ABSTRACT
Proteolytic activity is important in the lifecycles of parasites and their interactions with hosts. Cysteine proteases have been best studied in Giardia, but other protease classes have been implicated in growth and/or differentiation. In this study, we employed bioinformatics to reveal the complete set of putative proteases in the Giardia genome. We identified 73 peptidase homologs distributed over 5 catalytic classes in the genome. Serial analysis of gene expression of the G. lamblia lifecycle found thirteen protease genes with significant transcriptional variation over the lifecycle, with only one serine protease transcript upregulated late in encystation. The translated gene sequence of this encystation-specific transcript was most similar to eukaryotic subtilisin-like proprotein convertases (SPC), although the typical catalytic triad was not identified. Epitope-tagged gSPC protein expressed in Giardia under its own promoter was upregulated during encystation with highest expression in cysts and it localized to encystation-specific secretory vesicles (ESV). Total gSPC from encysting cells produced proteolysis in gelatin gels that co-migrated with the epitope-tagged protease in immunoblots. Immuno-purified gSPC also had gelatinase activity. To test whether endogenous gSPC activity is involved in differentiation, trophozoites and cysts were exposed to the specific serine proteinase inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF). After 21 h encystation, a significant decrease in ESV was observed with 1mM AEBSF and by 42 h the number of cysts was significantly reduced, but trophozoite growth was not inhibited. Concurrently, levels of cyst wall proteins 1 and 2, and AU1-tagged gSPC protein itself were decreased. Excystation of G. muris cysts was also significantly reduced in the presence of AEBSF. These results support the idea that serine protease activity is essential for Giardia encystation and excystation.
Subject(s)
Giardia lamblia/enzymology , Giardia lamblia/growth & development , Proprotein Convertases/genetics , Proprotein Convertases/metabolism , Amino Acid Sequence , Computational Biology/methods , Electrophoresis , Gelatin/metabolism , Gene Expression Profiling , Giardia lamblia/genetics , Immunoblotting , Microscopy, Confocal , Microscopy, Fluorescence , Molecular Sequence Data , Protein Structure, Tertiary , Secretory Vesicles/chemistryABSTRACT
Earlier, we found that three protein disulfide isomerases (PDI) from Giardia lamblia (gPDI) also have transglutaminase (TGase) activity in vitro. We now show that differentiating Giardia cells contain isopeptide bonds (epsilon(gamma-glutamyl)lysine), the biological product of TGase activity that results in irreversible crosslinking of proteins in vivo. HPLC analyses showed the highest isopeptide bond content in cells encysting for 21 h, indicating an important role for TGase early in encystation. We were not able to detect isopeptide bonds in water-resistant cysts, possibly because they could not be extracted. One of the hallmarks of early encystation is the formation of encystation secretory vesicles (ESV) that transport nascent cyst wall proteins (CWPs) to the outer cell surface. ImmunoEM and live-cell immunofluorescence assays of encysting parasites revealed that gPDIs 1-3 are located in ESV and that gPDI-2 is also novel in that it is localized on the cell surface. Cystamine, a widely used TGase inhibitor, caused a dose-dependent inhibition of ESV formation by 21 h, thereby preventing development of trophozoites into cysts. Since cystamine (0.5-1 mM) inhibited the TGase activity of recombinant gPDIs 1-3 in vitro, PDIs appear to be the physiologic targets of cystamine. We found that when parasites were treated with cystamine, CWPs were not processed normally. These data suggest that TGase-catalyzed reactions may be needed for either the machinery that processes CWP precursors or their recruitment to ESV.
Subject(s)
Giardia lamblia/enzymology , Giardia lamblia/growth & development , Transglutaminases/metabolism , Animals , Base Sequence , Cystamine/pharmacology , DNA, Protozoan/genetics , Enzyme Inhibitors/pharmacology , Giardia lamblia/drug effects , Giardia lamblia/genetics , Microscopy, Immunoelectron , Protein Disulfide-Isomerases/genetics , Protein Disulfide-Isomerases/metabolism , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Transglutaminases/antagonists & inhibitors , Transglutaminases/geneticsABSTRACT
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.
Subject(s)
Giardia lamblia/ultrastructure , Life Cycle Stages , Secretory Vesicles/ultrastructure , Animals , Cell Compartmentation , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/ultrastructure , Freeze Fracturing , Giardia lamblia/cytology , Giardia lamblia/physiology , Golgi Apparatus/metabolism , Golgi Apparatus/ultrastructure , Microscopy, Electron , Protozoan Proteins/metabolismABSTRACT
A phylogenetic analysis of protein disulfide isomerase (PDI) domain evolution was performed with the inclusion of recently reported PDIs from the amitochondriate protist Giardia lamblia, yeast PDIs that contain a single thioredoxin-like domain, and PDIs from a diverse selection of protists. We additionally report and include two new giardial PDIs, each with a single thioredoxin-like domain. Inclusion of protist PDIs in our analyses revealed that the evolutionary history of the endoplasmic reticulum may not be simple. Phylogenetic analyses support common ancestry of all eukaryotic PDIs from a thioredoxin ancestor and independent duplications of thioredoxin-like domains within PDIs throughout eukaryote evolution. This was particularly evident for Acanthamoeba PDI, Dictyostelium PDI, and mammalian erp5 domains. In contrast, gene duplication, instead of domain duplication, produces PDI diversity in G. lamblia. Based on our results and the known diversity of PDIs, we present a new hypothesis that the five single-domain PDIs of G. lamblia may reflect an ancestral mechanism of protein folding in the eukaryotic endoplasmic reticulum. The PDI complement of G. lamblia and yeast suggests that a combination of PDIs may be used as a redox chain analogous to that known for bacterial Dsb proteins.
Subject(s)
Evolution, Molecular , Giardia lamblia/genetics , Protein Disulfide-Isomerases/genetics , Amino Acid Sequence , Animals , Binding Sites/genetics , DNA, Protozoan/chemistry , DNA, Protozoan/genetics , Molecular Sequence Data , Phylogeny , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino AcidABSTRACT
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 (t1/2 < 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.
Subject(s)
Giardia lamblia/metabolism , Protozoan Proteins/metabolism , Secretory Vesicles/metabolism , Animals , Cell Wall/drug effects , Cell Wall/metabolism , Dithiothreitol/pharmacology , Giardia lamblia/drug effects , Protein Disulfide-Isomerases/metabolism , Protein Transport/drug effects , Protozoan Proteins/drug effects , Secretory Vesicles/drug effectsABSTRACT
We identified a novel gene encoding molecular chaperone HSP70 in the amitochondriate parasite Giardia lamblia. The predicted protein is similar to bacterial DnaK and mitochondrial HSP70s. The gene is transcribed and translated at a constant level during trophozoite growth and encystation. Alignment of the sequence with a data set of cytosolic, endoplasmic reticulum (ER), mitochondrial, and DnaK HSP70 homologs indicated that the sequence was extremely divergent and contained insertions unique to giardial HSP70s. Phylogenetic analyses demonstrated that this sequence was distinct from the cytosolic and ER forms and was most similar to proteobacterial and mitochondrial DnaKs. However, a specific relationship with the alpha proteobacterial and mitochondrial sequences was not strongly supported by phylogenetic analyses of this data set, in contrast to similar analyses of cpn60. These data neither confirm nor reject the possibility that this gene is a relic of secondary mitochondrial loss; they leave open the possibility that it was acquired in a separate endosymbiotic event.
Subject(s)
Escherichia coli Proteins , Genes, Protozoan , Giardia lamblia/genetics , HSP70 Heat-Shock Proteins/genetics , Protozoan Proteins/genetics , Amino Acid Sequence , Animals , Blotting, Northern , Blotting, Western , Giardia lamblia/physiology , HSP70 Heat-Shock Proteins/classification , HSP70 Heat-Shock Proteins/metabolism , Hot Temperature , Humans , Molecular Sequence Data , Oxidative Stress , Oxygen/metabolism , Phylogeny , Protozoan Proteins/metabolism , RNA, Protozoan/genetics , RNA, Protozoan/metabolism , Sequence AlignmentABSTRACT
To begin to characterize the components of the 20S proteasome of Giardia lamblia, we have cloned a genomic sequence encoding an alpha-chain (type alpha3/C9, predicted size 244 amino acid residues). Southern analysis indicated that a single gene codes for this protein, and a Northern blot exhibited a single signal at 850 nt. An antiserum against a C-terminal fragment of the alpha-chain expressed in Escherichia coli reacted with a single protein band of Mr 27,000 that was present at constant levels in trophozoites and encysting cells. On a 2D blot of the purified 20S proteasome, we identified the cross-reacting component as a single protein of IEP 6.0, in agreement with the IEP predicted by the coding sequence. Our data confirm that the G. lamblia 20S proteasome is typically eukaryotic in containing a set of diverged alpha-subunits.
Subject(s)
Cysteine Endopeptidases/chemistry , Cysteine Endopeptidases/genetics , Giardia lamblia/genetics , Multienzyme Complexes/chemistry , Multienzyme Complexes/genetics , Amino Acid Sequence , Animals , Blotting, Southern , Cysteine Endopeptidases/metabolism , Giardia lamblia/enzymology , Molecular Sequence Data , Multienzyme Complexes/metabolism , Proteasome Endopeptidase Complex , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Transcription, GeneticABSTRACT
Giardia lamblia is one of the most important causes of waterborne diarrheal disease worldwide, and giardiasis is the most common protozoan infection of the human small intestine. Symptomatic infection is characterized by diarrhea, abdominal pain, and malabsorption, leading to malnutrition and weight loss, particularly in children. The pathogen resides strictly in the lumen of the small intestine, and infection is typically not accompanied by significant mucosal inflammation. Clinical and experimental studies indicate that B cell-dependent host defenses, particularly IgA, are important for controlling and clearing Giardia infection, although B cell-independent mechanisms also contribute to this outcome. In contrast to antigiardial host defenses, much less is known about the pathophysiological mechanisms underlying the clinical symptoms of giardiasis, partly because of the current lack of suitable model systems. In addition to being an important human enteric pathogen, Giardia is an interesting model organism for gaining basic insights into genetic innovations that led to evolution of eukaryotic cells, since it belongs to the earliest diverging eukaryotic lineage known. The completion of the giardial genome project will increase understanding of the basic biology of the protozoan and will help us to better understand host pathogen-interactions as a basis for developing new vaccination and therapeutic strategies.
Subject(s)
Giardia lamblia/metabolism , Giardiasis/metabolism , Host-Parasite Interactions/immunology , Intestinal Mucosa/parasitology , Toxins, Biological/metabolism , Animals , Diarrhea/immunology , Diarrhea/metabolism , Diarrhea/parasitology , Giardiasis/immunology , Humans , Intestinal Mucosa/immunology , Intestinal Mucosa/metabolismABSTRACT
Since little is known of how the primitive protozoan parasite, Giardia lamblia, senses and responds to its changing environment, we characterized a giardial protein kinase A (gPKA) catalytic subunit with unusual subcellular localization. Sequence analysis of the 1080-base pair open reading frame shows 48% amino acid identity with the cyclic AMP-dependent kinase from Euglena gracilis. Northern analysis indicated a 1.28- kilobase pair transcript at relatively constant concentrations during growth and encystation. gPKA is autophosphorylated, although amino acid residues corresponding to Thr-197 and Ser-338 of human protein kinase A (PKA) that are important for autophosphorylation are absent. Kinetic analysis of the recombinant PKA showed that ATP and magnesium are preferred over GTP and manganese. Kinase activity of the native PKA has also been detected in crude extracts using kemptide as a substrate. A myristoylated PKA inhibitor, amide 14-22, inhibited excystation with an IC(50) of 3 microm, suggesting an important role of gPKA during differentiation from the dormant cyst form into the active trophozoite. gPKA localizes independently of cell density to the eight flagellar basal bodies between the two nuclei together with centrin, a basal body/centrosome-specific protein. However, localization of gPKA to marginal plates along the intracellular portions of the anterior and caudal pairs of flagella was evident only at low cell density and higher endogenous cAMP concentrations or after refeeding with fresh medium. These data suggest an important role of PKA in trophozoite motility during vegetative growth and the cellular activation of excystation.
Subject(s)
Cyclic AMP-Dependent Protein Kinases/chemistry , Cyclic AMP-Dependent Protein Kinases/physiology , Giardia lamblia/enzymology , Movement/physiology , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Amino Acids/metabolism , Animals , Blotting, Northern , Blotting, Southern , Blotting, Western , Catalysis , Cell Differentiation , Centrosome/metabolism , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , DNA, Complementary/metabolism , Flagella/metabolism , Gene Deletion , Gene Library , Guanosine Triphosphate/metabolism , Inhibitory Concentration 50 , Kinetics , Microscopy, Fluorescence , Molecular Sequence Data , Myristic Acids/metabolism , Oligopeptides/pharmacology , Open Reading Frames , Phosphorylation , Protein Isoforms , RNA, Messenger/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Signal Transduction , Time Factors , Transcription, GeneticABSTRACT
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.
Subject(s)
Databases, Factual , Genome, Protozoan , Giardia/genetics , AnimalsABSTRACT
Giardia lamblia infection of the human small intestine is a common protozoan cause of diarrheal disease worldwide. Although infection is luminal and generally self-limiting, and secretory Abs are thought to be important in host defense, other defense mechanisms probably affect the duration of infection and the severity of symptoms. Because intestinal epithelial cells produce NO, and its stable end products, nitrite and nitrate, are detectable mainly on the apical side, we tested the hypothesis that NO production may constitute a host defense against G. lamblia. Several NO donors, but not their control compounds, inhibited giardial growth without affecting viability, suggesting that NO is cytostatic rather than cytotoxic for G. lamblia. NO donors also inhibited giardial differentiation induced by modeling crucial environmental factors, i. e., encystation induced by bile and alkaline pH, and excystation in response to gastric pH followed by alkaline pH and protease. Despite the potent antigiardial activity of NO, G. lamblia is not simply a passive target for host-produced NO, but has strategies to evade this potential host defense. Thus, in models of human intestinal epithelium, G. lamblia inhibited epithelial NO production by consuming arginine, the crucial substrate used by epithelial NO synthase to form NO. These studies define NO and arginine as central components in a novel cross-talk between a luminal pathogen and host intestinal epithelium.
Subject(s)
Antiprotozoal Agents/pharmacology , Arginine/metabolism , Giardia lamblia/pathogenicity , Giardiasis/prevention & control , Growth Inhibitors/physiology , Intestinal Mucosa/metabolism , Intestinal Mucosa/parasitology , Nitric Oxide/biosynthesis , Animals , Binding, Competitive , Caco-2 Cells/enzymology , Caco-2 Cells/metabolism , Caco-2 Cells/parasitology , Cell Communication/immunology , Cell Polarity/immunology , Giardia lamblia/drug effects , Giardia lamblia/growth & development , Giardia lamblia/immunology , Giardiasis/immunology , Giardiasis/parasitology , Glutathione/analogs & derivatives , Glutathione/pharmacology , Growth Inhibitors/biosynthesis , HT29 Cells/enzymology , HT29 Cells/metabolism , HT29 Cells/parasitology , Humans , Intestinal Mucosa/enzymology , Nitric Oxide/antagonists & inhibitors , Nitric Oxide/metabolism , Nitric Oxide/physiology , Nitric Oxide Donors/pharmacology , Nitric Oxide Synthase/biosynthesis , Nitric Oxide Synthase Type II , Nitroso Compounds/pharmacology , S-NitrosoglutathioneABSTRACT
Giardia lamblia must encyst to survive in the environment and subsequently infect new hosts. We investigated the expression of glucosamine-6-phosphate isomerase (Gln6PI), the first enzyme required for biosynthesis of N-acetylgalactosamine, for the major cyst wall polysaccharide. We isolated two Gln6PI genes that encode proteins with large areas of identity, but distinctive central and terminal regions. Both recombinant enzymes have comparable kinetics. Interestingly, these genes have distinct patterns of expression. Gln6PI-A has a conventional, short 5' untranslated region (UTR), and is expressed at a low level during vegetative growth and encystation. The Gln6PI-B gene has two transcripts - one is expressed constitutively and the second species is highly upregulated during encystation. The non-regulated Gln6PI-B transcript has the longest 5'-UTR known for Giardia and is 5' capped or blocked. In contrast, the Gln6PI-B upregulated transcript has a short, non-capped 5'-UTR. A small promoter region (< 56 bp upstream from the start codon) is sufficient for the regulated expression of Gln6PI-B. Gln6PI-B also has an antisense overlapping transcript that is expressed constitutively. A shorter antisense transcript is detected during encystation. This is the first report of a developmentally regulated promoter in Giardia, as well as evidence for a potential role of 5' RNA processing and antisense RNA in differential gene regulation.
Subject(s)
5' Untranslated Regions/genetics , Gene Expression Regulation , Giardia lamblia/growth & development , Giardia lamblia/genetics , Promoter Regions, Genetic/genetics , RNA Processing, Post-Transcriptional , Aldose-Ketose Isomerases/genetics , Aldose-Ketose Isomerases/metabolism , Amino Acid Sequence , Animals , Base Sequence , Chromosome Mapping , Genes, Protozoan , Giardia lamblia/enzymology , Molecular Sequence Data , RNA Caps/metabolism , RNA, Antisense/metabolism , RNA, Messenger/metabolism , Recombinant Proteins/metabolism , Sequence Analysis, DNA , Transcription, GeneticABSTRACT
Protein-disulfide isomerase is essential for formation and reshuffling of disulfide bonds during nascent protein folding in the endoplasmic reticulum. The two thioredoxin-like active sites catalyze a variety of thiol-disulfide exchange reactions. We have characterized three novel protein-disulfide isomerases from the primitive eukaryote Giardia lamblia. Unlike other protein-disulfide isomerases, the giardial enzymes have only one active site. The active-site sequence motif in the giardial proteins (CGHC) is characteristic of eukaryotic protein-disulfide isomerases, and not other members of the thioredoxin superfamily that have one active site, such as thioredoxin and Dsb proteins from Gram-negative bacteria. The three giardial proteins have very different amino acid sequences and molecular masses (26, 50, and 13 kDa). All three enzymes were capable of rearranging disulfide bonds, and giardial protein-disulfide isomerase-2 also displayed oxidant and reductant activities. Surprisingly, the three giardial proteins also had Ca(2+)-dependent transglutaminase activity. This is the first report of protein-disulfide isomerases with a single active site that have diverse roles in protein cross-linking. This study may provide clues to the evolution of key functions of the endoplasmic reticulum in eukaryotic cells, protein disulfide formation, and isomerization.
Subject(s)
Giardia lamblia/enzymology , Protein Disulfide-Isomerases/genetics , Amino Acid Sequence , Animals , Base Sequence , DNA, Protozoan , Endoplasmic Reticulum/enzymology , Endoplasmic Reticulum/ultrastructure , Microscopy, Electron , Molecular Sequence Data , Protein Disulfide-Isomerases/metabolism , Sequence Homology, Amino Acid , Transglutaminases/metabolismABSTRACT
The molecular mechanisms for targeting and translocation of secreted proteins are highly conserved from bacteria to mammalian cells, although the machinery is more complex in higher eukaryotes. To investigate protein transport in the early-diverging eukaryote, Giardia lamblia, we cloned the gene encoding the alpha subunit (SRalpha) of the signal recognition particle (SRP) receptor. SRalpha is a small GTPase that functions in SRP-ribosome targeting to the ER. Sequence and phylogenetic analyses showed that SRalpha from G. lamblia is most homologous to SRalpha proteins from higher eukaryotes, although it lacks some conserved motifs. Specifically, giardial SRalpha has an N-terminal extension that enables SRalpha of higher eukaryotes to interact with a beta subunit that anchors it in the ER membrane. While the C-terminal regions are similar, giardial SRalpha lacks a prominent 13 amino acid regulatory loop that is characteristic of higher eukaryotic versions. Thus, giardial SRalpha resembles that of higher eukaryotes, but likely diverged before the advent of the regulatory loop. The 1.8 kb SRalpha transcript has extremely short untranslated regions (UTRs): a 1-2 nt 5'- and a 9 nt 3' UTR with the polyadenylation signal overlapping with the stop codon. RT-PCR, Northern and Western analyses showed that SRalpha is present at relatively constant levels during vegetative growth and encystation, even though there are extensive changes in endomembrane structures and secretory activity during encystation. Imnuno-EM showed that SRalpha localizes to ER-like structures, strengthening the observation of a typical ER in G. lamlia. Unexpectedly, SRalpha was also found in the lysosome-like peripheral vacuoles, suggesting unusual protein traffic in this early eukaryote. Our results indicate that the eukaryotic type of cotranslational transport appeared early in the evolution of the eukaryotic cell.
Subject(s)
Evolution, Molecular , GTP Phosphohydrolases/genetics , Giardia lamblia/genetics , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Peptide/genetics , Signal Recognition Particle , Amino Acid Sequence , Animals , Base Sequence , Biological Transport , Cell Compartmentation , Cell Differentiation , Cloning, Molecular , Endoplasmic Reticulum/chemistry , Eukaryotic Cells , Giardia lamblia/cytology , Lysosomes/chemistry , Molecular Sequence Data , Protein Biosynthesis , Protein Conformation , Protein Processing, Post-Translational , Protozoan Proteins/metabolism , Sequence Analysis , Sequence Homology, Amino AcidABSTRACT
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.
Subject(s)
Genome, Protozoan , Giardia lamblia/genetics , Sequence Analysis, DNA , Animals , Cosmids/genetics , Evolution, Molecular , Gene Library , Genes, Protozoan , Molecular Sequence Data , Phosphoric Monoester Hydrolases/genetics , Phosphotransferases/genetics , Protein Processing, Post-Translational , Protozoan Proteins/genetics , Protozoan Proteins/metabolismABSTRACT
Excystation of Giardia lamblia entails differentiation of dormant cysts into parasitic trophozoites. Despite its importance for infection, this transformation is not understood at the cellular or molecular levels. In these studies, we report that excystation entails detection of environmental stimuli across the tough extracellular cyst wall leading to highly coordinated physiological, structural, and molecular responses. We found that novel cytoplasmic rearrangements and changes in individual species of mRNA and in cytoplasmic pH occur within the cyst wall in the earliest stage of excystation, in response to conditions modeling cyst ingestion and passage into the human stomach. This suggests that cysts do not contain all the mRNA needed for excystation and emergence and supports our hypothesis that external stimuli, including hydrogen ions, may penetrate or be perceived across the cyst wall. In contrast, changes in cyst wall structure or proteins were detected only later in excystation, in the stage that models passage into the human small intestine, where trophozoites can emerge and survive. These findings show that excystation of G. lamblia is a highly complex and active process and provide important insights into its cellular and molecular components.
Subject(s)
Giardia lamblia/physiology , Transcription, Genetic , Animals , Blotting, Western , Giardia lamblia/genetics , Giardia lamblia/ultrastructure , Glycoproteins/chemistry , Glycoproteins/metabolism , Humans , Hydrogen-Ion Concentration , Intestine, Small/chemistry , Microscopy, Electron , Protozoan Proteins/chemistry , Protozoan Proteins/metabolism , RNA, Messenger/metabolism , Stomach/chemistry , Up-RegulationABSTRACT
Diplomonads, parabasalids, as represented by trichomonads, and microsporidia are three protist lineages lacking mitochondria that branch earlier than all other eukaryotes in small subunit rRNA and elongation factor phylogenies. The absence of mitochondria and plastids in these organisms suggested that they diverged before the origin of these organelles. However, recent discoveries of mitochondrial-like heat shock protein 70 and/or chaperonin 60 (cpn60) genes in trichomonads and microsporidia imply that the ancestors of these two groups once harbored mitochondria or their endosymbiotic progenitors. In this report, we describe a mitochondrial-like cpn60 homolog from the diplomonad parasite Giardia lamblia. Northern and Western blots reveal that the expression of cpn60 is independent of cellular stress and, except during excystation, occurs throughout the G. lamblia life cycle. Phylogenetic analyses position the G. lamblia cpn60 in a clade that includes mitochondrial and hydrogenosomal cpn60 proteins. The most parsimonious interpretation of these data is that the cpn60 gene was transferred from the endosymbiotic ancestors of mitochondria to the nucleus early in eukaryotic evolution, before the divergence of the diplomonads and trichomonads from other extant eukaryotic lineages. A more complicated explanation requires that these genes originated from distinct alpha-proteobacterial endosymbioses that formed transiently within these protist lineages.
Subject(s)
Chaperonin 60/genetics , Giardia lamblia/genetics , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , Evolution, Molecular , Mitochondria , Molecular Sequence Data , Phylogeny , SymbiosisABSTRACT
Encystation of Giardia lamblia is required for survival outside the host, whereas excystation initiates infection. The dormant cyst was considered an adaptation to external survival and passage through the stomach. However, we found previously that trophozoites which had recovered after completion of the life cycle had switched their major variant surface protein (VSP), called TSA 417, but neither the timing nor the molecular mechanism of switching had been elucidated. Here we demonstrate that TSA 417 predominates in cysts, but is downregulated during the stage of excystation that models cyst arrival in the small intestine. Transcripts of new VSPs appear late in encystation, and during and after excystation. Trophozoites appear to prepare for switching during encystation, when the major VSP on the cell surface diminishes and is internalized in lysosome-like vacuoles. As short-range DNA rearrangements were not detected, giardial VSP switching during differentiation appears to resemble the in situ switching of surface glycoproteins in African trypanosomes. We also report a unique extended 15 nucleotide polyadenylation signal in all VSP transcripts, but not in other known giardial genes. Antigenic variation during encystation-excystation may be a novel form of immune evasion that could help explain the common occurrence of reinfection by Giardia and other parasites with similar life cycles.
Subject(s)
Antigenic Variation , Antigens, Protozoan/genetics , Antigens, Surface/genetics , Giardia lamblia/genetics , Giardia lamblia/immunology , Protozoan Proteins , Animals , Cell Compartmentation , DNA, Protozoan , Eukaryotic Cells , Gene Rearrangement , Lysosomes , RNA, ProtozoanABSTRACT
Giardia lamblia trophozoites colonize the human small intestine, where they are exposed to high concentrations of conjugated bile acids. Previous work has shown that bile acids enhance trophozoite survival, multiplication, and differentiation into the cyst stage. Therefore, experiments were performed to test whether carrier-mediated uptake of conjugated bile acids is present in this primitive parasite. Uptake of both cholyltaurine (C-tau) and cholylglycine (C-gly) was increased manyfold after culturing trophozoites in medium lacking bile acids. Absence of uptake at 4 degrees C and inhibition by other conjugated bile acids provided additional evidence for carrier-mediated uptake. Uptake of C-tau was greater than that of C-gly under all experimental conditions and appeared to be mediated by a different carrier. The major evidence for different carriers is that C-tau uptake was Na(+)-dependent, while C-gly uptake was not. In addition, C-tau uptake was more strongly inhibited by DTNB and several organic anions than C-gly uptake. Radiolabeled C-tau and C-gly were each released rapidly from trophozoites at 37 degrees C but not at 4 degrees C, suggesting that release of conjugated bile acids was also carrier-mediated. These findings are consistent with the notion that multiple transporters for conjugated bile acids are present in a lower eukaryote. We speculate that intracellular bile acids may facilitate lipid trafficking and membrane biosynthesis.
Subject(s)
Bile Acids and Salts/metabolism , Giardia lamblia/metabolism , Animals , Biological Transport , Cattle , Dithionitrobenzoic Acid/pharmacology , Dithiothreitol/pharmacology , Giardia lamblia/drug effects , Glycocholic Acid/metabolism , Humans , Sulfhydryl Reagents/pharmacology , Taurocholic Acid/metabolismABSTRACT
Lactoferrin and its derived N-terminal peptide may be important host defenses against Giardia lamblia. We showed earlier that lactoferrin and the derived peptides have potent giardicidal activity in vitro. Using indirect immunofluorescence, we now demonstrate binding of lactoferrin and the peptides to the live trophozoite surface. Iron strongly inhibited binding of lactoferrin, and decreased binding of the peptides, while certain divalent metal ions decreased binding of all forms by about half. Lactoferrin and the peptides caused striking and complex morphologic changes in the trophozoite plasmalemma, endomembranes and cytoskeleton, and increased the electron density of the lysosome-like peripheral vacuoles.