Generation and characterization of monoclonal antibodies specific to Giardia lamblia lysosome-like organelles

FELIZIANI C; MERINO MC; TOUZ MC; ROPOLO AS

Santa Fe

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

Giardia lamblia is an early-diverging eukaryote that lacks a typical endosomal/lysosomal system. Instead, it possesses peripheral vacuoles (PVs) that function as both endosomes and lysosomes and are located underneath the plasma membrane of the trophozoites. In this work, we developed monoclonal antibodies (mAbs) directed against purified PVs, to provide a unique set of tools for understanding the assembly and function of these particular organelles. PVs purified by cell-fractionation were used as immunogen to subcutaneously immunize BALB/c mice.  The mice producing PV positive pAb were further euthanized and the spleen cells fused to the NSO myeloma cell line. Antibody-secreting hybridomas were screened with indirect immunofluorescence and dot-blotting using non-encysting trophozoites. Monoclonal antibodies against PVs, endoplasmic reticulum, ventral disc, and variant-specific surface proteins, were obtained. The development of mAbs against proteins from other organelles might be explained by their presence in the same PV-purified fractions. Also, there might be proteins that are sorted to the PVs at certain point in time for processing or degradation. Immunoblotting after cloning showed that each anti-PV mAb was able to recognize one particular protein. Further analysis utilizing proteomic strategies will allow us to characterize each protein in the context of their role in lysosome trafficking and function. Our studies, utilizing monoclonal antibodies directed against the PVs, might allow understanding the distinctive function of these unique organelles and whether G. lamblia has many of the endomembrane protein transport elements and sorting functions of higher cells. Giardia appears as a great system to study not only the evolution of the secretory pathway in eukaryotes but also “why” and “how” an organism may have lost some essential subcellular organelles when becoming a parasite, while maintaining the minimal machinery necessary for fundamental cellular functions.