Expressed Sequence Tag Analysis of the Erythrocytic Stage of Plasmodium berghei

Rodent malaria parasites, such as Plasmodium berghei, are practical and useful model organisms for human malaria research because of their analogies to the human malaria in terms of structure, physiology, and life cycle. Exploiting the available genetic sequence information, we constructed a cDNA library from the erythrocytic stages of P. berghei and analyzed the expressed sequence tag (EST). A total of 10,040 ESTs were generated and assembled into 2,462 clusters. These EST clusters were compared against public protein databases and 48 putative new transcripts, most of which were hypothetical proteins with unknown function, were identified. Genes encoding ribosomal or membrane proteins and purine nucleotide phosphorylases were highly abundant clusters in P. berghei. Protein domain analyses and the Gene Ontology functional categorization revealed translation/protein folding, metabolism, protein degradation, and multiple family of variant antigens to be mainly prevalent. The presently-collected ESTs and its bioinformatic analysis will be useful resources to identify for drug target and vaccine candidates and validate gene predictions of P. berghei.

Variation of the chloroquine resistance transporter [crt] gene in chloroquine-resistant and chloroquine-sensitive plasmodium berghei

The emergence and spread of chloroquine resistant Plasmodium falciparum in the world stimulated some investigators to consider different aspects of chloroquine resistance in human and rodent Plasmodia. Using animal Plasmodia, particularly primate and rodent Plasmodia can be useful model for human Plasmodia studies. In this study we have tried to consider and compare the sequence of chloroquine resistance transporter [crt] gene among chloroquine-resistant and chloroquine-sensitive strains of Plasmodium berghei. This experimental study was performed at the Malaria Laboratory of School of public health. DNA was extracted from two strains of P. berghei which their resistance and sensitivity had been demonstrated in mice with treatment by chloroquine. By using specific primer for crt gene some parts of this gene were amplified by PCR, and obtained fragments were then sequenced and compared. There were considerable differences in crt gene between two strains. Sequenced 1212 bp of crt gene fragment in the two strains showed 43 differences at nucleotides level and 16 differences in presumed coding amino acids. crt can be addressed as a considerable gene which involves in induction of resistance to chloroquine in P. berghei, as P. falciparum. The results increased such a promise that considering crt gene in chloroquine-sensitive and chloroquine-resistant P. berghei can prepare suitable and helpful fields for more understanding the molecular aspects of chloroquine-resistance in Plasmodia and reversing the effectiveness of 4-aminoquinolines particularly chloroquine for treatment of drug resistant Plasmodia

Molecular biology and biochemistry of malarial parasite pyrimidine biosynthetic pathway.

Metabolic pathways in the malarial parasite are markedly different from the host, eg, hemoglobin, fatty acids, folate and nucleic acids. Understanding of metabolic function will illuminate new chemotherapeutic targets for drug development, including the identification of target(s) for drugs in current use. The parasite-contained pyrimidine biosynthetic pathway is essential for growth and development in the human host. Plasmodium falciparum carbonic anhydrase, producing HCO3- as a pyrimidine precursor, was identified as alpha- type and the encoded gene was cloned and sequenced. The first six enzymes, catalyzing the conversion of HCO3-, ATP, L-aspartate and L-glutamine to uridine 5'-monophosphate (UMP), were partially characterized. The genes encoding these enzymes were identified in order, from the first to the sixth step, as CPSII (carbamyl phosphate synthase II), ATC (aspartate transcarbamylase), DHO (dihydroorotase), DHOD (dihydroorotate dehydrogenase, DHOD), OPRT (orotate phosphoribosyltransferase, OPRT), and OMPDC (orotidine 5'-monophosphate decarboxylase, OMPDC). Unlike its analogous parasitic protozoan, Trypanosoma, the organization of the malarial genes was not an operon-like cluster. The CPSII, DHO and OPRT genes were conserved to bacterial counterparts, whereas the ATC, DHOD and OMPDC were mosaic variations. The data support the mosaic pyrimidine pathway in the malarial parasite. The human host had five enzymes out of the six associated into two different multifunctional proteins, in that a single gene CPSII-ATC-DHO encoded the first three enzymes, and another gene OPRT-OMPDC encoded the last two enzymes. In the malarial parasite, the CPSII and ATC were not characterized. The DHO was partially characterized in Plasmodium berghei. The DHOD was well characterized in both P. falciparum and P. berghei. It was functionally expressed in Escherichia coli. The physical and kinetic properties of the recombinant pfDHOD were similar to the native enzyme. The OPRT and OMPDC were also partially characterized. These lines of evidence indicate that the malarial pyrimidine enzymes are mono-functional forms. In addition, the enzymatic activities inter-converting uracil, uridine and UMP of the pyrimidine salvage pathway, were demonstrated, and the gene encoding uridine phosphorylase was cloned. Our results suggest that the pyrimidine enzymes are possible new drug targets.

Stable genetic transformation of malaria parasites by homologous recombination

A transformaçäo genética de diferentes estágios dos parasitas da malária já é possível atualmente. Um conjunto crescente de marcadores seletivos estäo sendo desenvolvidos para permitir uma manipulaçäo genética mais completa de Plasmodium. "Gene targeting", que permite romper um gene ou introduzir alterações sutis na seqüência de um gene via recombinaçäo homóloga, é uma nova tecnologia usada no estudo de estrutura-funçäo de antígenos in vivo. A criaçäo de parasitas recombinantes carreando mutações pontuais em sítios conservados de TRAP de esporozoitas de P. berghei é usado como protótipo desta nova tecnologia.

The biology of malarial parasite in the mosquito: a review

The purpose of this review is to summarize the biology of Plasmodium in the mosquito including recent data to contribute to better understanding of the developmental interaction between mosquito and malarial parasite. The entire sporogonic cycle is discussed taking into consideration different parasite/vector interactions and factors affecting parasite development to the mosquito.