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.     

The median body of Giardia lamblia: an ultrastructural study

Giardia lamblia is an intestinal parasite of several mammals. The most striking feature of Giardia is the presence of a complex and unique cytoskeleton, and among its components the median body (MB) is the least defined microtubular structure. In the present study, we used a technique that allowed the removal of the plasma membrane and observation of cytoskeletal structures by both routine scanning electron microscopy (SEM) and field emission high resolution SEM. This technique permitted new observations such as details and insights of the median bodies, not previously described or controversial in the literature. Light microscopy after Panotic staining, immunofluorescence microscopy using several antibodies, and thin sections were also used to better characterized the Giardia MB. The new observations concerning the median bodies were : (1) they are not one or two structures, but varied in number, shape and position ; (2) they were found in mitotic and interphasic trophozoites, in disagreement with previous works ; (3) they were present in about 80 % of the cells, and not in 50 % of the cells, as previously described ; (4) they could be connected either to the plasma membrane, to the adhesive disc, and caudal flagella, and thus they are not completely free in the cells, as published before ; (5) they can protrude the cell surface ; (6) their microtubules react with several anti-tubulin and -beta giardin antibodies. These observations add new data on the scarce literature and to this largely understudied cell structure.     

Protein import, replication, and inheritance of a vestigial mitochondrion

Mitochondrial remnant organelles (mitosomes) that exist in a range of "amitochondrial" eukaryotic organisms represent ideal models for the study of mitochondrial evolution and for the establishment of the minimal set of proteins required for the biogenesis of an endosymbiosis-derived organelle. Giardia intestinalis, often described as the earliest branching eukaryote, contains double membrane-bounded structures involved in iron-sulfur cluster biosynthesis, an essential function of mitochondria. Here we present evidence that Giardia mitosomes also harbor Cpn60, mtHsp70, and ferredoxin and that despite their advanced state of reductive evolution they have retained vestiges of presequence-dependent and -independent protein import pathways akin to those that operate in mammalian mitochondria. Although import of IscU and ferredoxin is still reliant on their amino-terminal presequences, targeting of Giardia Cpn60, IscS, or mtHsp70 into mitosomes no longer requires cleavable presequences, a derived feature from their mitochondrial homologues. In addition, we found that division and segregation of a single centrally positioned mitosome tightly associated with the microtubular cytoskeleton is coordinated with the cell cycle, whereas peripherally located mitosomes are inherited into daughter cells stochastically.     

Tubulin diversity in trophozoites of<Emphasis Type="Italic"> Giardia lamblia</Emphasis>

Giardia lamblia is a diplomonad that parasitizes the small intestine of vertebrates. The trophozoite is multiflagellar and its cytoskeleton presents a complex organization of microtubular structures. One of these, the adhesive disk, consists of a microtubular spiral. The median body, whose function is not yet determined, is also composed by microtubules. The cell has eight flagella and two microtubule sheets, known as the funis. In this study we used several antibodies and immunofluorescence microscopy to help in the characterization of these structures. We observed that Giardia tubulin reacts with antibodies raised against very distinct immunogens. The antibodies used were against: (1) alpha-tubulin from chicken embryo brain, Trypanosoma brucei, sea urchin sperm, Paramecium, acetylated alpha-tubulin from Paramecium, and tyrosinated alpha-tubulin, (2) beta-tubulin from chicken embryo brain and Physarum polycephalum, and (3) an antibody with specificity to beta-tubulin having as immunogen the FtsZ bacterial protein. Each cytoskeletal structure of Giardia presented a distinct pattern of labeling by the several antibodies used. These data indicate that even being considered one of the most ancient of organisms, Giardia shares similarities (at least in relation to tubulin) with other organisms. They also open some questions about the organization and composition of its microtubular structures.     

Examination of a novel head-stalk protein family in Giardia lamblia characterised by the pairing of ankyrin repeats and coiled-coil domains

The intestinal pathogen Giardia lamblia possesses several unusual organelle features, including two equivalent nuclei, no mitochondria or peroxisomes, and a developmentally regulated rough endoplasmic reticulum and Golgi. Giardia also possesses a number of complex and unique cytoskeleton structures that dictate cell shape, motility and attachment. Our investigations of cytoskeletal proteins have revealed the presence of a new protein family. Proteins in this family contain both ankyrin repeats and coiled-coil domains; although these are common protein motifs, their pairing is unique, thus establishing a new class of head-stalk proteins. Examination of the G. lamblia genome shows evidence for at least 18 genes coding for proteins with a series of ankyrin repeats followed by a lengthy coiled-coil domain and at least an additional 14 genes coding for proteins with a prominent coiled-coil domain flanked by two series of ankyrin repeats. We have examined one of these proteins, Giardia Axoneme Associated Protein (GASP-180), in detail. GASP-180 is a 180 kDa protein containing five ankyrin repeats in a 200 amino acid N-terminal domain separated by a short spacer from an approximately 1375 amino acid coiled-coil domain. Using anti-peptide antibodies raised against a unique 20 amino acid sequence found at the C-terminus, we have determined that GASP-180 is present in cytoskeleton extractions of the parasite and localises to the proximal base of the anterior flagellar axonemes. The combination of the localisation and the structural and functional motifs of GASP-180 make it a strong candidate to participate in control of flagellar activity.     

Cell division of Giardia intestinalis: assembly and disassembly of the adhesive disc, and the cytokinesis

Trophozoites of Giardia are equipped with a special organelle of attachment, essential for parasite survival and pathogenicity, the ventral disc. Although its basic structure is well established, its reorganization and assembly during cell replication is poorly understood. We addressed some of these problems with aid of conventional, confocal and electron microscopy. We found that dividing Giardia alternates attached and free swimming phases in accordance with functional competence of the parent or newly assembled discs. The division started in attached cells by detachment of the disc microtubules from basal bodies. Shortening and eventual loss of the giardin microribbons, and unfolding of the microtubular layer resulting in collapse of the disc chamber and parasite detachment underlined gradual disassembly of the parent disc skeleton. Two daughter discs assembled on the dorsal side of the attached cell, with their ventral sides exposed on the parent cell surface and their microtubular skeletons growing in counter-clockwise direction. A depression between the assembling discs marked the cleavage plane. The splitting continued during the free-swimming phase with ventral-ventral axial symmetry in a plane of the daughter discs. Finally, the daughter cells with fully developed discs but still connected tail to tail by a cytoplasmic bridge, attached to a substrate and terminated the division by a process resembling adhesion-dependent cytokinesis. The mode of assembly of the daughter discs and plane of the division is compatible with maintenance of the left-right asymmetry of the Giardia cytoskeleton in progeny, which cannot be satisfactorily explained by alternative models proposed so far.     

Giardia lamblia: evaluation of the in vitro effects of nocodazole and colchicine on trophozoites

Giardia lamblia is the most commonly detected parasite in the intestinal tract of humans and other mammals causing giardiasis. Giardia presents several cytoskeletal structures with microtubules as major components such as the ventral adhesive disk, eight flagella axonemes, the median body and funis. Many drugs have already been tested as antigiardial agents, such as albendazole and mebendazole, which act by specifically inhibiting tubulin polymerization and hence microtubule assembly. In the present work, we used the microtubule inhibitors nocodazole and colchicine in order to investigate their direct and indirect effects on Giardia ultrastructure and attachment to the glass surface, respectively. Axenically grown G. lamblia trophozoites were treated with nocodazole or colchicine for different time intervals and analyzed by light and electron microscopy. It was observed that trophozoites became completely misshapen, detached from the glass surface and failed to complete cell division. The main alterations observed included disc fragmentation, presence of large vacuoles, and appearance of electrondense deposits made of tubulin. The cytokinesis was blocked, but not the karyokinesis, and membrane blebs were observed. These findings show that Giardia behavior and cytoskeleton are clearly affected by the commonly used microtubule targetting agents colchicine and nozodazole.     

Cell division of Giardia intestinalis: flagellar developmental cycle involves transformation and exchange of flagella between mastigonts of a diplomonad cell

Giardia intestinalis is a binucleated diplomonad possessing four pairs of flagella of distinct location and function. Its pathogenic potential depends on the integrity of a complex microtubular cytoskeleton that undergoes a profound but poorly understood reorganization during cell division. We examined the cell division of G. intestinalis with the aid of light and electron microscopy and immunofluorescence methods and present here new observations on the reorganization of the flagellar apparatus in the dividing Giardia. Our results demonstrated the presence of a flagellar maturation process during which the flagella migrate, assume different position, and transform to different flagellar types in progeny until their maturation is completed. For each newly assembled flagellum it takes three cell cycles to become mature. The mature flagellum of Giardia is the caudal one that possesses a privileged basal body at which the microtubules of the adhesive disk nucleate. In contrast to generally accepted assumption that each of the two diplomonad mastigonts develops separately, we found that they are developmentally linked, exchanging their cytoskeletal components at the early phase of mitosis. The presence of the flagellar maturation process in a metamonad protist Giardia suggests that the basal body or centriole maturation is a universal phenomenon that may represent one of the core processes in a eukaryotic cell.     

Synchronisation of Giardia lamblia: identification of cell cycle stage-specific genes and a differentiation restriction point

The intestinal parasite Giardia lamblia undergoes cell differentiations that entail entry into and departure from the replicative cell cycle. The pathophysiology of giardiasis depends directly upon the ability of the trophozoite form to replicate in the host upper small intestine. Thus, cell proliferation is tightly linked to disease. However, studies of cell cycle regulation in Giardia have been hampered by the inability to synchronise cultures. Here we report that Giardia isolates of the major human genotypes A and B can be synchronised using aphidicolin, a mycotoxin that reversibly inhibits replicative DNA polymerases in eukaryotic cells. Aphidicolin arrests Giardia trophozoites in the early DNA synthesis (S) phase of the cell cycle. We identified a set of cell cycle orthologues in the Giardia genome using bioinformatic analyses and showed that synchronised parasites express these genes in a cell cycle stage-specific manner. The synchronisation method also showed that during encystation, exit from the ordinary cell cycle occurs preferentially in G(2) and defines a restriction point for differentiation. Synchronisation opens up possibilities for further molecular and cell biological studies of chromosome replication, mitosis and segregation of the complex cytoskeleton in Giardia.     

Alpha-1 Giardin is an Annexin with Highly Unusual Calcium-Regulated Mechanisms

Alpha-giardins constitute the annexin proteome (group E annexins) in the intestinal protozoan parasite Giardia and, as such, represent the evolutionary oldest eukaryotic annexins. The dominance of alpha-giardins in the cytoskeleton of Giardia with its greatly reduced actin content emphasises the importance of the alpha-giardins for the structural integrity of the parasite, which is particularly critical in the transformation stage between cyst and trophozoite. In this study, we report the crystal structures of the apo- and calcium-bound forms of α1-giardin, a protein localised to the plasma membrane of Giardia trophozoites that has recently been identified as a vaccine target. The calcium-bound crystal structure of α1-giardin revealed the presence of a type III site in the first repeat as known from other annexin structures, as well as a novel calcium binding site situated between repeats I and IV. By means of comparison, the crystal structures of three different alpha-giardins known to date indicate that these proteins engage different calcium coordination schemes, among each other, as well as compared to annexins of groups A-D. Evaluation of the calcium-dependent binding to acidic phosphoplipid membranes revealed that this process is not only mediated but also regulated by the environmental calcium concentration. Uniquely within the large family of annexins, α1-giardin disengages from the phospholipid membrane at high calcium concentrations possibly due to formation of a dimeric species. The observed behaviour is in line with changing calcium levels experienced by the parasite during excystation and may thus provide first insights into the molecular mechanisms underpinning the transformation and survival of the parasite in the host.     

An insider's guide to the microtubule cytoskeleton of Giardia

Giardia intestinalis is a zoonotic, parasitic protist with a complex microtubule cytoskeleton critical for motility, attachment, intracellular transport, cell division and transitioning between its two life cycle stages – the cyst and the trophozoite. This review focuses on the structures of the primary elements of the microtubule cytoskeleton and cytoskeletal dynamics throughout this complex giardial life cycle. The giardial cytoskeleton has both highly dynamic elements and more stable MT structures, including several novel structures like the ventral disc that change conformation via unknown mechanisms. While our knowledge of the giardial cytoskeleton is primarily cytological, the completed Giardia genome and recently developed reverse genetic tools affords an opportunity to uncover the mechanisms of Giardia's cytoskeletal dynamics. Fundamental areas of giardial cytoskeletal biology remain to be explored, including high resolution imaging and compositional characterization of cytoskeletal structures required for elucidating the molecular mechanisms of cytoskeletal functioning.     

Giardia lamblia aurora kinase: a regulator of mitosis in a binucleate parasite

Giardia lamblia is a major cause of diarrhoeal disease worldwide. Since it has no known toxin, the ability of trophozoites to colonise the human small intestine is required for its pathogenesis. Mitosis in this protozoan parasite is a unique challenge because its two equivalent nuclei and complex cytoskeleton must be duplicated and segregated accurately. Giardial mitosis is a complex and rapid event that is poorly understood at the cellular and molecular levels. Higher eukaryotes have one to three members of the highly conserved Ser/Thr aurora kinase (AK) family that regulate key aspects of mitosis and cytokinesis. Giardia has a single AK orthologue (gAK) with 61% similarity to human AK A. In addition to the conserved active site residues, activation loop and destruction-box motifs characteristic of AKs, gAK contains a unique insert near the active site region. We epitope-tagged gAK at its C-terminus and expressed it under its own promoter. During interphase, gAK localises exclusively to the nuclei, but is not phosphorylated as shown by lack of staining with an antibody specific to phosphorylated AK A (pAK). In contrast, during mitosis pAK localises to the basal bodies/centrosomes and co-localises with tubulin to the spindle. During specific stages of mitosis, giardial pAK also localised dynamically to cytoskeletal structures unique to Giardia: the paraflagellar dense rods of the anterior flagella and the median body, whose functions are unknown, as well as to the parent attachment disc. Two AK inhibitors significantly decreased giardial growth and increased the numbers of cells arrested in cytokinesis. These inhibitors appeared to increase microtubule nucleation and cell-ploidy. Our data show that gAK is phosphorylated in mitosis and suggest that it plays an important role in the Giardia cell cycle. The pleiotropic localisation of AK suggests that it may co-ordinate the reorganisation and segregation of tubulin-containing structures in mitosis. We believe this is the first report of a signalling protein regulating cell division in Giardia.     

Giardiasis as a re-emerging infectious disease and its zoonotic potential

The reasons for considering giardiasis as a re-emerging infectious disease are presented, with emphasis on Giardia infections in child care centres, livestock and pets, and the role of zoonotic transmission. However, the aetiology and control of giardiasis is complicated by the genetic and phenotypic variability of Giardia species infective to mammals. Of particular significance has been the uncertainty about host specificity and the question of zoonotic transmission. The recent application of molecular characterisation procedures based on PCR has made an enormous contribution to an understanding of the genetic structure of Giardia populations, and this is reviewed in the context of the zoonotic transmission and molecular epidemiology of Giardia infections.     

Identification in the ancient protist Giardia lamblia of two eukaryotic translation initiation factor 4E homologues with distinctive functions

Eukaryotic translation initiation factor 4E (eIF4E) binds to the m(7)GTP of capped mRNAs and is an essential component of the translational machinery that recruits the 40S small ribosomal subunit. We describe here the identification and characterization of two eIF4E homologues in an ancient protist, Giardia lamblia. Using m(7)GTP-Sepharose affinity column chromatography, a specific binding protein was isolated and identified as Giardia eIF4E2. The other homologue, Giardia eIF4E1, bound only to the m(2,2,7)GpppN structure. Although neither homologue can rescue the function of yeast eIF4E, a knockdown of eIF4E2 mRNA in Giardia by a virus-based antisense ribozyme decreased translation, which was shown to use m(7)GpppN-capped mRNA as a template. Thus, eIF4E2 is likely the cap-binding protein in a translation initiation complex. The same knockdown approach indicated that eIF4E1 is not required for translation in Giardia. Immunofluorescence assays showed wide distribution of both homologues in the cytoplasm. But eIF4E1 was also found concentrated and colocalized with the m(2,2,7)GpppN cap, 16S-like rRNA, and fibrillarin in the nucleolus-like structure in the nucleus. eIF4E1 depletion from Giardia did not affect mRNA splicing, but the protein was bound to Giardia small nuclear RNAs D and H known to have an m(2,2,7)GpppN cap, thus suggesting a novel function not yet observed among other eIF4Es in eukaryotes.     

Expression of tubulin polyglycylation in Giardia lamblia

By means of immunofluorescence, immunoelectron microscopy and immunoblotting, we show that polyglycylation, a posttranslational modification of tubulin widely spread among eukaryotes, is present in the diplomonad, Giardia lamblia, a putative ancestral cell possessing a highly developed microtubular cytoskeleton. This modification was recently discovered in the ciliated protist, Paramecium, and was not found in the Euglenozoa, a lineage considered as ancient. We used two monoclonal antibodies (mAbs), TAP 952 and AXO 49, specifically recognizing mono- and polyglycylated tubulin isoforms, to detect this modification in Giardia extracts and to localize it in the different classes of microtubules within the cell. The alpha- and beta-tubulin subunits were recognized by the two mAbs, indicating that both tubulin subunits are glycylated, in agreement with lately reported mass spectrometry results. Noticeably, Giardia tubulin was much more reactive with AXO 49 than with TAP 952. In situ, AXO 49 intensely labeled the microtubules present in the four pairs of flagella and the median body, and lightly decorated the microtubules from the adhesive disc. In contrast, TAP 952 intensely labeled only the microtubules of the median body. The results indicate a differential expression of glycylated isoforms within various microtubular structures of Giardia lamblia. They also suggest that the complete set of enzymes required for polyglycylation is expressed in very divergent eukaryotes.     

The nuclei of <Emphasis Type="Italic">Giardia lamblia</Emphasis>—new ultrastructural observations

Giardia lamblia is a parasite possessing a complex cytoskeleton and an unusual morphology of bearing two nuclei. Here, the interphasic nuclei of trophozoites, using field emission scanning electron microscopy, routine scanning and transmission electron microscopy, immunocytochemistry, and 3D reconstruction, are presented. An approach using plasma-membrane extraction allowed the observation of the two nuclei still attached in their original positions. The observations are as follows: (1) Giardia nuclei and cytoskeleton were studied in demembranated cells by routine scanning electron microscopy and field emission; (2) both nuclei are anchored to basal bodies of the anterior flagella and to the descending posterior-lateral and ventral flagella, at the right and left nuclei, respectively, in cells attached by its ventral disc; (3) this attachment occurs by proteinaceous links, which were labeled by anti-actin and anti-centrin but not by anti-dynein or anti-tubulin antibodies; (4) fibrilar connections between the nuclei and the disc were also observed; and (5) nuclei exhibited a pendular movement when living cells were treated with cytochalasin, although the nuclei were still connected by their anterior region. Our analysis indicated that the nuclei have a defined position, and fibrils perform an anchoring system. This raises the possibility of a mechanism for nuclei-fidelity migration during mitosis.     

Localization of gamma-tubulin in interphase and mitotic cells of a unicellular eukaryote, Giardia intestinalis

Giardia intestinalis, a bi-nucleated amitochondrial flagellate, possesses a complex cytoskeleton based on several microtubular systems (flagella, adhesive disk, median body, funis, mitotic spindles). MTOCs of the individual systems have not been fully defined. By using monoclonal antibodies against a conserved synthetic peptide from the C-terminus of human gamma-tubulin we investigated occurrence and distribution of gamma-tubulin in interphase and mitotic Giardia cells. On the immunoblots of Giardia cytoskeletal extracts the antibodies bound to a single polypeptide of approximately 50 kDa. Immunostaining of the interphase cell demonstrated gamma-tubulin as four bright spots at the basis of four out of eight flagella. Gamma-tubulin label was associated with perikinetosomal areas of the ventral and posterolateral pairs of flagella which are formed de novo during cell division. Basal body regions of the anterolateral and caudal pairs of flagella which persist during the division and are integrated into the flagellar systems of the daughter cells did not show gamma-tubulin staining. At early mitosis, gamma-tubulin spots disappeared reappearing again at late mitosis in accord with reorientation of parent flagella and reorganization of flagellar apparatus during cell division. The antibody-detectable gamma-tubulin epitope was absent at the poles of both mitotic spindles. Albendazole-treated Giardia, in which spindle assembly was completely inhibited, showed the same gamma-tubulin staining pattern thus confirming that the fluorescent label is exclusively located in the basal body regions. Our results point to a role of gamma-tubulin in nucleation of microtubules of newly formed flagella and indicate unusual mitotic spindle assembly. Moreover, the demonstration of gamma-tubulin in Giardia shows ubiquity of this protein through the evolutionary history of eukaryotes.     

Cytotoxicity of monoclonal antibodies to a subset of Giardia isolates

Previous studies showed that some Giardia lamblia isolates differ and can be categorized on the basis of their DNA banding patterns after digestion with endonuclease restriction enzymes, surface antigens, and excretory-secretory (E-S) products. In the present study, monoclonal antibodies (McAb) were produced that reacted with one specific group of Giardia isolates. These McAb recognized a 170,000 dalton antigen, which was present on the surface of these Giardia and released into the medium as an E-S antigen. This antigen was previously characterized and found to distinguish this subgroup of Giardia. In addition, these McAb were cytotoxic only for this subgroup of Giardia. Immobilization occurred immediately, and killing was documented by 7 min. The mechanism(s) of killing remains unknown but was shown to be complement independent and did not occur with Fab'. These McAb identifies certain isolates and can be used to type Giardia.