A comparison of the Giardia lamblia trophozoite and cyst transcriptome using microarrays
© Faghiri and Widmer; licensee BioMed Central Ltd. 2011
Received: 5 January 2011
Accepted: 4 May 2011
Published: 4 May 2011
Compared with many protists, Giardia lamblia has a simple life cycle alternating between cyst and trophozoite. Most research on the molecular biology of Giardia parasites has focused on trophozoites and the processes of excystation and encystation, whereas cysts have attracted less interest. The striking morphological differences between the dormant cyst and the rapidly dividing and motile trophozoite implies profound changes in the metabolism as the parasite encysts in the host's intestine and excysts upon ingestion by a new host.
To investigate the magnitude of the transcriptional changes occurring during the G. lamblia life cycle we compared the transcriptome of G. lamblia trophozoites and cysts using single-color oligonucleotide microarrays. Cysts were found to possess a much smaller transcriptome, both in terms of mRNA diversity and abundance. Genes encoding proteins related to ribosomal functions are highly over-represented. The comparison of the transcriptome of cysts generated in culture or extracted from feces revealed little overlap, raising the possibility of significant biological differences between the two types of cysts.
The comparison of the G. lamblia cyst and trophozoite transcriptome showed that transcripts of most genes are present at a lower level in cysts. This global view of the cyst and trophozoite transcriptome complements studies focused on the expression of selected genes during trophozoite multiplication, encystation and excystation.
Giardia lamblia (G. duodenalis, G. intestinalis) is a diplomonad parasite which causes over 20,000 reported cases of giardiasis a year in the United States . In addition to its importance as a widespread human and animal pathogen, the long evolutionary history of the diplomonad lineage makes G. lamblia an interesting system for studying eukaryotic evolution and the evolution of parasitism. Research on G. lamblia is aided by the fact that the entire life cycle can be studied outside the host, and that the differentiation from cyst to trophozoite and the reverse process of encystation can be reproduced in vitro. Recently, the availability of the complete genome sequence [2–5] have facilitated genome-wide analyses.
Although many Giardia proteins and organelles have been studied in detail, genome-wide studies of the transcriptome and proteome have been few [6–11]. No microarray analyses of the transcriptome of cysts obtained from infected animals have to our knowledge been performed. Serial Analysis of Gene Expression (SAGE) was used to survey changes in the G. lamblia transcriptome during encystation and excystation . This study grouped about 10% of predicted G. lamblia genes into six clusters with related transcriptional profile. SAGE was also used to analyze the relative abundance of transcripts encoding cytoskeleton proteins . This analysis found that the level of mRNA transcripts encoding proteins localized in the adhesive decreases as the parasite encysts, and also found a lack of association between mRNA and protein level. Morf and co-workers focused on transcriptional changes associated with encystation . This study used micoarrays to identify genes which are induced during encystation and found evidence of transcriptional co-regulation mediated by a shared transcription factor binding motif in the promoter region of such genes.
The extensive morphological changes which take place during the parasite's life cycle have for years motivated the study of transcriptional regulation of selected genes during differentiation. Reverse-transcription PCR has been frequently used to monitor changes in the level of specific mRNA transcripts, such as those encoding enzymes involved in energy metabolism , recombination , structural functions  or regulatory functions .
We wished to compare on a global level the transcriptional landscape of trophozoites and cysts. We found that in cysts many genes are either not transcribed, or that the transcripts they encode are too rare to be detected with microarrays.
Analysis of the cyst transcriptome
Although the cyst and trophozoite transcriptome compared in these experiments both belonged to assemblage B, we investigated whether sequence polymorphism between the assemblage A sequence on which the G. lamblia microarray is based and assemblage B probe could reduce hybridization. Using the same single-color experimental design, we compared fluorescence values for microarrays hybridized with cDNA from assemblage A and B trophozoites (Additional file 1). Means of Cy3 fluorescence over all G. lamblia spots on the array for the assemblage B probe was 3.0 × 105, 2.2 × 105, and 2.9 × 105 fluorescence units, whereas for assemblage A probe mean fluorescence of 0.9 × 105, 1.5 × 105 and 3.2 × 105 were obtained. Thus, the fact that probe and array are derived from different assemblages does not influence the results. These results are consistent with the interpretation of Figure 1.
Gene ID and annotation of 14 most expressed cyst and trophozoite genes
ribosomal protein S10B
dynein light chain
ribosomal protein L18a
ribosomal protein S15A
variant surface protein
ribosomal protein L36-1
ribosomal protein L26
variant surface protein
ribosomal protein L10
variant surface protein
ribosomal protein S17
variant surface protein
Validation of microarray data
Giardia troph antigen GTA-1
endothelin-converting enzyme 2
mitotic spindle checkpt. MAD2
Summary of quantitative PCR validation
troph. 24 h
troph. 72 h
24 h troph/cysts*
troph antig GTA-1
Comparison of SAGE and microarray cyst transcriptome
Expression of histone and histone modifying enzymes
Histone and histone modification enzymes*
trophozoites (n = 3)
cysts (n = 6)
Histone acetyltransf. MYST2
Histone acetyltransf. B sub. 2
The fact that the entire life cycle of G. lamblia can be reproduced in vitro makes this species an attractive model to study the differentiation of cyst into trophozoite and the reverse process of encystation. Recently, genome-wide studies of G. lamblia transcriptional regulation have been undertaken [9, 12] but no global comparison of the cyst and trophozoite transcriptome has to our knowledge been published. The study of the trophozoite and cyst transcriptome is relevant to understanding the G. lamblia life cycle and the evolution of encysted forms which are essential to the survival of many enteric organisms. Given that cysts don't divide and are assumed to have little metabolic activity, it is likely that for many proteins in cysts no mRNA is present. Combined transcriptome and proteome analyses  will generate a more comprehensive view of the composition and metabolic activity of cysts.
Microarray and RT PCR data clearly show that the cyst transcriptome is much reduced in terms of abundance and complexity as compared to that of trophozoites. DAVID analysis of over-represented GO terms  suggests an overall resemblance in the composition of the transcriptome throughout the life cycle, but the analysis of highly expressed genes highlights significant differences.
As in most quantitative analyses, the comparison of microarray data required calibration against a benchmark. As described in Methods below, we used RNA quantity of as benchmark by using an equal amount of amplified RNA for preparing Cy3 labelled probes. The differences in transcript levels are thus to be interpreted as relative to total RNA extracted from cysts and trophozoites.
To what extent rRNA and tRNA which constitutes the bulk of cellular RNA varies is unknown. An alternative calibration would have been to normalize the data against the number of cysts, trophozoites or nuclei. This approach was discarded because of the possibility that extraction of RNA from cysts is less efficient than extraction from trophozoites. Had we chosen to normalize against cell number, it would have been difficult to assess whether differences between cyst and trophozoite were genuine or a result of cyst nucleic acid being more difficult to extract.
The experiments were constrained by the fact that G. lamblia microarrays are designed from the assemblage A genome and that the only source of cysts we could identify uses assemblage B. Because DNA sequence identity between assemblage A and B genome averages 77% , the possibility that analyzing assemblage B cyst cDNA with assemblage A microarrays could artificially reduce the hybridization signal was considered. Replicate microarray hybridizations were performed with cDNA originating from assemblage A and B trophozoites (Additional file 1). These controls showed no evidence of differential hybridization of cDNA originating from different assemblages under the hybridization conditions we used. This does not exclude that highly polymorphic transcripts were missed, but indicates that for the vast majority of genes annealing to the 70 mer microarray oligonucleotides was sufficiently stable to tolerate mismatches. Moreover, the vast majority of fluorescent signal from Arabidopsis control spots and empty spots present on the array were well below background (mean Cy3 fluorescence = 1552, n = 3860), confirming the specificity of the hybridization signal and demonstrating adequate stringency of the hybridization protocol.
Because we expected significant differences in the magnitude and diversity of cyst and trophozoite mRNA transcriptome we did not directly compare trophozoite and cyst transcriptome using a conventional 2-color microarray protocol. Two-color microarrays require normalization to eliminate the effect of differential labelling of dyes, which is typically accomplished with microarray analysis programs . These programs normalize Cy3 and Cy5 fluorescence based on the assumption that the samples being compared contain similar amounts of mRNA, as would be the cases with, say, healthy and diseased cells. Since we did not expect this assumption to hold, we chose to use only background-subtracted single-channel Cy3 fluorescence values. Since these data originated from calibrated amounts of Cy3 labelled probe, the resulting data are directly comparable. In the context of this study, an additional advantage of the single-dye design over a more conventional Cy3/Cy5 ratio is the feasibility to include fluorescence values below background, i.e., values equal zero. Since a large proportion of transcripts were not detected in cysts, the exclusion of ratios with a numerator or denominator equal zero would have excluded biologically relevant information.
The elevated expression of some genes observed in the microarray dataset confirms previous observations. For instance, we found high levels of ubiquitin mRNA in trophozoites and cysts, which is consistent with previous RT PCR analyses . The expression of ubiquitin in trophozoites is not unexpected, but the abundance of ubiquitin mRNA in cysts suggests extensive protein turn-over. Other top-ranking genes in cysts and trophozoites include histone. This observation is consistent with the constitutive expression of various histones during the trophozoite mitotic cycle , but had not been observed previously in cysts. The absence of mRNA encoding histone modifying enzymes suggests that histone modification does not occur in cysts, and is consistent with many genes not being transcribed in this phase of the life cycle. This interpretation is in agreement with the previously observed decrease of histone acetylation during trophozoite encystation and the predicted importance of epigenetic regulation of transcription in the life cycle of G. lamblia . Finally, we notice the unexpected expression in cysts of several genes encoding variant surface protein.
The comparison of SAGE and microarray data raises interesting questions regarding the properties of cysts produced in culture. Cysts encysted in vitro have been extensively characterized with respect to morphology, antigenic property , and cyst wall biosynthesis , as have many processes occurring during encystation. A direct comparison of the transcriptome and proteome of native cysts and cyst produced in vitro has to our knowledge not been performed. In light of the results presented here, such an analysis is warranted to assess to what extent cysts produced in vitro can serve as surrogates for native cysts. As RNA-Seq has become a more widely available technique for transcriptome profiling, an accurate comparison of the cyst transcriptome is now feasible.
The transcriptome of G. lamblia cysts and trophozoites was investigated using oligonucleotide microarrays. Although in both life cycle stages transcripts related to ribosomal function are overrepresented, clear quantitative differences were observed. This global comparison of the cyst and trophozoite transcriptome indicates that, in comparison to trophozoites, in cysts only about 5% of mRNA species are expressed at level detectable with microarrays.
G. lamblia cysts and trophozoites
G. lamblia cysts of assemblage B isolate H3 from experimentally infected gerbils were purchased from Waterborne (New Orleans, Louisiana). Cyst viability was assessed by monitoring exclusion of propidium iodide as described . Cysts were processed for RNA extraction within five days of shedding. Trophozoites of assemblage A isolate WB and assemblage B isolate GS were cultured in TYI-S-33 medium . Trophozoites grown for 24 h or 72 h were counted with a hemocytometer, pelleted by centrifugation and washed in PBS prior to RNA extraction.
RNA extraction, amplification and microarrays
Total RNA for microarray analysis was isolated using Trizol from trophozoites and cysts following 5 cycles of freeze/thawing. DNA was removed using the TurboDNase kit from Applied Biosystems/Ambion (Austin, Texas) and the RNA extracted with Qiagen RNeasy columns (Qiagen, Valencia, California) according to the RNA cleanup protocol. RNA quality was checked by running a portion of selected samples on an agarose gel and measuring absorbance at 260 nm and 280 nm. RNA was amplified in vitro with the WT-Ovation Pico RNA Amplification System (NuGEN, San Carlos, California). For the amplification reaction up to 5 μl of total RNA sample (50 ng) was used as substrate. A total of 2 μg cDNA was labelled using a Genomic DNA Enzymatic Labeling Kit from Agilent (Santa Clara, California).
Oligonucleotide microarrays were provided by the National Institutes of Allergy and Infectious Diseases (NIAID) Pathogen Functional Genomics Research Center. The arrays (Giardia lamblia microarray version 2) contain 19,230 elements consisting of duplicates of 70 mer oligomers derived from 9,115 predicted open-reading frames (ORFs) including the clearly indentified 6,470 ORFs of the genome of G. lamblia WB C6 (assemblage A). Also spotted on the slides are 500 Arabidopsis thaliana control oligomers. To prehybridize, slides were placed in a coplin jar containing 50 ml preheated prehybridization buffer (20× SSC, 10% SDS, 0.5 g BSA) and incubated at 42°C for 2 hr. Slides were then washed using filtered distilled water and isopropyl alcohol for 2 m and dried by centrifugation. To perform hybridization, labeled cDNA was dissolved in 50 μl of hybridization buffer (40% formamide, 5× SSC, 0.1% SDS, 0.1 M DTT). In some experiments 2 μl of universal microarray standard set was added to the probe mixture, and the probe denatured for 10 min at 95°C. a volume of 50 μl of probe was added to microarray slide and covered with LifterSlip coverslips (Erie Scientific, Portsmouth, New Hampshire). Slides were incubated in a 42°C water bath for 16-20 h. For post-hybridization wash slides were first submerged into a low stringency solution (2 × SSC, 0.1% SDS) preheated to 55°C and washed twice for 5 min each on a shaker. Slides were subsequently washed twice in medium stringency solution (0.1× SSC, 0.1% SDS), followed by two more 5-min washes at high stringency (0.1× SSC) at room temperature. Slides were dried in a centrifuge and scanned in an Agilent scanner.
Files in TIFF format generated by the scanner were imported into TIGR_Spotfinder software . Spots were manually curated to exclude artifactual spots and background cut-off was set at 5%. Cy3 fluorescence values output by Spotfinder were exported to Microsoft Excel. Fluorescence values from duplicate spots were averaged and the mean over six cyst biological replicates determined. Each cyst expression value used in the analyses is thus based on 12 individual fluorescence reading. For trophozoites, two microarray hybridizations were performed with GS trophozoites and three with WB trophozoites, for a total of four and eight fluorescence readings per gene. The DAVID suite of bioinformatics tools was used to identify functional annotations which are enriched as compared to the G. lamblia genome annotation. The program was accessed through the web interface at http://david.abcc.ncifcrf.gov/tools.jsp.
RT- PCR validation
cDNA amplified in vitro as described above was diluted 100-fold and 1 μl of this dilution was amplified by PCR. PCR was performed in 20-μl capillary tubes using a LightCycler (Roche Diagnostics, Indianapolis, Indiana) thermal cycler. Reaction mixtures contained 1× LC-Fast Start DNA master mix for SYBR Green I (Roche Diagnostics), 3 mM MgCl2, 20 pmol each of forward and reverse primers, and 1 μl of cDNA template. The primer sequences are shown in Table 2. The PCR program included a denaturation step of 10 min at 95°C followed by 45 cycles of 1 s at 95°C, annealing for 8-9 s, and a 8-s extension at 72°C. Following amplification, the PCR products were subjected to melting curve analysis by raising the temperature from 45 to 95°C at a rate of 0.05°C/s. During the initial optimization phase PCR products were also electrophoresed on agarose gels to ensure that products of the correct size were amplified. Because trophozoites and cysts originated from assemblage A and B, respectively, we verified that the PCR results were not affected by the genotype. Equivalent amounts of DNA from assemblage A isolate WB and assemblage B isolate GS were amplified in parallel using primers specific for portion of the ubiquitin, histone H2B and 14-3-3 protein shown in Table 2. No systematic bias that could be linked to the genotype was observed.
The comments and views detailed herein may not necessarily reflect the views of the WateReuse Research Foundation, its officers, directors, employees, affiliates or agents.
Microarray data were deposited in the GEO database [GPL:11228].
We gratefully acknowledge the WateReuse Research Foundation's financial, technical, and administrative assistance in funding and managing the project through which this information was discovered. This project was funded in part by the National Institute of Allergy and Infectious Diseases (grant AI083719). Giardia lamblia microarrays and universal standard probe were obtained through NIAID's Pathogen Functional Genomics Resource Center, managed and funded by the Division of Microbiology and Infectious Diseases, NIAID, NIH, DHHS and operated by the J. Craig Venter Institute. Our thanks to Phyllis Spatrick, UMass Worcester Genomics core facility, for help with microarray scanning and to the WateReuse Foundation Project Advisory Committee (Collin Balcombe, Walter Jakubowski, Paul Rochelle, Hal Stibbs, Shawn Thompson) for valuable advice and feedback.
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