Date Published: January 31, 2019
Publisher: Public Library of Science
Author(s): Kevin J. Hart, Jenna Oberstaller, Michael P. Walker, Allen M. Minns, Mark F. Kennedy, Ian Padykula, John H. Adams, Scott E. Lindner, Oliver Billker.
With relatively few known specific transcription factors to control the abundance of specific mRNAs, Plasmodium parasites may rely more on the regulation of transcript stability and turnover to provide sufficient gene regulation. Plasmodium transmission stages impose translational repression on specific transcripts in part to accomplish this. However, few proteins are known to participate in this process, and those that are characterized primarily affect female gametocytes. We have identified and characterized Plasmodium yoelii (Py) CCR4-1, a putative deadenylase, which plays a role in the development and activation of male gametocytes, regulates the abundance of specific mRNAs in gametocytes, and ultimately increases the efficiency of host-to-vector transmission. We find that when pyccr4-1 is deleted or its protein made catalytically inactive, there is a loss in the initial coordination of male gametocyte maturation and a reduction of parasite infectivity of the mosquito. Expression of only the N-terminal CAF1 domain of the essential CAF1 deadenylase leads to a similar phenotype. Comparative RNA-seq revealed that PyCCR4-1 affects transcripts important for transmission-related functions that are associated with male or female gametocytes, some of which directly associate with the immunoprecipitated complex. Finally, circular RT-PCR of one of the bound, dysregulated transcripts showed that deletion of the pyccr4-1 gene does not result in gross changes to its UTR or poly(A) tail length. We conclude that the two putative deadenylases of the CAF1/CCR4/NOT complex play critical and intertwined roles in gametocyte maturation and transmission.
Malaria remains one of the great global health problems today, with 216 million new infections and 445,000 deaths attributed to it annually . Resistance to frontline drugs is spreading, and understanding the development and transmission of the malaria parasite is important to bolster efforts to reduce or eliminate deaths due to this infection. For the parasite to transmit from a vertebrate host to the mosquito vector, a small percentage of the cells will differentiate from asexual forms and develop into sexual stage gametocytes, which can persist in an infectious state until a mosquito takes a blood meal. This event allows a small number of gametocytes to be taken up into the mosquito, but with far fewer parasites productively infecting it . Following two weeks of development within the mosquito, a small number of sporozoites will similarly be injected into a host by the mosquito as it takes another blood meal . In the effort to develop vaccines and drugs, transmission events have been identified as prime targets to prevent the spread of the parasite because they are population bottlenecks in the parasite life cycle. We and others have focused upon the transmitted gametocyte and sporozoite stages of Plasmodium parasites to identify and exploit their weaknesses. In both cases, very few parasites are transmitted, and thus these bottlenecks are excellent points of intervention. The identification of molecular processes that are important for the transmission of the parasite in one or both of these events, and their modes-of-action, are thus top priorities for the development of new therapeutics.
Plasmodium encodes few known specific transcription factors and a relatively over-represented number of RNA-binding proteins (10% of its predicted proteome) [11, 29]. One model suggests that Plasmodium has adapted these complementary regulatory mechanisms to achieve its preferred RNA homeostasis. Moreover, the malaria parasite also proactively transcribes a large number of genes before transmission, but does not produce the encoded proteins until transmission has occurred. This translational repressive mechanism has been shown to be imposed by members of the DOZI/CITH/ALBA complex, as well as by PUF2. Here, we demonstrate that the PyCCR4-1 and PyCAF1 members of the CAF1/CCR4/NOT complex play additional roles in either the preservation or expression of translationally repressed transcripts through direct and indirect means (Fig 6). Moreover, we find that PyCCR4-1 is also important for the development of the male gametocyte, as well as for the efficient transmission of gametocytes to the mosquito vector.
Extended versions of materials and methods are provided in S1 File.