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