Rodney D. Adam

Biology of Giardia lamblia

SUMMARY Giardia lamblia is a common cause of diarrhea in humans and other mammals throughout the world. It can be distinguished from other Giardia species by light or electron microscopy. The two major genotypes of G. lamblia that 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 that Giardia spp. are among the most primitive eukaryotes. G. lamblia probably 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 one vsp gene to another have not been associated with sequence alterations or gene rearrangements. The Giardia genome project promises to greatly increase our understanding of this interesting and enigmatic organism.

Genomic Minimalism in the Early Diverging Intestinal Parasite Giardia lamblia

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 Giardias requirement for a complex signal transduction network for coordinating differentiation. Lateral gene transfer from bacterial and archaeal donors has shaped Giardias 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: Overview and Update

Giardia is a protozoan flagellate that was first observed by Van Leeuwenhoek in 1681 and more fully described by Lamb in 1859. It was initially thought to be a commensal in humans, but it is now clearly recognized as a common cause of diarrhea and malabsorption. Giardia infects millions of people throughout the world in both epidemic and sporadic forms. It is transmitted through ingestion of contaminated water and food, person-to-person contact in child care centers, and male homosexual activity.

Coccidioidomycosis in the Acquired Immunodeficiency Syndrome

Of 27 patients with the acquired immunodeficiency syndrome (AIDS) in Tucson, Arizona, 7 had concurrent coccidioidomycosis. Early manifestations of infection in 6 patients included diffuse nodular pulmonary infiltrates and Coccidioides immitis in many extrathoracic sites. By comparison, a retrospective review of the cases of 300 patients hospitalized with coccidioidal infection identified only 13 patients without AIDS who had the same extent of infection, and only 3 of these patients had no immunosuppressing conditions. Antibodies for coccidioidal antigens at serum dilutions as high as 1:2048 were detected in 5 of the 7 patients with AIDS. Six had temporary responses to amphotericin B treatment, taken both alone and combined with ketoconazole, but all died within 14 months of their diagnosis of coccidioidomycosis. Because annual rates of coccidioidal infection in the Tucson area are 4% or less, the rate of 27% that we calculated, based on 7 patients having the infection during 26 years of risk for AIDS, suggests frequent reactivation of the infection or enhanced susceptibility to endemic exposure in persons with AIDS.

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 Giardia lamblia genome

Giardia lamblia is a protozoan parasite of humans and other mammals that is thought to be one of the most primitive extant eukaryotic organisms. Although distinctly eukaryotic, it is notable for its lack of mitochondria, nucleoli, and perixosomes. It has been suggested that Giardia spp. are pre-mitochondriate organisms, but the identification of genes in G. lamblia thought to be of mitochondrial origin has generated controversy regarding that designation. Giardi lamblia trophozoites have two nuclei that are identical in all ways that have been studied. They are polyploid with at least four, and perhaps eight or more, copies of each of five chromosomes per organism and have an estimated genome complexity of 1.2x10(7)bp of DNA, and GC content of 46%. There is evidence for recombination at the telomeres of some of the chromosomes, and multiple size variants of single chromosomes have been identified within cloned isolates. However, the internal regions of the chromosomes demonstrate no evidence of recombination. For example, there is no evidence for control of vsp gene expression by DNA recombination, and no evidence for rapid mutation in the vsp genes. Single pass sequences of approximately 9% of the G. lamblia genome have already been obtained. An ongoing genome project plans to obtain approximately 95% of the genome by a random approach, as well as a complete physical map using a bacterial artificial chromosome library. The results will facilitate a better understanding of the biology of Giardia spp. as well as their phylogenetic relationship to other primitive organisms.

The Two Nuclei of Giardia Each Have Complete Copies of the Genome and Are Partitioned Equationally at Cytokinesis

ABSTRACT Giardia lamblia is 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 of Giardia and 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.

Telomeric location of Giardia rDNA genes.

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.

Molecular Analysis of Household Transmission of Giardia lamblia in a Region of High Endemicity in Peru

BACKGROUND Giardia lamblia is ubiquitous in multiple communities of nonindustrialized nations. Genotypes A1, A2, and B (Nash groups 1, 2, and 3, respectively) are found in humans, whereas genotypes C and D are typically found in dogs. However, genotypes A and B have occasionally been identified in dogs. METHODS Fecal Giardia isolates from 22 families and their dogs, living in Pampas de San Juan, were collected over 7 weeks in 2002 and 6 weeks in 2003. Samples were genotyped, followed by sequencing and haplotyping of many of these isolates by using loci on chromosomes 3 and 5. RESULTS Human infections were all caused by isolates of genotypes A2 and B. Human coinfections with genotypes A2 and B were common, and the reassortment pattern of different subtypes of A2 isolates supports prior observations that suggested recombination among genotype A2 isolates. All dogs had genotypes C and/or D, with one exception of a dog with a mixed B/D genotype infection. CONCLUSIONS In a region of high endemicity where infected dogs and humans constantly commingle, different genotypes of Giardia are almost always found in dogs and humans, suggesting that zoonotic transmission is very uncommon.

The Giardia lamblia vsp gene repertoire: characteristics, genomic organization, and evolution

BackgroundGiardia 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.ResultsThe 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.ConclusionsThe 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.