Giardia lamblia (also called Giardia intestinalis) is one of the most common intestinal parasites of humans. To evade the host's immune response, Giardia undergoes antigenic variation-a process that allows the parasite to develop chronic and recurrent infections. From a repertoire of approximately 190 variant-specific surface protein (VSP)-coding genes, Giardia expresses only one VSP on the surface of each parasite at a particular time, but spontaneously switches to a different VSP by unknown mechanisms. Here we show that regulation of VSP expression involves a system comprising RNA-dependent RNA polymerase, Dicer and Argonaute, known components of the RNA interference machinery. Clones expressing a single surface antigen efficiently transcribe several VSP genes but only accumulate transcripts encoding the VSP to be expressed. Detection of antisense RNAs corresponding to the silenced VSP genes and small RNAs from the silenced but not for the expressed vsp implicate the RNA interference pathway in antigenic variation. Remarkably, silencing of Dicer and RNA-dependent RNA polymerase leads to a change from single to multiple VSP expression in individual parasites.

The protozoan Giardia lamblia is an obligate parasite of the mammalian small intestine. We studied the expression of a gene that encodes a protein component of the cyst wall, a complex structure assembled during the differentiation of trophozoites to cysts and which is critical to survival of the parasite outside its mammalian host. Transcripts from the cyst wall protein gene increase more than 100-fold during encystation, reaching a maximum between 5 and 24 hours after induction. Cyst wall protein expression also increases dramatically during encystation, and, prior to its incorporation into the nascent cyst wall, the protein is contained within the encystation-specific vesicles of encysting trophozoites. The sequence of the cloned gene predicts an acidic, leucine-rich polypeptide of M(r) 26,000 that contains 5.3 tandemly arranged copies of a degenerate 24-amino-acid repeat. A hydrophobic amino-terminal peptide probably serves as the initial signal that targets this protein to a secretory pathway involving vesicular localization during encystation and, ultimately, secretion to form the cyst wall.

Giardia lamblia trophozoites, like most intestinal parasitic protozoa, undergo fundamental biological changes to survive outside the intestine of their mammalian host by differentiating into infective cysts. This complex process entails the coordinated production, processing, and transport of cyst wall constituents for assembly into a protective cyst wall. Yet, little is known about this process and the identity of cyst wall constituents. We previously identified a 26-kDa cyst wall protein, CWP1. In the present work, using monoclonal antibodies to cyst wall antigens, we cloned the gene that encodes a novel 39-kDa cyst wall protein, CWP2. Expression of CWP1 and CWP2 was induced during encystation with identical kinetics. Soon after synthesis, these two proteins combine to form a stable complex, which is concentrated within the encystation-specific secretory granules before incorporation into the cyst wall. Both proteins contain five tandem copies of a 24-residue leucine-rich repeat, a motif implicated in protein-protein interactions. Unlike CWP1, CWP2 has an extremely basic 121-residue COOH-terminal extension that might be involved in the sorting of these proteins to the secretory granules.

A fundamental characteristic of eukaryotic cells is the presence of membrane-bound compartments and membrane transport pathways in which the Golgi complex plays a central role in the selective processing, sorting, and secretion of proteins. The parasitic protozoan Giardia lamblia belongs to the earliest identified lineage among eukaryotes and therefore offers unique insight into the progression from primitive to more complex eukaryotic cells. Here, we report that Giardia trophozoites undergo a developmental induction of Golgi enzyme activities, which correlates with the appearance of a morphologically identifiable Golgi complex, as they differentiate to cysts. Prior to this induction, no morphologically or biochemically identifiable Golgi complex exists within nonencysting cells. Remarkably, protein secretion in both nonencysting and encysting trophozoites is inhibited by brefeldin A, and brefeldin A-sensitive membrane association of ADP-ribosylation factor and beta-COP is observed. These results suggest that the secretory machinery of Giardia resembles that of higher eukaryotes despite the absence of a Golgi complex in nonencysting trophozoites. These findings have implications both for defining the minimal machinery for protein secretion in eukaryotes and for examining the biogenesis of Golgi structure and function.