A New Family of Giardial Cysteine-Rich Non-VSP Protein Genes and a Novel Cyst Protein

Abstract

Since the Giardia lamblia cyst wall is necessary for survival in the environment and host infection, we tested the hypothesis that it contains proteins other than the three known cyst wall proteins. Serial analysis of gene expression during growth and encystation revealed a gene, “HCNCp” (High Cysteine Non-variant Cyst protein), that was upregulated late in encystation, and that resembled the classic Giardia variable surface proteins (VSPs) that cover the trophozoite plasmalemma. HCNCp is 13.9% cysteine, with many “CxxC” tetrapeptide motifs and a transmembrane sequence near the C-terminus. However, HCNCp has multiple “CxC” motifs rarely found in VSPs, and does not localize to the trophozoite plasmalemma. Moreover, the HCNCp C-terminus differed from the canonical VSP signature. Full-length epitope-tagged HCNCp expressed under its own promoter was upregulated during encystation with highest expression in cysts, including 42 and 21 kDa C-terminal fragments. Tagged HCNCp targeted to the nuclear envelope in trophozoites, and co-localized with cyst proteins to encystation-specific secretory vesicles during encystation. HCNCp defined a novel trafficking pathway as it localized to the wall and body of cysts, while the cyst proteins were exclusively in the wall. Unlike VSPs, HCNCp is expressed in at least five giardial strains and four WB subclones expressing different VSPs. Bioinformatics identified 60 additional large high cysteine membrane proteins (HCMp) containing ≥20 CxxC/CxC's lacking the VSP-specific C-terminal CRGKA. HCMp were absent or rare in other model or parasite genomes, except for Tetrahymena thermophila with 30. MEME analysis classified the 61 gHCMp genes into nine groups with similar internal motifs. Our data suggest that HCNCp is a novel invariant cyst protein belonging to a new HCMp family that is abundant in the Giardia genome. HCNCp and the other HCMp provide a rich source for developing parasite-specific diagnostic reagents, vaccine candidates, and subjects for further research into Giardia biology.

Changes in the N -Glycome, Glycoproteins with Asn-Linked Glycans, of Giardia lamblia with Differentiation from Trophozoites to Cysts

“…These results are also consistent with the idea that secretion is less important to encysting Giardia than is development of membranous structures (8). Identification of VSPs in Giardia cysts is consistent with a previous demonstration of encystation-associated VSP-like proteins (9). Of great interest is the function of GRREAT proteins, which are very abundant in Giardia cysts and less abundant in Giardia trophozoites but have no homologs in other organisms.…”

The Transcriptional Response to Encystation Stimuli in Giardia lamblia Is Restricted to a Small Set of Genes

“…However, the intersecting data set of 13 positions contained all confirmed, highly regulated encystation genes, including CWPs and Myb. The HCNC protein identified in a previous study (5) did not appear in any of the data sets, and the three upregulated HCM protein family members are not associated with encystation according to these criteria. Genes in the intersecting data set show a comparable degree of signal increase in both protocols, with the exception of ORF 92729 (annotated as fatty acid elongase 1), which shows an induction difference of Ͼ2-fold between data sets.…”

Neogenesis and maturation of transient Golgi-like cisternae in a simple eukaryote

“…The giardial CWM appears to be of very low complexity: only three paralogous cyst wall proteins (CWP1-CWP3) and a simple β1-3 GalNAc homopolymer (Gillin et al, 1996;Jarroll et al, 2001;Lujan et al, 1995b;Sun et al, 2003) polymerize to a highly effective biological barrier on the surface of encysting cells. An epitope-tagged, membrane-anchored high cysteine non-variant cyst protein (HCNCp) (Davids et al, 2006) had been localized to the periphery of encysted cells, possibly at the interface between the plasma membrane and the cyst wall. In vitro, encystation is triggered by environmental cues, for example, low cholesterol concentration and elevated pH (Lujan et al, 1996), leading to de-repression of the genes coding for CWPs (Davis-Hayman et al, 2003).…”