Abstract
BackgroundPlasmodium knowlesi is one of five Plasmodium species known to cause malaria in humans and can result in severe illness and death. While a zoonosis in humans, this simian malaria parasite species infects macaque monkeys and serves as an experimental model for in vivo, ex vivo and in vitro studies. It has underpinned malaria discoveries relating to host-pathogen interactions, the immune response and immune evasion strategies. This study investigated differences in P. knowlesi gene expression in samples from ex vivo and in vitro cultures.MethodsGene expression profiles were generated using microarrays to compare the stage-specific transcripts detected for a clone of P. knowlesi propagated in the blood of a rhesus macaque host and then grown in an ex-vivo culture, and the same clone adapted to long-term in vitro culture. Parasite samples covering one blood-stage cycle were analysed at four-hour intervals. cDNA was generated and hybridized to an oligoarray representing the P. knowlesi genome. Two replicate experiments were developed from in vitro cultures. Expression values were filtered, normalized, and analysed using R and Perl language and applied to a sine wave model to determine changes in equilibrium and amplitude. Differentially expressed genes from ex vivo and in vitro time points were detected using limma R/Bioconductor and gene set enrichment analysis (GSEA).ResultsMajor differences were noted between the ex vivo and in vitro time courses in overall gene expression and the length of the cycle (25.5 hours ex vivo; 33.5 hours in vitro). GSEA of genes up-regulated ex vivo showed an enrichment of various genes including SICAvar, ribosomal- associated and histone acetylation pathway genes. In contrast, certain genes involved in metabolism and cell growth, such as porphobilinogen deaminase and tyrosine phosphatase, and one SICAvar gene, were significantly up-regulated in vitro.ConclusionsThis study demonstrates how gene expression in P. knowlesi blood-stage parasites can differ dramatically depending on whether the parasites are grown in vivo, with only one cycle of development ex vivo, or as an adapted isolate in long-term in vitro culture. These data bring emphasis to the importance of studying the parasite, its biology and disease manifestations in the context of the host.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-0612-8) contains supplementary material, which is available to authorized users.
Highlights
Plasmodium knowlesi is one of five Plasmodium species known to cause malaria in humans and can result in severe illness and death
A P. knowlesi infection was established in a naïve rhesus monkey and the maximum blood volume allowed by Institutional Animal Care and Use Committee (IACUC) guidelines and approved protocols (10 ml/kg/ month) was drawn to obtain ring-stage infected RBCs (iRBCs) for ex vivo culture and the collection of stage-specific samples every 4 hours over the parasite’s 24-hour intra-developmental cycle (IDC)
It would have been ideal from a scientific standpoint to obtain blood collections throughout the IDC, but, per IACUC guidelines, animals may only be sedated twice/day, and in addition to logistical limitations of the availability of animal resource staff over a 24-hr period, ex vivo cultures were by necessity initiated when the iRBCs were at the ring stage of development
Summary
Plasmodium knowlesi is one of five Plasmodium species known to cause malaria in humans and can result in severe illness and death. While the utility of P. falciparum and P. knowlesi in vitro blood-stage culture systems has been evident for many avenues of research, including genetic manipulation to study gene expression characteristics and determine functional traits, the biological states of parasites in host organisms can differ substantially from that of in vitro cultured parasites. This became evident for P. falciparum with recognition of the loss of non-essential gene segments in culture [16] and reviewed in [17]. Investigations have shown that even one altered parameter, such as glucose level, can result in a marked change in gene expression profiles in cultured P. falciparum, with 560 genes exhibiting a two-fold or greater change in the presence of 0.6 mM glucose compared to 11.1 mM glucose [20]
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