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Malaria, which is caused by protozoa of the genus Plasmodium, remains a major endemic public health problem worldwide. Since artemisinin combination therapies are used as a first-line treatment in all endemic regions, the emergence of parasites resistant to these regimens has become a serious problem. Differentiation-inducing factor 1 (DIF-1) is a chlorinated alkylphenone originally found in the cellular slime mold Dictyostelium discoideum. DIF-1 and its derivatives exhibit a range of biological activities. In the present study, we investigated the effects of 41 DIF derivatives on the growth of Plasmodium falciparum in vitro using four laboratory strains and 12 field isolates. Micromolar concentrations of several DIF derivatives strongly suppressed the growth of the four laboratory strains, including strains that exhibited resistance to chloroquine and artemisinin, as well as strains that were susceptible to these drugs. In addition, DIF-1(+2), the most potent derivative, strongly suppressed the growth of 12 field isolates. We also examined the effects of DIF-1(+2) on the activity of the rodent malarial parasite Plasmodium berghei in mice. Intraperitoneal administration of DIF-1(+2) over 4 days (50 or 70 mg/kg/day) significantly suppressed the growth of the parasite in the blood with no apparent adverse effects, and a dose of 70 mg/kg/day significantly prolonged animal survival. These results suggest that DIF derivatives, such as DIF-1(+2), could serve as new lead compounds for the development of antimalarial agents., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The ultrastructural features of axoneme organization within the cytoplasm and exflagellation were investigated in detail in microgametes of a malaria parasite, Plasmodium berghei, by electron and fluorescence microscopy. The kinetosomes (basal bodies) of the microgamete were characterized by an electron dense mass in which singlet microtubules (MTs) were embedded. Around the kinetosomes, several singlet and doublet MTs were recognized in transverse sections. Incomplete doublets with growing B-tubule were also observed. As precursors of the axoneme, arrays of over three doublets showed a tendency to encircle the central pair MTs. Some of the doublet MTs were already equipped with inner and outer dynein arms. In the microgamete, which lacks an intraflagellar transport (IFT) system, self-assembly of microtubular and associated components appeared to proceed stepwise from singlet MTs through arrays of one to nine doublet MTs, surrounding the central pair, to form the complete axoneme in a quite short time. At exflagellation, some extra doublets were occasionally included between the axoneme and the flagellar membrane. At high magnification, the outer dynein arm of the Plasmodium microgamete had a pistol-like shape representing a three-headed dynein molecule like that of other Alveolata.

In this study, we investigated the mutation tendency of a mutator rodent malaria parasite, Plasmodium berghei, with proofreading-deficient DNA polymerase δ. Wild-type and mutator parasites were maintained in mice for over 24 weeks, and the genome-wide accumulated mutations were determined by high-throughput sequencing. The mutator P. berghei had a significant preference for C/G to A/T substitutions; thus, its genome had a trend towards a higher AT content. The mutation rate was influenced by the sequence context, and mutations were markedly elevated at TCT. Some genes mutated repeatedly in replicate passage lines. In particular, knockout mutations of the AP2-G gene were frequent, which conferred strong growth advantages on parasites during the blood stage but at the cost of losing the ability to form gametocytes. This is the first report to demonstrate a biased mutation tendency in malaria parasites, and its results help to promote our basic understanding of Plasmodium genetics.

Background: Rab5 GTPase regulates membrane trafficking between the plasma membrane and endosomes and harbours a conserved C-terminal isoprenyl modification that is necessary for membrane recruitment. Plasmodium falciparum encodes three Rab5 isotypes, and one of these, Rab5b (PfRab5b), lacks the C-terminal modification but possesses the N-terminal myristoylation motif. PfRab5b was reported to localize to the parasite periphery. However, the trafficking pathway regulated by PfRab5b is unknown., Methods: A complementation analysis of Rab5 isotypes was performed in Plasmodium berghei. A constitutively active PfRab5b mutant was expressed under the regulation of a ligand-dependent destabilization domain (DD)-tag system in P. falciparum. The localization of PfRab5b was evaluated after removing the ligand followed by selective permeabilization of the membrane with different detergents. Furthermore, P. falciparum N-terminally myristoylated adenylate kinase 2 (PfAK2) was co-expressed with PfRab5b, and trafficking of PfAK2 to the parasitophorous vacuole membrane was examined by confocal microscopy., Results: PfRab5b complemented the function of PbRab5b, however, the conventional C-terminally isoprenylated Rab5, PbRab5a or PbRab5c, did not. The constitutively active PfRab5b mutant localized to the cytosol of the parasite and the tubovesicular network (TVN), a region that extends from the parasitophorous vacuole membrane (PVM) in infected red blood cells (iRBCs). By removing the DD-ligand, parasite cytosolic PfRab5b signal disappeared and a punctate structure adjacent to the endoplasmic reticulum (ER) and parasite periphery accumulated. The peripheral PfRab5b was sensitive to extracellular proteolysis after treatment with streptolysin O, which selectively permeabilizes the red blood cell plasma membrane, indicating that PfRab5b localized on the iRBC cytoplasmic face of the TVN. Transport of PfAK2 to the PVM was abrogated by overexpression of PfRab5b, and PfAK2 accumulated in the punctate structure together with PfRab5b., Conclusion: N-myristoylated Plasmodium Rab5b plays a role that is distinct from that of conventional mammalian Rab5 isotypes. PfRab5b localizes to a compartment close to the ER, translocated to the lumen of the organelle, and co-localizes with PfAK2. PfRab5b and PfAK2 are then transported to the TVN, and PfRab5b localizes on the iRBC cytoplasmic face of TVN. These data demonstrate that PfRab5b is transported from the parasite cytosol to TVN together with N-myristoylated PfAK2 via an uncharacterized membrane-trafficking pathway.

Gastrointestinal symptoms, such as abdominal pain and diarrhea, are frequently observed in patients with Plasmodium falciparum malaria. However, the correlation between malaria intestinal pathology and intestinal microbiota has not been investigated. In the present study, infection of C57BL/6 mice with P. berghei ANKA (PbA) caused intestinal pathological changes, such as detachment of epithelia in the small intestines and increased intestinal permeability, which correlated with development with experimental cerebral malaria (ECM). Notably, an apparent dysbiosis occurred, characterized by a reduction of Firmicutes and an increase in Proteobacteria. Furthermore, some genera of microbiota correlated with parasite growth and/or ECM development. By contrast, BALB/c mice are resistant to ECM and exhibit milder intestinal pathology and dysbiosis. These results indicate that the severity of cerebral and intestinal pathology coincides with the degree of alteration in microbiota. This is the first report demonstrating that malaria affects intestinal microbiota and causes dysbiosis.

Cerebral malaria is a fatal complication of malaria. Conventional methods for evaluating experimental cerebral malaria have several drawbacks. Therefore, we aimed to develop an easy-to-use method for evaluating experimental cerebral malaria using OKD48 (Keap1-dependent Oxidative stress Detector, No-48-luciferase) mice to evaluate oxidative stress. OKD48 mice infected with Plasmodium berghei ANKA strain (PbA) suffered from experimental cerebral malaria and oxidative stress was successfully detected in the brains of living OKD48 mice developing experimental cerebral malaria. Oxidative stress in the brain was dependent on the development of experimental cerebral malaria, as prevention of experimental cerebral malaria did not elicit oxidative stress. We provide a novel evaluation method for experimental cerebral malaria using oxidative stress indicator OKD48 mice., (Copyright © 2014 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

Plasmodium falciparum malaria imposes a serious public health concern throughout the tropics. Although genetic tools are principally important to fully investigate malaria parasites, currently available forward and reverse tools are fairly limited. It is expected that parasites with a high mutation rate can readily acquire novel phenotypes/traits; however, they remain an untapped tool for malaria biology. Here, we generated a mutator malaria parasite (hereinafter called a 'malaria mutator'), using site-directed mutagenesis and gene transfection techniques. A mutator Plasmodium berghei line with a defective proofreading 3' → 5' exonuclease activity in DNA polymerase δ (referred to as PbMut) and a control P. berghei line with wild-type DNA polymerase δ (referred to as PbCtl) were maintained by weekly passage in ddY mice for 122 weeks. High-throughput genome sequencing analysis revealed that two PbMut lines had 175-178 mutations and a 86- to 90-fold higher mutation rate than that of a PbCtl line. PbMut, PbCtl, and their parent strain, PbWT, showed similar course of infection. Interestingly, PbMut lost the ability to form gametocytes during serial passages. We believe that the malaria mutator system could provide a novel and useful tool to investigate malaria biology., (© The Author 2014. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.)

Although IL-12 is believed to contribute to protective immune responses, the role played by IL-23 (a member of the IL-12 family) in malaria is elusive. Here, we show that IL-23 is produced during infection with Plasmodium berghei NK65. Mice deficient in IL-23 (p19KO) had higher parasitemia and died earlier than wild-type (WT) controls. Interestingly, p19KO mice had lower numbers of IL-17-producing splenic cells than their WT counterparts. Furthermore, mice deficient in IL-17 (17KO) suffered higher parasitemia than the WT controls, indicating that IL-23-mediated protection is dependent on induction of IL-17 during infection. We found that macrophages were responsible for IL-17 production in response to IL-23. We observed a striking reduction in splenic macrophages in the p19KO and 17KO mice, both of which became highly susceptible to infection. Thus, IL-17 appears to be crucial for maintenance of splenic macrophages. Adoptive transfer of macrophages into macrophage-depleted mice confirmed that macrophage-derived IL-17 is required for macrophage accumulation and parasite eradication in the recipient mice. We also found that IL-17 induces CCL2/7, which recruit macrophages. Our findings reveal a novel protective mechanism whereby IL-23, IL-17, and macrophages reduce the severity of infection with blood-stage malaria parasites., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

It is generally accepted that the mitochondria play central roles in energy production of most eukaryotes. In contrast, it has been thought that Plasmodium spp., the causative agent of malaria, rely mainly on cytosolic glycolysis but not mitochondrial oxidative phosphorylation for energy production during blood stages. However, Plasmodium spp. possesses all genes necessary for the tricarboxylic acid (TCA) cycle and most of the genes for electron transport chain (ETC) enzymes. Therefore, it remains elusive whether oxidative phosphorylation is essential for the parasite survival. To elucidate the role of TCA metabolism and ETC in malaria parasites, we deleted the gene for flavoprotein (Fp) subunit, Pbsdha, one of four components of complex II, a catalytic subunit for succinate dehydrogenase activity. The Pbsdha(-) parasite grew normally at blood stages in mouse. In contrast, ookinete formation of Pbsdha(-) parasites in the mosquito stage was severely impaired. Finally, Pbsdha(-) ookinetes failed in oocyst formation, leading to complete malaria transmission blockade. These results suggest that malaria parasite may switch the energy metabolism from glycolysis to oxidative phosphorylation to adapt to the insect vector where glucose is not readily available for ATP production.

Malaria parasites perform sexual reproduction in mosquitoes where a pair of gametes fertilizes and differentiates into zygotes, and a single zygote produces several thousands of progeny infectious to next vertebrates. Although the parasite fertilization step has been considered as Achilles' heel of parasite life cycle and thus a critical target for blocking malaria transmission in the mosquito, its molecular mechanisms are largely unknown. Previously, we identified that GENERATIVE CELL SPECIFIC 1 (GCS1) is a reproduction factor in angiosperm. Subsequently, it was found that rodent malaria parasite, Plasmodium berghei and green algae, Chlamydomonas reinhardtii possess GCS1 homologues which also play essential roles in gamete interaction. Moreover, intensive database mining revealed that GCS1-like gene homologues exist in the genomes of various organisms. Thus, it appears that GCS1 is an ancient and highly conserved molecule functioning at gamete interaction. In this mini-review, we describe the mechanisms of gametogenesis and fertilization in malaria parasites, comparing with other eukaryotic reproduction, and also speculate GCS1 functions in gamete interaction. We discuss the possibility of whether malaria GCS1 is a novel type of transmission blocking vaccine, by which anti-malaria GCS1 antibody may halt parasite fertilization and subsequent developments in the mosquitoes., (Copyright (c) 2009 Elsevier B.V. All rights reserved.)

Malaria, which is caused by Plasmodium parasites, is transmitted by anopheline mosquitoes. When gametocytes, the precursor cells of Plasmodium gametes, are transferred to a mosquito, they fertilize and proliferate, which render the mosquito infectious to the next vertebrate host. Although the fertilization of malaria parasites has been considered as a rational target for transmission-blocking vaccines, the underlying mechanism is poorly understood. Here, we show that the rodent malaria parasite gene Plasmodium berghei GENERATIVE CELL SPECIFIC 1 (PbGCS1) plays a central role in its gametic interaction. PbGCS1 knockout parasites show male sterility, resulting in unsuccessful fertilization. Because such a male-specific function of GCS1 has been observed in angiosperms, this indicates, for the first time, that parasite sexual reproduction is controlled by a machinery common to flowering plants. Our present findings provide a new viewpoint for understanding the parasitic fertilization system and important clues for novel strategies to attack life-threatening parasites.

When malaria parasites enter to mosquitoes, they fertilize and differentiate to zygotes and ookinetes. The motile ookinetes cross the midgut cells and arrive to the basement membranes where they differentiate into oocysts. The midgut epithelium is thus a barrier for ookinetes to complete their life cycle in the mosquitoes. The ookinetes develop gliding motility to invade midgut cells successfully, but the molecular mechanisms behind are poorly understood. Here, we identified a single molecule with guanylate cyclase domain and N-terminal P-type ATPase like domain in the rodent malaria parasite Plasmodium berghei and named it PbGCbeta. We demonstrated that transgenic parasites in which the PbGCbeta gene was disrupted formed normal ookinetes but failed to produce oocyst. Confocal microscopic analysis showed that the disruptant ookinetes remained on the surface of the microvilli. The disruptant ookinetes showed severe defect in motility, resulting in failure of parasite invasion of the midgut epithelium. When the disruptant ookinetes were cultured in vitro, they transformed into oocysts and sporozoites. These results demonstrate that PbGCbeta is essential for ookinete motility when passing through the midgut cells, but not for further development of the parasites.

We found that infection of a rodent malaria, Plasmodium berghei, occurred when the sporozoites were injected into the skin, the muscle, the peritoneal cavity and the tail end. Mice, which were injected with sporozoites in the tail end and had the site cut 5 min later, did not develop malaria. We also found that mice developed malaria when malaria infective mosquitoes, Anopheles stephensi, were forced not to take blood but only to probe into the skin. Moreover, the mice probed by the infective mosquitoes were protected from malaria infection if the site was treated with Kyu (heat treatment) after the mosquitoes had probed. These findings indicate that malaria infection occurs not only by blood feeding of the infective mosquito but also by probing of the mosquito. Sporozoites injected into the skin remain at the injected site for at least 5 min, then migrate to the blood vessels and invade into the blood stream. At present, the mechanism is not clear, although we propose here the existence of the skin stage of malaria parasites before the liver stage and the blood stage.

Elucidation of the mechanisms of drug resistance in malaria parasites is crucial for combatting the emergence and spread of resistant parasites, which can be achieved by tracing resistance-associated mutations and providing useful information for drug development. Previously, we produced a novel genetic tool, a Plasmodium berghei mutator (PbMut), whose base substitution rate is 36.5 times higher than that of wild-type parasites. Here, we report the isolation of a mutant with reduced susceptibility to piperaquine (PPQ) from PbMut under PPQ pressure by sequential nine-cycle screening and named it PbMut-PPQ-R-P9. The ED50 of PbMut-PPQ-R-P9 was 1.79 times higher than that of wild-type parasites, suggesting that its PPQ resistance is weak. In the 1st screen, recrudescence occurred in the mice infected with PbMut but not in those infected with wild-type parasites, suggesting earlier emergence of PPQ-resistant parasites from PbMut. Whole-genome sequence analysis of PbMut-PPQ-R-P9 clones revealed that eight nonsynonymous mutations were conserved in all clones, including N331I in PbCRT , the gene encoding chloroquine resistance transporter (CRT). The PbCRT(N331I) mutation already existed in the parasite population after the 2nd screen and was predominant in the population after the 8th screen. An artificially inserted PbCRT(N331I) mutation gave rise to reduced PPQ susceptibility in genome-edited parasites (PbCRT-N331I). The PPQ susceptibility and growth rates of PbCRT-N331I parasites were significantly lower than those of PbMut-PPQ-R-P9, implying that additional mutations in the PbMut-PPQ-R9 parasites could compensate for the fitness cost of the PbCRT(N331I) mutation and contribute to reduced PPQ susceptibility. In summary, PbMut could serve as a novel genetic tool for predicting gene mutations responsible for drug resistance. Further study on PbMut-PPQ-R-P9 could identify genetic changes that compensate for fitness costs owing to drug resistance acquisition. [ABSTRACT FROM AUTHOR]

Abstract: The nuclear genome of the human malaria parasite Plasmodium falciparum encodes a homolog of the bacterial HU protein (PfHU). In this study, we characterised PfHU’s physiological function. PfHU, which is targeted exclusively to the parasite’s plastid, bound its natural target – the plastid DNA – sequence-independently and complemented lack of HU in Escherichia coli. The HU gene could not be knocked-out from the genome of Plasmodium berghei, implying that HU is important for the parasite’s survival. As the human cell lacks the HU homolog, PfHU is a potential target for drugs to control malaria. [Copyright &y& Elsevier]

Sexual reproduction of Plasmodium parasites takes place in anopheline mosquitoes, where male and female gametes fuse to form zygotes and then ookinetes. These processes are orchestrated by stage-specific protein expression, which is mediated in part by translational repression. Accumulating evidence shows that RNA binding proteins (RBPs) play crucial roles in these processes. Here, we report the characterization of P. berghei 103 (Pb103), which encodes a protein possessing double zinc finger domains (ZFs), an RBP. Reporter parasites expressing azami green fluorescent protein (AGFP) under the endogenous Pb103 gene promoter (Pb103-AGFP reporter) showed that the AGFP fluorescent signal was detected from gametes to ookinetes, while AGFP mRNA was translationally repressed in female gametocytes. The Pb103-disrupted parasites (Pb103(−)) grew and produced gametocytes with similar efficiencies to those of wild-type parasites. However, no oocysts were formed in mosquitoes fed Pb103(−). An in vitro fertilization assay showed abortion at the zygote stage in Pb103(−), suggesting that Pb103 plays a critical role in zygote/ookinete development. Cross-fertilization assays with Pb103(−) and male- or female-sterile parasites revealed that Pb103 was essential exclusively for female gametes. To identify the domains critical for zygote/ookinete development, transgenic parasites expressing partially deleted Pb103 were generated and assayed for ookinete maturation. As a result, deleting either of two ZFs but not the C-terminal region abolished zygote/ookinete development, highlighting the indispensable roles of ZFs in parasite sexual development, most likely via translational repression. [ABSTRACT FROM AUTHOR]

• PfDHPS-A437G is known as one of sulfadoxine-resistant mutations in P. falciparum. • PbDHPS-A394G in P. berghei corresponds to PfDHPS-A437G. • We generated a transgenic PbDHPS-A394G parasite and investigated the developments. • PbDHPS-A394G parasite grew in mice and produced oocysts with similar efficiency to wild-type. • The PbDHPS-A394G mutation may not impose fitness costs on the parasite during the examined stages. Genetic changes conferring drug resistance are generally believed to impose fitness costs to pathogens in the absence of the drug. However, the fitness of resistant parasites against sulfadoxine/pyrimethamine has been inconclusive in Plasmodium falciparum. This is because resistance is conferred by the complex combination of mutations in dihydropteroate synthase (dhps) and dihydrofolate reductase (dhfr), which makes it difficult to separately assess the extent and magnitude of the costs imposed by mutations in dhps and dhfr. To assess the fitness costs imposed by sulfadoxine resistance alone, we generated a transgenic rodent malaria parasite, P. berghei clone harboring an A394G mutation in dhps (PbDHPS-A394G), corresponding to the causative mutation for sulfadoxine resistance in P. falciparum (PfDHPS-A437G). A four-day suppressive test confirmed that the PbDHPS-A394G clone was resistant to sulfadoxine. PbDHPS-A394G and wild-type clones showed similar growth rates and gametocyte production. This observation was confirmed in competitive experiments in which PbDHPS-A394G and wild-type clones were co-infected into mice to directly assess the survival competition between them. In the mosquitoes, there were no significant differences in oocyst production between PbDHPS-A394G and wild-type. These results indicate that the PbDHPS-A394G mutation alters the parasites to sulfadoxine resistance but may not impose fitness disadvantages during the blood stages in mice and oocyst formation in mosquitoes. These results partly explain the persistence of the PfDHPS-A437G mutant in the natural parasite populations. [Display omitted] [ABSTRACT FROM AUTHOR]

The intraerythrocytic form of the human malaria parasite Plasmodium falciparum relies on glycolysis for its energy requirements. In glycolysis, lactate is an end product. It is therefore known that lactate accumulates in in vitro culture; however, its influence on parasite growth remains unknown. Here we investigated the effect of lactate on the development of P. falciparum during in vitro culture under lactate supplementation in detail. Results revealed that lactate retarded parasite development and reduced the number of merozoites in the schizont stage. These findings suggest that lactate has the potential to affect parasite development. [ABSTRACT FROM AUTHOR]

[Display omitted] The spread of malarial parasites resistant to first-line treatments such as artemisinin combination therapies is a global health concern. Differentiation-inducing factor 1 (DIF-1) is a chlorinated alkylphenone (1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl) hexan-1-one) originally found in the cellular slime mould Dictyostelium discoideum. We previously showed that some derivatives of DIF-1, particularly DIF-1(+2) (1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl) octan-1-one), exert potent antimalarial activities. In this study, we synthesised DIF-1(+3) (1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl) nonan-1-one). We then evaluated the effects of DIF-1(+3) in vitro on Plasmodium falciparum and in vivo over 7 days (50–100 mg/kg/day) in a mouse model of Plasmodium berghei. DIF-1(+3) exhibited a half-maximal inhibitory concentration of approximately 20–30 % of DIF-1(+2) in three laboratory strains with a selectivity index > 263, including in strains resistant to chloroquine and artemisinin. Parasite growth and multiplication were almost completely suppressed by treatment with 100 mg/kg DIF-1(+3). The survival time of infected mice was significantly increased (P = 0.006) with no apparent adverse effects. In summary, addition of an acyl group to DIF-1(+2) to prepare DIF-1(+3) substantially enhanced antimalarial activity, even in drug-resistant malaria, indicating the potential of applying DIF-1(+3) for malaria treatment. [ABSTRACT FROM AUTHOR]