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Plasmodium falciparum gene expression measured directly from tissue during human infection.

Van Tyne D, Tan Y, Daily JP, Kamiza S, Seydel K, Taylor T, Mesirov JP, Wirth DF, Milner DA - Genome Med (2014)

Bottom Line: Because these life cycle stages are not easily sampled due to deep tissue sequestration, measuring in vivo gene expression of parasites in the trophozoite and schizont stages has been a challenge.Finally, differential expression revealed a limited set of in vivo upregulated transcripts, which may indicate unique parasite genes involved in human clinical infections.Our study highlights the utility of a custom nCounter® P. falciparum probe set, validation of imputation within Plasmodium species, and documentation of in vivo schizont-stage expression patterns from human tissues.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, MA USA.

ABSTRACT

Background: During the latter half of the natural 48-h intraerythrocytic life cycle of human Plasmodium falciparum infection, parasites sequester deep in endothelium of tissues, away from the spleen and inaccessible to peripheral blood. These late-stage parasites may cause tissue damage and likely contribute to clinical disease, and a more complete understanding of their biology is needed. Because these life cycle stages are not easily sampled due to deep tissue sequestration, measuring in vivo gene expression of parasites in the trophozoite and schizont stages has been a challenge.

Methods: We developed a custom nCounter® gene expression platform and used this platform to measure malaria parasite gene expression profiles in vitro and in vivo. We also used imputation to generate global transcriptional profiles and assessed differential gene expression between parasites growing in vitro and those recovered from malaria-infected patient tissues collected at autopsy.

Results: We demonstrate, for the first time, global transcriptional expression profiles from in vivo malaria parasites sequestered in human tissues. We found that parasite physiology can be correlated with in vitro data from an existing life cycle data set, and that parasites in sequestered tissues show an expected schizont-like transcriptional profile, which is conserved across tissues from the same patient. Imputation based on 60 landmark genes generated global transcriptional profiles that were highly correlated with genome-wide expression patterns from the same samples measured by microarray. Finally, differential expression revealed a limited set of in vivo upregulated transcripts, which may indicate unique parasite genes involved in human clinical infections.

Conclusions: Our study highlights the utility of a custom nCounter® P. falciparum probe set, validation of imputation within Plasmodium species, and documentation of in vivo schizont-stage expression patterns from human tissues.

No MeSH data available.


Related in: MedlinePlus

The nCounter® platform has a large dynamic range and can be used with RNA extracted from various malaria patient sample types. (A, B) Linearity of total transcript counts versus absolute number of in vitro culture-adapted 3D7 parasites that were synchronized and isolated as (A) rings, or (B) schizonts. Horizontal lines indicate mean transcript counts among all genes. (C, D) Processed sample transcript counts versus pre-processing lysate transcript counts for (C) a mock filter paper sample, and (D) a mock formalin fixed paraffin embedded (FFPE) sample. (C) Culture-adapted 3D7 parasites were synchronized and grown to ring stage, and then were spotted onto Whatman filter paper. (D) Culture-adapted 3D7 parasites were synchronized and grown to trophozoite stage, and then parasitized red blood cells were clotted and fixed in formalin before paraffin embedding, sectioning, and processing.
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Fig1: The nCounter® platform has a large dynamic range and can be used with RNA extracted from various malaria patient sample types. (A, B) Linearity of total transcript counts versus absolute number of in vitro culture-adapted 3D7 parasites that were synchronized and isolated as (A) rings, or (B) schizonts. Horizontal lines indicate mean transcript counts among all genes. (C, D) Processed sample transcript counts versus pre-processing lysate transcript counts for (C) a mock filter paper sample, and (D) a mock formalin fixed paraffin embedded (FFPE) sample. (C) Culture-adapted 3D7 parasites were synchronized and grown to ring stage, and then were spotted onto Whatman filter paper. (D) Culture-adapted 3D7 parasites were synchronized and grown to trophozoite stage, and then parasitized red blood cells were clotted and fixed in formalin before paraffin embedding, sectioning, and processing.

Mentions: We developed a custom nCounter® malaria parasite gene probe set containing 328 genes, which were selected from a compendium of preexisting malaria expression microarray data sets (Methods). Genes selected for the probe set included genes that can distinguish between intraerythrocytic life cycle stages, highly differentially expressed genes from previous in vivo studies [8,9], landmark genes for imputation, and other genes of interest. This custom nCounter® probe set measured P. falciparum gene expression with excellent limit of detection along a 5-log dynamic range (Figure 1A and B). Transcripts were accurately counted from as few as 6,000 parasites, a volume that corresponds to 16 picoliters of parasitized red blood cells at 4% parasitemia. A range of sample types were tested and found to be suitable for analysis with nCounter®, including parasitized red blood cells spotted onto Whatman filter papers (Figure 1C), and formalin fixed paraffin embedded tissue blocks (Figure 1D). All data are deposited with NIH GEO (accession # GSE63260 and link: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE63260).Figure 1


Plasmodium falciparum gene expression measured directly from tissue during human infection.

Van Tyne D, Tan Y, Daily JP, Kamiza S, Seydel K, Taylor T, Mesirov JP, Wirth DF, Milner DA - Genome Med (2014)

The nCounter® platform has a large dynamic range and can be used with RNA extracted from various malaria patient sample types. (A, B) Linearity of total transcript counts versus absolute number of in vitro culture-adapted 3D7 parasites that were synchronized and isolated as (A) rings, or (B) schizonts. Horizontal lines indicate mean transcript counts among all genes. (C, D) Processed sample transcript counts versus pre-processing lysate transcript counts for (C) a mock filter paper sample, and (D) a mock formalin fixed paraffin embedded (FFPE) sample. (C) Culture-adapted 3D7 parasites were synchronized and grown to ring stage, and then were spotted onto Whatman filter paper. (D) Culture-adapted 3D7 parasites were synchronized and grown to trophozoite stage, and then parasitized red blood cells were clotted and fixed in formalin before paraffin embedding, sectioning, and processing.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4269068&req=5

Fig1: The nCounter® platform has a large dynamic range and can be used with RNA extracted from various malaria patient sample types. (A, B) Linearity of total transcript counts versus absolute number of in vitro culture-adapted 3D7 parasites that were synchronized and isolated as (A) rings, or (B) schizonts. Horizontal lines indicate mean transcript counts among all genes. (C, D) Processed sample transcript counts versus pre-processing lysate transcript counts for (C) a mock filter paper sample, and (D) a mock formalin fixed paraffin embedded (FFPE) sample. (C) Culture-adapted 3D7 parasites were synchronized and grown to ring stage, and then were spotted onto Whatman filter paper. (D) Culture-adapted 3D7 parasites were synchronized and grown to trophozoite stage, and then parasitized red blood cells were clotted and fixed in formalin before paraffin embedding, sectioning, and processing.
Mentions: We developed a custom nCounter® malaria parasite gene probe set containing 328 genes, which were selected from a compendium of preexisting malaria expression microarray data sets (Methods). Genes selected for the probe set included genes that can distinguish between intraerythrocytic life cycle stages, highly differentially expressed genes from previous in vivo studies [8,9], landmark genes for imputation, and other genes of interest. This custom nCounter® probe set measured P. falciparum gene expression with excellent limit of detection along a 5-log dynamic range (Figure 1A and B). Transcripts were accurately counted from as few as 6,000 parasites, a volume that corresponds to 16 picoliters of parasitized red blood cells at 4% parasitemia. A range of sample types were tested and found to be suitable for analysis with nCounter®, including parasitized red blood cells spotted onto Whatman filter papers (Figure 1C), and formalin fixed paraffin embedded tissue blocks (Figure 1D). All data are deposited with NIH GEO (accession # GSE63260 and link: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE63260).Figure 1

Bottom Line: Because these life cycle stages are not easily sampled due to deep tissue sequestration, measuring in vivo gene expression of parasites in the trophozoite and schizont stages has been a challenge.Finally, differential expression revealed a limited set of in vivo upregulated transcripts, which may indicate unique parasite genes involved in human clinical infections.Our study highlights the utility of a custom nCounter® P. falciparum probe set, validation of imputation within Plasmodium species, and documentation of in vivo schizont-stage expression patterns from human tissues.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, MA USA.

ABSTRACT

Background: During the latter half of the natural 48-h intraerythrocytic life cycle of human Plasmodium falciparum infection, parasites sequester deep in endothelium of tissues, away from the spleen and inaccessible to peripheral blood. These late-stage parasites may cause tissue damage and likely contribute to clinical disease, and a more complete understanding of their biology is needed. Because these life cycle stages are not easily sampled due to deep tissue sequestration, measuring in vivo gene expression of parasites in the trophozoite and schizont stages has been a challenge.

Methods: We developed a custom nCounter® gene expression platform and used this platform to measure malaria parasite gene expression profiles in vitro and in vivo. We also used imputation to generate global transcriptional profiles and assessed differential gene expression between parasites growing in vitro and those recovered from malaria-infected patient tissues collected at autopsy.

Results: We demonstrate, for the first time, global transcriptional expression profiles from in vivo malaria parasites sequestered in human tissues. We found that parasite physiology can be correlated with in vitro data from an existing life cycle data set, and that parasites in sequestered tissues show an expected schizont-like transcriptional profile, which is conserved across tissues from the same patient. Imputation based on 60 landmark genes generated global transcriptional profiles that were highly correlated with genome-wide expression patterns from the same samples measured by microarray. Finally, differential expression revealed a limited set of in vivo upregulated transcripts, which may indicate unique parasite genes involved in human clinical infections.

Conclusions: Our study highlights the utility of a custom nCounter® P. falciparum probe set, validation of imputation within Plasmodium species, and documentation of in vivo schizont-stage expression patterns from human tissues.

No MeSH data available.


Related in: MedlinePlus