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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

P. falciparumtranscriptional profiles can be measuredin vitroandin vivowith nCounter® using 328 genes. (A)In vitro intraerythrocytic gene expression measured in two different 3D7 ring-stage and schizont-stage cultures. Spearman correlations between nCounter® expression and in vitro expression [4], is plotted versus life cycle time point. Representative illustrations of parasite developmental stages are included from [26]. (B-E)In vivo sequestered parasite gene expression measured in postmortem blood and tissues from Malawian children that succumbed to cerebral malaria (B-D), or another cause of death (E). Spearman correlations between nCounter® expression and in vitro expression are plotted versus life cycle time point. Numbers in parentheses in the legend of each panel are the number of parasites counted in 10 high-power fields by microscopy.
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Fig2: P. falciparumtranscriptional profiles can be measuredin vitroandin vivowith nCounter® using 328 genes. (A)In vitro intraerythrocytic gene expression measured in two different 3D7 ring-stage and schizont-stage cultures. Spearman correlations between nCounter® expression and in vitro expression [4], is plotted versus life cycle time point. Representative illustrations of parasite developmental stages are included from [26]. (B-E)In vivo sequestered parasite gene expression measured in postmortem blood and tissues from Malawian children that succumbed to cerebral malaria (B-D), or another cause of death (E). Spearman correlations between nCounter® expression and in vitro expression are plotted versus life cycle time point. Numbers in parentheses in the legend of each panel are the number of parasites counted in 10 high-power fields by microscopy.

Mentions: In vitro and in vivo parasite life cycle stages, from both peripheral blood and human tissue, are easily and accurately predicted by 328 P. falciparum genes measured by the nCounter® custom malaria platform (Figure 2, Additional file 1). nCounter® transcript counts were compared to a previously published in vitro life cycle time course, where parasite transcriptional profiles were measured every hour over the 48-h intraerythrocytic life cycle [4]. Two independent cultures of in vitro culture-adapted 3D7 parasites, which were synchronized and harvested at either ring stage or schizont stage, showed peak correlation with life cycle time points corresponding to the appropriate stage (Figure 2A).Figure 2


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)

P. falciparumtranscriptional profiles can be measuredin vitroandin vivowith nCounter® using 328 genes. (A)In vitro intraerythrocytic gene expression measured in two different 3D7 ring-stage and schizont-stage cultures. Spearman correlations between nCounter® expression and in vitro expression [4], is plotted versus life cycle time point. Representative illustrations of parasite developmental stages are included from [26]. (B-E)In vivo sequestered parasite gene expression measured in postmortem blood and tissues from Malawian children that succumbed to cerebral malaria (B-D), or another cause of death (E). Spearman correlations between nCounter® expression and in vitro expression are plotted versus life cycle time point. Numbers in parentheses in the legend of each panel are the number of parasites counted in 10 high-power fields by microscopy.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: P. falciparumtranscriptional profiles can be measuredin vitroandin vivowith nCounter® using 328 genes. (A)In vitro intraerythrocytic gene expression measured in two different 3D7 ring-stage and schizont-stage cultures. Spearman correlations between nCounter® expression and in vitro expression [4], is plotted versus life cycle time point. Representative illustrations of parasite developmental stages are included from [26]. (B-E)In vivo sequestered parasite gene expression measured in postmortem blood and tissues from Malawian children that succumbed to cerebral malaria (B-D), or another cause of death (E). Spearman correlations between nCounter® expression and in vitro expression are plotted versus life cycle time point. Numbers in parentheses in the legend of each panel are the number of parasites counted in 10 high-power fields by microscopy.
Mentions: In vitro and in vivo parasite life cycle stages, from both peripheral blood and human tissue, are easily and accurately predicted by 328 P. falciparum genes measured by the nCounter® custom malaria platform (Figure 2, Additional file 1). nCounter® transcript counts were compared to a previously published in vitro life cycle time course, where parasite transcriptional profiles were measured every hour over the 48-h intraerythrocytic life cycle [4]. Two independent cultures of in vitro culture-adapted 3D7 parasites, which were synchronized and harvested at either ring stage or schizont stage, showed peak correlation with life cycle time points corresponding to the appropriate stage (Figure 2A).Figure 2

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