P. falciparum Life Cycle & Pathogenesis

of Malaria

Miller et al., Nature 2002

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

Molecular and genetic mechanisms underlying this diversity are poorly understood, but likely involve both host and pathogen biology

• Symptoms occur in the intraerythrocytic stage

• 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 32.3 +/- 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 32.3 +/- 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)

Gen

es

(n=

39

00

)

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 2005

1

2

3

195

Starvation(44, P=1.5X10-7)

General Tx mutants(23, P=2.8X10-5) 469

Stress (278, P=4.6X10-22)

350Glucose fermentation(168, P=2.3X10-23) 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 = 0.994

2 (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

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

continuous clinical variable (e.g . parasitemia, hematocrit,

cytokine level)

Human Clustered Gene Sets

Parasite Clustered Gene Sets

Hu

man

Gen

es

Para

sit

e G

en

es

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 Clusterin

g 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

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

R. Ordoñez was supported by the NIGMS Cell Decision Processes Grant #6914372