Genetic manipulation of unique transcription factors in Plasmodium falciparum

Abstract

Severe malaria is caused by the Plasmodium falciparum protozoan. This malaria-causing parasite has a complex life cycle including a proliferative asexual stage and a terminally differentiated gametocyte stage, which is important in transmission. The complexity of this life cycle requires strict regulation of gene expression on multiple levels. During the asexual stages, genes are expressed in a "just-in-time" manner, which is required for the rapid proliferation and changes between the asexual life cycle stages. There is also gene expression control in the gametocyte stages and especially during the transition into the gametocyte stages. This eukaryotic parasite has multiple levels of gene expression regulation including epigenetic, transcriptional, post-transcriptional, and post-translational. Transcriptional control includes the use of unique transcription factors to determine the rate of transcription as well as other regulatory elements such as enhancers and repressors. Two of these transcription factors, PfMyb1 and Pf3D7_0603600, were found to be expressed during early gametocyte stages. It is postulated that since a higher transcript abundance is observed in the early gametocyte stages, these transcription factors are required for gametocytogenesis. PfMyb1 has already been found to be essential during the asexual stages of the parasite where it regulates transcription and is directly involved in the regulation of certain cell cycle specific genes. Pf3D7_0603600 has, as of yet, not been studied in vivo but, through in silico studies, has been annotated as a putative transcription factor due to the presence of an ARID DNA binding domain. To study the regulatory function of these transcription factors, genetically recombinant P. falciparum lines were produced. For PfMyb1 a targeted gene disruption line was generated, and it was found to be essential in asexual stages which aligns with previous literature. Subsequently, a conditional knockdown line was produced to allow for further study of the importance of PfMyb1 in the gametocyte stages. This conditional knockout line was found to have a recombinant Pfmyb1 locus which expressed GFP. It was found to be localised to the nucleus of the parasite which is expected for a transcription factor. A partially integrated targeted gene disruption line was produced for PF3D7_0603600 and was found to be non- essential in asexual stages. In conclusion, to combat malaria the biology of the P. falciparum parasite should be well studied. These transcription factors are likely important for gametocytogenesis and should be further characterised using the recombinant conditional knockdown lines.