Molecular & Biochemical Parasitology 137 (2004) 161–168 Transcriptome of axenic liver stages of Plasmodium yoelii Qian Wang a , Stuart Brown b , David S. Roos c , Victor Nussenzweig a , Purnima Bhanot a,∗ a Department of Pathology, New York University School of Medicine, 550 First Avenue, MSB 131, New York, NY 10022, USA b Department of Cell Biology, New York University School of Medicine, New York, NY 10022, USA c Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA Received 15 March 2004; received in revised form 28 May 2004; accepted 1 June 2004 Available online 23 June 2004 Abstract Plasmodium liver stages or early exo-eythrocytic forms (EEFs) contain antigens that are essential for achieving sterile, protective immunity against malaria. Yet, attempts at identifying these antigens have been hampered by the challenge of obtaining large numbers of purified EEFs, uncontaminated with hepatocyte material. Using a recently described system for producing axenically cultured EEFs from Plasmodium yoelii, we have constructed a cDNA library and generated 1453 expressed sequence tags (ESTs) resulting in 652 unique transcripts. Analysis of the library provides insight into processes required for the initiation and development of Plasmodium liver stages, such as protein degradation, cell cycle progression and nutrient transport. Analysis of the gene expression profile of liver stages, as revealed by this library, suggests that liver stages represent a shift from “sporozoite-like” to “blood-stage-like”. This is the first study of the transcriptional repertoire of Plasmodium liver stages. © 2004 Elsevier B.V. All rights reserved. Keywords: Malaria; Plasmodium; Liver stages; Gene expression; cDNA library; Axenic culture 1. Introduction The apicomplexan parasite Plasmodium is the causative agent of malaria, one of the deadliest infectious diseases in the world. It is transmitted through the bite of an infected Anopheles mosquito that introduces parasite forms termed sporozoites, into the host. Sporozoites infect the liver where they undergo asexual replication to form exo-erthrocytic forms (EEFs). In the human parasite, Plasmodium falci- parum, EEF development takes about 7 days whereas in the rodent parasite, Plasmodium yoelii, it is completed in about 40 h after sporozoite invasion. The development of a sporozoite into an EEF occurs inside a membrane-bound parasitophorous vacuole and begins with the formation of a bulbous enlargement in the region containing the sporo- zoite nucleus [1]. As the transformation bulb grows larger Abbreviations: bp, base-pair; CS, circumsporozoite protein; EEF, exo-erythrocytic form; EST, expressed sequence tag; MSP-1, 7, merozoite surface protein 1, 7; PfEMP3, Plasmodium falciparum erythrocyte mem- brane protein 3; RESA, ring-infected erythrocyte surface antigen; SSP2, sporozoite surface protein 2; TRAP, thrombospondin related anonymous protein; UTR, untranslated region ∗ Corresponding author. Tel.: +1 212 263 5346; fax: +1 212 263-8179. E-mail address: bhanop01@med.nyu.edu (P. Bhanot). and the long, slender sporozoite body is subsumed into it, giving rise to a spherical EEF [2]. The EEF matures and fol- lowing a series of nuclear divisions, leads to the formation of 10,000–30,000 merozoites [3], depending on the parasite species. Plasmodium EEFs contain antigens that play a central role in achieving the sterile, protective immunity against malaria conferred by immunization with irradiation atten- uated sporozoites [4]. Irradiation attenuated sporozoites invade hepatocytes but experience an early block in devel- opment into EEFs [5]. Chemotherapy of immunized hosts with drugs that kill EEFs, decreases immunity conferred by irradiated sporozoites [6]. The cellular arm of this im- munity is mediated by IFN-, which specifically targets the development of early EEFs [7,8]. Therefore, it appears that antigens present in Plasmodium early liver stages confer immunity against malaria. Yet, few such antigens have been identified so far [9].The major obstacle has been the failure to obtain large quantities of purified EEFs either in vitro or in vivo. Another limitation is that P. falciparum EEFs grow in vitro only in primary hu- man hepatocytes. The rodent parasites, Plasmodium berghei and P. yoelii are more amenable to experimentation since their sporozoites infect cell lines of cultured hepatocytes. 0166-6851/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.molbiopara.2004.06.001