Daniel Palm

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.

Draft Genome Sequencing of Giardia intestinalis Assemblage B Isolate GS: Is Human Giardiasis Caused by Two Different Species?

Giardia intestinalis is a major cause of diarrheal disease worldwide and two major Giardia genotypes, assemblages A and B, infect humans. The genome of assemblage A parasite WB was recently sequenced, and the structurally compact 11.7 Mbp genome contains simplified basic cellular machineries and metabolism. We here performed 454 sequencing to 16× coverage of the assemblage B isolate GS, the only Giardia isolate successfully used to experimentally infect animals and humans. The two genomes show 77% nucleotide and 78% amino-acid identity in protein coding regions. Comparative analysis identified 28 unique GS and 3 unique WB protein coding genes, and the variable surface protein (VSP) repertoires of the two isolates are completely different. The promoters of several enzymes involved in the synthesis of the cyst-wall lack binding sites for encystation-specific transcription factors in GS. Several synteny-breaks were detected and verified. The tetraploid GS genome shows higher levels of overall allelic sequence polymorphism (0.5 versus <0.01% in WB). The genomic differences between WB and GS may explain some of the observed biological and clinical differences between the two isolates, and it suggests that assemblage A and B Giardia can be two different species.

A novel Myb-related protein involved in transcriptional activation of encystation genes in Giardia lamblia

Giardia lamblia is an important human intestinal parasite that survives outside of the host by differentiation of trophozoites into infectious cysts. Transcriptional regulation is key for encystation gene expression, but the mechanisms are unknown. Giardia genome database searches identified a myb ‐like gene ( gmyb2 ) whose expression increased during encystation. Epitope‐tagged gMyb2 localized to both nuclei. DNA binding and mutation analysis showed that gMyb2 binds specifically to C(T/A)ACAG, a c‐Myb‐like target sequence in the promoters of encystation‐induced genes encoding gMyb2, three cyst wall proteins and G6PI‐B, a key enzyme in cyst wall polysaccharide biosynthesis. gMyb2 binding sites were not found in the upstream regions of 31 other giardial genes. Deletion of the putative gMyb2 binding site greatly reduced encystation‐specific promoter activity of g6pi‐b . Fusion of gMyb2 binding sites to the constitutive ran promoter or g6pi‐b promoter deletion lacking the gMyb2 binding site in‐duced encystation‐specific expression. gMyb2 may play an important role in transcriptional regulation of encystation genes, and may help co‐ordinate synthesis of cyst wall proteins and polysaccharide. gMyb2 is the first giardial transcription factor to be functionally identified and the first that is associated with upregulation of encystation genes. This work provides a model for study of differential gene regulation in early diverging eukaryotic organisms.

Giardia lamblia-induced changes in gene expression in differentiated Caco-2 human intestinal epithelial cells.

ABSTRACT The parasitic protozoan Giardia lamblia is a worldwide cause of diarrhea, but the mechanism of disease remains elusive. The parasite colonizes the small intestinal epithelium, known to be a sensor for the presence of enteric pathogens, without invading or causing severe inflammation. In this study we investigated the epithelial cell response to G. lamblia. Differentiated Caco-2 cells were infected with G. lamblia isolate WB-A11, and the transcriptome of the intestinal cells was analyzed after 1.5, 6, and 18 h of interaction, using oligonucleotide microarrays. A large number of genes displayed changed expression patterns, showing the complexity of the interaction between G. lamblia and intestinal cells. A novel chemokine profile (CCL2, CCL20, CXCL1, CXCL2, and CXCL3) was induced that was different from the response induced by enteric pathogens causing intestinal inflammation. Several genes involved in stress regulation changed their expression. These findings indicate that the intestinal epithelium senses the G. lamblia infection, and this is important for induction of innate and adaptive immunity. The induced stress response can be important in the pathogenesis.

Organelle proteomics reveals cargo maturation mechanisms associated with Golgi-like encystation vesicles in the early-diverged protozoan Giardia lamblia

During encystation Giardia trophozoites secrete a fibrillar extracellular matrix of glycans and cyst wall proteins on the cell surface. The cyst wall material is accumulated in encystation-specific vesicles (ESVs), specialized Golgi-like compartments generated de novo, after export from the endoplasmic reticulum (ER) and before secretion. These large post-ER vesicles neither have the morphological characteristics of Golgi cisternae nor sorting functions, but may represent an evolutionary early form of the Golgi-like maturation compartment. Because little is known about the genesis and maturation of ESVs, we used a limited proteomics approach to discover novel proteins that are specific for developing ESVs or associated peripherally with these organelles. Unexpectedly, we identified cytoplasmic and luminal factors of the ER quality control system on two-dimensional electrophoresis gels, i.e. several proteasome subunits and HSP70-BiP. We show that BiP is exported to ESVs and retrieved via its C-terminal KDEL signal from ESVs. In contrast, cytoplasmic proteasome complexes undergo a developmentally regulated re-localization to ESVs during encystation. This suggests that maturation of bulk exported cyst wall material in the Golgi-like ESVs involves both continuous activity of ER-associated quality control mechanisms and retrograde Golgi to ER transport.

Comparison of the AF146527 and B1 Repeated Elements, Two Real-Time PCR Targets Used for Detection of Toxoplasma gondii

ABSTRACT Previous studies have reported the increased sensitivity of PCR targeting AF146527 over that of PCR targeting the B1 gene for diagnosis of toxoplasmosis. The present study suggests that the AF146527 element was absent in 4.8% of human Toxoplasma gondii-positive samples tested. The data argue that the B1 gene may be the preferred diagnostic target.

Transcriptome analyses of the Giardia lamblia life cycle

We quantified mRNA abundance from 10 stages in the Giardia lamblia life cycle in vitro using Serial Analysis of Gene Expression (SAGE). 163 abundant transcripts were expressed constitutively. 71 transcripts were upregulated specifically during excystation and 42 during encystation. Nonetheless, the transcriptomes of cysts and trophozoites showed major differences. SAGE detected co-expressed clusters of 284 transcripts differentially expressed in cysts and excyzoites and 287 transcripts in vegetative trophozoites and encysting cells. All clusters included known genes and pathways as well as proteins unique to Giardia or diplomonads. SAGE analysis of the Giardia life cycle identified a number of kinases, phosphatases, and DNA replication proteins involved in excystation and encystation, which could be important for examining the roles of cell signaling in giardial differentiation. Overall, these data pave the way for directed gene discovery and a better understanding of the biology of G. lamblia.

Synchronisation of Giardia lamblia : Identification of cell cycle stage-specific genes and a differentiation restriction point

The intestinal parasite Giardia lamblia undergoes cell differentiations that entail entry into and departure from the replicative cell cycle. The pathophysiology of giardiasis depends directly upon the ability of the trophozoite form to replicate in the host upper small intestine. Thus, cell proliferation is tightly linked to disease. However, studies of cell cycle regulation in Giardia have been hampered by the inability to synchronise cultures. Here we report that Giardia isolates of the major human genotypes A and B can be synchronised using aphidicolin, a mycotoxin that reversibly inhibits replicative DNA polymerases in eukaryotic cells. Aphidicolin arrests Giardia trophozoites in the early DNA synthesis (S) phase of the cell cycle. We identified a set of cell cycle orthologues in the Giardia genome using bioinformatic analyses and showed that synchronised parasites express these genes in a cell cycle stage-specific manner. The synchronisation method also showed that during encystation, exit from the ordinary cell cycle occurs preferentially in G(2) and defines a restriction point for differentiation. Synchronisation opens up possibilities for further molecular and cell biological studies of chromosome replication, mitosis and segregation of the complex cytoskeleton in Giardia.

Secretory antibodies against Giardia intestinalis in lactating Nicaraguan women

Secretory IgA (sIgA) antibodies are important in the host defence against the intestinal protozoan parasite Giardia intestinalis. However, few antigens have been identified. In this study 100 milk and saliva samples from lactating women, living in an endemic region (León, Nicaragua), were screened for the presence of antibodies against G. intestinalis. Most milk and saliva samples contained anti‐Giardia antibodies (59% and 52%, respectively), with a mean sIgA content 50 times higher in milk than in saliva. The positive samples reacted with trophozoite membrane, flagella and cytoplasmic antigens. Western blot analysis showed that milk and saliva anti‐Giardia sIgA recognized up to 16 different Giardia proteins in the molecular weight region 20–165 kDa. Two‐dimensional Western blotting showed that the major immunoreactive proteins were the same as the immunoreactive proteins identified by serum from acute giardiasis patients in a non‐endemic country. The major difference was a stronger reactivity against the variant surface proteins (VSPs) in the milk samples. Milk sIgAs also recognized recombinant Giardia proteins such as alpha‐1 giardin, ornithine carbamoyl transferase, VSP‐4EX, arginine deaminase and alpha‐enolase. These antigens will be important targets in the development of new immunodiagnostic tools and vaccines.

Gene expression changes during Giardia-host cell interactions in serum-free medium.

Serial Analysis of Gene Expression (SAGE) was used to quantify transcriptional changes in Giardia intestinalis during its interaction with human intestinal epithelial cells (IECs, HT-29) in serum free M199 medium. Transcriptional changes were compared to those in trophozoites alone in M199 and in TYI-S-33 Giardia growth medium. In total, 90 genes were differentially expressed, mainly those involved in cellular redox homeostasis, metabolism and small molecule transport but also cysteine proteases and structural proteins of the giardin family. Only 29 genes changed their expression due to IEC interaction and the rest were due to M199 medium. Although our findings generated a small dataset, it was consistent with our earlier microarray studies performed under different interaction conditions. This study has confined the number of genes in Giardia to a small subset that specifically change their expression due to interaction with IECs.