Interactions of Giardia lamblia with human intestinal mucus: enhancement of trophozoite attachment to glass

Giardia lamblia trophozoites frequently are associated with mucus in vivo. We investigated the effects of human intestinal mucus on parasite attachment and survival in vitro. All samples of mucus from the duodenum and ileum (from four humans and two rabbits) enhanced attachment at 100 micrograms/ml. Attachment increased with mucus concentrations from 1 to 1000 micrograms/ml but declined toward the unstimulated level at concentrations above 1000 micrograms/ml. Mucus from the small intestine also promoted the survival of the parasites during the 2-h incubation. In contrast, colonic mucus promoted survival, but inhibited attachment. Fractionation of mucus from the human small intestine by cesium chloride equilibrium density gradient ultracentrifugation revealed that both attachment- and survival-promoting activities were in the low density, protein-rich fraction. The high density fractions containing the mucins were devoid of activity. Thus, a non-mucin fraction of mucus from the human small intestine may promote colonization by G. lamblia.     

Intestinal mucus protects Giardia lamblia from killing by human milk

We have previously shown that nonimmune human milk kills Giardia lamblia trophozoites in vitro. Killing requires a bile salt and the activity of the milk bile salt-stimulated lipase. We now show that human small-intestinal mucus protects trophozoites from killing by milk. Parasite survival increased with mucus concentration, but protection was overcome during longer incubation times or with greater milk concentrations. Trophozoites preincubated with mucus and then washed were not protected. Protective activity was associated with non-mucin CsCl density gradient fractions. Moreover, it was heat-stable, non-dialyzable, and non-lipid. Whereas whole mucus inhibited milk lipolytic activity, protective mucus fractions did not inhibit the enzyme. Furthermore, mucus partially protected G. lamblia trophozoites against the toxicity of oleic acid, a fatty acid which is released from milk triglycerides by lipase. These studies show that mucus protects G. lamblia both by inhibiting lipase activity and by decreasing the toxicity of products of lipolysis. The ability of mucus to protect G. lamblia from toxic lipolytic products may help to promote intestinal colonization by this parasite.     

Cross-species transmission of Giardia spp.: inoculation of beavers and muskrats with cysts of human, beaver, mouse, and muskrat origin.

Giardia cysts isolated from humans, beavers, mice, and muskrats were tested in cross-species transmission experiments for their ability to infect either beavers or muskrats. Giardia cysts, derived from multiple symptomatic human donors and used for inoculation of beavers or muskrats, were shown to be viable by incorporation of fluorogenic dyes, excystation, and their ability to produce infections in the Mongolian gerbil model. Inoculation of beavers with 5 x 10(5) Giardia lamblia cysts resulted in the infection of 75% of the animals (n = 8), as judged by the presence of fecal cysts or intestinal trophozoites at necropsy. The mean prepatent period was 13.1 days. An infective dose experiment, using 5 x 10(1) to 5 x 10(5) viable G. lamblia cysts collected by fluorescence-activated cell sorting, demonstrated that doses of between, less than 50, and less than 500 viable cysts were required to produce infection in beavers. Scanning electron microscopy of beaver small intestine revealed that attachment of G. lamblia trophozoites produced lesions in the microvillous border. Inoculation of muskrats with G. lamblia cysts produced infections when the dose of cysts was equal to or greater than 1.25 x 10(5). The inoculation of beavers with Giardia ondatrae or Giardia muris cysts did not produce any infection; however, the administration to muskrats of Giardia cysts of beaver origin resulted in the infection of 62% of the animals (n = 8), with a prepatent period of 5 days. Our results demonstrated that beavers and muskrats could be infected with Giardia cysts derived from humans, but only by using large numbers of cysts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cross-species transmission of Giardia spp.: inoculation of beavers and muskrats with cysts of human, beaver, mouse, and muskrat origin

Giardia cysts isolated from humans, beavers, mice, and muskrats were tested in cross-species transmission experiments for their ability to infect either beavers or muskrats. Giardia cysts, derived from multiple symptomatic human donors and used for inoculation of beavers or muskrats, were shown to be viable by incorporation of fluorogenic dyes, excystation, and their ability to produce infections in the Mongolian gerbil model. Inoculation of beavers with 5 x 10(5) Giardia lamblia cysts resulted in the infection of 75% of the animals (n = 8), as judged by the presence of fecal cysts or intestinal trophozoites at necropsy. The mean prepatent period was 13.1 days. An infective dose experiment, using 5 x 10(1) to 5 x 10(5) viable G. lamblia cysts collected by fluorescence-activated cell sorting, demonstrated that doses of between, less than 50, and less than 500 viable cysts were required to produce infection in beavers. Scanning electron microscopy of beaver small intestine revealed that attachment of G. lamblia trophozoites produced lesions in the microvillous border. Inoculation of muskrats with G. lamblia cysts produced infections when the dose of cysts was equal to or greater than 1.25 x 10(5). The inoculation of beavers with Giardia ondatrae or Giardia muris cysts did not produce any infection; however, the administration to muskrats of Giardia cysts of beaver origin resulted in the infection of 62% of the animals (n = 8), with a prepatent period of 5 days. Our results demonstrated that beavers and muskrats could be infected with Giardia cysts derived from humans, but only by using large numbers of cysts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Giardia lamblia: electrophysiology and ultrastructure of cytopathology in cultured epithelial cells

The pathogenicity of Giardia lamblia is a subject of debate. Some studies of human biopsy material have mentioned the presence of trophozoites inside the intestinal mucosa, while in others, flagellates have only been found attached to the epithelium. To study the possible cytopathic effects of G. lamblia cultured under axenic conditions, trophozoites of the human 1/Portland and WB strains were placed in contact with monolayers of Madin Derby Canine Kidney cells, a well characterized cell strain with morphological and functional properties similar to those of a transporting epithelium. After 24 and 48 hr of interaction, the effect of the parasite on epithelial cells was assessed by transmission, scanning, and freeze fracture electron microscopy. In addition, the possible action of living trophozoites and sonicates of G. lamblia on the transepithelial resistance of MDCK monolayers mounted in Ussing chambers was analyzed for periods varying up to 48 hr. The results demonstrate that G. lamblia trophozoites do not invade epithelial monolayers. Furthermore, the parasites fail to produce cytoplasmic changes on target cells and have no effect on transepithelial resistance as judged both electrophysiologically and by the failure to open the occluding junctions that bind together epithelial cells. Damage induced by the parasites to cultured cells was limited to focal distortion or depletion of microvilli at the site of adhesion, which may progress to leave circular areas devoid of microvilli, different from the adhesion marks reported by others for G. muris. Therefore, under the in vitro conditions described here, giardias showed no toxic or invasive effect.     

The biology of Giardia spp.

Gardia spp. are flagellated protozoans that parasitize the small intestines of mammals, birds, reptiles, and amphibians. The infectious cysts begin excysting in the acidic environment of the stomach and become trophozoites (the vegetative form). The trophozoites attach to the intestinal mucosa through the suction generated by a ventral disk and cause diarrhea and malabsorption by mechanisms that are not well understood. Giardia spp. have a number of unique features, including a predominantly anaerobic metabolism, complete dependence on salvage of exogenous nucleotides, a limited ability to synthesize and degrade carbohydrates and lipids, and two nuclei that are equal by all criteria that have been tested. The small size and unique sequence of G. lamblia rRNA molecules have led to the proposal that Giardia is the most primitive eukaryotic organism. Three Giardia spp. have been identified by light lamblia, G. muris, and G. agilis, but electron microscopy has allowed further species to be described within the G. lamblia group, some of which have been substantiated by differences in the rDNA. Animal models and human infections have led to the conclusion that intestinal infection is controlled primarily through the humoral immune system (T-cell dependent in the mouse model). A major immunogenic cysteine-rich surface antigen is able to vary in vitro and in vivo in the course of an infection and may provide a means of evading the host immune response or perhaps a means of adapting to different intestinal environments.     

The biology of Giardia spp.

Gardia spp. are flagellated protozoans that parasitize the small intestines of mammals, birds, reptiles, and amphibians. The infectious cysts begin excysting in the acidic environment of the stomach and become trophozoites (the vegetative form). The trophozoites attach to the intestinal mucosa through the suction generated by a ventral disk and cause diarrhea and malabsorption by mechanisms that are not well understood. Giardia spp. have a number of unique features, including a predominantly anaerobic metabolism, complete dependence on salvage of exogenous nucleotides, a limited ability to synthesize and degrade carbohydrates and lipids, and two nuclei that are equal by all criteria that have been tested. The small size and unique sequence of G. lamblia rRNA molecules have led to the proposal that Giardia is the most primitive eukaryotic organism. Three Giardia spp. have been identified by light lamblia, G. muris, and G. agilis, but electron microscopy has allowed further species to be described within the G. lamblia group, some of which have been substantiated by differences in the rDNA. Animal models and human infections have led to the conclusion that intestinal infection is controlled primarily through the humoral immune system (T-cell dependent in the mouse model). A major immunogenic cysteine-rich surface antigen is able to vary in vitro and in vivo in the course of an infection and may provide a means of evading the host immune response or perhaps a means of adapting to different intestinal environments.     

Recent insights into the mucosal reactions associated with Giardia lamblia infections

Giardia lamblia is an intestinal protozoan parasite infecting humans and various other mammalian hosts. The most important clinical signs of giardiasis are diarrhoea and malabsorption. Giardia lamblia is able to undergo continuous antigenic variation of its major surface antigen, named VSP (variant surface protein). While intestinal antibodies, and more specifically anti-VSP IgA antibodies, were proven to be involved in modulating antigenic variation of the parasite the participation of the local antibody response in control of the parasite infection is still controversial. Conversely, previous studies based on experimental infections in mice showed that cellular immune mechanisms are essential for elimination of the parasite from its intestinal habitat. Furthermore, recent data indicated that inflammatory mast cells have a potential to directly, or indirectly, interfere in duodenal growth of G. lamblia trophozoites. However, this finding was challenged by other reports, which did not find a correlation between intestinal inflammation and resistance to infection. Since intestinal infiltration of inflammatory cells and/or CD8+T-cells were demonstrated to coincide with villus-shortening and crypt hyperplasia immunological reactions were considered to be a potential factor of pathogenesis in giardiasis. The contribution of physiological factors to pathogenesis was essentially assessed in vitro by co-cultivation of G. lamblia trophozoites with epithelial cell lines. By using this in vitro model, molecular (through surface lectins) and mechanical (through ventral disk) adhesion of trophozoites to the epithelium was shown to be crucial for increased epithelial permeability. This phenomenon as well as other Giardia-induced intestinal abnormalities such as loss of intestinal brush border surface area, villus flattening, inhibition of disaccharidase activities, and eventually also overgrowth of the enteric bacterial flora seem to be involved in the pathophysiology of giardiasis. However, it remains to be elucidated whether at least part of these pathological effects are causatively linked to the clinical manifestation of the disease.     

Lipid Requirements and Lipid Uptake by Giardia lamblia Trophozoites in Culture

To better understand the lipid requirements of Giardia lamblia trophozoites and the mechanisms of lipid uptake, we supplemented serum-free TYI-S-33 medium with lipids incorporated into different lipid carriers. We found that serum lipoproteins, β-cyclodextrins, and bile salts are able to supply cholesterol and phospholipids to Giardia and to support the multiplication of the parasite in vitro. The growth rates obtained with different lipoproteins or bile salts and lipid mixtures were similar to that in standard culture medium containing serum. Pulse labelling experiments using fluorescent lipid analogs demonstrated that Giardia can take up lipids from lipoproteins, β-cyclodextrins, or bile salt micelles, but with different kinetics, and that bile salts greatly facilitated lipid transfer from lipoproteins and cyclodextrins to the parasite surface. The binding of different radioiodinated lipoprotein classes to the trophozoite surface, inhibition of lipoprotein interiorization at 4°C or by cytochalasin D, and incorporation studies using fluorescent LDL suggested that a small component of lipid uptake by trophozoites was likely due to endocytosis of lipoproteins.     

Giardia lamblia rearranges F-actin and alpha-actinin in human colonic and duodenal monolayers and reduces transepithelial electrical resistance

The mechanisms of epithelial injury in giardiasis remain unknown. The effects of live Giardia lamblia on cellular G-actin, F-actin, alpha-actinin, and electrical resistance of human intestinal epithelial monolayers were investigated using SCBN and Caco2 cell lines grown on chamber slides or Transwell filter membranes. In separate experiments, some monolayers were also exposed to sonicated trophozoites, some to supernatant from live G. lamblia cultures, and some with or without the Ca2+ channel blocker verapamil. After 2, 24, or 48 hr of coincubation with G. lamblia, monolayers were assessed for cytoskeletal arrangement under fluorescence and confocal laser microscopy, and transepithelial electrical resistance was measured. Exposure to live G. lamblia trophozoites induced localized condensation of F-actin and loss of perijunctional alpha-actinin while G-actin remained unchanged. Confocal laser microscopy indicated that F-actin rearrangement was not affected by verapamil and was localized within the terminal web area. Coincubation of monolayers with G. lamblia lysates or with spent medium alone similarly rearranged F-actin. Verapamil alone did not alter F-actin. Electrical resistance of SCBN and Caco2 monolayers exposed to G. lamblia was significantly decreased versus controls regardless of whether live or lysed trophozoite samples were used. The results indicate that G. lamblia-induced epithelial injury is associated with F-actin and alpha-actinin rearrangements in the terminal web area via mechanisms independent of extracellular Ca2+. These alterations are associated with reduced transepithelial electrical resistance and are due at least in part to trophozoite products.     

Nitric oxide production by human intestinal epithelial cells and competition for arginine as potential determinants of host defense against the lumen-dwelling pathogen Giardia lamblia

Giardia lamblia infection of the human small intestine is a common protozoan cause of diarrheal disease worldwide. Although infection is luminal and generally self-limiting, and secretory Abs are thought to be important in host defense, other defense mechanisms probably affect the duration of infection and the severity of symptoms. Because intestinal epithelial cells produce NO, and its stable end products, nitrite and nitrate, are detectable mainly on the apical side, we tested the hypothesis that NO production may constitute a host defense against G. lamblia. Several NO donors, but not their control compounds, inhibited giardial growth without affecting viability, suggesting that NO is cytostatic rather than cytotoxic for G. lamblia. NO donors also inhibited giardial differentiation induced by modeling crucial environmental factors, i. e., encystation induced by bile and alkaline pH, and excystation in response to gastric pH followed by alkaline pH and protease. Despite the potent antigiardial activity of NO, G. lamblia is not simply a passive target for host-produced NO, but has strategies to evade this potential host defense. Thus, in models of human intestinal epithelium, G. lamblia inhibited epithelial NO production by consuming arginine, the crucial substrate used by epithelial NO synthase to form NO. These studies define NO and arginine as central components in a novel cross-talk between a luminal pathogen and host intestinal epithelium.     

Interactions of Giardia lamblia with Human Intestinal Mucus: Enhancement of Trophozoite Attachment to Glass1

Giardia lamblia trophozoites frequently are associated with mucus in vivo. We investigated the effects of human intestinal mucus on parasite attachment and survival in vitro. All samples of mucus from the duodenum and ileum (from four humans and two rabbits) enhanced attachment at 100 μm/ml. Attachment increased with mucus concentrations from 1 to 1000 μg/ml but declined toward the unstimulated level at concentrations above 1000 μg/ml. Mucus from the small intestine also promoted the survival of the parasites during the 2-h incubation. In contrast, colonic mucus promoted survival, but inhibited attachment. Fractionation of mucus from the human small intestine by cesium chloride equilibrium density gradient ultracentrifugation revealed that both attachment- and survival-promoting activities were in the low density, protein-rich fraction. The high density fractions containing the mucins were devoid of activity. Thus, a non-mucin fraction of mucus from the human small intestine may promote colonization by G. lamblia.     

Electron and video-light microscopy analysis of the in vitro effects of pyrantel pamoate on Giardia lamblia

Electron and video-light microscopy analysis of the in vitro effects of pyrantel pamoate on Giardia lamblia. Experimental Parasitology 97, 9-14. Giardia infection is predominant in the small intestine of vertebrates, where the trophozoites attach to epithelial cells and adversely affect the microvilli and other epithelial cell structures. Giardiasis, the disease caused by this protozoan, is very common in developing countries and mainly affects children. Drugs currently used to treat Giardia infection, such as some benzimidazole derivatives, were originally designed to treat helminthic infections. Many of the drugs are known to cause severe side effects and disturbances to the patient. Using transmission electron microscopy and video-light microscopy, we studied the effects of pyrantel pamoate, a drug commonly used in the treatment of helminthic infections in horses and ruminants, on Giardia lamblia trophozoites. Pyrantel pamoate was administered to Giardia cells in four different concentrations. Using video-light microscopy, we observed the decrease in flagella beating frequency and severe changes in the lateral flange and in the general aspect of the cell. Using transmission electron microscopy, we observed changes in the cytoplasm and peripheral vesicles. The flagella and adhesive disk structure were not affected. Apparently, the effects of pyrantel pamoate are irreversible.     

Giardia lamblia: the role of conjugated and unconjugated bile salts in killing by human milk

Killing of Giardia lamblia trophozoites by nonimmune human milk in vitro is dependent upon the presence of cholate which activates the milk bile salt-stimulated lipase to cleave fatty acids from milk triglycerides. In the present studies, conjugated bile salts, which predominate in vivo, displayed striking differences from unconjugated bile salts in ability to support killing by milk. Human milk killed greater than 99% of the parasites in the presence of cholate, but not glycocholate or taurocholate. In contrast, after brief sonication which disrupts milk fat globules, milk killed G. lamblia after addition of either conjugated or unconjugated bile salts. Whereas cholate stimulated milk lipase to cleave triglycerides of either unsonicated or sonicated human milk, glycocholate or taurocholate stimulated lipolysis only in sonicated milk. Since the concentration of bile salts in the small intestine fluctuates, the effect of this variable on killing was examined. Each bile salt at and above its critical micellar concentration increased Giardia survival of human milk probably because it sequestered released fatty acids in micelles. This partial protection could be overcome by increasing the milk concentration. Human hepatic and gall bladder bile and artificial bile also activated human milk to kill at low concentrations but partly protected the parasite at higher concentrations. These studies show that conjugated bile salts can activate the bile salt-stimulated lipase of sonicated human milk to release fatty acids; and kill G. lamblia. Conversely, bile salts in concentrations above their critical micellar concentration sequester fatty acids and interfere with killing. Thus, nonimmune host secretions such as milk and bile may affect the course of infection by G. lamblia.     

Synchronisation of Giardia lamblia: identification of cell cycle stage-specific genes and a differentiation restriction point

The intestinal parasite Giardia lamblia undergoes cell differentiations that entail entry into and departure from the replicative cell cycle. The pathophysiology of giardiasis depends directly upon the ability of the trophozoite form to replicate in the host upper small intestine. Thus, cell proliferation is tightly linked to disease. However, studies of cell cycle regulation in Giardia have been hampered by the inability to synchronise cultures. Here we report that Giardia isolates of the major human genotypes A and B can be synchronised using aphidicolin, a mycotoxin that reversibly inhibits replicative DNA polymerases in eukaryotic cells. Aphidicolin arrests Giardia trophozoites in the early DNA synthesis (S) phase of the cell cycle. We identified a set of cell cycle orthologues in the Giardia genome using bioinformatic analyses and showed that synchronised parasites express these genes in a cell cycle stage-specific manner. The synchronisation method also showed that during encystation, exit from the ordinary cell cycle occurs preferentially in G(2) and defines a restriction point for differentiation. Synchronisation opens up possibilities for further molecular and cell biological studies of chromosome replication, mitosis and segregation of the complex cytoskeleton in Giardia.     

Encystation of Giardia lamblia: a model for other parasites

To colonize the human small intestine, Giardia lamblia monitors a dynamic environment. Trophozoites attach to enterocytes that mature and die. The parasites must 'decide' whether to re-attach or differentiate into cysts that survive in the environment and re-activate when ingested. Other intestinal parasites face similar challenges. Study of these parasites is limited because they do not encyst in vitro. Giardia trophozoites were persuaded to encyst in vitro by mimicking physiological stimuli. Cysts are dormant, yet 'spring-loaded for action' to excyst upon ingestion. Giardial encystation has been studied from morphological, cell biological, biochemical, and molecular viewpoints. Yet important gaps remain and the mechanisms that co-ordinate responses to external signals remain enigmatic.     

Caspase-dependent apoptosis of the HCT-8 epithelial cell line induced by the parasite Giardia intestinalis

Giardia intestinalis is a flagellated protozoan which causes enteric disease worldwide. Giardia trophozoites infect epithelial cells of the proximal small intestine and can cause acute or chronic diarrhea. The mechanism of epithelial injury in giardiasis remains unknown. A number of enteric pathogens, including protozoan parasites, are able to induce enterocyte apoptosis. The aim of this work was to assess whether G. intestinalis strain WB clone C6 is able to induce apoptosis in the human intestinal epithelial cell line HCT-8, and to investigate the role of caspases in this process. Results demonstrated that the parasite induces cell apoptosis, as confirmed by DNA fragmentation analysis, detection of active caspase-3 and degradation of the caspase-3 substrate PARP [poly(ADP-ribose) polymerase]. Furthermore, G. intestinalis infection induces activation of both the intrinsic and the extrinsic apoptotic pathways, down-regulation of the antiapoptotic protein Bcl-2 and up-regulation of the proapoptotic Bax, suggesting a possible role for caspase-dependent apoptosis in the pathogenesis of giardiasis.     

Development of species-specific rDNA probes for Giardia by multiple fluorescent in situ hybridization combined with immunocytochemical identification of cyst wall antigens.

In this study, we describe the development of fluorescent oligonucleotide probes to variable regions in the small subunit of 16S rRNA in three distinct Giardia species. Sense and antisense probes (17-22 mer) to variable regions 1, 3, and 8 were labeled with digoxygenin or selected fluorochomes (FluorX, Cy3, or Cy5). Optimal results were obtained with fluorochome-labeled oligonucleotides for detection of rRNA in Giardia cysts. Specificity of fluorescent in situ hybridization (FISH) was shown using RNase digestion and high stringency to diminish the hybridization signal, and oligonucleotide probes for rRNA in Giardia lamblia, Giardia muris, and Giardia ardeae were shown to specifically stain rRNA only within cysts or trophozoites of those species. The fluorescent oligonucleotide specific for rRNA in human isolates of Giardia was positive for ten different strains. A method for simultaneous FISH detection of cysts using fluorescent antibody (genotype marker) and two oligonucleotide probes (species marker) permitted visualization of G. lamblia and G. muris cysts in the same preparation. Testing of an environmental water sample revealed the presence of FISH-positive G. lamblia cysts with a specific rDNA probe for rRNA, while negative cysts were presumed to be of animal or bird origin.     

A potentially important excretory-secretory product of Giardia lamblia

In this study we have reported the detailed characterization of a 58 kDa excretory-secretory product (ESP) of Giardia lamblia. The method of purification has been simplified which has improved the purification fold as well as the yield of the ESP. The binding efficacy of disialoganglioside (GD2) to the purified ESP was found to be maximum among all other gangliosides used. The N-terminal sequence of the immunoreactive 29 kDa peptide obtained from partial tryptic digest of the ESP was found to be AD-FVPQVST. The IgG against the purified ESP (IgGES) showed cross-reactivity with the binding subunit of the commercially available cholera toxin and also with two protein bands of western cottonmouth moccasin snake toxin. The ESP could accumulate fluid in the intestine of sealed adult mice and also induce morphological changes in HEp-2 cells. The crude extract of G. lamblia trophozoites preincubated with Escherichia coli revealed 8-fold augmentation in the cytopathic activity on HEp-2 cells as compared to that of crude preparation from trophozoites only.     

The Uptake and Metabolism of Cysteine by Giardia lamblia Trophozoites

ABSTRACT. The cysteine, cystine, methionine and sulfate uptake and cysteine metabolism of Giardia lamblia was studied. Initial experiments indicated that bathocuproine sulphonate (20 μM) added to Keister's modified TYI-S-33 medium supported the growth of G. lamblia at low L-cysteine concentration. This allowed the use of high specific activity radiolabeled L-cysteine for further studies. The analyses of L-cysteine uptake by G. lamblia indicate the presence of at least two different transport systems. The total cysteine uptake was non saturable, with a capacity of 3.7 pmoles per 10⁶ cells per min per μM of cysteine, and probably represent passive diffusion. However, cysteine transport was partially inhibited by L-methionine, D-cysteine and DL-homocysteine. indicating that another system specific for SH-containing amino acids is also present. Cysteine uptake was markedly decreased in medium without serum. In contrast to cysteine, the uptake of L-methionine and sulfate were carried out by saiurable systems with apparent K_m, of 71 and 72 μM, respectively, but the Vmax of the uptake of sulfate was six orders of magnitude lower than the Vmax of methionine uptake. Cystine was not incorporated into trophozoites. [³⁵S]-labeled L-cysteine and L-methionine, but not [³⁵S]sulfate, were incorporated into Giardia proteins, indicating that the parasite lacks the capacity to synthesize cysteine or methionine from sulfate. Neither cystathionine γ lyase nor crystathionine γ synthase activities was detected in homogenates of Giardia lamblia , suggesting that the transsulfuration pathway is not active and there is no conversion of methionine to cysteine. Our data indicate that cysteine is essential for Giardia because the parasite: a) cannot take up cystine, and b) cannot synthesize cysteine de novo.