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.     

Organelles of protein transport in Giardia lamblia

Giardia occupies a unique evolutionary position since it is considered to belong to the earliest known lineage to diverge from the eukaryotic line of descent. Although organelles of protein transport are thought to have evolved with the nuclear membrane, G. lamblia is reported to have no Golgi apparatus. Therefore, Frances Gillin, David Reiner and Michael McCaffery have investigated how it exports glycoproteins to the cyst wall during encystation and whether a Golgi might become evident during an active secretory phase. They have found both functional and morphological evidence of a Golgi in Giardia and have shown that trophozoites are capable of sophisticated protein recognition, sorting and trafficking. These studies suggest that membranous organelles of protein transport appeared early in the evolution of the eukaryotic cell.     

Codon Usage in Giardia lamblia

ABSTRACT 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 synonomous 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.     

Antigenic variation in Giardia lamblia

Clones of the WB isolate of Giardia lamblia were exposed to cytotoxic mAb 6E7 which reacts with a 170-kDa surface Ag. Surviving progeny occurred at a frequency of about 1 in 1000 and were resistant to the effects of mAb 6E7. Analysis of progeny and clones of these progeny by surface radiolabeling, surface immunofluorescence, and Western blotting failed to detect the 170-kDa Ag. Loss of this Ag was associated with the appearance of a series of new surface Ag. A cytotoxic mAb (5C1) was produced to one of the newly appearing antigens (approximately equal to 64 kDa) and Giardia resistant to the cytotoxic effects of 5C1 isolated. Neither the approximately equal to 64 kDa nor the 170 kDa Ag were present and were replaced by a second series of new Ag. These studies clearly establish the loss and subsequent replacement of two antigenically distinct epitopes on Giardia derived from a single organism.     

Antigenic variation in Giardia lamblia

Giardia lamblia undergo surface antigenic variation in vitro and in vivo. The presence of variant trophozoites can be detected in clones after exposure to cytotoxic monoclonal antibodies. Surviving Giardia (progeny) no longer possess the initial major surface antigen which is replaced by new antigens. Exposure of a clone from one progeny to another cytotoxic mAb specific to one newly appearing surface antigen resulted in the loss of this antigen and replacement by another set of antigens. The frequency of change was rapid (1:100-1:1000) and was dependent upon the isolate. The presence of variant populations in clones was confirmed by direct and indirect immunofluorescence using mAbs to major surface antigens of subsequent progeny. The putative amino acid sequence of a portion of one antigen revealed a cysteine-rich composition which was confirmed in this variant protein as well as others by preferential uptake of [35S]cysteine. The mechanism(s) responsible most likely involves genomic rearrangements since Southern blots revealed a family of related genes which changed frequently compared to other areas of the genome. However, expression-linked copies have not been detected. Loss and gain of surface antigens have also been found in gerbils and humans infected with defined clones, but there does not appear to be cyclical appearance of variant populations. The biological importance of antigenic variation is not known but it may contribute to chronic and/or repeated infections.     

Chromosome rearrangements in Giardia lamblia

Recent studies have shown that the genome of Giardia lamblia is plastic. Clinical isolates exhibit extensive karyotypic heterogeneity and chromosome rearrangements occur frequently, in vitro. In this review, Sylvie Le Blancq looks at genome organization and the impact of DNA rearrangement events.     

Protein trafficking in Giardia lamblia

Giardia, a protozoan parasite of humans and other vertebrates, is a common cause of intestinal disease worldwide. Besides its medical importance, Giardia is considered an excellent system to study the evolution of fundamental cellular processes because it belongs to the earliest branches of the eukaryotic lineage of descent. Giardia trophozoites lack organelles typical of higher eukaryotes such mitochondria, peroxisomes and compartments involved in intracellular protein trafficking and secretion, such as the Golgi apparatus and secretory granules. Nevertheless, the minimal machinery for protein transport and sorting is present in this parasite. When Giardia undergoes encystation, the biogenesis of secretory organelles necessary to transport cyst wall constituents to the cell surface takes place. Recent studies in both vegetative and encysting trophozoites have provided interesting information regarding the secretory pathway of this important human pathogen.     

A new set of vesicles in Giardia lamblia

In the present study it was demonstrated the existence of a new set of membrane-bounded vesicles in Giardia lamblia. They were found in dividing and non-dividing trophozoites studied by routine transmission electron microscopy, freeze-fracture and Thiéry's technique. Encysting cells were not studied. These vesicles appear different to the previously reported components of the Giardia endomembranous system, such as the endoplasmic reticulum (ER), lysosome-like peripheral vesicles (PV), and the encystation-specific vesicles (ESV) that appear during trophozoite differentiation into cysts. They measure 100-150 nm in diameter, and thus are smaller than the peripheral vesicles, and the encystation-specific vesicles (ESV). They were found in clusters, scattered throughout the cytoplasm, but preferentially located close to the nuclei, axonemes, median bodies, and ER profiles. These internal vesicles are roughly spherical, and their contents present different electron densities and are more electrondense than those of the peripheral vesicles. They appeared to be budding from the outer nuclear membrane envelope. These cytoplasmic vesicles were found only in cells with very good fixation. Only few cells in the same preparation exhibited these vesicles.     

A novel palmitoyl acyl transferase controls surface protein palmitoylation and cytotoxicity in Giardia lamblia

The intestinal protozoan parasite Giardia lamblia undergoes surface antigenic variation whereby one of a family of structurally related variant-specific surface proteins (VSPs) is replaced in a regulated process by another antigenically distinct VSP. All VSPs are type I membrane proteins that have a conserved hydrophobic sequence terminated by the invariant hydrophilic amino acids, CRGKA. Using transfected Giardia constitutively expressing HA-tagged VSPH7 and incubated with radioactive [3H]palmitate, we demonstrate that the palmitate is attached to the Cys in the conserved CRGKA tail. Surface location of mutant VSPs lacking either the CRGKA tail or its Cys is identical to that of wild-type VSPH7 but non-palmitoylated mutants fail to undergo complement-independent antibody specific cytotoxicity. In addition, membrane localization of non-palmitoylated mutant VSPH7 changes from a pattern similar to rafts to non-rafts. Palmitoyl transferases (PAT), responsible for protein palmitoylation in other organisms, often possess a cysteine-rich domain containing a conserved DHHC motif (DHHC-CRD). An open reading frame corresponding to a putative 50 kDa Giardia PAT (gPAT) containing a DHHC-CRD motif was found in the Giardia genome database. Expression of epitope-tagged gPAT using a tetracycline inducible vector localized gPAT to the plasma membrane, a pattern similar to that of VSPs. Transfection with gPAT antisense producing vectors inhibits gPAT expression and palmitoylation of VSPs in vitro confirming the function of gPAT. These results show that VSPs are palmitoylated at the cysteine within the conserved tail by gPAT and indicate an essential function of palmitoylation in control of VSP-mediated signalling and processing.     

Purine deoxynucleoside salvage in Giardia lamblia

Giardia lamblia is dependent on the salvage of preformed purines and pyrimidines, including deoxythymidine. Dependence on deoxynucleoside salvage is extremely unusual among eucaryotic cells (Moore, E. C., and Hurlbert, R. B. (1985) Pharmacol & Ther. 27, 167-196). The present study investigates the possibility that giardia lacks ribonucleotide reductase and depends entirely on deoxynucleoside salvage. A ribonucleotide reductase inhibitor, hydroxyurea, at concentrations up to 2 mM had no effect on the growth of giardia. This is 15-20 times the ED50 of hydroxyurea for the protozoans Trypanosoma cruzi, Trypanosoma gambiense, and Leishmania donovani. A lysate of giardia had no detectable ribonucleotide reductase. Although radiolabeled adenine, adenosine, guanine, and guanosine were readily incorporated into RNA by cultured cells, no adenine or adenosine and only trace amounts of guanine and guanosine were detectable in DNA. This is in contrast to deoxynucleosides, where 58% of deoxyadenosine and 10% of deoxyguanosine incorporated into nucleic acid were found in DNA. Phosphorylation of both deoxyadenosine and deoxyguanosine was catalyzed by a cell lysate of giardia when nucleoside kinase co-substrates were included in the assay but not when phosphotransferase co-substrates were present. The absence of detectable ribonucleotide reductase, the failure to incorporate purine nucleobases and nucleosides into DNA to any significant extent, the ready incorporation of deoxynucleosides into DNA, and the demonstration of a purine deoxynucleoside kinase suggest that giardia are dependent on the salvage of exogenous deoxynucleosides.     

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.     

Surface antigenic variation in Giardia lamblia

Giardia lamblia, a common intestinal dwelling protozoan and a cause of diarrhoea in humans and animals world-wide, undergoes surface antigenic variation. The variant-specific surface proteins (VSPs) are a family of related, highly unusual proteins that cover the entire surface of the parasite. VSPs are cysteine-rich proteins containing many CXXC motifs, one or two GGCY motifs, a conserved hydrophobic tail and a Zn finger motif. The biological role(s) of VSPs is unclear. As VSPs are resistant to the effects of intestinal proteases, they likely allow the organism to survive in the protease-rich small intestine. Although immune escape is commonly mentioned as the reason antigenic variation occurs, VSP expression changes in vivo even in the absence of an adaptive immune system suggesting the biological role of antigenic variation is more complex. The molecular mechanisms involved in antigenic variation are not known but appear to differ from those known to occur in other protozoa.     

A potentially important excretory-secretory product of Giardia lamblia

In this study we have reported the detailed characterization of a 58 kDa excretory-secretory product (ESP) of Giardia lamblia. The method of purification has been simplified which has improved the purification fold as well as the yield of the ESP. The binding efficacy of disialoganglioside (GD2) to the purified ESP was found to be maximum among all other gangliosides used. The N-terminal sequence of the immunoreactive 29 kDa peptide obtained from partial tryptic digest of the ESP was found to be AD-FVPQVST. The IgG against the purified ESP (IgGES) showed cross-reactivity with the binding subunit of the commercially available cholera toxin and also with two protein bands of western cottonmouth moccasin snake toxin. The ESP could accumulate fluid in the intestine of sealed adult mice and also induce morphological changes in HEp-2 cells. The crude extract of G. lamblia trophozoites preincubated with Escherichia coli revealed 8-fold augmentation in the cytopathic activity on HEp-2 cells as compared to that of crude preparation from trophozoites only.