INTRODUCTION

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Giardia lamblia (syn. Giardia intestinalis, Giardia duodenalis) is a flagellated unicellular eukaryotic microorganism that commonly causes diarrheal disease throughout the world. It is the most common cause of waterborne outbreaks of diarrhea in the United States (18) and is occasionally seen as a cause of food-borne diarrhea (47a, 227). In developing countries, there is a very high prevalence and incidence of infection, and data suggest that long-term growth retardation can result from chronic giardiasis (105). In certain areas of the world, water contaminated with G. lamblia cysts commonly causes travel-related giardiasis in tourists (33).

Giardia species have two major stages in the life cycle. Infection of a host is initiated when the cyst is ingested with contaminated water or, less commonly, food or through direct fecal-oral contact. The cyst is relatively inert, allowing prolonged survival in a variety of environmental conditions. After exposure to the acidic environment of the stomach, cysts excyst into trophozoites in the proximal small intestine. The trophozoite is the vegetative form and replicates in the small intestine, where it causes symptoms of diarrhea and malabsorption. After exposure to biliary fluid, some of the trophozoites form cysts in the jejunum and are passed in the feces, allowing completion of the transmission cycle by infecting a new host.

The clinical aspects of giardiasis have been reviewed recently (260), as has the host immune response to giardiasis (95) and epidemiology (106). This review will focus primarily on the biology of the organism and deal relatively little with the clinical disease or the host-parasite interaction. Since a previous review of Giardia species (3), considerable progress has been made in the understanding of the organism, and these new advances will be emphasized. Many of the major current advances have been facilitated by the ongoing progress of the G. lamblia genome project, based at the Marine Biological Laboratories and involving collaborators at the University of Illinois, University of Texas at E1 Paso, University of California at San Diego, and University of Arizona (210). The sequences as of June 2000 give at least a single-pass read of 85% of the genome, and the results are available at www.mbl.edu/Giardia.

CLASSIFICATION AND EVOLUTION OF GIARDIA SPECIES

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History of the Discovery and Species Designation of Giardia

Giardia was initially described by van Leeuwenhoek in 1681 as he was examining his own diarrheal stools under the microscope (66). The organism was described in greater detail by Lambl in 1859, who thought the organism belonged to the genus Cercomonas and named it Cercomonas intestinalis (172). Thereafter, some have named the genus after him while others have named the species of the human form after him (i.e., G. lamblia). In 1879, Grassi named a rodent organism now known to be a Giardia species, Dimorphus muris, apparently unaware of Lambl's earlier description. In 1882 and 1883, Kunstler described an organism in tadpoles (?G. agilis) that he named Giardia, the first time Giardia was used as a genus name. In 1888, Blanchard suggested the name Lamblia intestinalis (29), which Stiles then changed to G. duodenalis in 1902 (314). Subsequently, Kofoid and Christiansen proposed the names G. lamblia in 1915 (165) and G. enterica in 1920 (166). There continued to be controversy about the number of Giardia species for many years, with some investigators suggesting species names on the basis of host of origin and others focusing on morphology. For example, over 40 species names had been proposed on the basis of host of origin (169). Simon, on the other hand, used morphologic criteria to distinguish between G. lamblia and G. muris and accepted the name G. lamblia for the human form (304). In 1952, Filice published a detailed morphologic description of Giardia and proposed that three species names be used on the basis of the morphology of the median body: G. duodenalis, G. muris, and G. agilis (104). The species name G. lamblia became widely accepted through the 1970s. Since the 1980s, some have encouraged the use of the name G. duodenalis, and in the 1990s, the name G. intestinalis has been encouraged by other investigators (170). At this time there does not appear to be adequate reason to abandon the term G. lamblia, which has been widely accepted in the medical and scientific literature.

G. lamblia is pear shaped and has one or two transverse, claw-shaped median bodies; G. agilis is long and slender (100) and has a teardrop-shaped median body; and the G. muris trophozoite is shorter and rounder and has a small, rounded median body. G. lamblia is found in humans and a variety of other mammals, G. muris is found in rodents, and G. agilis is found in amphibians (Table 1).

For the Giardia isolates grouped with G. lamblia on the basis of morphologic criteria discernible by light microscopy, differences that can be detected by electron microscopy have allowed the description of additional species, G. psittaci from parakeets (78) and G. ardeae from herons (81). Another species, Giardia microti, has been suggested on the basis of host specificity for voles and muskrats, differences in the cyst as assessed by electron micrography (97), and by differences of the 18S rRNA sequences compared with G. lamblia of human origin (331).

Molecular classification tools have been of great value in understanding the pathogenesis and host range of Giardia isolates obtained from humans and a variety of other mammals. The first study of the molecular differences of G. lamblia isolates (26) was a zymodeme analysis of five axenized isolates, three from humans, one from a guinea pig, and one from a cat, using six metabolic enzymes. Zymodeme analysis consists of the typing of organisms based on the migration of a set of enzymes on a starch gel in the presence of an electric field. The migration depends on the size, structure, and isoelectric point of these enzymes. Since these properties are a function of the primary amino acid sequence, differences in the zymodemes should reflect differences in the sequences of the genes encoding these enzymes. In 1985, restriction fragment length polymorphism analysis of 15 isolates was performed using random probes (251). These studies resulted in the description of three groups; group 3 was so different from groups 1 and 2 that the suggestion of a separate species designation was made. Subsequently, a number of other molecular classification studies have been performed using zymodeme analysis (2, 16, 47, 212, 214, 234, 277, 316) and restriction fragment length polymorphism analysis (65, 85-87, 90, 137). Pulsed-field gel electrophoresis (PFGE) chromosome patterns have also been studied (7, 42, 143, 167, 291) but are of limited value for classification because of the frequent occurrence of chromosome rearrangements (4, 179). Likewise, classification by surface antigens (250) is limited by antigenic variation of the variant-specific proteins (VSPs) (6, 243). These studies have been very useful, but the conclusions that can be drawn from these types of data are limited by the semiquantitative nature of the data. To allow a more quantitative comparison of Giardia isolates, sequence comparisons of the small-subunit rRNA, triosephosphate isomerase (tim), and glutamate dehydrogenase (GDH) genes have been utilized in a number of subsequent studies (17, 92, 193, 229-231).

These studies have all confirmed the division of G. lamblia human isolates into two major genotypes (Table 2). The first consists of Nash groups 1 and 2, Mayrhofer assemblage A, groups 1 and 2, and the Polish isolates, while Nash group 3, Mayrhofer assemblage B, groups 3 and 4, and the Belgian isolates form the other major genotype. For example, the tim nucleotide sequences of the group 1 and 2 isolates diverged by 1% in the protein coding region and 2% in the flanking regions, while groups 1 and 3 diverged by 19% and the flanking regions were so dissimilar as to preclude their alignment (193). The small-subunit or 18S rRNA (SS rRNA) sequence shows a 1% divergence between groups 1 and 3 (333), reflecting its more highly conserved sequence. In addition to their marked genetic differences, the two genotypes may have a number of important biologic differences. For example, the GS isolate (group 3) was significantly more pathogenic in infections of human volunteers than was the WB isolate (group 1) (249). Group 3 organisms also appear to grow much more slowly in axenic culture than do genotype 1 organisms (155).

More recently, a number of additional assemblages (genotypes) have been proposed for Giardia isolates from a variety of mammals. These isolates are morphologically identical to human G. lamblia, but sequences of their protein-coding regions differ. These studies have allowed the identification of a dog isolate that is genetically distinct from human G. lamblia. Giardia isolates from dogs have been notoriously difficult to axenize, in comparison to isolates from humans (213), leading to the proposal that Giardia isolates from dogs were different from cat or human isolates. However, a few dog isolates have been axenized and characterized (17). To further evaluate the zoonotic potential of dog Giardia, suckling-mouse infections were established from 11 consecutive infected dogs. On the basis of sequence analysis, these were assigned to two assemblages (C and D) that were quite distinct from assemblages A and B (230). A PCR-based study of nine fecal isolates from dogs found that one of the nine was similar to human isolates while the other eight were different (138). These results suggest that most dog isolates are genetically distinct from those found in humans and have little or no potential for zoonotic transmission.

Separate assemblages (E through G) have also been proposed for hoofed livestock (92), cat (229), and rat (229) isolates (Table 2). This same sequence-based study demonstrated that G. microti was a member of this compilation of seven assemblages (229). Further studies of Giardia obtained from cattle have demonstrated that some of the isolates belong to the livestock assemblage (assemblage E) while others belong to assemblage A (genotype 1) and thus may have the potential for human infection (259). Assemblages C through G have not yet been isolated from humans, suggesting the likelihood that some genotypes of G. lamblia have a broad range of host specificity that includes humans while others appear to be more restricted in their host range and may not pose a risk of zoonotic transmission. Whether these seven assemblages should be considered separate species should await further data and consensus. To unify the designation of the G. lamblia genotypes, I propose an approach that takes into account all the currently available data. Priority should go to the first classification suggested by Nash et al. (251) in 1985. However, "group" has been used in different ways by different authors. In addition, it is clear from data published after the initial description that Nash groups 1 and 2 are closely related while group 3 is genetically distinct enough to allow consideration of a separate species or subspecies name as suggested (251). The assignment of species status to clonal organisms is problematic (324) and should be delayed until there is general agreement among investigators, but agreement on genotype designation should facilitate a better understanding and coordination of the literature. Therefore, I propose that genotype A be accepted as the designation for Nash group 1 (A-1), Nash group 2 (A-2), assemblage A, and the Polish isolates. Genotype B would then designate Nash group 3, assemblage B, and the Belgian isolates. It may also be appropriate to use a similar designation for assemblages C through G, since these also reflect significant genetic differences as well as host differences.

Traditionally, all living organisms have been classified as prokaryotes or eukaryotes, and some still argue for retaining the two major divisions (208). However, the most widely accepted classification now utilizes three major divisions, Archaea (archaebacteria), Bacteria (eubacteria), and Eukarya (eukaryotes) (353), which can then be divided into kingdoms. With either classification system, G. lamblia is clearly a eukaryotic organism and has been considered a member of the protozoa, the more "animal-like" of the unicellular eukaryotes. These protozoan organisms have traditionally been classified by their morphology into flagellates, ciliates, amebae (rhizopods), and sporozoa. Thus, G. lamblia was classified with the flagellated protozoans, including the kinetoplastids (e.g., Leishmania spp. and Trypanosoma spp.), parabasalids (e.g., Trichomonas vaginalis), and Dientamoeba (e.g., Dientamoeba fragilis) (185). Giardia has been placed in the order Diplomonadida (two karyomastigonts, each with four flagella, two nuclei, no mitochondria, and no Golgi complex; cysts are present, and it can be free-living or parasitic) and the family Hexamitidae (six or eight flagella, two nuclei, bilaterally symmetrical, and sometimes axostyles and median or parabasal bodies), along with the mole parasite Sppironucleus muris (145) and the free-living organism Hexamita inflata. Some of the higher orders of classification do not appear to be phylogenetically valid, such as the placement of all flagellated protozoans together. However, the family Hexamitidae does appear to be a monophyletic group (see below).

One recent classification system with one prokaryotic and five eukaryotic kingdoms has retained Protozoa as one of the six kingdoms (45), but most recent proposals have suggested abandoning the term "protozoa" in favor of the more general but at the same time more precise term "protista" (56, 57, 350). This review will use the term "protista," but it should be noted that in the clinical context, G. lamblia is usually referred to as a protozoan.

Recent classifications of the eukaryotic microbial organisms have depended primarily on molecular comparisons. An ideal molecular classification system would be based on a gene(s) that is required for all life and that is sufficiently highly conserved across all forms of life that accurate alignments allow comparison and classification of all organisms. In many ways, rRNA has been the most useful gene for molecular comparisons, because rRNA sequences are highly conserved across life and because the function of the rRNA is so central to the biology of the organism. Therefore, the most widely accepted classification scheme has been based on SS (18S) rRNA sequences. Based on comparisons of SS rRNA sequences, G. lamblia was proposed as one of the most primitive eukaryotic organisms (308), along with T. vaginalis and the microsporidia. The use of SS rRNA sequences to place Giardia as an early-branching eukaryote has been criticized because of the high G+C content of the SS rRNA of Giardia (75%) and because Giardia spp. are parasitic organisms; artifacts may be introduced by high rates of mutation accompanying host adaptation. An analysis of the early-branching eukaryotes suggested that the basal position of Giardia was an artifactual result of long-branch attraction due to a greater evolutionary rate of Giardia (315). Analysis of the large subunit of RNA polymerase II and reanalysis of the eF1 and eF2 sequences also supported the idea of the long-branch artifact (133). However, no such effect was shown for the eRF3 tree (142). In addition, the absence in G. lamblia of the highly conserved N-terminal domain of eRF3 found in other eukaryotes including T. vaginalis suggested the divergence of G. lamblia before the acquisition of the N-terminal domain (142). It should also be noted that the phylogenetic placement of Hexamita, a free-living organism in the same family (as determined by morphologic criteria), avoids artifacts due to high G+C content or parasitism. The G+C content of the Hexamita inflata SS rRNA is 51%, and H. inflata was found to be monophyletic with Giardia (183, 332). The classification of H. inflata with G. lamblia is also supported by a comparison of the glyceraldehyde-3-phosphate dehydrogenase sequences of G. lamblia, Trepomonas agilis, H. inflata, and Spironucleus sp. (287). A comparison of the SS rRNA sequences of Giardia, Hexamita, Trepomonas, and Spironucleus, all diplomonads, showed that all were phylogenetically related and that the last three comprised one clade while Giardia occupied another clade (46). These sequence-based comparisons have led to the following proposed classification system for Giardia: kingdom Protozoa, subkingdom Archezoa (includes the phyla Metamonada and Microsporidia), subphylum Eopharyngia, class Trepomonadea, subclass Diplozoa, and order Giardiida (includes the families Octomitidae and Giardiidae). In this classification system, the diplomonads are considered to comprise the subclass, Diplozoa, and Hexamita, Trepomonas, and Spironucleus are all members of the other diplozoan order, Distomatida (46).

An examination of the different genes used for the phylogenic classification of Giardia shows that the genes used in transcription and translation generally yield a basal position for Giardia, although artifact due to long-branch attraction has not been ruled out (Table 3). Interestingly, a phylogenetic tree of one of the genes thought to be of mitochondrial origin (cpn60) also suggests that Giardia is an early-branching eukaryote (107, 286). Of the other groups of genes, the metabolic genes do not yield a clear position, but some suggest a eubacterial origin for Giardia. Cytoskeletal genes give mixed results, with actin giving an early divergence but tubulin yielding a divergence that is later than that of Entamoeba histolytica. Among other classes of genes, HSP70 and cathepsin B phylogenies suggest an early divergence for Giardia. Thus, although the answers are not conclusive, most of the data suggest that G. lamblia and the other diplomonads are among the most basal of the extant eukaryotes.

The hypothesis that eukaryotes arose through endosymbiotic events, resulting in the origin of plastids and mitochondria and the use of oxidative phosphorylation as the major source of energy production, has become widely accepted (120). One version of this hypothesis is that a common ancestor of the archaebacteria and eukaryotes evolved a nucleus and cytoskeleton. A descendant then endocytosed a eubacterium, resulting in the development of a mitochondrion. The observation that certain amitochondriate eukaryotes (e.g., G. lamblia and other diplomonads as well as Trichomonas vaginalis) were basal on an 18S rRNA tree led to the suggestion that those organisms descended from Archezoa before the plastid symbiotic event (44, 46). However, the subsequent recognition of mitochondrial genes in the genomes of T. vaginalis (39) and G. lamblia (286) has led to the suggestion that the amitochondrial protists have secondarily lost their mitochondria.

An alternative version of the symbiotic hypothesis is that eukaryotes developed through a symbiotic association of an archaebacterium with a eubacterium (207). In this proposal, the eubacterial endosymbiont provided the pathways for organisms depending on the metabolism of pyruvate by mitochondrial respiration as well as those depending on pyruvate metabolism by pyruvate:ferredoxin oxidoreductase (PFOR), either in the cytosol (G. lamblia) or in hydrogenosomes (T. vaginalis). According to this proposal, the organism leading to G. lamblia subsequently lost the enzymatic components of mitochondrial respiration.

BIOCHEMISTRY AND METABOLISM

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Axenic Growth of Trophozoites

G. lamblia trophozoites obtained from a rabbit, chinchilla, and cat were first grown axenically (in the absence of exogenous cells) in 1970 (222). HSP-1 medium, a subsequent modification reported in 1976, contained phytone peptone, glucose, L-cysteine HCl, Hanks solution, and human serum (223). This study reported the first human G. lamblia isolate, Portland-1 (P-1). P-1 and WB, an isolate obtained from a symptomatic human who probably acquired his infection in Afghanistan (307), belong to the same genotype and have been used for many of the studies of G. lamblia. The growth medium has subsequently been modified, and currently the most commonly used medium is modified TYI-S-33 (159) (Table 4). Among the notable requirements for axenic growth are the absolute requirements for a low O₂ concentration, a high cysteine concentration, and the requirement for exogenous lipids, which are obtained from the serum component. When kept at 37°C, the trophozoites adhere to the glass wall of the tube in which they are grown. This adherence is dependent on glycolysis and on contraction of the proteins of the ventral disk (99). To date, the other species of Giardia (e.g., G. muris and G. agilis) have not been grown axenically.

Most eukaryotic organisms depend primarily on aerobic metabolism for their energy production. However, certain eukaryotes, including Trichomonas spp., Entamoeba spp., and Giardia spp., are characterized by their lack of mitochondria and cytochrome-mediated oxidative phosphorylation. They rely on fermentative metabolism (even when oxygen is present) for energy conservation. Glycolysis and its brief extensions generate ATP, with generation dependent only on substrate level phosphorylation. Glucose is not completely oxidized to CO₂ and H₂O as in aerobic metabolism but is incompletely catabolized to acetate, ethanol, alanine, and CO₂. The balance of end product formation is sensitive to the O₂ tension and glucose concentration in the medium. The proposed pathways of energy production from glucose and aspartate are shown in Fig. 1 to 3.

View larger version (17K): [in this window] [in a new window]   FIG. 1.   Metabolism of glucose to phosphoenolpyruvate. Many of the enzymes have been documented in terms of enzymatic activity, isolation of the enzyme, or cloning of the gene encoding the enzyme. The enzymes are labeled as follows: 1, hexokinase (188); 2, glucose phosphate isomerase (proposed); 3, pyrophosphate-dependent phosphofructokinase (186, 219, 274, 289); 4, fructose bisphosphate aldolase (128, 188); 5, triosephosphate isomerase (238); 6, glyceraldehyde-3-phosphate dehydrogenase (287); 7, phosphoglycerate kinase (proposed); 8, phosphoglyceromutase (proposed); 9, enolase (proposed). Certain enzymes (for steps 2, 7, 8, and 9) are suggested on the basis of pathways in other organisms but have not yet been proved for Giardia. This figure has been adapted from material presented in prior reviews (55, 146, 150) and updated from more recent literature as cited for individual enzymes.

View larger version (15K): [in this window] [in a new window]   FIG. 2.   Intermediary pathways to synthesis of pyruvate. 1, Pyruvate phosphate dikinase (132, 141); 2, pyruvate kinase (271) (the relative roles of pyruvate phosphate dikinase and pyruvate kinase in the conversion of phosphoenolpyruvate to pyruvate have not been determined); 3, phosphoenolpyruvate carboxyphosphotransferase; 4, malate dehydrogenase (188, 285); 5, malate dehydrogenase (decarboxylating) (188, 294); 6, alanine transaminase (72, 263); 7, aspartate transaminase (215). Pi, inorganic phosphate. This figure has been adapted from material presented in prior reviews (55, 215) and updated from more recent literature (see citations for individual enzymes).

View larger version (13K): [in this window] [in a new window]   FIG. 3.   End product synthesis from pyruvate. The enzymes are labeled as follows: 1, alanine aminotransferase (72, 263) (alanine is produced only under anaerobic conditions [265]); 2, GDH (263, 272, 359); 3, PFOR (327) (ferredoxin rather than NAD as the electron acceptor [326]); 4, acetyl-CoA synthetase (ADP forming) (293); 5, alcohol dehydrogenase E (ADHE) (has acetaldehyde dehydrogenase activity in the amino terminus which catalyzes the conversion of acetyl-CoA to acetaldehyde and alcohol dehydrogenase activity in the carboxy terminus which catalyzes the conversion of acetaldehyde to ethanol) (59, 292). Acetate is the major product under aerobic conditions; ethanol and alanine are preferentially produced under anaerobic conditions (265).

The metabolism of trophozoites is markedly affected by small changes in oxygen concentration. Under strictly anaerobic conditions, alanine is the major product of carbohydrate metabolism (72, 263, 265) (Fig. 2). Even with the addition of minimal amounts of O₂ (i.e., concentrations of <0.25 µM), ethanol production is stimulated and alanine production is inhibited (263). With further increases in O₂ concentration, ethanol and alanine production are inhibited. At O₂ concentrations of >46 µM, alanine production is completely inhibited and acetate and CO₂ are the predominant products of energy metabolism. These oxygen concentrations are likely to be relevant to the intestinal milieu in which the trophozoites replicate since the oxygen concentration in this environment is estimated to vary between 0 and 60 µM. Thus, the pathway of metabolism of pyruvate appears to be altered for differing anaerobic or microaerophilic environments.

Despite the anaerobic metabolism, trophozoites do produce some oxygen free radicals, and a mechanism for detoxification of oxygen is necessary. Detoxification of oxygen free radicals in aerobic organisms is typically accomplished by an enzymatic pathway beginning with superoxide dismutase. Superoxide dismutase activity has been detected by some (188), but other investigators did not detect superoxide dismutase, catalase, or peroxidase activities in G. lamblia (35) and proposed an H₂O-producing NADH oxidase as the major route of oxygen detoxification in G. lamblia (36).

Routine Giardia medium contains 50 mM glucose, and many of the metabolic studies of trophozoites have been performed with that medium. When the glucose concentration is reduced to 10 mM, there is little effect on trophozoite growth (299). At glucose concentrations below 10 mM, replication rates are reduced by nearly 50% and ethanol production is markedly reduced, alanine production is moderately reduced, and acetate production is unaffected. Thus, glucose promotes trophozoite growth but is not absolutely essential. The growth enhancement by glucose was not provided by other sugars.

In T. vaginalis, glycolytic enzymes (glucose to pyruvate) are found in the cytosol but those involved in pyruvate oxidation are localized in a separate organelle, the hydrogenosome (152). In Giardia, there is no metabolic compartmentalization; rather, all the reactions occur in the cytosol or on the cytosolic surfaces of membranes for PFOR-mediated reactions (188).

Glucose supplies the major source of energy derived from carbohydrates (147). Glucose is converted to pyruvate by the Embden-Meyerhof-Parnas and hexose monophosphate shunt pathways (Fig. 1). For most eukaryotic and prokaryotic organisms, the conversion of fructose-6-phosphate to fructose-1,6-bisphosphate is an irreversible and regulated step that is catalyzed by an ATP-dependent phosphofructokinase. However, for Giardia (219), as well as for T. vaginalis (221) and E. histolytica (279), this reaction is catalyzed by a pyrophosphate-dependent phosphofructokinase. In contrast to the ATP-dependent enzyme, this enzyme catalyzes a reversible reaction and is not a regulated enzyme (220). The pyrophosphate-dependent phosphofructokinase from Giardia has been cloned and characterized (274, 289). Triosephosphate isomerase, an enzyme that catalyzes a reversible conversion between dihydroxyacetone phosphate and D-glyceraldehyde-3-phosphate, was cloned by complementation in Escherichia coli and characterized (238).

Two enzymes that convert phosphoenolpyruvate into pyruvate, ATP-dependent pyruvate kinase (271), and pyrophosphate-dependent pyruvate phosphate dikinase (PPDK) have been identified (37, 132, 141). There is a potential energy advantage in the reaction mediated by PPDK, since two molecules of ATP can be generated by a coordinated reaction involving PPDK and adenylate kinase. Adenylate kinase (288) converts two ADP molecules into ATP + ADP by a reaction that is essentially energy neutral, and PPDK converts phosphoenolpyruvate plus AMP into pyruvate + ATP, resulting in the net generation of two ATP molecules during the conversion of phosphoenolpyruvate to pyruvate, rather than the one ATP produced by the pyruvate kinase reaction. Their relative roles in glycolysis have not yet been determined, but the higher specific activity of pyruvate kinase suggests that it may play a major role in glycolysis (271).

The conversion of pyruvate to acetyl coenzyme A (acetyl-CoA) (Fig. 3) is catalyzed by PFOR, which utilizes ferredoxin rather than NAD as the electron acceptor (188, 326, 327), in place of the pyruvate dehydrogenase complex found in aerobic eubacteria and eukaryotes. PFOR is also found in E. histolytica (280) and T. vaginalis (352).

Metronidazole is an antimicrobial agent with a broad spectrum of activity against anaerobic bacteria and protozoa. It is activated when its 5-nitro group is reduced by ferredoxin that has in turn been reduced by PFOR, generating a toxic nitro radical. Both metronidazole and sodium nitrite, another respiratory inhibitor thought to act by destroying the iron-sulfur center of PFOR, were toxic to G. muris trophozoites, but only sodium nitrite was toxic to cysts. It was proposed that they had different effects because of the inability of metronidazole to enter the cyst, but this proposal was not directly tested (262). In a study of different G. lamblia isolates, some of which were fully susceptible to metronidazole and some of which were relatively resistant, decreased ferredoxin levels were associated with drug resistance (192).

Acetyl-CoA can be converted directly to acetate by ADP-forming acetyl-CoA synthetase (293), resulting in the production of ATP from ADP as acetyl-CoA is converted to acetate. Alternatively, acetyl-CoA is converted to ethanol, using acetaldehyde as an intermediate, by the bifunctional enzyme alcohol dehydrogenase E (59, 292, 295). Alcohol dehydrogenase E has an acetaldehyde dehydrogenase activity in the amino terminus that catalyzes the conversion of acetyl-CoA to acetaldehyde and an alcohol dehydrogenase activity in the carboxy terminus that converts the acetaldehyde to ethanol.

Amino acids are becoming increasingly recognized as important components of the energy metabolism of G. lamblia. The uptake of aspartate, alanine, and arginine from the extracellular medium, as well as the documentation of glucose-independent metabolism, suggests the potential importance of amino acid metabolism for energy production in Giardia (215, 297, 299).

The arginine dihydrolase pathway is one potential source of energy (74, 297, 300) (Fig 4). This pathway is present in a number of prokaryotic organisms, but among eukaryotes it has been documented only in T. vaginalis (191) and G. lamblia. In the arginine dihydrolase pathway, arginine is converted to ornithine and ammonia with the generation of ATP from ADP by substrate-level phosphorylation. Ornithine is subsequently exported in exchange for extracellular arginine by a transporter mechanism (298).

View larger version (8K): [in this window] [in a new window]   FIG. 4.   Arginine dihydrolase pathway (74, 297, 300). The enzymes are labeled as follows: 1, arginine deiminase (163); 2, ornithine transcarbamoylase (297, 300); 3, carbamate kinase (226).

Aspartate is another potential source of energy. It is converted to oxaloacetate by aspartate transaminase, entering the intermediary pathway, where it is converted to pyruvate via a malate intermediate (215) (Fig. 2).

Alanine also appears to play an important role in allowing trophozoites to adapt to hypoosmotic challenge. With an isoosmolar extracellular environment, the intracellular alanine concentration is 50 mM (161, 270), and with a hypoosmolar challenge, the concentration of alanine rapidly decreases by an active transport mechanism. (In addition to alanine, potassium appears to play a role in osmoregulation of trophozoites [204].) The secretion of alanine occurs via an alanine transporter that also transports L-serine, glycine and L-threonine, L-glutamine, and L-asparagine (73, 258). This transporter acts as an antiport, exchanging intracellular alanine for these other amino acids from the extracellular environment (298).

Aside from the synthesis of alanine as a by-product of energy metabolism, the only other amino acid for which de novo synthesis has been documented is valine. Thus, Giardia lacks synthesis of most amino acids and depends on scavenging them from the intestinal milieu in which the trophozoite replicates.

One of the notable requirements for axenic growth of G. lamblia trophozoites is the absolute requirement for a relatively high concentration of cysteine (16 mM). Cysteine also provides a partial protection from the toxicity of oxygen that is not seen with other reducing agents, including cystine, and therefore appears to be a specific effect of cysteine (109, 110, 113). Cysteine is not synthesized de novo and is not synthesized from cystine (202). It appears to be imported into the cell by passive diffusion, although active transport may account for some of the acquisition of cysteine (202). The importance of free thiol groups on the surfaces of trophozoites was demonstrated by the toxicity of thiol-blocking agents that are unable to penetrate intact cells (116). This toxicity suggests that these agents are reacting with thiol groups on the trophozoite surfaces, killing the trophozoites. Cysteine appears to be the major thiol group present (34). When trophozoites are metabolically labeled with radiolabeled cysteine, most of the label is incorporated into the VSPs (6, 11), suggesting that these surface proteins may play a role in protection of the trophozoite from oxygen toxicity (see "Structure and biochemistry of the VSPs" below).

The growth of trophozoites predominantly in the duodenum and jejunum initially suggested the possible importance of bile in the growth of Giardia trophozoites. Short-term axenic growth in the absence of serum can be supported by bile (111). The biliary lipids cholesterol and phosphatidylcholine and the bile salts glycocholate and glycodeoxycholate will also support this growth (111). Serum is required for longer-term axenic growth, but it has been shown that the Cohn IV-1 fraction of bovine serum (enriched in alpha globulins, lipoproteins, and growth factors) can substitute for whole serum (194). In fact, insulin-like growth factor II, which is present in fraction IV-1, stimulates trophozoite growth and cysteine uptake. The same fraction from a number of other mammals was also effective in supporting growth, although in some cases antibody depletion was required.

G. lamblia trophozoites do not have the capacity of de novo synthesis of fatty acids (149), with the possible exception of certain minor fatty acids (75). However, free fatty acids are toxic to trophozoites, demonstrating 50% lethal doses of 2 to 12 µM (283). In fact, the toxicity of human milk for G. lamblia trophozoites appears to be mediated through products of milk lipolysis (129, 283). The trophozoites appear to satisfy their lipid requirements by obtaining cholesterol and phosphatidylcholine from the external environment (94, 111, 200). The cholesterol and phospholipids are supplied by lipoproteins, -cyclodextrins, and bile salts, with transfer of lipids to the parasite surface being facilitated by bile salts (200). It has also been suggested that a low level of endocytosis of lipids occurs (200). Conjugated bile acids appear to be taken up by a carrier-mediated mechanism that includes different carriers for cholyltaurine and cholylglycine (60).

The major fatty acids found in axenically grown trophozoites are palmitic acid, stearic acid, and oleic acid (75). Fatty acid desaturase activity, including desaturation of oleate to linoleate and linolenate, has been documented (75). Arachidonic acid is incorporated into neutral lipids, phospholipids, and a wide variety of cellular lipids (108), while palmitic acid, myristic acid, and oleic acid are transesterified primarily into phospholipids (28), including cellular phospholipids, (e.g., phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol) (149, 154, 228, 313). Interesterification also occurs with incorporation of conjugated fatty acids into phosphatidylglycerol (108). The toxicity of certain analogs of phosphatidylglycerol for trophozoites has been documented, although the mechanism of this toxicity has not been determined (108).

Isoprenoids are lipids derived from mevalonate that are commonly found in eukaryotic cells. The most notable end product is cholesterol, but isoprenoids are also incorporated into proteins such as the GTP-binding proteins by posttranslational modification. Isoprenylation of proteins has been demonstrated by the incorporation of radiolabeled mevalonate into trophozoite proteins (197). Incorporation of mevalonate and cell growth were inhibited in a reversible manner by competitive inhibitors of 3-hydroxy-3-methylglutoryl (HMG)-CoA reductase. Inhibitors of later steps of isoprenylation permanently inhibited cell growth.

Many of the pathogenic protozoa, including G. lamblia (339), depend on salvage pathways for obtaining purine nucleosides. In addition, G. lamblia (13), as well as T. vaginalis (340) and Tritrichomonas foetus (341), lack pyrimidine synthetic pathways and depends on salvage pathways for obtaining both purine and pyrimidine nucleosides.

Studies of purine metabolism using radiolabeled precursors have demonstrated the incorporation of adenine, adenosine, guanine, and guanosine into nucleotides but no incorporation of components of the de novo synthetic pathway, such as formate, glycine, hypoxanthine, inosine, or xanthine (23, 224, 339). The likely scenario is that the purine nucleosides (adenosine and guanosine) are imported by a transporter with broad specificity for nucleosides as well as deoxyribonucleosides (19, 63). The purine nucleosides are then broken down to the bases by their respective hydrolases (Fig. 5). Phosphoribosyl 1-pyrophosphate is synthesized by phosphoribosyl 1-pyrophosphate synthetase (171) and reacts with the salvaged purine bases to produce the nucleoside-5'-monophosphate in a reaction catalyzed by the respective monophosphate phosphoribosyltransferase (PRTase). The GPRTases from most eukaryotes utilize hypoxanthine or xanthine as substrate, but the G. lamblia GPRTase is highly specific for guanine (261, 311), indicating that guanine is the only source for guanine nucleotides. The amino acid sequence of the GPRTase enzyme shows less than 20% identity to the human enzyme (311). The crystallographic structure of the G. lamblia GPRTase has suggested possible reasons for the unique substrate of the G. lamblia enzyme, such as an aspartate substitution for leucine at position 181 (303).

View larger version (7K): [in this window] [in a new window]   FIG. 5.   Purine ribonucleoside salvage pathways. The enzymes are labeled as follows: 1, adenosine hydrolase (339); 2, adenine phosphoribosyltransferase (APRTase); (339); 3, guanosine hydrolase (339); 4, guanine phosphoribosyltransferase (GPRTase) (339).

G. lamblia trophozoites also depend on the salvage of exogenous thymidine, cytidine, and uridine for the synthesis of the pyrimidine nucleotides (13, 190) (Fig. 6). Uridine is probably imported by the thymidine transporter (62, 89) and broken down into uracil by a hydrolase (13) or phosphorylase (181). Cytidine and uridine can also be imported by a broad-specificity transporter (63), but most cytidine probably enters the synthetic pathway for UTP synthesis rather than being converted more directly to CTP (Fig. 6). The majority of CTP is probably generated from UTP by CTP synthetase (151). A low-affinity nucleobase transporter has also been documented (89). Although the transporter's function has not been determined, it may be involved in export of unusable (e.g., thymine) or excessive quantities of nucleobases.

View larger version (10K): [in this window] [in a new window]   FIG. 6.   Pyrimidine ribonucleoside salvage pathways. The enzymes are labeled as follows: 1, uracil phosphoribosyltransferase (UPRTase) (58); 2, uridine/thymine phosphorylase (181) (a uridine hydrolase [13] has not been confirmed); 3, uridine phosphotransferase (kinase) (13) (not confirmed [336]); 4, UMP kinase (151); 5, UDP kinase (151); 6, CTP synthetase (151, 187); 7, CDP kinase (151); 8, cytosine phosphoribosyltransferase (CPRTase) (13) (low level of activity [151]); 9, cytidine hydrolase (13); 10, cytidine deaminase (336). The initial pyrimidine salvage pathway was described in (reference) and has been updated from more recent literature (see the references cited for individual enzymes).

DNA synthesis also depends on the salvage of exogenous deoxynucleotides. G. lamblia trophozoites lack ribonucleotide reductase and do not incorporate purine bases or nucleosides into DNA. Rather, they depend on the salvage of exogenous purine deoxynucleosides (20) (Fig. 7). A single purine deoxynucleoside kinase for the synthesis of deoxynucleotides from their respective deoxynucleosides (deoxyadenosine and deoxyguanosine) and a pyrimidine deoxynucleoside kinase catalyze the production of the pyrimidine deoxynucleotides from the deoxynucleosides thymidine and cytosine (176).

View larger version (15K): [in this window] [in a new window]   FIG. 7.   Deoxynucleoside salvage pathways. The enzymes are labeled as follows: 1, pyrimidine deoxynucleoside kinase (176); 2, purine deoxynucleoside kinase (176).

CELL BIOLOGY

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Trophozoite Structure

The G. lamblia trophozoites are pear-shaped and are approximately 12 to 15 µm long and 5 to 9 µm wide. The cytoskeleton includes a median body, four pairs of flagella (anterior, posterior, caudal, and ventral), and a ventral disk (Fig. 8 and 9). Trophozoites have two nuclei without nucleoli that are located anteriorly and are symmetric with respect to the long axis (Fig. 9). Lysosomal vacuoles, as well as ribosomal and glycogen granules, are found in the cytoplasm. Golgi complexes become visible in encysting trophozoites but have not been confirmed to be present in vegetative trophozoites (117). However, stacked membranes suggestive of Golgi complexes have been demonstrated (174, 309).

View larger version (54K): [in this window] [in a new window]   FIG. 8.   Trophozoite coronal section. A coronal view of a trophozoite demonstrates the nuclei (N), endoplasmic reticulum (ER), flagella (F), and vacuoles (V). A mechanical suction is formed when the ventral disk (VD) attaches to an intestinal or glass surface. Components of the ventral disk include the bare area (BA), lateral crest (LC), and ventrolateral flange (VLF). A magnified view of the ventral disk is shown in Fig. 10.

View larger version (109K): [in this window] [in a new window]   FIG. 9.   Trophozoite cross section. A cross-sectional view of a trophozoite demonstrates the nuclei (N), flagella (F), vacuoles (V), and endoplasmic reticulum (ER).

G. lamblia trophozoites have two nuclei that are nearly identical in appearance (Fig. 8 and 9). They replicate at approximately the same time (351) and are both transcriptionally active (153) as determined by uridine incorporation into nuclear RNA. Both nuclei have approximately equal numbers of rDNA genes as determined by in situ hybridization using the rDNA probe (153). Both have approximately equal amounts of DNA as determined by the intensity of nuclear staining with 4',6-diamidino-2-phenylindole (DAPI) (153) or propidium iodide (25), although the possession of equal amounts of DNA by the two nuclei has been questioned (M. Frisardi and J. Samuelson, Woods Hole Molecular Parasitology Meeting, abstr. 263A, 1999, and abstr. 202C, 2000). It is generally assumed that the two nuclei have the same complement of genes and chromosomes, and results from our laboratory using fluorescence in situ hybridization with single-copy genes support this assumption (L. Yu, C. W. Birky, and R. D. Adam, unpublished observations). These features all differ from those of the ciliated protista that are binucleate, such as Tetrahymena and Paramecium (276). These organisms have a smaller micronucleus that contains the genomic DNA but is not transcriptionally active. DNA from the micronucleus is amplified into multiple copies, forming the macronucleus, from which transcription occurs. Giardia spp. and the other diplomonads appear to be unique in their possession of two nuclei that are identical by the above parameters.

The nuclei of higher eukaryotes have readily visible nucleoli, which are the sites of rRNA transcription. Fibrillarin is a major component of nucleoli and is required for pre-rRNA processing as well as for viability in Saccharomyces cerevisiae (144, 296, 325) Nucleoli have not been identified in G. lamblia nuclei, and antibody to G. lamblia fibrillarin diffusely stains the nuclei of G. lamblia, suggesting that rRNA transcription and processing are not localized to certain regions of the nuclei (240).

Axenic cultures have not been synchronized, and the division time is very brief, so studies of cellular and nuclear division have been limited to observations of trophozoite replication based on static microscopy. On the basis of these observations, it has been proposed that during cellular division, the nuclei move laterally followed by nuclear replication, resulting in trophozoites with four nuclei. The trophozoites then divide in the longitudinal plane in such a way as to maintain the left-right asymmetry (48, 104, 153). Electron micrographs of excysting organisms undergoing cytokinesis have shown cells that appear to be joined laterally or with the ventral disks opposing each other (130). Laterally joined cells would be consistent with the above mechanism of cytokinesis, but replication that results in opposing ventral disks would invert the left-right asymmetry with each division. Thus, it is not yet possible to be certain of the morphology of cytokinesis.

A number of drugs that arrest the cell cycle of mammalian cells have been tested for their effect on trophozoites. Colchicine (mammalian G₂+M) and gamma irradiation (mammalian G₂+M) had no effect, while hydroxyurea (mammalian G₂+M) and razoxane (mammalian G₁/S, sometimes G₂+M), arrested the cell cycle in the G₂+M phase (140). To date, axenic cultures of G. lamblia have not been synchronized.

The ventral disk is a unique and important component of the G. lamblia cytoskeleton. Grossly, it appears as a concave structure with a maximum depth of 0.4 µm covering the entire ventral surface. The edge narrows into a lateral crest, and a more flexible ventrolateral flange (Fig. 8) surrounds the disk (82). The disk contains the contractile proteins actinin, -actinin, myosin, and tropomyosin (103) as the biochemical basis for the contraction of the disk involved in adherence. Attachment depends on active metabolism and is inhibited by temperatures below 37°C, increased oxygen levels, or reduced cysteine concentrations (109, 113). Ultrastructurally, the ventral disk includes a set of microtubules containing 13 protofilaments and linked to the ventral membrane. These microtubules form the base of the microribbons (dorsal ribbons) that extend nearly perpendicular from the membrane (Fig. 8 and 10). The protein constituents of the dorsal ribbons (microribbons) include a set of giardins, which are alpha-coiled-helix proteins approximately 29 to 38 kDa in size. The giardins line the edges of the microribbons but are not found in the microtubules (273). Although 23 forms of these proteins have been separated by two-dimensional electrophoresis, the N-terminal sequences of some variants are identical, suggesting that post-translational modification accounts for some of the forms (273). Several giardins, ₁-giardin (273), ₂-giardin (15), -giardin (10, 134), and -giardin (257), have been cloned, and their sequences confirm the alpha-helical structure of these proteins. The ₁-giardin and ₂-giardin sequences demonstrate approximately 80% identity at the nucleotide and amino acid levels (15), while the other giardins have no significant sequence similarity. Of the two sequences published for the -giardin gene, the one found in reference (134) is the correct one except that the start codon is 13 amino acids upstream from the reported cDNA sequence (2 amino acids upstream from the reported genomic sequence) (10), yielding a slightly higher predicted molecular mass (R. Adam, unpublished results). A protein with immunologic cross-reactivity with -giardin, but significantly larger, has also been identified as a cytoskeletal protein (head stalk protein; HPSR2) (206). This 183-kDa protein has a long coiled-coil stalk and an N-terminal hydrolytic domain. Whether it has the same cellular distribution as the giardins has not been reported.

View larger version (45K): [in this window] [in a new window]   FIG. 10.   Close-up of the ventral disk. A magnified view of the ventral disk shows the microtubules (MT) and microribbons or dorsal ribbons (DR).

Tubulins are not found in the microribbons but are found in the microtubules (310). The microtubules of the ventral disk, as well as those of the flagella, are presumably composed of -tubulin. Posttranslational modifications of the G. lamblia tubulins include acetylation and polyglycylation (310, 345, 346). Benzimidazoles are compounds used primarily as anti-helminthic agents, but they also have significant in vitro activity against G. lamblia as well as in vivo efficacy for giardiasis (49, 68, 70, 122, 233). Their effect is thought to be mediated by their interaction with -tubulin. G. lamblia trophozoites exposed to albendazole (or other benzimidazoles) lose their ability to adhere despite normal flagellar movement (70). This suggests a difference between the tubulin found in the ventral disk and that found in the flagella. This, as well as the observation that adherence can occur in the absence of flagellar function (103), suggest that the ventral disk is important for adherence but the flagella are not. With more prolonged exposure (e.g. 24 h) to albendazole, the ventral disk becomes fragmented, with dislocation of the microribbons and microtubules of the ventral disk (49). Substantial electron-dense precipitates can be found in the microtubules and microribbons and to a lesser extent in the other tubulin-containing structures, such as the median body and flagella (49). The pronounced effect of albendazole on the microribbons, despite the apparent lack of tubulin in the microribbons, raises the possibility that the benzimidazoles are reacting with proteins other than tubulins, such as the giardins. Vinculin is a protein that binds -actinin and mediates the attachment of actin filaments to membrane sites. Its location in attachment regions of the ventral disk has led to the suggestion that it may be involved in the attachment process (241). Further functional studies are required to confirm or refute this proposal.

The trophozoite has four pairs of flagella that begin at two sets of basal bodies that are near the midline and anteroventral to the nucleus. They emerge from the anterior, posterior, caudal, and ventral regions of the trophozoite. Paraflagellar rods extend along one side of the two ventral flagella (102, 135). Nine pairs of microtubules encircle two microtubules to form the flagella (Fig. 8). The flagella appear to be important for motility but not for attachment. In addition, their early emergence through the cyst wall during the process of excystation suggests their importance in excystation (38) (see "Excystation" below).

The median body is a component of the cytoskeleton that is located in the midline and dorsal to the caudal flagella and consists of a group of microtubules in a tight bundle. It is unique to Giardia spp., and its morphology helps define the morphologic characteristics of the different Giardia species (Table 1). G. lamblia trophozoites typically have two median bodies that are shaped like claw hammers. The median body has been proposed as the assembly site for microtubules to be incorporated into the ventral disk (216). Caltractin/centrin, a calcium binding protein that is responsible for basal-body orientation, has been identified in the basal bodies as well as the paraflagellar rods and median bodies (21, 216). A 101-kDa coiled-coil protein has also been localized to the median body by immunofluorescence microscopy (205).

Endomembrane protein transport system. The endoplasmic reticulum (ER) and Golgi complex are part of a eukaryotic endomembrane system involved in protein folding and translocation. In higher eukaryotes, proteins destined for secretion have a signal sequence that directs them to the ER as they are translated in the ribosomes. The signal sequence binds to the signal recognition particle (SRP). This complex then binds to the SRP receptor (SR) on the cytoplasmic portion of the ER. The SR is a dimeric protein consisting of the membrane anchored SR and the GTPase, SR. After their translocation to the ER, chaperonins such as BiP (the HSP70 homologue found in the ER) aid in folding and further transport. Although structures consistent with ER had previously been identified by electron microscopy (EM), there was some doubt until recently about the existence of ER in G. lamblia. However, the cloning and characterization of SR, as well the identification of an extensive membrane system labeled with antibody to BiP (309), has clearly demonstrated the existence of the ER. One of the important aspects of protein folding includes the correct formation of disulfide bonds, which is accomplished in the lumen of the ER by protein disulfide isomerase. Three protein disulfide isomerase genes from G. lamblia have been cloned and characterized, and their products have been localized to the ER (162).

Endosome-lysosome vacuoles. Most eukaryotes have a system of endosomes and lysosomes that degrade and recycle endogenous proteins or those acquired by endocytosis or phagocytosis from the extracellular space. Early endosomes internalize endocytosed proteins to allow for their subsequent return to the cell membrane or transport to late endosomes (or, alternatively, maturation of early into late endosomes) followed by transport to and degradation by the lysosomes. Both are acidic, with an endosome pH of <6 and a lysosome pH of 5. Trophozoites have numerous vacuoles encompassing the periphery of the cell (Fig. 8 and 9), which fulfill at least some criteria of endosomes and lysosomes. These vacuoles are acidic, as shown by their uptake of acridine orange (101, 156). They concentrate exogenous ferritin and lucifer yellow, suggesting their potential role in endocytosis (30, 173). G. lamblia virus particles appear to be concentrated into the vacuoles by an endocytotic mechanism (323) (see "Giardia lamblia virus" below). Pulse-chase labeling with horseradish peroxidase showed early and persistent labeling of vacuoles, suggesting that there is no distinction between early and late endocytic vesicles as is found in higher eukaryotes (173). Labeling of a smaller portion of the vacuoles with chemicals that label the ER, such as glucose-6-phosphatase and zinc iodide-osmium tetroxide, and three-dimensional reconstructions (173), as well as EM using anti-BiP antibody (309), have suggested a continuity of these vacuoles with the ER. The vacuoles also contain a variety of hydrolase activities, such as acid phosphatase, proteinase, and RNase, indicating their lysosomal characteristics (98, 189). Thus, the vacuoles appear to function as early and late endosomes and lysosomes and may be functionally associated with the ER (173).

Proteasomes. While the lysosome degrades endocytosed proteins, the proteasome is a large complex that degrades improperly folded endogenous cytosolic proteins. The 20S proteasome complex is about 700 kDa and contains the protease activity. The complex can be converted into a 26S complex by the addition of proteins that add regulatory activity and bring the size to 2,000 kDa. Archaea have the simplest proteasome, consisting of two alpha and two beta heptamers, while the eukaryotic 20S proteasome contains seven different alpha and seven different beta subunits organized in a manner similar to the Archaea. Since G. lamblia has certain similarities to the Archaea but other eukaryotic features, it is of interest that the 20S proteasome of Giardia demonstrates a distinctly eukaryotic pattern with 14 subunits (77). Proteins are targeted to the proteasomes by ubiquitin, which appears to be present in Giardia as a single-copy gene, in contrast to the multiple gene copies found in other eukaryotes (168).

Encystation occurs after organisms have undergone nuclear replication but before cytokinesis; therefore, cysts contain four nuclei. They are approximately 5 by 7 to 10 µm in diameter and are covered by a wall that is 0.3 to 0.5 µm thick and composed of an outer filamentous layer and an inner membranous layer with two membranes. The outer portion of the cyst wall is covered by a web of 7- to 20-nm filaments (79, 80). Four major proteins have been identified in the outer cyst wall, 29, 75, 88, and 102 kDa in size (80). The sugar component of the outer portion is predominantly galactosamine in the form of N-acetylgalactosamine (GalNAc) (148). Earlier claims that the cyst wall is composed of chitin (N-acetylglucosamine) have been refuted (3).

As the environmentally stable form of the life cycle, cysts have a metabolic rate only 10 to 20% of that found in trophozoites (262). The respiration of both cysts and trophozoites is stimulated by ethanol, while trophozoites are stimulated only by glucose.

Promoting and inhibiting factors. As trophozoites replicate and colonize the intestinal surface, some encyst in the jejunum after exposure to biliary secretions. Encystation has been performed in vitro by exposing trophozoites to an environment that mimics that of the jejunum (114, 115, 301). Specific conditions that promoted encystation included a mildly alkalotic pH of 7.8 and conjugated bile salts plus fatty acids (114). Other investigators reported that expression of cyst wall protein, a marker of the encystation process, was detected 90 min after exposure to medium with lipoprotein-deficient serum (196). Encystation was abolished when cholesterol was added, leading to the proposal that encystation results from cholesterol starvation. The relative importance of cholesterol starvation and addition of bile salts and fatty acids for the induction of encystation remains controversial.

Events of encystation. Encystation can be divided into two phases, early and late. The timetables determined by various studies have differed somewhat, probably because of the different approaches used. After the initiation of encystation conditions, the organisms do not all enter the encystation process at the same time. Therefore, morphologic studies that examine the time course of the first encysting organisms tend to give earlier times for the encystation events than studies that depend on pooled organisms. These morphologic studies indicate that the early phase is complete within about 10 h and the late phase is complete by 16 h (83). For this discussion, we will assume that the early and late events from the various studies are comparable.