Reginald Kavishe Department of Pharmacology/Toxicology Radboud University Medical Centre NCMLS & Kilimanjaro Christian Medical Centre, Tanzania
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1 Reginald Kavishe Department of Pharmacology/Toxicology Radboud University Medical Centre NCMLS & Kilimanjaro Christian Medical Centre, Tanzania
2 ABC = ATP-binding cassette proteins Most are membrane transporters TMD TMD Membrane NBD NBD Ge-valvGpsGsGKstll ISgGQkqrvaiA IlllDEptsaID Walker A ABC signature Walker B Conserved domains - With 2 x 6 TM domains. Can be coded as ½ transporters gene - Efflux pump a wide range of substrates including GSH and GSH-conjugates
3 Role of GSH in heme detoxification in the malaria parasite Ribose5P 6PGDH 6PGL G6PDH G6P G6PD deficiency low GSH Hb protection against malaria X GSSG NADPH Heme - CQ-resistant parasites have increase GSH GSH metabolism NADP + - GSH-rich reticulocytes are preferred by P.vivax CQ & AQ GSH efflux pumps can be targets for drugs Haemozoin Host GR ABC transporter GSSG
4 Several ABC genes are present in the Plasmo genome: Aim: Identify and characterize putative P. falciparum ABC genes Objectives: Classify ABC transporters (a step towards identifying GSH pumps) Determine cellular localization of the ABCs in the parasite infected Determine cellular localization of the ABCs in the parasite infected RBCs
5 On the PlasmoDB, 16 ABC proteins were retrieved and classified. 11 were ABC transporters (Kavishe et al 2009) PF14_0455 IKPGTTCALVGHTGSGKTTISKLLYRF VKLSGGERQRISIARCLLKDPKI...VIFDEATSSLD... PFE1150w-b CDSKKTTAIVGETGSGKSTFMNLLLRF KSLSGGQKQRIAIARALLREPKI...LLLDEATSSLD... PFE1150w-a LKEGKTYAFVGESGCGKSTILKLIERL SKLSGGQKQRISIARAIMRNPKI...LILDEATSSLD... PF11_0466 IYKNTNNVIIGKSGGGKSTILKLILNM ELLTSSQKQKICIARALTRYPKI...LLLDESTSAID... PFC0125w FSNKYTYGILCYNDSGKNYLAKLAARL KILSGGQKQKISLARAIIKKPKI...LILDDAFSALD... PF13_0218 IKASEKVAIIGKSGSGKSTLWKLLTCN NNMSSGQKQRLSIIRSLMKDTPI...YIFDEITSFLD... PF13_0271 IENGEKVAIIGKSGSGKSSLIKLLLKF TKLSIGEKQRICIARCFLKDSKI...IVLDEHASNLD... PFL0495c LPHNKSVAIVGKSGSGKTTILNLLTKK TSLSGGQKQRIYLAQNLIKNNKI...LILDEPTSSLD... PFA0590w-b ALKNQKIGIVGKSGAGKSTILLSILGL SFLIGKAKSFNYIIRNHFPNNTV...LIISHHANTLSCCD PFL1410c-b ALKNQKIGIVGKSGAGKSTMILSILGL NFFTTDIKSFDYIIRTFFQNTTV...LIIAHDASTLSCCD PFA0590w-a LKRNSLAIIIGNVGSGKSAFFHSILGD LVNYNIPFNENYLQKCLMDDNNF.YLYLLDDIFTSLD... PFL1410c-a LKNNSVVIILGNVGSGKTIFFYSLLGQ TDNNNTSNNKNSCSKNLTEERNISYLYLFDDLFTSLD... PF14_0133 IYLGEKHTIMGRNGSGKSTLAKVIAGH..FSGGEKKRNEILQMLILKPTF...CILDETDSGLD... PF14_0244 LLPKTITVIMGPSGSGKTTLLNILSMK.GISGGQRKRLSIANEILSNPPL...LLMDEPTSGLD... PF11_0225-b VDMDSRIAICGVNGSGKTTLIKIILNL YVLSGGQKSKLALAILAYKNPNV...LILDEPSNHLD... PF08_0078-b INSGENVLLLGKNGIGKSTLFKILTNK KDKSGGEKVRILFLSLFLKKSNL...LLLDEINNNLD... PF08_0078-a LNKTECIGLIGNNGCGKTTLLNLIYEN CHLSNGYIIRVYLLLLLLSKSKL...LLIDEINNNLD... PF11_0225-a INVMNKYGLIGKNGIGKSTLLAKLARY NSLSGGMKMRLCLSRILFSNNDI...ILLDEPTNHLD... MAL13P1.344-b FSESEIFVLLGQNGSGKSTFIRLFAGL..LSGGELQKVAIIVTLAKNTNI...YLIDEPSAYLD... MAL13P1.344-a PKLGQILGLVGTNGIGKSTALKILSSK..LSGGELQRFALLMSIIGQSTN..VYMFDEPSSYLD... PF14_0321 FERGMRILVCGKNGAGKSTLLSIIAGK..LSKGERKKVQIMVNIIVRKDI...YIFDEATESLD... PFC0875w LRSNRIFVLLGENGSGKSTLINIITKM KNLMDEVKKKISIFICFLVKRDI...YILDEPFIALD..... **. ::.. :. Walker A ABC signature Walker B Classification of Pf ABC proteins based on NBD alignment
6 ABCB MDR group ABCI ABCG ABCC ABCE (MRP group) Possible GSH transporters ABCF ABCI
7 Polyclonal antibodies were raised against 3 of the ABCs 1. Pfmrp1 (full transporter) 2. Pfmrp2 (full transporter) 3. PfMDR5 (half transporter) Immunocytochemistry was performed to identify membrane Immunocytochemistry was performed to identify membrane location
8 Malaria life cycle stages Malaria infected blood Smear on cover slips 4% paraformaldehyde fixaction Quenching with 0.15% glycine Permeablization with 0.1% triton Block with normal goat serum 1 o Ab overnight 2 o Ab with florescent dye Nuclear dye Mount on slides Confocal imaging Olympus 1000
9 Localization PfMDR A R GREEN: glycophorin-a (RBC membrane) alexa 488 B RED: Pfmrp protein alexa 594 BLUE: nucleus / nuclei (DAPI) C R Arrow = food vacuole R = ring stage
10 Pfmrp A B GREEN: glycophorin-a (RBC membrane) alexa 488 RED: Pfmrp protein alexa 594 C BLUE: nucleus / nuclei (DAPI) Pfmrp A B R = ring stage S= schizont T= trophozoite
11 Antilocalisation with PfERC Pf13_0218 PfERC A B C A B Pfmrp1 GREEN: PfERC alexa 488 RED: Pfmrp protein alexa 594 BLUE: nucleus / nuclei (DAPI)
12 Pfmrp2 A T T T R PfERC B GREEN: PfERC alexa 488 RED: Pfmrp protein alexa 594 BLUE: nucleus / nuclei (DAPI)
13 Conclusion PfMDR5, pfmrp1 and pfmrp2 are located on the plasma membrane Expressed in all asexual stages of the parasite Membrane location could be GSH transporters PfMDR1 SNPs and/or gene duplication resistance to CQ SNPs on PfMRP1 have been associated with resistance vs quinoline resistance (Ursing et al,2006.acta Tropica) Adaptation of parasites in CQ and AQ result in upregulation of PfMRP1 and 2 Recent findings: PfMRP1 can efflux GSH conjugated drugs (Kumar et al., 2009) promising for antimalarial research
14 Acknowledgements: Department members: Dr. Jan B. Koenderink Prof. Frans Russel Ing. Jeroen van den Heuvel Collaborators: Dr. Adrian Luty Medical Microbiology, NCMLS Ing. Marga van Vegte Malaria Unit, NCMLS Ing. Huib Croes MIC staff, NCMLS Funding: NWO-WOTRO (PRIOR) Thanks for your attention
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