A unique mechanism of nuclear division in Giardia lamblia involves components of the ventral disk and the nuclear envelope

The fine structure of the binucleate, parasitic protist Giardia lamblia during interphase and divisional stages was studied by serial thin sectioning and three-dimensional reconstructions. The earlier sign of nuclear division is the development of a few peripheral areas of densely packed chromatin directly attached to the inner nuclear envelope. An intracytoplasmic sheet of ventral disk components grows from the cell periphery towards one of the nuclei, apparently constricting this nucleus, which becomes located at a ventral bulge. After the basal bodies become duplicated, a full nuclear division occurs in trophozoites, giving two pairs of parent-daughter nuclei. This full division occurs in a dorsal-ventral direction, with the resulting nuclear pairs located at the sides of the two sets of basal bodies. A new ventral disk is formed from the disk-derived sheets in the cell harboring the four nuclei. Cytokinesis is polymorphic, but at early stages is dorsal-to-dorsal. Encysting trophozoites show the development of Golgi cisternae stacks and dense, specific secretory granules. 3-D reconstructions show that cysts contain a single pair of incompletely strangled nuclei. The dividing Giardia lacks a typical, microtubular spindle either inside or outside the nuclei. The nuclear envelope seems to be the only structure involved in the final division of the parent-daughter nuclei.

A unique mechanism of nuclear division in Giardia lamblia involves components of the ventral disk and the nuclear envelope

The fine structure of the binucleate, parasitic protist Giardia lamblia during interphase and divisional stages was studied by serial thin sectioning and three-dimensional reconstructions. The earlier sign of nuclear division is the development of a few peripheral areas of densely packed chromatin directly attached to the inner nuclear envelope. An intracytoplasmic sheet of ventral disk components grows from the cell periphery towards one of the nuclei, apparently constricting this nucleus, which becomes located at a ventral bulge. After the basal bodies become duplicated, a full nuclear division occurs in trophozoites, giving two pairs of parent-daughter nuclei. This full division occurs in a dorsal-ventral direction, with the resulting nuclear pairs located at the sides of the two sets of basal bodies. A new ventral disk is formed from the disk-derived sheets in the cell harboring the four nuclei. Cytokinesis is polymorphic, but at early stages is dorsal-to-dorsal. Encysting trophozoites show the development of Golgi cisternae stacks and dense, specific secretory granules. 3-D reconstructions show that cysts contain a single pair of incompletely strangled nuclei. The dividing Giardia lacks a typical, microtubular spindle either inside or outside the nuclei. The nuclear envelope seems to be the only structure involved in the final division of the parent-daughter nuclei. (AU)

Changes in Trypanosoma cruzi phospholipid turnover induced by parasite contact with cell membranes.

To investigate the possibility that cell contact could initiate a series of signals in both the host cell and the flagellate protozoan Trypanosoma cruzi, we studied [32P]-phospholipid turnover during parasite interaction with cellular membranes in vitro. Lipid alterations were produced in the parasite during the initial period of contact with the plasma membranes of human erythrocytes. In the presence of calcium an increment in phosphatidylethanolamine was observed with a concomitant decrease in phosphatidic acid fractions, whereas these modifications were not observed in the absence of calcium. There was an evident decrease in phosphatidylcholine and a shift in the phosphatidylinositol/lysophosphatidylethanolamine fraction among the phospholipids of major turnover in the absence or presence of calcium. Among the minor labeled species, lysophosphatidylcholine reached levels that duplicated control values, whereas the amounts of lysophosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate diminished by over 50%. All of these variations indicate that the parasite's contact with plasma membranes induces changes involving T. cruzi phospholipids and suggest the participation of these compounds in the activation of intracellular mechanisms that might be important during the life cycle of this parasite.

Identification of surface molecules on salivary glands of the mosquito, Aedes aegypti, by a panel of monoclonal antibodies.

Malaria transmission by the mosquito vector requires sporozoite invasion into mosquito salivary glands. Parasites probably enter the glands by specific receptor-ligand interactions with molecules on the surface of the glands. We have undertaken the characterization of salivary gland surface molecules of Aedes aegypti to identify candidate receptors for Plasmodium gallinaceum sporozoite invasion. Monoclonal antibodies (mAbs) were generated against antigen enriched for salivary gland membranes and basal lamina. A panel of 44 mAbs were generated that bound to surface molecules of mosquito tissues. Twenty-four mAbs bound exclusively to salivary glands, six bound to salivary glands and ovaries, one bound to salivary gland and midgut, and 13 bound to all tissues tested. We present data on the immunolocalization and biochemical characteristics of the antigens. Many of the salivary gland-specific mAbs bound preferentially to the median and distal lateral lobes of the salivary glands, indicating that there are anatomical region-specific biochemical differences on the gland surface. These lobes of the salivary glands are the preferential sites of malaria sporozoite invasion. Therefore, antigens specific for these regions are promising candidate receptors for sporozoite invasion. The present identification of surface molecules of mosquito salivary glands by means of monoclonal antibodies represents the first description of individual molecules on the mosquito salivary gland surface. This work lays the basis for further studies on the molecular mechanisms involved in malaria sporozoite invasion of mosquito salivary glands.

Ganglioside treatment of acute Trypanosoma cruzi infection in mice promotes long-term survival and parasitological cure.

Ganglioside treatment of mice during their acute infection with Trypanosoma cruzi promoted long-term survival and clearance of parasites from the bloodstream and organs. Additionally, such treatment completely prevented the clinical manifestations of the infection, and progression into the chronic stages of the disease, for at least 18 months post-infection. Trypanosoma cruzi must invade nucleated cells to survive and reproduce within the mammalian host, and it has been suggested that ganglioside treatment inhibits the parasite's phospholipase A2 enzymes (PLA2), which are involved in membrane destabilization. However, since total brain gangliosides were not toxic to the parasite, either in xenic or axenic cultures, it seems unlikely that their action in vivo relates to their inhibition of PLA2. Other possible mechanisms of action are discussed.

Detection of microsporidia spore-specific antigens by monoclonal antibodies.

Microsporidia (phylum Microspora) are unicellular parasites commonly found in invertebrates, fish, and laboratory animals; however, microsporidiosis is an emerging problem in patients with the acquired immunodeficiency syndrome (AIDS). The infective stage of these parasites is the spore, which possesses a rigid cell wall that protects the parasite outside its host. Little is known about their antigenic composition. Sensitive, reliable, and easily performed methods for identification and speciation are generally not available. Here, we report the production of 21 MAbs specific to spore antigens of several species of Microsporidia. MAbs were generated to purified spores of Encephalitozoon intestinalis and Encephalitozoon hellem, and their reactivities were tested against spores and intracellular developing forms of E. intestinalis, E. hellem, Encephalitozoon cuniculi, and Vittaforma corneae. Both species-specific and broad-reactivity MAbs were produced. Five MAbs reacted against the spores of all four species tested: 7 with 3 species, 6 with 2 species, 1 with E. intestinalis, and 4 with the polar tube of all species. Immunoelectron microscopy confirmed the reactivity of specific MAbs to the spore wall or the polar tube. These MAbs reacted to a few antigens as determined by Western blot, and none of the epitopes were periodate-sensitive. These MAbs may be useful in the diagnosis and speciation of Microsporidia as well in the purification, cloning, and detection of these antigens.

The molecular mechanisms of giardia encystation.

The protozoan parasite Giardia lamblia is transmitted as an environmentally resistant cyst. The encystation process is attracting attention not only from the viewpoint of disease transmission, but also as a model for differentiation. Here, Hugo Luján, Michael Mowatt and Theodore Nash discuss molecular events underlying this process, including the induction of expression and transport of cyst wall proteins and the induction of Golgi-like activity. They also propose that the signal for encystation derives from cholesterol deprivation in the lower small intestine.

Mechanisms of Giardia lamblia differentiation into cysts.

Microbiologists have long been intrigued by the ability of parasitic organisms to adapt to changes in the environment. Since most parasites occupy several niches during their journey between vectors and hosts, they have developed adaptive responses which allow them to survive under adverse conditions. Therefore, the life cycles of protozoan and helminthic parasites are excellent models with which to study numerous mechanisms involved in cell differentiation, such as the regulation of gene expression, signal transduction pathways, and organelle biogenesis. Unfortunately, many of these studies are very difficult because the conditions needed to elicit developmental changes in parasites remain undetermined in most cases. Recently, several interesting findings were reported on the process of differentiation of Giardia lamblia trophozoites into cysts. G. lamblia is a flagellated protozoan that inhabits the upper small intestine of its vertebrate host and is a major cause of enteric disease worldwide. It belongs to the earliest identified lineage among eukaryotes and therefore offers a unique insight into the progression from primitive to more complex eukaryotic cells. The discovery of a specific stimulus that induces trophozoites to differentiate into cysts, the identification and characterization of encystation-specific molecules, the elucidation of novel biochemical pathways, and the development of useful reagents and techniques have made this parasite an excellent model with which to study differentiation in eukaryotic cells. In this review, we summarize the most recent fundings on several aspects of Giardia differentiation and discuss the significance of these findings within the context of current knowledge in the field.

Cholesterol starvation induces differentiation of the intestinal parasite Giardia lamblia.

Giardia lamblia, like most human intestinal parasitic protozoa, sustains fundamental morphological and biochemical changes to survive outside the small intestine of its mammalian host by differentiating into an infective cyst. However, the stimulus that triggers this differentiation remains totally undefined. In this work, we demonstrate the induction of cyst formation in vitro when trophozoites are starved for cholesterol. Expression of cyst wall proteins was detected within encystation-specific secretory vesicles 90 min after the cells were placed in lipoprotein-deficient TYI-S-33 medium. Four cloned lines derived from two independent Giardia isolates were tested, and all formed cysts similarly. Addition of cholesterol, low density or very low density lipoproteins to the lipoprotein-deficient culture medium, inhibited the expression of cyst wall proteins, the generation of encystation-specific vesicles, and cyst wall biogenesis. In contrast, high density lipoproteins, phospholipids, bile salts, or fatty acids had little or no effect. These results indicate that cholesterol starvation is necessary and sufficient for the stimulation of Giardia encystation in vitro and, likely, in the intestine of mammalian hosts.

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, beta-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, beta-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 degrees 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.

Increased expression of the molecular chaperone BiP/GRP78 during the differentiation of a primitive eukaryote.

Giardia lamblia, a major cause of intestinal disease worldwide, is a parasitic protozoan that represents the earliest branch of the eukaryotic lineage. Trophozoites, which possess two nuclei but lack mitochondria, peroxisomes and a typical Golgi apparatus, colonize the small intestine of the vertebrate host where they may differentiate into infective cysts. Encystation is a regulated process characterized by the biosynthesis, secretion and formation of a protective extracellular cyst wall. In previous studies, we demonstrated the biogenesis of the Golgi apparatus during encystation and identified two leucine-rich proteins (CWPs), which localize within encystation-specific secretory granules before their incorporation into the cyst wall. Here, we used immunological, biochemical and molecular biological approaches to analyze the expression of BiP/GRP78, an endoplasmic reticulum (ER)-resident chaperone, during the Giardia life cycle. A monoclonal antibody specific for Giardia BiP permitted the visualization of the ER of this protozoan and showed that BiP expression increased simultaneously with the increased expression of CWPs during encystation. However, in contrast to the 140-fold increase in levels of CWP transcripts, the steady-state level of BiP mRNA did not increase during encystation. Furthermore, potent inducers of BiP expression in higher eukaryotic cells, including agents that perturb the ER environment, did not affect BiP expression in Giardia. These results, when considered together with the profound changes that occur in the secretory pathway during Giardia encystation, indicate an important role for this molecular chaperone during the differentiation of this primitive eukaryote.

Identification of a novel Giardia lamblia cyst wall protein with leucine-rich repeats. Implications for secretory granule formation and protein assembly into the cyst wall.

Giardia lamblia trophozoites, like most intestinal parasitic protozoa, undergo fundamental biological changes to survive outside the intestine of their mammalian host by differentiating into infective cysts. This complex process entails the coordinated production, processing, and transport of cyst wall constituents for assembly into a protective cyst wall. Yet, little is known about this process and the identity of cyst wall constituents. We previously identified a 26-kDa cyst wall protein, CWP1. In the present work, using monoclonal antibodies to cyst wall antigens, we cloned the gene that encodes a novel 39-kDa cyst wall protein, CWP2. Expression of CWP1 and CWP2 was induced during encystation with identical kinetics. Soon after synthesis, these two proteins combine to form a stable complex, which is concentrated within the encystation-specific secretory granules before incorporation into the cyst wall. Both proteins contain five tandem copies of a 24-residue leucine-rich repeat, a motif implicated in protein-protein interactions. Unlike CWP1, CWP2 has an extremely basic 121-residue COOH-terminal extension that might be involved in the sorting of these proteins to the secretory granules.

Isoprenylation of proteins in the protozoan Giardia lamblia.

We report the ability of Giardia lamblia to modify several of its cellular proteins by isoprenylation. Trophozoites cultured in the presence of [3H]mevalonate synthesized radiolabeled proteins of approx. 50 and 21-26 kDa. Chemical analysis indicated that farnesyl and geranylgeranyl isoprenoids comprised the majority of the radiolabel covalently associated with trophozoite proteins. In addition, antibodies to human p21ras immunoprecipitated mevalonate-labelled species of approx. 21 kDa. Inhibitors of several enzymatic steps of the mevalonate pathway dramatically affected Giardia metabolism. Protein isoprenylation and cell growth were blocked by compactin and mevinolin, competitive inhibitors of HMG-CoA reductase, the rate-limiting enzyme in isoprenoid biosynthesis. In the presence of these inhibitors, Giardia growth was restored by the addition of mevalonate to the culture medium. In contrast, cell growth was blocked irreversibly by inhibitors of subsequent steps in the protein isoprenylation pathway. Trophozoite growth inhibition by limonene, perillic acid, perillyl alcohol and N-acetyl-S-farnesyl-L-cysteine was not reversed after the addition of mevalonate, dolichol, ubiquinone or cholesterol to the medium. These observations constitute the first description of protein isoprenylation in any protozoan and indicate that this post-translational modification is an important step in the regulation of the growth of this primitive eukaryote.

Purification of a variant-specific surface protein of Giardia lamblia and characterization of its metal-binding properties.

Giardia lamblia, an intestinal parasite of humans and other vertebrates, undergoes surface antigenic variation by modulating the expression of different variant-specific surface proteins (VSP). VSPs are cysteine-rich surface proteins that bind zinc and other heavy metals in vitro. We developed an immunoaffinity chromatographic method to purify a VSP in order to determine its biochemical properties. The sequences of two different proteolytic fragments agreed with the sequence deduced from the cloned gene, and amino-terminal sequence indicated the removal of a 14-residue signal peptide, consistent with the transport of VSP to the cell surface. The protein is not glycosylated and has an isoelectric point of 5.3. X-ray microanalyses indicated that the major metals in Giardia trophozoites, as well as purified VSP, are zinc and iron. The zinc concentration in Giardia cells was found to be 0.43 mM and the iron concentration 0.80 mM when compared with standard samples (zinc) or calculated from a known physical constants (iron). We propose that metal coordination stabilizes VSPs, rendering them resistant to proteolytic attack in the upper small intestine. Moreover, the ability to bind ions by Giardia may play a role in nutritional deficiency and/or malabsorption in heavily infected persons.

Developmental induction of Golgi structure and function in the primitive eukaryote Giardia lamblia.

A fundamental characteristic of eukaryotic cells is the presence of membrane-bound compartments and membrane transport pathways in which the Golgi complex plays a central role in the selective processing, sorting, and secretion of proteins. The parasitic protozoan Giardia lamblia belongs to the earliest identified lineage among eukaryotes and therefore offers unique insight into the progression from primitive to more complex eukaryotic cells. Here, we report that Giardia trophozoites undergo a developmental induction of Golgi enzyme activities, which correlates with the appearance of a morphologically identifiable Golgi complex, as they differentiate to cysts. Prior to this induction, no morphologically or biochemically identifiable Golgi complex exists within nonencysting cells. Remarkably, protein secretion in both nonencysting and encysting trophozoites is inhibited by brefeldin A, and brefeldin A-sensitive membrane association of ADP-ribosylation factor and beta-COP is observed. These results suggest that the secretory machinery of Giardia resembles that of higher eukaryotes despite the absence of a Golgi complex in nonencysting trophozoites. These findings have implications both for defining the minimal machinery for protein secretion in eukaryotes and for examining the biogenesis of Golgi structure and function.

Developmentally regulated expression of a Giardia lamblia cyst wall protein gene.

The protozoan Giardia lamblia is an obligate parasite of the mammalian small intestine. We studied the expression of a gene that encodes a protein component of the cyst wall, a complex structure assembled during the differentiation of trophozoites to cysts and which is critical to survival of the parasite outside its mammalian host. Transcripts from the cyst wall protein gene increase more than 100-fold during encystation, reaching a maximum between 5 and 24 hours after induction. Cyst wall protein expression also increases dramatically during encystation, and, prior to its incorporation into the nascent cyst wall, the protein is contained within the encystation-specific vesicles of encysting trophozoites. The sequence of the cloned gene predicts an acidic, leucine-rich polypeptide of M(r) 26,000 that contains 5.3 tandemly arranged copies of a degenerate 24-amino-acid repeat. A hydrophobic amino-terminal peptide probably serves as the initial signal that targets this protein to a secretory pathway involving vesicular localization during encystation and, ultimately, secretion to form the cyst wall.

Serum Cohn fraction IV-1 supports the growth of Giardia lamblia in vitro.

Bovine serum Cohn fractions were substituted for whole bovine serum in TYI-S-33 medium. Only fraction IV-1 supported attachment, cysteine uptake, and growth of Giardia lamblia. Among the mammalian sources tested, only goat and horse fractions showed activities comparable to that of the bovine fraction. Agglutinating immunoglobulins were identified as deleterious agents in human, rat, rabbit, and dog fractions.

Insulin-like growth factors stimulate growth and L-cysteine uptake by the intestinal parasite Giardia lamblia.

Giardia lamblia, a parasitic protozoan responsible for diarrhea and malabsorption in humans, grows axenically only in media that contain serum and a high concentration of L-cysteine. During our attempts to grow Giardia in the absence of serum, we found that: (a) human insulin-like growth factors (especially IGF-II), but not insulin, promote the growth and L-cysteine uptake by G. lamblia trophozoites; (b) the growth stimulation was inhibited by alpha IR3, an anti-type 1 IGF receptor monoclonal antibody, but an anti-type 2 IGF receptor antibody had no effect; and (c) IGFs act on Giardia through a type 1 IGF receptor-like protein, which can bind IGF-II with higher affinity than IGF-I, and most likely possesses intrinsic phosphotyrosine kinase activity.

Intermembrane lipid transfer during Trypanosoma cruzi-induced erythrocyte membrane destabilization.

The ability of Trypanosoma cruzi to induce erythrocyte membrane destabilization in vitro was studied. Epimastigote forms adhered to human erythrocytes and caused fusion or lysis of the red cells, depending on the conditions of the interaction. Red cells were fused in the presence of calcium, while haemolysis was induced in the absence of the cation. Dextran 60 C facilitated fusion but delayed lysis. Optimum pH and temperature for fusion were 7.4 and 37 degrees C, respectively. Lipid alterations were produced in the plasma membrane of the red cell during the interaction with the parasite. A Ca(2+)-independent increase of lysophospholipids and free fatty acids was common to both the lysis and fusion processes. A relative increase of 1,2-diacylglycerides was unique to the fusion process and these changes were dependent on Ca2+. The transfer of free fatty acids and lysophospholipids from T. cruzi to erythrocyte membranes was demonstrated using parasites pre-labelled with radioactive phospholipids. Pre-treatment of parasites with exogenous phospholipase A2 abolished the fusogenicity, while lysis was increased. Neither fusion nor haemolysis occurred when the parasites were pre-treated with fatty acid free albumin, phospholipase A2 inhibitors or when these compounds were present in the medium during the parasite-erythrocyte interaction. Our results suggest that T. cruzi induces erythrocyte membrane destabilization in vitro by transfer of lipid material in a calcium independent manner and that this ion is necessary for other membrane alterations that lead to erythrocyte fusion.