Hugo D. Luján

Antigenic variation in Giardia lamblia is regulated by RNA interference

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 ∼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.

DEVELOPMENTALLY REGULATED EXPRESSION OF A GIARDIA LAMBLIA CYST WALL PROTEIN GENE

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 Mr, 26000 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.

INCREASED EXPRESSION OF THE MOLECULAR CHAPERONE BIP/GRP78 DURING THE DIFFERENTIATION OF A PRIMITIVE EUKARYOTE

Summary— Giardia lamblia, a major cause of intestinal disease worldwide, is a parasitic protozoan that represents the earliest branch of the eukaryotic lineage. Trophozoites, which possess two nuclei but lack mitochondria, peroxisomes and a typical Golgi apparatus, colonize the small intestine of the vertebrate host where they may differentiate into infective cysts. Encystation is a regulated process characterized by the biosynthesis, secretion and formation of a protective extracellular cyst wall. In previous studies, we demonstrated the biogenesis of the Golgi apparatus during encystation and identified two leucine‐rich proteins (CWPs), which localize within encystation‐specific secretory granules before their incorporation into the cyst wall. Here, we used immunological, biochemical and molecular biological approaches to analyze the expression of BiP/GRP78, an endoplasmic reticulum (ER)‐resident chaperone, during the Giardia life cycle. A monoclonal antibody specific for Giardia BiP permitted the visualization of the ER of this protozoan and showed that BiP expression increased simultaneously with the increased expression of CWPs during encystation. However, in contrast to the 140‐fold increase in levels of CWP transcripts, the steady‐state level of BiP mRNA did not increase during encystation. Furthermore, potent inducers of BiP expression in higher eukaryotic cells, including agents that perturb the ER environment, did not affect BiP expression in Giardia. These results, when considered together with the profound changes that occur in the secretory pathway during Giardia encystation, indicate an important role for this molecular chaperone during the differentiation of this primitive eukaryote.

Isoprenylation of proteins in the protozoan Giardia lamblia.

We report the ability of Giardia lamblia to modify several of its cellular proteins by isoprenylation. Trophozoites cultured in the presence of [3H]mevalonate synthesized radiolabeled proteins of approx. 50 and 21-26 kDa. Chemical analysis indicated that farnesyl and geranylgeranyl isoprenoids comprised the majority of the radiolabel covalently associated with trophozoite proteins. In addition, antibodies to human p21ras immunoprecipitated mevalonate-labelled species of approx. 21 kDa. Inhibitors of several enzymatic steps of the mevalonate pathway dramatically affected Giardia metabolism. Protein isoprenylation and cell growth were blocked by compactin and mevinolin, competitive inhibitors of HMG-CoA reductase, the rate-limiting enzyme in isoprenoid biosynthesis. In the presence of these inhibitors, Giardia growth was restored by the addition of mevalonate to the culture medium. In contrast, cell growth was blocked irreversibly by inhibitors of subsequent steps in the protein isoprenylation pathway. Trophozoite growth inhibition by limonene, perillic acid, perillyl alcohol and N-acetyl-S-farnesyl-L-cysteine was not reversed after the addition of mevalonate, dolichol, ubiquinone or cholesterol to the medium. These observations constitute the first description of protein isoprenylation in any protozoan and indicate that this post-translational modification is an important step in the regulation of the growth of this primitive eukaryote.

Active and passive mechanisms drive secretory granule biogenesis during differentiation of the intestinal parasite Giardia lamblia.

The parasitic protozoan Giardia lamblia undergoes important changes to survive outside the intestine of its host by differentiating into infective cysts. During encystation, three cyst wall proteins (CWPs) are specifically expressed and concentrated within encystation-specific secretory vesicles (ESVs). ESVs are electron-dense secretory granules that transport CWPs before exocytosis and extracellular polymerization into a rigid cyst wall. Because secretory granules form at the trans-Golgi in higher eukaryotes and because Giardia lacks an identifiable Golgi apparatus, the aim of this work was to investigate the molecular basis of secretory granule formation in Giardia by examining the role of CWPs in this process. Although CWP1, CWP2, and CWP3 are structurally similar in their 26-kDa leucine-rich overlapping region, CWP2 is distinguished by the presence of a 13-kDa C-terminal basic extension. In non-encysting trophozoites, expression of different CWP chimeras showed that the CWP2 basic extension is necessary for biogenesis of ESVs, which occurs in a compartment derived from the endoplasmic reticulum. Nevertheless, the CWP2 basic extension per se is insufficient to trigger ESV formation, indicating that other domains in CWPs are also required. We found that CWP2 is a key regulator of ESV formation by acting as an aggregation factor for CWP1 and CWP3 through interactions mediated by its conserved region. CWP2 also acts as a ligand for sorting via its C-terminal basic extension. These findings show that granule biogenesis requires complex interactions among granule components and membrane receptors.

Lipid Requirements and Lipid Uptake by Giardia lamblia Trophozoites in Culture

To better understand the lipid requirements of Giardia lamblia trophozoites and the mechanisms of lipid uptake, we supplemented serum‐free TYI‐S‐33 medium with lipids incorporated into different lipid carriers. We found that serum lipoproteins, β‐cyclodextrins, and bile salts are able to supply cholesterol and phospholipids to Giardia and to support the multiplication of the parasite in vitro. The growth rates obtained with different lipoproteins or bile salts and lipid mixtures were similar to that in standard culture medium containing serum. Pulse labelling experiments using fluorescent lipid analogs demonstrated that Giardia can take up lipids from lipoproteins, β‐cyclodextrins, or bile salt micelles, but with different kinetics, and that bile salts greatly facilitated lipid transfer from lipoproteins and cyclodextrins to the parasite surface. The binding of different radioiodinated lipoprotein classes to the trophozoite surface, inhibition of lipoprotein interiorization at 4°C or by cytochalasin D, and incorporation studies using fluorescent LDL suggested that a small component of lipid uptake by trophozoites was likely due to endocytosis of lipoproteins.

Characterization of SNAREs Determines the Absence of a Typical Golgi Apparatus in the Ancient Eukaryote Giardia lamblia

Giardia is a eukaryotic protozoal parasite with unusual characteristics, such as the absence of a morphologically evident Golgi apparatus. Although both constitutive and regulated pathways for protein secretion are evident in Giardia, little is known about the mechanisms involved in vesicular docking and fusion. In higher eukaryotes, soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) of the vesicle-associated membrane protein and syntaxin families play essential roles in these processes. In this work we identified and characterized genes for 17 SNAREs in Giardia to define the minimal set of subcellular organelles present during growth and encystation, in particular the presence or not of a Golgi apparatus. Expression and localization of all Giardia SNAREs demonstrate their presence in distinct subcellular compartments, which may represent the extent of the endomembrane system in eukaryotes. Remarkably, Giardia SNAREs, homologous to Golgi SNAREs from other organisms, do not allow the detection of a typical Golgi apparatus in either proliferating or differentiating trophozoites. However, some features of the Golgi, such as the packaging and sorting function, seem to be performed by the endoplasmic reticulum and/or the nuclear envelope. Moreover, depletion of individual genes demonstrated that several SNAREs are essential for viability, whereas others are dispensable. Thus, Giardia requires a smaller number of SNAREs compared with other eukaryotes to accomplish all of the vesicle trafficking events that are critical for the growth and differentiation of this important human pathogen.

The uptake and metabolism of cysteine by Giardia lamblia trophozoites

ABSTRACT. The cysteine, cystine, methionine and sulfate uptake and cysteine metabolism of Giardia lamblia was studied. Initial experiments indicated that bathocuproine sulphonate (20 μM) added to Keisters modified TYI‐S‐33 medium supported the growth of G. lamblia at low L‐cysteine concentration. This allowed the use of high specific activity radiolabeled L‐cysteine for further studies. The analyses of L‐cysteine uptake by G. lamblia indicate the presence of at least two different transport systems. The total cysteine uptake was non saturable, with a capacity of 3.7 pmoles per 106 cells per min per μM of cysteine, and probably represent passive diffusion. However, cysteine transport was partially inhibited by L‐methionine, D‐cysteine and DL‐homocysteine. indicating that another system specific for SH‐containing amino acids is also present. Cysteine uptake was markedly decreased in medium without serum. In contrast to cysteine, the uptake of L‐methionine and sulfate were carried out by saiurable systems with apparent Km, of 71 and 72 μM, respectively, but the Vmax of the uptake of sulfate was six orders of magnitude lower than the Vmax of methionine uptake. Cystine was not incorporated into trophozoites. [35S]‐labeled L‐cysteine and L‐methionine, but not [35S]sulfate, were incorporated into Giardia proteins, indicating that the parasite lacks the capacity to synthesize cysteine or methionine from sulfate. Neither cystathionine γ lyase nor crystathionine γ synthase activities was detected in homogenates of Giardia lamblia, suggesting that the transsulfuration pathway is not active and there is no conversion of methionine to cysteine. Our data indicate that cysteine is essential for Giardia because the parasite: a) cannot take up cystine, and b) cannot synthesize cysteine de novo.

Regulation of Antigenic Variation in Giardia lamblia

Antigenic variation, a clonal phenotypic variation developed by microorganisms, involves the permanent switching of homologous, antigenically different cell surface molecules. In pathogenic microorganisms, antigenic variation is often described as a mechanism to evade the host immune system and therefore is responsible for the generation of chronic and/or recurrent infections. However, antigenic variation has also been involved in expanding host diversity and differential courses of the diseases. The intestinal protozoan parasite Giardia lamblia undergoes antigenic variation through the continuous exchange of approximately 200 variant-specific surface proteins. Here we review the principal issues regarding the significance of antigenic variation during Giardia infections, the particular features of the variant-specific surface proteins, and the current knowledge on the mechanisms that regulate this process, as well as the relevance of disrupting antigenic variation as a novel approach to design effective antiparasitic vaccines.

Functional characterization of methionine sulfoxide reductase A from Trypanosoma spp.

Methionine is an amino acid susceptible to being oxidized to methionine sulfoxide (MetSO). The reduction of MetSO to methionine is catalyzed by methionine sulfoxide reductase (MSR), an enzyme present in almost all organisms. In trypanosomatids, the study of antioxidant systems has been mainly focused on the involvement of trypanothione, a specific redox component in these organisms. However, no information is available concerning their mechanisms for repairing oxidized proteins, which would be relevant for the survival of these pathogens in the various stages of their life cycle. We report the molecular cloning of three genes encoding a putative A-type MSR in trypanosomatids. The genes were expressed in Escherichia coli, and the corresponding recombinant proteins were purified and functionally characterized. The enzymes were specific for L-Met(S)SO reduction, using Trypanosoma cruzi tryparedoxin I as the reducing substrate. Each enzyme migrated in electrophoresis with a particular profile reflecting the differences they exhibit in superficial charge. The in vivo presence of the enzymes was evidenced by immunological detection in replicative stages of T. cruzi and Trypanosoma brucei. The results support the occurrence of a metabolic pathway in Trypanosoma spp. involved in the critical function of repairing oxidized macromolecules.