Proteomics in Malaria Parasites: Packed with Potential! Janette Reader University of Pretoria Proteomics in Malaria Parasites: Packed with Potential! Janette Reader University of Pretoria Department of Biochemistry

MALARIA The most devastating tropical infectious disease 300 million people infected annually 1 million deaths 88% children under the age of 5 90% of disease burden in sub-Saharan Africa PROBLEM: Increasing drug and insecticide resistance of the malaria parasite and mosquito vector

BIOLOGICAL CONTROL Parasite control Vector control Monitoring & Diagnostics Vaccines (transmission blocking)

Parasite Vacuole Erythrocyte Mitochondrion Apicoplast Nucleus Transporters Food vacuole ER & Golgi 23 megabase 5332 transcripts ~5300 proteins BIOLOGICAL MECHANISMS FOR PARASITE CONTROL Genome Transcriptome Proteome

Genome-wide questions Functional genomics tools Biological and mechanistic insights Transcriptomics Proteomics Interactomics Bioinformatics Transcriptional machinery Regulation of transcription Transcriptional inheritance Posttranscriptional regulation Posttranslational repression Protein function Relationships Regulatory mechanisms Lifecycle development (stage- specific expression) Reproduction genes (strategy-specific expression) Drug resistance mechanisms Mechanism of drug action Reponses to environmental stressors Drug target specification Host-specific adaptation and expression Identification of vaccine targets Virulence determinants Severe disease progression in vivo Metabolic pathways Identity determination of hypothetical proteins Cell cycle regulators Sex determinants Chemical validation of drug targets Mode-of-action of inhibitory compounds Improved drug target action Gene expression regulators (transcription and translation) Virulence factors Specialised organel function and metabolism Damage compensation Drug target identification Birkholtz et al, Malaria J 2006

GMEP Teng et al, 2009 Florens et al, 2002 Bozdech et al, 2003 FUNCTIONAL GENOMICS

GMEP GENE REGULATION IN P. FALCIPARUM Genome of P. falciparum Mb Encodes for  5300 genes 14 chromosomes  80% A+T nucleotide content Linear mitochondrial genome  6 kb Circular apicoplast genome  35 kb 60% of the predicted open reading frames show no sequence similarity to genes from other organisms Transcripts are only produced when needed during the life cycle of the parasite in a ‘just-in- time’ fashion Bozdech et al, 2003

PROTEOME OF P. FALCIPARUM Predicted to have about 5300 proteins Comprehensive description of only 46% of proteome (~2400 proteins) Proteins –Have long hydrophobic stretches (insoluble) –Have amino acid repeats (Lysine and Asparagine) –Are comparatively large –Are non-homologous –Highly charged Proteome is complex: stage-specific expression of proteins associated with biological and metabolic changes 6% common to all stages Florens et al, 2002

Smit et al., J Prot Res, 2010 Clark et al., Biochem J, 2008 Van Brummelen et al, JBC, 2010 INFORMATION GAINED FROM FUNCTIONAL GENOMICS

GMEP STRATEGY IN OUR LAB Birkholtz et al., 2008

Panpumthong (2006), et al (~200 spots) Nirmalan (2004), et al Makanga (2005), et al Aly (2007), et al (~200 spots) Wu (2006), et al PROTEOMIC PROTOCOLS AVAILIBLE

EXPERIMENTAL LAYOUT Isolation of proteins Concentration with 2D-Quant Determination of proteins with difference in abundance using PD Quest Identification of proteins — Plasmo2D — Mass Spectrometry

QUANTIFICATION OF PROTEINS Work done by Dr Salome Smit Bradford Bicinchoninic Acid (BCA) Lowry 2D Quant kit

EXPERIMENTAL LAYOUT Isolation of proteins Concentration with 2D-Quant Determination of proteins with difference in abundance using PD Quest Identification of proteins — Plasmo2D — Mass Spectrometry

CCBMS Silver Flamingo PinkSYPRO Ruby OPTIMIZATION OF PROTEOMICS Sensitivity of stains on Plasmodial proteins Work done by Dr Salome Smit Smit et al., J Prot Res, 2010 CCB MS Silver Flamingo PinkSYPRO Ruby

Smit et al., J Prot Res, 2010 SUMMARY OF STAIN PERFORMANCE ON 1-D AND 2-DE WITH PLASMODIAL PROTEINS

Smit et al., 2010 Ring stage Trophozoite stage STAGE SPECIFIC PROTEINS Spots detected (PD Quest) Nr cut for MS Nr identified by MS Identification success rate (%) Ring stage Trophozoite stage Total Smit et al., J Prot Res, 2010

APPLICATION OF THIS METHOD IN OTHER STUDIES IN P. FALCIPARUM Co-inhibition of AdoMetDC/ODC in P. falciparum (Tharina Van Brummelen) Mono-functional inhibition of AdoMetDC in P. falciparum (Salome Smit) Mono-functional inhibition of ODC in P. falciparum (Katherine Clark) Herbicide derived compounds tested on P. falciparum (Janette Reader & Jeff Verlinden)

ACKNOWLEDGEMENTS Prof L Birkholtz Prof AI Louw Dr Salome Smit Malaria team – Past and Present