentina de Protozoología.; 2009

Revista Médica de Rosario. ISSN 0327-5019. Vol 75, pag 30.

The differentiation of Giardia into the cyst form allows the parasite to survive in an unfavorable environment, thereby increasing the likelihood of the infection of a new host. In fact, the mechanism by which Giardia traffics essential proteins to Pheripheral Vacuoles, (PVs) as well as regulates their secretion has important implications in the control of the parasite dissemination and disease. We have already described the participation of the heterotetrameric clathrin-adaptor protein AP2 complex in lysosomal protein trafficking, primarily during endocytosis. This complex localize in the PVs, cytoplasm, and plasma membrane in growing trophozoites. Targeted disruption of the gene encoding gµ2 (the media subunit of AP2) in growing and encysting trophozoites, results in a major decrease in the cell growth and differentiation suggesting a distinct mechanism in which AP2 is directly involved in both endocytosis and vesicular trafficking. In order to further analyze the role of gAP2 during encystation, µ2 expressing or not cells were analyzed by IFA using anti-CWP1 mAb (that detects Cyst Wall Protein 1 in mature cysts), microscopy, and flow cytometry. IFA and microscopical analysis of cyst revealed almost a complete absence of cysts in µ2-not-expressing cells compared with µ2-expressing ones. Moreover, quantitative analysis of cyst by FACS showed that the silencing of gm2 drastically reduced the number of cysts. Immunoelecto-microscopy of encysting trophozoites showed that the PVs containing gAP2 enfolds the Encystation Secretory Vesicles (ESVs), where an interchange of material between both organelles could occurs. Later during encystation, gAP2 was present to the plasma membrane remaining on the inner side of it, just where the CWPs were released to form the filamentous cyst wall. To better detail the participation of AP2 during this process, we analyzed the localization of gm2 together with the ESVs formation and CWPs release by IFA and confocal microscopy. Even though gm2 expression does not increase during encystation, our findings support the hypothesis that gAP2 is important in Giardia survival being involved in the transport of cyst wall proteins to the surface for cyst development. Nevertheless, the restricted localization of gAP2 may also be important in the endocytosis of cyst wall molecules during membrane recycling after release. Protein trafficking during encystation needs to be explored in detail to a better understanding of the molecular bases involved in surviving and dissemination of this important human parasite.