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

Kelly N Dubois,Marla Abodeely,Judy Sakanari,Charles S Craik,Malinda Lee,James H Mckerrow,Mohammed Sajid

doi:10.1074/jbc.m802133200

Kelly N Dubois, Marla Abodeely + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m802133200

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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.

Highlights

IntroductionTrophozoites can form infective cysts that are passed in the host feces and ingested by another host to propagate the life cycle [1]
Grant AI35707 from the Tropical Disease Research Unit
There Are Twenty-seven Clan CA Cysteine Proteases in the Genome of Giardia lamblia—Prior to the completion of the Giardia genome, only four cysteine proteases from Giardia had been identified [10, 11]. Three of these genes encode cathepsin B-like cysteine proteases, the fourth a cathepsin C-like protease. Using these papain family enzymes as a query, the Giardia genome was mined for additional genes coding for cysteine proteases

Summary

Introduction

Trophozoites can form infective cysts that are passed in the host feces and ingested by another host to propagate the life cycle [1]. In response to environmental cues, trophozoites produce abundant cyst wall proteins that are packaged into encystationspecific vesicles (ESVs).. 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 [8, 9]. To address the question of which gene product(s) was responsible for key events in the life cycle, such as cyst wall processing, we analyzed the transcription levels of all twenty-

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