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Dual RNA-sequencing of host and pathogen during Plasmodium berghei infection of hepatocytes in vitro

ABSTRACT: We report the dual RNA-sequencing of host and pathogen transcriptomes during Plasmodium berghei liver-stage development in vitro. Unlike traditional transcriptomic approaches that analyze RNA reads separately from host and pathogen, a dual-approach maps the mixed reads to each annotated genome within samples of pathogen-infected host cells. This is a powerful method, as host and pathogen transcriptomes can be analyzed simultaneously. We have taken advantage of this dual-RNA sequencing approach in order to gain insight into Plasmodium liver stage development within host hepatocytes. Huh7.5.1 hepatocytes were infected in vitro with P. berghei sporozoites freshly dissected from infected Anopheles stephansi mosquitos, and cells were collected throughout liver-stage development. This included samples collected at time zero (uninfected hepatocytes and sporozoites before infection), time 24 hours post infection (when the sporozoites have transformed into trophozoites), and time 48-50 hours post infection (when the trophozoites have transformed into liver-stage schizonts).

ORGANISM(S): Plasmodium berghei Homo sapiens mixed sample

PROVIDER: GSE78931 | GEO | 2018/03/06

REPOSITORIES: GEO

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Project description:After entering their mammalian host via the bite of an Anopheles mosquito, Plasmodium sporozoites migrate to the liver where they traverse several hepatocytes before invading the one inside which they will develop and multiply into thousands of merozoites. Although this constitutes an essential step in malaria infection, the requirements of Plasmodium parasites in liver cells and how they use the host cell for their own survival and development are poorly understood. To gain new insights into the molecular host-parasite interactions that take place during malaria liver infection, we have used high-throughput microarray technology to determine the transcriptional profile of P. yoelii-infected hepatocytes that were collected from P. yoelii-infected mice 24 and 40 h after infection. This in vivo microarray expression was compared with the microarray analysis of in vitro infected hepatoma cells infected with closely related rodent malaria parasite P. berghei. Differential expression patterns for host genes identify genes and pathways involved in the host response to rodent Plasmodium parasites. Keywords: gene expression Total RNA from sorted liver stage (LS) infected hepatocytes were isolated from PyGFP-infected BALB/c mice as described in Tarun et al (2008). RNA from LS-infected hepatocytes were isolated at two time points post-infection (pi): 24 hr pi (LS24) and 40 hr pi (LS40). As control, RNA from hepatocytes isolated from mock-infected mice (infected with salivary gland extract) at the same time points was also isolated following the same procedure. Total RNA was then subjected to two rounds of linear amplification using the Amino Allyl Message Amp II aRNA Amplification Kit (Ambion) according to manufaturerâs directions. The quality of total and amplified RNAs were examined with the Agilent 2100 BioanalyzerTM (Agilent Technologies) and the quantity of the RNA samples was assessed using a Nanodrop ND-1000 spectrophotometer prior to microarray hybridization. For each timepoint two independent biological replicates were obtained.

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Dual RNA-sequencing of host and pathogen during Plasmodium berghei infection of hepatocytes in vitro

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