Proteomics in Malaria Parasites: Packed with Potential!
Janette ReaderUniversity 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)
ParasiteVacuole
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 1955-1969
Teng et al, 2009
Florens et al, 2002Bozdech et al, 2003
FUNCTIONAL GENOMICS
GMEP 1955-1969
GENE REGULATION IN P. FALCIPARUM
• Genome of P. falciparum - 22.8 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 stagesFlorens et al, 2002
Smit et al., J Prot Res, 2010 Clark et al., Biochem J, 2008Van Brummelen et al, JBC, 2010
INFORMATION GAINED FROM FUNCTIONAL GENOMICS
GMEP 1955-1969
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 PROTEINSWork 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
CCB MS Silver
Flamingo PinkSYPRO Ruby
OPTIMIZATION OF PROTEOMICSSensitivity 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 328 77 73 95Trophozoite stage
272 63 52 83
Total 600 140 125 90
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