NSR-seq transcriptional profiling enables identification of a gene signature of Plasmodium falciparum parasites infecting children

Abstract

Malaria caused by Plasmodium falciparum results in approximately 1 million annual deaths worldwide, with young children and pregnant mothers at highest risk. Disease severity might be related to parasite virulence factors, but expression profiling studies of parasites to test this hypothesis have been hindered by extensive sequence variation in putative virulence genes and a preponderance of host RNA in clinical samples. We report here the application of RNA sequencing to clinical isolates of P. falciparum, using not-so-random (NSR) primers to successfully exclude human ribosomal RNA and globin transcripts and enrich for parasite transcripts. Using NSR-seq, we confirmed earlier microarray studies showing upregulation of a distinct subset of genes in parasites infecting pregnant women, including that encoding the well-established pregnancy malaria vaccine candidate var2csa. We also describe a subset of parasite transcripts that distinguished parasites infecting children from those infecting pregnant women and confirmed this observation using quantitative real-time PCR and mass spectrometry proteomic analyses. Based on their putative functional properties, we propose that these proteins could have a role in childhood malaria pathogenesis. Our study provides proof of principle that NSR-seq represents an approach that can be used to study clinical isolates of parasites causing severe malaria syndromes as well other blood-borne pathogens and blood-related diseases.

Plasmodium Helical Interspersed Subtelomeric (PHIST) Proteins, at the Center of Host Cell Remodeling

“…Genes annotated "phista-like" or as "phist" genes were grouped together as "phist others." Yellow boxes to the right of the heat map indicate PHIST proteins present in the early gametocyte proteome (35), differentially expressed phist genes in pregnancy-associated malaria (29,49,50,84,92,112) and in cerebral malaria (32,34), or if variant expression has been reported (55). Green to red colors represent fold changes (log fold changes from Ϫ3 to 3).…”

Erythrocyte Lysis and Xenopus laevis Oocyte Rupture by Recombinant Plasmodium falciparum Hemolysin III

“…The cDNA encodes a polypeptide of 282 amino acids with a predicted molecular mass of 33 kDa. PfHly III gene transcripts were detected in the erythrocytic stages, in gametocytes, and in patient samples from pregnant women and also children (9,10). Mass spectrometry detected PfHly III in gametocytes (11).…”

Expression of the Plasmodium falciparum Clonally Variant clag3 Genes in Human Infections

“…Previous studies of clag3 expression were conducted with culture-adapted parasites. Genome-wide transcriptomic analysis of P. falciparum field isolates [27][28][29] could not reliably characterize the expression patterns of these genes because the sequences of clag3.1 and clag3.2 are almost identical and the regions that are more distinct between the 2 genes are highly polymorphic between parasite isolates [13]. Here we developed a procedure to analyze clag3 expression in natural human infections and used it to study the expression of these genes in clinical malaria cases and after parasite adaptation to culture conditions or to blasticidin S pressure.…”