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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