Proteomic Approaches to Studying Drug Targets and Resistance in Plasmodium

Abstract

Ever increasing drug resistance by Plasmodium falciparum , the most virulent of human malaria parasites, is creating new challenges in malaria chemotherapy. The entire genome sequences of P. falciparum and the rodent malaria parasite, P. yoelii yoelii are now available. Extensive genome sequence data from other Plasmodium species including another important human malaria parasite, P. vivax are also available. Powerful research techniques coupled to genomic resources are needed to help identify new drug and vaccine targets against malaria. Applied to Plasmodium, proteomics combines high-resolution protein or peptide separation with mass spectrometry and computer software to rapidly identify large numbers of proteins expressed from various stages of parasite development. Proteomic methods can be applied to study sub-cellular localization, cell function, organelle composition, changes in protein expression patterns in response to drug exposure, drug-protein binding and validation of data from genomic annotation and transcript expression studies. Recent high-throughput proteomic approaches have provided a wealth of protein expression data on P. falciparum, while smaller-scale studies examining specific drug-related hypotheses are also appearing. Of particular interest is the study of mechanisms of action and resistance of drugs such as the quinolines, whose targets currently may not be predictable from genomic data. Coupling the Plasmodium sequence data with bioinformatics, proteomics and RNA transcript expression profiling opens unprecedented opportunities for exploring new malaria control strategies. This review will focus on pharmacological research in malaria and other intracellular parasites using proteomic techniques, emphasizing resources and strategies available for Plasmodium.

Comparison of protein patterns between Plasmodium falciparum mutant clone T9/94-M1-1(b3) induced by pyrimethamine and the original parent clone T9/94

“…Few proteomic research have been undertaken to elucidate the mechanisms of action or resistance of antimalarial drugs in P. falciparum [9][10][11] . In the present study, the proteomic approach was applied to analyze the pattern of protein expression in the schizont stage of P. falciparum following being subjected to selective pressure by the antifolate drug pyrimethamine.…”

“…Red blood cell pellet was washed in 1 mL of 10 mM Tris-HCl (pH 7.4) containing 1 暳 protease inhibitor Cocktail (Roche Co. Ltd.) until red cell ghost was colorless. Pellet was then re-suspended in 500 毺 L of lysis buffer (8 M urea, 2 M thiourea, 1 % CHAPS, 65 mM DTT, 0.5 % ampholyte pH [3][4][5][6][7][8][9][10] and sample was vortexed and sonicated on ice four times, eight seconds each (21 % amplitude, 8 sec, interspersed with 9 sec), followed by centrifugation at 13 000 暳 g for 1 h (4 曟 ), and the supernatant was subjected to 2-DE. Quantification of protein concentrations of the extracts was performed by using Bradford reagent (BioRad Co. Ltd.).…”

“…The extract of parasite protein (100 毺 g) was mixed with rehydration buffer (8 M urea, 1 % CHAPS, 15 mM dithiothretol, 0.001% bromophenol blue). Protein mixture (125 毺 L) was applied onto 7 cm IPG strips in an isoelectric focusing (IEF) system (BioRad Co. Ltd., USA), and was subjected to pH gradient (3)(4)(5)(6)(7)(8)(9)(10) with an electrical charge using ampholytes as carrier. IEF was performed initially at 250 v for 15 min, followed by 4 000 v for 1 h, and terminated with 4 000-20 000 v-h.…”

Comparative Genomics and Systems Biology of Malaria Parasites Plasmodium

“…The proteomes of the P. falciparum asexual trophozoites and schizonts, sexual stage gametocytes, and gametes were revealed by a high accuracy LC-MS/MS analysis [163], and the proteomes of the sporozoite, merozoite, trophozoite and gametocyte stages were elucidated using MudPIT [164]. Potential drug targets, such as proteases and transporters, as well as vaccine targets were found to be expressed at specific stages and subcellular locations in multiple Plasmodium strains [46, 47, 110, 165–169]. MudPIT also enabled the identification of surface antigens and epitopes expressed in the sporozoite and intrahepatic stages [170] and proteins on the surface of parasite-infected erythrocytes (PIESPs), which may play a role in pathogenesis and immunity [171].…”

Recent Progress in Functional Genomic Research in Plasmodium falciparum

“…Although it can be difficult to obtain sufficient material and to prevent contamination from host cells, two large-scale studies using high-throughput proteomics have detected many stage-specific predicted gene products consistent with results from transcript profiling studies [90,91]. This genomics-based approach has also been widely applied in studies of drug targets [92-94], organelle composition [95], stage- and sex-specific gene functions [23,96,97], validation of data from genomic annotation, post-translational modifications [98,99]. With the completion of its human and insect host genome project, genomic, metabolomic and proteomic analyses of host-pathogen interactions have shed light on many malaria genes’ functions [100-102].…”