A Novel Pax-like Protein Involved in Transcriptional Activation of Cyst Wall Protein Genes in Giardia lamblia

Giardia lamblia differentiates into infectious cysts to survive outside of the host. It is of interest to identify factors involved in up-regulation of cyst wall proteins (CWPs) during this differentiation. Pax proteins are important regulators of development and cell differentiation in Drosophila and vertebrates. No member of this gene family has been reported to date in yeast, plants, or protozoan parasites. We have identified a pax-like gene (pax1) encoding a putative paired domain in the G. lamblia genome. Epitope-tagged Pax1 localized to nuclei during both vegetative growth and encystation. Recombinant Pax1 specifically bound to the AT-rich initiator elements of the encystation-induced cwp1 to -3 and myb2 genes. Interestingly, overexpression of Pax1 increased cwp1 to -3 and myb2 gene expression and cyst formation. Deletion of the C-terminal paired domain or mutation of the basic amino acids of the paired domain resulted in a decrease of the transactivation function of Pax1. Our results indicate that the Pax family has been conserved during evolution, and Pax1 could up-regulate the key encystation-induced genes to regulate differentiation of the protozoan eukaryote, G. lamblia.     

Protein trafficking in Giardia lamblia

Giardia, a protozoan parasite of humans and other vertebrates, is a common cause of intestinal disease worldwide. Besides its medical importance, Giardia is considered an excellent system to study the evolution of fundamental cellular processes because it belongs to the earliest branches of the eukaryotic lineage of descent. Giardia trophozoites lack organelles typical of higher eukaryotes such mitochondria, peroxisomes and compartments involved in intracellular protein trafficking and secretion, such as the Golgi apparatus and secretory granules. Nevertheless, the minimal machinery for protein transport and sorting is present in this parasite. When Giardia undergoes encystation, the biogenesis of secretory organelles necessary to transport cyst wall constituents to the cell surface takes place. Recent studies in both vegetative and encysting trophozoites have provided interesting information regarding the secretory pathway of this important human pathogen.     

Effect of Chlorine on Giardia lamblia Cyst Viability

The effect of chlorine concentration on Giardia lamblia cyst viability was tested under a variety of conditions. The ability of Giardia cysts to undergo excystation was used as the criterion of viability. The experimental variables employed included temperature (25, 15, and 5°C), pH (6, 7, and 8), chlorine-cyst contact time (10, 30, and 60 min), and chlorine concentration (1 to 8 mg/liter). In the pH range studied, cyst survival generally was observed to increase as buffer pH increased. Water temperature coupled with chlorination proved to be important in cyst survival. Results of these experiments at the three temperatures studied can be summarized as follows: at 25°C, exposure to 1.5 mg/liter for 10 min killed all cysts at pH 6, 7, and 8. At 15°C, 2.5 mg of chlorine per liter for 10 min killed all cysts at pH 6, but at pH 7 and 8 small numbers of cysts remained viable after 30 min but not after 60 min. At 5°C, 1 mg of chlorine per liter for 60 min failed to kill all the cysts at any pH tested. At this temperature, 2 mg of chlorine per liter killed all cysts after 60 min at pH 6 and 7, but not at pH 8. A chlorine concentration of 4 mg/liter killed all the cysts at all three pH values after 60 min, but not after 30 min. A chlorine concentration of 8 mg/liter killed all Giardia cysts at pH 6 and 7 after contact for 10 min, and at pH 8 after 30 min. This study points up the role of temperature, pH, and chlorine demand in the halogen treatment of drinking water to destroy cysts. It also raises an epidemiological problem, namely: low water temperatures, where killing of Giardia requires relatively high chlorine concentrations and long contact times, are (i) to be expected in many areas where epidemic waterborne giardiasis has been reported and (ii) particularly conducive to the long-term survival of Giardia cysts.     

Vacuolar Protein Sorting Receptor in Giardia lamblia

In Giardia, lysosome-like peripheral vacuoles (PVs) need to specifically coordinate their endosomal and lysosomal functions to be able to successfully perform endocytosis, protein degradation and protein delivery, but how cargo, ligands and molecular components generate specific routes to the PVs remains poorly understood. Recently, we found that delivering membrane Cathepsin C and the soluble acid phosphatase (AcPh) to the PVs is adaptin (AP1)-dependent. However, the receptor that links AcPh and AP1 was never described. We have studied protein-binding to AcPh by using H6-tagged AcPh, and found that a membrane protein interacted with AcPh. This protein, named GlVps (for Giardia lamblia Vacuolar protein sorting), mainly localized to the ER-nuclear envelope and in some PVs, probably functioning as the sorting receptor for AcPh. The tyrosine-binding motif found in the C-terminal cytoplasmic tail domain of GlVps was essential for its exit from the endoplasmic reticulum and transport to the vacuoles, with this motif being necessary for the interaction with the medium subunit of AP1. Thus, the mechanism by which soluble proteins, such as AcPh, reach the peripheral vacuoles in Giardia appears to be very similar to the mechanism of lysosomal protein-sorting in more evolved eukaryotic cells.     

Protein SUMOylation is Involved in Cell‐cycle Progression and Cell Morphology in Giardia lamblia

The unicellular protozoa Giardia lamblia is a food‐ and waterborne parasite that causes giardiasis. This illness is manifested as acute and self‐limited diarrhea and can evolve to long‐term complications. Successful establishment of infection by Giardia trophozoites requires adhesion to host cells and colonization of the small intestine, where parasites multiply by mitotic division. The tight binding of trophozoites to host cells occurs by means of the ventral adhesive disc, a spiral array of microtubules and associated proteins such as giardins. In this work we show that knock down of the Small Ubiquitin‐like MOdifier (SUMO) results in less adhesive trophzoites, decreased cell proliferation and deep morphological alterations, including at the ventral disc. Consistent with the reduced proliferation, SUMO knocked‐down trophozoites were arrested in G1 and in S phases of the cell cycle. Mass spectrometry analysis of anti‐SUMO immunoprecipitates was performed to identify SUMO substrates possibly involved in these events. Among the identified SUMOylation targets, α‐tubulin was further validated by Western blot and confirmed to be a SUMO target in Giardia trophozoites.     

Rab11 and Actin Cytoskeleton Participate in Giardia lamblia Encystation,Guiding the Specific Vesicles to the Cyst Wall

Background

Giardia passes through two stages during its life cycle, the trophozoite and the cyst. Cyst formation involves the synthesis of cyst wall proteins (CWPs) and the transport of CWPs into encystation-specific vesicles (ESVs). Active vesicular trafficking is essential for encystation, but the molecular machinery driving vesicular trafficking remains unknown. The Rab proteins are involved in the targeting of vesicles to several intracellular compartments through their association with cytoskeletal motor proteins.

Methodology and Principal Findings

In this study, we found a relationship between Rab11 and the actin cytoskeleton in CWP1 transport. Confocal microscopy showed Rab11 was distributed throughout the entire trophozoite, while in cysts it was translocated to the periphery of the cell, where it colocalized with ESVs and microfilaments. Encystation was also accompanied by changes in rab11 mRNA expression. To evaluate the role of microfilaments in encystation, the cells were treated with latrunculin A. Scanning electron microscopy showed this treatment resulted in morphological damages to encysted parasites. The intensity of fluorescence-labeled Rab11 and CWP1 in ESVs and cyst walls was reduced, and rab11 and cwp1 mRNA levels were down-regulated. Furthermore, knocking down Rab11 with a hammerhead ribozyme resulted in an up to 80% down-regulation of rab11 mRNA. Although this knockdown did not appear lethal for trophozoites and did not affect cwp1 expression during the encystation, confocal images showed CWP1 was redistributed throughout the cytosol.

Conclusions and Significance

Our results indicate that Rab11 participates in the early and late encystation stages by regulating CWP1 localization and the actin-mediated transport of ESVs towards the periphery. In addition, alterations in the dynamics of actin affected rab11 and cwp1 expression. Our results provide new information about the molecules involved in Giardia encystation and suggest that Rab11 and actin may be useful as novel pharmacological targets.     

A new set of vesicles in Giardia lamblia

In the present study it was demonstrated the existence of a new set of membrane-bounded vesicles in Giardia lamblia. They were found in dividing and non-dividing trophozoites studied by routine transmission electron microscopy, freeze-fracture and Thiéry's technique. Encysting cells were not studied. These vesicles appear different to the previously reported components of the Giardia endomembranous system, such as the endoplasmic reticulum (ER), lysosome-like peripheral vesicles (PV), and the encystation-specific vesicles (ESV) that appear during trophozoite differentiation into cysts. They measure 100-150 nm in diameter, and thus are smaller than the peripheral vesicles, and the encystation-specific vesicles (ESV). They were found in clusters, scattered throughout the cytoplasm, but preferentially located close to the nuclei, axonemes, median bodies, and ER profiles. These internal vesicles are roughly spherical, and their contents present different electron densities and are more electrondense than those of the peripheral vesicles. They appeared to be budding from the outer nuclear membrane envelope. These cytoplasmic vesicles were found only in cells with very good fixation. Only few cells in the same preparation exhibited these vesicles.     

Novel protein-disulfide isomerases from the early-diverging protist Giardia lamblia.

Protein-disulfide isomerase is essential for formation and reshuffling of disulfide bonds during nascent protein folding in the endoplasmic reticulum. The two thioredoxin-like active sites catalyze a variety of thiol-disulfide exchange reactions. We have characterized three novel protein-disulfide isomerases from the primitive eukaryote Giardia lamblia. Unlike other protein-disulfide isomerases, the giardial enzymes have only one active site. The active-site sequence motif in the giardial proteins (CGHC) is characteristic of eukaryotic protein-disulfide isomerases, and not other members of the thioredoxin superfamily that have one active site, such as thioredoxin and Dsb proteins from Gram-negative bacteria. The three giardial proteins have very different amino acid sequences and molecular masses (26, 50, and 13 kDa). All three enzymes were capable of rearranging disulfide bonds, and giardial protein-disulfide isomerase-2 also displayed oxidant and reductant activities. Surprisingly, the three giardial proteins also had Ca(2+)-dependent transglutaminase activity. This is the first report of protein-disulfide isomerases with a single active site that have diverse roles in protein cross-linking. This study may provide clues to the evolution of key functions of the endoplasmic reticulum in eukaryotic cells, protein disulfide formation, and isomerization.     

Analysis of a Repeat-Containing Family of Giardia lamblia Variant-Specific Surface Protein Genes: Diversity Through Gene Duplication and Divergence

ABSTRACT. Giardia lamblia trophozoites express on their surfaces one of a set of cysteine-rich antigenically variant proteins, called variant-specific surface proteins, which comprise the majority of proteins detected by surface labeling. While these VSP proteins may be immunodominant proteins important in the host immune response to G. lamblia , the ability to switch expression from one VSP to another may provide a means for the trophozoites to avoid the host immune response. The first VSP characterized, VSPA6 (from the A6 clone of the WB isolate, originally termed CRP170), contains 18–23 copies of a 65 amino acid repeat. We have now used the repeat as a probe to isolate from a WBA6 genomic library two genes related to vspA6 (called vspA6-S1, vspA6-S2). Sequence analysis of the vspA6-S1 gene revealed nearly two complete copies of the 195 bp repeat and substantial nucleotide and translated amino acid similarity in the coding regions 5'and 3'to the repeats. The vspA6-S2 gene, while still related, showed greater divergence from vspA6 than vspA6-S1 in the nonrepeat coding region and contained nearly four copies of a 201 bp repeat that was 75% identical to the 195 bp vspA6 repeat. These results suggest that gene duplication followed by divergence has played a key role in the generation of the vsp gene repertoire.     

Antigenic variation in Giardia lamblia

Giardia lamblia undergo surface antigenic variation in vitro and in vivo. The presence of variant trophozoites can be detected in clones after exposure to cytotoxic monoclonal antibodies. Surviving Giardia (progeny) no longer possess the initial major surface antigen which is replaced by new antigens. Exposure of a clone from one progeny to another cytotoxic mAb specific to one newly appearing surface antigen resulted in the loss of this antigen and replacement by another set of antigens. The frequency of change was rapid (1:100-1:1000) and was dependent upon the isolate. The presence of variant populations in clones was confirmed by direct and indirect immunofluorescence using mAbs to major surface antigens of subsequent progeny. The putative amino acid sequence of a portion of one antigen revealed a cysteine-rich composition which was confirmed in this variant protein as well as others by preferential uptake of [35S]cysteine. The mechanism(s) responsible most likely involves genomic rearrangements since Southern blots revealed a family of related genes which changed frequently compared to other areas of the genome. However, expression-linked copies have not been detected. Loss and gain of surface antigens have also been found in gerbils and humans infected with defined clones, but there does not appear to be cyclical appearance of variant populations. The biological importance of antigenic variation is not known but it may contribute to chronic and/or repeated infections.     

Chromosome rearrangements in Giardia lamblia

Recent studies have shown that the genome of Giardia lamblia is plastic. Clinical isolates exhibit extensive karyotypic heterogeneity and chromosome rearrangements occur frequently, in vitro. In this review, Sylvie Le Blancq looks at genome organization and the impact of DNA rearrangement events.     

The cytoskeleton of Giardia lamblia

Giardia lamblia is a ubiquitous intestinal pathogen of mammals. Evolutionary studies have also defined it as a member of one of the earliest diverging eukaryotic lineages that we are able to cultivate and study in the laboratory. Despite early recognition of its striking structure resembling a half pear endowed with eight flagella and a unique ventral disk, a molecular understanding of the cytoskeleton of Giardia has been slow to emerge. Perhaps most importantly, although the association of Giardia with diarrhoeal disease has been known for several hundred years, little is known of the mechanism by which Giardia exacts such a toll on its host. What is clear, however, is that the flagella and disk are essential for parasite motility and attachment to host intestinal epithelial cells. Because peristaltic flow expels intestinal contents, attachment is necessary for parasites to remain in the small intestine and cause diarrhoea, underscoring the essential role of the cytoskeleton in virulence. This review presents current day knowledge of the cytoskeleton, focusing on its role in motility and attachment. As the advent of new molecular technologies in Giardia sets the stage for a renewed focus on the cytoskeleton and its role in Giardia virulence, we discuss future research directions in cytoskeletal function and regulation.     

Characterization of Microtubule-Binding and Dimerization Activity of Giardia lamblia End-Binding 1 Protein

End-binding 1 (EB1) proteins are evolutionarily conserved components of microtubule (MT) plus-end tracking protein that regulate MT dynamics. Giardia lamblia, with two nuclei and cytoskeletal structures, requires accurate MT distribution for division. In this study, we show that a single EB1 homolog gene of G. lamblia regulates MT dynamics in mitosis. The haemagglutinin-tagged G. lamblia EB1 (GlEB1) localizes to the nuclear envelopes and median bodies, and is transiently present in mitotic spindles of dividing cells. Knockdown of GlEB1 expression using the morpholinos-based anti-EB1 oligonucleotides, resulted in a significant defect in mitosis of Giardia trophozoites. The MT-binding assays using recombinant GlEB1 (rGlEB1) proteins demonstrated that rGlEB1_102–238, but not rGlEB1_1–184, maintains an MT-binding ability comparable with that of the full length protein, rGlEB1_1–238. Size exclusion chromatography showed that rGlEB1 is present as a dimer formed by its C-terminal domain and a disulfide bond. In vitro-mutagenesis of GlEB1 indicated that an intermolecular disulfide bond is made between cysteine #13 of the two monomers. Complementation assay using the BIM1 knockout mutant yeast, the yeast homolog of mammalian EB1, indicated that expression of the C13S mutant GlEB1 protein cannot rescue the mitotic defect of the BIM1 mutant yeast. These results suggest that dimerization of GlEB1 via the 13th cysteine residues plays a role during mitosis in Giardia.     

A potentially important excretory-secretory product of Giardia lamblia

In this study we have reported the detailed characterization of a 58 kDa excretory-secretory product (ESP) of Giardia lamblia. The method of purification has been simplified which has improved the purification fold as well as the yield of the ESP. The binding efficacy of disialoganglioside (GD2) to the purified ESP was found to be maximum among all other gangliosides used. The N-terminal sequence of the immunoreactive 29 kDa peptide obtained from partial tryptic digest of the ESP was found to be AD-FVPQVST. The IgG against the purified ESP (IgGES) showed cross-reactivity with the binding subunit of the commercially available cholera toxin and also with two protein bands of western cottonmouth moccasin snake toxin. The ESP could accumulate fluid in the intestine of sealed adult mice and also induce morphological changes in HEp-2 cells. The crude extract of G. lamblia trophozoites preincubated with Escherichia coli revealed 8-fold augmentation in the cytopathic activity on HEp-2 cells as compared to that of crude preparation from trophozoites only.     

A single-stranded RNA copy of the Giardia lamblia virus double-stranded RNA genome is present in the infected Giardia lamblia.

An isolate of Giardia lamblia infected with the double-stranded RNA virus (GLV) has two major species of RNA that are not present in an uninfected isolate. One of these species is the previously characterized double-stranded RNA genome of GLV (1). The second species of RNA appears to be a full length copy of one strand of the double-stranded RNA genome. This full length single-stranded RNA is not present in viral particles isolated from the growth medium. The cellular concentration of the single-stranded RNA changes during exponential and stationary phases of cell growth in a fashion consistent with a viral replicative intermediate or mRNA. The single-stranded species does not appear to be polyadenylated.