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At least 19 recordsLinked to original sources
PubMed1998

Episomal and integrated maintenance of foreign DNA in Giardia lamblia.

Giardia lamblia is an early diverging eukaryote which causes gastrointestinal disease throughout the world. Different subgroups of Giardia have been defined based on several biochemical and genetic criteria. We have developed a method for stably introducing DNA into the nuclei of the parasite using puromycin acetyltransferase (pac) as a dominant selectable marker. Transfected circular DNAs were maintained as episomes in the isolate WB, a representative of one Giardia subgroup. When input DNAs were linearized, integration was observed to occur by homologous recombination producing gene replacements in this isolate. In isolate GS, which represents a different subgroup, both linear and circular transfected DNAs were integrated into the genome by homologous recombination. In GS, linear DNA again produced gene replacements, while circular DNA produced duplicative integration events. The failure of GS to replicate episomes may reflect differences in the structure or recognition of DNA replication origins between these subgroups. A plasmid shuttle vector was also developed for expression of other genes in Giardia lamblia. Utilizing the green fluorescent protein as a reporter gene in the WB isolate, we show that gene expression from this vector correlated with plasmid copy number over a range of two orders of magnitude. Together these tools should greatly enhance our ability to study both the basic biology and the pathogenesis of this ubiquitous parasite.

Acetyltransferases↗
PubMed1990

Frequency of variant antigens in Giardia lamblia.

Giardia lamblia undergoes antigenic variation. The rate of antigenic variation and the size of the variant antigen repertoire were estimated in clones of Giardia lamblia which reexpresses surface variant antigens that are characteristics of its parent. Calculations were based on determinations of the number of trophozoites expressing defined or nondefined epitopes as well as the total number of trophozoites in newly established clones. The rate of appearance of variant antigens containing defined epitopes was expressed as the number of generations until the first trophozoite expressing a defined epitope appeared. In clones of isolate WB, tested because their major surface variant antigens were largely nondefined, variants expressing epitopes recognized by Mabs 6E7 or 3F6 appeared after approximately 12 generations. Variants expressing epitopes recognized by Mab 5C1 appeared at about 13 generations, significantly greater than for the other epitopes. The rate of antigenic variation was studied in another isolate, GS/M, whose surface epitope repertoire differs from that of isolate WB. A single epitope recognized by Mab G10/4 was tested. Trophozoites reexpressing this epitope first appeared after about 6.5 generations, significantly less than in WB. Therefore, the single epitope studied in isolate GS/M is reexpressed much more frequently than those of WB. In isolate WB, the epitopes recognized by Mab 6E7 and 3F6 tended to appear at the same time. The median number of variant antigens in WB was estimated to lie between 20.5 and 184.

Analysis of Variance↗
PubMed1992

Isolation and characterization of a NADP-dependent glutamate dehydrogenase gene from the primitive eucaryote Giardia lamblia.

Giardia lamblia is believed to be the earliest branching derivative from the eucaryotic lineage. Genomic and cDNA clones encoding the giardia NADP-dependent glutamate dehydrogenase have been isolated and characterized. Southern hydridization using genomic DNA indicates that the gene encoding this activity is unique and single copy. Primer extension, S1 nuclease protection, and genomic and cDNA sequence analysis demonstrate that gene transcripts are initiated within a conserved AT-rich sequence element immediately preceding the ATG translation initiation codon and the short 5' untranslated region is not extended by transsplicing. The open reading frame is 1350 nucleotides in length and encodes a protein of 449 amino acids. The reading frame is not interrupted by introns and the primary transcript is probably not subjected to RNA editing. In the strictly anaerobic metabolism of giardia, NADP-dependent glutamate dehydrogenase activity participates along with alanine aminotransferase, in the cyclic dissipation of reducing equivalents (NADPH) through the conversion of pyruvate to alanine. The deduced amino acid sequence of the giardia protein exhibits substantial homology to numerous fungal and eubacterial NADP-dependent glutamate dehydrogenases. Comparisons of alignment gap positions and amino acid identities indicate that the giardia sequence is at least as similar or more similar to the eubacterial sequence than it is to the fungal sequence. This supports the hypothesis that giardia diverged very early from the eucaryotic lineage.

Amino Acid Sequence↗
PubMedDate not supplied

Evidence for adhesive activity of the ventrolateral flange in Giardia lamblia.

Giardia lamblia is an intestinal protozoan that inhabits the intestinal tract of man and other mammals by attaching to the mucosal surface via the contractile activity of an attachment organelle called the ventral adhesive disk. We have investigated the presence of other attachment mechanisms in G. lamblia trophozoites by using microfabricated substrates that sterically interfere with formation of the hypothesized "negative pressure" under the ventral adhesive disk that would mediate attachment to a substratum. Pillars measuring 1 microm high and 2 microm in diam. were constructed in microarrays with spacings smaller than the diameter of the ventral adhesive disk. Using high resolution field emission scanning electron microscopy, the attachment of trophozoites to the tops of pillars in the microfabricated substrates was investigated. Firm adhesion of trophozoites was observed to be mediated by direct attachment of the ventrolateral flange membrane to the tops of microfabricated pillars. Attachment to microfabricated surfaces was 16% of that observed for attachment mediated by the ventral adhesive disk (4.4 +/- 1.5 cells/100 micro2 micropillar surface vs. 25.9 +/- 3.1 cells/100 micro2 flat substrate, p < 0.0001) This is the first report of trophozoite adhesion to a substratum by a mechanism other than the direct attachment of the ventral adhesive disk, and provides experimental evidence that the ventrolateral flange may play a role in trophozoite adhesion. A hypothesis is presented describing how the adhesive nature of the ventrolateral flange might be involved in normal attachment of G. lamblia trophozoites to a substratum.

Animals↗
PubMed1992

Human giardiasis: correlation of specific secretory IgA levels in duodenal fluid to the severity of disease and infestation by Giardia lamblia.

Giardia lamblia specific secretory immunoglobulin A (sIgA) levels in the duodenal fluid of adult giardiasis cases are reported for the first time. The sIgA levels in the study group were found to be significantly higher (p less than 0.01) than in the 20 age- and sex-matched controls comprising cases classified as non-ulcerative dyspepsia who did nor reveal any G. lamblia in their stools and the duodenal fluid. An inverse relationship between the clinical severity of giardiasis and the level of sIgA in the duodenal fluid was noted. Cases with a higher trophozoite load in duodenal aspirate tended to be associated with envanescent G. lamblia-specific antibodies.

Adult↗
PubMed2002

A novel Myb-related protein involved in transcriptional activation of encystation genes in Giardia lamblia.

Giardia lamblia is an important human intestinal parasite that survives outside of the host by differentiation of trophozoites into infectious cysts. Transcriptional regulation is key for encystation gene expression, but the mechanisms are unknown. Giardia genome database searches identified a myb-like gene (gmyb2) whose expression increased during encystation. Epitope-tagged gMyb2 localized to both nuclei. DNA binding and mutation analysis showed that gMyb2 binds specifically to C(T/A)ACAG, a c-Myb-like target sequence in the promoters of encystation-induced genes encoding gMyb2, three cyst wall proteins and G6PI-B, a key enzyme in cyst wall polysaccharide biosynthesis. gMyb2 binding sites were not found in the upstream regions of 31 other giardial genes. Deletion of the putative gMyb2 binding site greatly reduced encystation-specific promoter activity of g6pi-b. Fusion of gMyb2 binding sites to the constitutive ran promoter or g6pi-b promoter deletion lacking the gMyb2 binding site in-duced encystation-specific expression. gMyb2 may play an important role in transcriptional regulation of encystation genes, and may help co-ordinate synthesis of cyst wall proteins and polysaccharide. gMyb2 is the first giardial transcription factor to be functionally identified and the first that is associated with upregulation of encystation genes. This work provides a model for study of differential gene regulation in early diverging eukaryotic organisms.

5' Flanking Region↗
PubMed1996

Purification and characterization of the acetate forming enzyme, acetyl-CoA synthetase (ADP-forming) from the amitochondriate protist, Giardia lamblia.

Giardia lamblia, an amitochondriate eukaryote, contains acetyl-CoA synthetase (ADP-forming), an enzyme known only from one other eukaryote (Entamoeba histolytica) and a few anaerobic prokaryotes. The enzyme has been purified about 350-fold. The activity in the direction of acetate formation was dependent on ADP and inorganic phosphate. The reverse reaction could not be detected. Succinyl-CoA, propionyl-CoA and dADP were utilized with lower efficiency. The enzyme did not utilize AMP plus PPi thus differs from the broadly distributed acetyl-CoA synthetase (AMP-forming). The enzyme is responsible for acetate production accompanied by ATP generation, thus plays an important role in G. lamblia metabolism.

Acetate-CoA Ligase↗
PubMed2000

Development of a tetracycline controlled gene expression system in the parasitic protozoan Giardia lamblia.

Giardia lamblia is a very common intestinal protozoan pathogen of humans. Recent development of gene transfection systems in G. lamblia has allowed constitutive expression of selected genes in the organism. To extend the uses of DNA transfection in G. lamblia, an inducible gene expression system was developed by integrating the bacterial tet operator-repressor elements into an episomal DNA transfection vector. Tetracycline-responsive promoters with insertions of multiple tet operator sequences in the vicinity of a synthetic ran promoter were tested for their inducibility of a luciferase reporter gene expression. Stable cell lines transfected with individual plasmid constructs were established under drug selection. By assaying luciferase activity in transfected cells in response to tetracycline, an inducible promoter with insertion of two tet operators downstream of the adjacent synthetic ran promoter was found to confer a 10-fold inducibility in gene expression with co-expression of the tet-repressor driven by a gdh promoter. To further improve its inducibility, several other synthetic promoter contexts were also tested to increase expression of the tet-repressor gene. An optimal inducibility of 50-fold was obtained when a synthetic alpha-giardin promoter was used. Fine tuning of luciferase expression was achieved by adjusting the concentration of tetracycline and duration of drug exposure. The inducible gene expression system provides us an easy way to manipulate the level of gene expression in G. lamblia in a controllable manner that could not previously be achieved.

Animals↗
PubMed1994

Presence and cellular distribution of a 60-kDa protein related to mitochondrial hsp60 in Giardia lamblia.

Giardia lamblia trophozoites contain a 60-kDa protein recognized in immunoblots by antibody to mammalian hsp60, a protein localized in mitochondria in eukaryotic cells. The cellular distribution of this protein is evaluated by immunofluorescence microscopy using monoclonal antibody to human hsp60, polyclonal antibody to rodent hsp60, and 2 monoclonal antibodies to mycobacterial 65-kDa antigen, a prokaryotic hsp60 homolog. All of these antibodies, except 1, which is specific for prokaryotic hsp60, give a punctate labeling pattern throughout the cytoplasm, indicating that the 60-kDa protein is concentrated at discrete sites in the cytoplasm. The polyclonal hsp60 antiserum reveals additional punctate labeling colocalized on axonemes of the anterior flagella. Postembedment immunogold labeling and electron microscopy confirm that the antigen is clustered at foci in the cytoplasm but show no evidence of association with a membranous organelle. The hsp60 reactivity is also observed on anterior axonemes within the cytoplasm and on the adhesive disc. Hoechst 33258 DNA staining as well as electron microscopy give no evidence of endosymbionts, so a false positive due to a prokaryotic hsp60 homolog is unlikely. The presence of an hsp60-related protein in G. lamblia raises interesting questions concerning its origin.

Animals↗
PubMed1994

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.

Animals↗
PubMed2004

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.

Acid Phosphatase↗
PubMed2003

The cytoskeleton of Giardia lamblia.

Giardia lamblia is a ubiquitous intestinal pathogen of mammals. Evolutionary studies have also defined it as a member of one of the earliest diverging eukaryotic lineages that we are able to cultivate and study in the laboratory. Despite early recognition of its striking structure resembling a half pear endowed with eight flagella and a unique ventral disk, a molecular understanding of the cytoskeleton of Giardia has been slow to emerge. Perhaps most importantly, although the association of Giardia with diarrhoeal disease has been known for several hundred years, little is known of the mechanism by which Giardia exacts such a toll on its host. What is clear, however, is that the flagella and disk are essential for parasite motility and attachment to host intestinal epithelial cells. Because peristaltic flow expels intestinal contents, attachment is necessary for parasites to remain in the small intestine and cause diarrhoea, underscoring the essential role of the cytoskeleton in virulence. This review presents current day knowledge of the cytoskeleton, focusing on its role in motility and attachment. As the advent of new molecular technologies in Giardia sets the stage for a renewed focus on the cytoskeleton and its role in Giardia virulence, we discuss future research directions in cytoskeletal function and regulation.

Animals↗
PubMed1996

Amplification, expression, and packaging of a foreign gene by giardiavirus in Giardia lamblia.

Giardia lamblia is an intestinal protozoan parasite and one of the earliest eukaryotic divergents. The trophozoite multiplies via asexual binary fission and lacks all natural means of lateral gene transfer. A system is developed here for long-term expression of a foreign gene in this organism by exploiting recombinant virions derived from the giardiavirus (GLV), a double-stranded RNA virus that infects many Giardia isolates. An in vitro transcript of the cloned GLV cDNA, comprising the firefly luciferase-encoding region flanked by 5' and 3' fragments of GLV positive-strand RNA, was electroporated into GLV-infected trophozoites. Luciferase activity in electroporated cells peaked on day 2 at levels 6 orders of magnitude above background. Expression of this foreign gene remained at 80% of its peak level after 30 days in the absence of selective pressure. The chimeric RNA was replicated as double-stranded RNA and packaged into virus-like particles. The recombinant virions were partially purified from the wild-type helper virus by CsCl equilibrium density-gradient centrifugation and used to superinfect Giardia trophozoites. At multiplicities of infection of 100 or higher, these chimeric virions were able to initiate new rounds of expression of luciferase activity in the superinfected cells. Thus, the engineered virion can be successfully used to introduce and efficiently express a heterologous gene in this eukaryotic microorganism.

Animals↗
PubMed2002

The pivotal role of guanine phosphoribosyltransferase in purine salvage by Giardia lamblia.

Giardia lamblia is an anaerobic binucleate flagellated protozoan known to lack de novo synthesis of purine nucleotides. Our previous metabolic studies indicated two major parallel pathways mediated by adenine phosphoribosyltransferase (APRT) and guanine phosphoribosyltransferase (GPRT) that constitute the primary route of purine salvage in this organism. To verify further that these enzymes play a pivotal role in replenishing the purine ribonucleotide pool and are required for replicative growth of Giardia, a knock-out of GPRT gene expression in this organism was attempted. A hammerhead ribozyme flanked by two arms of GPRT antisense RNA (GPRZ) was designed, synthesized and found capable of cleaving a GPRT mRNA fragment in vitro at the designated site. GPRZ cDNA was then cloned into a viral vector pC631pac, derived from the genome of giardiavirus (GLV), and its transcript was introduced into GLV-infected Giardia trophozoites by electroporation. Stable transformants selected under increasing concentrations of puromycin displayed parallel increases in ribozyme levels and associated decreases in GPRT mRNA levels, GPRT enzyme activity and replicative cell growth to less than 10-20% of wild-type levels. Metabolite analyses showed specific depletion of the guanine ribonucleotide pools in parallel with slower cell growth. We conclude that GPRT plays an essential role in supplying guanine nucleotides required for growth and multiplication of Giardia, emphasizing its potential as a bona fide target for antigiardiasis chemotherapy.

Animals↗
PubMed1996

Cell biology of the primitive eukaryote Giardia lamblia.

Giardia lamblia is an extremely primitive or early-diverging eukaryote that has been considered to have no typical ER or Golgi apparatus, although it is a complex and highly developed cell. Both the trophozoite and cyst have unusual surface proteins that enable these stages to survive in very different and hostile environments. We found that G. lamblia forms novel encystation-specific secretory vesicles and can sort cyst wall proteins to a regulated secretory pathway distinct from the constitutive pathway used to transport the variable cysteine-rich protein to the trophozoite surface. Our studies, utilizing novel ultrastructural methods that preserve the endomembranes, as well as IEM, support the idea that G. lamblia has many of the endomembrane protein transport elements and sorting functions of higher cells and that these appeared very early in the evolution of eukaryotic cells.

Animals↗
PubMed1995

Virus-mediated expression of firefly luciferase in the parasitic protozoan Giardia lamblia.

Giardia lamblia, a prevalent human pathogen and one of the lineages that branched earliest from prokaryotes, can be infected with a double-stranded RNA virus, giardiavirus (GLV). The 6,277-bp viral genome has been previously cloned (A.L. Wang, H.-M. Yang, K.A. Shen, and C.C. Wang, Proc. Natl. Acad. Sci. USA 90:8595-8599, 1993; C.-H. Wu, C.C. Wang, H.M. Yang, and A.L. Wang, Gene, in press) and was converted to a transfection vector for G. lamblia in the present study. By flanking the firefly luciferase gene with the 5' and 3' untranslated regions (UTRs) of the GLV genome, transcript of the construct was synthesized in vitro with T7 polymerase and used to transfect G. lamblia WB trophozoites already infected with GLV (WBI). Optimal electroporation conditions used for the transfection were set at 1,000 V/cm and 500 microF, which resulted in expression of significant luciferase activity up to 120 h after electroporation. Furthermore, the mRNA and the antisense RNA of the luciferase gene were both detected by reverse transcription and PCR from 6 to 120 h postelectroporation, whereas no antisense RNA of luciferase was observed in the electroporated virus-free Giardia WB trophozoites. The mRNA of luciferase was detectable in the virus-free trophozoites by reverse transcription and PCR only up to 20 h after the electroporation, indicating that the introduced mRNA was replicated only by the viral RNA-dependent RNA polymerase inside the WBI cells. This expression of luciferase was dependent on the presence of UTRs on both ends of the viral genome transcript, including a putative packaging site that was apparently indispensable for luciferase expression. This is the first time that a viral vector in the form of mRNA URTs has been successfully used in transfecting a protozoan.

Animals↗
PubMed2003

Tubulin diversity in trophozoites of Giardia lamblia.

Giardia lamblia is a diplomonad that parasitizes the small intestine of vertebrates. The trophozoite is multiflagellar and its cytoskeleton presents a complex organization of microtubular structures. One of these, the adhesive disk, consists of a microtubular spiral. The median body, whose function is not yet determined, is also composed by microtubules. The cell has eight flagella and two microtubule sheets, known as the funis. In this study we used several antibodies and immunofluorescence microscopy to help in the characterization of these structures. We observed that Giardia tubulin reacts with antibodies raised against very distinct immunogens. The antibodies used were against: (1) alpha-tubulin from chicken embryo brain, Trypanosoma brucei, sea urchin sperm, Paramecium, acetylated alpha-tubulin from Paramecium, and tyrosinated alpha-tubulin, (2) beta-tubulin from chicken embryo brain and Physarum polycephalum, and (3) an antibody with specificity to beta-tubulin having as immunogen the FtsZ bacterial protein. Each cytoskeletal structure of Giardia presented a distinct pattern of labeling by the several antibodies used. These data indicate that even being considered one of the most ancient of organisms, Giardia shares similarities (at least in relation to tubulin) with other organisms. They also open some questions about the organization and composition of its microtubular structures.

Animals↗
PubMed2001

Microbes and microbial toxins: paradigms for microbial-mucosal interactions I. Pathophysiological aspects of enteric infections with the lumen-dwelling protozoan pathogen Giardia lamblia.

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.

Animals↗