Co‐ordinated programme of gene expression during asexual intraerythrocytic development of the human malaria parasite <i>Plasmodium falciparum</i> revealed by microarray analysis

Mamoun, Choukri Ben; Gluzman, Ilya Y.; Hott, Christian; MacMillan, Sandra; Amarakone, Aloka; Anderson, Dustin L.; Carlton, Jane M.; Dame, John B.; Chakrabarti, Debopam; Martin, Rodger K.; Brownstein, Bernard H.; Goldberg, Daniel E.

doi:10.1046/j.1365-2958.2001.02222.x

Cited by 143 publications

(94 citation statements)

References 43 publications

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“…2). The predominant Pfpka-c expression in asexual blood stages described here and by Li and Cox [23] may in fact be restricted to schizonts, as a recent microarraybased study of gene expression also detected Pfpka-c transcripts at this stage [24]. The developmental downregulation of Pfpka-c levels in sexual stages may render regulation of the kinase more sensitive to fluctuations in cAMP, thereby explaining the effect of this compound on gametocytogenesis in P. falciparum cultures [6][7][8].…”

Section: Discussionmentioning

confidence: 81%

The H89 cAMP‐dependent protein kinase inhibitor blocks Plasmodium falciparum development in infected erythrocytes

Syin

Parzy

Traincard

et al. 2001

European Journal of Biochemistry

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In Plasmodium falciparum, the causative agent of human malaria, the catalytic subunit gene of cAMP-dependent protein kinase (Pfpka-c ) exists as a single copy. Interestingly, its expression appears developmentally regulated, being at higher levels in the pathogenic asexual stages than in the sexual forms of parasite that are responsible for transmission to the mosquito vector. Within asexual parasites, PfPKA activity can be readily detected in schizonts. Similar to endogenous PKA activity of noninfected red blood cells, the parasite enzyme can be stimulated by cAMP and inhibited by protein kinase inhibitor. Importantly, ex vivo treatment of infected erythrocytes with the classical PKA-C inhibitor H89 leads to a block in parasite growth. This suggests that the PKA activities of infected red blood cells are essential for parasite multiplication. Finally, structural considerations suggest that drugs targeting the parasite, rather than the erythrocyte enzyme, might be developed that could help in the fight against malaria.Keywords: parasite; PKA; inhibition; H89.The emergence and dissemination of drug-resistant malaria parasites represents one of the most important public health problems in many parts of the world today. New antimalarials are urgently required, whose rational design and development requires the identification of potential therapeutic targets. This in turns rests on a better understanding of the molecular mechanisms controlling the progression of the complex life cycle of malaria parasites, especially Plasmodium falciparum, the species responsible for the lethal form of the disease. All Plasmodium species are intracellular parasites during infection of their vertebrate hosts. Sporozoites inoculated into a host during a bite by an infected Anopheles mosquito soon invade hepatocytes, within which intense asexual division takes place (exoerythrocytic schizogony), yielding up to 40 000 merozoites in the case of P. falciparum (http:// www.malaria.org). Upon schizont rupture, these merozoites invade red blood cells, where additional rounds of asexual replication occur (erythrocytic schizogony, the phase responsible for the pathogenesis of the disease). Some merozoites, instead of undergoing a further asexual cycle, arrest their cell cycle and differentiate into male or female gametocytes. Unlike asexual forms, these sexual forms are infective to the Anopheles vector.cAMP is involved in the regulation of development of several microorganisms, and cAMP-dependent pathways exist in most eukaryotic cells. For example, in the slime mold Dictyostelium discoideum, cAMP acts as a signal for the aggregation and differentiation of cells into a multicellular organism (reviewed in [1]). In most instances, cAMP exerts its action by binding to the regulatory subunit complexed to the catalytic subunit in an inactive holoenzyme of the protein kinase A (PKA or cAMPdependent protein kinase), thereby releasing the active catalytic subunit (PKA-C), whose substrates can include other protein kinases and transcription factors. P...

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Section: Discussionmentioning

confidence: 81%

The H89 cAMP‐dependent protein kinase inhibitor blocks Plasmodium falciparum development in infected erythrocytes

Syin

Parzy

Traincard

et al. 2001

European Journal of Biochemistry

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“…Microarrays will be the method of choice for expression analysis in asexual and sexual blood stage parasites where the acquisition of sufficient RNA is not a limitation. Although whole genome microarrays are not yet available, partial arrays from mung bean genomic libraries (37) or blood stage cDNA libraries (38) have been used successfully to study gene expression in blood stages. However, microarray analysis of gene expression in ookinetes, early oocysts, sporozoites, and EEF of mammalian Plasmodia will be difficult because large quantities of these stages are not available.…”

Section: Discussionmentioning

confidence: 99%

Exploring the transcriptome of the malaria sporozoite stage

Kappe

Gardner

Brown

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

123

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Most studies of gene expression in Plasmodium have been concerned with asexual and͞or sexual erythrocytic stages. Identification and cloning of genes expressed in the preerythrocytic stages lag far behind. We have constructed a high quality cDNA library of the Plasmodium sporozoite stage by using the rodent malaria parasite P. yoelii, an important model for malaria vaccine development. The technical obstacles associated with limited amounts of RNA material were overcome by PCR-amplifying the transcriptome before cloning. Contamination with mosquito RNA was negligible. Generation of 1,972 expressed sequence tags (EST) resulted in a total of 1,547 unique sequences, allowing insight into sporozoite gene expression. The circumsporozoite protein (CS) and the sporozoite surface protein 2 (SSP2) are well represented in the data set. A BLASTX search with all tags of the nonredundant protein database gave only 161 unique significant matches (P(N) < 10 ؊4 ), whereas 1,386 of the unique sequences represented novel sporozoite-expressed genes. We identified ESTs for three proteins that may be involved in host cell invasion and documented their expression in sporozoites. These data should facilitate our understanding of the preerythrocytic Plasmodium life cycle stages and the development of preerythrocytic vaccines.Plasmodium yoelii yoelii ͉ expressed sequence tag P rotozoan parasites of the genus Plasmodium are the causative agents of malaria, the most devastating parasitic disease in humans. The parasites occur in distinct morphological and antigenic stages as they progress through a complex life cycle, thwarting decades of efforts to develop an effective malaria vaccine. Plasmodium is transmitted via the bite of an infected Anopheles mosquito, which releases the sporozoite stage into the skin. Sporozoites enter the bloodstream and, on reaching the liver, invade hepatocytes and develop into exo-erythrocytic forms (EEF). After multiple cycles of DNA replication, the EEF contains thousands of merozoites (liver schizont) that are released into the blood stream and initiate the erythrocytic cycle (asexual blood stage) that causes the disease malaria. Changes in life cycle stages are accompanied by major changes in gene expression and therefore by major changes in antigenic composition. The form of the parasite best studied is the asexual blood stage, mainly because of its comparatively easy experimental accessibility. Therefore, most Plasmodium proteins that have been well characterized are expressed during the erythrocytic cycle, among them some major erythrocytic-stage vaccine candidates such as merozoite surface protein-1 (MSP-1) and apical membrane antigen-1 (AMA-1; ref. 1). Erythrocytic-stage vaccines are aimed at inducing an immune response that suppresses or eradicates parasite load in the blood. In contrast, preerythrocytic vaccines are aimed at eliciting an immune response that destroys the sporozoites and the EEF, thereby preventing progression of the parasite to the blood stage. The feasibility of a preerythrocytic v...

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“…In this asexual cycle a single merozoite leads to the development of 10 or more new merozoites percell cycle (Roberts and Janovy, 2000). Each stage has been well characterized for gene expression, biochemistry and morphology (Ben Mamoun et al, 2001), but strikingly little is understood about the natural variation in, and regulation of, these processes. Development of in vitro culturing methods provided powerful experimental access to P. falciparum and was closely followed by development of methods to infer parasite viability and proliferation (Trager and Jensen, 1976;Richards and Maples, 1979;Trager and Jensen, 1997).…”

Section: Introductionmentioning

confidence: 99%

Quantitative dissection of clone-specific growth rates in cultured malaria parasites

Reilly

Wang

Steuter

et al. 2007

International Journal for Parasitology

105

125

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Measurement of parasite proliferation in cultured red blood cells underpins many facets of malaria research, from drug sensitivity assays to assessing the impact of experimentally altered genes on parasite growth, virulence and fitness. Pioneering efforts to grow Plasmodium falciparum in cultured red blood cells revolutionized malaria research and spurred the development of semi-high throughput growth assays using radio-labeled hypoxanthine (Hx), an essential nucleic acid precursor, as a reporter of whole-cycle proliferation (Trager and Jensen, 1976;Desjardins et al., 1979). The isotopic Hx assay remains the standard quantitative growth assay with which newernon-radioactive procedures based on fluorescent DNA dyes or ELISA are compared. All of these readouts are surrogate reporters of changes in bulk parasitemias, reflecting proliferation over entire asexual reproductive cycles. While quantitatively robust and amenable to semi-high-throughput applications, these methods are blind to the underlying developmental and cellular events of growth in human red blood cells. Modern whole-genome tools including gene knockouts, mutagenesis and small molecule screens promise to reveal much about basic parasite biology; however methods to precisely quantify the within-cycle growth process are needed. Here we elaborate on the classical growth index, i.e.changes in parasitemia, by quantifying sub-phenotypes of a rapid proliferator, the multi-drug resistant clone Dd2, and a standard wild type clone, HB3. These data illustrate differences in cycle duration, merozoite production, and invasion rate and efficiency that underpin Dd2's average twofold proliferation advantage over HB3 per erythrocytic cycle. The ability to refine growth phenotypes will inform the search for molecular determinants of differential parasite growth rates and broaden our understanding of killing mechanisms and cellular targets of antimalarial drugs.

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Co‐ordinated programme of gene expression during asexual intraerythrocytic development of the human malaria parasite Plasmodium falciparum revealed by microarray analysis

Cited by 143 publications

References 43 publications

The H89 cAMP‐dependent protein kinase inhibitor blocks Plasmodium falciparum development in infected erythrocytes

The H89 cAMP‐dependent protein kinase inhibitor blocks Plasmodium falciparum development in infected erythrocytes

Exploring the transcriptome of the malaria sporozoite stage

Quantitative dissection of clone-specific growth rates in cultured malaria parasites

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