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The transcriptional response to encystation stimuli in Giardia lamblia is restricted to a small set of genes.

ABSTRACT: The protozoan parasite Giardia lamblia undergoes stage differentiation in the small intestine of the host to an environmentally resistant and infectious cyst. Encystation involves the secretion of an extracellular matrix comprised of cyst wall proteins (CWPs) and a ?(1-3)-GalNAc homopolymer. Upon the induction of encystation, genes coding for CWPs are switched on, and mRNAs coding for a Myb transcription factor and enzymes involved in cyst wall glycan synthesis are upregulated. Encystation in vitro is triggered by several protocols, which call for changes in bile concentrations or availability of lipids, and elevated pH. However, the conditions for induction are not standardized and we predicted significant protocol-specific side effects. This makes reliable identification of encystation factors difficult. Here, we exploited the possibility of inducing encystation with two different protocols, which we show to be equally effective, for a comparative mRNA profile analysis. The standard encystation protocol induced a bipartite transcriptional response with surprisingly minor involvement of stress genes. A comparative analysis revealed a core set of only 18 encystation genes and showed that a majority of genes was indeed upregulated as a side effect of inducing conditions. We also established a Myb binding sequence as a signature motif in encystation promoters, suggesting coordinated regulation of these factors.

SUBMITTER: Morf L

PROVIDER: S-EPMC2950437 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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The transcriptional response to encystation stimuli in Giardia lamblia is restricted to a small set of genes.

Morf Laura L Spycher Cornelia C Rehrauer Hubert H Fournier Catharine Aquino CA Morrison Hilary G HG Hehl Adrian B AB

Eukaryotic cell 20100806 10

The protozoan parasite Giardia lamblia undergoes stage differentiation in the small intestine of the host to an environmentally resistant and infectious cyst. Encystation involves the secretion of an extracellular matrix comprised of cyst wall proteins (CWPs) and a β(1-3)-GalNAc homopolymer. Upon the induction of encystation, genes coding for CWPs are switched on, and mRNAs coding for a Myb transcription factor and enzymes involved in cyst wall glycan synthesis are upregulated. Encystation in vi ...[more]

PMID: 20693303

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Project description:BackgroundEncystation is one of the two processes comprising the life cycle of Giardia lamblia, a protozoan pathogen with tetraploid genome. Giardia lamblia Myb2 (GlMyb2) is a distinct encystation-induced transcription factor whose binding sites are found in the promoter regions of many encystation-induced genes, including its own.MethodsTwo sequential CRISPR/Cas9 experiments were performed to remove four glmyb2 alleles. The expression level of G. lamblia cyst wall protein 1 (GlCWP1), a well-known target gene of GlMyb2, was measured via western blotting and immunofluorescence assays. Chromatin immunoprecipitation experiments using anti-GlMyb2 antibodies were performed on the encysting G. lamblia cells. Quantitative real-time PCR was performed to confirm an expression of candidate GlMyb2-regulated genes by comparing the transcript level for each target candidate in wild-type and knockout mutant Giardia. The promoter region of glcwp1 was analyzed via deletion and point mutagenesis of the putative GlMyb2 binding sites in luciferase reporters.ResultsCharacterization of the null glmyb2 mutant indicated loss of functions related to encystation, i.e. cyst formation, and expression of GlCWP1. The addition of the wild-type glmyb2 gene to the null mutant restored the defects in encystation. Chromatin immunoprecipitation experiments revealed dozens of target genes. Nineteen genes were confirmed as GlMyb2 regulons, which include the glmyb2 gene, six for cyst wall proteins, five for signal transduction, two for transporter, two for metabolic enzymes, and three with unknown functions. Detailed analysis on the promoter region of glcwp1 defined three GlMyb2 binding sites important in its encystation-induced expression.ConclusionsOur data confirm that GlMyb2 acts as a transcription activator especially during encystation by comparing the glmyb2 knockout mutant with the wild type. Further investigation using glmyb2 null mutant will provide knowledge regarding transcriptional apparatus required for the encystation process of G. lamblia.

Project description:UnlabelledEncystation of the common intestinal parasite Giardia lamblia involves the production, trafficking, and secretion of cyst wall material (CWM). However, the molecular mechanism responsible for the regulation of these sequential processes remains elusive. Here, we examined the role of GlRac, Giardia's sole Rho family GTPase, in the regulation of endomembrane organization and cyst wall protein (CWP) trafficking. Localization studies indicated that GlRac is associated with the endoplasmic reticulum (ER) and the Golgi apparatus-like encystation-specific vesicles (ESVs). Constitutive GlRac signaling increased levels of the ER marker PDI2, induced ER swelling, reduced overall CWP1 production, and promoted the early maturation of ESVs. Quantitative analysis of cells expressing constitutively active hemagglutinin (HA)-tagged GlRac (HA-Rac(CA)) revealed fewer but larger ESVs than control cells. Consistent with the phenotype of premature maturation of ESVs in HA-Rac(CA)-expressing cells, constitutive GlRac signaling resulted in increased CWP1 secretion and, conversely, morpholino depletion of GlRac blocked CWP1 secretion. Wild-type cells unexpectedly secreted large quantities of CWP1 into the medium, and free CWP1 was used cooperatively during cyst formation. These results, in part, could account for the previously reported observation that G. lamblia encysts more efficiently at high cell densities. These studies of GlRac show that it regulates encystation at several levels, and our findings support its coordinating role as a regulator of CWP trafficking and secretion. The central role of GlRac in regulating membrane trafficking and the cytoskeleton, both of which are essential to Giardia parasitism, further suggests its potential as a novel target for drug development to treat giardiasis.ImportanceThe encystation process is crucial for the transmission of giardiasis and the life cycle of many protists. Encystation for Giardia lamblia involves the assembly of a protective cyst wall via sequential production, trafficking, and secretion of cyst wall material. However, the regulatory pathways that coordinate cargo maturation and secretion remain unknown. Here, we asked whether the signaling activities of G. lamblia's single Rho family GTPase, GlRac, might have a regulatory role in the encystation process. We show that GlRac localizes to endomembranes and its signaling activities regulate the production of cyst wall protein 1 (CWP1), the maturation of encystation-specific vesicles (ESVs), and secretion of CWP1. We also show that secreted CWP1 is available for the development of cysts at the population level, a finding that in part could explain why Giardia encystation proceeds more efficiently at high cell densities.

Project description:BackgroundGiardia passes through two stages during its life cycle, the trophozoite and the cyst. Cyst formation involves the synthesis of cyst wall proteins (CWPs) and the transport of CWPs into encystation-specific vesicles (ESVs). Active vesicular trafficking is essential for encystation, but the molecular machinery driving vesicular trafficking remains unknown. The Rab proteins are involved in the targeting of vesicles to several intracellular compartments through their association with cytoskeletal motor proteins.Methodology and principal findingsIn this study, we found a relationship between Rab11 and the actin cytoskeleton in CWP1 transport. Confocal microscopy showed Rab11 was distributed throughout the entire trophozoite, while in cysts it was translocated to the periphery of the cell, where it colocalized with ESVs and microfilaments. Encystation was also accompanied by changes in rab11 mRNA expression. To evaluate the role of microfilaments in encystation, the cells were treated with latrunculin A. Scanning electron microscopy showed this treatment resulted in morphological damages to encysted parasites. The intensity of fluorescence-labeled Rab11 and CWP1 in ESVs and cyst walls was reduced, and rab11 and cwp1 mRNA levels were down-regulated. Furthermore, knocking down Rab11 with a hammerhead ribozyme resulted in an up to 80% down-regulation of rab11 mRNA. Although this knockdown did not appear lethal for trophozoites and did not affect cwp1 expression during the encystation, confocal images showed CWP1 was redistributed throughout the cytosol.Conclusions and significanceOur results indicate that Rab11 participates in the early and late encystation stages by regulating CWP1 localization and the actin-mediated transport of ESVs towards the periphery. In addition, alterations in the dynamics of actin affected rab11 and cwp1 expression. Our results provide new information about the molecules involved in Giardia encystation and suggest that Rab11 and actin may be useful as novel pharmacological targets.

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The transcriptional response to encystation stimuli in Giardia lamblia is restricted to a small set of genes.

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The transcriptional response to encystation stimuli in Giardia lamblia is restricted to a small set of genes.

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