Genetic and chemical interrogation of the transcription factor PfMyb1 during plasmodium falciparum proliferation and differentiation

Abstract

Eukaryotic cellular function and differentiation are mediated by programmed transcription factor cascades, which drive the expression of critically important genes while suppressing others. Plasmodium falciparum is no exception to this, and the parasite undergoes drastic morphological and functional differentiation events as it progresses through its complex life cycle, mediated by exceptionally well-controlled gene expression profiles. However, the parasite displays anomalies to typical eukaryotic gene regulation mechanisms as it has a relative paucity of transcription factors compared to eukaryotes of similar genome size. While most transcriptional regulators in P. falciparum cluster within the apicomplexan apetala 2 family of transcription factors, a small number of additional proposed transcription factors are present in the genome, including zinc finger proteins and helix-turn-helix proteins such as members of the Myb transcription factor family. P. falciparum Myb1 (PfMyb1) is essential for asexual parasite proliferation but is also expressed in gametocytes, suggesting a binary role during both asexual replication and gametocyte differentiation. However, the latter has not been investigated. In the current study, chemical and genetic interrogation approaches were used to elucidate the role of this transcription factor both to asexual and gametocyte forms of P. falciparum. Genetic interrogation included both conditional knockout and knockdown of pfmyb1, using DiCre and glmS gene manipulation systems, respectively. Conditional knockdown of pfmyb1 transcripts to >80% proved ineffective as small amounts of the transcription factor can apparently still sustain asexual parasite proliferation. However, complete knockout of the gene abrogated the ability of asexual parasites to progress through the intraerythrocytic developmental cycle, confirming the essential requirement of PfMyb1 during these stages. Chemical interrogation strategies were used as an alternative and complementary strategy to evaluate the importance of PfMyb1 to various developmental stages of P. falciparum. Putative Myb inhibitors displayed potent activity against asexual parasites, confirming that interfering with Myb1 function affects asexual proliferation, consistent with the essential role of this transcriptional regulator in these replicative forms of the parasite. In the sexual gametocyte stages, neither conditional knockout of pfmyb1 nor chemical interference of PfMyb1 severely affected the formation and development of mature stage V gametocytes, suggesting that PfMyb1 does not play an important role during these stages. However, several PfMyb1 target genes are not required for gametocytogenesis but are important for gamete formation. Hence, we posit that PfMyb1 may be required for gamete formation in P. falciparum with a particular effect on male gametes. Overall, this project evaluated the importance of the PfMyb1 transcription factor during P. falciparum proliferation and differentiation. We show that PfMyb1 is required for the asexual stages but not for the formation and development of gametocytes in vitro. This contributes to our growing knowledge of the impeccable transcriptional regulation employed by P. falciparum parasites