Giardia lamblia is a medically important protozoan parasite with a basal position in the eukaryotic lineage and is an interesting model to explain the evolution of biochemical events in eukaryotic cells. G. lamblia trophozoites undergo significant changes in order to survive outside the intestine of their host by differentiating into infective cysts. In the present study, we characterize the previously identified Orf-C4 (G. lamblia open reading frame C4) gene, which is considered to be specific to G. lamblia. It encodes a 22 kDa protein that assembles into high-molecular-mass complexes during the entire life cycle of the parasite. ORF-C4 localizes to the cytoplasm of trophozoites and cysts, and forms large spherical aggregates when overexpressed. ORF-C4 overexpression and down-regulation do not affect trophozoite viability; however, differentiation into cysts is slightly delayed when the expression of ORF-C4 is down-regulated. In addition, ORF-C4 protein expression is modified under specific stress-inducing conditions. Neither orthologous proteins nor conserved domains are found in databases by conventional sequence analysis of the predicted protein. However, ORF-C4 contains a region which is similar structurally to the α-crystallin domain of sHsps (small heat-shock proteins). In the present study, we show the potential role of ORF-C4 as a small chaperone which is involved in the response to stress (including encystation) in G. lamblia.

INTRODUCTION

Giardia lamblia is a flagellated parasite of humans and other vertebrates and is one of the main causes of intestinal diarrhoea worldwide [1]. Although G. lamblia's phylogenetic position is still under debate [2,3], the unique biological characteristics of this early branching eukaryote are certainly fascinating since it shares genetic, metabolic and functional characteristics with prokaryotes [1,3].

G. lamblia alternates between two morphologically and metabolically distinct forms during its life cycle. Trophozoites colonize the host upper small intestine and proliferate while they are attached to epithelial cells. To survive outside the intestine, they differentiate into environmentally resistant cysts, which are eliminated with faeces, transmitting the disease among different hosts [1]. Encystation is an adaptive mechanism that involves the up-regulation of specific genes, such as those encoding CWPs (cyst wall proteins) [4,5], as well as the biogenesis of ESVs (encystation-specific secretory vesicles) that transport cyst-wall components that are assembled extracellularly into a protective cell wall [6,7].

Orf-C4 (G. lamblia open reading frame C4, which corresponds to ORF 50803_13747 in the Giardia Genome Database) is a gene considered unique to G. lamblia since no closely related genes have been found in sequences databases [810]. Although it was initially described as specific to G. lamblia assemblage B [8], we [11] and others [9,10] have demonstrated that it is present and expressed in the other human-infecting genotype, the morphologically indistinguishable assemblage A. Taking advantage of this fact, Orf-C4 has been successfully used in RFLP (restriction-fragment-length polymorphism) PCR and oligonucleotide microarray analyses to detect and differentiate G. lamblia from other pathogenic waterborne protozoa found in biological samples, and to genetically discriminate betweeen assemblages A and B, based on an almost 30% dissimilarity in their gene sequences [9,10].

The broad expression of Orf-C4 in G. lamblia and its presence as a single copy in the genome suggest an active role of its product in the biology of the parasite. In the present work, we have characterized Orf-C4, which codes for ORF-C4, a cytoplasmic 22 kDa protein that assembles into high-molecular-mass complexes. We discuss its putative role as an α-crystallin-type sHsp [small Hsp (heat-shock protein)] involved in the response to stressful conditions in G. lamblia.

MATERIALS AND METHODS

G. lamblia cultivation and encystation in vitro

Trophozoites belonging to G. lamblia assemblage A (WB isolate, clones 9B10, C6 and 1267) [12,13] were cultured and induced to encyst as described previously [6]. For stress experiments, trophozoites were cultured either in growth medium at 42 °C (for 1 h or 16 h) or at 37 °C (16 h) in growth medium lacking L-cysteine, with the addition of 50 μg/ml brefeldin A or 10 μM ionophore A23187 as described previously [14].

Screening of a cDNA expression library

An assemblage A (WB isolate, clone 1267) G. lamblia cDNA expression library, constructed in λgt22A using polyadenylated RNA from different stages of encystation, was screened by using a pool of mouse polyclonal antibodies generated against encystation organelles as described previously [4].

DNA sequencing and computer-based analyses

DNA sequencing of the clones obtained after library screening was performed at Macrogen (Seoul, Korea). Similarity searches and analyses were performed using BLAST (http://www.ncbi.nlm.nih.gov/BLAST/), SMART (http://smart.embl-heidelberg.de/) and software available at ExPASy (http://ca.expasy.org/). Analyses of Giardia DNA sequences were performed using the BLAST server and the GiardiaDB (http://www.giardiadb.org/giardiadb/) [15]. Local sequence and secondary-structure similarity prediction was performed by HMM (hidden Markov model) by comparison of two different HMMs with HHPred [16], using the pdb70_13Dec07 database, five psiblast max iterations and scoring the predicted secondary structure compared with the predicted secondary structure. Comparative protein-structure modelling of ORF-C4 and Saccharomyces cerevisiae Hsp26 by satisfying spatial restraints was performed with Modeller [17] and three-dimensional structure validation was carried out with MolProbity [18]. For this purpose, the crystal structure of Triticum aestivum Hsp16.9B was used as a template structure (PDB code 1GME, chain A) [19]; only the C-terminal region was modelled as a result of heterogeneity in the N-terminal regions. Wheat Hsp16.9B, ORF-C4 and yeast Hsp26 protein sequences were aligned with MUSCLE [20] and manually curated with Genedoc [21], taking into account secondary structure information. Molecular graphics images were produced using the UCSF Chimera package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco [supported by National Institutes of Health (NIH) grant P41 RR-01081] [22].

Northern blot assays

Total RNA was extracted from trophozoites in different stages of the life cycle. Immobilization and hybridization with radiolabelled probes was performed as described previously [23]. Probes corresponding to G. lamblia dipeptidyl-peptidase IV (GenBank Nucleotide Sequence Database accession number AF293412 [23]) and Cwp2 (GenBank® Nucleotide Sequence Database accession number U28965 [4]) genes were used as controls.

Expression of recombinant ORF-C4 and anti-(ORF-C4) polyclonal antibody generation

The Orf-C4 DNA fragment was amplified with the primers C4B_F (5′-CGGGATCCATGTTCAACCCGAGAC-3′) and C4S_R (5′-TCCCCCGGGTTACTTCTGAATCGT-3′) and cloned into pGEX-4T3. The resulting glutathione transferase fusion protein was expressed in Escherichia coli (Amersham Biosciences) and purified following the manufacturer's instructions. The corresponding band was purified from polyacrylamide gels and emulsified with Freund's adjuvant (Sigma). Giardia-free Balb/c mice, maintained in a SPF (specific pathogen free) facility, were immunized subcutaneously (day 0 and day 14). Antiserum was collected and the specificity of the mouse polyclonal anti-(ORF-C4) antibody was checked by immunofluorescence and Western blotting (results not shown).

Overexpression and down-regulation of ORF-C4

The Orf-C4 sense DNA fragment was tagged with a C-terminal influenza HA (haemagglutinin) epitope by in-frame cloning into the pTubHA.pac plasmid [6], generating HA-tagged ORF-C4 (ORF-C4–HA). The fragment was amplified with the primers C4_F (5′-CATGCCATGGTCAACCCAAGGCACGCC-3′) and C4_R (5′-CAAGATATCCTTCTGAATCGTAATAGG-3′), containing NcoI and EcoRV restriction endonuclease sites respectively. To down-regulate the expression of endogenous Orf-C4, the reverse complement sequence of Orf-C4 was amplified with the primers C4AS_F (5′-CAAGATATCATGTTCAACCCAAGGCAC-3′) and C4AS_R (5′-CATGCCATGGTTCTGAATCGTAATAGG-3′) containing EcoRV and NcoI restriction endonuclease sites respectively, and cloned into the pTubHA.pac vector. Using this approach, the coding region was inserted inversely, leading to the generation of antisense transcripts and resulting in diminished expression of the transcript of interest, as has been demonstrated previously [6]. Overexpression and down-regulation of ORF-C4 was verified by Western blotting and immunofluorescence assays (see the Results section for details). Transfection of G. lamblia trophozoites was performed by electroporation and stable cell lines were selected with 100 μM puromycin as described previously [6]. Transgenic trophozoites were cultivated in growth medium or induced to encyst as described above. Cysts were recovered from the medium and counted in a Coulter Z1 cell counter (Beckman Coulter).

Western blotting and immunofluorescence assays

Cells cultured in growth or encystation medium were harvested and processed for Western blotting as described previously [4]. Immunofluorescence assays were performed as described previously [4,6] using FITC or TRITC (tetramethylrhodamine β-isothiocyanate)-conjugated goat anti-mouse IgG secondary antibodies (Sigma). Specimens were viewed on a Leica IRBE fluorescence microscope or a Zeiss LSM5 Pascal laser-scanning confocal microscope (Plan-Apochromat, ×630 magnification). Primary antibodies used were as follows: mouse monoclonal anti-CWP2 antibody (7D2 [4]), mouse monoclonal anti-CWP1 antibody (5-3C [5]), mouse monoclonal anti-HA antibody (Sigma) and mouse polyclonal anti-(ORF-C4) antibody.

Transmission electron microscopy

Electron microscopy was performed essentially as described previously [6].

Biosynthetic labelling and immunoprecipitation

Assays were performed essentially as described previously [4]. ORF-C4–HA-transfected trophozoites were metabolically labelled with radioactive cysteine and methionine for 1 h and then disrupted in lysis buffer. The cell lysate was pre-cleared using Protein A/G–Sepharose beads (Santa Cruz Biotechnology) and then subsequently subjected to immunoprecipitation using an anti-HA antibody or a specific mouse polyclonal anti-(ORF-C4) antibody (results not shown). After incubating overnight at 4 °C, Protein A/G–Sepharose was added, and the incubation was continued for 4 h. The immunoprecipitates were washed in lysis buffer before being separated by SDS/PAGE under reducing conditions.

Immunofluorescence-based proteinase K protection assay

The assay was performed as described previously [6]. Briefly, trophozoites fixed in 4% (w/v) paraformaldehyde were selectively permeabilized for 15 min at 4 °C with either 100 μg/ml saponin, 50 μg/ml digitonin or 0.05% Tween 20, and then incubated with or without 5 units/ml proteinase K (Sigma) for 1 or 2 h at room temperature (23–25 °C). Cells were subsequently incubated with ice-cold methanol to inactivate proteinase K and to further permeabilize them, and then washed and processed for immunofluorescence.

RESULTS

ORF-C4 sequence analyses

To search for molecules involved in G. lamblia differentiation into cysts, we screened an assemblage A (WB isolate, clone 1267) cDNA encystation library with mouse polyclonal antibodies generated against a protein extract of trophozoites that had been encysting for 1, 2, 4, 6, 8, 12 and 24 h. From this screening, we isolated several cDNA clones coding for Orf-C4. The assembled nucleotide sequence (GenBank® Nucleotide Sequence Database accession number AF293413) results in a predicted protein of 198 amino acids (Figure 1A). ORF-C4 (GenBank® Entrez Protein sequence database accession number AAK97083) displays 72.7% identity at the amino-acid level with the protein described in assemblage B previously [8] and 99.5% identity at the amino-acid level with other assemblage A sequences [9,10].

Structure of G. lamblia ORF-C4

Figure 1
Structure of G. lamblia ORF-C4

(A) Alignment of the amino-acid sequences of G. lamblia ORF-C4 (WB isolate, clone 1267), S. cerevisiae Hsp26 (yHSP26) and T. aestivum Hsp16.9B (wHSP16.9B) (GenBank™ Entrez Protein accession numbers AAK97083, P15992 and Q41560 respectively), which was generated taking into consideration structural information published previously. The secondary structure of T. aestivum Hsp16.9B is depicted above the alignment according to the crystal structure data (PDB code 1GME, chain A [19]). ORF-C4 putative amidation sites are underlined with a broad line, putative phosphorylation sites by casein kinase II are underlined with a fine line and protein kinase C sites are marked with arrowheads. The polyserine, AXXXNG(I/V)L and (I/V)X(I/V) motifs are located at amino acid residues 25–33, 168–175 and 192–194 of the ORF-C4 sequence respectively. (B) Three-dimensional structure comparison showing amino acids 47–151 from T. aestivum Hsp16.9 (wHSP16.9; 1GME, chain A structure), 62–213 from S. cerevisiae Hsp26 (yHSP26) and 80–194 from ORF-C4. ORF-C4 and S. cerevisiae Hsp26 structures were modelled with Modeller.

Figure 1
Structure of G. lamblia ORF-C4

(A) Alignment of the amino-acid sequences of G. lamblia ORF-C4 (WB isolate, clone 1267), S. cerevisiae Hsp26 (yHSP26) and T. aestivum Hsp16.9B (wHSP16.9B) (GenBank™ Entrez Protein accession numbers AAK97083, P15992 and Q41560 respectively), which was generated taking into consideration structural information published previously. The secondary structure of T. aestivum Hsp16.9B is depicted above the alignment according to the crystal structure data (PDB code 1GME, chain A [19]). ORF-C4 putative amidation sites are underlined with a broad line, putative phosphorylation sites by casein kinase II are underlined with a fine line and protein kinase C sites are marked with arrowheads. The polyserine, AXXXNG(I/V)L and (I/V)X(I/V) motifs are located at amino acid residues 25–33, 168–175 and 192–194 of the ORF-C4 sequence respectively. (B) Three-dimensional structure comparison showing amino acids 47–151 from T. aestivum Hsp16.9 (wHSP16.9; 1GME, chain A structure), 62–213 from S. cerevisiae Hsp26 (yHSP26) and 80–194 from ORF-C4. ORF-C4 and S. cerevisiae Hsp26 structures were modelled with Modeller.

BLAST analyses of the ORF-C4 sequences did not display high sequence similarity to any known genes from other organisms. However, when the HHPred server was used for sensitive protein-similarity detection and structure prediction by comparison of two HMMs, ORF-C4 showed local secondary structure and sequence similarity to the Hsp20/α-crystallin family domain of two sHsps. The top hit, with a probability of 59.1%, an E-value of 1.7, a P-value of 1e−04 and an identity of 22%, is the α-crystallin domain of Hsp16.9B from T. aestivum (PDB code 1GME, chain A [19]) (Figure 1A). The second hit corresponds to Hsp16.5 from the hyperthermophilic archaea Methanococcus jannaschii (PDB code 1SHS, chain A [24]).

As a result of the significant but inconclusive similarity detected by the HHPred server, we performed ORF-C4 three-dimensional structure modelling, followed by structure validation. Wheat Hsp16.9B α-crystallin domain was used as the template structure and a fungal sHsp (S. cerevisiae Hsp26) was modelled for comparison. The three structures consist of two antiparallel β-sheets and a large loop, though ORF-C4 has small insertions between β-sheets 3 and 4 and between β-sheets 6 and 7, and yeast Hsp26 has a large insertion between β-sheets 3 and 4 (β-sheets numbered according to results published previously [19]) (Figure 1). Ramachandran plots show that all structures have a similar percentage of amino acids in favoured and allowed regions and this therefore strengthens the idea that a domain similar to α-crystallin is present in ORF-C4 (see Supplementary Figure S1 at http://www.bioscirep.org/bsr/029/bsr0290025add.htm).

The presence of the α-crystallin domain, a C-terminal region of approx. 90 residues involved in extensive subunit–subunit contacts, defines the family of α-crystallin-type Hsps or sHsps sHsps are small and diverse chaperones that assemble into high-molecular-mass oligomers and prevent aggregation of proteins under a wide range of stress conditions, among multiple other functions [2527]. The extent of amino-acid sequence similarity in the α-crystallin domain is variable, ranging from 20% between distant members of the family to over 60% between closely related members [28]. The modelled structures of yeast sHsp and ORF-C4 are structurally similar to the crystallized structures of wheat and archaea sHsps (among the few crystallized sHsp structures), although they are distantly related from an evolutionary standpoint [19,24] (Figure 1B).

ORF-C4 α-crystallin-like domain possesses an ASYANKIV motif, which is very similar to the AXXXNG(I/V)L consensus motif found in sHsps [25,27] (Figure 1A). An N-terminal region and a C-terminal tail flank the ORF-C4 α-crystallin-like domain (Figure 1A). In sHsps, both regions are of variable length, possess considerable sequence diversity and are involved in the assembly of large aggregates [29,30]. Finally, ORF-C4 possesses the sHsp conserved C-terminal (I/V)X(I/V) motif, which participates in multimerization or quaternary structure organization [31].

ORF-C4 is a 22 kDa protein that assembles into high-molecular-mass complexes

To determine the subcellular localization of ORF-C4, a polyclonal antibody was raised in mice against the recombinant protein expressed in E. coli. Indirect immunofluorescence assays were carried out in non-encysting, pre-encysting and encysting trophozoites and in cysts. In all stages of differentiation, ORF-C4 displays a cytoplasmic localization (Figure 2A).

ORF-C4 is a 22 kDa cytoplasmic protein that assembles into high-molecular mass complexes

Figure 2
ORF-C4 is a 22 kDa cytoplasmic protein that assembles into high-molecular mass complexes

(A) Immunolocalization of ORF-C4 (C4) in the cytoplasm of trophozoites and cysts using a polyclonal anti-(ORF-C4) antibody. Cyst walls are visualized with the monoclonal antibody 7D2, which recognizes CWP2. (B) Northern blot assays performed with an Orf-C4 probe and RNA extracted from non-encysting trophozoites (N), pre-encysting trophozoites (P) and trophozoites induced to encyst for 4 or 16 h. Fragments of the constitutively expressed dipeptidyl-peptidase IV gene (dpp) and the developmentally induced CWP2 gene (cwp2) were used as controls. (C) ORF-C4 expression in Western blot assays. Under reducing conditions (RED), ORF-C4 is detected by blotting with a mouse polyclonal anti-(ORF-C4) antibody as a doublet of 22 kDa (C4/22) and a 60–66 kDa band (C4/66) (left-hand panel, 12% polyacrylamide gel). Under non-reducing condition (NRED), ORF-C4 is expressed as high-molecular-mass complexes above 200 kDa (C4) (right-hand panel, 6% polyacrylamide gel). Molecular masses are shown on the right-hand side (in kDa). (D) ORF-C4 expression during the life cycle. Western blot assays were performed under reducing (RED) and non-reducing (NRED) conditions after resolution with 10% polyacrylamide gels of non-encysting trophozoites (N), pre-encysting trophozoites (P) and trophozoites induced to encyst for 1, 2, 4, 8 and 20 h. Blotting was performed using a mouse polyclonal anti-(ORF-C4) antibody. At this gel concentration, the 22 kDa doublet is not resolved and ORF-C4 high-molecular-mass complexes are visualized as a smear trapped in the stacking gel. CWP1 was detected as a control under reducing conditions. Molecular masses are shown on the right-hand side (in kDa). The divide between the stacking and resolution gels is indicated (arrow).

Figure 2
ORF-C4 is a 22 kDa cytoplasmic protein that assembles into high-molecular mass complexes

(A) Immunolocalization of ORF-C4 (C4) in the cytoplasm of trophozoites and cysts using a polyclonal anti-(ORF-C4) antibody. Cyst walls are visualized with the monoclonal antibody 7D2, which recognizes CWP2. (B) Northern blot assays performed with an Orf-C4 probe and RNA extracted from non-encysting trophozoites (N), pre-encysting trophozoites (P) and trophozoites induced to encyst for 4 or 16 h. Fragments of the constitutively expressed dipeptidyl-peptidase IV gene (dpp) and the developmentally induced CWP2 gene (cwp2) were used as controls. (C) ORF-C4 expression in Western blot assays. Under reducing conditions (RED), ORF-C4 is detected by blotting with a mouse polyclonal anti-(ORF-C4) antibody as a doublet of 22 kDa (C4/22) and a 60–66 kDa band (C4/66) (left-hand panel, 12% polyacrylamide gel). Under non-reducing condition (NRED), ORF-C4 is expressed as high-molecular-mass complexes above 200 kDa (C4) (right-hand panel, 6% polyacrylamide gel). Molecular masses are shown on the right-hand side (in kDa). (D) ORF-C4 expression during the life cycle. Western blot assays were performed under reducing (RED) and non-reducing (NRED) conditions after resolution with 10% polyacrylamide gels of non-encysting trophozoites (N), pre-encysting trophozoites (P) and trophozoites induced to encyst for 1, 2, 4, 8 and 20 h. Blotting was performed using a mouse polyclonal anti-(ORF-C4) antibody. At this gel concentration, the 22 kDa doublet is not resolved and ORF-C4 high-molecular-mass complexes are visualized as a smear trapped in the stacking gel. CWP1 was detected as a control under reducing conditions. Molecular masses are shown on the right-hand side (in kDa). The divide between the stacking and resolution gels is indicated (arrow).

Northern blot analyses show that Orf-C4 transcripts are present during the entire life cycle of the parasite and their levels rise slightly during early encystation, and decrease to their original levels later during this process (Figure 2B). In contrast, CWP2 mRNA levels change dramatically from being almost absent in non-encysting trophozoites to very high levels at 16 h of encystation, accumulating throughout the encystation process (Figure 2B) [4].

Protein expression was analysed by Western blotting. Under reducing conditions, the anti-(ORF-C4) polyclonal antibody recognizes a major band of the expected size (22 kDa), as well as a closely migrating band and a 60–66 kDa band in all stages of differentiation (Figures 2C and 2D). This 2-mercaptoethanol/SDS-resistant 60–66 kDa band is clearly suggestive of a complex of the 22 kDa form, since the association of chaperones to other molecules should be disrupted by this treatment. Besides, when metabolic labelling with a mixture of radiolabelled cysteine and methionine was carried out in HA-tagged ORF-C4 transgenic cells (see below), followed by immunoprecipitation using a monoclonal anti-HA antibody or a polyclonal anti-(ORF-C4) antibody (results not shown), reducing SDS/PAGE resulted in only two bands that corresponded to the higher 66 kDa band and the monomer of 22 kDa. Under non-reducing conditions, native ORF-C4 migrates as high-molecular-mass complexes greater than 200 kDa, and this form possibly represents high-ordered C4 complexes interacting with other protein(s) (Figures 2C and 2D).

Although mRNA transcripts seem to be induced slightly earlier during differentiation, protein expression during encystation does not show major changes when compared with the developmentally induced component of the cyst wall, CWP1 (Figure 2D).

ORF-C4 forms large aggregates when overexpressed in G. lamblia

To gain insights into the biological function of ORF-C4, we generated transgenic trophozoites constitutively expressing the C-terminal HA-tagged version of ORF-C4 (ORF-C4–HA) under the control of the α-tubulin promoter. HA-tagging has been proven to be effective for studying G. lamblia protein expression without modifying the folding, localization or function of proteins [4].

ORF-C4–HA expression varies from a diffuse cytoplasmic pattern to granular expression in immunofluorescence studies (Figure 3A). In general, we found only a few large, spherical structures of up to 1 μm in diameter, and often found many small ones scattered throughout the cytoplasm. These structures are unusual, since ORF-C4 does not have a signal peptide that might direct it to the endomembranous system nor putative transmembrane domains or lipid binding/attachment sequences. Some of the granule-like structures are highly dense, resembling the homogeneous ESVs which transport CWPs in encysting trophozoites; however, most of them have a clear space in the centre and are visible as hollow spheres or rings (Figures 4A–4C). These structures do not co-localize with ESVs in encysting trophozoites and are observed in transgenic trophozoites and cysts, regardless of the stage of differentiation (Figure 3A). The large structures generated by ORF-C4 overexpression were not labelled when specific endoplasmic reticulum, peripheral vesicle or plasma membrane markers were used in immunofluorescence assays (results not shown), indicating that they are not formed from those organelles. As shown by electron microscopy, these granule-like structures are not surrounded by membranes and are typically localized near the cell surface or lysosome-like peripheral vacuoles (Figure 4C), and also near the funis, ventral parts and almost on the ventral disc.

Overexpression of ORF-C4 in G. lamblia

Figure 3
Overexpression of ORF-C4 in G. lamblia

(A) HA-tagged ORF-C4 (ORF-C4–HA) G. lamblia trophozoites were cultured in growth, pre-encystation or encystation medium for different times and then processed for immunofluorescence assays using monoclonal anti-HA or 7D2 (anti-CWP2) antibodies. ORF-C4–HA (C4) (green) is expressed in the cytoplasm and granules in the whole life cycle. ORF-C4–HA granules and CWP2 (red) do not co-localize in encysting trophozoites nor in cysts, where CWP2 is localized to ESVs and cyst walls respectively. (B) ORF-C4–HA expression in Western blot assays under reducing (RED) and non-reducing (NRED) conditions using a monoclonal anti-HA antibody (10% polyacrylamide gel). The divide between the stacking and resolution gels is indicated (arrow). Molecular masses are shown on the right-hand side (in kDa). C4/22, monomeric ORF-C4; C4/31, ORF-C4 associated with other proteins; C4/66, multimeric ORF-C4.

Figure 3
Overexpression of ORF-C4 in G. lamblia

(A) HA-tagged ORF-C4 (ORF-C4–HA) G. lamblia trophozoites were cultured in growth, pre-encystation or encystation medium for different times and then processed for immunofluorescence assays using monoclonal anti-HA or 7D2 (anti-CWP2) antibodies. ORF-C4–HA (C4) (green) is expressed in the cytoplasm and granules in the whole life cycle. ORF-C4–HA granules and CWP2 (red) do not co-localize in encysting trophozoites nor in cysts, where CWP2 is localized to ESVs and cyst walls respectively. (B) ORF-C4–HA expression in Western blot assays under reducing (RED) and non-reducing (NRED) conditions using a monoclonal anti-HA antibody (10% polyacrylamide gel). The divide between the stacking and resolution gels is indicated (arrow). Molecular masses are shown on the right-hand side (in kDa). C4/22, monomeric ORF-C4; C4/31, ORF-C4 associated with other proteins; C4/66, multimeric ORF-C4.

Characterization of ORF-C4–HA granule-like structures

Figure 4
Characterization of ORF-C4–HA granule-like structures

(A) Light or (B) confocal microscopy showing a granule with a hollow sphere or ring appearance, generated by transgenic expression of HA-tagged ORF-C4 in trophozoites. Scale bar in (B), 1 μm. (C) Transmission electron microscopy analysis of ORF-C4–HA granules (left-hand panels, arrows) or native ESVs generated during encystation (right panel, extracted from previous work [6]). F, flagella; N, nucleus; PV, lysosome-like peripheral vesicles; VD, ventral disk. Scale bar, 1 μm. (D) Immunofluorescence-based proteinase K protection assay. ORF-C4–HA expressing cells were permeabilized (with digitonin, saponin or Tween 20), incubated in the presence (+PK) or absence (-PK) of proteinase K, and then subjected to immunofluorescence using a monoclonal anti-HA antibody.

Figure 4
Characterization of ORF-C4–HA granule-like structures

(A) Light or (B) confocal microscopy showing a granule with a hollow sphere or ring appearance, generated by transgenic expression of HA-tagged ORF-C4 in trophozoites. Scale bar in (B), 1 μm. (C) Transmission electron microscopy analysis of ORF-C4–HA granules (left-hand panels, arrows) or native ESVs generated during encystation (right panel, extracted from previous work [6]). F, flagella; N, nucleus; PV, lysosome-like peripheral vesicles; VD, ventral disk. Scale bar, 1 μm. (D) Immunofluorescence-based proteinase K protection assay. ORF-C4–HA expressing cells were permeabilized (with digitonin, saponin or Tween 20), incubated in the presence (+PK) or absence (-PK) of proteinase K, and then subjected to immunofluorescence using a monoclonal anti-HA antibody.

To study whether ORF-C4 is packaged into membrane-enclosed vesicles, we performed immunofluorescence-based proteinase K protection assays. For this, cells were selectively permeabilized with different detergents: saponin for plasma-membrane permeabilization, digitonin for both plasma-membrane and nuclear-membrane permeabilization, and Tween 20, which permeabilizes all cellular membranes. Trophozoites were then incubated in the presence or absence of proteinase K and examined by immunofluorescence. Protein digestion by proteinase K results in a loss of fluorescence, unless the proteins of interest are shielded by membranes. All permeabilized ORF-C4–HA-expressing cells showed a typical cytoplasmic and granular expression pattern in the absence of proteinase K treatment (Figure 4D, left-hand panel). When permeabilized cells were treated with proteinase K, cytoplasmic fluorescence disappeared upon digestion, but cytoplasmic granules were hard to digest even after Tween 20 treatment (Figure 4D, right-hand panel). As a control, CWPs were detected in membrane-enclosed ESVs of encysting trophozoites when they were permeabilized by saponin or digitonin before proteinase K treatment, but no fluorescence was observed when all the membranes inside the cells were permeabilized by Tween 20 and then incubated with proteinase K (results not shown). We therefore suggest that ORF-C4 forms tightly packaged non-membrane-enclosed aggregates. This protein–protein aggregation in transgenic cells was supported by Western blotting (Figure 3B). Bands of approx. 22, 31–33 and 60–66 kDa are observed under reducing conditions. Even though these bands are also detected in low concentrations under non-reducing conditions, ORF-C4–HA (as well as native ORF-C4) is predominantly found formed into high-molecular-mass complexes which are retained in the stacking gel (compare Figure 3B and Figure 2C).

ORF-C4 overexpression and down-regulation during encystation

We studied the effects of ORF-C4 overexpression during G. lamblia encystation, in particular on events such as CWP expression, ESV formation and cyst biogenesis. Interestingly, we did not find any remarkable differences in CWP expression patterns between ORF-C4-transfected and non-transfected trophozoites by immunofluorescence (Figure 5A) or Western blotting (Figure 5B). Furthermore, ESV and cyst morphology was identical to that in controls (Figure 5A) and total cyst production was not modified (Figure 5C).

Effects of ORF-C4 overexpression and down-regulation on the expression of CWPs

Figure 5
Effects of ORF-C4 overexpression and down-regulation on the expression of CWPs

G. lamblia ORF-C4–HA transfected (+) and non-transfected (−) trophozoites were cultured in either growth medium (N) or in encystation medium for 1, 6, 24 and 48 h. Native CWP expression was then analysed in trophozoites or cysts by (A) immunofluorescence or by (B) Western blot assays using an 5-3C (anti-CWP1) monoclonal antibody. The blue and red immunofluorescence staining corresponds to DAPI (4′,6-diamidino-2-phenylindole) and anti-CWP1 antibody staining respectively. (C) Cyst production in non-transfected (striped bars), ORF-C4–HA-transfected (white bars) and down-regulated (grey bars) trophozoites cultured in encystation medium for various time points. Results are means±S.D. (n=3).

Figure 5
Effects of ORF-C4 overexpression and down-regulation on the expression of CWPs

G. lamblia ORF-C4–HA transfected (+) and non-transfected (−) trophozoites were cultured in either growth medium (N) or in encystation medium for 1, 6, 24 and 48 h. Native CWP expression was then analysed in trophozoites or cysts by (A) immunofluorescence or by (B) Western blot assays using an 5-3C (anti-CWP1) monoclonal antibody. The blue and red immunofluorescence staining corresponds to DAPI (4′,6-diamidino-2-phenylindole) and anti-CWP1 antibody staining respectively. (C) Cyst production in non-transfected (striped bars), ORF-C4–HA-transfected (white bars) and down-regulated (grey bars) trophozoites cultured in encystation medium for various time points. Results are means±S.D. (n=3).

The role of ORF-C4 was further tested by down-regulating its expression using antisense technology, as performed successfully for many other G. lamblia genes previously [4]. Using this strategy, we found that lowering the expression of ORF-C4 does not affect either encystation or cell viability (Figure 5C). Nevertheless, at the earliest time points of the encystation process, the number of ESVs (results not shown) per encysting trophozoites was reduced compared with the control, and the beginning of cyst-wall formation was delayed (Figure 5C). The time at which this effect is observed coincides with the time when ORF-C4 is up-regulated at the RNA level during encystation.

ORF-C4 response to stress

To test if ORF-C4 expression is triggered or modified by stressful conditions, as has been shown for sHsps from most organisms [27], we examined the effects of some commonly used cellular stress-inducing agents (Figure 6). Expression was not affected by heat-shock or an increase in intracellular calcium levels by ionophore A23187. Cells died when exposed to 42 °C overnight. Interestingly, when trophozoites were deprived of cysteine or exposed to brefeldin A, the 22 kDa form of ORF-C4 was no longer detected, and only the 60–66 kDa band was identified by Western blotting.

ORF-C4 response to stress conditions

Figure 6
ORF-C4 response to stress conditions

Trophozoites were cultured either in growth medium under stress conditions (see the Materials and methods section for details) or in encystation medium at pH 7.0 or pH 7.8. Expression of ORF-C4 and CWP2 was analysed by Western blotting using a polyclonal anti-ORF-C4 antibody and a monoclonal antibody 7D2. C4/22, monomeric ORF-C4; C4/66, multimeric ORF-C4.

Figure 6
ORF-C4 response to stress conditions

Trophozoites were cultured either in growth medium under stress conditions (see the Materials and methods section for details) or in encystation medium at pH 7.0 or pH 7.8. Expression of ORF-C4 and CWP2 was analysed by Western blotting using a polyclonal anti-ORF-C4 antibody and a monoclonal antibody 7D2. C4/22, monomeric ORF-C4; C4/66, multimeric ORF-C4.

DISCUSSION

As an early-divergent protist and because of its parasitic life style, Giardia possesses unique features relevant to understanding the evolution of eukaryotic cells and its potential impact on human disease [1,2]. In the present study, we present evidence that supports the role of ORF-C4 as a putative α-crystallin-type sHsp.

sHsps comprise a protein family of widespread small chaperones (12–47 kDa). Overall sequence similarity between sHsps is low and the common attribute of this family is confined to the α-crystallin domain [2527]. In this regard, ORF-C4 (although previously considered specific to G. lamblia) is a small protein (22 kDa) which contains a domain that is structurally similar to the α-crystallin domain of sHsp, and assembles into high-molecular-mass oligomers.

A hallmark of sHsps is their tendency to assemble into large oligomeric complexes. sHsp monomers are hypothesized to form dimers or trimers (that could correspond to the 60–66 kDa form in ORF-C4), which serve as building blocks for multimeric structures with multiple subunits (up to 40) with a molecular-mass range of 150–1000 kDa [19,24,25]. Associated with sHsp sequence divergence, oligomeric structures present different symmetries and degrees of order [3234]; oligomerization appears to be a highly dynamic process resulting in homo- or hetero-oligomers [3234] and its regulation is associated with post-translational modifications, such as phosphorylation and amidation [30,35]. Apart from the sHsp-conserved motifs involved in oligomerization, ORF-C4 presents putative sites of amidation and phosphorylation and a polyserine motif, similar to the polythreonine motif found in Toxoplasma gondii Hsp29 [36], which is predicted to be a phosphorylation site. Beyond this, we have been unable to demonstrate phosphorylation in ORF-C4 using monoclonal anti-phosphoserine antibodies or by immunoprecipitation of radioactive-phosphate-labelled trophozoites (results not shown).

The non-membrane-enclosed granules generated by the overexpression of ORF-C4 may be a product of protein aggregation occurring only under unregulated expression of this protein. This observation has been already made in many α-crystallin sHsps, either under natural or stressful conditions or generated by mutation or overexpression. For instance, α-crystallins and E. coli IpB form aggregates of micrometre size [33,37], and Hsp25 forms granules after heat-shock treatment [32]. sHsps are a structural component of HSGs (heat-shock granules), large cytoplasmic chaperone complexes found in heat-stressed plant cells that result from an ordered process of sHsp auto-aggregation or recruitment [38]. Mutation or disruption of sHsps is associated with inclusion body formation in CHO (Chinese-hamster ovary) cells [39]. Moreover, in the protozoan T. gondii, overexpression of sHsp20 leads to unusual filamentous pattern formation along the body [40]. Granule-like formation in G. lamblia could be produced by insolubility due to high protein levels, promiscuous association with other proteins, changes in cytoskeleton organization or unpaired post-translational modifications. Our hypothesis is that ORF-C4 overexpression may be considered a stressful condition in G. lamblia, leading to a semi-ordered aggregation of proteins that mostly results in hollow spheres. Nevertheless, these structures are highly different to the multilamellar bodies found to be generated in trophozoites after oxidative stress [41].

All cells have developed mechanisms to resist stressful and harmful conditions. Most sHsps are involved in protection against heat-induced denaturation by prevention of protein aggregation and the response to stress of various origins [25,27]. Others take part in cellular processes, such as membrane quality control, cell growth and signal transduction by different pathways, such as control of redox status and modulation of cytoskeleton dynamics; in parasitic organisms, sHsps play roles in cell adaptation to environmental changes or in the process of differentiation [36].

In G. lamblia, several reports support the presence of a stress response and chaperones involved in protein folding and maturation, such as the endoplasmic reticulum-resident chaperone BiP [14,42]. In the present study, we showed that ORF-C4 is expressed in the cell cytoplasm during the entire life cycle of the parasite as a monomer or in complexes, and that it could be involved in the response to stress. When trophozoites were deprived of cysteine, an essential amino acid for G. lamblia involved in redox control and cell attachment, among other processes [1,43], or exposed to brefeldin A, an inhibitor of cytoplasmic coat-protein recruitment to membranes and subsequently an inhibitor of protein transport from the endoplasmic reticulum [44], the formation of 60–66 kDa complexes was favoured. In agreement with this idea, ORF-C4 possesses the (I/V)X(I/V) motif that is structurally and functionally important for chaperone activity in sHsps [45]. Alternatively, ORF-C4 may have other functions. It lacks glycine and leucine residues in the α-crystallin motif [AXXXNG(I/V)L] that, when mutated, are associated with a reduction or abolition of the chaperone activity of sHsps, besides resulting in unstable oligomerization [46,47].

We also found that ORF-C4 underexpression only produces a slight effect on the velocity of differentiation, suggesting that it is not essential for this process, that its function is redundant in the cell or that it can be compensated for by other proteins. In most organisms, sHsps seem to be dispensable, working in networks with other cellular chaperones [27]. The fact that the expression of Orf-C4 transcripts is increased by a small amount at the early stages of encystation might indicate that, when the entire cell is committed to differentiation, this protein plays a role that may later be performed by other still undefined molecules. The earliest biochemical events which occur during encystation are the rapid activation of a cytoplasmic pathway responsible for production of the carbohydrate portion of the cyst wall, together with de novo biogenesis of the ESVs that transport the protein components of this protective extracellular organelle [6,7]. When trophozoites confront the stimulus for encystation, the rapid synthesis of the molecules involved in these early events must be highly controlled to avoid unnecessary synthesis of cyst wall molecules or to shut down other cellular activities that may lead to cell death. The potential role of ORF-C4 as a chaperone could be important in ensuring the co-ordination in time and space of these molecular events.

In summary, the universal occurrence of sHsps in all kingdoms might be indicative of their early phylogenetic origin. As a result of its basal evolutionary position, G. lamblia has a simplified molecular machinery, cytoskeletal structure and metabolic network compared with later diverging lineages. In the present work, our results suggest that ORF-C4 is a G. lamblia protein that displays features of a sHsp and constitutes the first described member of the α-crystallin-type sHsp family that plays a role in the stress response of this early branching eukaryote.

Abbreviations

     
  • CWP

    cyst wall protein

  •  
  • ESV

    encystation-specific secretory vesicle

  •  
  • HA

    haemagglutinin

  •  
  • HMM

    hidden Markov model

  •  
  • Hsp

    heat-shock protein

  •  
  • ORF-C4

    Giardia lamblia open reading frame C4 protein

  •  
  • sHsp

    small Hsp

FUNDING

This work was supported by the Agencia Nacional para la Promoción de la Ciencia y la Tecnología [grant number PICT-01-15009]; the Consejo Nacional de Investigaciones Científicas y Técnicas [grant number PIP/98 No 910]; and the Catholic University of Córdoba [grant number 2008-023]. M.J.N., R.Q., C.G.P. and N.G. are recipients of CONICET (Consejo Nacional de Investigaciones Científicas y Técnicas) fellowships, and A.M.P. was a recipient of a Fulbright Commission fellowship. H.D.L. is a member of the Scientist Career of CONICET and is an International Research Scholar of the Howard Hughes Medical Institute.

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Author notes

1

These authors contributed equally to this work.

The nucleotide sequence data reported for Giardia lamblia C4 protein gene Orf-C4 will appear in the DDBJ, EMBL, GenBank™ and GSDB Nucleotide Sequence Databases under the accession number AF293413.

Supplementary data