Characterization of a new extra-axonemal structure in the Giardia intestinalis flagella.

The inner structure of the flagella of Giardia intestinalis is similar to that of other organisms, consisting of nine pairs of outer microtubules and a central pair containing radial spokes. Although the 9+2 axonemal structure is conserved, it is not clear whether subregions, including the transition zone, are present in the flagella of this parasite. Giardia axonemes originate from basal bodies and have a lengthy cytosolic portion before becoming active flagella. The region of the emergence of the flagellum is not accompanied by any membrane specialization, as seen in other protozoa. Although Giardia is an intriguing model of study, few works focused on the ultrastructural analysis of the flagella of this parasite. Here, we analyzed the externalization region of the G. intestinalis flagella using ultra-high resolution scanning microscopy (with electrons and ions), atomic force microscopy in liquid medium, freeze fracture, and electron tomography. Our data show that this region possesses a distinctive morphological feature - it extends outward and takes on a ring-like shape. When the plasma membrane is removed, a structure surrounding the axoneme becomes visible in this region. This new extra-axonemal structure is observed in all pairs of flagella of trophozoites and remains attached to the axoneme even when the interconnections between the axonemal microtubules are disrupted. High-resolution scanning electron microscopy provided insights into the arrangement of this structure, contributing to the characterization of the externalization region of the flagella of this parasite.

Nanoarchitecture of the ventral disc of Giardia intestinalis as revealed by high-resolution scanning electron microscopy and helium ion microscopy.

The parasitic protozoan Giardia intestinalis, the causative agent of giardiasis, presents a stable and elaborated cytoskeleton, which shapes and supports several intracellular structures, including the ventral disc, the median body, the funis, and four pairs of flagella. Giardia trophozoite is the motile form that inhabits the host small intestine and attaches to epithelial cells, leading to infection. The ventral disc is considered one important element of adhesion to the intestinal cells. It is adjacent to the plasma membrane in the ventral region of the cell and consists of a spiral layer of microtubules and microribbons. In this work, we studied the organization of the cytoskeleton in the ventral disc of G. intestinalis trophozoites using high-resolution scanning electron microscopy or helium ion microscopy in plasma membrane-extracted cells. Here, we show novel morphological details about the arrangement of cross-bridges in different regions of the ventral disc. Results showed that the disc is a non-uniformly organized structure that presents specific domains, such as the margin and the ventral groove region. High-resolution scanning electron microscopy allowed observation of the labeling pattern for several anti-tubulin antibodies using secondary gold particle-labeled antibodies. Labeling in the region of the emergence of the microtubules and supernumerary microtubules using an anti-acetylated tubulin antibody was observed. Ultrastructural analysis and immunogold labeling for gamma-tubulin suggest that disc microtubules originate from a region bounded by the bands of the banded collar and merge with microtubules formed at the perinuclear region. Actin-like filaments and microtubules of the disc are associated, showing an interconnection between elements of the cytoskeleton of the trophozoite.

Giardia intestinalis coiled-coil cytolinker protein 259 interacts with actin and tubulin.

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 intestinalis can interact, change its shape and internalize large particles and microorganisms.

Giardia intestinalis is a parasitic protozoan that inhabits its vertebrate hosts' upper small intestine and is the most common cause of waterborne diarrhoea worldwide. Giardia trophozoites present few organelles, and among them, they possess peripheral vesicles (PVs), which are considered an endosomal-lysosomal system. All experimental procedures carried out until now indicate that Giardia ingests macromolecules by fluid-phase and receptor-mediated endocytic pathways. Still, there is no description concerning the interaction and ingestion of large materials. Here, we tested Giardia's capacity to interact with large particles; once, in vivo, it inhabits an environment with a microbiota. We tested protozoan interaction with yeasts, bacteria, latex beads, ferritin and albumin, in different times of interaction and used several microscopy techniques (light microscopy, scanning electron microscopy and transmission electron microscopy) to follow their fate. Giardia interacted with all of the materials we tested. Projections of the plasma membrane similar to pseudopods were seen. As albumin, small markers were found in the PVs while the larger materials were not seen there. Large vacuoles containing large latex beads were detected intracellularly. Thus, we observed that: (1) Giardia interacts with large materials; (2) Giardia can display an amoeboid shape and exhibit membrane projections when in contact with microorganisms and large inorganic materials; (3) the region of the exit of the ventral flagella is very active when in contact with large materials, although all cell surface also present activity in the interactions; (4) intracellular vacuoles, which are not the PVs, present ingested large beads.

Exosome Biogenesis in the Protozoa Parasite Giardia lamblia: A Model of Reduced Interorganellar Crosstalk.

: Extracellular vesicles (EVs) facilitate intercellular communication and are considered a promising therapeutic tool for the treatment of infectious diseases. These vesicles involve microvesicles (MVs) and exosomes and selectively transfer proteins, lipids, mRNAs, and microRNAs from one cell to another. While MVs are formed by extrusion of the plasma membrane, exosomes are a population of vesicles of endosomal origin that are stored inside the multivesicular bodies (MVBs) as intraluminal vesicles (ILVs) and are released when the MVBs fuse with the plasma membrane. Biogenesis of exosomes may be driven by the endosomal sorting complex required for transport (ESCRT) machinery or may be ESCRT independent, and it is still debated whether these are entirely separate pathways. In this manuscript, we report that the protozoan parasite, Giardia lamblia, although lacking a classical endo-lysosomal pathway, is able to produce and release exosome-like vesicles (ElV). By using a combination of biochemical and cell biology analyses, we found that the ElVs have the same size, shape, and protein and lipid composition as exosomes described for other eukaryotic cells. Moreover, we established that some endosome/lysosome peripheral vacuoles (PVs) contain ILV during the stationary phase. Our results indicate that ILV formation and ElV release depend on the ESCRT-associated AAA+-ATPase Vps4a, Rab11, and ceramide in this parasite. Interestingly, EIV biogenesis and release seems to occur in Giardia despite the fact that this parasite has lost most of the ESCRT machinery components during evolution and is unable to produce ceramide de novo. The differences in protozoa parasite EV composition, origin, and release may reveal functional and structural properties of EVs and, thus, may provide information on cell-to-cell communication and on survival mechanisms.

On the molecular and cellular effects of omeprazole to further support its effectiveness as an antigiardial drug.

Research on Giardia lamblia has accumulated large information about its molecular cell biology and infection biology. However, giardiasis is still one of the commonest parasitic diarrheal diseases affecting humans. Additionally, an alarming increase in cases refractory to conventional treatment has been reported in low prevalence settings. Consequently, efforts directed toward supporting the efficient use of alternative drugs, and the study of their molecular targets appears promising. Repurposing of proton pump inhibitors is effective in vitro against the parasite and the toxic activity is associated with the inhibition of the G. lamblia triosephosphate isomerase (GlTIM) via the formation of covalent adducts with cysteine residue at position 222. Herein, we evaluate the effectiveness of omeprazole in vitro and in situ on GlTIM mutants lacking the most superficial cysteines. We studied the influence on the glycolysis of Giardia trophozoites treated with omeprazole and characterized, for the first time, the morphological effect caused by this drug on the parasite. Our results support the effectiveness of omeprazole against GlTIM despite of the possibility to mutate the druggable amino acid targets as an adaptive response. Also, we further characterized the effect of omeprazole on trophozoites and discuss the possible mechanism involved in its antigiardial effect.

Giardia lamblia: Identification of peroxisomal-like proteins.

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.

Proteomic and ultrastructural analysis of the effect of a new nitazoxanide-N-methyl-1H-benzimidazole hybrid against Giardia intestinalis.

In an effort to develop alternative drugs for the treatment of giardiasis our research group has synthesized and evaluated a novel nitazoxanide and N-methyl-1H-benzimidazole hybrid molecule, named CMC-20. It showed an IC50 of 0.010 µM on Giardia intestinalis, lower than the IC50 values of 0.015, 0.037 and 1.224 µM for nitazoxanide, albendazole and metronidazole, respectively. In addition, we report studies carried out on its mechanism of action and effect at the ultrastructural level on G. intestinalis. The proteomic analysis of trophozoites treated with CMC-20 revealed significant changes in the expression level of proteins of the cytoskeleton, alpha and beta tubulin, alpha-1, beta giardin and axoneme-associated protein, among other molecules. Ultrastructural studies demonstrated that CMC-20 induces morphological changes on the parasite that loses its characteristic pear shape. Uncommon large bulbous structure at the flagella end, and parasites showing flange membrane bending and a concave depression in the ventral region, resembling an encystation process, were also observed. In addition, some apoptotic and autophagic-like features, such as membrane blebbing, intense vacuolation, chromatin condensation and multilamellar bodies were detected. Phosphatidylserine externalization was determined as an apoptotic marker by flow cytometry and immunofluorescence microscopy; however, a typical ladder-like DNA fragmentation profile was not detected. Although it was found that CMC-20 triggers the encystation process, damage to the cyst wall indicates loss of viability.

Helium ion microscopy and ultra-high-resolution scanning electron microscopy analysis of membrane-extracted cells reveals novel characteristics of the cytoskeleton of Giardia intestinalis.

Giardia intestinalis presents a complex microtubular cytoskeleton formed by specialized structures, such as the adhesive disk, four pairs of flagella, the funis and the median body. The ultrastructural organization of the Giardia cytoskeleton has been analyzed using different microscopic techniques, including high-resolution scanning electron microscopy. Recent advances in scanning microscopy technology have opened a new venue for the characterization of cellular structures and include scanning probe microscopy techniques such as ultra-high-resolution scanning electron microscopy (UHRSEM) and helium ion microscopy (HIM). Here, we studied the organization of the cytoskeleton of G. intestinalis trophozoites using UHRSEM and HIM in membrane-extracted cells. The results revealed a number of new cytoskeletal elements associated with the lateral crest and the dorsal surface of the parasite. The fine structure of the banded collar was also observed. The marginal plates were seen linked to a network of filaments, which were continuous with filaments parallel to the main cell axis. Cytoplasmic filaments that supported the internal structures were seen by the first time. Using anti-actin antibody, we observed a labeling in these filamentous structures. Taken together, these data revealed new surface characteristics of the cytoskeleton of G. intestinalis and may contribute to an improved understanding of the structural organization of trophozoites.

In vitro activity of 'Mexican Arnica' Heterotheca inuloides Cass natural products and some derivatives against Giardia intestinalis.

Giardiasis is a gastrointestinal disease that affects humans and other animals caused by parasitic protists of the genus Giardia. Giardia intestinalis (Syn. Giardia lamblia; Giardia duodenalis) infections can cause acute or chronic diarrhoea, dehydration, abdominal discomfort and weight loss. Metronidazole is the most widely used drug for treating giardiasis. Although effective, metronidazol has undesirable secondary effects. Plants used in traditional medicine as antidiarrhoeals or antiparasitics may represent alternative sources for new compounds to treat giardiasis. Heterotheca inuloides Cass. (Asteraceae/Compositae) plant is widely used in Mexican traditional medicine. The following secondary metabolites were isolated from H. inuloides flowers: 7-hydroxy-3,4-dihydrocadalene (1), 7-hydroxycadalene (2), 3,7-dihydroxy-3(4H)-isocadalen-4-one (3), 1R,4R-hydroxy-1,2,3,4-tetrahydrocadalen-15-oic acid (4), quercetin (5), quercetin-3,7,3'-trimethyl ether (6), quercetin-3,7,3',4'-tetramethyl ether (7) and eriodictyol-7,4'-dimethyl ether (8). The activity of these compounds against Giardia intestinalis trophozoites was assessed in vitro as was the activity of the semisynthetic compounds 7-acetoxy-3,4-dihydrocadalene (9), 7-benzoxy-3,4-dihydrocadalene (10), 7-acetoxycadalene (11), 7-benzoxycadalene (12), quercetin pentaacetate (13) and 7-hydroxycalamenene (14). Among these, 7-hydroxy-3,4-dihydrocadalene (1) and 7-hydroxycalamenene (14) were the most active, whereas the remaining compounds showed moderate or no activity. The G. intestinalis trophozoites exposed to compound 1 showed marked changes in cellular architecture along with ultrastructural disorganization. The aim of this study was to evaluate the giardicidal activity of selected H. inuloides metabolites and some semisynthetic derivatives using an in vitro experimental model of giardiasis.

Effects of metronidazole analogues on Giardia lamblia: experimental infection and cell organization.

The chemotherapeutic agents used for the treatment of giardiasis are often associated with adverse side effects and are refractory cases, due to the development of resistant parasites. Therefore the search for new drugs is required. We have previously reported the giardicidal effects of metronidazole (MTZ) and its analogues (MTZ-Ms, MTZ-Br, MTZ-N(3), and MTZ-I) on the trophozoites of Giardia lamblia. Now we evaluated the activity of some giardicidal MTZ analogues in experimental infections in gerbils and its effects on the morphology and ultrastructural organization of Giardia. The giardicidal activity in experimental infections showed ED(50) values significantly lower for MTZ-I and MTZ-Br when compared to MTZ. Transmission electron microscopy was employed to approach the mechanism(s) of action of MTZ analogues upon the protozoan. MTZ analogues were more active than MTZ in changing significantly the morphology and ultrastructure of the parasite. The analogues affected parasite cell vesicle trafficking, autophagy, and triggered differentiation into cysts. These results coupled with the excellent giardicidal activity and lower toxicity demonstrate that these nitroimidazole derivates may be important therapeutic alternatives for combating giardiasis. In addition, our results suggest a therapeutic advantage in obtaining synthetic metronidazole analogues for screening of activities against other infectious agents.

A new set of carbohydrate-positive vesicles in encysting Giardia lamblia.

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.

Heterogeneity in the sensitivity of microtubules of Giardia lamblia to the herbicide oryzalin.

Giardia lamblia is a protozoan that inhabits the small intestine of vertebrates, attaching to the epithelial cells by means of cytoskeletal elements. G. lamblia trophozoites possess several microtubular structures, namely the adhesive disc, the median body, the funis and the four pairs of flagella. Several drugs that target cytoskeletal proteins have been used in the study of cytoskeletal function and dynamics. In this work, we used oryzalin, which binds to alpha-tubulin, as a tool to study the Giardia cytoskeleton. The trophozoites were treated with oryzalin, and its effects were analysed by immunofluorescency, transmission and scanning electron microscopies. Oryzalin inhibited Giardia proliferation. Treated cells were not able to complete cell division and had flagella showing extensive shortening. Strikingly, the drug did not interfere with the adhesive disc, in contrast to what happens when other drugs are used.

Encystation process of Giardia lamblia: morphological and regulatory aspects.

One important step in the life cycle of the pathogenic protozoan Giardia lamblia is the transformation of the proliferative form, the trophozoite, into the non-proliferative cyst. This process, known as encystation, can be triggered in vitro. Morphological analysis showed that during trophozoite-cyst transformation, major changes take place: modification of the protozoan shape, internalization of the flagella, fragmentation of the adhesive disk, and appearance of encystation vesicles (ESVs), which later on fuse with the plasma membrane forming the cell wall. Sites of attachment of these vesicles to the inner portion of the protozoan plasma membrane were observed 6 h after the beginning of the encystation process. These sites were only visible when we used high-resolution scanning electron microscopy to study Giardia surface. In order to analyze the involvement of protein kinases and phosphatases on the encystation process, inhibitors of these enzymes were added to the culture medium, and their effect on the differentiation process was determined using light, immunofluorescence, and electron microscopy. Significant inhibition was observed with LY294002, an inhibitor of PI3 kinase; genistein, an inhibitor of tyrosine kinase; and staurosporine, at concentrations, which inhibit protein kinase C. Okadaic acid, an inhibitor or protein phosphatase, and wortmannin, an inhibitor of PI3K, did not interfere with the encystation process. However, they induced the appearance of large and pleomorphic forms where several nuclei and disorganization of the peripheral vesicles were observed.

Cell death induction in Giardia lamblia: effect of beta-lapachone and starvation.

Giardia lamblia is a protozoan that parasitizes the small intestine of vertebrates. It is a cause of intestinal infection and diarrhea and infects millions of people worldwide. This protozoan presents many characteristics common to eukaryotic cells but it lacks organelles found in most eukaryotes (e.g., peroxisomes, typical Golgi complex and mitochondria). Also it presents mitosomes, a relic organelle that appears to be a mitochondrial remnant. Cell death in Giardia was induced by the drug beta-Lapachone and by starvation. Giardia behavior was followed by scanning, transmission and fluorescence microscopy, quantification of cell metabolism using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide), changes in lipid rafts, using DiIC(16) and cholera toxin. Cell shrinkage, chromatin condensation, membrane blebbing and vacuolization provided ultrastructural evidence of apoptosis, whereas the myelinic figures in large vacuoles and LC-3 staining suggested an autophagic process. Lipids rafts were altered by drug treatment and co-localized with regions containing membrane blebbing. The treatment with beta-Lap induced encystation. A search for sequence similarities in databases and protein alignments was carried out. Although Giardia is an amitochondrial organism, it presented some autophagic-like cell death characteristics and several, but not all, apoptotic characteristics, induced by beta-Lapachone and starvation.

Giardia lamblia: a report of drug effects under cell differentiation.

The Giardia lamblia life cycle is characterized by two phases during which two major cell differentiation processes take place: encystation and excystation. During encystation, the trophozoites transform into cysts, the resistance form. Once ingested by a susceptible host, the cysts are stimulated to excyst in the stomach, and the excysted trophozoites adhere to the epithelium of the upper small intestine. Our work analyses the effects of four benzimidazole derivatives during Giardia differentiation into cysts and evaluates the excystation efficiency of water resistant cysts. Albendazole (AB) showed the most significant results by inhibiting encystation about 30% and a decreasing rate of excystation efficiency. The ultrastructural organization of the cyst adhesive disk was notably affected by AB treatment. Although other benzimidazoles showed some effect on encystation, they were not able to inhibit the excystation process. It is known that the benzimidazoles affect the cytoskeleton of many organisms but how it interferes in Giardia differentiation processes is our main focus. The importance of studying Giardia's differentiation under drug action is reinforced by the following arguments: (1) Cysts eliminated by hosts undergoing treatment could still be potentially infective; (2) once the host has been treated, it would be desirable that the shedding of cysts into the environment is avoided; (3) the prevention of Giardia dissemination is a question of extreme importance mainly in underdeveloped countries, where poor sanitary conditions are related to high rates of giardiasis. This report concerns the importance of keeping the environment free from infective cysts and on Giardia's drug resistance and differentiating abilities.

Giardia duodenalis: effects of an ozonized sunflower oil product (Oleozon) on in vitro trophozoites.

The ozonized sunflower oil product (Oleozon) was investigated to explore its cytotoxic activity on Giardia duodenalis in vitro cultivated trophozites. Oleozon produced inactivation of Giardia trophozoites in a dose- and cell density-dependent manner. Thirty microliter of Oleozon with peroxide index value of 500 equivalent-mmol of activated oxygen per kilogram were used to achieve a 100% inhibition (<-4.00 log unit) of trophozoites from an initial inoculum of 15x10(4) cells. This potent effect was confirmed by transmission electron microscopy where morphological deterioration of superficial structures mainly in the ventral disc, and formation of a great number of micro vesicles in the cytoplasm were found. We concluded that a direct chemical-oxidation attack by the active substances from Oleozon is one of the causes of the parasitocidal effect of this product. We suggest that the dose and cell density-dependent effect must be taken into account when prescription of this product for giardiasis treatment in humans.

Giardia lamblia behavior during encystment: how morphological changes in shape occur.

Giardia is an intestinal parasite that undergoes adaptation for survival outside the host. Different stages in the Giardia cyst formation include distinctive changes in the trophozoite shape and polarization, from the characteristic flattened dorsal-ventral axis found in motile trophozoites to a rounded appearance and also the appearance of a "tail-like" appendage in later stages of cyst formation. In addition, the flagella disappear and the cyst is oval or rounded and immotile. Since we found no clear information describing how the cells change shape and how the flagella disappear, we applied videomicroscopy, scanning and transmission electron microscopy to follow the gradual modifications that occur in the trophozoite, including alterations in the cell shape, the manner of flagella internalization and changes in disc behavior. Based on the data presented here, it was possible to construct a temporal sequence of changes during Giardia encystation. In this article we show how the membrane growth of the flange contributes to cell shape changes during encystment. In addition, an operculum and flagella internalization is shown. There is a video as a supplement showing these modifications. In other procedure, the plasma membrane was removed and the disc was seen by high resolution scanning electron microscopy where the modifications of the disc spiral can be followed.

Characterization of SNAREs determines the absence of a typical Golgi apparatus in the ancient eukaryote Giardia lamblia.

Giardia is a eukaryotic protozoal parasite with unusual characteristics, such as the absence of a morphologically evident Golgi apparatus. Although both constitutive and regulated pathways for protein secretion are evident in Giardia, little is known about the mechanisms involved in vesicular docking and fusion. In higher eukaryotes, soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) of the vesicle-associated membrane protein and syntaxin families play essential roles in these processes. In this work we identified and characterized genes for 17 SNAREs in Giardia to define the minimal set of subcellular organelles present during growth and encystation, in particular the presence or not of a Golgi apparatus. Expression and localization of all Giardia SNAREs demonstrate their presence in distinct subcellular compartments, which may represent the extent of the endomembrane system in eukaryotes. Remarkably, Giardia SNAREs, homologous to Golgi SNAREs from other organisms, do not allow the detection of a typical Golgi apparatus in either proliferating or differentiating trophozoites. However, some features of the Golgi, such as the packaging and sorting function, seem to be performed by the endoplasmic reticulum and/or the nuclear envelope. Moreover, depletion of individual genes demonstrated that several SNAREs are essential for viability, whereas others are dispensable. Thus, Giardia requires a smaller number of SNAREs compared with other eukaryotes to accomplish all of the vesicle trafficking events that are critical for the growth and differentiation of this important human pathogen.

Effects of a putrescine analog on Giardia lamblia.

The protozoan Giardia lamblia is the most frequent intestinal parasite of first-world countries and a major cause of waterborne disorder often referred to as traveler's diarrhea. We have previously noticed that the putrescine analog 1,4-diamino-2-butanone (DAB) remarkably inhibits the growth of anaerobic trichomonad and Trypanosoma cruzi parasites. Here, we examined the role of polyamines in Giardia cells using this putrescine analog. DAB impaired parasite proliferation dose-dependently. The analog induced increased flagella numbers and sometimes four ventral disks as well as asymmetrical division, indicating truncated or deregulated cytokinesis. Electron microscopy analysis revealed that DAB also triggered the encystment process. Oxidative stress was evaluated by measuring lipid peroxidation by thiobarbituric acid reactive substances (TBARS) detection. Trophozoites incubated either with 1 mM of DAB or putrescine for 18 h displayed increased lipoperoxide levels. Addition of 200 microM aminoguanidine, a polyamine/diamine oxidase inhibitor, partially reverted the DAB, but not the putrescine effects, indicating that the DAB effects are due, at least in part, to DAB oxidation end products. These data indicate that polyamines play a role in Giardia cell division, differentiation, and antioxidant defenses.