The Activity of a Developmentally Regulated Cysteine Proteinase Is Required for Cyst Wall Formation in the Primitive EukaryoteGiardia lamblia

Abstract

Giardia is an intestinal parasite that belongs to the earliest diverging branch of the eukaryotic lineage of descent. Giardia undergoes adaptation for survival outside the host's intestine by differentiating into infective cysts. Encystation involves the synthesis and transport of cyst wall constituents to the plasma membrane for release and extracellular organization. Nevertheless, little is known about the molecular events related to cyst wall biogenesis in Giardia. Among the components of the cyst wall there are two proteins that we have previously identified and characterized: CWP1 (26 kDa) and CWP2 (39 kDa). Expression of these proteins is coordinately induced, and both concentrated within encystation-specific secretory vesicles before their extracellular polymerization. Although highly similar to each other at the amino terminus, CWP2 includes a COOHterminal 121-amino acid extension. Here, we show that this extension, rich in basic residues, is cleaved from CWP2 before cyst wall formation by an intracellular cysteine proteinase activity, which is induced during encystation like CWPs. Specific inhibitors prevent release of cyst wall materials, abolishing cyst wall formation. We also report the purification, cloning, and characterization of the encystation-specific cysteine proteinase responsible for the proteolytic processing of CWP2, which is homologue to lysosomal cathepsin C. Encystation-specific cysteine proteinase ESCP possesses unique characteristics compared with cathepsins from higher eukaryotes, such as a transmembrane domain and a short cytoplasmic tail. These features make this enzyme the most divergent cathepsin C identified to date and provide new insights regarding cyst wall formation in Giardia.

Organelle Proteomics Reveals Cargo Maturation Mechanisms Associated with Golgi-like Encystation Vesicles in the Early-diverged Protozoan Giardia lamblia

“…This indicates that the compartments themselves and presumably also their cargo undergo a maturation process during which CWPs are post-translationally modified. Evidence for cargo modification was inferred from the presence of protein-disulfide isomerases in ESVs (18), cleavage of the CWP2 C terminus by an encystation-specific proteases in vitro (19), or phosphorylation of newly synthesized CWPs (20). In particular, the formation and reshuffling of intra-and intermolecular disulfide bonds is essential for establishing a cyst wall, because treatment with dithiothreitol (DTT) reversibly abolished cyst wall formation and appeared to dissolve ESVs (21).…”

The Unique Endosomal/Lysosomal System of Giardia lamblia

“…The first description of hydrolase activity in the PVs came from studies in which acid phosphatase activity was tested, showing a cytochemical localization in these vacuoles as well as in the ER and nuclear envelope cisternae (Feely and Dyer, 1987). The presence of hydrolase activities in the PVs was also proved for cysteine proteases and RNases, demonstrating their lysosomal characteristics (Lindmark, 1988;Ward et al, 1997;Touz et al, 2002b). In addition, their potential role in endocytosis was demonstrated by the uptake of exogenous ferritin and Lucifer yellow (Bockman and Winborn, 1968;Lanfredi-Rangel et al, 1998).…”

“…These PVs, distinguished by their localization, are about 150 nm in diameter with variable oval shapes and contain a core of low electron density (Figure 3). They are acidic, as shown by the uptake of acridine orange and the lysosomal markers Lyso-sensor and Lysotracker (Lanfredi-Rangel et al, 1998;Touz et al, 2002b;Touz et al, 2003). The first description of hydrolase activity in the PVs came from studies in which acid phosphatase activity was tested, showing a cytochemical localization in these vacuoles as well as in the ER and nuclear envelope cisternae (Feely and Dyer, 1987).…”

“…Although the 1-2 and 2 mRNA transcripts change little during the completion of the cell cycle (Marti et al, 2003b;Rivero et al, 2010), the role of the corresponding AP complexes appears essential for the adaptation of the parasite. AP-1 is not critical for Giardia trophozoite survival and multiplication, but it is necessary for cyst formation, acting indirectly in this process by transporting a transmembrane protein to the PVs (Touz et al, 2002b;Touz et al, 2003;Touz et al, 2004). In contrast, AP-2 is essential for Giardia growth and survival, being involved in the endocytosis of essential molecules (e.g., exogenous lipids) (Rivero et al, 2010) and in the fragmentation of ESVs into small transport vesicles containing cyst wall proteins during encystation (Rivero & Touz, unpublished).…”

“…Another characteristic of these PVs is that they contain hydrolytic activity resembling the function performed by lysosomes. These vacuoles have a high lytic capacity and a low luminal pH, both properties of mature lysosomes (Ward et al, 1997;Touz et al, 2002b).…”