Identification of an immunogenic protein of Giardia lamblia using monoclonal antibodies generated from infected mice

The humoral immune response plays an important role in the clearance of Giardia lamblia. However, our knowledge about the specific antigens of G. lamblia that induce a protective immune response is limited. The purpose of this study was to identify and characterise the immunogenic proteins of G. lamblia in a mouse model. We generated monoclonal antibodies (moAbs) specific to G. lamblia (1B10, 2C9.D11, 3C10.E5, 3D10, 5G8.B5, 5F4, 4C7, 3C5 and 3C6) by fusing splenocytes derived from infected mice. Most of these moAbs recognised a band of ± 71 kDa (5G8 protein) and this protein was also recognised by serum from the infected mice. We found that the moAbs recognised conformational epitopes of the 5G8 protein and that this antigen is expressed on the cell surface and inside trophozoites. Additionally, antibodies specific to the 5G8 protein induced strong agglutination (> 70-90%) of trophozoites. We have thus identified a highly immunogenic antigen of G. lamblia that is recognised by the immune system of infected mice. In summary, this study describes the identification and partial characterisation of an immunogenic protein of G. lamblia. Additionally, we generated a panel of moAbs specific for this protein that will be useful for the biochemical and immunological characterisation of this immunologically interesting Giardia molecule.     

Cardiolipin, a lipid found in mitochondria, hydrogenosomes and bacteria was not detected in Giardia lamblia

Giardia lamblia is a protozoan parasite with many characteristics common among eukaryotic cells, but lacking other features found in most eukaryotes. Cardiolipin is a phospholipid located exclusively in energy transducing membranes and it was identified in mitochondria, bacteria, hydrogenosomes and chloroplasts. In eukaryotes, cardiolipin is the only lipid that is synthesized in the mitochondria. Biochemical procedures (TLC, HPLC) and fluorescent tools (NAO) were applied in order to search for cardiolipin in G. lamblia. In addition, BLAST searches were used to find homologs of enzymes that participate in the cardiolipin synthesis. Cardiolipin synthase was searched in the Giardia genome, using Saccharomyces cerevisiae and Mycoplasma penetrans sequences as bait. However, a good match to G. lamblia related proteins was not found. Here we show that mitosomes of G. lamblia apparently do not contain cardiolipin, which raises the discussion for its endosymbiotic origin and for the previous proposal that Giardia mitosomes are modified mitochondria.     

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.     

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.     

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.     

An Improved PCR-RFLP Assay for Detection and Genotyping of Asymptomatic Giardia lamblia Infection in a Resource-Poor Setting

Laboratory workers, in resource-poor countries, still consider PCR detection of Giardia lamblia more costly and more time-consuming than the classical parasitological techniques. Based on 2 published primers, an in-house one-round touchdown PCR-RFLP assay was developed. The assay was validated with an internal amplification control included in reactions. Performance of the assay was assessed with DNA samples of various purities, 91 control fecal samples with various parasite load, and 472 samples of unknown results. Two cysts per reaction were enough for PCR detection by the assay with exhibited specificity (Sp) and sensitivity (Se) of 100% and 93%, respectively. Taking a published small subunit rRNA reference PCR test results (6%; 29/472) as a nominated gold standard, G. lamblia was identified in 5.9% (28/472), 5.2%, (25/472), and 3.6% (17/472) by PCR assay, RIDA^® Quick Giardia antigen detection test (R-Biopharm, Darmstadt, Germany), and iodine-stained smear microscopy, respectively. The percent agreements (kappa values) of 99.7% (0.745), 98.9% (0.900), and 97.7% (0.981) were exhibited between the assay results and that of the reference PCR, immunoassay, and microscopy, respectively. Restriction digestion of the 28 Giardia-positive samples revealed genotype A pattern in 12 and genotype B profile in 16 samples. The PCR assay with the described format and exhibited performance has a great potential to be adopted in basic clinical laboratories as a detection tool for G. lamblia especially in asymptomatic infections. This potential is increased more in particular situations where identification of the parasite genotype represents a major requirement as in epidemiological studies and infection outbreaks.     

Phosphorylation of Serine 148 in Giardia lamblia End‐binding 1 Protein is Important for Cell Division

Giardia lamblia is a unicellular organism, showing a polarity with two nuclei and cytoskeletal structures. Accurate positioning of these organelles is essential for division of G. lamblia, which is poorly understood. Giardia lamblia end‐binding 1 (GlEB1) protein and G. lamblia aurora kinase (GlAK) have been shown to modulate microtubule (MT) distribution during cytokinesis. A direct association between GlEB1 and GlAK was demonstrated. Like GlEB1, GlAK was also found at nuclear envelopes and median bodies of G. lamblia. In vitro kinase assays using Giardia lysates immunoprecipitated with anti‐GlAK antibodies or recombinant GlAK suggested that GlEB1 is a substrate of GlAK. Site‐directed mutagenesis indicated that threonine‐205 in GlAK was auto‐phosphorylated and that GlAK phosphorylated serine (Ser)‐148 in GlEB1. Ectopic expression of a mutant GlEB1 (with conversion of Ser‐148 into alanine of GlEB1) resulted in an increased number of Giardia cells with division defects. Treatment of G. lamblia with an AK inhibitor triggered cytokinesis defects, and ectopic expression of a phospho‐mimetic mutant GlEB1 (with conversion of Ser‐148 into aspartate) rescued the defects in Giardia cell division caused by the AK inhibitor. These results suggested that phosphorylation of GlEB1 played a role in cytokinesis in G. lamblia.     

Macrophages expressing arginase 1 and nitric oxide synthase 2 accumulate in the small intestine during Giardia lamblia infection

Nitric oxide (NO) has been shown to inhibit Giardia lamblia in vitro and in vivo. This study sought to determine if Giardia infection induces arginase 1 (ARG1) expression in host macrophages to reduce NO production. Stimulations of RAW 264.7 macrophage-like cells with Giardia extract induced arginase activity. Real-time PCR and immunohistochemistry showed increased ARG1 and nitric oxide synthase 2 (NOS2) expression in mouse intestine following infection. Flow cytometry demonstrated increased numbers of macrophages positive for both ARG1 and NOS2 in lamina propria following infection, but there was no evidence of increased expression of ARG1 in these cells.     

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.     

Genotyping of Giardia in Dutch patients and animals: a phylogenetic analysis of human and animal isolates

Giardia duodenalis (syn. Giardia lamblia, Giardia intestinalis) is a protozoan organism that can infect the intestinal tract of many animal species including mammals. Genetic heterogeneity of G. duodenalis is well described but the zoonotic potential is still not clear. In this study, we analysed 100 Giardia DNA samples directly isolated from human stool specimens, to get more insight in the different G. duodenalis assemblages present in the Dutch human population. Results showed that these human isolates could be divided into two main Assemblages A and B within the G. duodenalis group on the basis of PCR assays specific for the Assemblages A and B and the DNA sequences of 18S ribosomal RNA and the glutamate dehydrogenase (gdh) genes. Genotyping results showed that G. duodenalis isolates originating from Dutch human patients belonged in 35% of the cases to Assemblage A (34/98) and in 65% of the cases to Assemblage B (64/98) whereas two human cases remained negative in all assays tested. In addition, we compared these human samples with animal samples from the Netherlands and human and animal samples from other countries. A phylogenetic analysis was carried out on the DNA sequences obtained from these Giardia and those available in GenBank. Using gdh DNA sequence analysis, human and animal Assemblage A and B Giardia isolates could be identified. However, phylogenetic analysis revealed different sub-clustering for human and animal isolates where host-species-specific assemblages (C, D, E, F and G) could be identified. The geographic origin of the human and animal samples was not a discriminating factor.     

Structural Basis for Inactivation of Giardia lamblia Carbamate Kinase by Disulfiram

Carbamate kinase from Giardia lamblia is an essential enzyme for the survival of the organism. The enzyme catalyzes the final step in the arginine dihydrolase pathway converting ADP and carbamoyl phosphate to ATP and carbamate. We previously reported that disulfiram, a drug used to treat chronic alcoholism, inhibits G. lamblia CK and kills G. lamblia trophozoites in vitro at submicromolar IC₅₀ values. Here, we examine the structural basis for G. lamblia CK inhibition of disulfiram and its analog, thiram, their activities against both metronidazole-susceptible and metronidazole-resistant G. lamblia isolates, and their efficacy in a mouse model of giardiasis. The crystal structure of G. lamblia CK soaked with disulfiram revealed that the compound thiocarbamoylated Cys-242, a residue located at the edge of the active site. The modified Cys-242 prevents a conformational transition of a loop adjacent to the ADP/ATP binding site, which is required for the stacking of Tyr-245 side chain against the adenine moiety, an interaction seen in the structure of G. lamblia CK in complex with AMP-PNP. Mass spectrometry coupled with trypsin digestion confirmed the selective covalent thiocarbamoylation of Cys-242 in solution. The Giardia viability studies in the metronidazole-resistant strain and the G. lamblia CK irreversible inactivation mechanism show that the thiuram compounds can circumvent the resistance mechanism that renders metronidazole ineffectiveness in drug resistance cases of giardiasis. Together, the studies suggest that G. lamblia CK is an attractive drug target for development of novel antigiardial therapies and that disulfiram, an FDA-approved drug, is a promising candidate for drug repurposing.     

COX-2 is required to mediate crosstalk of ROS-dependent activation of MAPK/NF-κB signaling with pro-inflammatory response and defense-related NO enhancement during challenge of macrophage-like cell line with Giardia duodenalis

Giardia duodenalis, the causative agent of giardiasis, is among the most important causes of waterborne diarrheal diseases around the world. Giardia infection may persist over extended periods with intestinal inflammation, although minimal. Cyclooxygenase (COX)-2 is well known as an important inducer of inflammatory response, while the role it played in noninvasive Giardia infection remains elusive. Here we investigated the regulatory function of COX-2 in Giardia-induced pro-inflammatory response and defense-related nitric oxide (NO) generation in macrophage-like cell line, and identified the potential regulators. We initially found that Giardia challenge induced up-regulation of IL-1β, IL-6, TNF-α, prostaglandin (PG) E2, and COX-2 in macrophages, and pretreatment of the cells with COX-2 inhibitor NS398 reduced expressions of those pro-inflammatory factors. It was also observed that COX-2 inhibition could attenuate the up-regulated NO release and inducible NO synthase (iNOS) expression induced by Giardia. We further confirmed that Giardia-induced COX-2 up-regulation was mediated by the phosphorylation of p38 and ERK1/2 MAPKs and NF-κB. In addition, inhibition of reactive oxygen species (ROS) by NAC was shown to repress Giardia-induced activation of MAPK/NF-κB signaling, up-regulation of COX-2 and iNOS, increased levels of PGE2 and NO release, and up-expressions of IL-1β, IL-6, and TNF-α. Collectively, in this study, we revealed a critical role of COX-2 in modulating pro-inflammatory response and defense-related NO production in Giardia-macrophage interactions, and this process was evident to be controlled by ROS-dependent activation of MAPK/NF-κB signaling. The results can deepen our knowledge of anti-Giardia inflammatory response and host defense mechanisms.     

Baicalein inhibits IL-1β- and TNF-α-induced inflammatory cytokine production from human mast cells via regulation of the NF-κB pathway

Background

Human mast cells are multifunctional cells capable of a wide variety of inflammatory responses. Baicalein (BAI), isolated from the traditional Chinese herbal medicine Huangqin (Scutellaria baicalensis Georgi), has been shown to have anti-inflammatory effects. We examined its effects and mechanisms on the expression of inflammatory cytokines in an IL-1β- and TNF-α-activated human mast cell line, HMC-1.

Methods

HMC-1 cells were stimulated either with IL-1β (10 ng/ml) or TNF-α (100 U/ml) in the presence or absence of BAI. We assessed the expression of IL-6, IL-8, and MCP-1 by ELISA and RT-PCR, NF-κB activation by electrophoretic mobility shift assay (EMSA), and IκBα activation by Western blot.

Results

BAI (1.8 to 30 μM) significantly inhibited production of IL-6, IL-8, and MCP-1 in a dose-dependent manner in IL-1β-activated HMC-1. BAI (30 μM) also significantly inhibited production of IL-6, IL-8, and MCP-1 in TNF-α-activated HMC-1. Inhibitory effects appear to involve the NF-κB pathway. BAI inhibited NF-κB activation in IL-1β- and TNF-α-activated HMC-1. Furthermore, BAI increased cytoplasmic IκBα proteins in IL-1β- and TNF-α-activated HMC-1.

Conclusion

Our results showed that BAI inhibited the production of inflammatory cytokines through inhibition of NF-κB activation and IκBα phosphorylation and degradation in human mast cells. This inhibitory effect of BAI on the expression of inflammatory cytokines suggests its usefulness in the development of novel anti-inflammatory therapies.     

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.     

Differential gene expression in Giardia lamblia under oxidative stress: Significance in eukaryotic evolution

Giardia lamblia is a unicellular, early branching eukaryote causing giardiasis, one of the most common human enteric diseases. Giardia, a microaerophilic protozoan parasite has to build up mechanisms to protect themselves against oxidative stress within the human gut (oxygen concentration 60 μM) to establish its pathogenesis. G. lamblia is devoid of the conventional mechanisms of the oxidative stress management system, including superoxide dismutase, catalase, peroxidase, and glutathione cycling, which are present in most eukaryotes. NADH oxidase is a major component of the electron transport chain of G. lamblia, which in concurrence with disulfide reductase, protects oxygen-labile proteins such as pyruvate: ferredoxin oxidoreductase against oxidative stress by sustaining a reduced intracellular environment. It also contains the arginine dihydrolase pathway, which occurs in a number of anaerobic prokaryotes, includes substrate level phosphorylation and adequately active to make a major contribution to ATP production.     

Evaluation of ��-Tubulin as an Antigenic and Molecular Probe to Detect Giardia lamblia

The α/β-tubulin heterodimer is the basic subunit of microtubules in eukaryotes. Polyclonal antibodies specific to recombinant α-tubulin of Giardia lamblia were made, and found effective as a probe to specifically detect G. lamblia by immunofluorescence assays. Nucleotide sequences of α-tubulin genes were compared between G. lamblia WB and GS strains, prototypes of assemblage A and assemblage B, respectively. A set of primers was designed and used to amplify a portion of the α-tubulin gene from G. lamblia. PCR-RFLP analysis of this α-tubulin PCR product successfully differentiated G. lamblia into 2 distinct groups, assemblages A and B. The results indicate that α-tubulin can be used as a molecular probe to detect G. lamblia.     

Identification of zoonotic Giardia genotypes in fish

Apart from a single record in a shark, there have been no published studies conducted on Giardia genotypes in fish. The present study investigated the prevalence of Giardia in cultured fingerlings (n = 227), wild freshwater (n = 227) and wild marine/estuarine species (n = 255) of fish in Western Australia by PCR amplification at the 18S rRNA, glutamate dehydrogenase (gdh), triose phosphate isomerase (tpi) and beta-giardin (bg) loci. Results revealed a low prevalence of Giardia, 3.8% (27/709), in fish hosts. The zoonotic Giardia species, Giardia duodenalis assemblages A, B as well as G. duodenalis assemblage E and Giardia microti were detected. The identification of zoonotic species of Giardia highlights the public health importance of investigating parasites within fish host species.     

The Cre/loxP system in Giardia lamblia: genetic manipulations in a binucleate tetraploid protozoan

The bacteriophage-derived Cre/loxP system is a valuable tool that has revolutionised genetic and cell biological research in many organisms. We implemented this system in the intestinal parasite Giardia lamblia, an evolutionarily diverged protozoan whose binucleate and tetraploid genome organisation severely limits the application of reverse genetic approaches. We show that Cre-recombinase is functionally expressed in G. lamblia and demonstrate “recycling” of selectable markers. Providing the means for more complex and versatile genetic modifications, this technique massively increases the scope of functional investigations in G. lamblia and other protozoa with similar limitations with respect to genetic manipulation.     

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

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