ORF-C4 from the early branching eukaryote Giardia lamblia displays characteristics of alpha-crystallin small heat-shock proteins

Giardia lamblia is a medically important protozoan parasite with a basal position in the eukaryotic lineage and is an interesting model to explain the evolution of biochemical events in eukaryotic cells. G. lamblia trophozoites undergo significant changes in order to survive outside the intestine of their host by differentiating into infective cysts. In the present study, we characterize the previously identified Orf-C4 (G. lamblia open reading frame C4) gene, which is considered to be specific to G. lamblia. It encodes a 22 kDa protein that assembles into high-molecular-mass complexes during the entire life cycle of the parasite. ORF-C4 localizes to the cytoplasm of trophozoites and cysts, and forms large spherical aggregates when overexpressed. ORF-C4 overexpression and down-regulation do not affect trophozoite viability; however, differentiation into cysts is slightly delayed when the expression of ORF-C4 is down-regulated. In addition, ORF-C4 protein expression is modified under specific stress-inducing conditions. Neither orthologous proteins nor conserved domains are found in databases by conventional sequence analysis of the predicted protein. However, ORF-C4 contains a region which is similar structurally to the alpha-crystallin domain of sHsps (small heat-shock proteins). In the present study, we show the potential role of ORF-C4 as a small chaperone which is involved in the response to stress (including encystation) in G. lamblia.     

An unusual triosephosphate isomerase from the early divergent eukaryote Giardia lamblia

Recombinant triosephosphate isomerase from the parasite Giardia lamblia (GlTIM) was characterized and immunolocalized. The enzyme is distributed uniformly throughout the cytoplasm. Size exclusion chromatography of the purified enzyme showed two peaks with molecular weights of 108 and 55 kDa. Under reducing conditions, only the 55-kDa protein was detected. In denaturing gel electrophoresis without dithiothreitol, the enzyme showed two bands with molecular weights of 28 and 50 kDa; with dithiotretitol, only the 28-kDa protein was observed. These data indicate that GlTIM may exist as a tetramer or a dimer and that, in the former, the two dimers are covalently linked by disulfide bonds. The kinetics of the dimer were similar to those of other TIMs. The tetramer exhibited half of the kcat of the dimer without changes in the Km. Studies on the thermal stability and the apparent association constants between monomers showed that the tetramer was slightly more stable than the dimer. This finding suggests the oligomerization is not related to enzyme thermostability as in Thermotoga maritima. Instead, it could be that oligomerization is related to the regulation of catalytic activity in different states of the life cycle of this mesophilic parasite.     

Identification of 20 snoRNA-like RNAs from the primitive eukaryote, Giardia lamblia

From a specialized cDNA library of Giardia lamblia, 20 snoRNA-like RNAs, including 16 box C/D sRNAs and four box H/ACA sRNAs, were first identified. The sRNAs were predicted to guide a total of 11 2′-O-methylation and four pseudouridylation sites on the G. lamblia rRNAs, respectively. By using primer extension assay, seven methylation sites were precisely mapped in the G. lamblia 16S rRNA, despite its high GC content. All of the sRNA genes locate on the small intergenic regions of the G. lamblia genome and seem to be independently transcribed from their own promoters. Particularly, a cluster composed of GlsR17 and GlsR18 genes is transcribed as a dicistronic precursor, implying a mechanism of endonuclease cleavage for the maturation of the two sRNAs. The systematic identification of the sRNAs in G. lamblia has provided valuable information about the characteristics of the two major families of small guide RNAs in one of the most primitive eukaryotes and would contribute to the understanding of the evolution of small non-messenger RNA genes from prokaryotes to eukaryotes.     

Calcium signaling in excystation of the early diverging eukaryote,Giardia lamblia

Excystation of Giardia lamblia, which initiates infection, is a poorly understood but dramatic differentiation induced by physiological signals from the host. Our data implicate a central role for calcium homeostasis in excystation. Agents that alter cytosolic Ca(2+) levels (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-tetra(acetyloxymethyl) ester, a Ca(2+) channel blocker, Ca(2+) ionophores, and thapsigargin) strongly inhibit excystation. Treatment of Giardia with thapsigargin raised intracellular Ca(2+) levels, and peak Ca(2+) responses increased with each stage of excystation, consistent with the kinetics of inhibition. Fluorescent thapsigargin localized to a likely Ca(2+) storage compartment in cysts. The ability to sequester ions in membrane-bounded compartments is a hallmark of the eukaryotic cell. These studies support the existence of a giardial thapsigargin-sensitive Ca(2+) storage compartment resembling the sarcoplasmic/endoplasmic reticulum calcium ATPase pump-leak system and suggest that it is important in regulation of differentiation and appeared early in the evolution of eukaryotic cells. Calmodulin antagonists also blocked excystation. The divergent giardial calmodulin localized to the eight flagellar basal bodies/centrosomes, like protein kinase A. Inhibitor kinetics suggest that protein kinase A signaling triggers excystation, whereas calcium signaling is mainly required later, for parasite activation and emergence. Thus, the basal bodies may be a cellular control center to coordinate the resumption of motility and cytokinesis in excystation.     

Reversible interruption of Giardia lamblia cyst wall protein transport in a novel regulated secretory pathway

To survive in the environment and infect a new host, Giardia lamblia secretes an extracellular cyst wall using a poorly understood pathway. The two cyst wall proteins (CWPs) form disulphide-bonded heterodimers and are exported via novel encystation-specific secretory vesicles (ESVs). Exposure of eukaryotic cells to dithiothreitol (DTT) blocks the formation of disulphide bonds in nascent proteins that accumulate in the endoplasmic reticulum (ER) and induces an unfolded protein response (UPR). Proteins that have exited the ER are not susceptible. Exposure to DTT inhibits ESV formation by > 85%. Addition of DTT to encysting cells causes rapid ( t _1/2 < 10 min), reversible disappearance of ESVs, correlated with reduction of CWPs to monomers and reformation of CWP oligomers upon removal of DTT. Neither CWPs nor ESVs are affected by mercaptoethanesulphonic acid, a strong reducing agent that does not penetrate cells. DTT does not inhibit the overall protein secretory pathway, and recovery does not require new protein synthesis. We found evidence of protein disulphide isomerases in the ESV and the surface of encysting cells, in which they may catalyse initial CWP folding and recovery from DTT. This is the first suggestion of non-CWP proteins in ESVs and of enzymes on the giardial surface. DTT treatment did not stimulate a UPR, suggesting that Giardia may have diverged before the advent of this conserved form of ER quality control.     

Identification and characterization of a Giardia lamblia group-specific gene.

Giardia lamblia consist of heterogeneous isolates that can be divided into at least three groups. Differential screening of a cDNA library with isolate-specific antisera identified a gene which is expressed and found only in Group 3 isolates. This gene, GLORF-C4, is 597 bp in length and predicts a deduced protein of 198 amino acids that is characterized by a polyserine motif. Giardia can also be grouped by their ability to express certain variant-specific surface proteins (VSPs), expression of which is restricted among groups. In Southern blots, probes specific to two VSPs were used to characterize isolates. Failure to detect VSP genes correlated with inability to express the same VSP. Analysis of isolates with these new probes complements and confirms the groupings previously suggested using other criteria. These genetic differences should allow differentiation of isolates and permit the application of basic epidemiological techniques to determine the manner of spread and the presence of animal reservoirs.     

Stress-induced tRNA-derived RNAs: a novel class of small RNAs in the primitive eukaryote Giardia lamblia

Giardia lamblia is an early diverging and evolutionarily successful protozoan as it can enter into a dormant cyst stage from a vegetative trophozoite. During dormant stage, its metabolic rate decreases dramatically. However, to date, the regulatory molecules participating in the initiation and maintenance of this process have not been fully investigated. In this study, we have identified a class of abundant small RNAs named sitRNAs, which are approximately 46 nucleotides in length and accumulate in G. lamblia encysting cultures. Remarkably, they are derived from the 3' portion of fully matured tRNAs by cleavage of the anticodon left arm, with the 3' terminal CCA triplex still connected. During differentiation, only a limited portion of mature tRNAs is cleaved, but this cleavage occurs almost in the entire tRNA family. sitRNAs begin to accumulate as early as 3 h after initiation of encystation and are maintained at a relatively stable level during the whole process, exhibiting an expression peak at around 24 hr. Our studies further show that sitRNAs can be induced by several other stress factors, and in the case of serum deprivation, both tRNAs and sitRNAs degrade rapidly, with the accumulation of tRNA being halved. Our results may provide new insight into a novel mechanism for stressed G. lamblia to regulate gene expression globally.     

Sorting of cyst wall proteins to a regulated secretory pathway during differentiation of the primitive eukaryote, Giardia lamblia

Giardia lamblia, which belongs to the earliest identified lineage to diverge from the eukaryotic line of descent, is one of many protists reported to lack a Golgi apparatus. Our recent finding of a developmentally regulated secretory pathway in G. lamblia makes it an ideal organism with which to test the hypothesis that the Golgi may be more readily demonstrated in actively secreting cells. These ultrastructural studies now show that a regulated pathway of transport and secretion of cyst wall antigens via a novel class of large, osmiophilic secretory vesicles, the encystation-specific vesicles (ESV), is assembled during encystation of G. lamblia. Early in encystation, cyst antigens are localized in simple Golgi membrane stacks and concentrated within enlarged Golgi cisternae which appear to be precursors of ESV. This would represent an unusual mechanism of secretory vesicle biogenesis. Later in differentiation, cyst antigens are localized within ESV, which transport them to the plasma membrane and release them by exocytosis to the nascent cell wall. ESV are not observed after completion of the cyst wall. In contrast to the regulated transport of cyst wall proteins, we demonstrate a distinct constitutive lysosomal pathway. During encystation, acid phosphatase activity is localized in endoplasmic reticulum, Golgi, and small constitutive peripheral vacuoles which function as lysosomes. However, acid phosphatase activity is not detectable in ESV. These studies show that G. lamblia, an early eukaryote, is capable of carrying out Golgi-mediated sorting of proteins to distinct regulated secretory and constitutive lysosomal pathways.     

Active and passive mechanisms drive secretory granule biogenesis during differentiation of the intestinal parasite Giardia lamblia

The parasitic protozoan Giardia lamblia undergoes important changes to survive outside the intestine of its host by differentiating into infective cysts. During encystation, three cyst wall proteins (CWPs) are specifically expressed and concentrated within encystation-specific secretory vesicles (ESVs). ESVs are electron-dense secretory granules that transport CWPs before exocytosis and extracellular polymerization into a rigid cyst wall. Because secretory granules form at the trans-Golgi in higher eukaryotes and because Giardia lacks an identifiable Golgi apparatus, the aim of this work was to investigate the molecular basis of secretory granule formation in Giardia by examining the role of CWPs in this process. Although CWP1, CWP2, and CWP3 are structurally similar in their 26-kDa leucine-rich overlapping region, CWP2 is distinguished by the presence of a 13-kDa C-terminal basic extension. In non-encysting trophozoites, expression of different CWP chimeras showed that the CWP2 basic extension is necessary for biogenesis of ESVs, which occurs in a compartment derived from the endoplasmic reticulum. Nevertheless, the CWP2 basic extension per se is insufficient to trigger ESV formation, indicating that other domains in CWPs are also required. We found that CWP2 is a key regulator of ESV formation by acting as an aggregation factor for CWP1 and CWP3 through interactions mediated by its conserved region. CWP2 also acts as a ligand for sorting via its C-terminal basic extension. These findings show that granule biogenesis requires complex interactions among granule components and membrane receptors.     

Identification of nucleoli in the early branching protist Giardia duodenalis

Giardia duodenalis has been described as 'anucleolated'. In this work we analysed the subcellular distribution of several nucleolar markers in Giardia nuclei using silver and immunostaining techniques for electron and confocal laser microscopy as well as expression of epitope-tagged proteins in transgenic trophozoites. We identified anteronuclear fibrogranular structures corresponding to nucleolar organising regions with recruited ribonucleoprotein complexes, rRNA and epitope-tagged fibrillarin and rRNA-pseudouridine synthase (CBF5). Recombinant fibrillarin and CBF5 were targeted to this subcompartment. This study demonstrates the presence of nucleoli in G. duodenalis and provides a model to analyse minimal requirements for nucleolar assembly and maintenance in eukaryotic cells.     

Purification and characterization of guanine phosphoribosyltransferase from Giardia lamblia

Giardia lamblia, a flagellated parasitic protozoan and the causative agent of giardiasis, lacks de novo purine biosynthesis and exists on salvage of adenine and guanine by adenine phosphoribosyltransferase and guanine phosphoribosyltransferase. Guanine phosphoribosyltransferase from G. lamblia crude extracts has been purified to apparent homogeneity by Sephacryl S-200 gel filtration followed by C-8-GMP-agarose and 2',3'-GMP-agarose affinity chromatography, resulting in an overall recovery of 77% and a purification of 83,000-fold. The molecular weight of the native enzyme as estimated by gel filtration and isokinetic sucrose gradients was found to be 58,000-63,000, with a subunit molecular weight of approximately 29,000, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Mono P chromatofocusing chromatography gives rise to a major activity peak eluting from the column at a pH of 6.75 and two minor activity peaks at pH of 5.3 and 5.2. Hypoxanthine and xanthine can be recognized by the enzyme as substrates but at Km values 20 times higher than that observed with guanine. G. lamblia guanine phosphoribosyltransferase is immunologically distinct from human hypoxanthine-guanine phosphoribosyltransferase and Escherichia coli xanthine-guanine phosphoribosyltransferase, and G. lamblia DNA fragments are incapable of hybridizing with mouse neuroblastoma hypoxanthine-guanine phosphoribosyltransferase DNA or E. coli xanthine phosphoribosyltransferase DNA under relatively relaxed conditions. All evidence presented suggests that G. lamblia guanine phosphoribosyltransferase may be qualified as a potential target for antigiardiasis chemotherapy.     

The superoxide reductase from the early diverging eukaryote Giardia intestinalis

Unlike superoxide dismutases (SODs), superoxide reductases (SORs) eliminate superoxide anion (O₂^•−) not through its dismutation, but via reduction to hydrogen peroxide (H₂O₂) in the presence of an electron donor. The microaerobic protist Giardia intestinalis, responsible for a common intestinal disease in humans, though lacking SOD and other canonical reactive oxygen species-detoxifying systems, is among the very few eukaryotes encoding a SOR yet identified. In this study, the recombinant SOR from Giardia (SOR_Gi) was purified and characterized by pulse radiolysis and stopped-flow spectrophotometry. The protein, isolated in the reduced state, after oxidation by superoxide or hexachloroiridate(IV), yields a resting species (T_final) with Fe³⁺ ligated to glutamate or hydroxide depending on pH (apparent pK_a = 8.7). Although showing negligible SOD activity, reduced SOR_Gi reacts with O₂^•− with a pH-independent second-order rate constant k₁ = 1.0 × 10⁹ M^− 1 s^− 1 and yields the ferric-(hydro)peroxo intermediate T₁; this in turn rapidly decays to the T_final state with pH-dependent rates, without populating other detectable intermediates. Immunoblotting assays show that SOR_Gi is expressed in the disease-causing trophozoite of Giardia. We propose that the superoxide-scavenging activity of SOR in Giardia may promote the survival of this air-sensitive parasite in the fairly aerobic proximal human small intestine during infection.     

Identification and characterization of taglin, a mannose 6-phosphate binding, trypsin-activated lectin from Giardia lamblia

We have previously reported the presence of a cell surface associated lectin activity in Giardia lamblia, a human protozoan parasite that is a significant cause of diarrheal disease worldwide [Lev, B., Ward, H., Keusch, G. T., & Pereira, M. E. A. (1986) Science (Washington, D.C.) 232, 71-73]. This lectin is specifically activated in vitro by a host protease, trypsin, which is secreted in vivo at the site of infection. The activated lectin agglutinates cells to which the parasite adheres in vivo and binds specifically to isolated brush border membranes of these cells. These findings suggest that this lectin may be of importance in the host-parasite interaction. We now report the identification of this lectin, which we have named taglin (to denote trypsin-activated Giardia lectin), and describe some of its properties. A monoclonal antibody that inhibits the hemagglutinating activity of taglin recognizes a protein of 28,000/30,000 kdaltons in Western blots of Giardia lysates. This finding was confirmed by direct demonstration of lectin activity with the technique of erythrocyte binding to proteins electroblotted to nitrocellulose, which revealed specific red cell binding to giardial protein bands in the same molecular weight range as those recognized by the monoclonal antibody. This study also elucidates the binding of taglin to terminal phosphomannosyl residues. The involvement of cell surface phosphate in binding of taglin to erythrocytes is shown by the abolition of lectin activity by alkaline phosphatase treatment of the erythrocytes. Taglin also requires divalent cations, Ca2+ or Mn2+, for hemagglutinating activity and is active within a narrow pH range of 6-7.     

Adaptor protein complex 1 mediates the transport of lysosomal proteins from a Golgi-like organelle to peripheral vacuoles in the primitive eukaryote Giardia lamblia

Giardia lamblia is an early branching protist that possesses peripheral vacuoles (PVs) with characteristics of lysosome-like organelles, located underneath the plasma membrane. In more evolved cells, lysosomal protein trafficking is achieved by cargo recognition involving adaptor protein (AP) complexes that recognize specific amino acid sequences (tyrosine and/or dileucine motifs) within the cytoplasmic tail of membrane proteins. Previously, we reported that Giardia has a tyrosine-based sorting system, which mediates the targeting of a membrane-associated cysteine protease (encystation-specific cysteine protease, ESCP) to the PVs. Here, we show that Giardia AP1 mediates the transport of ESCP and the soluble acid phosphatase (AcPh) to the PVs. By using the yeast two-hybrid assay we found that the ESCP tyrosine-based motif interacts specifically with the medium subunit of AP1 (Gimicroa). Hemagglutinin-tagged Gimicroa colocalizes with ESCP and AcPh and coimmunoprecipitates with clathrin, suggesting that protein trafficking toward the PVs is clathrin-adaptin dependent. Targeted disruption of Gimicroa results in mislocalization of ESCP and AcPh but not of variant-specific surface proteins. Our results suggest that, unlike mammalian cells, only AP1 is involved in anterograde protein trafficking to the PVs in Giardia. Moreover, even though Giardia trophozoites lack a morphologically discernible Golgi apparatus, the presence of a clathrin-adaptor system suggests that this parasite possess a primitive secretory organelle capable of sorting proteins similar to that of more evolved cells.     

Fine structure of the biogenesis of Giardia lamblia encystation secretory vesicles

Synthesis, transport, and assembly of the extracellular cyst wall is the hallmark of Giardia lamblia encystation. Much is known of the biochemical pathways and their regulation. However, from a cell biology point of view, the biogenesis of the encystation specific vesicles (ESVs) that transport cyst wall proteins to the periphery of the cell is poorly understood. Therefore, we exploited a number of complementary ultrastructural approaches to test the hypothesis that the formation of ESVs utilizes a novel regulated secretory pathway. We analyzed parasites at different stages of encystation in vitro by electron microscopy of thin sections, freeze fracture replicas, and three-dimensional reconstruction from serial sections of cells fixed for cytochemical localization of the endoplasmic reticulum (ER) marker, glucose 6-phosphatase. We also used a stereological approach to determine the area occupied by the ER, clefts, ESVs, and cyst wall. Taken together, our kinetic data suggest that some ER cisternae first dilate to form clefts, which enlarge into the ESVs. Living non-encysting and early-encysting trophozoites were labeled around the periphery of both nuclei with C(6)-NBD-ceramide. At 18-21 h, outward migration of some ESVs frequently caused protrusions at the periphery of encysting trophozoites. The presence of lysosome-like peripheral vesicles between the ESV and plasma membrane of the cell was confirmed using acridine orange, an acidic compartment marker. Our data suggest that G. lamblia has a novel secretory pathway in which certain functions of the ER and Golgi co-localize spatially and temporally. These studies will increase understanding of the evolutionary appearance of regulated secretory pathways for assembly of a primitive extracellular matrix in an early diverging eukaryote.     

Giardia lamblia: detection and characterization of calmodulin

Calmodulin was detected in Giardia lamblia by radioimmunoassay and cyclic AMP phosphodiesterase activation. This protein was purified to apparent homogeneity by fast protein liquid chromatography with a yield of 260 ng of calmodulin/mg of protozoan protein. Purity was established by gel electrophoresis, gel filtration, and ion exchange chromatography. The parasite calmodulin has properties characteristic of calmodulin isolated from other eukaryotes, e.g., an apparent molecular weight of 16.7 kD; activation in calcium dependent manner of bovine heart cyclic nucleotide phosphodiesterase; and sensitivity to known calmodulin antagonists.