Nek8445, a protein kinase required for microtubule regulation and cytokinesis in Giardia lamblia.

Giardia has 198 Nek kinases whereas humans have only 11. Giardia has a complex microtubule cytoskeleton that includes eight flagella and several unique microtubule arrays that are utilized for parasite attachment and facilitation of rapid mitosis and cytokinesis. The need to regulate these structures may explain the parallel expansion of the number of Nek family kinases. Here we use live and fixed cell imaging to uncover the role of Nek8445 in regulating Giardia cell division. We demonstrate that Nek8445 localization is cell cycle regulated and this kinase has a role in regulating overall microtubule organization. Nek8445 depletion results in short flagella, aberrant ventral disk organization, loss of the funis, defective axoneme exit, and altered cell shape. The axoneme exit defect is specific to the caudal axonemes, which exit from the posterior of the cell, and this defect correlates with rounding of the cell posterior and loss of the funis. Our findings implicate a role for the funis in establishing Giardia's cell shape and guiding axoneme docking. On a broader scale our results support the emerging view that Nek family kinases have a general role in regulating microtubule organization.

The structural organization of Giardia intestinalis cytoskeleton.

Giardia intestinalis, the causative agent of giardiasis, has complex cytoskeleton organization with structures involved in motility, adhesion, cell division, and cell differentiation. Microtubules are key components of the cytoskeleton and are the main elements of the ventral disc, median body, funis, in addition to four pairs of flagella. These cytoskeletal elements are basically stable microtubule arrangements. Although tubulins are the main proteins of these elements, molecular and biochemical analyses of Giardia trophozoites have revealed the presence of several new and not yet characterized proteins in these structures, which may contribute to their nanoarchitecture (mainly in the ventral disc). Despite these findings, morphological data are still required for understanding the organization and biogenesis of the cytoskeletal structures. In the study of this complex and specialized network of filaments in Giardia, two distinct and complementary approaches have been used in recent years: (a) transmission electron microscopy tomography of conventionally processed as well as cryo-fixed samples and (b) high-resolution scanning electron microscopy and helium ion microscopy in combination with new plasma membrane extraction protocols. In this review we include the most recent studies that have allowed better understanding of new Giardia components and their association with other filamentous structures of this parasite, thus providing new insights in the role of the cytoskeletal structures and their function in Giardia trophozoites.

Report of Giardia assemblages and giardiasis in residents of Guilan province-Iran.

Giardia duodenalis is considered a highly diverse organism that infects a variety of mammalian hosts. Giardiasis is a significant public health problem in Iran. The purpose of this study was to investigate the occurrence of Giardia duodenalis (G. lamblia, G. intestinalis) infections in humans residing in the Guilan province of Iran. Stool samples were collected during 12 months from 8356 individuals that had been referred to certain hospitals in the capital city of Rasht in the Guilan province, of which 4126 were males and 4230 were females. The samples were separated into three groups according to patient age: group A 1-9 years old (n = 483); group B 10-19 years old (n = 491); and group C greater than 20 years old (n = 7382). The wet mount technique was performed directly on 8356 fecal samples for microscopy. Samples were examined using a saline and iodine direct smear technique in order to confirm the presence of G. duodenalis. The results indicated that 2.5% (206/8356) of the samples were identified as positive for G. duodenalis. A total of 30% of the infected patients (n = 62) had no symptoms. In symptomatic cases, the most common symptoms (46%, n = 95) were abdominal cramps and bloating. Twenty-four percent of patients (n = 50) had cramps, bloating, nausea, and diarrhea. Sixty positive samples were sent for G. duodenalis genotyping based on the amplification of the gdh gene. Forty-one PCR products were successfully selected and sequenced, where 38 (92.6%) samples were identified as genotype A/subgenotype II and in three samples (7.4%) genotype B/subgenotype IV. Genotype A-II had a dominant prevalence as compared to the genotype B-IV samples that were identified in the study. Based on the samples provided by the regional teaching hospitals and subsequent sample analysis, the authors concluded that assemblage A-II is most likely the most common Giardia subgroup infection in the Guilan region. Assemblages have been reported in both humans and animals; however, further studies need to investigate the role of domestic animals and water reservoirs as potential sources of Giardia infection in the Guilan region.

Nicotinamide induces G2 cell cycle arrest in Giardia duodenalis trophozoites and promotes changes in sirtuins transcriptional expression.

Giardia duodenalis is a flagellated unicellular eukaryotic microorganism that commonly causes diarrheal disease throughout the world. Treatment of giardiasis is limited to nitroheterocyclic compounds as metronidazole and benzimidazoles as albendazole, where remarkably treatment failure is relatively common. Consequently, the need for new options to treat this disease is underscored. We predicted by a bioinformatic approach that nicotinamide inhibits Giardia sirtuins by the nicotinamide exchange pathway, and since sirtuins are involved in cell cycle control, they could be related with arrest and decrease of viability. When trophozoites were treated with nicotinamide (NAM), a strong arrest of Giardia trophozoites in G2 phase was observed and at the same time changes in transcriptional expression of sirtuins were produced. Interestingly, the G2 arrest is not related to double-strand breaks, which strengthens the role of sirtuins in the control of the Giardia cell cycle. Results with NAM-treated trophozoites as predicted demonstrate antigiardial effects and thus open new options for the treatment of giardiasis, either with the combination of nicotinamide with another antigiardial drug, or with the design of specific inhibitors for Giardia sirtuins.

Role of gamma-giardin in ventral disc formation of Giardia lamblia.

BACKGROUND: Giardia lamblia, a protozoan pathogen causing diarrheal outbreaks, has characteristic cytoskeletal structures including eight flagella, a median body and a ventral disc. Gamma-giardin is a unique component protein of the cytoskeleton of this protozoan. RESULTS: Through comparative proteomic analysis between different stages of the cell cycle, G. lamblia γ-giardin (Glγ-giardin) was identified as an upregulated protein in the G2-phase. Increased Glγ-giardin expression in G2 was confirmed by western blot and real-time polymerase chain reaction analyses. Knockdown of this protein using a morpholino affected the formation of ventral discs, especially the microribbons of the discs, but exerted little effect on the binding ability of G. lamblia. The number of cells with four nuclei was increased in Glγ-giardin-knockdown cells. Expression of Glγ-giardin was decreased during encystation, in contrast with the G2-phase. CONCLUSIONS: Knockdown experiments demonstrated that Glγ-giardin is a component of the trilaminar structure of the ventral disc. Expression of Glγ-giardin is induced in the G2-phase prior to active cell division, whereas its expression decreases during encystation, a dormant stage of G. lamblia.

Development of CRISPR/Cas9-mediated gene disruption systems in Giardia lamblia.

Giardia lamblia becomes dormant by differentiation into a water-resistant cyst that can infect a new host. Synthesis of three cyst wall proteins (CWPs) is the fundamental feature of this differentiation. Myeloid leukemia factor (MLF) proteins are involved in cell differentiation, and tumorigenesis in mammals, but little is known about its role in protozoan parasites. We developed a CRISPR/Cas9 system to understand the role of MLF in Giardia. Due to the tetraploid genome in two nuclei of Giardia, it could be hard to disrupt a gene completely in Giardia. We only generated knockdown but not knockout mutants. We found that knockdown of the mlf gene resulted in a significant decrease of cwp gene expression and cyst formation, suggesting a positive role of MLF in encystation. We further used mlf as a model gene to improve the system. The addition of an inhibitor for NHEJ, Scr7, or combining all cassettes for gRNA and Cas9 expression into one plasmid resulted in improved gene disruption efficiencies and a significant decrease in cwp gene expression. Our results provide insights into a positive role of MLF in inducing Giardia differentiation and a useful tool for studies in Giardia.

Alterations on growth and cell organization of Giardia intestinalis trophozoites after treatment with KH-TFMDI, a novel class III histone deacetylase inhibitor.

Giardia trophozoites have developed resistance mechanisms to currently available compounds, leading to treatment failures. In this context, the development of new additional agents is mandatory. Sirtuins, which are class III NAD+-dependent histone deacetylases, have been considered important targets for the development of new anti-parasitic drugs. Here, we evaluated the activity of KH-TFMDI, a novel 3-arylideneindolin-2-one-type sirtuin inhibitor, on G. intestinalis trophozoites. This compound decreased the trophozoite growth presenting an IC50 value lower than nicotinamide, a moderately active inhibitor of yeast and human sirtuins. Light and electron microscopy analysis showed the presence of multinucleated cell clusters suggesting that the cytokinesis could be compromised in treated trophozoites. Cell rounding, concomitantly with the folding of the ventro-lateral flange and flagella internalization, was also observed. These cells eventually died by a mechanism which lead to DNA/nuclear damage, formation of multi-lamellar bodies and annexin V binding on the parasite surface. Taken together, these data show that KH-TFMDI has significant effects against G. intestinalis trophozoites proliferation and structural organization and suggest that histone deacetylation pathway should be explored on this protozoon as target for chemotherapy.

The minimal ESCRT machinery of Giardia lamblia has altered inter-subunit interactions within the ESCRT-II and ESCRT-III complexes.

The ESCRT pathway functions at different subcellular membranes to induce their negative curvature, and it has been largely characterized in model eukaryotes belonging to Opisthokonta. But searches of the genomes of many nonopisthokonts belonging to various supergroups indicate that some of them may harbour fewer ESCRT components. Of the genomes explored thus far, one of the most minimal set of ESCRT components was identified in the human pathogen Giardia lamblia, which belongs to Excavata. Here we report that an ESCRT-mediated pathway most likely operates at the peripheral vesicles, which are located at the cell periphery and the bare zone of this protist. Functional comparison of all the identified putative giardial ESCRT components, with the corresponding well-characterized orthologues from Saccharomyces cerevisiae, indicated that only some of the ESCRT components could functionally substitute for the corresponding yeast proteins. While GlVps25, GlVps2, and all three paralogues of GlVps4, tested positive in functional complementation assays, GlVps22, GlVps20, and GlVps24 did not. Binary interactions of either GlVps22 or GlVps25, with other ESCRT-II components from Giardia or yeast indicate that the giardial Vps36 orthologue is either completely missing or highly diverged. Interactions within the giardial ESCRT-III components also differ from those in yeast; while GlVps46a interacts preferentially with Vps24 compared to Vps2, GlVps46b, like the yeast orthologue, interacts with both.

Myosin-independent cytokinesis in Giardia utilizes flagella to coordinate force generation and direct membrane trafficking.

Devoid of all known canonical actin-binding proteins, the prevalent parasite Giardia lamblia uses an alternative mechanism for cytokinesis. Unique aspects of this mechanism can potentially be leveraged for therapeutic development. Here, live-cell imaging methods were developed for Giardia to establish division kinetics and the core division machinery. Surprisingly, Giardia cytokinesis occurred with a median time that is ∼60 times faster than mammalian cells. In contrast to cells that use a contractile ring, actin was not concentrated in the furrow and was not directly required for furrow progression. Live-cell imaging and morpholino depletion of axonemal Paralyzed Flagella 16 indicated that flagella-based forces initiated daughter cell separation and provided a source for membrane tension. Inhibition of membrane partitioning blocked furrow progression, indicating a requirement for membrane trafficking to support furrow advancement. Rab11 was found to load onto the intracytoplasmic axonemes late in mitosis and to accumulate near the ends of nascent axonemes. These developing axonemes were positioned to coordinate trafficking into the furrow and mark the center of the cell in lieu of a midbody/phragmoplast. We show that flagella motility, Rab11, and actin coordination are necessary for proper abscission. Organisms representing three of the five eukaryotic supergroups lack myosin II of the actomyosin contractile ring. These results support an emerging view that flagella play a central role in cell division among protists that lack myosin II and additionally implicate the broad use of membrane tension as a mechanism to drive abscission.

Morphological and physiological characteristics of a virulent and zoonotic assemblage A Giardia duodenalis canine strain.

Giardiasis is an intestinal parasitosis that affects millions of people worldwide and is considered a zoonotic disease. Frequently in contact with humans, dogs are the main host involved in this zoonotic transmission. Here, we compared some aspects of Giardia duodenalis biology between two strains: a recently isolated dog strain (BHFC1) and a human reference strain (Portland-1). Growth curve analysis revealed that BHFC1 trophozoites multiply faster than the human isolate Portland-1 in axenic culture, but has a lower rate of cysts formation. Scanning electron microscopy revealed that BHFC1 trophozoites have the same conventional shape and morphological structures expected for G. duodenalis trophozoites, but presented a more prominent flange. For the best of our knowledge, this work is the first description of morphological aspects and encystation process of a G. duodenalis strain isolated from a dog. Since BHFC1 and Portland-1 have been maintained in axenic cultures for different periods of time, differences observed in growth, encystation rates and flange size may be attributed to adaptation of Portland-1 to axenic culture and lack of the environmental pressures. BHFC1 can be useful as tool for better understanding of Giardia duodenalis biology.

Giardia intestinalis mitosomes undergo synchronized fission but not fusion and are constitutively associated with the endoplasmic reticulum.

BACKGROUND: Mitochondria of opisthokonts undergo permanent fission and fusion throughout the cell cycle. Here, we investigated the dynamics of the mitosomes, the simplest forms of mitochondria, in the anaerobic protist parasite Giardia intestinalis, a member of the Excavata supergroup of eukaryotes. The mitosomes have abandoned typical mitochondrial traits such as the mitochondrial genome and aerobic respiration and their single role known to date is the formation of iron-sulfur clusters. RESULTS: In live experiments, no fusion events were observed between the mitosomes in G. intestinalis. Moreover, the organelles were highly prone to becoming heterogeneous. This suggests that fusion is either much less frequent or even absent in mitosome dynamics. Unlike in mitochondria, division of the mitosomes was absolutely synchronized and limited to mitosis. The association of the nuclear and the mitosomal division persisted during the encystation of the parasite. During the segregation of the divided mitosomes, the subset of the organelles between two G. intestinalis nuclei had a prominent role. Surprisingly, the sole dynamin-related protein of the parasite seemed not to be involved in mitosomal division. However, throughout the cell cycle, mitosomes associated with the endoplasmic reticulum (ER), although none of the known ER-tethering complexes was present. Instead, the ER-mitosome interface was occupied by the lipid metabolism enzyme long-chain acyl-CoA synthetase 4. CONCLUSIONS: This study provides the first report on the dynamics of mitosomes. We show that together with the loss of metabolic complexity of mitochondria, mitosomes of G. intestinalis have uniquely streamlined their dynamics by harmonizing their division with mitosis. We propose that this might be a strategy of G. intestinalis to maintain a stable number of organelles during cell propagation. The lack of mitosomal fusion may also be related to the secondary reduction of the organelles. However, as there are currently no reports on mitochondrial fusion in the whole Excavata supergroup, it is possible that the absence of mitochondrial fusion is an ancestral trait common to all excavates.

Giardia duodenalis Rad52 protein: biochemical characterization and response upon DNA damage.

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.

Synchronization of Pathogenic Protozoans.

Protozoans are single-celled eukaryotes and many of the best-studied protozoans are parasitic to humans (e.g., Plasmodium falciparum causing malaria and Trypanosoma brucei causing sleeping sickness). These organisms are distantly related to humans but with retained eukaryotic type of cellular processes, making them good model systems for studies of the evolution of basic processes like the cell cycle. Giardia intestinalis causes 250 million cases of diarrhea yearly and is one of the earliest diverging protozoans. It is possible to synchronize its cell cycle using compounds that inhibit different steps of the cell cycle and the detailed protocol is described here.

Cyst-Wall-Protein-1 is fundamental for Golgi-like organelle neogenesis and cyst-wall biosynthesis in Giardia lamblia.

The genome of the protozoan parasite Giardia lamblia is organized in two diploid nuclei, which has so far precluded complete analysis of gene function. Here we use a previously developed Cre/loxP-based knock-out and selection marker salvage strategy in the human-derived isolate WB-C6 to eliminate all four copies of the Cyst-Wall-Protein-1 locus (CWP1). Because these loci are silenced in proliferating trophozoites and highly expressed only in encysting cells, CWP1 ablation allows functional characterization of a conditional phenotype in parasites induced to encyst. We show that encysting Δcwp1 cells are unable to establish the stage-regulated trafficking machinery with Golgi-like encystation-specific vesicles required for cyst-wall formation but show morphological hallmarks of cyst development and karyokinesis. This 'pseudocyst' phenotype is rescued by transfection of Δcwp1 cells with an episomally maintained CWP1 expression vector. Genome editing in genera Giardia and Trypanosoma are the only reported examples addressing questions on pathogen transmission within the Excavata supergroup.

Specific histone modifications play critical roles in the control of encystation and antigenic variation in the early-branching eukaryote Giardia lamblia.

During evolution, parasitic microorganisms have faced the challenges of adapting to different environments to colonize a variety of hosts. Giardia lamblia, a common cause of intestinal disease, has developed fascinating strategies to adapt both outside and inside its host's intestine, such as trophozoite differentiation into cyst and the switching of its major surface antigens. How gene expression is regulated during these adaptive processes remains undefined. Giardia lacks some typical eukaryotic features, like canonical transcription factors, linker histone H1, and complex promoter regions; suggesting that post-transcriptional and translational control of gene expression is essential for parasite survival. However, epigenetic factors may also play critical roles at the transcriptional level. Here, we describe the most common post-translational histone modifications; characterize enzymes involved in these reactions, and analyze their association with the Giardia's differentiation processes. We present evidence that NAD+-dependent and NAD+-independent histone deacetylases regulate encystation; however, a unique NAD+-independent histone deacetylase modulate antigenic switching. The rates of acetylation of H4K8 and H4K16 are critical for encystation, whereas a decrease in acetylation of H4K8 and methylation of H3K9 occur preferentially during antigenic variation. These results show the complexity of the mechanisms regulating gene expression in this minimalistic protozoan parasite.

Morphological Studies of Nucleologenesis in Giardia lamblia.

The nucleolus is a nuclear organelle involved in ribosome biogenesis. In most eukaryotes this structure disperses during prophase through anaphase and reorganizes at telophase by a process known as nucleologenesis. This process involves new transcription of ribosomal DNA at the nucleolar organizer region and the formation of prenucleolar bodies fusing to it. In Giardia lamblia, for a long time considered the only anucleolated eukaryote, a very small nucleolus has been recently described. In order to evaluate whether nucleologenesis is also present in Giardia, we analyzed the distribution of nucleolar material during telophase using different light and electron microscopy techniques including silver staining for the nucleolar organizer. Results indicate that in G. lamblia, nucleolar elements persist mainly as an intranuclear peripheral organelle during all stages of division, including telophase, however, no prenucleolar bodies are detected in the nucleoplasm. Therefore, in the parasite, nucleolar material is present throughout cell division including telophase and formation of prenucleolar bodies may not be required for nucleologenesis.

A detailed look at the cytoskeletal architecture of the Giardia lamblia ventral disc.

Giardia lamblia is a protistan parasite that infects and colonizes the small intestine of mammals. It is widespread and particularly endemic in the developing world. Here we present a detailed structural study by 3-D negative staining and cryo-electron tomography of a unique Giardia organelle, the ventral disc. The disc is composed of a regular array of microtubules and associated sheets, called microribbons that form a large spiral, held together by a myriad of mostly unknown associated proteins. In a previous study we analyzed by cryo-electron tomography the central microtubule portion (here called disc body) of the ventral disc and found a large portion of microtubule associated inner (MIPs) and outer proteins (MAPs) that render these microtubules hyper-stable. With this follow-up study we expanded our 3-D analysis to different parts of the disc such as the ventral and dorsal areas of the overlap zone, as well as the outer disc margin. There are intrinsic location-specific characteristics in the composition of microtubule-associated proteins between these regions, as well as large differences between the overall architecture of microtubules and microribbons. The lateral packing of microtubule-microribbon complexes varies substantially, and closer packing often comes with contracted lateral tethers that seem to hold the disc together. It appears that the marginal microtubule-microribbon complexes function as outer, laterally contractible lids that may help the cell to clamp onto the intestinal microvilli. Furthermore, we analyzed length, quantity, curvature and distribution between different zones of the disc, which we found to differ from previous publications.

Dog's genotype of Giardia duodenalis in human: first evidence in Europe.

The unicellular parasite Giardia duodenalis has been divided to eight assemblages (A-H) from which A and B have the most important zoonotic potential. All remaining genotypes have a strong commitment to various host animals. We present here the first clinical case of a human infection with the dog-specific genotype C of G. duodenalis in Slovakia. The patient, 44-year-old woman, suffered from long-term diarrhoea, abdominal pain, anorexia, weight loss, severe itching and dermatitis in the perianal area. The initial microscopic diagnosis was completed by a nested polymerase chain reaction (PCR) which revealed the first evidence of human giardiasis caused by the dog-specific genotype of G. duodenalis on a European scale. A possible role of dogs in zoonotic transmission of giardiasis and its epidemiological and public health relevance is accentuated.

Probing the Biology of Giardia intestinalis Mitosomes Using In Vivo Enzymatic Tagging.

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.

Infections by Intestinal Coccidia and Giardia duodenalis.

The coccidians Cryptosporidium spp, Cyclospora cayetanensis, and Cystoisospora belli and the flagellate Giardia duodenalis are pathogenic protozoa associated with gastrointestinal manifestations. Diagnosis relies heavily on microscopy, and although ova-and-parasite examinations can detect Giardia and Cystoisospora, Cryptosporidium and Cyclospora often require specific diagnostic requests. Approved non-microscopy methods are available for Giardia and Cryptosporidium, although negative results are frequently followed by microscopic assays. Polymerase chain reaction-based methods are not frequently used for diagnosis of Giardia and Cryptosporidium and have been used primarily for epidemiologic or outbreak investigations of Giardia and Cryptosporidium.