Iwanaga Lab/Research Center for Infectious Disease Control  Department of Molecular Protozoology

Malaria is a mosquito-borne infectious disease, and is caused by Plasmodium species. Its burden exceeds 200 million infections every year, resulting in more than 400,000 deaths annually. Plasmodium parasites have a complex life cycle between host animals and mosquitos. They express specific genes at each developmental stage during the life cycle and those stage-specific genes are essential for invasion, parasitizing, and multiplication. We aimed to determine the mechanisms through which the parasite regulates gene expression stage-specifically. To this end, we focused on the sequence-specific transcriptional factors and characterized them by genetic engineering techniques and next generation sequencing. The expected result will be useful for understanding the molecular basis of the parasite’s life cycle, and also for exploring the drug target and vaccine antigens. 

 

  • Transcriptional regulation of Plasmodium parasites

 Plasmodium parasites have a complex life cycle between host animals and mosquito vectors (see: https://www.cdc.gov/malaria/about/biology/index.html). During the course of their life cycle, the parasites change their morphology markedly and infect specific cells of the host animal and the vector. Stage-specific gene expression allows morphological change and acquirement of infectivity to cells, and is thus essential for the completion of this complex life cycle. Transcriptional regulation plays an important role in this stage-specific gene expression. However, the transcriptional factors had not been identified even after completion of whole genome sequencing, and the mechanism of transcriptional regulation was not elucidated. We firstly demonstrated that the Apetala2 (AP2) protein family is the sequence-specific transcriptional factor of Plasmodium parasites. AP2 transcription factors express stage-specifically, and further, directly and comprehensively control transcription of all genes involved in stage formation at each developmental stage. Our group attempted to identify target genes of all AP2 transcription factors using next generation sequencing technology, such as chromatin-immunoprecipitation sequencing. Based on the obtained information, we attempted to elucidate the mechanism of transcriptional regulation for the entire life cycle. 

 

  • From Plasmodium artificial chromosome to synthetic Plasmodium parasites

 In the previous study, we generated Plasmodium artificial chromosome (9.0 kbp), which consists of centromere, telomere, and replication origin. It segregates into daughter parasites with more than 99.9% efficiency during nuclear division. Owing to the centromere, it is maintained as a single copy. In addition, its telomere functions properly, which protects the ends. We further combined artificial chromosomes of Plasmodium parasite and budding yeast and generated a shuttle artificial chromosome, which behaves like an actual chromosome in both living organisms. We then attempted to synthesize the parasite’s genome in budding yeast and transplant the resultant synthetic genome into Plasmodium parasite. The transgenic parasites, into which the synthetic genome was transplanted, would be the first synthetic eukaryotic cells, and would be utilized for synthetic biological research. Furthermore, it would be utilized in designing artificial attenuated parasites, which would be a safe live vaccine for malaria.  

 

  • Fig. 1: Chromatin-immunoprecipitation sequencing analysis shows that AP2 transcription factor binds to the promoter region of target genes specifically. The information on target genes is useful for our understanding of the formation of the developmental stage. This figure shows the summary of the target genes in ookinete, which is the mosquito midgut invasion stage.

  • Fig. 2: Plasmodium artificial chromosome consists of a centromere, telomere, and replication origin. The shuttle artificial chromosome, yPAC, is generated by combining Plasmodium and yeast artificial chromosomes (YAC). The genome of Plasmodium parasites can be synthesized using YAC in budding yeast. Synthetic Plasmodium parasites can be generated by transplanting synthetic genome into parasites.

Staff

  • Prof.: Shiroh Iwanaga
  • SA Asst. Prof.: Toshiyuki Mori

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