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In vivo and in vitro characterization of a Plasmodium liver stage-specific promoter.

ABSTRACT: Little is known about stage-specific gene regulation in Plasmodium parasites, in particular the liver stage of development. We have previously described in the Plasmodium berghei rodent model, a liver stage-specific (lisp2) gene promoter region, in vitro. Using a dual luminescence system, we now confirm the stage specificity of this promoter region also in vivo. Furthermore, by substitution and deletion analyses we have extended our in vitro characterization of important elements within the promoter region. Importantly, the dual luminescence system allows analyzing promoter constructs avoiding mouse-consuming cloning procedures of transgenic parasites. This makes extensive mutation and deletion studies a reasonable approach also in the malaria mouse model. Stage-specific expression constructs and parasite lines are extremely valuable tools for research on Plasmodium liver stage biology. Such reporter lines offer a promising opportunity for assessment of liver stage drugs, characterization of genetically attenuated parasites and liver stage-specific vaccines both in vivo and in vitro, and may be key for the generation of inducible systems.

SUBMITTER: De Niz M

PROVIDER: S-EPMC4398466 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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In vivo and in vitro characterization of a Plasmodium liver stage-specific promoter.

De Niz Mariana M Helm Susanne S Horstmann Sebastian S Annoura Takeshi T Del Portillo Hernando A HA Khan Shahid M SM Heussler Volker T VT

PloS one 20150415 4

Little is known about stage-specific gene regulation in Plasmodium parasites, in particular the liver stage of development. We have previously described in the Plasmodium berghei rodent model, a liver stage-specific (lisp2) gene promoter region, in vitro. Using a dual luminescence system, we now confirm the stage specificity of this promoter region also in vivo. Furthermore, by substitution and deletion analyses we have extended our in vitro characterization of important elements within the prom ...[more]

PMID: 25874388

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Project description:During the blood meal of a Plasmodium-infected mosquito, 10 to 100 parasites are inoculated into the skin and a proportion of these migrate via the bloodstream to the liver where they infect hepatocytes. The Plasmodium liver stage, despite its clinical silence, represents a highly promising target for antimalarial drug and vaccine approaches. Successfully invaded parasites undergo a massive proliferation in hepatocytes, producing thousands of merozoites that are transported into a blood vessel to infect red blood cells. To successfully develop from the liver stage into infective merozoites, a tight regulation of gene expression is needed. Although this is a very interesting aspect in the biology of Plasmodium, little is known about gene regulation in Plasmodium parasites in general and in the liver stage in particular. We have functionally analyzed a novel promoter region of the rodent parasite Plasmodium berghei that is exclusively active during the liver stage of the parasite. To prove stage-specific activity of the promoter, GFP and luciferase reporter assays have been successfully established, allowing both qualitative and accurate quantitative analysis. To further characterize the promoter region, the transcription start site was mapped by rapid amplification of cDNA ends (5'-RACE). Using promoter truncation experiments and site-directed mutagenesis within potential transcription factor binding sites, we suggest that the minimal promoter contains more than one binding site for the recently identified parasite-specific ApiAP2 transcription factors. The identification of a liver stage-specific promoter in P. berghei confirms that the parasite is able to tightly regulate gene expression during its life cycle. The identified promoter region might now be used to study the biology of the Plasmodium liver stage, which has thus far proven problematic on a molecular level. Stage-specific expression of dominant-negative mutant proteins and overexpression of proteins normally active in other life cycle stages will help to understand the function of the proteins investigated.

Project description:Plasmodium 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.Gene 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).Major 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.This 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.

Dataset Information

In vivo and in vitro characterization of a Plasmodium liver stage-specific promoter.

Publications

In vivo and in vitro characterization of a Plasmodium liver stage-specific promoter.

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