Plasmodium falciparum Parasites Oct/28/08 Feng, Chi-wei Supervised - PowerPoint PPT Presentation

1 A Regulatable Transgene Expression System for Cultured Plasmodium falciparum Parasites Oct/28/08 Feng, Chi-wei Supervised by Prof. Ching-Tsan Huang, PhD

2 Plasmodium falciparum 8~24 merozoits rupture By the courtesy of Bannister, L. H. et al. and Stockbower, R.

3 The Life Cycle of Human Malaria Parasite, Plasmodium falciparum Erythrocytic cycle By Courtesy of Brian M. Greenwood et al., “ Malaria: progress, perils, and prospects for eradication ” , The Journal of Clinical Investigation, Vol.112, Number 4, April2008

4 Blood Stages of P. falciparum Multiple invasion 1. Ring 3. Schizont 2. Trophozoite By Courtesy of Dr. Chairat Uthaipibull

5 Current developments Protein overexpression Annotated genome data base Gene knockout technique by Homologous Recombination By courtesy of Y Wu. et al By courtesy of Daily JP. et al

6 The 2 shortcomings • The oversized episome with AT-rich sequence is not favorable by E.coli in multiplication. • Limited copy number of episome and low efficiency of promoter.

7 Previous efforts • P.falciparum gene expression regulation by: the Atc-inducible system, FKBP12 system, use of ribozyme actions • Complexity of system in manipulation • Narrow utility that’s hardly widespread

8 Strategy Bidirectional Selectable Promoter marker Drives both Regulation of selectable expression by marker and concentration Transgene of drug A regulatable expression system by a mini-sized plasmid

9 General plasmid with promoter (1 Gene+1 Promoter)x N= Plasmid in large size

10 Bidirectional Promoters • A series of gene promoting and silencing experiments in studies in genes of interested in Plasmodium species Introns with promoter activity in Bidirectional manner • PFC0005w • PFB1055c • PFD0020c

11 1. Stable transgenic construct in minimized size

12 Trangenic Construct- pHLIDH Pst I The VLH region was replaced by HL pHLIDH Kpn I

13 Trangenic Construct- pHLIDH (H: hrp2 3’UTR, L: firefly luciferase )

14 Promoter activities of the Introns The intron PFC0005w shows the highest promotion activity according to luciferase activity among the three. Luminescence was expressed per 1% parasitaemia

15 Coordination in promoting actitivity : dfhr : luciferase : spb1 : msp1 RT-PCR pHLIDH with PFC0005w is suitable for late gene expression

16 2. Regulation in transgenic expression level

17 Trangenic Construct- pHBIRH (H: hrp2 3’UTR, B: blasticidin-s-deaminase; bsd, R: Renilla luciferase)

18 Blasticidin-s Formula: C 17 H 26 N 8 O 5 , Aminohydrolase substrate Isolated from Streptomyces griseochromogenes

19 Promoter activities of the Introns The promoter PFC0005w shows the highest activity according to luciferase activity among the two.

20 Luciferase activity vs. Drug conc. Blasticidin-s conc. Increase in drug concentration : 2 μ g/ml : 5 μ g/ml : 10 μ g/ml : 20 μ g/ml Luciferase expression is stage-specific, and yet coordinate to drug concentration. Luminescence was expressed per 1% parasitaemia

21 Increase in Copy number Blasticidin-s conc. : 2 μ g/ml : 5 μ g/ml : 10 μ g/ml : 20 μ g/ml By DNA extraction, Increase in bsd and Renilla luciferase expression is due to change in copy number of construct.

22 Increase in gene expression Blasticidin-s conc. : 2 μ g/ml : 5 μ g/ml : 10 μ g/ml : 20 μ g/ml The relative amount of bsd and Renilla luciferase mRNA directly correlated with blasticidin concentration in the late stage.

23 Conclusion- Pros and Cons • Transgene expression will be brought about due to bsd expression, which give rise to ability of regulation. • Simplicity and stability as a bidirectional construct in smaller size. • Applicability in Protein Localization studies • Adjustment of the level of gene expression only by copy number. Cannot be switched off completely.

24 Bacterial Plasmid P.falciparum system: Oversize and Complexity Bidirectional Activity Regulation in gene Protein over-expression, expression level trafficking in the parasite, etc

25 Thank you for your Attention! Special Gratitude to Prof. HUANG

26 Definition • Parasitaemia: the ratio of a group of RBC being invaded be parasite(s). Regular ratio~1% eg. A group of 1000 rbc within which 20 are invaded yields parasitaemia of 2%.

27 Problems in previous efforts • ATc-inducible system: tetracycline analogue called anhydrotetracycline. This system’s regulatable in blood stages. ✖Tedious in 2 different selection cycles ✖Very large in Construct size • FKBP12 system: Engineered destabilizing domain of human FK506 binding protein (ddFKBP), promoting degradation of fused protein. But in the presence of Shld1 , degradation is reduced. ✖Efficacy is limited to the protein investigated

28 Blasticidin-s deaminase • Blasticidin-S aminohydrolase • EC 3.5.4.23 PDB access: 1WN5/1WN6 • Blasticidin-S + H 2 O = deaminohydroxyblasticidin-S + NH 3 o