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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

In vivoparasite gene expression clusters by patient. (A) Hierarchical clustering of expression of 328 genes measured by nCounter® from 13 tissue samples collected at autopsy, collected from five patients and three organs. Clustering demonstrates that samples cluster by patient, rather than by organ, suggesting that parasite physiology within a patient is conserved. (B) Hierarchical clustering of global expression profiles imputed from nCounter® (n = 3,696 genes) shows that within-patient clusters remain intact. Note that two outliers in the first analysis (P1, Brain and P2, Heart) are further delineated as true outliers after imputation.
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Fig4: In vivoparasite gene expression clusters by patient. (A) Hierarchical clustering of expression of 328 genes measured by nCounter® from 13 tissue samples collected at autopsy, collected from five patients and three organs. Clustering demonstrates that samples cluster by patient, rather than by organ, suggesting that parasite physiology within a patient is conserved. (B) Hierarchical clustering of global expression profiles imputed from nCounter® (n = 3,696 genes) shows that within-patient clusters remain intact. Note that two outliers in the first analysis (P1, Brain and P2, Heart) are further delineated as true outliers after imputation.

Mentions: We used the nCounter® platform and global imputation to ask whether in vivo parasite samples cluster by organ or by patient (Figure 4), and to detect genes that are overexpressed by parasites sequestered in human tissues (Table 1). Hierarchical clustering of in vivo sequestered parasites from autopsy tissue samples showed that samples cluster by patient, rather than by organ, regardless of whether clustering is based on gene expression measured by the nCounter® platform (Figure 4A), or imputed gene expression (Figure 4B). Differential gene expression analysis between rank-normalized imputed in vivo and in vitro microarray data revealed a shared set of 39 genes that were among the top 100 overexpressed genes in all three of the analyzed patients (Table 1). This set of in vivo overexpressed genes included 10 genes with maximal expression during sexual and mosquito stages, 10 genes with maximal in vitro expression during trophozoite and schizont stages, seven genes that are not expressed in vitro, and six genes that code for ribosomal proteins. These data indicate that parasites sequestered in human tissues may exist in a unique physiological state.Figure 4


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)

In vivoparasite gene expression clusters by patient. (A) Hierarchical clustering of expression of 328 genes measured by nCounter® from 13 tissue samples collected at autopsy, collected from five patients and three organs. Clustering demonstrates that samples cluster by patient, rather than by organ, suggesting that parasite physiology within a patient is conserved. (B) Hierarchical clustering of global expression profiles imputed from nCounter® (n = 3,696 genes) shows that within-patient clusters remain intact. Note that two outliers in the first analysis (P1, Brain and P2, Heart) are further delineated as true outliers after imputation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: In vivoparasite gene expression clusters by patient. (A) Hierarchical clustering of expression of 328 genes measured by nCounter® from 13 tissue samples collected at autopsy, collected from five patients and three organs. Clustering demonstrates that samples cluster by patient, rather than by organ, suggesting that parasite physiology within a patient is conserved. (B) Hierarchical clustering of global expression profiles imputed from nCounter® (n = 3,696 genes) shows that within-patient clusters remain intact. Note that two outliers in the first analysis (P1, Brain and P2, Heart) are further delineated as true outliers after imputation.
Mentions: We used the nCounter® platform and global imputation to ask whether in vivo parasite samples cluster by organ or by patient (Figure 4), and to detect genes that are overexpressed by parasites sequestered in human tissues (Table 1). Hierarchical clustering of in vivo sequestered parasites from autopsy tissue samples showed that samples cluster by patient, rather than by organ, regardless of whether clustering is based on gene expression measured by the nCounter® platform (Figure 4A), or imputed gene expression (Figure 4B). Differential gene expression analysis between rank-normalized imputed in vivo and in vitro microarray data revealed a shared set of 39 genes that were among the top 100 overexpressed genes in all three of the analyzed patients (Table 1). This set of in vivo overexpressed genes included 10 genes with maximal expression during sexual and mosquito stages, 10 genes with maximal in vitro expression during trophozoite and schizont stages, seven genes that are not expressed in vitro, and six genes that code for ribosomal proteins. These data indicate that parasites sequestered in human tissues may exist in a unique physiological state.Figure 4

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