Plasmodium sporozoites are transmitted from infected mosquitoes to mammals, and must navigate the host skin and vasculature to infect the liver. This journey requires distinct proteomes. Here, we report the dynamic transcriptomes and proteomes of both oocyst sporozoites and salivary gland sporozoites in both rodent-infectious Plasmodium yoelii parasites and human-infectious Plasmodium falciparum parasites. The data robustly define mRNAs and proteins that are upregulated in oocyst sporozoites (UOS) or upregulated in infectious sporozoites (UIS) within the salivary glands, including many that are essential for sporozoite functions in the vector and host. Moreover, we find that malaria parasites use two overlapping, extensive, and independent programs of translational repression across sporozoite maturation to temporally regulate protein expression. Together with gene-specific validation experiments, these data indicate that two waves of translational repression are implemented and relieved at different times during sporozoite maturation, migration and infection, thus promoting their successful development and vector-to-host transition.

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 is a disease caused by Plasmodium parasites, which are transmitted during an infectious blood meal by anopheline mosquitoes. Transmission of the sexual stages of the parasite to mosquitoes requires the proper regulation of specific mRNAs. While much work has been done to characterize regulation of mRNAs in female gametocytes, little has been done to assess this regulation in male gametocytes. Here, we demonstrate that PyCCR4-1, a member of the CAF1/CCR4/NOT RNA metabolic complex, acts upon transcripts both directly and indirectly in gametocytes, and results in a reduction of male gametocytemia. In gametocytes lacking PyCCR4-1, as well as those expressing a catalytically dead variant, the initial coordinated wave of male gametocyte activation is lost, and these parasites are ~4-fold less able to productively infect mosquitoes. We find that the deletion of the C-terminal portion of CAF1 in both Plasmodium yoelii and Plasmodium falciparum phenocopies the deletion of pyccr4-1. We also find that the CAF1/CCR4/NOT complex is directly binding some of these transcripts and is likely acting both directly upon mRNAs and indirectly to modulate transcript abundance. These findings demonstrate that the combined effects of the CAF1/CCR4/NOT complex upon specific mRNAs are important for both male and female gametocytes, and that this regulation is required for efficient transmission to the mosquito vector.

Productive transmission of malaria parasites hinges upon the execution of key transcriptional and posttranscriptional regulatory events. While much is now known about how specific transcription factors activate or repress sexual commitment programs, far less is known about the production of a preferred mRNA homeostasis following commitment and through the host-to-vector transmission event. Here, we show that in Plasmodium parasites, the NOT1 scaffold protein of the CAF1/CCR4/Not complex is duplicated, and one paralogue is dedicated for essential transmission functions. Moreover, this NOT1-G paralogue is central to the sex-specific functions previously associated with its interacting partners, as deletion of not1-g in Plasmodium yoelii leads to a comparable or complete arrest phenotype for both male and female parasites. We show that, consistent with its role in other eukaryotes, PyNOT1-G localizes to cytosolic puncta throughout much of the Plasmodium life cycle. PyNOT1-G is essential to both the complete maturation of male gametes and to the continued development of the fertilized zygote originating from female parasites. Comparative transcriptomics of wild-type and pynot1-g⁻ parasites shows that loss of PyNOT1-G leads to transcript dysregulation preceding and during gametocytogenesis and shows that PyNOT1-G acts to preserve mRNAs that are critical to sexual and early mosquito stage development. Finally, we demonstrate that the tristetraprolin (TTP)-binding domain, which acts as the typical organization platform for RNA decay (TTP) and RNA preservation (ELAV/HuR) factors is dispensable for PyNOT1-G’s essential blood stage functions but impacts host-to-vector transmission. Together, we conclude that a NOT1-G paralogue in Plasmodium fulfills the complex transmission requirements of both male and female parasites.

Plasmodium sporozoites are transmitted from an infected mosquito to mammals in which they infect the liver. The infectivity profile of sporozoites changes as they egress from oocysts on the mosquito midgut into the hemocoel, and then invade the salivary glands, where they maintain a poised and infectious state until transmission occurs. Upon transmission, the sporozoite must then navigate the host skin, vasculature, and liver. All of these feats require distinct repertoires of proteins and capabilities that are coordinated in an appropriate temporal manner. Here, we report the comprehensive and dynamic transcriptomes and proteomes of both oocyst sporozoite and salivary gland sporozoite stages in both rodent-infectious Plasmodium yoelii parasites and human-infectious Plasmodium falciparum parasites. These data robustly define mRNAs and proteins that are U pregulated in O ocyst S porozoites (UOS) or U pregulated in Infectious S porozoites (UIS), which include critical gene products for sporozoite functions, as well as many of unknown importance that are similarly regulated. Moreover, we found that Plasmodium uses two overlapping, extensive, and independent programs of translational repression across sporozoite maturation to temporally regulate specific genes necessary to successfully navigate the mosquito vector and mammalian host environments. Finally, gene-specific validation experiments of selected, translationally repressed transcripts in P. yoelii confirmed the interpretations of the global transcriptomic and proteomic datasets. Together, these data indicate that two waves of translational repression are implemented and relieved at different times in sporozoite maturation to promote its successful life cycle progression.

With relatively few known specific transcription factors to control the abundance of specific mRNAs, Plasmodium parasites also regulate the stability and turnover of transcripts to provide more comprehensive 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 PyCCR4-1, 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, which prevents PyCCR4-1 association with the complex, 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 PyCCR4-1 does not have gross changes in UTR or poly(A) tail length. We conclude that general and transmission-specialized members of the CAF1/CCR4/NOT complex play critical and intertwined roles in gametocyte maturation and transmission. AUTHOR SUMMARY: Malaria is a disease caused by Plasmodium parasites, which are transmitted during an infectious blood meal by anopheline mosquitoes. Transmission of the sexual stages of the parasite to mosquitoes requires the proper regulation of specific mRNAs. While much work has been done to characterize regulation of mRNAs in female gametocytes, little has been done to assess this regulation in male gametocytes. Here, we demonstrate that PyCCR4-1, a member of the CAF1/CCR4/NOT RNA metabolic complex, acts upon transcripts both directly and indirectly in both male and female parasites, and results in a reduction of male gametocytemia. In gametocytes lacking PyCCR4-1, as well as those expressing a catalytically dead variant, the initial coordinated wave of male gametocyte activation is lost, and these parasites are less able to productively infect mosquitoes. We find that PyCCR4-1 requires its association with PyCAF1 and by proxy, the rest of the complex, in order to perform its functions based upon experiments in both Plasmodium yoelii and Plasmodium falciparum . We also find that the CAF1/CCR4/NOT complex is directly binding some of these transcripts and is likely acting both directly and indirectly to modulate transcript abundance. These findings demonstrate that the combined effects of the CAF1/CCR4/NOT complex upon specific mRNAs are important for both male and female gametocytes, and that this regulation is required for efficient transmission to the mosquito vector.

Urinary incontinence (UI), the involuntary leakage of urine, is a common condition that imposes significant physical, emotional, and financial burdens. Injury to the external urethral sphincter (EUS) contributes to myogenic UI by damaging the skeletal muscle responsible for urinary control. This study evaluated the engraftment and therapeutic potential of human muscle-derived cells (hMDCs) using acute and chronic skeletal muscle injury models. Immunocompromised female rats received cardiotoxin (CTX) or volumetric muscle loss (VML) injuries to the tibialis anterior muscle. Animals were treated with 100,000 or 500,000 passage 3 or passage 6 hMDCs injected one day after CTX injury or six weeks after VML injury. VivoTrack imaging and human leukocyte antigen staining confirmed successful engraftment of hMDCs at the injection site. A total of 500,000 passage 3 hMDCs reduced muscle force, whereas later-passage 6 cells had no effect on force production. All treatment groups exhibited small, newly regenerated muscle fibers. In chronic VML injuries, hMDC engraftment using 500,000 passage 6 cells enhanced regenerative characteristics despite limited fusion with host muscle fibers. These findings suggest that hMDCs improved regenerative characteristics in a fibrotic VML model but did not restore contractile force. This work supports the therapeutic potential of hMDCs and provides a foundation for future clinical strategies to treat urinary incontinence.