Essential role of a Plasmodium berghei heat shock protein (PBANKA_0938300) in gametocyte development

The continued existence of Plasmodium parasites in physiologically distinct environments during their transmission in mosquitoes and vertebrate hosts requires effector proteins encoded by parasite genes to provide adaptability. Parasites utilize their robust stress response system involving heat shock proteins for their survival. Molecular chaperones are involved in maintaining protein homeostasis within a cell during stress, protein biogenesis and the formation of protein complexes. Due to their critical role in parasite virulence, they are considered targets for therapeutic interventions. Our results identified a putative P. berghei heat shock protein (HSP) belonging to the HSP40 family (HspJ62), which is abundantly induced upon heat stress and expressed during all parasite stages. To determine the role HspJ62, a gene-disrupted P. berghei transgenic line was developed (ΔHspJ62), which resulted in disruption of gametocyte formation. Such parasites were unable to form subsequent sexual stages because of disrupted gametogenesis, indicating the essential role of HspJ62 in gametocyte formation. Transcriptomic analysis of the transgenic line showed downregulation of a number of genes, most of which were specific to male or female gametocytes. The transcription factor ApiAP2 was also downregulated in ΔHspJ62 parasites. Our findings suggest that the downregulation of ApiAP2 likely disrupts the transcriptional regulation of sexual stage genes, leading to impaired gametogenesis. This finding also highlights the critical role that HspJ62 indirectly plays in the development of P. berghei sexual stages and in facilitating the conversion from the asexual blood stage to the sexual stage. This study characterizes the HspJ62 protein as a fertility factor because parasites lacking it are unable to transmit to mosquitoes. This study adds an important contribution to ongoing research aimed at understanding gametocyte differentiation and formation in parasites. The molecule adds to the list of potential drug targets that can be targeted to inhibit parasite sexual development and consequently parasite transmission.

The Plasmodium NOT1-G paralogue is an essential regulator of sexual stage maturation and parasite transmission

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.

Monoclonal antibodies block transmission of genetically diverse Plasmodium falciparum strains to mosquitoes

Malaria parasite transmission to mosquitoes relies on the uptake of sexual stage parasites during a blood meal and subsequent formation of oocysts on the mosquito midgut wall. Transmission-blocking vaccines (TBVs) and monoclonal antibodies (mAbs) target sexual stage antigens to interrupt human-to-mosquito transmission and may form important tools for malaria elimination. Although most epitopes of these antigens are considered highly conserved, little is known about the impact of natural genetic diversity on the functional activity of transmission-blocking antibodies. Here we measured the efficacy of three mAbs against leading TBV candidates (Pfs48/45, Pfs25 and Pfs230) in transmission assays with parasites from naturally infected donors compared to their efficacy against the strain they were raised against (NF54). Transmission-reducing activity (TRA) was measured as reduction in mean oocyst intensity. mAb 45.1 (α-Pfs48/45) and mAb 4B7 (α-Pfs25) reduced transmission of field parasites from almost all donors with IC80 values similar to NF54. Sequencing of oocysts that survived high mAb concentrations did not suggest enrichment of escape genotypes. mAb 2A2 (α-Pfs230) only reduced transmission of parasites from a minority of the donors, suggesting that it targets a non-conserved epitope. Using six laboratory-adapted strains, we revealed that mutations in one Pfs230 domain correlate with mAb gamete surface binding and functional TRA. Our findings demonstrate that, despite the conserved nature of sexual stage antigens, minor sequence variation can significantly impact the efficacy of transmission-blocking mAbs. Since mAb 45.1 shows high potency against genetically diverse strains, our findings support its further clinical development and may inform Pfs48/45 vaccine design.

Chrysomyxa rhododendri (European rhododendron rust).

C. rhododendri is a heteroecious rust fungus; an obligate parasite completing stages of its life cycle on different plants. Mating of haploid strains occurs on species of Picea, followed by the production of asexual aeciospores that infect Rhododendron species. Another asexual form producing urediniospores occurs on Rhododendron, followed by the production of teliospores (the sexual stage). All stages are known from Europe. The fungus was reported in 1954 on Rhododendron in the northwestern USA, but the aecial form has not been found in North America. The fungus is a Regulated Pest for the USA; it has been introduced into the UK, New Zealand and Australia. As an invasive species, this rust is damaging on species of Picea and Rhododendron. As latent infections on Rhododendron can be overlooked, accidental introduction of the rust may occur through the importation of these popular ornamental plants (Savile, 1973).

Puccinia gladioli.

P. gladioli is a heteroecious rust fungus, an obligate parasite with alternating life stages on different plants. The asexual form occurs on species of Valerianella, producing aeciospores that then infect Gladiolus species. The production of teliospores, the sexual stage, on Gladiolus, completes the cycle. Both stages are known in Europe, North Africa and southwestern Asia, but only the aecial form has been reported in North America, and only on the west coast. The fungus is a Regulated Pest for the USA (Wise et al., 2004) and is absent from South Africa and Australia, where other Gladiolus species are native or naturalized. Although not a significant problem in its native range, this rust fungus could be damaging as an invasive in other temperate areas. Small amounts of infection may be overlooked; therefore accidental introduction of the rust could occur through importation of infected germplasm by the horticultural industry or flower enthusiasts.

Cronartium flaccidum (Scots pine blister rust).

C. flaccidum is a heteroecious rust fungus, completing different stages of its life cycle on different plants. Mating of haploid strains occurs on species of Pinus, followed by the production of aeciospores, which infect various species of herbaceous dicotyledons. An asexual stage producing urediniospores occurs on the dicotyledonous plants, followed by the production of teliospores, the sexual stage, that germinate to form basidiospores that infect pines thus completing the cycle. A closely-related autoecious rust, Endocronartium (Peridermium) pini, only infects Pinus hosts. C. flaccidum is known from Europe and parts of northern and eastern Asia; it is a Regulated Pest for the USA (USDA/APHIS, 2008). As an invasive in other temperate areas, this rust could be damaging on native and introduced pines or the alternate host species. The infections on pines develop slowly, therefore the fungus might be overlooked, such that accidental introduction of the rust could occur through the importation of conifer [Pinopsida] seedlings or trees.

The transcriptome of circulating sexually committed Plasmodium falciparum ring stage parasites forecasts malaria transmission potential

Malaria is spread by the transmission of sexual stage parasites, called gametocytes. However, with Plasmodium falciparum, gametocytes can only be detected in peripheral blood when they are mature and transmissible to a mosquito, which complicates control efforts. Here, we identify the set of genes overexpressed in patient blood samples with high levels of gametocyte-committed ring stage parasites. Expression of all 18 genes is regulated by transcription factor AP2-G, which is required for gametocytogenesis. We select three genes, not expressed in mature gametocytes, to develop as biomarkers. All three biomarkers we validate in vitro using 6 different parasite lines and develop an algorithm that predicts gametocyte production in ex vivo samples and volunteer infection studies. The biomarkers are also sensitive enough to monitor gametocyte production in asymptomatic P. falciparum carriers allowing early detection and treatment of infectious reservoirs, as well as the in vivo analysis of factors that modulate sexual conversion.

Plasmodium falciparum artemisinin-resistant K13 mutations confer a sexual-stage transmission advantage that can be overcome with atovaquone-proguanil

ABSTRACTContaining the spread of artemisinin (ART)-resistant Plasmodium falciparum will be assisted by improved understanding of its human-to-mosquito transmission. We compared gametocyte dynamics among field isolates containing K13 mutations conferring ART resistance and K13 wild-type parasites. In Pailin, Cambodia, the male to female gametocyte ratio was higher among K13 mutant infections compared to K13 wild-type infections. We also investigated the effects of artesunate and atovaquone-proguanil on the transmissibility of an ART-resistant K13 mutant strain, Cam3.IIR539T, in a volunteer infection study. Gametocyte production was higher after a single dose of artesunate (2 mg/kg) in volunteers infected with ART-resistant compared to ART-sensitive parasites. Despite the presence of gametocytes in volunteers infected with ART-resistant parasites, there was no infection observed in Anopheles stephensi mosquitoes after atovaquone-proguanil treatment. We report transmission determinants of ART-resistant infections that could be advantageous over ART-sensitive infections. Moreover, we show additional benefits of treating ART-resistant infections with atovaquone-proguanil treatment.