Plasmodium male gametocyte development and transmission are critically regulated by the two putative deadenylases of the CAF1/CCR4/NOT complex

Abstract

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.

Fig 1. In pyccr4-1^-, dCCR4-1, and PyCAF1ΔC parasites, a semi-synchronous coordination of gametocyte maturation is lost and parasite transmissibility is reduced.

A) The number of mature male gametocytes was determined by flow cytometry of sulfadiazine-treated and DDD/BIP-stained P. yoelii parasites and is plotted as a fraction of total gametocytes. * denotes a p-value <0.05. B,D,F) The number of centers-of-movement/exflagellation centers were quantified daily by microscopy on a 400x field of RBCs taken from mice infected with 10,000 (B, F) or 1,000 (D) blood stage parasites. Fewer wild-type and dCCR4-1 parasites were injected to better preserve animal health over the course of the experiment in accordance with our IACUC protocol (D). Plotted are three biological replicates with three technical replicates each. Error bars represent the standard error of the mean. C) The number of oocysts per infected mosquito on day seven post-infectious blood meal are plotted. Data represents at least 20 dissected mosquitoes per biological replicate conducted in triplicate. Error bars represent the standard error of the mean. E) Parasitemia was measured microscopically by Giemsa-stained thin blood smears. Plotted are three biological replicates with three technical replicates each. Error bars represent the standard error of the mean.

Fig 2. PfCAF1ΔC has decreased gametocyte conversion and exflagellation compared to wild type NF54 parasites.

A) P. falciparum ring stage parasitemia and B) total gametocytemia counted in 10,000 RBCs were averaged over a minimum of two biological replicates. Ring stages were used to represent asexual parasitemia as they were most easily distinguishable from dead/dying asexual forms. C) Conversion rates were calculated as described previously [59] by taking the stage II-gametocytemia on Day T and dividing by ring stage parasitemia on Day T-2. D) Exflagellation events were counted under a 40x objective for 10 fields of view. Error bars represent standard error of the mean. Statistical differences between wild-type and PfCAF1ΔC parasites were assessed via a paired Wilcoxon test. * = p<0.05, ** = p<0.01.

Fig 3. PyCCR4-1::GFP is expressed in cytosolic granules in asexual and sexual blood stage parasites.

Representative images of parasites treated with DAPI and antibodies to GFP (to detect PyCCR4-1::GFP) or to stage-specific cellular markers (ACP, alpha-tubulin, or human DDX6 that cross-reacts with DOZI) are shown. Scale bars are 5 or 10 microns.

Fig 4. Transcripts with sex-specific or transmission-related functions are modulated by PyCCR4-1.

A) A volcano plot showing statistically significant increases (blue), decreases (red), or no change (black) is provided. While few transcripts go up in abundance (from antigenically variant genes), nearly all affected transcripts decrease in abundance, thus indicating that PyCCR4-1 may play a role in preserving these mRNAs. B) In pyccr4-1^- gametocytes, 24% of differentially abundant transcripts are translationally repressed in female P. falciparum gametocytes and another one third of the transcripts are enriched in the male P. berghei gamete proteome [50, 69]. * = p<0.01 by Fisher test. C) In pyccr4-1^- gametocytes, transcripts that are differentially expressed in male and female P. berghei gametocytes are equally affected.

Fig 5. The CAF1/CCR4/NOT complex associates with some dysregulated transcripts but doesn’t grossly affect UTR/poly(A) tail length.

A) Immunoprecipitation of PyCCR4-1::GFP allowed detection of the association of three selected transcripts that are affected by pyccr4-1^- (top), whereas other affected transcripts do not associate with PyCCR4-1::GFP (bottom, left). Two control transcripts that do not change upon deletion of pyccr4-1 also do not interact with PyCCR4-1::GFP (bottom right). Shown are input and elution samples of a constitutive GFP-expressing clone and a PyCCR4-1::GFP clone. Amplicons and primer dimer bands are indicated with arrows. B) Circularized RT-PCR (cRT-PCR) allows for joining of the 5’ and 3’ ends of RNA following the removal of proteins and the 5’ 7-methylguanosine cap. C and D) Analysis of p28 by cRT-PCR was conducted in both wild-type and pyccr4-1^- parasites to detect effects by PyCCR4-1 upon UTR and poly(A) tail length in purified gametocytes. Primers were designed within the coding sequences (B) or near the poly(A) tail (C) as defined by sequencing of cRT-PCR products from panel B, which allow observations of UTR and poly(A) tail lengths respectively. Three biological replicates and a No Template Control (NTC) are shown with NEB 100bp molecular weight ladder in parallel. Extended data is available in S5 and S6 Figs.

Fig 6. A model for the roles of PyCCR4-1 and PyCAF1 in gametocyte maturation and host-to-vector transmission is presented.

The CAF1/CCR4/NOT complex in Plasmodium as identified through crosslinking IP-MS is shown. Contacts are inferred through previous studies in model organisms and sequence conservation of the interaction domains [, , –72].