Transcriptional Profiling of Plasmodium falciparum Parasites from Patients with Severe Malaria Identifies Distinct Low vs. High Parasitemic Clusters

Background: In the past decade, estimates of malaria infections have dropped from 500 million to 225 million per year; likewise, mortality rates have dropped from 3 million to 791,000 per year. However, approximately 90% of these deaths continue to occur in sub-Saharan Africa, and 85% involve children less than 5 years of age. Malaria mortality in children generally results from one or more of the following clinical syndromes: severe anemia, acidosis, and cerebral malaria. Although much is known about the clinical and pathological manifestations of CM, insights into the biology of the malaria parasite, specifically transcription during this manifestation of severe infection, are lacking. Methods and Findings: We collected peripheral blood from children meeting the clinical case definition of cerebral malaria from a cohort in Malawi, examined the patients for the presence or absence of malaria retinopathy, and performed whole genome transcriptional profiling for Plasmodium falciparum using a custom designed Affymetrix array. We identified two distinct physiological states that showed highly significant association with the level of parasitemia. We compared both groups of Malawi expression profiles with our previously acquired ex vivo expression profiles of parasites derived from infected patients with mild disease; a large collection of in vitro Plasmodium falciparum life cycle gene expression profiles; and an extensively annotated compendium of expression data from Saccharomyces cerevisiae. The high parasitemia patient group demonstrated a unique biology with elevated expression of Hrd1, a member of endoplasmic reticulum-associated protein degradation system. Conclusions: The presence of a unique high parasitemia state may be indicative of the parasite biology of the clinically recognized hyperparasitemic severe disease syndrome

Metagene projection of 58 Malawi <i>ex vivo</i> samples, 43 Senegal <i>ex vivo</i> samples, as well as yeast and <i>in vitro</i> treated <i>P. falciparum</i> compendia onto the two-cluster Malawi model.

<p>(a) Metagene projection of 58 Malawi samples onto the two-cluster Malawi model. F1 and F2 represent the two metagene axes with F1 corresponding to the “low parasitemia” Cluster A (orange) and F2 corresponding to the “high parasitemia” Cluster B (blue). (b-d) Metagene projection of 43 Senegal samples (from Daily <i>et al</i>.) by cluster designation. Senegal Clusters 1 and 3 project onto the “low parasitemia” Malawi Cluster A while Senegal Cluster 2 projects either on Cluster A or at the transition between Clusters A and B. (e) Metagene projection of 1,439 yeast expression profiles onto the Malawi space. Enrichments of yeast experiments in Clusters A and B are consistent with projections of Senegal samples. There is a distinct space in Cluster B that is not covered by any yeast experiments, thus representing novel biology in the high parasitemia Malawi samples.</p

Heatmap showing the enrichment scores from single sample GSEA analysis using our general gene set categories.

<p>Heatmap showing the enrichment scores from single sample GSEA analysis using our general gene set categories.</p

A summary of the parameters for the patients included in the study.

<p>A summary of the parameters for the patients included in the study.</p

Enrichment of general categories derived from metagene projections of 1,439 yeast expression profiles onto the two-cluster Malawi model.

<p>Additionally, we used the manual and automated (gene set-based) annotations of the yeast samples to determine yeast conditions enriched in Clusters A and B. We observed that the yeast experiments fully cover the “low parasitemia” Cluster A and only a subset – the lower parasitemia region – of the “high parasitemia” Cluster B. Enriched in Cluster B are fermentation (happy growing yeast), perturbation in protein biosynthesis, and knock-outs in yeast peptome dextrose medium. Enriched in Cluster A are stress, starvation, amino acid and nitrogen starvation, respiration, and limiting growth.</p

Association of clinical variables to Clusters A and B.

<p>Patients in Cluster B have significantly higher levels of parasitemia and hematocrit, while a higher proportion of patients in Cluster A use bed nets.</p

Results from GSEA analysis using our general gene set categories.

<p>Results from GSEA analysis using our general gene set categories.</p

Heatmap showing expression profiles after identification of two distinct physiological states, the “low parasitemia” Cluster A (orange) and the “high parasitemia” Cluster B (blue).

<p>Samples were sorted by parasitemia within each class. Parasitemia is indicated at the top in log10 scale, ranging from low (white) to high (black). Genes are sorted by their degree of differential expression between clusters A and B.</p