A Micronemal Protein, Scot1, Is Essential for Apicoplast Biogenesis and Liver Stage Development in Plasmodium berghei.

Plasmodium sporozoites invade hepatocytes, transform into liver stages, and replicate into thousands of merozoites that infect erythrocytes and cause malaria. Proteins secreted from micronemes play an essential role in hepatocyte invasion, and unneeded micronemes are subsequently discarded for replication. The liver-stage parasites are potent immunogens that prevent malarial infection. Late liver stage-arresting genetically attenuated parasites (GAPs) exhibit greater protective efficacy than early GAP. However, the number of late liver-stage GAPs for generating GAPs with multiple gene deletions is limited. Here, we identified Scot1 (Sporozoite Conserved Orthologous Transcript 1), which was previously shown to be upregulated in sporozoites, and by endogenous tagging with mCherry, we demonstrated that it is expressed in the sporozoite and liver stages in micronemes. Using targeted gene deletion in Plasmodium berghei, we showed that Scot1 is essential for late liver-stage development. Scot1 KO sporozoites grew normally into liver stages but failed to initiate blood-stage infection in mice due to impaired apicoplast biogenesis and merozoite formation. Bioinformatic studies suggested that Scot1 is a metal-small-molecule carrier protein. Remarkably, supplementation with metals in the culture of infected Scot1 KO cells did not rescue their phenotype. Immunization with Scot1 KO sporozoites in C57BL/6 mice confers protection against malaria via infection. These proof-of-concept studies will enable the generation of P. falciparum Scot1 mutants that could be exploited to generate GAP malaria vaccines.

An axonemal intron splicing program sustains Plasmodium male development.

Differentiation of male gametocytes into flagellated fertile male gametes relies on the assembly of axoneme, a major component of male development for mosquito transmission of the malaria parasite. RNA-binding protein (RBP)-mediated post-transcriptional regulation of mRNA plays important roles in eukaryotic sexual development, including the development of female Plasmodium. However, the role of RBP in defining the Plasmodium male transcriptome and its function in male gametogenesis remains incompletely understood. Here, we performed genome-wide screening for gender-specific RBPs and identified an undescribed male-specific RBP gene Rbpm1 in the Plasmodium. RBPm1 is localized in the nucleus of male gametocytes. RBPm1-deficient parasites fail to assemble the axoneme for male gametogenesis and thus mosquito transmission. RBPm1 interacts with the spliceosome E complex and regulates the splicing initiation of certain introns in a group of 26 axonemal genes. RBPm1 deficiency results in intron retention and protein loss of these axonemal genes. Intron deletion restores axonemal protein expression and partially rectifies axonemal defects in RBPm1-null gametocytes. Further splicing assays in both reporter and endogenous genes exhibit stringent recognition of the axonemal introns by RBPm1. The splicing activator RBPm1 and its target introns constitute an axonemal intron splicing program in the post-transcriptional regulation essential for Plasmodium male development.

An ApiAp2 Transcription Factor with a Dispensable Role in Plasmodium berghei Life Cycle.

Malaria parasites have a complex life cycle and undergo replication and population expansion within vertebrate hosts and mosquito vectors. These developmental transitions rely on changes in gene expression and chromatin reorganization that result in the activation and silencing of stage-specific genes. The ApiAp2 family of DNA-binding proteins plays an important role in regulating gene expression in malaria parasites. Here, we characterized the ApiAp2 protein in Plasmodium berghei, which we termed Ap2-D. In silico analysis revealed that Ap2-D has three beta-sheets followed by a helix at the C-terminus for DNA binding. Using gene tagging with 3XHA-mCherry, we found that Ap2-D is expressed in Plasmodium blood stages and is present in the parasite cytoplasm and nucleus. Surprisingly, our gene deletion study revealed a completely dispensable role for Ap2-D in the entirety of the P. berghei life cycle. Ap2-D KO parasites were found to grow in the blood successfully and progress through the mosquito midgut and salivary glands. Sporozoites isolated from mosquito salivary glands were infective for hepatocytes and achieved similar patency as WT in mice. We emphasize the importance of genetic validation of antimalarial drug targets before progressing them to drug discovery.

LC3B labeling of the parasitophorous vacuole membrane of Plasmodium berghei liver stage parasites depends on the V-ATPase and ATG16L1.

The protozoan parasite Plasmodium, the causative agent of malaria, undergoes an obligatory stage of intra-hepatic development before initiating a blood-stage infection. Productive invasion of hepatocytes involves the formation of a parasitophorous vacuole (PV) generated by the invagination of the host cell plasma membrane. Surrounded by the PV membrane (PVM), the parasite undergoes extensive replication. During intracellular development in the hepatocyte, the parasites provoke the Plasmodium-associated autophagy-related (PAAR) response. This is characterized by a long-lasting association of the autophagy marker protein, and ATG8 family member, LC3B with the PVM. LC3B localization at the PVM does not follow the canonical autophagy pathway since upstream events specific to canonical autophagy are dispensable. Here, we describe that LC3B localization at the PVM of Plasmodium parasites requires the V-ATPase and its interaction with ATG16L1. The WD40 domain of ATG16L1 is crucial for its recruitment to the PVM. Thus, we provide new mechanistic insight into the previously described PAAR response targeting Plasmodium liver stage parasites.

Regulation of sexual commitment in malaria parasites - a complex affair.

Malaria blood stage parasites commit to either one of two distinct cellular fates while developing within erythrocytes of their mammalian host: they either undergo another round of asexual replication or they differentiate into nonreplicative transmissible gametocytes. Depending on the state of infection, either path may support or impair the ultimate goal of human-to-human transmission via the mosquito vector. Malaria parasites therefore evolved strategies to control investments into asexual proliferation versus gametocyte formation. Recent work provided fascinating molecular insight into shared and unique mechanisms underlying the control and environmental modulation of sexual commitment in the two most widely studied malaria parasite species, Plasmodium falciparum and P. berghei. With this review, we aim at placing these findings into a comparative mechanistic context.

PbARID-associated chromatin remodeling events are essential for gametocyte development in Plasmodium.

Gametocyte development of the Plasmodium parasite is a key step for transmission of the parasite. Male and female gametocytes are produced from a subpopulation of asexual blood-stage parasites, but the mechanisms that regulate the differentiation of sexual stages are still under investigation. In this study, we investigated the role of PbARID, a putative subunit of a SWI/SNF chromatin remodeling complex, in transcriptional regulation during the gametocyte development of P. berghei. PbARID expression starts in early gametocytes before the manifestation of male and female-specific features, and disruption of its gene results in the complete loss of gametocytes with detectable male features and the production of abnormal female gametocytes. ChIP-seq analysis of PbARID showed that it forms a complex with gSNF2, an ATPase subunit of the SWI/SNF chromatin remodeling complex, associating with the male cis-regulatory element, TGTCT. Further ChIP-seq of PbARID in gsnf2-knockout parasites revealed an association of PbARID with another cis-regulatory element, TGCACA. RIME and DNA-binding assays suggested that HDP1 is the transcription factor that recruits PbARID to the TGCACA motif. Our results indicated that PbARID could function in two chromatin remodeling events and paly essential roles in both male and female gametocyte development.

Stearoyl-CoA desaturase regulates organelle biogenesis and hepatic merozoite formation in Plasmodium berghei.

Plasmodium is an obligate intracellular parasite that requires intense lipid synthesis for membrane biogenesis and survival. One of the principal membrane components is oleic acid, which is needed to maintain the membrane's biophysical properties and fluidity. The malaria parasite can modify fatty acids, and stearoyl-CoA Δ9-desaturase (Scd) is an enzyme that catalyzes the synthesis of oleic acid by desaturation of stearic acid. Scd is dispensable in P. falciparum blood stages; however, its role in mosquito and liver stages remains unknown. We show that P. berghei Scd localizes to the ER in the blood and liver stages. Disruption of Scd in the rodent malaria parasite P. berghei did not affect parasite blood stage propagation, mosquito stage development, or early liver-stage development. However, when Scd KO sporozoites were inoculated intravenously or by mosquito bite into mice, they failed to initiate blood-stage infection. Immunofluorescence analysis revealed that organelle biogenesis was impaired and merozoite formation was abolished, which initiates blood-stage infections. Genetic complementation of the KO parasites restored merozoite formation to a level similar to that of WT parasites. Mice immunized with Scd KO sporozoites confer long-lasting sterile protection against infectious sporozoite challenge. Thus, the Scd KO parasite is an appealing candidate for inducing protective pre-erythrocytic immunity and hence its utility as a GAP.

Antimalarial potential of homeopathic medicines against schizont maturation of Plasmodium berghei in short-term in vitro culture

In vitro assessment of antimalarial drug susceptibility of Plasmodium has been a major research success, which has paved the way for the understanding of parasite and rapid screening of antimalarial drugs for their effectiveness. In the present study a preliminary screening to check the antiplasmodial activity of mother tincture (ϕ) and various potencies (6C, 30C, 200C) of homeopathic medicines Cinchona officinalis/china (Chin.), Chelidonium majus (Chel.) and Arsenicum album (Ars.) were done by assessing the in vitro schizont maturation inhibition assay. A significant reduction in the growth of intraerythrocytic stages of P. berghei was observed with decreasing dilution of ϕ and various potencies of Chin., Chel. and Ars. exhibiting a dose dependent effect. Maximum schizont maturation inhibition was observed by Chin. ϕ (1:1), Chin. 30 (1:1, 1:2) and Chel. 30 (1:1) i.e. 80%. The standard drug CQ at 10 µM concentration exhibited 95.4±1.6% inhibition of schizont maturation. Ars. 30 (1:1) also have been found to possess strong antiplasmodial efficacy with 75.5±2.6% schizont inhibition. The presence of free merozoites in Ars. 200 with weak schizonticidal inhibition activity (40-45%) also pointed towards the ability of parasite to survive in the given drug pressure.

O estudo in vitro da susceptibilidade de Plasmodium a drogras antimaláricas representa um grande avanço nas pesquisas, abrindo novas rotas para o entendimento do parasite e da efetividade de drogas antiomaláricas. Nesse trabalho, realizamos um estudo preliminar da atividade antiplasmódica da tintura mãe (ϕ) e várias potências (6 cH, 30 cH, 200 cH) dos medicamentos homeopáticos China officinalis (Chin), Chelidonium majus (Chel) e Arsenicum album (Ars), através do estudo in vitro da inibição da maturação de esquizontes. Observamos uma redução significativa do crescimento do estágio intra-eritrócito do P. berghei conforme a tintura mãe e demais potências de Chin, Chel e Ars foram diluídas, observando-se um efeito dependente da dose. O máximo de inibição na maturação dos esquizontes (80%) foi observado com Chin ϕ (1:1), Chin 30 cH (1:1, 1:2) and Chel 30 cH (1:1). A droga Cloroquina (CQ), usada como controle, em uma concentração de 10µM, exibiu (95.4 ± 1.6) % de inibição. Ars 30cH (1:1) também apresentou uma forte eficácia antiplasmódica com (75.5 ± 2.6) % de inibição de esquizontes. A presence de merozoites livres com Ars 200 cH e uma fraca atividade inibidora (40-45%) indicam a habilidade do parasita em sobreviver na presença dessa droga.

Effect of dietary iron on the course of Plasmodium berghei malaria in young rats

Clinical and experimental evidence suggests that iron-deficient hosts are less susceptible to severe malaria and that iron supplementation aggravates infection. In the present study, 60 weanling Wistar rats were fed standard diets with different iron concentrations: 21 mg/kg (group 1), 45 mg/kg (group 2) and 113 mg/kg (group 3). Ferrous sulfate (FeSO4 x 7H2O) was added to the normal-iron and iron-supplemented diets (groups 2 and 3, respectively). Data are reported as mean +/- SEM. After 16 days of regimen, eight rats from each group were killed to measure serum iron concentration (SI) and transferrin saturation capacity (TSC). At this moment, rats from group 1 were underweight and their dietary intake was significantly lower than that of animals from the other groups. Severe iron deficiency (SI = 49.2 +/- 4.5 micrograms/100 ml and TSC = 8.3 +/- 0.7 per cent ) was observed in rats from group 1, while the animals from the other groups were iron-sufficient (group 2: SI = 186.5 +/- 28.5 micrograms/100 ml and TSC = 27.3 +/- 3.4 per cent ; group 3: SI = 137.3 +/- 18.2 micrograms/100 ml and TSC = 21.3 +/- 2.3 per cent ). Nine animals from each group were then infected with the malaria parasite Plasmodium berghei, whereas three animals from each group were used as noninfected controls. Parasitemias ( per cent of infected red blood cells) peaked 7 days post-infection in animals from groups 2 and 3 (mean values of 2.4 per cent and 1.7 per cent , respectively), but in animals from group 1 parasitemias increased until the 9th day post-infection (mean at peak, 2.3 per cent ) and parasite clearance was significantly slower than in the other groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Antimalarial chemotherapy with natural products and chemically defined molecules

In the present work we have described the in vivo antimalarial actrivity of six different plants. Two of them (Verninia brasiliana and Eupatorium squalidum) were tested in a randomic approach among 273 crude extracts from plants; four (Acanhospermum australe, Esenbeckia febrifuga, Lisianthus specious and Tachia guianensis) were selected after screening 22 crude extracts from different medicinal and some of them showed antimalarial activity in vitro. Some aspects of recent research with natural products aiming to produce drugs are discussed.