Kelly N. DuBois Marla Abodeely Judy Sakanari Charles S. Craik Malinda Lee James H. McKerrow Mohammed Sajid
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 aninfectious 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 Giardiagenome 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 proteasegene 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 andprocessed 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 parasiteworld wide. 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. 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, vegetativetrophozoites 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.
The process of encystation is a coordinated secretion of cyst wallmaterials to the periphery of a cell to form the cyst wall. In response to environmental cues, trophozoites produce abundant cyst wall proteins that are packaged into encystation-specific vesicles (ESVs).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. The expressionof many proteins is up-regulated during the encystation process.
Cysteine proteases have been found to be essential to the life cycles of several parasitic organisms, catalyzing diverse processes such as parasite immunevasion, tissue invasion, and encystment/excystment in addition to well established roles in protein processing and catabolism. Indeed, in G. lamblia indispensable roles forcysteine proteases have been documented in the processes of encystation and excystation. Ward et al. validated a role for cysteine endoprotease activity in the excystation process by demonstrating that excystation was inhibited by the addition of small molecule cysteine protease inhibitors to the excystation media. Touz et al. implicated a cysteine exoprotease in the process of encystation....