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Eiler et al. used cryo-electron microscopy to determine a 2.49 Å resolution structure of Giardia lamblia 80S ribosome bound to tRNA, mRNA, and the anti-protozoal drug emetine. The structure reveals some critical aspects of translation in G. lamblia, including the lack of ribosomal protein RACK1, and how emetine blocks translation by interacting with both the ribosome and mRNA., Competing Interests: Declaration of interests The author declares no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Giardia lamblia is a deeply branching protist and a human pathogen. Its unusual biology presents the opportunity to explore conserved and fundamental molecular mechanisms. We determined the structure of the G. lamblia 80S ribosome bound to tRNA, mRNA, and the antibiotic emetine by cryo-electron microscopy, to an overall resolution of 2.49 Å. The structure reveals rapidly evolving protein and nucleotide regions, differences in the peptide exit tunnel, and likely altered ribosome quality control pathways. Examination of translation initiation factor binding sites suggests these interactions are conserved despite a divergent initiation mechanism. Highlighting the potential of G. lamblia to resolve conserved biological principles; our structure reveals the interactions of the translation inhibitor emetine with the ribosome and mRNA, thus providing insight into the mechanism of action for this widely used antibiotic. Our work defines key questions in G. lamblia and motivates future experiments to explore the diversity of eukaryotic gene regulation., Competing Interests: Declaration of interests OSR is a member of the Molecular Cell Scientific Advisory Board and the Cell Reports Scientific Advisory Board., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Giardiasis is the most prevalent diarrheal disease globally and affects humans and animals. It is a significant problem in developing countries, the number one cause of travelers' diarrhea and affects children and immunocompromised individuals, especially HIV-infected individuals. Giardiasis is treated with antibiotics (tinidazole and metronidazole) that are also used for other infections such as trichomoniasis. The ongoing search for new therapeutics for giardiasis includes characterizing the structure and function of proteins from the causative protozoan Giardia lamblia. These proteins include hypothetical proteins that share 30% sequence identity or less with proteins of known structure. Here, the atomic resolution structure of a 15.6 kDa protein was determined by molecular replacement. The structure has the two-layer αβ-sandwich topology observed in the prototypical endoribonucleases L-PSPs (liver perchloric acid-soluble proteins) with conserved allosteric active sites containing small molecules from the crystallization solution. This article is an educational collaboration between Hampton University and the Seattle Structural Genomics Center for Infectious Disease., (open access.)

Giardia intestinalis is a human parasite that causes a diarrheal disease in developing countries. G. intestinalis has a cytoskeleton (CSK) composed of microtubules and microfilaments, and the Giardia genome does not code for the canonical CSK-binding proteins described in other eukaryotic cells. To identify candidate actin and tubulin cross-linking proteins, we performed a BLAST analysis of the Giardia genome using a spectraplakins consensus sequence as a query. Based on the highest BLAST score, we selected a 259-kDa sequence designated as a cytoskeleton linker protein (CLP259). The sequence was cloned in three fragments and characterized by immunoprecipitation, confocal microscopy, and mass spectrometry (MS). CLP259 was located in the cytoplasm in the form of clusters of thick rods and colocalized with actin at numerous sites and with tubulin in the median body. Immunoprecipitation followed by mass spectrometry revealed that CLP259 interacts with structural proteins such as giardins, SALP-1, axonemal, and eight coiled-coils. The vesicular traffic proteins detected were Mu adaptin, Vacuolar ATP synthase subunit B, Bip, Sec61 alpha, NSF, AP complex subunit beta, and dynamin. These results indicate that CLP259 in trophozoites is a CSK linker protein for actin and tubulin and could act as a scaffold protein driving vesicular traffic.

Giardia lamblia , a single-celled eukaryote, colonizes and thrives in the small intestine of humans. Because of its compact and reduced genome, Giardia has adapted a "minimalistic" life style, as it becomes dependent on available resources of the small intestine. Because Giardia expresses fewer sphingolipid (SL) genes-and glycosphingolipids are critical for encystation-we investigated the SL metabolic cycle in this parasite. A tandem mass spectrometry (MS/MS) analysis reveals that major SLs in Giardia include sphingomyelins, sphingoid bases, ceramides, and glycosylceramides. Many of these lipids are obtained by Giardia from the growth medium, remodeled at their fatty acyl chains and end up in the spent medium. For instance, ceramide-1-phosphate, a proinflammatory molecule that is not present in the culture medium, is generated from sphingosine (abundant in the culture medium) possibly by remodeling reactions. It is then subsequently released into the spent medium. Thus, the secretion of ceramide-1-phospate and other SL derivatives by Giardia could be associated with inflammatory bowel disease observed in acute giardiasis. Additionally, we found that the levels of SLs increase in encysting Giardia and are differentially regulated throughout the encystation cycle. We propose that SL metabolism is important for this parasite and, could serve as potential targets for developing novel anti-giardial agents.

Giardia lamblia , one of the most common protozoal infections of the human intestine, is an important worldwide cause of diarrheal disease, malabsorption, malnutrition, delayed cognitive development in children, and protracted postinfectious syndromes. Despite its medical importance, no human vaccine is available against giardiasis. A crude veterinary vaccine has been developed, and experimental vaccines based on expression of multiple variant-specific surface proteins have been reported, but poorly defined vaccine components and excessive antigen variability are problematic for pharmaceutical vaccine production. To expand the repertoire of antigen candidates for vaccines, we reasoned that surface proteins may provide an enriched source of such antigens since key host effectors, such as secretory IgA, can directly bind to such antigens in the intestinal lumen and interfere with epithelial attachment. Here, we have applied a proteomics approach to identify 23 novel surface antigens of G. lamblia that show >90% amino acid sequence identity between the two human-pathogenic genetic assemblages (A and B) of the parasite. Surface localization of a representative subset of these proteins was confirmed by immunostaining. Four selected proteins, uridine phosphorylase-like protein-1, protein 21.1 (GL50803_27925), α1-giardin, and α11-giardin, were subsequently produced in recombinant form and shown to be immunogenic in mice and G. lamblia -infected humans and confer protection against G. lamblia infection upon intranasal immunization in rodent models of giardiasis. These results demonstrate that identification of conserved surface antigens provides a powerful approach for overcoming a key rate-limiting step in the design and construction of an effective vaccine against giardiasis., (Copyright © 2019 American Society for Microbiology.)

Giardia lamblia is one of the most common protozoan infectious agents in the world and is responsible for diarrheal disease and chronic postinfectious illness. During the host-parasite interaction, proteases are important molecules related to virulence, invasion, and colonization, not only for Giardia but also for other parasites. We aimed to characterize the cysteine protease activity detected in trophozoite lysates. This proteolytic activity showed the ability to cleave NH-terminal sequences with either a recognition sequence for a viral protease or a recognition sequence for thrombin. This cleavage activity was detected in nonencysting trophozoites and increased with the progression of encystation. This activity was also detected in excretion/secretion products of axenic trophozoites and in trophozoites cocultured with differentiated Caco-2 cells. Based on size exclusion chromatography, we obtained a fraction enriched in low- to medium-molecular-weight proteins that was capable of exerting this cleavage activity and aggregating human platelets. Finally, our results suggest that this proteolytic activity is shared with other protozoan parasites., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Intestinal and free-living protozoa, such as Giardia lamblia, express a dense coat of variant-specific surface proteins (VSPs) on trophozoites that protects the parasite inside the host's intestine. Here we show that VSPs not only are resistant to proteolytic digestion and extreme pH and temperatures but also stimulate host innate immune responses in a TLR-4 dependent manner. We show that these properties can be exploited to both protect and adjuvant vaccine antigens for oral administration. Chimeric Virus-like Particles (VLPs) decorated with VSPs and expressing model surface antigens, such as influenza virus hemagglutinin (HA) and neuraminidase (NA), are protected from degradation and activate antigen presenting cells in vitro. Orally administered VSP-pseudotyped VLPs, but not plain VLPs, generate robust immune responses that protect mice from influenza infection and HA-expressing tumors. This versatile vaccine platform has the attributes to meet the ultimate challenge of generating safe, stable and efficient oral vaccines.

Giardia intestinalis is a protozoan parasite and the causative agent of giardiasis, a common diarrheal disease. Cysteine protease (CP) activities have been suggested to be involved in Giardia's pathogenesis and we have recently identified and characterized three secreted Giardia CPs; CP14019, CP16160 and CP16779. Here we have studied the cleavage specificity of these CPs using substrate phage display and recombinant protein substrates. The phage display analyses showed that CP16160 has both chymase and tryptase activity and a broad substrate specificity. This was verified using recombinant protein substrates containing different variants of the cleavage sites. Phage display analyses of CP14019 and CP16779 failed but the substrate specificity of CP14019 and CP16779 was tested using the recombinant substrates generated for CP16160. CP16160 and CP14019 showed similar substrate specificity, while CP16779 has a slightly different substrate specificity. The consensus sequence for cleavage by CP16160, obtained from phage display analyses, was used in an in silico screen of the human intestinal proteome for detection of potential targets. Immunoglobulins, including IgA and IgG and defensins (α-HD6 and β-HD1) were predicted to be targets and they were shown to be cleaved by the recombinant CPs in vitro. Our results suggest that the secreted Giardia CPs are key players in the interaction with host cells during Giardia infections since they can cleave several components of the human mucosal defense machinery., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The international spread of infectious diseases is a global problem of health security. Vaccination is one of the most successful and profitable health interventions. Oral immunization has significant advantages over the widely used parental vaccines. Intestinal and free-living protozoa express on their surface a dense layer of proteins that protect them from hostile environmental conditions. The use of variable surface proteins (VSPs), such as those of the intestinal protozoan Giardia lamblia, is a feasible mechanism for the generation of oral vaccines, since they are highly immunogenic as well as resistant to changes in pH and proteases. In a recently published article, we showed that these properties of VSPs can be exploited to protect and enhance the immunogenicity of vaccine antigens, thus enabling their oral administration. We recently generated an oral vaccine against influenza virus composed of virus-like particles (VLPs) containing VSPs of G. lamblia and the HA antigen (viral hemagglutinin) in its envelope. When administered orally to mice, these coated particles elicit HA-specific humoral (systemic and local) and cellular responses, without the need of any additional adjuvant. Treated mice are protected against viral challenge as well as against the development of tumors expressing the HA vaccine antigen.

Giardiasis is a common diarrheal disease caused by the protozoan parasite Giardia intestinalis. Cysteine proteases (CPs) are acknowledged as virulence factors in Giardia but their specific role in the molecular pathogenesis of disease is not known. Herein, we aimed to characterize the three main secreted CPs (CP14019, CP16160 and CP16779), which were identified by mass spectrometry in the medium during interaction with intestinal epithelial cells (IECs) in vitro. First, the CPs were epitope-tagged and localized to the endoplasmic reticulum and cytoplasmic vesicle-like structures. Second, we showed that recombinant CPs, expressed in Pichia pastoris, are more active in acidic environment (pH 5.5-6) and we determined the kinetic parameters using fluorogenic substrates. Third, excretory-secretory proteins (ESPs) from Giardia trophozoites affect the localization of apical junctional complex (AJC) proteins and recombinant CPs cleave or re-localize the AJC proteins (claudin-1 and -4, occludin, JAM-1, β-catenin and E-cadherin) of IECs. Finally, we showed that the ESPs and recombinant CPs can degrade several chemokines, including CXCL1, CXCL2, CXCL3, IL-8, CCL2, and CCL20, which are up-regulated in IECs during Giardia-host cell interactions. This is the first study that characterizes the role of specific CPs secreted from Giardia and our results collectively indicate their roles in the disruption of the intestinal epithelial barrier and modulating immune responses during Giardia infections.

The protozoan parasite Giardia lamblia has traditionally been reported as lacking peroxisomes, organelles involved in fatty acid metabolism and detoxification of reactive oxygen species. We here report the finding with transmission electron microscopy of an oxidase activity in cytoplasmic vesicles of trophozoites and cysts of G. lamblia. These vesicles were positive to 3,3'-diaminobenzidine and to cerium chloride staining. In addition, using bioinformatic tools, two peroxisomal proteins were identified in the G. lamblia proteome: acyl-CoA synthetase long chain family member 4 (ACSL-4) and peroxin-4 (PEX-4). With confocal and immunoelectron microscopy using polyclonal antibodies both proteins were identified in cytoplasmic vesicles of trophozoites. Altogether, our results suggest for the first time the presence of peroxisomal-like proteins in the cytoplasm of G. lamblia., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Recently developed tissue-hydrogel methods for specimen expansion now enable researchers to perform super-resolution microscopy with ∼65 nm lateral resolution using ordinary microscopes, standard fluorescent probes, and inexpensive reagents. Here we use the combination of specimen expansion and the optical super-resolution microscopy technique structured illumination microscopy (SIM) to extend the spatial resolution to ∼30 nm. We apply this hybrid method, which we call ExSIM, to study the cytoskeleton of the important human pathogen Giardia lamblia including the adhesive disc and flagellar axonemes. We determined the localization of two recently identified disc-associated proteins, including DAP86676 , which localizes to disc microribbons, and the functionally unknown DAP16263 , which primarily localizes to dorsal microtubules of the disc overlap zone and the paraflagellar rod of ventral axonemes. Based on its strong performance in revealing known and unknown details of the ultrastructure of Giardia, we find that ExSIM is a simple, rapid, and powerful super-resolution method for the study of fixed specimens, and it should be broadly applicable to other biological systems of interest.

Flavohemoglobins are microbial enzymes that counter nitrosative stress, but the details of their underlying enzymatic activities and structure-function relationships are not completely understood. These enzymes have been identified in Gram-negative bacteria, certain fungi, and the parasitic protist Giardia intestinalis (gFlHb) which, despite lacking the ability to make heme, encodes several hemeproteins. To gain knowledge about the biophysical properties of the active site of gFlHb, we used resonance Raman spectroscopy to probe the wild-type protein and variants at globin domain positions E11, E7, and B10 on the distal, ligand-binding side of the heme. The heme of gFlHb has a peroxidase-like environment resembling that of the well-characterized E. coli flavohemoglobin HMP. We provide evidence that gFlHb has two Fe-His stretching modes, a feature that also occurs in type I/II-peroxidases in which a proximal histidine with strong imidazolate character and a nearby carboxylic acid residue can exist as a tautomeric pair depending on the position of a shared proton. Characterization of the distal variants Tyr30Phe, Gln54Leu, and Leu59Ala shows that TyrB10 and GlnE7 but not LeuE11 are implicated in stabilisation of bound exogenous ligands such as CO and O 2 . Our work revealed that several biophysical properties of the heme active site of gFlHb are highly conserved compared to HMP and suggest that they are conserved across the flavohemoglobin family.

Giardia duodenalis is a flagellated binucleated protozoan that colonizes the small intestine in mammals, causing giardiasis, acute or chronic diarrhea. DNA double strand break either endogenously or exogenously generated is a major insult to DNA and its repair by homologous recombination (HR) is crucial for genomic stability. During HR, Rad52 plays key roles in the loading of the Rad51 recombinase, and the annealing of the second double-strand break end to the displaced strand of the D-loop structure. Among the functions found in vitro in yeast and human Rad52 protein are: ssDNA or dsDNA binding activity, ability to anneal bare or RPA coated-ssDNA, as well as multimeric ring formation. In this work, we searched for conserved domains in a putative Rad52 protein from G. duodenalis (GdRad52). Its coding sequence was cloned, expressed and purified to study its biochemical properties. rGdRad52 binds to dsDNA and ssDNA, with greater affinity for the latter. Likewise, rGdRad52 promotes annealing of DNA uncoated and coated with GdRPA1. rGdRad52 interacts with GdDMC1B and with GdRPA1 protein as shown in far western blotting assay. Additionally, rGdRad52 formed multimeric rings as observed by electronic microscopy. Finally, GdRad52 is over expressed in response upon DNA damage inflicted on trophozoites., (© The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

To study prokaryotic expression and subcellular localization of α -13 giardin in Giardia lamblia trophozoites, α -13 giardin gene was amplified and cloned into prokaryotic expression vector pET-28a(+). The positive recombinant plasmid was transformed into E. coli BL21(DE3) for expression by using IPTG and autoinduction expression system (ZYM-5052). The target protein was validated by SDS-PAGE and Western blotting and purified by Ni-NTA Resin. Rabbits were immunized with purified fusion proteins for preparation of polyclonal antibody; then the intracellular location of α -13 giardin was determined by fluorescence immunoassay. The results showed that the length of α -13 giardin gene was 1038 bp, encoding a polypeptide of 345 amino acids. The expressed product was a fusion protein with about 40 kDa largely present in soluble form. The target protein accounted for 21.0% of total proteins after being induced with IPTG, while it accounted for 28.8% with ZYM-5052. The anti- α 13-giardin polyclonal antibody possessed good antigenic specificity as well as excellent binding activity with recombinant α -13 giardin. Immunofluorescence assays revealed that α -13 giardin was localized in the cytoplasm of G. lamblia trophozoite, suggesting that it is a cytoplasm-associated protein. The present study may lay a foundation for further functional research on α -13 giardin of G. lamblia ., Competing Interests: The authors declare that they have no competing interests.

Giardiasis is widely acknowledged to be a neglected disease in need of new therapeutics to address toxicity and resistance issues associated with the limited available treatment options. We examined seven protein kinases in the Giardia lamblia genome that are predicted to share an unusual structural feature in their active site. This feature, an expanded active site pocket resulting from an atypically small gatekeeper residue, confers sensitivity to "bumped" kinase inhibitors (BKIs), a class of compounds that has previously shown good pharmacological properties and minimal toxicity. An initial phenotypic screen for biological activity using a subset of an in-house BKI library found that 5 of the 36 compounds tested reduced trophozoite growth by at least 50% at a concentration of 5 μM. The cellular localization and the relative expression levels of the seven protein kinases of interest were determined after endogenously tagging the kinases. Essentiality of these kinases for parasite growth and infectivity were evaluated genetically using morpholino knockdown of protein expression to establish those that could be attractive targets for drug design. Two of the kinases were critical for trophozoite growth and attachment. Therefore, recombinant enzymes were expressed, purified and screened against a BKI library of >400 compounds in thermal stability assays in order to identify high affinity compounds. Compounds with substantial thermal stabilization effects on recombinant protein were shown to have good inhibition of cell growth in wild-type G. lamblia and metronidazole-resistant strains of G. lamblia. Our data suggest that BKIs are a promising starting point for the development of new anti-giardiasis therapeutics that do not overlap in mechanism with current drugs., Competing Interests: The authors have declared that no competing interests exist.

To study subcellular localization of α18- and α12-giardin in Giardia lamblia trophozoites, the α18- and α12-giardin genes were amplified from G. lamblia assemblage A, respectively. The PCR products were cloned into the prokaryotic expression vector pET-28a(+), and the positive recombinant plasmids were transformed into E. coli Rosetta (DE3) strain for the expression, and expressed α18- and α12-giardin fusion protein were purified by Ni-Agarose resin, respectively. Mice were immunized with purified fusion proteins for preparation of polyclonal antibody, and then the subcellular localization of α18- and α12-giardin was determined by fluorescence immunoassay. Results showed that the concentrations of purified α18- and α12-giardin fusion proteins were 1.20 and 0.86 mg/ml, respectively. The titers of anti-α18- and anti-α12-giardin polyclonal antibody were both as high as 1:25600 dilutions. Immunofluorescent analysis showed that α18- and α12-giardin proteins were mainly localized at four pairs of flagella and the cytoplasm of G. lamblia trophozoites, suggesting that α18- and α12-giardin are the flagella and cytoplasm-associated proteins, respectively. The above information would lay the foundation for research about the crystal structure and biological function of α18- and α12-giardin.

Giardia lamblia is a protozoan that causes diarrheal diseases in humans. Cytoskeletal structures of Giardia trophozoites must be finely reorganized during cell division. To identify Giardia proteins which interact with microtubules (MTs), Giardia lysates were incubated with in vitro-polymerized MTs and then precipitated by ultracentifugation. A hypothetical protein (GL50803_8405) was identified in the precipitated fraction with polymerized MTs and was named GlMBP1 (G. lamblia microtubule-binding protein 1). Interaction of GlMBP1 with MTs was confirmed by MT binding assays using recombinant GlMBP1 (rGlMBP1). In vivo expression of GlMBP1 was shown by a real-time PCR and western blot analysis using anti-rGlMBP1 antibodies. Transgenic G. lamblia trophozoites were constructed by integrating a chimeric gene encoding hemagglutinin (HA)-tagged GlMBP1 into a Giardia chromosome. Immunofluorescence assays of this transgenic G. lamblia, using anti-HA antibodies, revealed that GlMBP1 mainly localized at the basal bodies, axonemes, and median bodies of G. lamblia trophozoites. This result indicates that GlMBP1 is a component of the G. lamblia cytoskeleton., Competing Interests: We have no conflict of interest related to this work.

Introduction: Giardia intestinalis is an early divergent organism that was recently shown to have introns. The machinery responsible for the removal of introns in higher eukaryotes is the spliceosome, which consists of five ribonucleoproteins. Each of these ribonucleoproteins has a small nuclear RNA, a set of seven Sm proteins (B, D1, D2, D3, E, F and G) and several specific proteins. Some genes that encode spliceosome proteins have been bioinformatically identified in the parasite genome. Although it is assumed that the spliceosome is responsible for splicing in this parasite, biochemical characterization is lacking. Objective. To inhibit two G. intestinalis spliceosome protein genes in order to determine whether this inhibition affects parasite growth or encystation. Materials and methods. Antisense sequences of the genes encoding the spliceosomal parasite proteins SmB and SmD3 were cloned into a specific G. intestinalis vector. G. intestinalis individuals were subsequently transfected with the recombinant vectors and those parasites that incorporated the vector were selected. A decrease in mRNA levels by real-time PCR was confirmed and the growth and encystation in wild and transfected parasites was assessed. Results. A decrease of 40% and 70% of SmB and SmD3 mRNA levels, respectively, was observed. Growth and encystation in these parasites were not affected. Conclusion. Decrease of SmB and SmD3 mRNA levels does not affect the parasite, indicating that the spliceosome remains functional or that splicing is not essential for parasite viability.

Among the few organisms that cannot make the iron cofactor heme, some nonetheless possess heme proteins. This includes the protozoan parasite Giardia intestinalis, which encodes five known heme proteins: a flavohemoglobin and four members of the cytochrome b5 family. Giardia flavohemoglobin closely resembles those of the Enterobacteriaceae in structure and function, acting as a nitric oxide dioxygenase that is induced when trophozoites are exposed to reactive nitrogen species. The Giardia cytochromes b5 are soluble proteins having relatively low reduction potentials and lack several features that are expected to promote rapid electron transfer with redox partners. Only one potential electron donor, and no electron acceptors, have yet been identified in the Giardia genome, and the roles of these cytochromes are presently unknown. The answer may lie in the sequences that flank the heme-binding core of these proteins which could serve to localize them within the cell through reversible post-translational modifications and to promote specific protein-protein interactions., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Several series of tests have shown that fresh, intact samples of Giardia duodenalis and Cryptosporidium parvum (oo)cysts are not marked by fluorescent probes such as carboxyfluorcein-succinimidyl-diacetate-ester (CFDA-SE), C12-resazurin and SYTOX® Green, probably because of their robust cell walls. These dyes fail to indicate the viability of such protozoa and allow negative responses to be recorded from living and infectious samples. Cryptosporidium parvum showed stronger isolation from chemicals, with living oocysts remaining unstained by the probe for up to 90 days after extraction. However, in further fluorescence decay (FD) experiments run with G. duodenalis samples stained using CFDA-SE (comprising living, non-stressed but aged cysts, heat-killed samples and UV-C-stressed samples) each showed a different FD decay profile, here studied in seven series of tests of five replicates each. The FD profiles were fitted by double-exponential decay kinetics, with the decay constant k2 being five times higher than k1. This FD procedure is fast and can be easily reproduced in 10 steps, taking ~ 1 h of laboratory work for already purified samples., (Copyright © 2015 John Wiley & Sons, Ltd.)

Superoxide reductase (SOR), which is commonly found in prokaryotic organisms, affords protection from oxidative stress by reducing the superoxide anion to hydrogen peroxide. The reaction is catalyzed at the iron centre, which is highly conserved among the prokaryotic SORs structurally characterized to date. Reported here is the first structure of an SOR from a eukaryotic organism, the protozoan parasite Giardia intestinalis (GiSOR), which was solved at 2.0 Å resolution. By collecting several diffraction data sets at 100 K from the same flash-cooled protein crystal using synchrotron X-ray radiation, photoreduction of the iron centre was observed. Reduction was monitored using an online UV-visible microspectrophotometer, following the decay of the 647 nm absorption band characteristic of the iron site in the glutamate-bound, oxidized state. Similarly to other 1Fe-SORs structurally characterized to date, the enzyme displays a tetrameric quaternary-structure arrangement. As a distinctive feature, the N-terminal loop of the protein, containing the characteristic EKHxP motif, revealed an unusually high flexibility regardless of the iron redox state. At variance with previous evidence collected by X-ray crystallography and Fourier transform infrared spectroscopy of prokaryotic SORs, iron reduction did not lead to dissociation of glutamate from the catalytic metal or other structural changes; however, the glutamate ligand underwent X-ray-induced chemical changes, revealing high sensitivity of the GiSOR active site to X-ray radiation damage.

Despite lacking mitochondria and a known pathway for heme biosynthesis the micro-aerotolerant anaerobic protozoan parasite Giardia intestinalis encodes four members of the cytochrome b5 family of electron transfer proteins, three of which are small, single-domain proteins. While these are similar in size and fold to their better-known mammalian counterparts the Giardia proteins have distinctly lower reduction potentials, ranging from -140 to -171 mV compared to +6 mV for the bovine microsomal protein. This difference is accounted for by a more polar heme environment in the Giardia proteins, as mutation of a conserved heme pocket tyrosine residue to phenylalanine in the Giardia cytochrome b5 isotype-I (gCYTb5-I Y61F) raises its reduction potential by nearly 100 mV. All three isotypes have UV-visible spectra consistent with axial coordination of the heme by a pair of histidine residues, but electron paramagnetic spectroscopy indicates that the planes of their imidazole rings are nearly perpendicular rather than coplanar as observed in mammalian cytochrome b5, which may be due to geometrical constraints imposed by a one-residue shorter spacing between the ligand pair in the Giardia proteins. Although no function has yet to be ascribed to any Giardia cytochrome b5, the presence of similar sequences in many other eukaryotes indicates that these represent an under-characterized class of low reduction potential family members., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Gastrointestinal infection due to intestinal parasites is an enormous health problem in developing countries and its reliable diagnosis is demanding. Therefore, this study aimed at evaluating a commercially available immunochromatographic assay (ICA) for the detection of cryptosporidia, Giardia duodenalis, and Entamoeba histolytica/dispar for its usefulness in the Greater Cairo Region, Egypt. Stool samples of 104 patients who presented between October 2012 and March 2013 with gastrointestinal symptoms or for the exclusion of parasites at Kasr-Al-Ainy University Medical School were examined by light microscopy of wet mounts and the triple ICA. Microscopy revealed in 20% of the patients [95% confidence interval (CI), 13.5-29.0%] parasites with Hymenolepis nana, E. histolytica/dispar and Blastocystis hominis being the most frequent ones, but was not able to detect G. duodenalis and cryptosporidia, whereas ICA was positive in 21% (95% CI, 14.3-30.0%) and detected E. histolytica/dispar in 12.5% (95% CI, 7.3-20.4%), cryptosporidia in 6.7% (95% CI, 3.1-13.5%) and G. duodenalis in 15.4% (95% CI, 9.6-23.6%) of the patients. Detection of one or more pathogens was associated with access to water retrieved from a well or pump (p = 0.01). Patients between 20 and 29 years of age (p = 0.08) and patients with symptoms of 5 days or longer (p = 0.07) tended to have a higher risk to be infected than patients of other age groups or with shorter-lasting symptoms. In conclusion, the ICA was easy to perform and timesaving. Importantly, it enabled the detection of cryptosporidia, which cannot be found microscopically in unstained smears, demonstrated a higher sensitivity for the detection of G. duodenalis than microscopy, and was more specific for distinguishing E. histolytica/dispar from apathogenic amoeba.

Giardia intestinalis parasites contain mitosomes, one of the simplest mitochondrion-related organelles. Strategies to identify the functions of mitosomes have been limited mainly to homology detection, which is not suitable for identifying species-specific proteins and their functions. An in vivo enzymatic tagging technique based on the Escherichia coli biotin ligase (BirA) has been introduced to G. intestinalis; this method allows for the compartment-specific biotinylation of a protein of interest. Known proteins involved in the mitosomal protein import were in vivo tagged, cross-linked, and used to copurify complexes from the outer and inner mitosomal membranes in a single step. New proteins were then identified by mass spectrometry. This approach enabled the identification of highly diverged mitosomal Tim44 (GiTim44), the first known component of the mitosomal inner membrane translocase (TIM). In addition, our subsequent bioinformatics searches returned novel diverged Tim44 paralogs, which mediate the translation and mitosomal insertion of mitochondrially encoded proteins in other eukaryotes. However, most of the identified proteins are specific to G. intestinalis and even absent from the related diplomonad parasite Spironucleus salmonicida, thus reflecting the unique character of the mitosomal metabolism. The in vivo enzymatic tagging also showed that proteins enter the mitosome posttranslationally in an unfolded state and without vesicular transport., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The structures of three aspartate aminotransferases (AATs) from eukaryotic pathogens were solved within the Seattle Structural Genomics Center for Infectious Disease (SSGCID). Both the open and closed conformations of AAT were observed. Pyridoxal phosphate was bound to the active site via a Schiff base to a conserved lysine. An active-site mutant showed that Trypanosoma brucei AAT still binds pyridoxal phosphate even in the absence of the tethering lysine. The structures highlight the challenges for the structure-based design of inhibitors targeting the active site, while showing options for inhibitor design targeting the N-terminal arm.

The prevalence of Giardia duodenalis in humans is partly owed to its direct and simple life cycle, as well as the formation of the environmentally resistant and infective cysts. Proteomic and transcriptomic studies have previously analysed the encystation process using the well-characterised laboratory genomic strain, WB C6. This study presents the first quantitative study of encystation using pathogenically relevant and alternative assemblage A strains: the human-derived BRIS/82/HEPU/106 (H-106)and avian-derived BRIS/95/HEPU/2041 (B-2041). We utilised tandem MS/MS with a label-free quantitative approach to compare cysts and trophozoite life stages for strain variation, as well as confirm universal encystation markers of assemblage A. A total of 1061 non-redundant proteins were identified from both strains, including trophozoite- and cyst-specific proteomes and life-stage differentially expressed proteins. Additionally, 24 proteins previously classified in the literature as encystation-specific were confirmed as strain-independent markers of encystation. Functional cluster analysis of differentially expressed proteins saw significant overlap between strains, including protein trafficking and localisation in cysts, NEK kinase function, and carbohydrate metabolism in trophozoites. Two significant points of strain specific adaptations in cysts were also identified. B-2041 possessed major up-regulation of the ankyrin repeat protein 21.1 family compared to H-106. Furthermore, cysts of B-2041 retained near-complete VSP variant diversity between cysts and trophozoites, while H-106 lost 45% of its VSP variant diversity between life cycle stages, a constriction previously observed in studies of WB C6. This is the first report of strain variation in the cyst stage in G. duodenalis, and highlights cyst variation and its impacts on reinfection and life cycle success., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Giardia duodenalis is a well-known protozoan parasite of humans and other mammals. The repair of DNA double strand breaks (DSBs) is crucial for genomic stability and homologous recombination is one of the primary mechanisms used by cells to repair DNA. The Mre11 complex is comprised by Mre11, an endonuclease and 3'-5' exonuclease known to resect ends during homologous recombination, and Rad50, a member of the structural maintenance of chromosomes (SMC) family of ATPases. In this work we cloned, expressed and characterized the catalytic activities of the giardial Mre11 (GdMre11) and Rad50 (GdRad50) proteins. Our results show that while purified recombinant GdMre11 and GdRad50 proteins bind DNA, GdMre11 contains a 3'-5' exonuclease and purified recombinant GdRad50 has ATPase activity. The predicted structure for GdMre11 revealed a conserved Mn(2+) dependent binding pocket. We also explored the expression of giardial mre11 and rad50 genes after ionizing radiation, and our results indicate that both specific transcripts were increased after 1-2 h while their protein levels were found to be significantly increased 4 h after gamma radiation treatment. These proteins were localized in the nuclei before and after irradiation. The implication of these observations is discussed., (Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Rapid and sensitive detection of waterborne pathogens in drinkable and recreational water sources is crucial for treating and preventing the spread of water related diseases, especially in resource-limited settings. Here we present a field-portable and cost-effective platform for detection and quantification of Giardia lamblia cysts, one of the most common waterborne parasites, which has a thick cell wall that makes it resistant to most water disinfection techniques including chlorination. The platform consists of a smartphone coupled with an opto-mechanical attachment weighing ~205 g, which utilizes a hand-held fluorescence microscope design aligned with the camera unit of the smartphone to image custom-designed disposable water sample cassettes. Each sample cassette is composed of absorbent pads and mechanical filter membranes; a membrane with 8 μm pore size is used as a porous spacing layer to prevent the backflow of particles to the upper membrane, while the top membrane with 5 μm pore size is used to capture the individual Giardia cysts that are fluorescently labeled. A fluorescence image of the filter surface (field-of-view: ~0.8 cm(2)) is captured and wirelessly transmitted via the mobile-phone to our servers for rapid processing using a machine learning algorithm that is trained on statistical features of Giardia cysts to automatically detect and count the cysts captured on the membrane. The results are then transmitted back to the mobile-phone in less than 2 minutes and are displayed through a smart application running on the phone. This mobile platform, along with our custom-developed sample preparation protocol, enables analysis of large volumes of water (e.g., 10-20 mL) for automated detection and enumeration of Giardia cysts in ~1 hour, including all the steps of sample preparation and analysis. We evaluated the performance of this approach using flow-cytometer-enumerated Giardia-contaminated water samples, demonstrating an average cyst capture efficiency of ~79% on our filter membrane along with a machine learning based cyst counting sensitivity of ~84%, yielding a limit-of-detection of ~12 cysts per 10 mL. Providing rapid detection and quantification of microorganisms, this field-portable imaging and sensing platform running on a mobile-phone could be useful for water quality monitoring in field and resource-limited settings.

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.

The 14-3-3s are a family of dimeric evolutionary conserved pSer/pThr binding proteins that play a key role in multiple biological processes by interacting with a plethora of client proteins. Giardia duodenalis is a flagellated protozoan that affects millions of people worldwide causing an acute and chronic diarrheal disease. The single giardial 14-3-3 isoform (g14-3-3), unique in the 14-3-3 family, needs the constitutive phosphorylation of Thr214 and the polyglycylation of its C-terminus to be fully functional in vivo. Alteration of the phosphorylation and polyglycylation status affects the parasite differentiation into the cyst stage. To further investigate the role of these post-translational modifications, the crystal structure of the g14-3-3 was solved in the unmodified apo form. Oligomers of g14-3-3 were observed due to domain swapping events at the protein C-terminus. The formation of filaments was supported by TEM. Mutational analysis, in combination with native PAGE and chemical cross-linking, proved that polyglycylation prevents oligomerization. In silico phosphorylation and molecular dynamics simulations supported a structural role for the phosphorylation of Thr214 in promoting target binding. Our findings highlight unique structural features of g14-3-3 opening novel perspectives on the evolutionary history of this protein family and envisaging the possibility to develop anti-giardial drugs targeting g14-3-3.

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., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Giardia lamblia is a protozoan parasite that presents both trophozoite and cyst forms. In this study, the distribution of the different sugar residues and the origin of the carbohydrate components of the cyst wall were studied using transmission electron microscopy, ultrastructural cytochemistry for carbohydrate detection and immunocytochemistry. Immunofluorescence microscopy using anti-cyst wall protein 1 (CWP1) and gold- and fluorescent-conjugated lectins, such as WGA and DBA, were also used. Interestingly, a population of carbohydrate-containing vesicles, distinct from the encystation-specific vesicles (ESVs) was found in the encysting cells and was named encystation carbohydrate-positive vesicles (ECVs). The differences between the ECVs and the ESVs were: (1) they are electron-translucent, whereas ESVs are electron dense; (2) they do not react with antibodies against cyst wall proteins; (3) the contents are positive for carbohydrates, whereas ESVs display a negative reaction; and (4) they exhibit a positive labeling for DBA indicating the presence of N-acetyl-galactosamine, whereas ESVs are negative. To evaluate if ECVs could be vesicles involved in the endocytic pathway, endocytic markers were used. No co-localization of these markers with ECVs was observed. We suggest that the ECVs may represent a new structure involved in cyst wall formation., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

Force-separation measurements between Giardia lamblia cysts and an inorganic oxide (silicate glass) have been obtained by using an atomic force microscope (AFM). The cysts are compressible on the scale of the loads applied during force measurement, with the surface compressibility expressed in terms of an interfacial spring constant (K(int)). The force of interaction prior to this Hookean region, on approach, is long-range and repulsive. The long-range force has been compared to models of the electrical double layer as well as an electrosteric layer. The comparison has led to the conclusion that the cyst surface can be described as a polyelectrolyte brush at intermediate separations (5-115 nm from linear compliance) with an electrical double layer often observed at larger separations. The dependence of the interaction force on surface retraction suggests that tethering between the cyst and siliceous surface can occur. The variation of the interaction with pH and upon variation with ionic strength has also been assessed. The information gained from the measurement of the interaction between G. lamblia and this model sandlike surface informs water treatment processes. Similar studies have been performed by us for the Cryptosporidium parvum (C. parvum) oocyst system to which this work is compared.

Objective: To construct rapidly a full-length cDNA library from nanogram amounts total RNA of Giardia lamblia (G. lamblia) trophozoites stocked in RNA stabilization reagent., Methods: Total RNA of Giardia was extracted using Trizol reagent. A full-length cDNA library of G. lamblia trophozoites was constructed by a long-distance PCR (LD-PCR) method. The recombinant rate and the coverage rate of full-length clones of the library were evaluated. The inserted fragments were identified and sequenced by PCR amplification., Results: The titer of cDNA library was 3.85 × 10(7) pfu/mL. The length of inserted fragments ranged from 0.4 to 2.5 kb, and the recombination efficiency accounted for 100% (20/20). The coverage rate of full-length clones is high (17/20)., Conclusions: The RNA stabilization reagent may be used to fix the cells and prevent the RNA in cells even though delivered under normal atmospheric temperature. The long-distance PCR can be used to construct a full-length cDNA library rapidly and it needs less RNA than the traditional method from mRNA., (Copyright © 2012 Hainan Medical College. Published by Elsevier B.V. All rights reserved.)

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., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The genome of the human intestinal parasite Giardia lamblia contains only a single aminoacyl-tRNA synthetase gene for each amino acid. The Giardia prolyl-tRNA synthetase gene product was originally misidentified as a dual-specificity Pro/Cys enzyme, in part owing to its unexpectedly high off-target activation of cysteine, but is now believed to be a normal representative of the class of archaeal/eukaryotic prolyl-tRNA synthetases. The 2.2 Å resolution crystal structure of the G. lamblia enzyme presented here is thus the first structure determination of a prolyl-tRNA synthetase from a eukaryote. The relative occupancies of substrate (proline) and product (prolyl-AMP) in the active site are consistent with half-of-the-sites reactivity, as is the observed biphasic thermal denaturation curve for the protein in the presence of proline and MgATP. However, no corresponding induced asymmetry is evident in the structure of the protein. No thermal stabilization is observed in the presence of cysteine and ATP. The implied low affinity for the off-target activation product cysteinyl-AMP suggests that translational fidelity in Giardia is aided by the rapid release of misactivated cysteine.

Immunolocalization of β- and δ-giardin in Giardia duodenalis trophozoites revealed that both giardins are strictly associated with the ventral disk (VD). Optical sectioning of the immunolabeled VD, together with quantitative colocalization of δ- and β-giardin immunoreactivity, demonstrated that δ-giardin is primarily localized to the ventral side, and β-giardin is localized to the dorsal side of the VD.

Dicer is a multidomain enzyme that generates small RNAs for gene silencing in eukaryotes. Current understanding of Dicer structure is restricted to simple forms of the enzyme, whereas that of the large and complex Dicer in metazoans is unknown. Here we describe a new domain localization strategy developed to determine the structure of human Dicer by EM. A rearrangement of the nuclease core, compared to the archetypal Giardia lamblia Dicer, explains how metazoan Dicers generate products that are 21-23 nucleotides in length. The helicase domains form a clamp-like structure adjacent to the RNase III active site, facilitating recognition of pre-miRNA loops or translocation on long dsRNAs. Drosophila melanogaster Dicer-2 shows similar features, revealing that the three-dimensional architecture is conserved. These results illuminate the structural basis for small RNA production in eukaryotes and provide a versatile new tool for determining structures of large molecular machines.

Giardia intestinalis is a ubiquitous parasitic protist that is the causative agent of giardiasis, one of the most common protozoan diarrheal diseases in the world. Giardia trophozoites attach to the intestinal epithelium using a specialized and elaborate microtubule structure, the ventral disc. Surrounding the ventral disc is a less characterized putatively contractile structure, the lateral crest, which forms a continuous perimeter seal with the substrate. A better understanding of ventral disc and lateral crest structure, conformational dynamics, and biogenesis is critical for understanding the mechanism of giardial attachment to the host. To determine the components comprising the ventral disc and lateral crest, we used shotgun proteomics to identify proteins in a preparation of isolated ventral discs. Candidate disc-associated proteins, or DAPs, were GFP-tagged using a ligation-independent high-throughput cloning method. Based on disc localization, we identified eighteen novel DAPs, which more than doubles the number of known disc-associated proteins. Ten of the novel DAPs are associated with the lateral crest or outer edge of the disc, and are the first confirmed components of this structure. Using Fluorescence Recovery After Photobleaching (FRAP) with representative novel DAP::GFP strains we found that the newly identified DAPs tested did not recover after photobleaching and are therefore structural components of the ventral disc or lateral crest. Functional analyses of the novel DAPs will be central toward understanding the mechanism of ventral disc-mediated attachment and the mechanism of disc biogenesis during cell division. Since attachment of Giardia to the intestine via the ventral disc is essential for pathogenesis, it is possible that some proteins comprising the disc could be potential drug targets if their loss or disruption interfered with disc biogenesis or function, preventing attachment.

Dielectrophoresis (DEP) is the motion of polarizable particles in the presence of nonuniform electric fields. This novel electrokinetic technique has successfully been employed in many miniaturized systems for the manipulation and detection of microbes. This review article depicts the application of dielectrophoresis for the monitoring of microorganisms in microfluidic devices for environmental applications. The research studies described here are mainly conceived for water- and air-monitoring assessments, and are classified considering the target aimed to detect, concentrate, and/or separate, including chemical and toxicant agents, and microorganisms ranging from virus to protozoa. Dielectrophoresis has also played an important role in biofilm formation studies. This review article comprises mainly studies published from 2000 to present. Even in this relatively short time frame, there have been many significant contributions of this powerful and nascent technique related to environmental monitoring; thus, unveiling its great potential for future research directions., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (Published by Elsevier Ltd.)

A specific fatty acid binding protein was isolated from Giardia lamblia, using an affinity column with butyric acid acting as a ligand in place of stearic acid. This method has proved to be more efficient than the one previously described using stearic acid as ligand. The purified fraction showed 8 electrophoretic bands of proteins, with molecular weights ranging between 8 and 80 kDa. This pattern is a consequence of the aggregation of a protein with a molecular weight of 8,215 Da, corresponding to the lower molecular weight band, the only one capable of binding to fatty acids. The labeled oleic acid bound to these purified proteins was replaced by a 100-fold greater concentration of taurocholate, glycocholate, deoxycholate, palmitic acid, and arachidonic acid, having a greater displacement of the bile salts than the free fatty acids.

The morphology of the endomembrane system of Giardia lamblia appears to be significantly different from higher eukaryotes. Therefore, the molecular mechanisms controlling vesicular trafficking are also likely to be altered. Since FYVE domain is a known regulator of endosomal trafficking, the authors used BLAST search to identify FYVE domain(s) in G. lamblia. A 990 amino acid long putative FYVE domain-containing ORF was identified, which contains all the conserved sequence elements in the ligand binding pocket. Phylogenetic analysis reveals that this domain is significantly diverged. The authors have shown that the corresponding gene is expressed in G. lamblia trophozoites and cysts. In spite of this phylogenetic divergence, in vitro biochemical assay indicates that this domain preferentially binds to phosphatidylinositol 3-phosphate {PtdIns(3)P}and in vivo expression of the GFP-tagged G. lamblia FYVE domain in S. cerevisiae, displays its selective localization to PtdIns(3)P-enriched endosomes. This is the first study to characterize a PtdIns(3)P effector protein in this early-diverged eukaryote.

As Giardia lamblia is unable to synthesize cholesterol de novo, this steroid might be obtained from the host's intestinal milieu by endocytosis of lipoproteins. In this work, we identified a putative Giardia lamblia low-density lipoprotein receptor-related proteins (GlLRP), a type I membrane protein, which shares the substrate N-terminal binding domain and a FXNPXY-type endocytic motif with human LRPs. Expression of tagged GlLRP showed that it was localized predominantly in the endoplasmic reticulum, lysosomal-like peripheral vacuoles and plasma membrane. However, the FXNPXY-deleted GlLRP was retained at the plasma membrane suggesting that it is abnormally transported and processed. The low-density lipoprotein and chylomicrons interacted with GlLRP, with this interaction being necessary for lipoprotein internalization and cell proliferation. Finally, we show that GlLRP binds directly to the medium subunit of Giardia adaptor protein 2, indicating that receptor-mediated internalization occurs through an adaptin mechanism., (© 2011 Blackwell Publishing Ltd.)

Alpha8-giardin (α8-giardin) is a member of the multi-gene α-giardin family in the intestinal parasitic protozoan, Giardia lamblia. This gene family shares an ancestry with the annexin super family, whose common characteristic is calcium dependent binding to membranes that contain acidic phospholipids. In the present study, the antigenicity, hydrophilicity, flexibility, surface probability, and secondary structure of α8-giardin amino acids were predicted by bioinformatics applications. A specific anti-peptide antiserum, anti-P3, was used to determine the intracellular location of α8-giardin with confocal immunofluorescence microscopy and immunoelectron microscopy. The results indicated that α8-giardin was located on the plasma membrane and flagella, but not on the ventral disk. Reduction of α8-giardin transcript levels by ribozyme-mediated cleavage decreased trophozoite motility and growth rate, indicating the functional importance of α8-giardin to Giardia trophozoite biology.

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 beta-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 (approximately 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 (approximately 1.2 microm), 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.

Background: Giardia lamblia parasitizes the human small intestine to cause diarrhea and malabsorption. It undergoes differentiation from a pathogenic trophozoite form into a resistant walled cyst form. Few cyst proteins have been identified to date, including three cyst wall proteins (CWPs) and one High Cysteine Non-variant Cyst protein (HCNCp). They are highly expressed during encystation and are mainly targeted to the cyst wall., Methodology and Principal Findings: To identify new cyst wall proteins, we searched the G. lamblia genome data base with the sequence of the Cryptosporidium parvum oocyst wall protein as a query and found an Epidermal Growth Factor (EGF)-like Cyst Protein (EGFCP1). Sequence analysis revealed that the EGF-like repeats of the EGFCP1 are similar to those of the tenascin family of extracellular matrix glycoproteins. EGFCP1 and HCNCp have a higher percentage of cysteine than CWPs, but EGFCP1 has no C-terminal transmembrane region found in HCNCp. Like CWPs and HCNCp, the EGFCP1 protein (but not transcript) was expressed at higher levels during encystation and it was localized to encystation-specific vesicles in encysting trophozoites. Like HCNCp, EGFCP1 was localized to the encystation-specific vesicles, cyst wall and cell body of cysts, suggesting that they may share a common trafficking pathway. Interestingly, overexpression of EGFCP1 induced cyst formation and deletion of the signal peptide from EGFCP1 reduced its protein levels and cyst formation, suggesting that EGFCP1 may help mediate cyst wall synthesis. We also found that five other putative EGFCPs have similar expression profiles and similar locations and that the cyst formation was induced upon their overexpression., Conclusions and Significance: Our results suggest that EGFCPs may function like cyst wall proteins, involved in differentiation of G. lamblia trophozoites into cysts. The results lead to greater understanding of parasite cyst walls and provide valuable information that helps develop ways to interrupt the G. lamblia life cycle.