Identification of the Major Cysteine Protease of Giardia and Its Role in Encystation

Abstract

Giardia lamblia is a protozoan parasite and the earliest branching clade of eukaryota. The Giardia life cycle alternates between an asexually replicating vegetative form and an infectious cyst form. Encystation and excystation are crucial processes for the survival and transmission of Giardia. Cysteine proteases in Giardia have been implicated in proteolytic processing events that enable the continuance of the life cycle throughout encystation and excystation. Using quantitative real-time PCR, the expression of twenty-seven clan CA cysteine protease genes in the Giardia genome was measured during both vegetative growth and encystation. Giardia cysteine protease 2 was the most highly expressed cysteine protease during both life cycle stages measured, with a dramatic expression increase during encystation. The mRNA transcript for Giardia cysteine protease 2 was 7-fold up-regulated during encystation and was greater than 3-fold higher than any other Giardia protease gene product. Recombinant Giardia cysteine protease 2 was expressed, purified, and biochemically characterized. The activity of the recombinant cysteine protease 2 protein was confirmed to be identical to the dominant cysteine protease activity found in G. lamblia lysates. Giardia cysteine protease 2 was co-localized with cyst wall protein in encystation-specific vesicles during encystation and processed cyst wall protein 2 to the size found in Giardia cyst walls. These data suggest that Giardia cysteine protease 2 is not only the major cysteine endoprotease expressed in Giardia, but is also central to the encystation process.Giardia lamblia is a protozoan parasite that inhabits the upper small intestine of many vertebrate hosts and is the most commonly isolated intestinal parasite world wide (1). In addition to its medical importance, Giardia is of interest as a model cell system because it represents the most early branching clade of eukaryota (2, 3). Giardia has a simple two-stage life cycle consisting of a vegetative replicating trophozoite and an infectious cyst. Infection is initiated with cyst ingestion by a vertebrate host. After passage through the acidic host stomach, vegetative trophozoites emerge from the cyst by the process of excystation, asexually divide by binary fission, establish the duodenal infection, and give rise to the characteristic symptoms of giardiasis. Trophozoites can form infective cysts that are passed in the host feces and ingested by another host to propagate the life cycle (1).The process of encystation is a coordinated secretion of cyst wall materials to the periphery of a cell to form the cyst wall (4, 5). In response to environmental cues, trophozoites produce abundant cyst wall proteins that are packaged into encystationspecific vesicles (ESVs). 3 These vesicles grow, mature, and eventually traffic to the plasma membrane of the trophozoite, where cyst wall precursor material is secreted to form the environmentally stable cyst wall (4, 6, 7). The expression of many proteins is up-regulated during the encystation proc...

Encystation commitment inGiardia duodenalis: a long and winding road

“…Each CWP is encoded by a single copy gene which share structural features: CWF-localized CWP1 and CWP2 are ≈ 26 kDa with five LRR tandem repeats while CWP3 is 27.3 kDa and has four and a possible fifth LRR tandem repeat. CWP2 is synthesized as a 39 kDa-sized precursor that is processed by a cysteine protease releasing the distinctive 13 kDa-basic C-terminal extension (TCWP2; Touz et al, 2002;DuBois et al, 2008). All CWPs have a peptide signal sequence at the N-terminus, possibly recognized by a cognate giardial receptor (Svärd et al, 1999) that targets CWP to endoplasmic reticulum (ER), and a cysteine-rich region after the LRR tandem repeats where all 14 cysteine residues are positionally conserved (Mowatt et al, 1995;Luján et al, 1995;Sun et al, 2003).…”

“…. Post-translational modifications of cargo A critical modification of CWP2 is its processing by a cathepsin B-like cysteine protease (CP2, orf EAA 41050), the most up-regulated of 25 clan CA proteases expressed by Giardia (DuBois et al, 2008), likely releasing the 26 kDa fragment (and hence CWP complexes) to the ESV lumen. CWP2 processing would be also performed by another ESV-contained cathepsin B-like cysteine protease (orf EAA 37074; DuBois et al, 2008) but not so for the peripheral vesicle (PV)-contained cathepsin C-like encystation-specific cysteine protease (ESCP, orf EAA 36907;Touz et al, 2002).…”

“…Post-translational modifications of cargo A critical modification of CWP2 is its processing by a cathepsin B-like cysteine protease (CP2, orf EAA 41050), the most up-regulated of 25 clan CA proteases expressed by Giardia (DuBois et al, 2008), likely releasing the 26 kDa fragment (and hence CWP complexes) to the ESV lumen. CWP2 processing would be also performed by another ESV-contained cathepsin B-like cysteine protease (orf EAA 37074; DuBois et al, 2008) but not so for the peripheral vesicle (PV)-contained cathepsin C-like encystation-specific cysteine protease (ESCP, orf EAA 36907;Touz et al, 2002). Near to the plasma membrane, ESV appears to carry out two processes: a) fusion with PV (Luján & Touz, 2003) that acidifies the cargo to limit CP2 activity and/or allow ESCP activity and favor other undefined processing mechanisms preceding the release of CW material on the cell surface; or b) fission (dispersal) into small secretory vesicles that eventually fuse with the plasma membrane to discharge its content (Hehl & Marti, 2004).…”

Giardia duodenalis: New Research Developments in Pathophysiology, Pathogenesis, and Virulence Factors

“…Cathepsin-like cysteine proteases are key components of the pathogenesis of several protozoan parasitic disorders, and the Giardia genome contains genes for 23 of those proteases [94,95,104,105]. Cathepsin cysteine proteases contain an active site cysteine and histidine residue.…”

“…This reduction of CXCL8 leads to decreased neutrophil chemotaxis and was the result of secreted G. duodenalis cathepsin B-like proteases that degraded CXCL8. The G. duodenalis genome contains genes for at least 23 cathepsin cysteine proteases, the majority of which have no described function [94,95]. However, several G. duodenalis cathepsin proteases are upregulated upon exposure to intestinal epithelial cells [87,96].…”