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Giardia duodenalis captured the attention of Leeuwenhoek in 1681 while he was examining his own diarrheal stool, but, ironically, it did not really gain attention as a human pathogen until the 1960s, when outbreaks were reported. Key technological advances, including in vitro cultivation, genomic and proteomic databases, and advances in microscopic and molecular approaches, have led to an understanding that this is a eukaryotic organism with a reduced genome rather than a truly premitochondriate eukaryote. This has included the discovery of mitosomes (vestiges of mitochondria), a transport system with many of the features of the Golgi apparatus, and even evidence for a sexual or parasexual cycle. Cell biology approaches have led to a better understanding of how Giardia survives with two nuclei and how it goes through its life cycle as a noninvasive organism in the hostile environment of the lumen of the host intestine. Studies of its immunology and pathogenesis have moved past the general understanding of the importance of the antibody response in controlling infection to determining the key role of the Th17 response. This work has led to understanding of the requirement for a balanced host immune response that avoids the extremes of an excessive response with collateral damage or one that is unable to clear the organism. This understanding is especially important in view of the remarkable ranges of early manifestations, which range from asymptomatic to persistent diarrhea and weight loss, and longer-term sequelae that include growth stunting in children who had no obvious symptoms and a high frequency of postinfectious irritable bowel syndrome (IBS).

Giardia lamblia (syn G. intestinalis, G. duodenalis) is the most common pathogenic intestinal parasite of humans worldwide and is a frequent cause of endemic and epidemic diarrhea. G. lamblia is divided into eight genotypes (A–H) which infect a wide range of mammals and humans, but human infections are caused by Genotypes A and B. To unambiguously determine the relationship among genotypes, we sequenced GS and DH (Genotypes B and A2) to high depth coverage and compared the assemblies with the nearly completed WB genome and draft sequencing surveys of Genotypes E (P15; pig isolate) and B (GS; human isolate). Our results identified DH as the smallest Giardia genome sequenced to date, while GS is the largest. Our open reading frame analyses and phylogenetic analyses showed that GS was more distant from the other three genomes than any of the other three were from each other. Whole-genome comparisons of DH_A2 and GS_B with the optically mapped WB_A1 demonstrated substantial synteny across all five chromosomes but also included a number of rearrangements, inversions, and chromosomal translocations that were more common toward the chromosome ends. However, the WB_A1/GS_B alignment demonstrated only about 70% sequence identity across the syntenic regions. Our findings add to information presented in previous reports suggesting that GS is a different species of Giardia as supported by the degree of genomic diversity, coding capacity, heterozygosity, phylogenetic distance, and known biological differences from WB_A1 and other G. lamblia genotypes.

Pneumocystis spp. infect the lungs of multiple mammalian species and cause disease in immunosuppressed individuals. The Pneumocystis isolates that have been studied to date fall into two major clades, those from primates and those from rodents. Within each of these clades, different species have been described on the basis of host specificity and differences in sequence and morphology. Here, we demonstrate that dexamethasone immunosuppression consistently results in histologically apparent lung infection in gerbils (28/35 animals). Sequence analysis of the 18S, 5.8S and internal transcribed spacer regions of the rDNA and a portion of the mitochondrial large subunit rDNA demonstrated that this gerbil Pneumocystis is grouped with other rodent Pneumocystis spp., but is distinct from them. Our results suggest that gerbil Pneumocystis differs sufficiently from Pneumocystis species found in other rodents to be considered a separate species.

The genome of the eukaryotic protist Giardia lamblia, an important human intestinal parasite, is compact in structure and content, contains few introns or mitochondrial relics, and has simplified machinery for DNA replication, transcription, RNA processing, and most metabolic pathways. Protein kinases comprise the single largest protein class and reflect Giardia's requirement for a complex signal transduction network for coordinating differentiation. Lateral gene transfer from bacterial and archaeal donors has shaped Giardia's genome, and previously unknown gene families, for example, cysteine-rich structural proteins, have been discovered. Unexpectedly, the genome shows little evidence of heterozygosity, supporting recent speculations that this organism is sexual. This genome sequence will not only be valuable for investigating the evolution of eukaryotes, but will also be applied to the search for new therapeutics for this parasite.

Giardia lambliais medically important as a cause of diarrhea and malabsorption throughout the world and is thought to be one of the earliest-branching eukaryotes on a phylogenetic tree. Nevertheless, the mechanisms of inheritance are largely unknown. The trophozoites ofGiardiaand other diplomonads are interesting in their possession of two nuclei that are identical or similar in several respects. They replicate at nearly the same time, have similar quantities of DNA, and are both transcriptionally active. We used fluorescence in situ hybridization to demonstrate that genes from each of the five chromosomes are found in both nuclei, confirming that each nucleus has at least one complete copy of the genome. This raises a second question. The alleles of a gene in different nuclei are expected to accumulate different mutations, but surprisingly, the degree of heterozygosity in a clone is very low. One possible mechanism for eliminating sequence differences between nuclei is that each daughter cell receives two copies of the same nucleus at cell division. We used trophozoites with a plasmid transfected into a single nucleus to demonstrate that the two nuclei are partitioned equationally at cytokinesis. The mechanism(s) by which homozygosity is maintained will require further investigation.

SUMMARYGiardia lambliais a common cause of diarrhea in humans and other mammals throughout the world. It can be distinguished from otherGiardiaspecies by light or electron microscopy. The two major genotypes ofG. lambliathat infect humans are so different genetically and biologically that they may warrant separate species or subspecies designations. Trophozoites have nuclei and a well-developed cytoskeleton but lack mitochondria, peroxisomes, and the components of oxidative phosphorylation. They have an endomembrane system with at least some characteristics of the Golgi complex and encoplasmic reticulum, which becomes more extensive in encysting organisms. The primitive nature of the organelles and metabolism, as well as small-subunit rRNA phylogeny, has led to the proposal thatGiardiaspp. are among the most primitive eukaryotes.G. lambliaprobably has a ploidy of 4 and a genome size of approximately 10 to 12 Mb divided among five chromosomes. Most genes have short 5′ and 3′ untranslated regions and promoter regions that are near the initiation codon. Trophozoites exhibit antigenic variation of an extensive repertoire of cysteine-rich variant-specific surface proteins. Expression is allele specific, and changes in expression from onevspgene to another have not been associated with sequence alterations or gene rearrangements. TheGiardiagenome project promises to greatly increase our understanding of this interesting and enigmatic organism.

Cyclospora cayetanensis is an apicomplexan protozoan parasite which has emerged as an important cause of epidemic and endemic diarrhea. Water-borne as well as food-borne outbreaks have occurred, including a large number of U.S. cases associated with raspberries imported from Guatemala. Molecular markers exist for tracing the epidemiology of many of the bacterial pathogens associated with water-borne or food-borne diarrhea, such as serotyping and pulsed-field electrophoresis. However, there are currently no molecular markers available for C. cayetanensis . The intervening transcribed spacer (ITS) regions between the small- and large-subunit rRNA genes demonstrate much greater sequence variability than the small-subunit rRNA sequence itself and have been useful for the molecular typing of other organisms. Thus, ITS1 variability might allow the identification of different genotypes of C. cayetanensis . In order to determine the degree of ITS1 variability among C. cayetanensis isolates, the ITS1 sequences of C. cayetanensis isolates from a variety of sources, including raspberry-associated cases, cases from Guatemala, and pooled and individual isolates from Peru, were obtained. The ITS1 sequences of all five raspberry-associated isolates were identical, consistent with their origin from a single source. In contrast, one of the two Guatemala isolates and two Peruvian isolates contained multiple ITS1 sequences. These multiple sequences could represent multiple clones from a single clinical source or, more likely, variability of the ITS1 region within the genome of a single clone.

Giardia lamblia (also Giardia duodenalis, Giardia intestinalis) isolates have been variably divided into two or three genotypes by different investigators. We have compared the triose phosphate isomerase sequences of the three genotypes (Groups 1, 2, and 3) described by Nash and shown that Groups 1 and 2 are similar, while Group 3 is markedly different from Groups 1 and 2, indicating that Group1/2 and Group 3 correspond to the two major genotypes identified by other investigators. We have also analysed three Chinese isolates and showed that two fit into Group 3, while the third contained a mixture of Groups 1 and 3 isolates. These results confirm the relatedness of G. lamblia isolates from throughout the world, and established the feasibility of using DNA amplification and sequence analysis for detecting mixed isolates.

The surfaces of Giardia lamblia trophozoites contain one of a set of variant-specific surface proteins. The genes encoding these proteins are highly conserved at the 3' terminus, but frequently demonstrate little similarity in the remainder of the coding region. This report describes a family of vsp genes highly similar to a repeat-containing vsp gene (vspC5) at the 5' coding and flanking regions, but which diverge abruptly from vspC5 in the first repeat and do not themselves contain full copies of the repeat. This observation suggests the possibility that recombination among different vsp genes may have played a role in development of the vsp gene repertoire.

The Giardia lamblia genome consists of 12 Mb divided among 5 chromosomes ranging in size from approximately 1 to 4 Mb. The assembled contigs of the genotype A1 isolate, WB, were previously mapped along the 5 chromosomes on the basis of hybridization of plasmid clones representing the contigs to chromosomes separated by PFGE. In the current report, we have generated an MluI optical map of the WB genome to improve the accuracy of the physical map. This has allowed us to correct several assembly errors and to better define the extent of the subtelomeric regions that are not included in the genome assembly.

Our understanding of the transcription and recombination of Giardia has evolved substantially with recent experimental, genomic and population genetic data. Earlier assumptions that Giardia is an asexual organism have been called into serious question by the fi nding of the meiotic genes in the genome, the documentation that genetic material can be transferred from one nucleus to another during the process of encystation, and the evidence from population genetic data supporting the occurrence of meiotic recombination within Genotype A2. The different genotypes vary signifi cantly in the way they handle introduction of plasmid DNA and their levels of allelic heterozygosity, indicating differences in their processes of DNA recombination. Transcription in Giardia is typical of that of other eukaryotes with its use of a 5′ cap, polyadenylated tail, and its (rare) use of introns. However, it follows the minimalist approach that Giardia follows in many areas of metabolism, with an apparently abbreviated set of transcription factors and promoters that vary signifi cantly from those of other eukaryotes and may also be simplifi ed in their structure and function. The answers to how Giardia handles transcription and recombination with its minimalist approach may also shed light on the more complex systems used by other organisms.

Giardia lamblia trophozoites demonstrate variable expression of a repertoire of cysteine-rich surface antigens in vitro and in vivo. The size of the repertoire has been estimated at 20 to 184, and specific variants can be detected after approximately 12 generations of in vitro growth for the WB isolate. In earlier studies, we cloned a portion of the gene for a 170-kDa surface antigen (CRP170) and demonstrated by DNA sequencing that it was cysteine rich (12%) and contained 2.6 copies of a tandemly repeated 195-bp pair sequence. The clone hybridized to multiple bands on a Southern blot of G. lamblia DNA in a pattern that was variable among the cloned lines but did not correlate with expression of CRP170. We have now cloned a nearly full length cDNA as well as genomic clones for CRP170 from the WBA6 cloned isolate. In addition, we have isolated a cDNA clone from the WB1269 line (expressing CRP72), an antigenic variant which was derived from WBA6. Sequence analysis of the CRP170 and CRP72 genes revealed marked C-terminal amino acid homology, suggesting a conserved functional role such as membrane anchoring. The CRP170 repeat oligonucleotide hybridized to a stairstep of bands approximately 6 kb in size on HindIII-digested WBA6 DNA representing the expressed copy(ies) of CRP170. In contrast, there was no hybridization to a fragment of similar size in WB1269, suggesting that WB1269 trophozoites have lost the expressed copy of the CRP170 gene.

Giardia lamblia trophozoites contain at least five sets of chromosomes that have been categorized by chromosome-specific probes. Pulsed field separations of G. lamblia chromosomes also demonstrated minor bands in some isolates which stained less intensely with ethidium than the major chromosomal bands. Two of the minor bands of the E11 clone of the ISR isolate, MBa and MBb, were similar to each other and to chromosomal band I by hybridization to total chromosomal DNA and by hybridization of specific probes. In order to determine the extent of this similarity, I have developed a panel of probes for many of the Pacl restriction fragments and have shown that most of the Pacl and Notl fragments found in MBa are also present in MBb. The differences are found in both telomeric regions. At one end, MBb contains a 300 kb region not found in MBa. At the other end of MBb is a 160 kb region containing the rDNA repeats which is bounded on one end by the telomeric repeat and on the other by sites for multiple enzymes that do not digest the rDNA repeats. The corresponding region of MBa is 23 kb in size. The size difference is consistent with the eightfold greater number of rDNA repeats in MBb than MBa and suggests that 30% of the size difference is accounted for by different numbers of copies of the rDNA repeat. MBa of another ISR clone (ISR G5) is 150 kb larger in size than MBa of ISR E11. The data suggest that MBa and MBb are homologous chromosomes of different sizes and that a portion of the size difference is accounted for by different copy numbers of the rDNA repeat.

SummaryGiardia lamblia (syn. Giardia intestinalis, Giardia duodenalis) is an enteric protozoan parasite with two nuclei, and it might be one of the earliest branching eukaryotes [1]. 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.

Giardia lamblia, a protozoan parasite inhabiting the small intestine, is a common infection worldwide that frequently results in chronic diarrhea, malabsorption and upper gastrointestinal symptoms. Giardia undergoes surface antigenic variation in humans and animal model infections, a phenomenon that may account for both chronicity of infections and the relatively broad mammalian host specificity with genotypically identical organisms found in humans, cats, beavers, and other mammals. The variant-specific surface proteins (VSPs) are an unusual family of related cysteine-rich proteins, from 50 kD to over 200 kD in size, that coat the surface of the trophozoite. Only one VSP of the estimated 150 or more vsp genes is expressed on an individual at any specific time. However, the repertoires of vsp genes may differ depending on the genetic group. VSPs switch spontaneously every 6–12 generations although some Giardia also switch during encystation/excystation. All VSPs have a high cysteine content of about 12 % cysteines that are mostly present as CXXC motifs as well as a highly conserved C-terminus, a surface Zn finger motif, a GGCY motif and other common features. The biological function(s) of VSPs are uncertain but they undergo both immune and biological selection. The molecular mechanism(s) involved in antigenic variation are unknown but because there is the absence of gene movement or gene mutations and only one of 4 practically identical alleles (Giardia are tetraploid) is expressed; epigenetic processes are likely involved. Further studies of the mechanism of antigenic variation and the biological role of the VSPs promise to contribute to our understanding of G. lamblia and its pathogenesis.

Animals are commonly considered to be potential sources for Giardia lamblia infections in humans, but the extent of zoonotic transmission of G. lamblia remains controversial because of inadequate understanding of its epidemiology. A better understanding of the epidemiology of G. lamblia may be facilitated by a more effective means for classifying G. lamblia isolates. To develop a sequence-based classification system, the gene encoding the metabolic enzyme triose phosphate isomerase (tim) was sequenced from a number of G. lamblia isolates of various host origins. Restriction enzymes were identified that can distinguish among isolates without the need for sequencing, simplifying the application of this approach to the epidemiologic investigation of giardiasis. Isolates from a previously reported epidemic of giardiasis were accurately classified by this technique, further verifying its utility for epidemiologic investigation.

Giardia lamblia trophozoites express on their surfaces one of a set of cysteine-rich antigenically variant proteins, called variant-specific surface proteins, which comprise the majority of proteins detected by surface labeling. While these VSP proteins may be immunodominant proteins important in the host immune response to G. lamblia, the ability to switch expression from one VSP to another may provide a means for the trophozoites to avoid the host immune response. The first VSP characterized, VSPA6 (from the A6 clone of the WB isolate, originally termed CRP170), contains 18-23 copies of a 65 amino acid repeat. We have now used the repeat as a probe to isolate from a WBA6 genomic library two genes related to vspA6 (called vspA6-S1, vspA6-S2). Sequence analysis of the vspA6-S1 gene revealed nearly two complete copies of the 195 bp repeat and substantial nucleotide and translated amino acid similarity in the coding regions 5' and 3' to the repeats. The vspA6-S2 gene, while still related, showed greater divergence from vspA6 than vspA6-S1 in the nonrepeat coding region and contained nearly four copies of a 201 bp repeat that was 75% identical to the 195 bp vspA6 repeat. These results suggest that gene duplication followed by divergence has played a key role in the generation of the vsp gene repertoire.

Giardia lamblia trophozoites undergo antigenic variation of a variant-specific surface protein (VSP). All VSPs that have been reported have had high cysteine contents, including numerous copies of a CXXC motif. The first vsp gene described (vspA6; from the cloned line, WBA6), contained 21 copics of a 195 base pair tandem repeat, but other reported VSPs have not contained repeats. In this report, we describe the vsp gene from WBC5, a cloned line derived from WBA6. The vspC5 gene contains short 5′ and 3′ regions flanking 26 copies of a 105-bp tandem repeat, which comprises 93% of the coding region. In addition to the copy containing 26 repeats, the genome contains other copies of the vspC5 with fewer copies of the repeat. The sequences flanking the repeats are identical, and all copies map to the same location on chromosomal Band 5. suggesting that multiple alleles of the vspC5 gene are present.

The surfaces of Giardia lamblia trophozoites demonstrate variable expression of a set of cysteine-rich surface proteins, called variant-specific surface proteins (VSP). The cloned Giardia line, WBA6, expresses a 170 kD VSP (VSPA6 or CRP170) which contains approximately 18 to 23 copies of a 65 amino acid repeat. We have cloned the expressed vspA6 gene containing 23 repeats from a genomic library as well as copies of the vspA6 gene with only 8 or 9 repeats from both WBA6 and from WB1269, a cloned line derived from WBA6 which has lost the expressed copy of the gene. The recombinant clones containing the genes with only 8 or 9 repeats have 8 nucleotide substitutions in the coding region. All the recombinant clones map to the same chromosomal location, yet RNA sequencing and comparison with the transcript size indicate that only the clone with 23 repeats contains a gene producing a stable transcript. The most likely interpretation of these data is that G.lamblia trophozoites contain multiple alleles of the vspA6 gene of which only one is expressed.

Background Giardia lamblia trophozoites colonize the intestines of susceptible mammals and cause diarrhea, which can be prolonged despite an intestinal immune response. The variable expression of the variant-specific surface protein (VSP) genes may contribute to this prolonged infection. Only one is expressed at a time, and switching expression from one gene to another occurs by an epigenetic mechanism. Results The WB Giardia isolate has been sequenced at 10× coverage and assembled into 306 contigs as large as 870 kb in size. We have used this assembly to evaluate the genomic organization and evolution of the vsp repertoire. We have identified 228 complete and 75 partial vsp gene sequences for an estimated repertoire of 270 to 303, making up about 4% of the genome. The vsp gene diversity includes 30 genes containing tandem repeats, and 14 vsp pairs of identical genes present in either head to head or tail to tail configurations (designated as inverted pairs), where the two genes are separated by 2 to 4 kb of non-coding DNA. Interestingly, over half the total vsp repertoire is present in the form of linear gene arrays that can contain up to 10 vsp gene members. Lastly, evidence for recombination within and across minor clades of vsp genes is provided. Conclusions The data we present here is the first comprehensive analysis of the vsp gene family from the Genotype A1 WB isolate with an emphasis on vsp characterization, function, evolution and contributions to pathogenesis of this important pathogen.

Giardia lamblia telomeres have been isolated from a library enriched for repaired chromosome ends by (i) screening with a Plasmodium falciparum telomere and (ii) differential hybridization with Bal 31-digested and total G. lamblia DNA. Analysis of three clones isolated by this strategy has identified multiple tandem repeats of the 5-mer TAGGG. An oligonucleotide containing these repeats recognizes Bal 31-sensitive bands in Southern hybridizations and detects all G. lamblia chromosomes in pulsed-field gel electrophoresis separations. An abrupt transition from the G. lamblia rDNA sequence to telomeric repeats has been found in all three clones. In two of the clones the transition occurs at the same site, near the beginning of the large subunit rDNA sequence. In the third clone the transition occurs at a site in the intergenic spacer sequence between the rDNA genes. Hybridization of an rDNA probe to a pulsed-field separation of G. lamblia chromosomes indicates that rDNA genes are present on several chromosomes but vary in location from isolate to isolate. These results suggest that rRNA genes are clustered at telomeric locations in G. lamblia and that these clusters are mobile.

Background Giardia intestinalis is a protist found in freshwaters worldwide, and is the most common cause of parasitic diarrhea in humans. The phylogenetic position of this parasite is still much debated. Histones are small, highly conserved proteins that associate tightly with DNA to form chromatin within the nucleus. There are two classes of core histone genes in higher eukaryotes: DNA replication-independent histones and DNA replication-dependent ones. Results We identified two copies each of the core histone H2a, H2b and H3 genes, and three copies of the H4 gene, at separate locations on chromosomes 3, 4 and 5 within the genome of Giardia intestinalis, but no gene encoding a H1 linker histone could be recognized. The copies of each gene share extensive DNA sequence identities throughout their coding and 5' noncoding regions, which suggests these copies have arisen from relatively recent gene duplications or gene conversions. The transcription start sites are at triplet A sequences 1–27 nucleotides upstream of the translation start codon for each gene. We determined that a 50 bp region upstream from the start of the histone H4 coding region is the minimal promoter, and a highly conserved 15 bp sequence called the histone motif (him) is essential for its activity. The Giardia core histone genes are constitutively expressed at approximately equivalent levels and their mRNAs are polyadenylated. Competition gel-shift experiments suggest that a factor within the protein complex that binds him may also be a part of the protein complexes that bind other promoter elements described previously in Giardia. Conclusion In contrast to other eukaryotes, the Giardia genome has only a single class of core histone genes that encode replication-independent histones. Our inability to locate a gene encoding the linker histone H1 leads us to speculate that the H1 protein may not be required for the compaction of Giardia's small and gene-rich genome.