Gametocyte Stages Research Articles

Excystation of Eimeria tenella sporozoites impaired by antibody recognizing gametocyte/oocyst antigens GAM22 and GAM56.

Eimeria tenella is the causative agent of coccidiosis in poultry. Infection of the chicken intestine begins with ingestion of sporulated oocysts releasing sporocysts, which in turn release invasive sporozoites. The monoclonal antibody E2E5 recognizes wall-forming body type II (WFBII) in gametocytes and the WFBII-derived inner wall of oocysts. Here we describe that this antibody also binds to the stieda body of sporocysts and significantly impairs in vitro excystation of sporozoites. Using affinity chromatography and protein sequence analysis, E2E5 is shown to recognize EtGAM56, the E. tenella ortholog of the Eimeria maxima gametocyte-specific GAM56 protein. In addition, this antibody was used to screen a genomic phage display library presenting E. tenella antigens as fusion proteins with the gene VIII product on the surfaces of phagemid particles and identified the novel 22-kDa histidine- and proline-rich protein EtGAM22. The Etgam22 mRNA is expressed predominantly at the gametocyte stage, as detected by Northern blotting. Southern blot analysis in combination with data from the E. tenella genome project revealed that Etgam22 is an intronless multicopy gene, with approximately 12 to 22 copies in head-to-tail arrangement. Conspicuously, Etgam56 is also intronless and is localized adjacent to another gam56-like gene, Etgam59. Our data suggest that amplification is common for genes encoding oocyst wall proteins.

A pre-PEXEL histidine-rich protein II erythrocyte binding peptide as a new way for anti-malarial vaccine development

The Plasmodium falciparum malaria parasite produces several proteins characterised by an unusually high histidine content in infected red blood cells (iRBC). The histidine-rich protein II (HRP-II) is synthesised throughout the parasite’s asexual and gametocyte stages, transported through the parasitophorous vacuole (PV) to iRBC cytosol and membrane and released to the bloodstream via a PEXEL motif. Immunogenicity and protection-inducing studies were begun with an RBC high activity binding peptide (HABP) from this protein named 6800 (preceding the PEXEL motif) in the experimental Aotus monkey model. Modifying critical residues (determined by glycine scanning in this HABP) induced immunogenicity and protection against experimental challenge. Native 6800 did not bind to any HLA-DRβ 1 ∗ molecule, but these modified HABPs acquired the ability to specifically bind to HLA-DRβ 1 ∗0701. 1H NMR studies revealed that whilst 6800 had a random structure, modified immunogenic and protection-inducing 24230 displayed very short α-helical segments allowing appropriate binding to the MHCII-pep-TCR complex. Modifications in conserved HABPs preceding PEXEL motifs thus open up new avenues for subunit-based, multi-component synthetic anti-malarial vaccine development.

Improved synchronous production of Plasmodium falciparum gametocytes in vitro

The sexual stages of the Plasmodium falciparum life cycle are attractive targets for vaccines and transmission blocking drugs. Difficulties in culturing and obtaining large amounts of sexual stage P. falciparum parasites, particularly early stages, have often limited research progress in this area. We present a new protocol which simplifies the process of stimulating gametocytogenesis leading to improved synchronous gametocyte production. This new method can be adapted to enrich for early stage gametocytes (I and II) with a higher degree of purity than has previously been achieved, using MACS® magnetic affinity columns. The protocol described lends itself to large scale culturing and harvesting of synchronous parasites suitable for biochemical assays, northern blots, flow cytometry, microarrays and proteomic analysis.

Transcription and coregulation of multigene families in Plasmodium falciparum

Subtelomeric regions in the Plasmodium genome are enriched with members of three major multigene families: var, rif and stevor. In this article, we discuss a recent study by Sharp and colleagues that focused on the transcribed repertoire of var and stevor genes in asexual and sexual Plasmodium parasites. Transcription of these two multigene families seems to be unlinked in asexual parasites. Parasites in the gametocyte stages transcribe members of the upsC subset of var genes, which indicates the intriguing possibility of their involvement in gametocyte adhesion.

Pfnek3: An atypical activator of a MAP kinase in Plasmodium falciparum

The canonical mitogen-activated protein kinase (MAPK) signal cascade was previously suggested to be atypical in the malaria parasite. This raises queries on the existence of alternative mediators of plasmodial MAPK pathways. This study describes, Pfnek3, a malarial protein kinase belonging to the NIMA (Never in Mitosis, Aspergillus) family. Endogenous Pfnek3 is expressed during late asexual to gametocyte stages and lacks some classical protein kinase sequence motifs. Moreover, Pfnek3 is phylogenetically distant from mammalian NIMA-kinases. Recombinant Pfnek3 was able to phosphorylate and stimulate a malarial MAPK (Pfmap2). Contrastingly, this was not observed with two other kinases, Pfmap1 and human MAPK1, suggesting that the Pfnek3–Pfmap2 interaction may be specific for Pfmap2 regulation. In summary, our data reveal a malarial NIMA-kinase with the potential to regulate a MAPK. Possessing biochemical properties divergent from classical mammalian NIMA-kinases, Pfnek3 could potentially be an attractive target for parasite-selective anti-malarials.

A ubiquitous Plasmodium protein displays a unique surface labeling pattern in sporozoites

The Plasmodium sporozoite is infective for mosquito salivary glands and vertebrate host tissues. Although it is a key developmental stage of the malaria parasite, relatively few sporozoite surface or secreted proteins have been identified and characterized. Herein, we describe the molecular and cellular characterization of a novel surface molecule that is preferentially-expressed in salivary gland sporozoites as compared to oocyst and hemolymph sporozoites. This molecule, designated the sporozoite and erythrocytic stages (SES) protein (formerly known as Pg4), exhibits a spiral surface labeling pattern that spans over a known sporozoite surface antigen, the circumsporozoite protein, with only minor co-localization. SES consists of 551 amino acids encoding a putative 63.2 kDa protein that has been shown to be expressed not only on particular sporozoite stages, but also during the asexual and gametocyte stages. This novel protein also has three domains of unknown function that are conserved in at least eight Plasmodium spp. that represent human, avian, non-human primate, and rodent malarias.

Recombinant Expression and Biochemical Characterization of the Unique Elongating β-Ketoacyl-Acyl Carrier Protein Synthase Involved in Fatty Acid Biosynthesis of Plasmodium falciparum Using Natural and Artificial Substrates

The human malaria parasite Plasmodium falciparum synthesizes fatty acids by using a type II synthase that is structurally different from the type I system found in eukaryotes. Because of this difference and the vital role of fatty acids, the enzymes involved in fatty acid biosynthesis of P. falciparum represent interesting targets for the development of new antimalarial drugs. beta-Ketoacyl-acyl carrier protein (ACP) synthase (PfFabBF), being the only elongating beta-ketoacyl-ACP synthase in P. falciparum, is a potential candidate for inhibition. In this study we present the cloning, expression, purification, and characterization of PfFabBF. Soluble protein was obtained when PfFabBF was expressed as a NusA fusion protein in Escherichia coli BL21(DE3)-CodonPlus-RIL cells under conditions of osmotic stress. The fusion protein was purified by affinity and ion exchange chromatography. Various acyl-P. falciparum acyl carrier protein (PfACP) substrates were tested for their specific activities, and their kinetic parameters were determined. Activity of PfFabBF was highest with C(4:0)- to C(10:0)-acyl-PfACPs and decreased with use of longer chain acyl-PfACPs. Consistent with the fatty acid synthesis profile found in the parasite cell, no activity could be detected with C(16:0)-PfACP, indicating that the enzyme is lacking the capability of elongating acyl chains that are longer than 14 carbon atoms. PfFabBF was found to be specific for acyl-PfACPs, and it displayed much lower activities with the corresponding acyl-CoAs. Furthermore, PfFabBF was shown to be sensitive to cerulenin and thiolactomycin, known inhibitors of beta-ketoacyl-ACP synthases. These results represent an important step toward the evaluation of P. falciparum beta-ketoacyl-ACP synthase as a novel antimalaria target.

Population biology and antimalarial resistance: The transmission of antimalarial drug resistance in Plasmodium falciparum

Malaria morbidity and mortality continue to increase across sub-Saharan Africa. This is largely as a result of the continued use of chloroquine and sulfadoxine-pyrimethamine, despite widespread resistance. Although eliminating the asexual stages of Plasmodium falciparum is the focus of treatment of individual symptomatic patients, at a population level, reducing the carriage of gametocytes – the sexual stage responsible for infection of the mosquito vector – is necessary to limit the transmission of malaria parasites and the spread of antimalarial resistance. The probability of a mosquito being infected depends on the prevalence, duration and density of viable gametocyte carriage in the human host, although additional humoral and leukocyte factors also affect transmissibility. There is a log–sigmoid relationship between gametocyte density in the patients’ blood and infectivity to the mosquito. The infectivity and thus transmission potential associated with a particular antimalarial treatment can be characterised as a function of blood gametocyte density and time, summing these over the acute and all subsequent recrudescences of that infection. Gametocyte carriage and infectivity to mosquitoes is consistently higher in patients infected with drug resistant compared with drug sensitive malaria parasites. It is the ratio of transmission potential in drug resistant versus sensitive infections that drives the spread of resistance. Early access to highly effective antimalarial treatment reduces the risk of disease progression and limits gametocyte carriage. The remarkable spread of sulfadoxine-pyrimethamine (SP) resistance across vast regions results from the very high post-treatment prevalence and density of gametocyte carriage following SP treatment. In areas of low intensity malaria transmission, the gametocyte-reducing effect of widespread use of artemisinin-based combination therapy has resulted in a sustained decrease in malaria transmission and a decrease in the spread of resistance. Malaria treatment policy should be based primarily on therapeutic efficacy against asexual stages, but should also consider transmission reduction potential. Artemisinin-based combination therapies are the only antimalarials currently available which rapidly reduce both asexual and gametocyte stages of the P. falciparum lifecycle.

Characterization of PfPuf2, Member of the Puf Family RNA-Binding Proteins from the Malaria ParasitePlasmodium falciparum

Puf proteins are a family of evolutionarily conserved translational regulators in eukaryotes. The malaria parasite has two Puf proteins (PfPuf1 and PfPuf2) that share 25% homology in the RNA binding domain. Here we confirmed the preferential expression of PfPuf2 in gametocyte stages using Northern analysis. The transcriptional initiation site of this gene, mapped using RNA ligase-mediated rapid amplification of cDNA end and primer extension, is located approximately 300 bp upstream from the translational start codon. The 3' end of PfPuf2 is located approximately 250 bp downstream from the stop codon. The total length of the RNA is approximately 2.1 kb, consistent with the mRNA size determined by Northern analysis. Recombinant PfPuf2 proteins expressed in bacteria were purified and used to produce polyclonal antibodies. Western blot further established the preferential synthesis of PfPuf2 in gametocyte stages. Using the Nanos-responsive elements (NRE) in the Hunchback mRNA of Drosophila melanogaster as an artificial target sequence, we tested the binding of PfPuf2 Puf domain to this sequence using the yeast three-hybrid system. The results showed that PfPuf2 Puf domain bound specifically to NRE, suggesting that PfPuf2 may be involved in translational regulation of target genes using a conserved mechanism of the Puf family proteins.

Quantification of Plasmodium falciparum gametocytes in differential stages of development by quantitative nucleic acid sequence-based amplification.

Two quantitative nucleic acid sequence-based amplification assays (QT-NASBA) based on Pfs16 and Pfs25, have been developed to quantify sexual stage commitment and mature gametocytes of Plasmodium falciparum. Pfs16 mRNA is expressed in all sexual forms including sexually committed ring stages while expression of Pfs25 mRNA is restricted to late stage gametocytes. Both assays showed a sensitivity of one sexual stage parasite/microl of blood. Blood samples from experimentally infected non-immune human volunteers were tested for Plasmodium falciparum by standard microscopy, a previously developed asexual 18S rRNA QT-NASBA, Pfs16 and Pfs25 mRNA QT-NASBA. Pfs16 QT-NASBA was positive in 9 out of 10 volunteers within 48 h after first detection of 18S rRNA, mostly before or at the day of positive microscopy. In contrast, the Pfs25 mRNA QT-NASBA was negative during the 28 days of follow-up, but consistently positive in gametocyte samples from naturally infected Kenyan patients. These data suggest that sexual stage commitment can occur early in the blood-stage infection without successful maturation into infectious gametocytes. In conclusion, Pfs16 and Pfs25 QT-NASBA assays in combination with a previously developed asexual stage QT-NASBA allow for the separate quantification of all developmental stages present in the circulation. The application of sexual stage QT-NASBA assays may contribute to a better understanding of the biology and epidemiology of malaria transmission.

Characterisation of the antigenic and immunogenic properties of bacterially expressed, sexual stage antigens of the coccidian parasite, Eimeria maxima

Coccidiosis in poultry is caused by the intestinal parasite Eimeria; it causes significant financial losses to the commercial poultry industry worldwide. CoxAbic ® is the first commercially available subunit vaccine against coccidiosis. The vaccine consists of affinity purified sexual stage (gametocyte) antigens (APGA) isolated from Eimeria maxima. Production of this vaccine is time-consuming and laborious and, therefore, a recombinant subunit vaccine substitute for CoxAbic ® is desirable. The genes encoding the two immunodominant components of CoxAbic ®, gam56 and gam82, were cloned into the bacterial expression vector, pTRCHisB, and the proteins expressed and purified. Both recombinant proteins were recognised by protective chicken antibodies that were raised to APGA, by immunoblotting. In a competitive ELISA, a combination of the recombinant proteins inhibited the binding of anti-APGA antibodies to APGA by 76%, which was comparable to the inhibition of 98% observed when APGA was used as the competing protein in the assay. In two breeds of chicken (Australorp and Cobb500), the recombinant proteins alone, or in combination, elicited a dose-dependent, antibody response that recognised APGA by ELISA, and gametocytes by immunoblotting. Together, the results suggested that the development of a recombinant subunit vaccine that maintains the antigenic and immunogenic properties of the native protein vaccine, CoxAbic ®, is feasible.

Molecular aspects of malaria pathogenesis

Plasmodium falciparum being the most lethal plasmodiae is still a major cause of the disease burden and mortality in malaria endemic areas. Due to the wide spread drug resistance in combination with poor socio-economic situation in the vast majority of the endemic countries, malaria is today a great global challenge. The scientific community is, however, progressing. The 23 Mb genome of P. falciparum has been decoded and publicly available. Data of transcriptional profiling at certain developmental stages have already been generated. More than 50% of P. falciparum genes are transcribed constitutively in all the developmental stages of parasite life cycle. Functional disruption of these genes might have implications for parasite growth and development. Available microarray data indicate that P. falciparum preferentially expresses rif and stevor gene families at gametocyte and sporozoite stages while var genes are predominantly expressed at the erythrocytic stage. Gene regulation mechanisms of the variant gene families in P. falciparum are still not understood though some regulatory elements have been proposed. The occurrence of severe malaria is determined by both parasite and human host factors. Sequestration and antigenic variation are two of the evasion mechanisms utilized by P. falciparum in order to escape the human host defences. Understanding the molecular mechanisms underlying these phenomena is of a major importance and interest in malaria research. Here, we summarize and highlight the recent progress in molecular aspects of severe malaria.

Multiple Splice Variants Encode a Novel Adenylyl Cyclase of Possible Plastid Origin Expressed in the Sexual Stage of the Malaria Parasite Plasmodium falciparum

We report the characterization of an unusual adenylyl cyclase gene from Plasmodium falciparum, here designated PfACalpha. The level of mRNA expression is maximum during development of gametocytes (the sexual blood stage of the parasite life cycle). The gene is highly interrupted by 22 introns, and reverse transcriptase-PCR analysis revealed that there are multiple mRNA splice variants. One intron has three alternative 3'-splice sites that confer the potential to encode distinct forms of the enzyme using alternative start codons. Deduced amino acid sequences predict membrane-spanning regions, the number of which can vary between two and six depending on the splice variant. Expression of a synthetic form of two of these variants in Xenopus oocytes and in Dictyostelium adenylyl cyclase-deficient mutants, confirms that PfACalpha is a functional adenylyl cyclase. These results identify a novel mechanism in P. falciparum for the generation of multiple isoforms of a key, membrane-bound signaling molecule from a single genomic copy. Comparisons of the catalytic domains of PfACalpha and a second putative P. falciparum adenylyl cyclase (PfACbeta) with those from other species reveal an unexpected similarity with adenylyl cyclases from certain prokaryotes including the cyanobacteria (blue green algae). In addition, the presence of an unusual active site substitution in a position that determines substrate specificity, also characteristic of these prokaryotic forms of the enzyme, further suggests a plastid origin for the Plasmodium cyclases.

Integrative analysis of intraerythrocytic differentially expressed transcripts yields novel insights into the biology of Plasmodium falciparum

BackgroundThe intraerythrocytic development of Plasmodium falciparum, the most virulent human malaria parasite involves asexual and gametocyte stages. There has been a significant increase in disparate datasets derived from genomic and post-genomic analysis of the parasite that necessitates delivery of integrated analysis from which biological processes important to the survival of the parasite can be determined.MethodsIn order to resolve genes associated with stage differentially expressed transcripts, we have developed and implemented an integrative approach that combines evidence from P. falciparum expressed sequence tags (ESTs), genomic, microarray, proteomic and gene ontology data.ResultsA total of 143 gametocyte-overexpressed and 51 asexual-overexpressed transcripts were identified. A subset of 74 genes associated with these transcripts showed evidence of stage-correlated protein expression, of which 53 have not been experimentally characterised. Our study has revealed (1) possible regulatory mechanisms in malaria parasites' gametocyte maturation, (2) correlation between EST and microarray data for a P. falciparum gene family to present unique EST-derived information, (3) candidate drug and antigenic targets on which computational and experimental studies can be performed, and (4) the need for more empirical studies on gene and protein expression in malaria parasites.ConclusionApplying different domains of data to the same underlying gene set has yielded novel insights into the biology of the parasite and presents an approach to appraise critically the data quality of post-genomic datasets from malaria parasites.

Male-specific expression of the paralog of malaria transmission-blocking target antigen Pfs230, PfB0400w

Malaria transmission requires that Plasmodium parasites circulating in the vertebrate host develop into male and female gametocytes, which are then taken up by a mosquito to undergo fertilization and further development into infectious sporozoites. To understand the malaria specific events involved in this process, the gene products involved require identification and characterization. This work demonstrates that antibodies generated against the paralog of malaria transmission-blocking antigen Pfs230, PfB0400w, react only with stage V male gametocytes, not gametes or asexual parasites. In contrast, Pfs230 is expressed on the surface of all gametocytes and remains associated with emerged gametes as one of the primary surface antigens for several hours. Consistent with the localization findings, a high molecular weight band is recognized by anti-PfB0400w antibodies on western blots of extracts of late stage gametocytes, not asexual parasites, early (stage II/III) gametocytes, or gametes. PfB0400w mRNA is also not observed in asexual parasites. The transcript levels peak in stage III/IV gametocytes, then sharply decline in gametes. This work identifies a novel male-specific protein with an expression pattern that is distinctly different than its paralog.

Plasmodium falciparum and P. vivax gametocyte-specific exoantigens stimulate proliferation of TCR gammadelta+ lymphocytes.

Immune modulation of Plasmodium vivax and P. falciparum gametocytes occurs over the course of erythrocytic infection. The response is linked to proliferative and inflammatory responses, which may be stimulated by stage-specific gametocyte proteins. Stage-specific exoantigens were purified from supernatants of P. falciparum and P. vivax gametocyte cultures, and either primary or secondary postinfection lymphocytes were stimulated for proliferation. Five of 25 exoantigens purified from P. falciparum gametocyte cultures and 6 of 28 exoantigens isolated from P. vivax were gametocyte stage specific. Metabolic labeling of soluble P. falciparum gametocyte proteins confirmed synthesis and secretion of 5 stage-specific exoantigens, with molecular masses of 118, 62, 52, 37, and 33 kDa. Purified gametocyte exoantigens within the range of 50 to 100 kDa stage-specifically stimulated proliferation of lymphocytes from postprimary P. falciparum infections, and from postprimary and secondary P. vivax infection patients with homologous purified exoantigens. T-cell receptor (TCR)gammadelta+, and CD3+ CD8+ and CD3+ CD4- CD8- T cells were specifically upregulated from P. falciparum primary- and P. vivax secondary-infection lymphocytes, respectively, using gametocyte stage-specific exoantigens. CD25+ was the major activation marker expressed by CD3+ and gammadelta T cells when stimulated with gametocyte exoantigens. None of the T cell markers was significantly upregulated using gametocyte stage-specific exoantigens with primary-infection P. vivax lymphocytes.

Malaria and mosquito gene expression

It has long been known that sexual development of Plasmodium falciparum occurs in the hindgut of the Anopheles gambiae mosquito. However, knowledge regarding the molecular basis of midgut–parasite interaction has been lacking. The identification of genes expressed during invasion and development of the sexual stage could shed light on host–parasite interactions. Furthermore, an understanding of the interactions between parasite and mosquito might also lead to the development of new strategies to interrupt malaria transmission.To this end, a recent study by Bonnet and colleagues1xTranscripts of the malaria vector Anopheles gambiae that are differentially regulated in the midgut upon exposure to invasive stages of Plasmodium falciparum. Bonnet, S. et al. Cell. Microbiol. 2001; 3: 449–458Crossref | PubMed | Scopus (25)See all References1 used differential display to identify mosquito genes expressed during the ingestion of a blood meal. As the in vitro production of gametocytes of P. falciparum is difficult, infection of mosquitoes was carried out in an endemic area of Cameroon, Africa, by experimental feeding on the blood of volunteers known to be infected with P. falciparum. The study focused specifically on the identification of genes differentially expressed in the midgut of mosquitoes fed on either parasite-free blood or on blood of P. falciparum gametocyte or asexual carriers. Five transcripts were identified: four corresponded to genes selectively upregulated by the presence of gametocyte-stage parasites, and one corresponded to a gene overexpressed in the presence of both gametocyte and asexual stages of P. falciparum. Seven additional transcripts were identified in the midgut of An. gambiae after a blood meal containing P. falciparum. Identified transcripts included rRNA genes, trypsin1xTranscripts of the malaria vector Anopheles gambiae that are differentially regulated in the midgut upon exposure to invasive stages of Plasmodium falciparum. Bonnet, S. et al. Cell. Microbiol. 2001; 3: 449–458Crossref | PubMed | Scopus (25)See all References1 and a profilin homolog. Analysis of the other transcripts revealed no known homology to other proteins. This is the first demonstration that ingestion of gametocyte-stage parasites of P. falciparum affects expression of An. gambiae midgut genes in a developmental-stage-dependent manner. The identification of mosquito genes involved in digestion of the blood meal or sexual development of malaria parasites might lead to the development of transgenic mosquitoes that are refractory to these parasites, possibly reducing the transmission of malaria.

Epitopes for modified band 3 monoclonal antibody 1C4 are not exposed on the malaria-infected red blood cell surface

Red blood cells (RBC) infected with Plasmodium falciparum asexual trophozoites and stage IB–V gametocytes are sequestered to the vital organs during infection. In vitro models of infected RBC adherence reveal that the mature asexual parasites and early stage gametocytes (stages I–IIA) bind to platelet glycoprotein CD36 under static assay conditions [1], [2]. At least one parasite-derived surface protein, variant surface antigen PfEMP1, functions as a ligand for this stage-specific binding [3], [4]. A second adhesion ligand, parasite-induced modified erythrocyte anion transporter protein band 3, has also been implicated in mediating adherence of trophozoite- [5] and all stages of gametocyte-infected RBC to CD36.

Ultrastructure and function of mitochondria in gametocytic stage of Plasmodium falciparum.

Morphological properties of the mitochondrial organelles in the asexual and sexual gametocytic stages of Plasmodium falciparum have been analyzed and found to be markedly different. From in vitro cultures of both stages in human erythrocytes, it has been demonstrated that the asexual stages contained a defined double-membrane organelle having a few tubular-like cristae. The numbers of mitochondria in the gametocytes were found to be approximately 6 organelles per parasite, and they showed a greater density of the cristae than that of the asexual stage parasite. The organelles of the gametocytes were successfully purified by differential centrifugation following Percoll density gradient separation with the results of approximately 7% yields and approximately 5 folds. The gametocytic organelles contained much more activities of mitochondrial electron transporting enzymes (i.e., cytochrome c reductase, cytochrome c oxidase) than the asexual stage organelles. Mitochondrial function as measured by oxygen consumption were found to be different between these two stages organelles. Their rates of oxygen consumption were relatively low, as compared to those of human leukocyte and mouse liver mitochondria. In contrast to the coupled mammalian mitochondria, the gametocytic organelles were in the uncoupling state between oxidation and phosphorylation reactions during their respiration. However, they were sensitive to inhibitors of the electron transport system, e.g., antimycin A, cyanide. Our results suggest that the mitochondria of the gametocytic stages are metabolically active and still underdeveloped, although their inner membranes are extensively folded. The biochemical significance of the unique structure of the mitochondria in these developing stages in host erythrocytes remains to be elucidated.

Shotgun DNA microarrays and stage-specific gene expression in Plasmodium falciparum malaria.

Malaria infects over 200 million individuals and kills 2 million young children every year. Understanding the biology of malarial parasites will be facilitated by DNA microarray technology, which can track global changes in gene expression under different physiological conditions. However, genomes of Plasmodium sp. (and many other important pathogenic organisms) remain to be fully sequenced so, currently, it is not possible to construct gene-specific microarrays representing complete malarial genomes. In this study, 3648 random inserts from a Plasmodium falciparum mung bean nuclease genomic library were used to construct a shotgun DNA microarray. Through differential hybridization and sequencing of relevant clones, large differences in gene expression were identified between the blood stage trophozoite form of the malarial parasite and the sexual stage gametocyte form. The present study lengthens our list of stage-specific transcripts in malaria by at least an order of magnitude above all previous studies combined. The results offer an unprecedented number of leads for developing transmission blocking agents and for developing vaccines directed at blood stage antigens. A significant fraction of the stage-selective transcripts had no sequence homologues in the current genome data bases, thereby underscoring the importance of the shotgun approach. The malarial shotgun microarray will be useful for unravelling additional important aspects of malaria biology and the general approach may be applied to any organism, regardless of how much of its genome is sequenced.