P. falciparum Life Cycle & Pathogenesis of Malaria

1. P. falciparum Life Cycle & Pathogenesis of Malaria • Symptoms occur in the intraerythrocytic stage http://www.cdc.gov/malaria/biology/life_cycle.htm • Molecular and genetic mechanisms underlying this diversity are poorly understood, but likely involve both host and pathogen biology Miller et al., Nature 2002

2. Study Design Screen 1900 Patients Velingara, Senegal Isolate human/parasite RNA directly from blood draw Hybridize 2 different chips • 43 samples hybridized to custom P. falciparum (3D7) chip • 28 samples also hybridized to HG_U133A chip • Diverse age range: 8.3 +/- 6.9 years • Illness severity: parasitemia 5.5% +/- 6.2%, hematocrit 32.3 +/- 6.8

3. NMF Parasite Clusters & Patient Clinical Correlates 1 (n=8) 2 (n=17) 3 (n=18) Samples (n=43) Samples (n=43, NMF clustered) >3 Genes (n=3900) NMF = Nonnegative Matrix Factorization

4. Gene Set Enrichment Analysis Subramanian et al., PNAS 2005 • The parasites look similar in each patient blood sample, early ring stage, however, GSEA identified gene sets differentially expressed between clusters • Major metabolic shift • Cluster 1: Starvation • Cluster 2: Glycolytic Metabolism • More like in vitro model 1 2 3

5. Cross-species Projections Glucose fermentation(168, P=2.3X10-23) 350 P. falciparum array S. cerevisiae array 195 Starvation(44, P=1.5X10-7) General Tx mutants(23, P=2.8X10-5) Stress (278, P=4.6X10-22) 469 • Large S. cerevisiae expression compendium projected onto the expression space defined by the 3 P. falciparum NMF clusters • Cluster 1 resembles a starvation response, while Cluster 3 resembles an environmental stress response, consistent with elevated markers of inflammation measured in patient sera

6. GSEA & NMF:Human Expression Profile These are not same three clusters seen in the parasite. k = 3; cophenetic coefficient = 0.994 1 (n=8) 2 (n=16) 3 (n=4) • Clustering of human profiles did not match parasite clusters • Gene sets related to carbon sources were not enriched • e.g. Fatty acid, nitrogen, & glycolytic metabolism • Enrichment in many other gene sets (FDR≤0.05) • e.g. DNA replication, RNA transcription, and DNA repair

7. Methods continuous clinical variable (e.g . parasitemia, hematocrit, cytokine level) blue: negatively correlated, red: positively correlated • GSEA revealed gene sets with inflammatory response and oxidative phosphorylation gene signatures in human and gene sets related to cell cycle and virulence in parasite Human Clustered Gene Sets Parasite Clustered Gene Sets Parasite Genes Human Genes

8. Host-Pathogen Interaction Human Expression Profile of Clinically Correlated Genes P. falciparum Expression Profile of Clinically Correlated Genes 1 (n=8) 2 (n=16) 3 (n=4) 1 (n=8) 2 (n=17) 3 (n=18) NMF Clustering of Parasite Expression Profile k=2; cophenetic coefficient=0.988 k=3; cophenetic coefficient=0.999 k=4 cophenetic coefficient=0.992 • Can these metabolic shifts be explained by the host environment? • NMF on parasite profile using gene set reflects the previously identified clusters

9. Conclusions & Future Work • Can we identify targets for updating previous models? • Previously unknown physiological diversity revealed in the in vivo biology of the malaria parasite • Update in vitro models by varying carbon sources and monitoring response to cytokines • Is there a biological story driving transcriptional changes? • Role of chromatin/global transcriptional mechanisms in mediating transcriptional shift • Host immune response possibly driving metabolic shifts in P. falciparum • R. Ordoñez was supported by the NIGMS Cell Decision Processes Grant #6914372