DNA microarray analysis of protozoanparasite gene expression: outcomescorrelate with mechanisms ofregulationRobert Duncan

Division of Emerging and Transfusion Transmitted Diseases, Center for Biologics Evaluation and Research (CBER),

Food and Drug Administration (FDA), 1401 Rockville Pk, Rockville, MD 20852, USA

DNA microarray analysis has been successfully applied

to most of the protozoan parasites that cause human

disease, but has not made equal progress in all cases.

The results for kinetoplastid parasites (Leishmania and

Trypanosoma) are primarily at the stage of validation

and new gene discovery. By contrast, the results for api-

complexan parasites (Plasmodium and Toxoplasma)

have advanced to the analysis of coordinate regulation

of clusters of genes. This difference in progress relates

to the more complete genome sequence identified for

the apicomplexans and, more significantly, to the differ-

ences in the regulation of gene expression between

these two groups.

DNA microarray is a high-density microscopic arrange-ment of immobilized samples of nucleic acid on a glassslide. Hybridization with fluorescent cDNAs synthesizedfrom RNA samples of interest permits the evaluation ofgene expression on a genomic scale [1–3]. This techniquehas been successfully applied to a range of biologicalquestions including the study of human cancer [4], themetamorphosis of fruit flies [5] and the dissection of theyeast cell cycle [6]. DNA microarrays of various typeshave been developed and applied to studying the majorprotozoan parasites that cause human disease [7,8]. Thesophistication of the information derived from thesestudies varies by species, primarily owing to the under-lying biology of these diverse organisms (despite beingcommonly discussed as a single group) and the extent ofprogress in genome sequencing for each species. DNAmicroarray analysis has also been applied to helminthparasites [9,10], but their evaluation is beyond the scope ofthis article.

Progress in microarray analysis

Microarray analysis holds the promise of tracking theexpression pattern of a large collection of genes simul-taneously. Expression patterns might change over time, orin response to different environmental conditions, or withstages of internal physiological change, or between strains

or cell types. This kind of analysis could produce data thatsuggest the coordinate regulation of groups of genes, andcould shed light on how multiple genes and their productsintegrate into pathways to determine the functioning ofcells and organisms. This promise has been fulfilled to itsgreatest extent in the human, mouse and yeast systems,where knowledge of the genome sequence and microarraystudies are extensive [11]. By contrast, in the initial stageof application of microarray technology, where a largecollection of characterized genes is not available, infor-mation from microarray studies largely provides vali-dation of the method and selection of clones for furthercharacterization. The presentations of results that charac-terize these different stages in the development of micro-array analysis are illustrated in Figure 1. At the validationstage (Figure 1a), data from microarray images is com-pared with traditional methods of gene expression analy-sis such as northern blots or reverse transcriptase (RT)-PCR. At a more sophisticated level (Figure 1b), genescan be grouped by their patterns of expression andfunction to examine coordinate regulation and physiologi-cal pathways.

Microarray progress with protozoan parasites

As a tool to discover new genes and to identify coordinateexpression of families of genes, the microarray has alreadyproven effective for the apicomplexan parasites Plasmo-dium and Toxoplasma. However, results to date for thekinetoplastid parasites Trypanosoma and Leishmaniahave not advanced substantially beyond the stage ofvalidation and new gene discovery.

Although the first Plasmodium microarray studiespresented largely validation data and new gene discovery(Table 1; [12]), subsequent studies began to show patternsof coordinate regulation. For instance, in a microarray ofknown Plasmodium falciparum cDNAs used to assessgene expression at five different stages of erythrocytedevelopment (Table 1; [13]), cluster analysis revealedcommon patterns of expression of groups of genes involvedin carbohydrate metabolism, adhesion/invasion and trans-lation machinery. This analysis demonstrated how mul-tiple components of a pathway are expressed at theCorresponding author: Robert Duncan (duncan@cber.fda.gov).

Opinion TRENDS in Parasitology Vol.20 No.5 May 2004

www.sciencedirect.com 1471-4922/$ - see front matter. Published by Elsevier Ltd. doi:10.1016/j.pt.2004.02.008

Figure 1. Microarray data presentation. At the initial phase of development of microarray analysis for an organism (a), images of fluorescent hybridization (as shown in the

top row for hypothetical genes 1 through 4) are compared with northern blot hybridizations of total RNA from the two stages being evaluated using the genes as radioactive

probes (as shown in the lower two rows). This type of analysis validates the microarray technique and can lead to discovery of new genes. At a more advanced stage, when

the DNA samples on the array have been sequenced and assigned functions (b), results from multiple microarray hybridizations can be combined and displayed graphically

with cluster analysis. In this hypothetical example, the mean relative signal intensity compared with a reference sample for genes 1 through 26 is indicated for two distinct

life stages and two intermediate time points during the transition between the stages. The scale to the right assigns a relative expression level to each color. Across the top

are hypothetical functional groups that might cluster together and below is an interpretation of the expression pattern indicated by the colored rectangles for each func-

tional group. Abbreviations: stage 1-Gr, stage 1 RNA sample labeled with green fluorophore; stage 2-Rd, stage 2 RNA sample labeled with red fluorophore.

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TRENDS in Parasitology

Relativeexpression level

Table 1. Microarray studies of protozoan parasites

Species Microarray type Array clones Results Genome sequenced Ref.

Plasmodium falciparum Genomic 3648 New genes 100%a [12]

P. falciparum CDNA 944 New genes; coordinate regulation 100% [13]

P. falciparum Oligo 6272 Coordinate regulation 100% [14]

Toxoplasma gondii CDNA 4319 New genes; coordinate regulation 100%b [15]

Trypanosoma brucei Genomic 21 024 New genes 100%c [17]

Trypanosoma cruzi Genomic and cDNA 4321 New genes WGSd [18]

Leishmania major Genomic 10 464 New genes Nearly completede [19]

L. major CDNA 2091 New genes Nearly completed [21]

L. major Genomic 10 479 New genes Nearly completed [20]

Leishmania donovani Genomic 2304 New genes 3%f [8]

aComplete sequence released with annotation (http://www.plasmodb.org).bTenfold coverage, not annotated (http://www.toxodb.org/about_data.shtml).cRelease 1, December 16, 2003 (http://www.genedb.org/genedb/tryp/index.jsp).dWhole genome shotgun (WGS) approach has yielded 759.07 Mb of sequence (19-fold coverage) that is searchable, but not completely assembled or annotated (http://www.

tigr.org/tdb/e2k1/tca1/wgsStatus.shtml).e7837 of the estimated 8000 genes are annotated, complete genome near release (http://www.genedb.org/genedb/leish/index.jsp).fThere is no genome-sequencing project for L. donovani. There are 573 entries in the NCBI Entrez nucleotide database (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi) with

‘Leishmania donovani’ in the ‘Organism’ field with an average length, computed by sampling, of 1625. Thus, 573 £ 1625 ¼ 931 125 nucleotides in total. If there were no

redundancy (which there certainly is), then ,3% of the estimated 33.6 Mb genome has been reported. However, significant genomic information about L. donovani can be

obtained from L. major sequence because of the high level of nucleotide identity (,85% or more for protein coding sequence and some flanking sequences).

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appropriate stage in a way not possible by earlier tech-niques that analyzed gene expression one by one. Asfurther advancement, a more sophisticated Plasmodiummicroarray spotted with open-reading frame (ORF)-specificoligonucleotides (Table 1; [14]) showed coordinated expres-sion of ribosomal proteins, tRNA synthetases, initiationand elongation factors, helicases and chaperones thatexpands the understanding of protein translation activityin the trophozoite stage. The authors intend to extend thisanalysis to the global gene-expression profile of the 48 herythrocytic life cycle with 1 h resolution [14].

The application of microarray analysis to Toxoplasmagondii gene expression has also moved beyond validationand gene discovery alone. Changes in gene expressionbetween the rapidly dividing tachyzoite stage and theslow-growing, immune-system-avoiding bradyzoites wereevaluated on a cDNA array (Table 1; [15]). Cluster analysisof these results identified coordinate bradyzoite expressionof clones that encode cell-surface proteins that could aidin immune avoidance. Clustering also showed a groupof bradyzoite-downregulated clones encoding metabolicenzymes that reinforces arguments for differences in sugarmetabolism in this stage. Transiently expressed clonesalso cluster, which may be essential to the developmentalprocess. The same microarray, used to compare global geneexpression in wild-type cell lines with chemically induced,tachyzoite-to-bradyzoite differentiation-defective cell lines(Table 1; [16]), allowed the construction of a model ofhierarchical gene activation during bradyzoite develop-ment. These results set the stage to focus on genes thatoccupy key positions in the developmental pathway ofToxoplasma.

In contrast to the apicomplexan microarray reports,studies of kinetoplastids have not advanced significantlybeyond validation of the data and discovery of new genes.A genomic microarray study of Trypanosoma brucei, thepathogen causing African sleeping sickness (Table 1; [17]),compared gene expression in cultured procyclic (insect)stagetrypanosomes with cultured blood-stage (mammalian-infecting form) trypanosomes. Hybridizations identified75% of the clones as expressed sequence, with ,300 clonesdifferentially expressed. The microarray results were veri-fied by the expression pattern of known genes andnorthern blots or semi-quantitative RT–PCR for newclones. The results established the effectiveness of themicroarray as a method to assess expression of a largecollection of potential genes and the 20 new, differen-tially regulated genes described represents a substantialadvance in the knowledge of T. brucei genomics. However,as the authors suggest, more functional analysis will beneeded before the patterns of expression can be inter-preted as coordinate regulation.

The other Trypanosome that causes human disease,Trypanosoma cruzi, is the most recent protozoan parasiteto be examined by microarray analysis. The study reportedchanges in gene expression as the circulating trypomasti-gote form was induced to differentiate axenically intothe normally intracellular amastigote form (Table 1; [18]).A total of 37 new putative genes were assigned tofunctional categories and array results were validated bycomparison to quantitative RT–PCR, but no coordinate

regulation was discussed. The authors commented thatthe modest changes in mRNA abundance could be due tothe post-transcriptional control of gene expression in thisparasite.

In the other kinetoplastid genus, Leishmania, severalstudies in two species have used microarray analysis.Spotted arrays showed changes in RNA abundance fornumerous clones between significantly different stages inthe life cycles of the parasites; yet the quantitativedifferences have been modest and the incomplete annota-tion of their genomes limits the conclusions that can bedrawn about coordinate gene regulation. In Leishmaniamajor, which is the species that causes cutaneous leish-maniasis, a randomly sheared genomic microarray wasapplied to the changes in gene expression in culturedparasites differentiating from the rapidly growing, pro-cyclic stage to the infectious, metacyclic stage, both ofwhich are found in the insect vector in nature (Table 1; [19]).The microarray results showed upregulation in procyclicsof genes that are known to be active in that stage, such asthose encoding b-tubulin, histones and ribosomal proteins,supporting the validity of the technique. In metacyclics,HASP/geneB, SHERP/geneD, META1 and HSP70 wereamong those found to be upregulated. Many moredifferentially regulated clones were identified that didnot have homology to proteins of known function. Thevalidity of the microarray results was further affirmed bycomparison with northern blots, with 33/47 of the clonestested showing regulation as predicted [19]. Thus, thetechnique was established as an effective tool to find newgenes that are expressed in a pattern that suggests stage-specific importance. However, no pattern of coordinateexpression of functionally related or pathway-specificgenes was described.

A second application of the same randomly shearedgenomic clones of L. major was also used to evaluate geneexpression as procyclic promastigotes differentiated inculture into metacyclic promastigotes (Table 1; [20]).Though extensive homology searches using clones withstatistically significant expression patterns assignedputative genes to functional categories, the major contri-bution of this study is in validation of the array and newgene discovery, more than in coordinate regulation.

Using a L. major cDNA array (Table 1; [21]), the geneexpression in one reference sample of day 5 culturedL. donovani promastigotes was compared with L. majorLV39 RNA samples from days 3, 5, 7 and 10 of pro-mastigote culture and to lesion-derived amastigotes (theparasite stage found in mammalian macrophages). Theclones were clustered into ten patterns of expressionacross the five samples, although the functional signifi-cance of these clusters is unclear. This analysis identified147/1092 (13.5%) unique genes that were significantlyupregulated in amastigotes. The results were well vali-dated, new genes were discovered and the 147 amastigote-expressed genes were identified. Taking the microarrayresults in a more applied direction, genes selected asamastigote expressed were spliced into DNA vaccineconstructs and some of those tested protected mice fromlive parasite challenge, encouraging the authors to pursuea high-throughput strategy to screen all the potential

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amastigote-expressed genes [21]. Therefore, this micro-array made a major contribution to the rapid analysis ofgenome scale information, but has not advanced ourunderstandingofcoordinategeneexpressioninLeishmania.

Gene expression in Leishmania donovani, which causesvisceral leishmaniasis, was compared between culturedpromastigotes and in vitro-differentiated, axenic amasti-gotes on a genomic microarray (Table 1; [8]). The resultsidentified many clones as expressed sequence that havepotential to be new genes due to the small amount of theL. donovani genome sequenced (Table 1). Of the expressedclones, 10% showed changes in abundance by a factor of 1.5or more. Validation of microarray results with northernblots and comparison with expression patterns of knowngenes was presented. However, the changes in geneexpression were small (very few more than twofold) andthere were not enough clones with assigned biologicalfunction to draw conclusions about patterns of coordinateregulation. The array will be expanded to 10 000 genomicclones, but the particular biology of the kinetoplastidparasites might defy use of the microarray to understandcoordinate regulation of gene expression.

Why differences in progress?

Results from the application of the microarray to kineto-plastid parasites remain at the level of validation andidentification of new genes, whereas microarray resultshave achieved a more global analysis of coordinate expres-sion in apicomplexan parasites. This difference can beattributed to the more complete sequencing and annota-tion of the apicomplexan genomes, but perhaps moresignificant is the basic differences in the mechanisms ofgene expression between these two groups.

Apicomplexans initiate monocistronic transcriptionfrom promoters structurally similar to higher eukaryotes[22–24]. Thus, mRNA abundance (the quantity measuredin a microarray hybridization) can be regulated at ini-tiation of transcription. The changes in abundance ofregulated mRNAs reported in microarray publicationsare commonly from fourfold to as high as 28-fold forPlasmodium and Toxoplasma [13–15]. By contrast,kinetoplastids transcribe protein-coding genes as largepolycistronic molecules, initiated without defined pro-moters, and subsequently process them into maturemRNA [25–27]. The abundance of a particular mRNAspecies in kinetoplastids is controlled largely by its stability,which is regulated by sequences in the 30-untranslatedregion [28–31]. Consequently, changes in abundance areoften not as pronounced as in organisms with generegulation at the level of transcription initiation such asbacteria, apicomplexans and higher eukaryotes. The smallchanges in mRNA abundance in kinetoplastids is indi-cated by the fact that microarray publications reportreproducible changes in gene expression as low as 1.2-fold[20] and few are greater than threefold [8,18,19,21]. Thesemodest changes in mRNA abundance are between lifecycle stages that are as distinct in terms of morphology,mitotic activity and biochemistry as are the stagesexamined for apicomplexans. Recognizing that geneexpression is ultimately determined by the abundance offunctionally active protein, the problem of assessing gene

expression that underlies the key phenotypes determiningpathogenesis in kinetoplastids may be overcome by other,protein-based methods. Unfortunately, such techniquesare likely to lengthen substantially the process fromscreening to the identification and isolation of new genes.However, greater differences in mRNA abundance mightalso be revealed in the pool of actively translatedmessages, which can be enriched by centrifugal isolationof polyribosome-associated RNA. This approach is sup-ported by the observation that, in the kinetoplastidT. cruzi, mRNA for the metacyclogenin gene is presentin differentiating cells, but is absent from proliferat-ing cells, when the polyribosome fraction is isolated;whereas, it is equally represented in these two cell typeswhen total RNA is examined [32]. Labeling and hybrid-ization of polyribosome-associated mRNA or other, as-yet-undiscovered, novel approaches might allow microarraysto reveal the patterns of gene expression that will lead to abetter understanding of kinetoplastid physiology andpathogenesis. Additionally, continued progress in genomesequencing and annotation will allow production of micro-arrays comprised of better-characterized gene probes thatwill improve the information content of microarray data.

Perspective

The next few years should be exciting as microarrays areapplied to questions of gene expression that have beendifficult to address previously. Complex phenotypes arelikely to be the summation of the effect of multiple genes.Screening techniques that have looked for such genes inthe past have either measured small groups of genes afew at a time or have measured differential RNA levelsthat were not reproducible. With the microarray, largeunbiased collections of genes can be screened simul-taneously for their role in crucial traits or stages anda manageable number selected for further analysis.Furthermore, especially in the apicomplexan parasites,the analysis of coordinate expression of groups of func-tionally related genes can begin to unravel the mysteries ofprotozoan parasite biology and the diseases caused bythese organisms.

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The Drugs for Neglected Diseases Initiative:a call for concerted action

In 1999, Medecins Sans Frontieres (http://www.msf.org/) received the Nobel Peace Prize. The prize money sponsored the launch, in July

2003, of the Drugs for Neglected Diseases initiative (DNDi)*, which aims to research and develop new drugs or new formulations of

existing drugs for patients suffering from the most neglected communicable diseases. The first International Meeting of DNDi since its

launch was held in Malaysia, 6–10 February 2004. Recognizing that there has been very little progress in the management of these

diseases for decades, in addition to little current research and development of new health tools, DNDi intends to initiate and coordinate

drug development in collaboration with the international research community, the public sector, the pharmaceutical industry, and other

relevant parties. This not-for-profit model, driven by the needs of the poor, and built upon solid North–South and South–South

collaborations, plans to develop new medicines and stimulate relevant public responsibility and leadership.

The inequity and inadequacy of the current market driven research and development agenda is illustrated by the management of

kala azar and African trypanosomiasis. These two of the most neglected infections are among the initial focus of the DNDi.

Both diseases are on the rise, affect the very poor almost exclusively and have antiquated, toxic treatments. Antimony and

arsenic were introduced as specific treatments for kala azar and African trypanosomiasis, respectively, nearly a century ago. Arsenicals

kill ~5% of those receiving treatment for sleeping sickness. These treatments are also in short supply and are failing because of

resistance problems. There has been a considerable investment in biological research on the kinetoplastids, reflected by the 25 000

references in the scientific literature, but this has yet to be translated into a benefit for patients. This sad state of affairs is remediable, but

it will take concerted effort. DNDi will employ a variety of means to achieve its objectives including invitations for needs-driven research

projects, especially in conjunction with disease-endemic countries. To succeed, DNDi will need the active participation and commitment

of scientists. Researchers are best placed to identify, among their published and unpublished findings, the potential benefits arising

from their scientific endeavours. Please think about these problems, support and, if possible, participate in this worthy initiative,

to provide ‘the best science to the most neglected’.

For more information on DNDi, go to: http://www.dndi.org or email: info@dndi.org

*The founding members of The Drugs for Neglected Diseases Initiative include: Medecins Sans Frontieres; Oswaldo Cruz

Foundation/Far Manguinhos, Brazil; Indian Council of Medical Research; Kenya Medical Research Institute; Institut Pasteur; the Ministry

of Health of Malaysia; and WHO/TDR as a permanent observer.

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