Population genetics provides evidence for recombination in Giardia

Giardia lamblia (syn. Giardia intestinalis, Giardia duodenalis) is an enteric protozoan parasite with two nuclei, and it might be one of the earliest branching eukaryotes. However, the discovery of at least rudimentary forms of certain features, such as Golgi and mitochondria, has refuted the proposal that its emergence from the eukaryotic lineage predated the development of certain eukaryotic features. The recent recognition of many of the genes known to be required for meiosis in the genome has also cast doubt on the idea that Giardia is primitively asexual, but so far there has been no direct evidence of sexual reproduction in Giardia, and population data have suggested clonal reproduction. We did a multilocus sequence evaluation of the genotype A2 reference strain, JH, and five genotype A2 isolates from a highly endemic area in Peru. Loci from different chromosomes yielded significantly different phylogenetic trees, indicating that they do not share the same evolutionary history; within individual loci, tests for recombination yielded significant statistical support for meiotic recombination. These observations provide genetic data supportive of sexual reproduction in Giardia.     

The biology of Giardia spp.

Gardia spp. are flagellated protozoans that parasitize the small intestines of mammals, birds, reptiles, and amphibians. The infectious cysts begin excysting in the acidic environment of the stomach and become trophozoites (the vegetative form). The trophozoites attach to the intestinal mucosa through the suction generated by a ventral disk and cause diarrhea and malabsorption by mechanisms that are not well understood. Giardia spp. have a number of unique features, including a predominantly anaerobic metabolism, complete dependence on salvage of exogenous nucleotides, a limited ability to synthesize and degrade carbohydrates and lipids, and two nuclei that are equal by all criteria that have been tested. The small size and unique sequence of G. lamblia rRNA molecules have led to the proposal that Giardia is the most primitive eukaryotic organism. Three Giardia spp. have been identified by light lamblia, G. muris, and G. agilis, but electron microscopy has allowed further species to be described within the G. lamblia group, some of which have been substantiated by differences in the rDNA. Animal models and human infections have led to the conclusion that intestinal infection is controlled primarily through the humoral immune system (T-cell dependent in the mouse model). A major immunogenic cysteine-rich surface antigen is able to vary in vitro and in vivo in the course of an infection and may provide a means of evading the host immune response or perhaps a means of adapting to different intestinal environments.     

The biology of Giardia spp.

Gardia spp. are flagellated protozoans that parasitize the small intestines of mammals, birds, reptiles, and amphibians. The infectious cysts begin excysting in the acidic environment of the stomach and become trophozoites (the vegetative form). The trophozoites attach to the intestinal mucosa through the suction generated by a ventral disk and cause diarrhea and malabsorption by mechanisms that are not well understood. Giardia spp. have a number of unique features, including a predominantly anaerobic metabolism, complete dependence on salvage of exogenous nucleotides, a limited ability to synthesize and degrade carbohydrates and lipids, and two nuclei that are equal by all criteria that have been tested. The small size and unique sequence of G. lamblia rRNA molecules have led to the proposal that Giardia is the most primitive eukaryotic organism. Three Giardia spp. have been identified by light lamblia, G. muris, and G. agilis, but electron microscopy has allowed further species to be described within the G. lamblia group, some of which have been substantiated by differences in the rDNA. Animal models and human infections have led to the conclusion that intestinal infection is controlled primarily through the humoral immune system (T-cell dependent in the mouse model). A major immunogenic cysteine-rich surface antigen is able to vary in vitro and in vivo in the course of an infection and may provide a means of evading the host immune response or perhaps a means of adapting to different intestinal environments.     

Identification, characteristic and phylogenetic analysis of type II DNA topoisomerase gene in Giardia lamblia

The genes encoding type II DNA topoisomerases were investigated in Giardia lamblia genome, and a type IIA gene,GlTop 2 was identified. It is a single copy gene with a 4476 bp long ORF without intron. The deduced amino acid sequence shows strong homology to eukaryotic DNA Top 2. However, some distortions were found, such as six insertions in the ATPase domain and the central domain, a -100 aa longer central domain; a ~200 aa shorter C-terminal domain containing rich charged residues. These features revealed by comparing with Top 2 of the host, human, might be helpful in exploiting drug selectivity for antigiardial therapy. Phylogenetic analysis of eukaryotic enzymes showed that kinetoplastids, plants, fungi, and animals were monophyletic groups, and the animal and fungi lineages shared a more recent common ancestor than either did with the plant lineage; microsporidia grouped with fungi. However, unlike many previous phylogenetic analyses, the ““amitochondriate““ G. lamblia was not the earliest branch but diverged after mitochondriate kinetoplastids in our trees. Both the finding of typical eukaryotic type IIA topoisomerase and the phylogenetic analysis suggest G, lamblia is not possibly as primitive as was regarded before and might diverge after the acquisition of mitochondria. This is consistent with the recent discovery of mitochondrial remnant organelles in G. lamblia.     

Phylogenetic positions of several amitochondriate protozoa-Evidence from phylogenetic analysis of DNA topoisomerase II

Several groups of parasitic protozoa, as represented by Giardia, Trichomonas, Entamoeba and Microsporida, were once widely considered to be the most primitive extant eukaryotic group―Archezoa. The main evidence for this is their 'lacking mitochondria' and possessing some other primitive features between prokaryotes and eukaryotes, and being basal to all eukaryotes with mitochondria in phylogenies inferred from many molecules. Some authors even proposed that these organisms diverged before the endosymbiotic origin of mitochondria within eukaryotes. This view was once considered to be very significant to the study of origin and evolution of eukaryotic cells (eukaryotes). However, in recent years this has been challenged by accumulating evidence from new studies. Here the sequences of DNA topoisomerase II in G. lamblia, T. vaginalis and E. histolytica were identified first by PCR and sequencing, then combining with the sequence data of the microsporidia Encephalitozoon cunicul and other eukaryotic groups of different evolutionary positions from GenBank, phylogenetic trees were constructed by various methods to investigate the evolutionary positions of these amitochondriate protozoa. Our results showed that since the characteristics of DNA topoisomerase II make it avoid the defect of 'long-branch attraction' appearing in the previous phylogenetic analyses, our trees can not only reflect effectively the relationship of different major eukaryotic groups, which is widely accepted, but also reveal phylogenetic positions for these amitochondriate protozoa, which is different from the previous phylogenetic trees. They are not the earliest-branching eukaryotes, but diverged after some mitochondriate organisms such as kinetoplastids and mycetozoan; they are not a united group but occupy different phylogenetic positions. Combining with the recent cytological findings of mitochondria-like organelles in them, we think that though some of them (e.g. diplomonads, as represented by Giardia) may occupy a very low evolutionary position, generally these organisms are not as extremely primitive as was thought before; they should be polyphyletic groups diverging after the endosymbiotic origin of mitochondrion to adapt themselves to anaerobic parasitic life.     

Identification of a Giardia krr1 homolog gene and the secondarily anucleolate condition of Giaridia lamblia

Giaridia lamblia was long considered to be one of the most primitive eukaryotes and to lie close to the transition between prokaryotes and eukaryotes, but several supporting features, such as lack of mitochondrion and Golgi, have been challenged recently. It was also reported previously that G. lamblia lacked nucleolus, which is the site of pre-rRNA processing and ribosomal assembling in the other eukaryotic cells. Here, we report the identification of the yeast homolog gene, krr1, in the anucleolate eukaryote, G. lamblia. The krr1 gene, encoding one of the pre-rRNA processing proteins in yeast, is actively transcribed in G. lamblia. The deduced protein sequence of G. lamblia krr1 is highly similar to yeast KRR1p that contains a single-KH domain. Our database searches indicated that krr1 genes actually present in diverse eukaryotes and also seem to present in Archaea. However, only the eukaryotic homologs, including that of G. lamblia, have the single-KH domain, which contains the conserved motif KR(K)R. Fibrillarin, another important pre-rRNA processing protein has also been identified previously in G. lamblia. Moreover, our database search shows that nearly half of the other nucleolus-localized protein genes of eukaryotic cells also have their homologs in Giardia. Therefore, we suggest that a common mechanism of pre-RNA processing may operate in the anucleolate eukaryote G. lamblia and in the other eukaryotes and that like the case of "lack of mitochondrion," "lack of nucleolus" may not be a primitive feature, but a secondarily evolutionary condition of the parasite.     

Phylogenetic positions of several amitochondriate protozoa

Several groups of parasitic protozoa, as represented by Giardia, Trichomonas, Entamoeba and Microsporida, were once widely considered to be the most primitive extant eukaryotic group―Archezoa. The main evidence for this is their ‘lacking mitochondria’ and possessing some other primitive features between prokaryotes and eukaryotes, and being basal to all eukaryotes with mitochondria in phylogenies inferred from many molecules. Some authors even proposed that these organisms diverged before the endosymbiotic origin of mitochondria within eukaryotes. This view was once considered to be very significant to the study of origin and evolution of eukaryotic cells (eukaryotes). However, in recent years this has been challenged by accumulating evidence from new studies. Here the sequences of DNA topoisomerase II in G. lamblia, T. vaginalis and E. histolytica were identified first by PCR and sequencing, then combining with the sequence data of the microsporidia Encephalitozoon cunicul and other eukaryotic groups of different evolutionary positions from GenBank, phylogenetic trees were constructed by various methods to investigate the evolutionary positions of these amitochondriate protozoa. Our results showed that since the characteristics of DNA topoisomerase II make it avoid the defect of ‘long-branch attraction’ appearing in the previous phylogenetic analyses, our trees can not only reflect effectively the relationship of different major eukaryotic groups, which is widely accepted, but also reveal phylogenetic positions for these amitochondriate protozoa, which is different from the previous phylogenetic trees. They are not the earliest-branching eukaryotes, but diverged after some mitochondriate organisms such as kinetoplastids and mycetozoan; they are not a united group but occupy different phylogenetic positions. Combining with the recent cytological findings of mitochondria-like organelles in them, we think that though some of them (e.g. diplo-monads, as represented by Giardia) may occupy a very low evolutionary position, generally these organisms are not as extremely primitive as was thought before; they should be poly-phyletic groups diverging after the endosymbiotic origin of mitochondrion to adapt themselves to anaerobic parasitic life.     

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.     

Genetic Exchange Within and Between Assemblages of Giardia duodenalis

ABSTRACT. Meiotic sex evolved early in the history of eukaryotes. Giardia duodenalis (syn. Giardia lamblia, Giardia intestinalis ), a parasitic protist belonging to an early diverging lineage of eukaryotes, shows no cytological or physiological evidence of meiotic or sexual processes. Recent molecular analyses challenge the idea that G. duodenalis is a strictly clonal organism by providing evidence of recombination between homologous chromosomes within one subgroup (Assemblage A) of this species as well as genetic transfer from one subgroup to another (Assemblage A–B). Because recombination is not well documented and because it is not known whether the observed inter-assemblage transfer represents true reciprocal genetic exchange or a non-sexual process, we analyzed genic sequences from all major subgroups (Assemblages A–G) of this species. For all assemblages, we detected molecular signatures consistent with meiotic sex or genetic exchange, including low levels of heterozygosity, as indicated by allelic sequence divergence within isolates, and intra- and inter-assemblage recombination. The identification of recombination between assemblages suggests a shared gene pool and calls into question whether it is appropriate to divide the genetically distinct assemblages of G. duodenalis into a species complex.     

The profile of snoRNA-derived microRNAs that regulate expression of variant surface proteins in Giardia lamblia

In the current investigation, we analysed all the known small nucleolar RNAs (snoRNAs) in the deeply branching protozoan parasite Giardia lamblia for potential microRNAs (miRNAs) that might be derived from them. Two putative miRNAs have since been identified by Northern blot, primer extension, 3' RACE and co-immunoprecipitation with Giardia Argonaute (GlAgo), and designated miR6 and miR10. Giardia Dicer (GlDcr) is capable of processing the snoRNAs into the corresponding miRNAs in vitro. Potential miR6 and miR10 binding sites in Giardia genome were predicted bio-informatically. A miR6 binding site was found at the 3' untranslated regions (UTR) of 44 variant surface protein (vsp) genes, whereas a miR10 binding site was identified at the 3' end of 159 vsp open-reading frames. Thirty-three of these vsp genes turned out to contain binding sites for both miR6 and miR10. A reporter mRNA tagged with the 3' end of vsp1267, which contains the target sites for both miRNAs, was translationally repressed by both miRNAs in Giardia. Episomal expression of an N-terminal c-myc tagged VSP1267 was found significantly repressed by introducing either miR6 or miR10 into the cells and the repressive effects were additive. When the 2'-O-methyl antisense oligos (ASOs) of either miR6 or miR10 was introduced, however, there was an enhancement of tagged VSP1267 expression suggesting an inhibition of the repressive effects of endogenous miR6 or miR10 by the ASOs. Of the total 220 vsp genes in Giardia, we have now found 178 of them carrying putative binding sites for all the miRNAs that have been currently identified, suggesting that miRNAsare likely the regulators of VSP expression in Giardia.     

Spontaneous chromosome rearrangements in the protozoan Giardia lamblia: estimation of mutation rates.

Subcloned lines of the WB strain of Giardia lamblia contain polymorphic ribosomal RNA (rRNA) encoding chromosomes (Le Blancq et al., Nucl. Acids Res. 1991, 19, 4405-4412). We show that in a continuously propagated culture of G.lamblia trophozoites the proportion of trophozoites with rearranged rRNA encoding chromosomes gradually increases, consistent with the high mutation rate of about 1% per cell per division cycle. This conclusion is based on the finding in one experiment that after about 8 division cycles 20% of the population consisted of independent mutants, while after approximately 100 division cycles 87.5% of the population were independent mutants. In a second experiment, approximately 38% and 71.5% of the trophozoites were independent mutants after approximately 9 and approximately 100 division cycles, respectively. The data show that the genome of the WB strain of G.lamblia has a highly recombinogenic phenotype. Extensive karyotype heterogeneity has also been observed among recently isolated G.lamblia strains obtained from a defined geographic area (Korman et al., J. Clin. Invest. 1992, 89, 1725-1733) suggesting that a high mutation rate might also occur in vivo.     

Phylogenetic positions of several amitochondriate protozoa

Several groups of parasitic protozoa, as represented by Giardia, Trichomonas, En-tamoeba and Microsporida, were once widely considered to be the most primitive extant eukaryotic group—Archezoa. The main evidence for this is their ‘lacking mitochondria’ and possessing some other primitive features between prokaryotes and eukaryotes, and being basal to all eukaryotes with mitochondria in phylogenies inferred from many molecules. Some authors even proposed that these organisms diverged before the endosymbiotic origin of mitochondria within eukaryotes. This view was once considered to be very significant to the study of origin and evolution of eukaryotic cells (eukaryotes). However, in recent years this has been challenged by accumulating evidence from new studies. Here the sequences of DNA topoisomerase II in G. lamblia, T. vaginalis and E. histolytica were identified first by PCR and sequencing, then combining with the sequence data of the microsporidia Encephalitozoon cunicul and other eukaryotic groups of different evolutionary positions from GenBank, phylogenetic trees were constructed by various methods to investigate the evolutionary positions of these amitochondriate protozoa. Our results showed that since the characteristics of DNA topoisomerase II make it avoid the defect of ‘long-branch attraction’ appearing in the previous phylogenetic analyses, our trees can not only reflect effectively the relationship of different major eukaryotic groups, which is widely accepted, but also reveal phylogenetic positions for these amitochondriate protozoa, which is different from the previous phylogenetic trees. They are not the earliest-branching eukaryotes, but diverged after some mitochondriate organisms such as kinetoplastids and mycetozoan; they are not a united group but occupy different phylogenetic positions. Combining with the recent cytological findings of mitochondria-like organelles in them, we think that though some of them (e.g. diplomonads, as represented by Giardia) may occupy a very low evolutionary position, generally these organisms are not as extremely primitive as was thought before; they should be polyphyletic groups diverging after the endosymbiotic origin of mitochondrion to adapt themselves to anaerobic parasitic life.     

Evolution of the eukaryotic translation termination system: origins of release factors

Accurate translation termination is essential for cell viability. In eukaryotes, this process is strictly maintained by two proteins, eukaryotic release factor 1 (eRF1), which recognizes all stop codons and hydrolyzes peptidyl-tRNA, and eukaryotic release factor 3 (eRF3), which is an elongation factor 1alpha (EF-1alpha) homolog stimulating eRF1 activity. To retrace the evolution of this core system, we cloned and sequenced the eRF3 genes from Trichomonas vaginalis (Parabasalia) and Giardia lamblia (Diplomonada), which are generally thought to be "early-diverging eukaryotes," as well as those from two ciliates (Oxytricha trifallax and Euplotes aediculatus). We also determined the sequence of the eRF1 gene for G. lamblia. Surprisingly, the G. lamblia eRF3 appears to have only one domain, corresponding to EF-1alpha, while other eRF3s (including the T. vaginalis protein) have an additional N-terminal domain, of 66-411 amino acids. Considering this novel eRF3 structure and our extensive phylogenetic analyses, we suggest that (1) the current translation termination system in eukaryotes evolved from the archaea-like version, (2) eRF3 was introduced into the system prior to the divergence of extant eukaryotes, including G. lamblia, and (3) G. lamblia might be the first eukaryotic branch among the organisms considered.     

Characterisation of the subtelomeric regions of Giardia lamblia genome isolate WBC6

Giardia trophozoites are polyploid and have five chromosomes. The chromosome homologues demonstrate considerable size heterogeneity due to variation in the subtelomeric regions. We used clones from the genome project with telomeric sequence at one end to identify six subtelomeric regions in addition to previously identified subtelomeric regions, to study the telomeric arrangement of the chromosomes. The subtelomeric regions included two retroposons, one retroposon pseudogene, and two vsp genes, in addition to the previously identified subtelomeric regions that include ribosomal DNA repeats. The presence of vsp genes in a subtelomeric region suggests that telomeric rearrangements may contribute to the generation of vsp diversity. These studies of the subtelomeric regions of Giardia may contribute to our understanding of the factors that maintain stability, while allowing diversity in chromosome structure.     

Viruses of parasitic protozoa

Recently, specific viruses have been identified among the parasitic protozoa Trichomonas vaginalis, Giardia lamblia, Leishmania braziliensis, the Eimeria spp and the Babesia spp. These viruses share many features: they are all RNA viruses and most, if not all, doublestranded (ds) RNA viruses with nonsegmented genomes ranging between 5 and 7 kilobases (kb); they are spherical or icosahedral with an average diameter of 30-40 nm. The giardiavirus is one of the best characterized and can infect virus free G. lamblia trophozoites in its freed, pure form. The replicative intermediate of the giardiavirus genome has been isolated from infected cells, and can be introduced into G. lamblia by electroporation to produce giardiavirus, thus raising the possibility of its being used as a specific genetic transfection vector for the parasite.     

A phylogenomic inventory of meiotic genes; evidence for sex in Giardia and an early eukaryotic origin of meiosis

Sexual reproduction in eukaryotes is accomplished by meiosis, a complex and specialized process of cell division that results in haploid cells (e.g., gametes). The stereotypical reductive division in meiosis is a major evolutionary innovation in eukaryotic cells, and delineating its history is key to understanding the evolution of sex. Meiosis arose early in eukaryotic evolution, but when and how meiosis arose and whether all eukaryotes have meiosis remain open questions. The known phylogenetic distribution of meiosis comprises plants, animals, fungi, and numerous protists. Diplomonads including Giardia intestinalis (syn. G. lamblia) are not known to have a sexual cycle; these protists may be an early-diverging lineage and could represent a premeiotic stage in eukaryotic evolution. We surveyed the ongoing G. intestinalis genome project data and have identified, verified, and analyzed a core set of putative meiotic genes-including five meiosis-specific genes-that are widely present among sexual eukaryotes. The presence of these genes indicates that: (1) Giardia is capable of meiosis and, thus, sexual reproduction, (2) the evolution of meiosis occurred early in eukaryotic evolution, and (3) the conserved meiotic machinery comprises a large set of genes that encode a variety of component proteins, including those involved in meiotic recombination.