P. falciparum Life Cycle & Pathogenesis of Malaria Miller et al., Nature  Molecular and genetic mechanisms underlying this diversity are poorly understood, but likely involve both host and pathogen biology Symptoms occur in the intraerythrocytic stage

Study Design  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 /- 6.8  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 /- 6.8 Screen 1900 Patients Hybridize 2 different chips Isolate human/parasite RNA directly from blood draw Velingara, Senegal

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

Gene Set Enrichment Analysis  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  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 Subramanian et al., PNAS

195 Starvation (44, P=1.5X10 -7 ) General Tx mutants (23, P=2.8X10 -5 ) 469 Stress (278, P=4.6X ) 350 Glucose fermentation (168, P=2.3X ) P. falciparum array S. cerevisiae array  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  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 Cross-species Projections

GSEA & NMF: Human Expression Profile  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  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 3 (n=4) k = 3; cophenetic coefficient = (n=16) 1 (n=8) These are not same three clusters seen in the parasite.

Methods  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 Clustered matrix of genes vs gene sets Created gene set from the union of 15 leading edges Identified “leading edge genes” that contributed to each enrichment (FWER≤0.05) GSEA using clinical correlate as phenotype continuous clinical variable (e.g. parasitemia, hematocrit, cytokine level) Human Clustered Gene SetsParasite Clustered Gene Sets Human Genes Parasite Genes blue: negatively correlated, red: positively correlated

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

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 #