Lipid metabolism in Giardia: a post-genomic perspective.

Giardia lamblia, a protozoan parasite, infects a wide variety of vertebrates, including humans. Studies indicate that this anaerobic protist possesses a limited ability to synthesize lipid molecules de novo and depends on supplies from its environment for growth and differentiation. It has been suggested that most lipids and fatty acids are taken up by endocytic and non-endocytic pathways and are used by Giardia for energy production and membrane/organelle biosynthesis. The purpose of this article is to provide an update on recent progress in the field of lipid research of this parasite and the validation of lipid metabolic pathways through recent genomic information. Based on current cellular, biochemical and genomic data, a comprehensive pathway has been proposed to facilitate our understanding of lipid and fatty acid metabolism/syntheses in this waterborne pathogen. We envision that the current review will be helpful in identifying targets from the pathways that could be used to design novel therapies to control giardiasis and related diseases.

Knob-positive and knob-negative Plasmodium falciparum differ in expression of a strain-specific malarial antigen on the surface of infected erythrocytes.

We have investigated the expression of a strain-specific malarial antigen on the surface of erythrocytes infected with knobless (K-) variants of knob-positive (K+) strains of Plasmodium falciparum. Aotus blood infected with K+ or K- parasites derived from two independent geographical isolates (Malayan camp and Santa Lucia) was surface iodinated by the lactoperoxidase method. Infected and uninfected erythrocytes were then separated by a new procedure involving equilibrium density sedimentation on a Percoll gradient containing sorbitol. Strain-specific antigens were readily identified on the surface of erythrocytes infected with either of the K+ strains by their characteristic size and detergent solubility. These proteins were not detected on the surface of erythrocytes infected with either of the K- variants nor on uninfected erythrocytes isolated from K+- or K- -infected blood. These results are consistent with a role for the strain-specific surface antigen in cytoadherence of P. falciparum-infected erythrocytes. Our findings represent the second biochemical difference (with the knob-associated histidine-rich protein) between K+ and K- P. falciparum.

Endocytosis in Entamoeba histolytica. Evidence for a unique non-acidified compartment.

Our studies on endocytosis in Entamoeba histolytica trophozoites suggest that there are two vacuolar compartments in this organism. The first compartment consists of large vacuoles (greater than 2 microns diameter). As measured by the fluid phase markers, fluorescein isothiocyanate (FITC)-dextran and horseradish peroxidase (HRP), this compartment is a rapid equilibrium with the external milieu and is constantly exchanging (1-2 h) its contents with the external medium. The contents of these vacuoles are not acidified. This together with the absence of degradation of fluid phase markers clearly differentiates these vacuoles from lysosomes of eucaryotes. By labeling externally disposed peptides on the surface membrane of trophozoites with 125I, we could show that the surface membrane was rapidly internalized over a 2-h period and then reached a plateau. All major 125I surface proteins, with the exception of a set of peptides in the 40,000 molecular weight range, were interiorized and approximately 60% of the total radiolabel were found to be in the internal membrane fraction at any given time. The kinetics of this process were similar to those for the uptake of fluid phase markers and are best explained by cycling of the surface membrane into the vacuolar compartment(s) and then back to the cell surface. The second vacuolar compartment consisted of small vesicles (less than 2 microns diameter) with acidified contents as indicated by acridine orange uptake. The endocytic nature of these vesicles was shown by their slow (days) labeling with FITC-dextran, and spectral analysis of internalized FITC-dextran confirmed that this second compartment is acidified (pH 5.2).

Plasma membrane of Entamoeba histolytica.

Axenically propagated Entamoeba histolytica (HK9:NIH strain) were employed as starting material for the isoation of plasma membrane by a novel procedure. In the absence of known enzymatic markers, the externally disposed polypeptides of intact amoebae were iodinated and the incorporated label used to monitor membrane separation and recovery. 12 major plasma membrane polypeptides (12 x 10(3)-200 x 10(3) mol wt) were labeled and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Each of these was a glycoprotein. Preincubation of amoebae with concanavalin A stabilized the plasma membranes as large sheets, facilitating its separation by low-speed centrifugation. Dissociation of the lectin with alpha-methyl mannoside, followed by additional homogenization led to vesiculation and further purification. The isolated plasma membrane was recovered in high yield (28%) and enriched 30-fold in terms of incorporated iodide. All iodinated surface glycoproteins of the intact organism were present in the plasma membrane fraction. A Ca++-dependent ATPase was enriched in the plasma membrane to a similar extent, but over one-half of the total activity was associated with internal, unlabeled membranes, suggesting a dual localization of this activity. The isolated plasma membrane was enriched in cholesterol and had a cholesterol:molar ratio of 0.87. It also contained larger amounts of an unusual phospholipid--ceramide aminoethyl phosphonate--a phospholipase-resistant species.

Synthetic peptides from the circumsporozoite proteins of Plasmodium falciparum and Plasmodium knowlesi recognize the human hepatoma cell line HepG2-A16 in vitro.

Several lines of evidence have emphasized the importance of the malaria circumsporozoite (CS) protein as a factor in sporozoite invasion of the hepatocyte; however, the specific mechanism of cell recognition and invasion has not been explained. In this study we present evidence that a highly conserved region of the CS protein immediately adjacent to the repeat region, the N1 region, specifically recognizes receptors on the human hepatoma cell line HepG2-A16 under conditions where invasion by sporozoites can occur. Peptides consisting of sequences from the repeat region or of the more extensive N2 region showed no such specific association. Antibody against the N1 peptide could inhibit sporozoite invasion in vitro. Covalent coupling of radiolabeled N1 peptide to HepG2-A16 cells identified two hepatic cell proteins to be closely associated with the peptide. We suggest that these proteins could act as receptors or mediators, via the N1 region of the CS protein, for the P. falciparum sporozoite in the process of invasion of the hepatocyte.

Transport of an Mr approximately 300,000 Plasmodium falciparum protein (Pf EMP 2) from the intraerythrocytic asexual parasite to the cytoplasmic face of the host cell membrane.

The profound changes in the morphology, antigenicity, and functional properties of the host erythrocyte membrane induced by intraerythrocytic parasites of the human malaria Plasmodium falciparum are poorly understood at the molecular level. We have used mouse mAbs to identify a very large malarial protein (Mr approximately 300,000) that is exported from the parasite and deposited on the cytoplasmic face of the erythrocyte membrane. This protein is denoted P. falciparum erythrocyte membrane protein 2 (Pf EMP 2). The mAbs did not react with the surface of intact infected erythrocytes, nor was Pf EMP 2 accessible to exogenous proteases or lactoperoxidase-catalyzed radioiodination of intact cells. The mAbs also had no effect on in vitro cytoadherence of infected cells to the C32 amelanotic melanoma cell line. These properties distinguish Pf EMP 2 from Pf EMP 1, the cell surface malarial protein of similar size that is associated with the cytoadherent property of P. falciparum-infected erythrocytes. The mAbs did not react with Pf EMP 1. In one strain of parasite there was a significant difference in relative mobility of the 125I-surface-labeled Pf EMP 1 and the biosynthetically labeled Pf EMP 2, further distinguishing these proteins. By cryo-thin-section immunoelectron microscopy we identified organelles involved in the transit of Pf EMP through the erythrocyte cytoplasm to the internal face of the erythrocyte membrane where the protein is associated with electron-dense material under knobs. These results show that the intraerythrocytic malaria parasite has evolved a novel system for transporting malarial proteins beyond its own plasma membrane, through a vacuolar membrane and the host erythrocyte cytoplasm to the erythrocyte membrane, where they become membrane bound and presumably alter the properties of this membrane to the parasite's advantage.

Secretion of a malarial histidine-rich protein (Pf HRP II) from Plasmodium falciparum-infected erythrocytes.

Plasmodium falciparum-infected erythrocytes (IRBCs) synthesize several histidine-rich proteins (HRPs) that accumulate high levels of [3H]histidine but very low levels of amino acids such as [3H]isoleucine or [35S]methionine. We prepared a monoclonal antibody which reacts specifically with one of these HRPs (Pf HRP II) and studied the location and synthesis of this protein during the parasite's intracellular growth. With the knob-positive Malayan Camp strain of P. falciparum, the monoclonal antibody identified a multiplet of protein bands with major species at Mr 72,000 and 69,000. Pf HRP II synthesis began with immature parasites (rings) and continued through the trophozoite stage. The Mr 72,000 band of Pf HRP II, but not the faster moving bands of the multiplet, was recovered as a water-soluble protein from the culture supernatant of intact IRBCs. Approximately 50% of the total [3H]histidine radioactivity incorporated into the Mr 72,000 band was extracellular between 2 and 24 h of culture. Immunofluorescence and cryothin-section immunoelectron microscopy localized Pf HRP II to several cell compartments including the parasite cytoplasm, as concentrated "packets" in the host erythrocyte cytoplasm and at the IRBC membrane. Our results provide evidence for an intracellular route of transport for a secreted malarial protein from the parasite through several membranes and the host cell cytoplasm.

Entamoeba histolytica: a eukaryote without glutathione metabolism.

Entamoeba histolytica was found to grow normally without producing glutathione and the main enzymes of glutathione metabolism, indicating that glutathione is not essential for many eukaryotic processes. This parasitic amoeba is an unusual eukaryote whose special features may help define the crucial functions of glutathione in those eukaryotes that do use it. Since Entamoeba histolytica lacks mitochondria and the usual aerobic respiratory pathways, the finding that it grows without glutathione and other evidence support the hypothesis that a primary function of glutathione in eukaryotes involves protection against oxygen toxicity associated with mitochondria and suggest that eukaryotes may have acquired glutathione metabolism at the time that they acquired mitochondria.

Susceptibility of Entamoeba histolytica to oxygen intermediates.

To explore the susceptibility of the extracellular protozoan, Entamoeba histolytica, to toxic oxygen intermediates, trophozoites were exposed to fluxes of O2, H2O2, and OH. generated enzymatically by the glucose oxidase and xanthine oxidase reactions. HM-1 trophozoites were resistant to O2, but were readily killed by H2O2 alone. OH. and 1O2 were not required for effective amebicidal activity. The addition of peroxidase and halide enhanced trophozoite killing by H2O2. Sonicates of amebae contained virtually no catalase and little glutathione peroxidase activity which may contribute to susceptibility to H2O2. Coupled with our previous studies with Toxoplasma gondii and Leishmania spp. these observations indicate that there is a broad spectrum of susceptibility of intra- and extracellular pathogenic protozoa to killing by oxygen intermediates.

Identification of parasite proteins in a membrane preparation enriched for the surface membrane of erythrocytes infected with Plasmodium knowlesi.

A subcellular fraction enriched in erythrocyte membranes has been isolated from rhesus monkey erythrocytes infected with Plasmodium knowlesi. Infected cells were lysed by centrifugation through a zone of hypotonic buffer and membranes isolated by equilibrium density gradient centrifugation in the same tube. The purified membrane fraction was shown to include the erythrocyte surface membrane by several methods: electron microscopy, identification of Coomassie Blue stained erythrocyte membrane proteins, identification of band 3 with a monoclonal antibody, and identification of radioiodinated cell surface proteins. The resulting ghosts were shown to be specifically reactive with monkey sera against the variant surface antigens of P. knowlesi by indirect immunofluorescence and membrane agglutination. No reactivity was seen with a monoclonal antibody (13C11) against the intracellular schizont surface. A number of metabolically labelled parasite proteins were enriched in this membrane function, including peptides of 277, 208, 173, 153, 134, 109, 80, 60 and 48 kDa and the variant surface antigens of variable molecular mass (180-207 kDa). These proteins were distinct from the major parasite proteins of total infected erythrocytes and isolated merozoites. The major glucosamine labelled glycoprotein of the internal schizont (230 kDa) was not found in this fraction. Moreover, no fragment of this parasite glycoprotein was found in this membrane fraction, indicating that no part of this molecule is transported to the erythrocyte surface. In contrast, the variant antigen of P. knowlesi, known to be on the erythrocyte surface, could be readily identified as peptides unique to specific cloned parasite lines. We propose that the other nine parasite proteins found within this membrane fraction represent a starting point for the identification of other parasite proteins transported to the surface membrane of the infected erythrocyte.

Sequence survey of the Giardia lamblia genome.

The parasitic protozoan Giardia lamblia represents one of the earliest diverging lineages in the evolutionary history of eukaryotic organisms as well as an important human pathogen. A representative sampling of gene sequences from this early diverging protozoan could provide insights into genotypic and phenotypic innovations associated with the origin of eukaryotes. Currently, known giardial gene sequences are heavily biased toward a few gene families, including variant surface proteins (VSPs), structural proteins, and ribosomal RNA genes. One-pass sequences of Giardia genomic DNA were obtained using vector flanking priming sequences on the ends of cosmids in two independent libraries. Comparisons of 2304 of these sequences against the GenBank database identified 205 potential giardial genes with BLAST scores P(n) < 10(9). These coding regions encompass a wide range of metabolic, repair, and signaling enzymes, and include some genes not predicted by our current understanding of Giardia biochemistry. The efficiency of identification of putative genes is consistent with earlier findings that coding regions in the Giardia genome are densely packed and do not appear to contain introns. Our current results suggest that direct genome sequencing is an efficient method for identifying giardial genes for evolutionary and biochemical studies.

Developmentally regulated transcripts and evidence of differential mRNA processing in Giardia lamblia.

Although encystation and excystation are crucial to transmission of Giardia lamblia, little is known about the regulation of these very distinct differentiation processes. Fingerprinting of giardial mRNA populations throughout the time course of differentiation demonstrated complex patterns in mRNA differential display. Certain transcripts appeared or increased, while others decreased or disappeared at specific times, in response to physiologic stimuli that mimic key stages in parasite descent through the host gastrointestinal tract. This approach has allowed the direct identification of critical stages in differentiation, as well as isolation of genes which may be crucial to the development of G. lamblia. One stage-specific single copy gene (ENC6) whose transcript is greatly upregulated during encystation was analyzed further. Partial sequence analysis revealed no correspondence with known genes. 3'-rapid amplification of cDNA ends (3'-RACE) analysis of ENC6 transcripts at various times of encystation revealed two polyadenylation sites. The more proximal site, 10 nucleotides past the single classic AGTAAA sequence, was utilized only during encystation and its transcript increased approximately 16-fold during the first 24 h of encystation. In contrast, a slightly divergent polyadenylation site 288 nucleotides downstream from the open reading frame (ORF) was used during both vegetative growth and encystation, although its transcript was present at low levels. These studies are the first evidence of differential mRNA processing in G. lamblia and suggest a potential role of the 3'-untranslated region (3'-UTR) in modulating gene expression during differentiation of this primitive eukaryote.

Localization of the major Plasmodium falciparum glycoprotein on the surface of mature intraerythrocytic trophozoites and schizonts.

The subcellular location of the major malarial glycoprotein in erythrocytes infected with schizonts of Plasmodium falciparum has been studied by two methods. In the first, glycoproteins were labelled with [3H]glucosamine or [3H]isoleucine during in vitro culture. Trypsin treatment of intact infected erythrocytes caused no major qualitative or quantitative changes in [3H]glucosamine labelled glycoproteins or [3H]isoleucine labelled proteins separated by sodium dodecylsulfate polyacrylamide gel electrophoresis. However, in the presence of Triton X-100 the labelled glycoproteins and proteins were completely cleaved by trypsin. In the second method, two monoclonal antibodies which specifically immunoprecipitate the major 195 kDa glycoprotein failed to react on indirect immunofluorescence with intact non-fixed schizont-infected erythrocytes, but reacted strongly with saponin released schizonts indicating specificity for the surface of mature intracellular parasites. Immunoelectronmicroscopy using ferritin-conjugated secondary antibody confirmed the location of the epitope(s) recognized by these monoclonals on the surface of intracellular parasites. Ferritin particles were not associated with knob-bearing erythrocyte membranes. The results indicate that only a small proportion or none of the 195 kDa glycoprotein is on the surface of the infected erythrocyte and that the largest proportion is expressed on the surface of mature intraerythrocytic parasites.

Localization of Plasmodium falciparum histidine-rich protein 1 in the erythrocyte skeleton under knobs.

Plasmodium falciparum parasites that induce knobs in the host erythrocyte membrane (K+ phenotype) synthesize a 90 kDa histidine-rich protein (PfHRP-1), whereas knobless variants do not. A monoclonal antibody (mAb 89) to PfHRP-1, in combination with cryo-thin section immunoelectron microscopy, localized the antigen in the parasitophorous vacuolar space and vesicles within the erythrocyte cytosol. Additional immunoelectron microscopic studies showed that PfHRP-1 was also associated with submembranous electron-dense material under knobs and with microfilaments of the host erythrocyte skeletal network. Immunofluorescence and immunoelectron microscopy of intact, non-fixed K+ infected erythrocytes using mAb 89 and a rabbit antiserum raised against purified PfHRP-1, failed to identify any surface exposed epitopes. These antibodies also failed to block cytoadherence of infected erythrocytes to C32 melanoma cells or to affect macrophage phagocytosis of infected erythrocytes.

Expression of Plasmodium berghei circumsporozoite antigen on the surface of exoerythrocytic schizonts and merozoites.

The intracellular distribution of circumsporozoite (CS) antigen was traced by immunoelectron microscopy in cultures of Plasmodium berghei exoerythrocytic (EE) schizonts with monoclonal antibody (Mab) 3D11 to the immunodominant repeat region of the P. berghei CS protein. CS antigen was localized on the parasitophorous vacuole (PV) membrane and pellicular complex of recently invaded sporozoites and on electron-dense masses of sloughed CS antigen in the host cell cytoplasm. CS antigen persisted throughout the complete EE cycle of P. berghei on the surface of EE schizonts and was incorporated into the plasma membrane of budding EE merozoites. Erythrocytic merozoites were not labeled by Mab 3D11, indicating that these 2 populations of merozoites differ in antigenic composition. Significant internal labeling occurred in 50 hr EE schizonts in association with the limiting membranes of peripheral vesicles and short, tube-like structures attached to their outer surfaces. These vesicles contained an electron-dense flocculent material also present in the PV space. Association of CS antigen with the limiting membranes of these vesicles suggests that they either develop as endocytotic invaginations of the schizont plasma membrane or transport newly synthesized CS antigen from the endoplasmic reticulum and Golgi of developing EE schizonts to the parasite surface.

The Giardia genome project database.

The Giardia genome project database provides an online resource for Giardia lamblia (WB strain, clone C6) genome sequence information. The database includes edited single-pass reads, the results of BLASTX searches, and details of progress towards sequencing the entire 12 million-bp Giardia genome. Pre-sorted BLASTX results can be retrieved based on keyword searches and BLAST searches of the high throughput Giardia data can be initiated from the web site or through NCBI. Descriptions of the genomic DNA libraries, project protocols and summary statistics are also available. Although the Giardia genome project is ongoing, new sequences are made available on a bi-monthly basis to ensure that researchers have access to information that may assist them in the search for genes and their biological function. The current URL of the Giardia genome project database is www.mbl.edu/Giardia.

GP49, an invariant GPI-anchored antigen of Giardia lamblia.

Giardia lamblia is a primitive protozoan and a major cause of waterborne enteric disease throughout tropical and temperate zones. The ability to grow the infective trophozoites in culture as well as the discovery of the method of in vitro encystation made it possible to study the biology of this primitive protozoan and to characterize the surface antigens. Giardia trophozoites are exposed to high concentrations of fatty acids in the human small intestine. This raises the possibility that intestinal fatty acids may become incorporated into Giardia. Therefore, we determined the pattern of fatty acylation of Giardia surface molecules. By metabolic labeling with radiolabeled fatty acids we identified a single glycosylphosphatidylinositol (GPI)-anchored surface protein in Giardia. GP49 differs from the cysteine-rich variable surface antigens described previously. The presence of a GPI anchor in GP49 was supported by the metabolic incorporation of [14C]-ethanolamine, [3H]-myoinositol and fatty acids into the protein. This was confirmed by chemical and enzymatic cleavage experiments. Most interestingly, GP49 was found to be present in different isolates of Giardia and thus can be considered as an invariant surface antigen. Although the biological function of GP49 is not known, recently we have found that intact and soluble GP49 altered the electrolyte fluxes which regulate fluid secretion in the cultured human intestinal epithelial cell line, T84. These studies indicate that the GPI-anchored invariant antigen of Giardia may play an important role in the pathophysiology of giardiasis.

Ultrastructural localization of Plasmodium falciparum circumsporozoite protein in newly invaded hepatoma cells.

The fate and disposition of the circumsporozoite (CS) protein of Plasmodium falciparum was investigated during hepatoma cell invasion with several sera raised against defined CS peptides, including both repeat and nonrepeat regions spanning approximately 60% of the P. falciparum CS gene product. Distribution of the protein, as revealed by immunoelectron microscopy, was limited to the surface of the sporozoite both before and after invasion. In particular, no CS protein antigen was detected in association with either the parasitophorous vacuole membrane or the host cell surface.