Characterisation of alpha-1 giardin: an immunodominant Giardia lamblia annexin with glycosaminoglycan-binding activity

Alpha-1 giardin is an immunodominant protein in the intestinal protozoan parasite Giardia lamblia. The Triage((R)) parasite panel, used to detect copro-antigens in stool from giardiasis patients, reacts with an epitope between amino acids 160 and 200 in alpha-1 giardin. This region of the protein is also highly immunogenic during human infections. Alpha-1 giardin is related to annexins and like many other annexins it was shown to be plasma membrane associated. Immunoelectron and immunofluorescence microscopy revealed that some alpha-1 giardin are displayed on the surface of recently excysted cells. Recombinant alpha-1 giardin displayed a Ca(2+)-dependent binding to glycosaminoglycans (GAGs), in particular heparan sulphate, a common GAG in the intestinal tract. Recombinant alpha-1 giardin bound to thin sections of human small intestine, a binding which could be inhibited by adding increasing concentrations of sulphated sugars. A surface associated trypsin activated Giardia lectin (taglin) has been suggested to be important for G. lamblia attachment. In this study we show that a monoclonal antibody that inhibits taglin recognises alpha-1 and alpha-2 giardin. Thus, alpha-1 giardin is a highly immunoreactive GAG-binding protein, which may play a key role in the parasite-host interaction. Our results further show a conserved function of annexins from lower to higher eukaryotes.     

Codon usage in Giardia lamblia.

A codon usage table for the intestinal parasite Giardia lamblia was generated by analysis of the nucleotide sequences of eight genes comprising 3,135 codons. Codon usage revealed a biased use of synonymous codons with a preference for NNC codons (42.1%). The codon usage of G. lamblia more closely resembles that of the prokaryote Halobacterium halobium (correlation coefficient r = 0.73) rather than that of other eukaryotic protozoans, i.e. Trypanosoma brucei (r = 0.434) and Plasmodium falciparum (r = -0.31). These observations are consistent with the view that G. lamblia represents the first line of descent from the ancestral cells that first took on eukaryotic features.     

Detection of Giardia lamblia by immunofluorescence.

High-titer immune sera to cysts of Giardia lamblia, produced in guinea pigs, were labeled with fluorescein isothiocyanate. The resulting conjugates were used to detect G. lamblia in stool specimens by fluorescence microscopy. The sera also reacted with cysts of Chilomastix mesnili, but the two organisms could be differentiated by their size.     

Electron microscopy of Giardia lamblia cysts.

The flagellated protozoan Giardia lamblia is a recognized public health problem. Intestinal infection can result in acute or chronic diarrhea with associated symptoms in humans. As part of a study to evaluate removal of G. lamblia cysts from drinking water by the processes of coagulation and dual-media filtration, we developed a methodology by using 5.0-microns-porosity membrane filters to evaluate the filtration efficiency. We found that recovery rates of G. lamblia cysts by membrane filtration varied depending upon the type and diameter of the membrane filter. Examination of membrane-filtered samples by scanning electron microscopy revealed flexible and flattened G. lamblia cysts on the filter surface. This feature may be responsible for the low recovery rates with certain filters and, moreover, may have implications in water treatment technology. Formation of the cyst wall is discussed. Electron micrographs of cysts apparently undergoing binary fission and cysts exhibiting a possible bacterial association are shown.     

Novel protein-disulfide isomerases from the early-diverging protist Giardia lamblia.

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.     

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.     

Segmented alpha-helical coiled-coil structure of the protein giardin from the Giardia cytoskeleton

The parasitic flagellate Giardia is the source of a filamentous protein, giardin, which binds to microtubules. The primary sequence of one giardin chain has been decoded from the base sequences of cDNAs isolated by antibody screens of a library constructed in the expression vector lambda gt11. The amino acid sequence favours a continuous alpha-helical fold for the protein without any inserts of a non-helical character. Analysis of apolar residue positions revealed 35 repeating heptads consistent with coiled-coil structure. This conformation relates giardin to the alpha-type fibrous proteins (k-m-e-f class) like tropomyosin and myosin (also found in Giardia). The giardin sequence has a regular series of skip residues like those at certain positions in the rod section of nematode myosin where the internal apolar seam of the coiled coil is shifted on the helix surface. The skips divide the giardin coil into quasi-equivalent structural segments about 4 nm in length, which might be domains for combining with tubulin subunits in the microtubule surface lattice.     

Crystal structure of a macrophage migration inhibitory factor from Giardia lamblia

Macrophage migration inhibitory factor (MIF) is a eukaryotic cytokine that affects a broad spectrum of immune responses and its activation/inactivation is associated with numerous diseases. During protozoan infections MIF is not only expressed by the host, but, has also been observed to be expressed by some parasites and released into the host. To better understand the biological role of parasitic MIF proteins, the crystal structure of the MIF protein from Giardia lamblia (Gl-MIF), the etiological agent responsible for giardiasis, has been determined at 2.30 Å resolution. The 114-residue protein adopts an α/β fold consisting of a four-stranded β-sheet with two anti-parallel α-helices packed against a face of the β-sheet. An additional short β-strand aligns anti-parallel to β4 of the β-sheet in the adjacent protein unit to help stabilize a trimer, the biologically relevant unit observed in all solved MIF crystal structures to date, and form a discontinuous β-barrel. The structure of Gl-MIF is compared to the MIF structures from humans (Hs-MIF) and three Plasmodium species (falciparum, berghei, and yoelii). The structure of all five MIF proteins are generally similar with the exception of a channel that runs through the center of each trimer complex. Relative to Hs-MIF, there are differences in solvent accessibility and electrostatic potential distribution in the channel of Gl-MIF and the Plasmodium-MIFs due primarily to two “gate-keeper” residues in the parasitic MIFs. For the Plasmodium MIFs the gate-keeper residues are at positions 44 (Y?R) and 100 (V?D) and for Gl-MIF it is at position 100 (V?R). If these gate-keeper residues have a biological function and contribute to the progression of parasitemia they may also form the basis for structure-based drug design targeting parasitic MIF proteins.     

Giardia lamblia expresses a proteobacterial-like DnaK homolog

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.     

Isoenzyme comparison of axenic Giardia lamblia strains.

We obtained isoenzyme patterns by polyacrylamide gradient gel electrophoresis (PGGE) of water-soluble protein fractions prepared from trophozoites of 11 axenic G. lamblia strains. The strains were isolated from animals and humans (both symptomatic and asymptomatic) from various geographic locations. Isoenzymes were also separated by isoelectric focusing. Of 12 enzymes attempted, eight exhibited well-defined and reproducible isoenzyme patterns by PGGE, based on which the strains were grouped into four zymodemes. Although the 11 strains were grouped into four zymodemes based on PGGE, no correlation between zymodeme and the known characteristics of the strains existed. Thus, a high degree of characteristic sharing appears to occur among genetically different G. lamblia strains.     

Excretory-secretory products of Giardia lamblia

The surface of Giardia lamblia strain WB was radioiodinated with either 1,3,4,6-tetrachloro-3 alpha, 6 alpha-diphenylglycoluril (IODOGEN) or lactoperoxidase, and the labeled membrane components as well as the released excretory-secretory (E-S) products were identified. The surface of G. lamblia was easily labeled, and G. lamblia excretory-secretory (E-S) products were identified in the medium. Over 70% of the label on the cell surface was released over 24 hr. The major E-S product released was polydisperse (m.w. 225 to 94,000), protease VI and periodate-sensitive, chloroform-methanol insoluble, and failed to adhere to a series of carbohydrate-binding lectins or to diethylaminoethyl (DEAE) cellulose. Hydrophobicity was suggested by adherence to phenyl-Sepharose. The major E-S product of another G. lamblia isolate, Portland-1 (P-1), was antigenically different. A previous study showed that strain P-1 lacked a major antigenic component of strain WB. In the present study, this material was identified as the major secretory product of WB by crossed immunoelectrophoresis.     

Circannual incidence of Giardia lamblia

A circannual rhythm of Giardia lamblia positive stools was found by examination of records from three clinical laboratories in central Arkansas for the period 1980-1986. Cosinor analysis of monthly Giardia incidence based on stool specimen records from approximately 12,000 patients over the 7-year period revealed a circannual rhythm (P less than 0.001) on the basis of percent positive patients/month, with a computive acrophase occurring in late summer and minimum values in the winter. Patients involved in the study were primarily from the central Arkansas metropolitan areas, southern delta regions and northern mountainous regions of the state. Analysis of the data on the basis of total positive Giardia patients/month also revealed a circannual rhythm with the acrophase again occurring in late summer. The overall mean for percent positive stool specimens for the 7-year period was 5.3%, compared with the national average of 3.8% for G. lamblia positive stools. The data indicate that there may be a "Giardia season" in Arkansas since they could not be explained on the basis of day-care age distribution, or geographic origin. Awareness by epidemiologists, public health officials and other health care professionals of this circannual incidence of giardiasis is important for the prevention, diagnosis and treatment of this infectious disorder.     

Mechanisms of Giardia lamblia differentiation into cysts.

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

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.     

Inactivation of Giardia lamblia cysts with ozone.

Giardia lamblia cysts were inactivated in water with ozone at pH 7.0 and 5 and 25 degrees C. The concentration-time products for 99% inactivation were 0.53 and 0.17 mg-min/liter at 5 and 25 degrees C, respectively. These products were significantly lower than those reported for chlorine.