Identification of Giardia lamblia DHHC Proteins and the Role of Protein S-palmitoylation in the Encystation Process

Protein S-palmitoylation, a hydrophobic post-translational modification, is performed by protein acyltransferases that have a common DHHC Cys-rich domain (DHHC proteins), and provides a regulatory switch for protein membrane association. In this work, we analyzed the presence of DHHC proteins in the protozoa parasite Giardia lamblia and the function of the reversible S-palmitoylation of proteins during parasite differentiation into cyst. Two specific events were observed: encysting cells displayed a larger amount of palmitoylated proteins, and parasites treated with palmitoylation inhibitors produced a reduced number of mature cysts. With bioinformatics tools, we found nine DHHC proteins, potential protein acyltransferases, in the Giardia proteome. These proteins displayed a conserved structure when compared to different organisms and are distributed in different monophyletic clades. Although all Giardia DHHC proteins were found to be present in trophozoites and encysting cells, these proteins showed a different intracellular localization in trophozoites and seemed to be differently involved in the encystation process when they were overexpressed. dhhc transgenic parasites showed a different pattern of cyst wall protein expression and yielded different amounts of mature cysts when they were induced to encyst. Our findings disclosed some important issues regarding the role of DHHC proteins and palmitoylation during Giardia encystation.     

Mining the Giardia genome and proteome for conserved and unique basal body proteins

Giardia lamblia is a flagellated protozoan parasite and a major cause of diarrhoea in humans. Its microtubular cytoskeleton mediates trophozoite motility, attachment and cytokinesis, and is characterised by an attachment disk and eight flagella that are each nucleated in a basal body. To date, only 10 giardial basal body proteins have been identified, including universal signalling proteins that are important for regulating mitosis or differentiation. In this study, we have exploited bioinformatics and proteomic approaches to identify new Giardia basal body proteins and confocal microscopy to confirm their localisation in interphase trophozoites. This approach identified 75 homologs of conserved basal body proteins in the genome including 65 not previously known to be associated with Giardia basal bodies. Thirteen proteins were confirmed to co-localise with centrin to the Giardia basal bodies. We also demonstrate that most basal body proteins localise to additional cytoskeletal structures in interphase trophozoites. This might help to explain the roles of the four pairs of flagella and Giardia-specific organelles in motility and differentiation. A deeper understanding of the composition of the Giardia basal bodies will contribute insights into the complex signalling pathways that regulate its unique cytoskeleton and the biological divergence of these conserved organelles.     

Identification of α-11 giardin as a flagellar and surface component of Giardia lamblia

Giardia lamblia is a protozoan pathogen with distinct cytoskeletal structures, including median bodies and eight flagella. In this study, we examined components comprising G. lamblia flagella. Crude flagellar extracts were prepared from G. lamblia trophozoites, and analyzed by two-dimensional (2-D) gel electrophoresis. The 19 protein spots were analyzed by MALDI–TOF mass spectrometry, identifying ten metabolic enzymes, six distinct giardins, Giardia trophozoite antigen 1, translational initiation factor eIF-4A, and an extracellular signal-regulated kinase 2. Among the identified proteins, we studied α-11 giardin which belongs to a group of cytoskeletal proteins specific to Giardia. Western blot analysis and real-time PCR indicated that expression of α-11 giardin is not significantly increased during encystation of G. lamblia. Immunofluorescence assays using anti-α-11 giardin antibodies revealed that α-11 giardin protein mainly localized to the plasma membranes and basal bodies of the anterior flagella of G. lamblia trophozoites, suggesting that α-11 giardin is a genuine component of the G. lamblia cytoskeleton.     

Ultrastructural Evidence for the Presence of Bacteria,Viral-like Particles,and Mycoplasma-like Organisms Associated with Giardia spp.1

ABSTRACT. Giardia trophozoites and cysts, isolated from mammalian and avian hosts, were examined by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and by fluorescent light microscopy for the presence of microbial symbionts. Mycoplasma-like organisms were observed on the surfaces of trophozoites isolated from the prairie vole, laboratory rat, and beaver. Intracellular bacteria were observed by TEM in the trophozoites and cysts of G. microti and by fluorescence microscopy in trophozoites and cysts of Giardia spp. isolated from beaver, muskrat, great-blue heron, and the green heron. Trophozoites of G. muris from rat small intestine contained viral-like particles measuring 60 nm in diameter. These observations suggest that biological associations between Giardia spp. and diverse microbes may be more common than formerly appreciated. It also raises the possibility of transmission of these apparent symbionts, via the Giardia cyst, to other mammalian hosts including man.     

Giardia Cyst Wall Protein 1 Is a Lectin That Binds to Curled Fibrils of the GalNAc Homopolymer

The infectious and diagnostic stage of Giardia lamblia (also known as G. intestinalis or G. duodenalis) is the cyst. The Giardia cyst wall contains fibrils of a unique β-1,3-linked N-acetylgalactosamine (GalNAc) homopolymer and at least three cyst wall proteins (CWPs) composed of Leu-rich repeats (CWP_LRR) and a C-terminal conserved Cys-rich region (CWP_CRR). Our goals were to dissect the structure of the cyst wall and determine how it is disrupted during excystation. The intact Giardia cyst wall is thin (∼400 nm), easily fractured by sonication, and impermeable to small molecules. Curled fibrils of the GalNAc homopolymer are restricted to a narrow plane and are coated with linear arrays of oval-shaped protein complex. In contrast, cyst walls of Giardia treated with hot alkali to deproteinate fibrils of the GalNAc homopolymer are thick (∼1.2 µm), resistant to sonication, and permeable. The deproteinated GalNAc homopolymer, which forms a loose lattice of curled fibrils, is bound by native CWP1 and CWP2, as well as by maltose-binding protein (MBP)-fusions containing the full-length CWP1 or CWP1_LRR. In contrast, neither MBP alone nor MBP fused to CWP1_CRR bind to the GalNAc homopolymer. Recombinant CWP1 binds to the GalNAc homopolymer within secretory vesicles of Giardia encysting in vitro. Fibrils of the GalNAc homopolymer are exposed during excystation or by treatment of heat-killed cysts with chymotrypsin, while deproteinated fibrils of the GalNAc homopolymer are degraded by extracts of Giardia cysts but not trophozoites. These results show the Leu-rich repeat domain of CWP1 is a lectin that binds to curled fibrils of the GalNAc homopolymer. During excystation, host and Giardia proteases appear to degrade bound CWPs, exposing fibrils of the GalNAc homopolymer that are digested by a stage-specific glycohydrolase.     

A Detailed, Hierarchical Study of Giardia lamblia's Ventral Disc Reveals Novel Microtubule-Associated Protein Complexes

Giardia lamblia is a flagellated, unicellular parasite of mammals infecting over one billion people worldwide. Giardia''s two-stage life cycle includes a motile trophozoite stage that colonizes the host small intestine and an infectious cyst form that can persist in the environment. Similar to many eukaryotic cells, Giardia contains several complex microtubule arrays that are involved in motility, chromosome segregation, organelle transport, maintenance of cell shape and transformation between the two life cycle stages. Giardia trophozoites also possess a unique spiral microtubule array, the ventral disc, made of approximately 50 parallel microtubules and associated microribbons, as well as a variety of associated proteins. The ventral disc maintains trophozoite attachment to the host intestinal epithelium. With the help of a combined SEM/microtome based slice and view method called 3View® (Gatan Inc., Pleasanton, CA), we present an entire trophozoite cell reconstruction and describe the arrangement of the major cytoskeletal elements. To aid in future analyses of disc-mediated attachment, we used electron-tomography of freeze-substituted, plastic-embedded trophozoites to explore the detailed architecture of ventral disc microtubules and their associated components. Lastly, we examined the disc microtubule array in three dimensions in unprecedented detail using cryo-electron tomography combined with internal sub-tomogram volume averaging of repetitive domains. We discovered details of protein complexes stabilizing microtubules by attachment to their inner and outer wall. A unique tri-laminar microribbon structure is attached vertically to the disc microtubules and is connected to neighboring microribbons via crossbridges. This work provides novel insight into the structure of the ventral disc microtubules, microribbons and associated proteins. Knowledge of the components comprising these structures and their three-dimensional organization is crucial toward understanding how attachment via the ventral disc occurs in vivo.     

Giardipain-1, a protease secreted by Giardia duodenalis trophozoites,causes junctional,barrier and apoptotic damage in epithelial cell monolayers

The adhesion of Giardia duodenalis trophozoites to intestinal epithelial cells allows the onset and maintenance of giardiasis. During these interactions, epithelial cells can be committed to apoptosis by enzymes secreted by the parasites, including cysteine proteases that are increasingly identified as virulence factors in parasitic protozoa. In this work, a monoclonal antibody (mAb1G3) raised against G. duodenalis surface components was found to react with a 25?kDa protein expressed in the cell surface and flagella of G. duodenalis trophozoites. When trophozoites expressing this protein were cultured with IEC-6 intestinal epithelial cell monolayers, a dynamic release of this protein was observed with mAbIG3. Proteomic analysis identified the protein as a mature cathepsin B-like (gCatB) enzyme, whose proteolytic activity, detected in zymograms, was eliminated by CatB inhibitor E-64. This protein was named giardipain-1 due to its functional papain-like features and was purified by affinity chromatography using mAbIG3. Upon exposure to the purified, mature and secreted forms of giardipain-1, IEC-6 epithelial cell monolayers displayed membrane blebbing and phosphatidylserine exposure on the outer cell surface, indicating an apoptotic process. In Madin Darby Canine Kidney (MDCK) cell monolayers, giardipain-1 leads to the appearance of pore-like regions and of gaps along cell–cell junctions, to decreased transepithelial electrical resistance (TER), caspase-3 activation and poly-ADP-ribose polymerase (PARP) fragmentation. At early times during exposure, giardipain-1 co-localized at cell–cell junctions, associated with occludin and induced the delocalization and degradation of tight junction proteins occludin and claudin-1. The damage caused to epithelial monolayers by giardipain-1 was blocked by pre-incubation with the CatB B Inhibitor E-64. Furthermore, silencing the giardipain-1 gene in trophozoites lowered the proteolytic activity of giardipain-1 and reduced the damage in IEC-6 monolayers. The damage observed appears to be specific to giardipain activity since almost no damage was observed when IEC-6 monolayers were incubated with papain, a non-related cysteine protease. Hence this study suggests that giardipain-1 triggers, in epithelial cells, degradation of cell–cell junctional components and apoptotic damage, supporting the notion of giardiapain-1 as a virulence factor of Giardia.     

Analysis of sugar chain-binding specificity of tomato lectin using lectin blot: recognition of high mannose-type N-glycans produced by plants and yeast

The sugar chain-binding specificity of tomato lectin (LEA) against glycoproteins was investigated qualitatively using lectin blot analysis. Glycoproteins containing tri- and tetra-antennary complex-type N-glycans were stained with LEA. Unexpectedly, glycoproteins containing high mannose-type N-glycans and a horseradish peroxidase were stained with LEA. LEA blot analysis of the glycoproteins accompanied by treatment with exoglycosidase revealed that the binding site of LEA for the complex-type N-glycans was the N-acetyllactosaminyl side chains, whereas the proximal chitobiose core appeared to be the binding site of LEA for high mannose-type N-glycans. Despite these results, the glycoproteins did not inhibit the hemagglutinating activity of LEA. Among the chitin-binding lectins compared, potato tuber lectin showed specificity similar to LEA on lectin blot analysis, while Datura stramonium lectin and wheat germ agglutinin (WGA) did not interact with glycoproteins containing high mannose-type N-glycans, except that RNase B was stained by WGA. Based on these observations, LEA blot analysis was applied to sugar chain analysis of tomato glycoproteins. The most abundant LEA-reactive glycoprotein was purified from the exocarp of ripe tomato fruits, and was identified as the tomato anionic peroxidase1 (TAP1). These results suggest that LEA interacts with glycoproteins produced by tomatoes, which participate in biological activities in tomato plants.     

Giardia intestinalis: Aphidicolin influence on the trophozoite cell cycle

This study is a thorough examination of the effects of the DNA polymerase inhibitor aphidicolin on the nuclear cycle and cell cycle progression characteristics, as well as their reversibility, in Giardia intestinalis. Giardia trophozoites are arrested in the G1/S-junction after aphidicolin treatment according to their DNA content. However, cell growth continues and trophozoites arrested with aphidicolin resemble cells in the G2 phase and trophozoites in ageing cultures. Extensive treatment with aphidicolin causes side effects and we detected positive signals for phosphorylated histone H2A, which, in mammalian cells, is involved in a signalling pathway triggered as a reaction to double stranded DNA breaks. These results suggest that aphidicolin causes dissociation of the nuclear and cytoplasmic cycles, a phenomenon that has also been described for other inhibitors in mammalian cell lines. Thus, if aphidicolin is used for synchronization of Giardia trophozoites, this fact must be accounted for, and treatment with aphidicolin must be minimal.     

Involvement of lectin pathway activation in the complement killing of Giardia intestinalis

Giardia intestinalis (syn. G. lamblia, G. duodenalis) is a flagellated unicellular eukaryotic microorganism that commonly causes diarrheal disease throughout the world. In humans, the clinical effects of Giardia infection range from the asymptomatic carrier state to a severe malabsorption syndrome possibly due to different virulence of the Giardia strain, the number of cysts ingested, the age of the host, and the state of the host immune system at the time of infection.The question about how G. intestinalis is controlled by the organism remains unanswered. Here, we investigated the role of the complement system and in particular, the lectin pathway during Giardia infections. We present the first evidence that G. intestinalis activate the complement lectin pathway and in doing so participate in eradication of the parasite. We detected rapid binding of mannan-binding lectin, H-ficolin and L-ficolin to the surface of G. intestinalis trophozoites and normal human serum depleted of these molecules failed to kill the parasites. Our finding provides insight into the role of lectin pathway in the control of G. intestinalis and about the nature of surface components of parasite.     

Clathrin-dependent pathways and the cytoskeleton network are involved in ceramide endocytosis by a parasitic protozoan, Giardia lamblia

Although identified as an early-diverged protozoan, Giardia lamblia shares many similarities with higher eukaryotic cells, including an internal membrane system and cytoskeleton, as well as secretory pathways. However, unlike many other eukaryotes, Giardia does not synthesize lipids de novo, but rather depends on exogenous sources for both energy production and organelle or membrane biogenesis. It is not known how lipid molecules are taken up by this parasite and if endocytic pathways are involved in this process. In this investigation, we tested the hypothesis that highly regulated and selective lipid transport machinery is present in Giardia and necessary for the efficient internalization and intracellular targeting of ceramide molecules, the major sphingolipid precursor. Using metabolic and pathway inhibitors, we demonstrate that ceramide is internalized through endocytic pathways and is primarily targeted into perinuclear/endoplasmic reticulum membranes. Further investigations suggested that Giardia uses both clathrin-dependent pathways and the actin cytoskeleton for ceramide uptake, as well as microtubule filaments for intracellular localization and targeting. We speculate that this parasitic protozoan has evolved cytoskeletal and clathrin-dependent endocytic mechanisms for importing ceramide molecules from the cell exterior for the synthesis of membranes and vesicles during growth and differentiation.     

A comparison of the Giardia lamblia trophozoite and cyst transcriptome using microarrays

Background  

Compared with many protists, Giardia lamblia has a simple life cycle alternating between cyst and trophozoite. Most research on the molecular biology of Giardia parasites has focused on trophozoites and the processes of excystation and encystation, whereas cysts have attracted less interest. The striking morphological differences between the dormant cyst and the rapidly dividing and motile trophozoite implies profound changes in the metabolism as the parasite encysts in the host's intestine and excysts upon ingestion by a new host.     

Spin label studies of microsomal membranes from Acanthamoeba castellanii in different states of differentiation

Two fatty acid spin labels—[I(1,14)], stearic acid bearing a paramagnetic nitroxide group on carbon 16, and [I(12,3)], stearic acid bearing a paramagnetic nitroxide group on carbon 5—have been used to compare the physical properties of lipid in rough and smooth microsomal membranes from trophozoites and cysts of Acanthamoeba castellanii. Arrhenius plots of rotational correlation times (τ_c) calculated from the spectra for I(1,14) showed an abrupt discontinuity in slope for membranes from both trophozoites and cysts. This occurred at temperatures ranging from ?3 to 1 °C for smooth microsomes and from 8 to 11 °C for rough microsomes for both cysts and amoebae. The value of τ_c at 29 °C, the culturing temperature, in effect scores fluidity of the membrane matrix, and did not show any significant difference for either rough or smooth microsomes during the transition from exponential to stationary phase growth. However, smooth microsomes from cysts showed a 14% increase in fluidity relative to trophozoites, and the fluidity of rough microsomes from cysts tended to be lower. An order parameter (S) calculated from spectra for I(12,3) did not change as a function of encystment for the smooth membranes and increased only slightly for rough microsomes. The activation energy (E_a) for Arrhenius plots of τ_c above the inflection temperature increased as a result of encystment, indicating a greater degree of molecular interaction within the cyst membranes. Moreover, the τ_c plots for both rough and smooth microsomal membranes from trophozoites tended to converge at 29 °C, the growth temperature, whereas plots for cyst membranes were virtually parallel, bracketing those for the trophozoite membranes. This suggests that the trophozoite is able to regulate its membrane fluidity and that cysts, which are resting cells, have lost this regulatory capacity.     

Triazoxins: Novel nucleosides with anti-Giardia activity

Novel nucleoside analogues named “triazoxins” were synthesized. Of these, two analogues were found to be highly effective against Giardia lamblia, an intestinal parasite and a major cause of waterborne infection, worldwide. While compound 7 reduced the growth of trophozoites in culture (IC₅₀, ~5 μM), compound 21 blocked the in vitro cyst production (IC₅₀ ~5 μM). Compound 21 was also effective against trophozoites (IC₅₀, ~36 μM). A third analogue (compound 8) was effective against both trophozoites (IC₅₀, ~36 μM) and cysts (IC₅₀, ~20 μM) although at higher concentration. Thus triazoxin analogues are unique and exhibit morphology (i.e., trohozoites or cysts) -specific effects against Giardia.     

Morphology of the Cyst of Giardia microti by Light and Electron Microscopy1

ABSTRACT. Cysts of Giardia microti, isolated from feces and intestinal contents of Microtus ochrogaster, were examined by light and electron microscopy. These cysts differed morphologically from cysts of other G. duodenalis morphological types in that these cysts often contained two apparently differentiated trophozoites with mature ventral discs. Cysts more closely resembling those reported for G. lamblia and G. muris were in greater abundance in preparations made from intestinal contents and were interpreted as immature cysts. “Multiple fission” cysts, reported in G. muris and G. microti by earlier workers, were not observed; however, endosymbiotic bacteria were found in the cysts of G. microti and may have been responsible for reports of multiple fission in the cysts of Giardia.     

New insights regarding the biology of Giardia lamblia

Giardia lamblia is one of the most common causes of intestinal disease in humans. To adapt to environments both inside and outside of the host's small intestine, this protozoan parasite undergoes significant developmental changes during its life cycle. In this review, we analyze and discuss the most recent findings regarding the process of Giardia trophozoites differentiation into infective cysts as well as the mechanism of antigenic variation, which allows the parasite to cause chronic and recurrent infections in susceptible individuals.     

Modeling Long-Term Host Cell-Giardia lamblia Interactions in an In Vitro Co-Culture System

Globally, there are greater than 700,000 deaths per year associated with diarrheal disease. The flagellated intestinal parasite, Giardia lamblia, is one of the most common intestinal pathogens in both humans and animals throughout the world. While attached to the gastrointestinal epithelium, Giardia induces epithelial cell apoptosis, disrupts tight junctions, and increases intestinal permeability. The underlying cellular and molecular mechanisms of giardiasis, including the role lamina propria immune cells, such as macrophages, play in parasite control or clearance are poorly understood. Thus far, one of the major obstacles in ascertaining the mechanisms of Giardia pathology is the lack of a functionally relevant model for the long-term study of the parasite in vitro. Here we report on the development of an in vitro co-culture model which maintains the basolateral-apical architecture of the small intestine and allows for long-term survival of the parasite. Using transwell inserts, Caco-2 intestinal epithelial cells and IC-21 macrophages are co-cultured in the presence of Giardia trophozoites. Using the developed model, we show that Giardia trophozoites survive over 21 days and proliferate in a combination media of Caco-2 cell and Giardia medium. Giardia induces apoptosis of epithelial cells through caspase-3 activation and macrophages do not abrogate this response. Additionally, macrophages induce Caco-2 cells to secrete the pro-inflammatory cytokines, GRO and IL-8, a response abolished by Giardia indicating parasite induced suppression of the host immune response. The co-culture model provides additional complexity and information when compared to a single-cell model. This model will be a valuable tool for answering long-standing questions on host-parasite biology that may lead to discovery of new therapeutic interventions.     

Giardia lamblia: Immunogenicity and intracellular distribution of GHSP-115, a member of the Giardia head-stalk family of proteins

Giardia lamblia, a protozoan causing diarrheal outbreaks, is one of the main pathogens monitored in developed countries. Immunoscreening of G. lamblia expression library using the monoclonal antibodies (mAb) against G. lamblia, identified a subset of antigenic proteins in this protozoan, which are proteins belonging to GHSP (Giardia head-stalk protein), GHSP115, GHSP138, and GHSP180. In order to map the epitope region of GHSP115, the corresponding open reading frame was dissected into three parts and expressed as recombinant proteins with histidine tags. Western blot analysis of these recombinant proteins with mAbs reacting with GHSP115 indicated that one-third of the C-terminus of GHSP115 showed immunoreactivity with the mAb. Intracellular location of GHSP115 was examined both in trophozoites and encysting cells of G. lamblia by an immunofluorescence assay, indicating that location of GHSP115 varies during encystation. These results suggest that GHSP115 is an abundant and antigenic protein, which is differentially localized during life cycle of G. lamblia.