Search

Bovine lactoferrin (bLF) is a 77 kDa glycoprotein that is abundant in bovine breast milk and exerts various bioactive functions, including antibacterial and antiviral functions. Few studies have explored bLF activity against parasites. We found that bLF affects hemozoin synthesis by binding to heme, inhibiting heme iron polymerization necessary for Plasmodium berghei ANKA survival in infected erythrocytes, and also binds to hemozoin, causing it to disassemble. In a challenge test, bLF administration inhibited the growth of murine malaria parasites compared to untreated group growth. To determine whether the iron content of bLF affects the inhibition of malaria growth, we tested bLFs containing different amounts of iron (apo-bLF, native-bLF, and holo-bLF), but found no significant difference in their effects. This indicated that the active sites were located within the bLFs themselves. Further studies showed that the C-lobe domain of bLF can inhibit hemozoin formation and the growth of P. berghei ANKA. Evaluation of pepsin degradation products of the C-lobe identified a 47-amino-acid section, C-1, as the smallest effective region that could inhibit hemozoin formation. This study highlights bLF's potential as a novel therapeutic agent against malaria, underscoring the importance of its non-iron-dependent bioactive sites in combating parasite growth., (© 2024. The Author(s).)

The emergence of clinically drug-resistant malaria parasites requires the urgent development of new drugs. Mosquitoes are vectors of multiple pathogens and have developed resistance mechanisms against them, which often involve antimicrobial peptides (AMPs). An-cecB is an AMP of the malaria-transmitting mosquito genus Anopheles , and we herein report its antimalarial activity against Plasmodium falciparum 3D7, the artemisinin-resistant strain 803, and the chloroquine-resistant strain Dd2 in vitro . We also demonstrate its anti-parasite activity in vivo , using the rodent malaria parasite Plasmodium berghei (ANKA). We show that An-cecB displays potent antimalarial activity and that its mechanism of action may occur through direct killing of the parasite or through interaction with infected red blood cell membranes. Unfortunately, An-cecB was found to be cytotoxic to mammalian cells and had poor antimalarial activity in vivo . However, its truncated peptide An-cecB-1 retained most of its antimalarial activity and avoided its cytotoxicity in vitro . An-cecB-1 also showed better antimalarial activity in vivo . Mosquito-derived AMPs may provide new ideas for the development of antimalarial drugs against drug-resistant parasites, and An-cecB has potential use as a template for antimalarial peptides., Competing Interests: The authors declare no conflict of interest.

Parasite-derived new permeation pathways (NPPs) expressed at the red blood cell (RBC) membrane enable Plasmodium parasites to take up nutrients from the plasma to facilitate their survival. Thus, NPPs represent a potential novel therapeutic target for malaria. The putative channel component of the NPP in the human malaria parasite P. falciparum is encoded by mutually exclusively expressed clag3.1/3.2 genes. Complicating the study of the essentiality of these genes to the NPP is the addition of three clag paralogs whose contribution to the P. falciparum channel is uncertain. Rodent malaria P. berghei contains only two clag genes, and thus studies of P. berghei clag genes could significantly aid in dissecting their overall contribution to NPP activity. Previous methods for determining NPP activity in a rodent model have utilised flux-based assays of radioisotope-labelled substrates or patch clamping. This study aimed to ratify a streamlined haemolysis assay capable of assessing the functionality of P. berghei NPPs. Several isotonic lysis solutions were tested for their ability to preferentially lyse infected RBCs (iRBCs), leaving uninfected RBCs (uRBCs) intact. The osmotic lysis assay was optimised and validated in the presence of NPP inhibitors to demonstrate the uptake of the lysis solution via the NPPs. Guanidinium chloride proved to be the most efficient reagent to use in an osmotic lysis assay to establish NPP functionality. Furthermore, following treatment with guanidinium chloride, ring-stage parasites could develop into trophozoites and schizonts, potentially enabling use of guanidinium chloride for parasite synchronisation. This haemolysis assay will be useful for further investigation of NPPs in P. berghei and could assist in validating its protein constituents.

Malaria is a serious vector-borne disease characterized by periodic episodes of high fever and strong immune responses that are coordinated with the daily synchronized parasite replication cycle inside RBCs. As immune cells harbor an autonomous circadian clock that controls various aspects of the immune response, we sought to determine whether the intensity of the immune response to Plasmodium spp., the parasite causing malaria, depends on time of infection. To do this, we developed a culture model in which mouse bone marrow-derived macrophages are stimulated with RBCs infected with Plasmodium berghei ANKA (iRBCs). Lysed iRBCs, but not intact iRBCs or uninfected RBCs, triggered an inflammatory immune response in bone marrow-derived macrophages. By stimulating at four different circadian time points (16, 22, 28, or 34 h postsynchronization of the cells' clock), 24-h rhythms in reactive oxygen species and cytokines/chemokines were found. Furthermore, the analysis of the macrophage proteome and phosphoproteome revealed global changes in response to iRBCs that varied according to circadian time. This included many proteins and signaling pathways known to be involved in the response to Plasmodium infection. In summary, our findings show that the circadian clock within macrophages determines the magnitude of the inflammatory response upon stimulation with ruptured iRBCs, along with changes of the cell proteome and phosphoproteome., (Copyright © 2024 The Authors.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

RTS,S is the first malaria vaccine recommended for implementation among young children at risk. However, vaccine efficacy is modest and short-lived. To mitigate the risk of cerebral malaria (CM) among children under the age of 5, it is imperative to develop new vaccines. EVs are potential vaccine candidates as they obtain the ability of brain-targeted delivery and transfer plasmodium antigens and immunomodulators during infections. This study extracted EVs from BALB/c mice infected with Plasmodium yoelii 17XNL (P.y17XNL). C57BL/6J mice were intravenously immunized with EVs (EV-I.V. + CM group) or subcutaneously vaccinated with the combination of EVs and CpG ODN-1826 (EV + CPG ODN-S.C. + CM group) on days 0 and 20, followed by infection with Plasmodium berghei ANKA (P.bANKA) on day 20 post-second immunization. We monitored Parasitemia and survival rate. The integrity of the Blood-brain barrier (BBB) was examined using Evans blue staining.The levels of cytokines and adhesion molecules were evaluated using Luminex, RT-qPCR, and WB. Brain pathology was evaluated by hematoxylin and eosin and immunohistochemical staining. The serum levels of IgG, IgG1, and IgG2a were analyzed by enzyme-linked immunosorbent assay. Compared with those in the P.bANKA-infected group, parasitemia increased slowly, death was delayed (day 10 post-infection), and the survival rate reached 75 %-83.3 % in the EV-I.V. + ECM and EV + CPG ODN-S.C. + ECM groups. Meanwhile, compared with the EV + CPG ODN-S.C. + ECM group, although parasitemia was almost the same, the survival rate increased in the EV-I.V. + ECM group.Additionally, EVs immunization markedly downregulated inflammatory responses in the spleen and brain and ameliorated brain pathological changes, including BBB disruption and infected red blood cell (iRBC) sequestration. Furthermore, the EVs immunization group exhibited enhanced antibody responses (upregulation of IgG1 and IgG2a production) compared to the normal control group. EV immunization exerted protective effects, improving the integrity of the BBB, downregulating inflammation response of brain tissue, result in reduces the incidence of CM. The protective effects were determined by immunological pathways and brain targets elicited by EVs. Intravenous immunization exhibited better performance than subcutaneous immunization, which perhaps correlated with EVs, which can naturally cross BBB to play a better role in brain protection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Malaria is still the deadliest parasitic disease caused by Plasmodium spp. Due to drug resistance and their unpleasant side effects, of conventional researchers are enormously seeking to achieve antimalarial drugs with more curative effective, less toxic and cost-affordable drugs using more advanced technology such as nanodrugs., Purpose: The present study aimed to examine the antimalarial effects of a novel synthesized nonochloroquine-loaded curcumin relying on dendrimer G2 in susceptible mice., Methods: Antimalarial activity and toxicity of the nanocomposite were examined on BALB/C mice with microscopy, checking RBCs morphology and related enzymatic activity rate., Results: The maximum inhibitory effect of the nanocomposite was seen at 10 mg/kg, killing 98% of P. berghei compared to sole chloroquine, whereas ED50 was reported at 5.5 mg/kg. The safety of the synthesized nanocomposite was confirmed with biochemical tests with no detrimental effects on mice. The sustainability and longevity of the nanodrug increased significantly with the NDC-CQ assay compared to the control groups., Conclusion: The study showed that nonochloroquine-loaded curcumin had a promising inhibitory effect on P. berghei growth in infected mice compared to standard drugs. However, further studies and clinical trials with large samples are recommended to study different aspects of using nanodrug., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Background: Malaria caused by Plasmodium species is a prominent public health concern worldwide, and the infection of a malarial parasite is transmitted to humans through the saliva of female Anopheles mosquitoes. Plasmodium invasion is a rapid and complex process. A critical step in the blood-stage infection of malarial parasites is the adhesion of merozoites to red blood cells (RBCs), which involves interactions between parasite ligands and receptors. The present study aimed to investigate a previously uncharacterized protein, PbMAP1 (encoded by PBANKA_1425900), which facilitates Plasmodium berghei ANKA (PbANKA) merozoite attachment and invasion via the heparan sulfate receptor., Methods: PbMAP1 protein expression was investigated at the asexual blood stage, and its specific binding activity to both heparan sulfate and RBCs was analyzed using western blotting, immunofluorescence, and flow cytometry. Furthermore, a PbMAP1-knockout parasitic strain was established using the double-crossover method to investigate its pathogenicity in mice., Results: The PbMAP1 protein, primarily localized to the P. berghei membrane at the merozoite stage, is involved in binding to heparan sulfate-like receptor on RBC surface of during merozoite invasion. Furthermore, mice immunized with the PbMAP1 protein or passively immunized with sera from PbMAP1-immunized mice exhibited increased immunity against lethal challenge. The PbMAP1-knockout parasite exhibited reduced pathogenicity., Conclusions: PbMAP1 is involved in the binding of P. berghei to heparan sulfate-like receptors on RBC surface during merozoite invasion., (© 2023. BioMed Central Ltd., part of Springer Nature.)

During vertebrate infection, obligate intracellular malaria parasites develop within a parasitophorous vacuole, which constitutes the interface between the parasite and its hepatocyte or erythrocyte host cells. To traverse this barrier, Plasmodium spp. utilize a dual-function pore formed by EXP2 for nutrient transport and, in the context of the PTEX translocon, effector protein export across the vacuole membrane. While critical to blood-stage survival, less is known about EXP2/PTEX function in the liver stage, although major differences in the export mechanism are suggested by absence of the PTEX unfoldase HSP101 in the intrahepatic vacuole. Here, we employed the glucosamine-activated glmS ribozyme to study the role of EXP2 during Plasmodium berghei liver-stage development in hepatoma cells. Insertion of the glmS sequence into the exp2 3' untranslated region (UTR) enabled glucosamine-dependent depletion of EXP2 after hepatocyte invasion, allowing separation of EXP2 function during intrahepatic development from a recently reported role in hepatocyte invasion. Postinvasion EXP2 knockdown reduced parasite size and largely abolished expression of the mid- to late-liver-stage marker LISP2. As an orthogonal approach to monitor development, EXP2- glmS parasites and controls were engineered to express nanoluciferase. Activation of glmS after invasion substantially decreased luminescence in hepatoma monolayers and in culture supernatants at later time points corresponding to merosome detachment, which marks the culmination of liver-stage development. Collectively, our findings extend the utility of the glmS ribozyme to study protein function in the liver stage and reveal that EXP2 is important for intrahepatic parasite development, indicating that PTEX components also function at the hepatocyte-parasite interface. IMPORTANCE After the mosquito bite that initiates a Plasmodium infection, parasites first travel to the liver and develop in hepatocytes. This liver stage is asymptomatic but necessary for the parasite to transition to the merozoite form, which infects red blood cells and causes malaria. To take over their host cells, avoid immune defenses, and fuel their growth, these obligately intracellular parasites must import nutrients and export effector proteins across a vacuole membrane in which they reside. In the blood stage, these processes depend on a translocon called PTEX, but it is unclear if PTEX also functions during the liver stage. Here, we adapted the glmS ribozyme to control expression of EXP2, the membrane pore component of PTEX, during the liver stage of the rodent malaria parasite Plasmodium berghei. Our results show that EXP2 is important for intracellular development in the hepatocyte, revealing that PTEX components are also functionally important during liver-stage infection.

Protein export and host membrane remodeling are crucial for multiple Plasmodium species to establish a niche in infected hosts. To better understand the contribution of these processes to successful parasite infection in vivo, we sought to find and characterize protein components of the intraerythrocytic Plasmodium berghei-induced membrane structures (IBIS) that form in the cytoplasm of infected erythrocytes. We identified proteins that immunoprecipitate with IBIS1, a signature member of the IBIS in P. berghei-infected erythrocytes. In parallel, we also report our data describing proteins that co-precipitate with the PTEX (Plasmodium translocon of exported proteins) component EXP2. To validate our findings, we examined the location of three candidate IBIS1-interactors that are conserved across multiple Plasmodium species, and we found they localized to IBIS in infected red blood cells and two further colocalized with IBIS1 in the liver-stage parasitophorous vacuole membrane. Successful gene deletion revealed that these two tryptophan-rich domain-containing proteins, termed here IPIS2 and IPIS3 (for intraerythrocytic Plasmodium-induced membrane structures), are required for efficient blood-stage growth. Erythrocytes infected with IPIS2-deficient schizonts in particular fail to bind CD36 as efficiently as wild-type P. berghei-infected cells and therefore fail to effectively sequester out of the circulating blood. Our findings support the idea that intra-erythrocytic membrane compartments are required across species for alterations of the host erythrocyte that facilitate interactions of infected cells with host tissues., Competing Interests: The authors have declared that no competing interests exist.

The Plasmodium parasite has to cross various immunological barriers for successful infection. Parasites have evolved mechanisms to evade host immune responses, which hugely contributes to the successful infection and transmission by parasites. One way in which a parasite evades immune surveillance is by expressing molecular mimics of the host molecules in order to manipulate the host responses. In this study, we report a Plasmodium berghei hypothetical protein, Pb TIP (PbANKA_124360.0), which is a Plasmodium homolog of the human T-cell immunomodulatory protein (TIP). The latter possesses immunomodulatory activities and suppressed the host immune responses in a mouse acute graft- versus -host disease (GvHD) model. The Plasmodium berghei protein, Pb TIP, is expressed on the merozoite surface and exported to the host erythrocyte surface upon infection. It is shed in the blood circulation by the activity of an uncharacterized membrane protease(s). The shed Pb TIP could be detected in the host serum during infection. Our results demonstrate that the shed Pb TIP exhibits binding on the surface of macrophages and reduces their inflammatory cytokine response while upregulating the anti-inflammatory cytokines such as TGF-β and IL-10. Such manipulated immune responses are observed in the later stage of malaria infection. Pb TIP induced Th2-type gene transcript changes in macrophages, hinting toward its potential to regulate the host immune responses against the parasite. Therefore, this study highlights the role of a Plasmodium -released protein, Pb TIP, in immune evasion using macrophages, which may represent the critical strategy of the parasite to successfully survive and thrive in its host. This study also indicates the human malaria parasite TIP as a potential diagnostic molecule that could be exploited in lateral flow-based immunochromatographic tests for malaria disease diagnosis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kalia, Anand, Quadiri, Bhattacharya, Sahoo and Singh.)

Experimental cerebral malaria (ECM) is a severe complication of Plasmodium berghei ANKA (PbA) infection in mice, characterized by CD8 + T-cell accumulation within the brain. Whilst the dynamics of CD8 + T-cell activation and migration during extant primary PbA infection have been extensively researched, the fate of the parasite-specific CD8 + T cells upon resolution of ECM is not understood. In this study, we show that memory OT-I cells persist systemically within the spleen, lung and brain following recovery from ECM after primary PbA-OVA infection. Whereas memory OT-I cells within the spleen and lung exhibited canonical central memory (Tcm) and effector memory (Tem) phenotypes, respectively, memory OT-I cells within the brain post-PbA-OVA infection displayed an enriched CD69 + CD103 - profile and expressed low levels of T-bet. OT-I cells within the brain were excluded from short-term intravascular antibody labelling but were targeted effectively by longer-term systemically administered antibodies. Thus, the memory OT-I cells were extravascular within the brain post-ECM but were potentially not resident memory cells. Importantly, whilst memory OT-I cells exhibited strong reactivation during secondary PbA-OVA infection, preventing activation of new primary effector T cells, they had dampened reactivation during a fourth PbA-OVA infection. Overall, our results demonstrate that memory CD8 + T cells are systemically distributed but exhibit a unique phenotype within the brain post-ECM, and that their reactivation characteristics are shaped by infection history. Our results raise important questions regarding the role of distinct memory CD8 + T-cell populations within the brain and other tissues during repeat Plasmodium infections., (© 2021 The Authors. Immunology published by John Wiley & Sons Ltd.)

Malaria parasites exhibit a complex lifecycle, requiring extensive asexual replication in the liver and blood of the vertebrate host, and in the haemocoel of the insect vector. Yet, they must also undergo a single round of sexual reproduction, which occurs in the vector's midgut upon uptake of a blood meal. Sexual reproduction is obligate for infection of the vector and thus, is essential for onwards transmission to new hosts. Sex in malaria parasites involves several bottlenecks in parasite number, making the stages involved attractive targets for blocking disease transmission. Malaria parasites have evolved a suite of adaptations ("strategies") to maximise the success of sexual reproduction and transmission, which could undermine transmission-blocking interventions. Yet, understanding parasite strategies may also reveal novel opportunities for such interventions. Here, we outline how evolutionary and ecological theories, developed to explain reproductive strategies in multicellular taxa, can be applied to explain two reproductive strategies (conversion rate and sex ratio) expressed by malaria parasites within the vertebrate host., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Autophagy, a lysosome-dependent degradative process, does not appear to be a major degradative process in malaria parasites and has a limited repertoire of genes. To better understand the autophagy process, we investigated Plasmodium falciparum Atg18 (PfAtg18), a PROPPIN family protein, whose members like S. cerevisiae Atg18 (ScAtg18) and human WIPI2 bind PI3P and play an essential role in autophagosome formation. Wild type and mutant PfAtg18 were expressed in P. falciparum and assessed for localization, the effect of various inhibitors and antimalarials on PfAtg18 localization, and identification of PfAtg18-interacting proteins. PfAtg18 is expressed in asexual erythrocytic stages and localized to the food vacuole, which was also observed with other Plasmodium Atg18 proteins, indicating that food vacuole localization is likely a shared feature. Interaction of PfAtg18 with the food vacuole-associated PI3P is essential for localization, as PfAtg18 mutants of PI3P-binding motifs neither bound PI3P nor localized to the food vacuole. Interestingly, wild type ScAtg18 interacted with PI3P, but its expression in P. falciparum showed complete cytoplasmic localization, indicating additional requirement for food vacuole localization. The food vacuole multi-drug resistance protein 1 (MDR1) was consistently identified in the immunoprecipitates of PfAtg18 and P. berghei Atg18, and also interacted with PfAtg18. In contrast with PfAtg18, ScAtg18 did not interact with MDR1, which, in addition to PI3P, could play a critical role in localization of PfAtg18. Chloroquine and amodiaquine caused cytoplasmic localization of PfAtg18, suggesting that these target PfAtg18 transport pathway. Thus, PI3P and MDR1 are critical mediators of PfAtg18 localization., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Background: Daily periodicity in the diverse activities of parasites occurs across a broad taxonomic range. The rhythms exhibited by parasites are thought to be adaptations that allow parasites to cope with, or exploit, the consequences of host activities that follow daily rhythms. Malaria parasites (Plasmodium) are well-known for their synchronized cycles of replication within host red blood cells. Whilst most species of Plasmodium appear sensitive to the timing of the daily rhythms of hosts, and even vectors, some species present no detectable rhythms in blood-stage replication. Why the intraerythrocytic development cycle (IDC) of, for example Plasmodium chabaudi, is governed by host rhythms, yet seems completely independent of host rhythms in Plasmodium berghei, another rodent malaria species, is mysterious., Methods: This study reports a series of five experiments probing the relationships between the asynchronous IDC schedule of P. berghei and the rhythms of hosts and vectors by manipulating host time-of-day, photoperiod and feeding rhythms., Results: The results reveal that: (i) a lack coordination between host and parasite rhythms does not impose appreciable fitness costs on P. berghei; (ii) the IDC schedule of P. berghei is impervious to host rhythms, including altered photoperiod and host-feeding-related rhythms; (iii) there is weak evidence for daily rhythms in the density and activities of transmission stages; but (iv), these rhythms have little consequence for successful transmission to mosquitoes., Conclusions: Overall, host rhythms do not affect the performance of P. berghei and its asynchronous IDC is resistant to the scheduling forces that underpin synchronous replication in closely related parasites. This suggests that natural variation in the IDC schedule across species represents different parasite strategies that maximize fitness. Thus, subtle differences in the ecological interactions between parasites and their hosts/vectors may select for the evolution of very different IDC schedules.

The Apicomplexa phylum groups important human and animal pathogens that cause severe diseases, encompassing malaria, toxoplasmosis, and cryptosporidiosis. In common with most organisms, apicomplexans rely on heme as cofactor for several enzymes, including cytochromes of the electron transport chain. This heme derives from de novo synthesis and/or the development of uptake mechanisms to scavenge heme from their host. Recent studies have revealed that heme synthesis is essential for Toxoplasma gondii tachyzoites, as well as for the mosquito and liver stages of Plasmodium spp. In contrast, the erythrocytic stages of the malaria parasites rely on scavenging heme from the host red blood cell. The unusual heme synthesis pathway in Apicomplexa spans three cellular compartments and comprises enzymes of distinct ancestral origin, providing promising drug targets. Remarkably given the requirement for heme, T. gondii can tolerate the loss of several heme synthesis enzymes at a high fitness cost, while the ferrochelatase is essential for survival. These findings indicate that T. gondii is capable of salvaging heme precursors from its host. Furthermore, heme is implicated in the activation of the key antimalarial drug artemisinin. Recent findings established that a reduction in heme availability corresponds to decreased sensitivity to artemisinin in T. gondii and Plasmodium falciparum, providing insights into the possible development of combination therapies to tackle apicomplexan parasites. This review describes the microeconomics of heme in Apicomplexa, from supply, either from de novo synthesis or scavenging, to demand by metabolic pathways, including the electron transport chain., (© 2020 Federation of European Biochemical Societies.)

Plasmodium , the malaria parasite, undergoes a complex life cycle alternating between a vertebrate host and a mosquito vector of the genus Anopheles In red blood cells of the vertebrate host, Plasmodium multiplies asexually or differentiates into gamete precursors, the male and female gametocytes, responsible for parasite transmission. Sexual stage maturation occurs in the midgut of the mosquito vector, where male and female gametes egress from the host erythrocytes to fuse and form a zygote. Gamete egress entails the successive rupture of two membranes surrounding the parasite, the parasitophorous vacuole membrane and the erythrocyte plasma membrane. In this study, we used the rodent model parasite Plasmodium berghei to design a label-free quantitative proteomic approach aimed at identifying gender-related proteins differentially released/secreted by purified mature gametocytes when activated to form gametes. We compared the abundance of molecules secreted by wild type gametocytes of both genders with that of a transgenic line defective in male gamete maturation and egress. This enabled us to provide a comprehensive data set of egress-related molecules and their gender specificity. Using specific antibodies, we validated eleven candidate molecules, predicted as either gender-specific or common to both male and female gametocytes. All of them localize to punctuate, vesicle-like structures that relocate to cell periphery upon activation, but only three of them localize to the gametocyte-specific secretory vesicles named osmiophilic bodies. Our results confirm that the egress process involves a tightly coordinated secretory apparatus that includes different types of vesicles and may put the basis for functional studies aimed at designing novel transmission-blocking molecules., Competing Interests: Conflict of interest—Authors declare no competing interests., (© 2020 Grasso et al.)

Invasion of hepatocytes by Plasmodium sporozoites initiates the pre-erythrocytic step of a malaria infection. Subsequent development of the parasite within hepatocytes and exit from them is essential for starting the disease-causing erythrocytic cycle. Identification of signaling pathways that operate in pre-erythrocytic stages provides insight into a critical step of infection and potential targets for chemoprotection from malaria. We demonstrate that P. berghei homologs of Calcium Dependent Protein Kinase 1 (CDPK1), CDPK4 and CDPK5 play overlapping but distinct roles in sporozoite invasion and parasite egress from hepatocytes. All three kinases are expressed in sporozoites. All three are required for optimal motility of sporozoites and consequently their invasion of hepatocytes. Increased cGMP can compensate for the functional loss of CDPK1 and CDPK5 during sporozoite invasion but cannot overcome loss of CDPK4. CDPK1 and CDPK5 expression is downregulated after sporozoite invasion. CDPK5 reappears in a subset of late stage liver stages and is present in all merosomes. Chemical inhibition of CDPK4 and depletion of CDPK5 in liver stages implicate these kinases in the formation and/or release of merosomes from mature liver stages. Furthermore, depletion of CDPK5 in merosomes significantly delays initiation of the erythrocytic cycle without affecting infectivity of hepatic merozoites. These data suggest that CDPK5 may be required for the rupture of merosomes. Our work provides evidence that sporozoite invasion requires CDPK1 and CDPK5, and suggests that CDPK5 participates in the release of hepatic merozoites., Competing Interests: The authors have declared that no competing interests exist.

Male and female Plasmodium gametocytes ingested by the Anopheles mosquitoes during a blood meal egress from the red blood cells by rupturing the two surrounding membranes, the parasitophorous vacuole and the red blood cell membranes. Proteins of the so-called osmiophilic bodies, (OBs), secretory organelles resident in the cytoplasm, are important players in this process. Once gametes emerge, the female is ready to be fertilized while the male develops into motile flagellar gametes. Here, we describe the function(s) of PBANKA_1115200, which we named Gamete Egress Protein (GEP), a protein specific to malaria parasites. GEP is restricted to gametocytes, expressed in gametocytes of both genders and partly localizes to the OBs. A mutant lacking the protein shows aberrant rupture of the two surrounding membranes, while OBs discharge is delayed but not aborted. Moreover, we identified a second function of GEP during exflagellation since the axonemes of the male flagellar gametes were not motile. Genetic crossing experiments reveal that both genders are unable to establish infections in mosquitoes and thus the lack of GEP leads to a complete block in Plasmodium transmission from mice to mosquitoes. The combination of our results reveals essential and pleiotropic functions of GEP in Plasmodium gametogenesis., (© 2020 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Ethnopharmacological Relevance: In spite of worldwide efforts, malaria remains one of the most devastating illnesses in the world. The huge number of lives it takes and the resistance of malaria parasites to current drugs necessitate the search for new effective antimalarial drugs. Medicinal plants have been the major source of such drugs and A. pirottae is one of these plants used traditionally for the treatment of malaria in Ethiopia., Aim: This study was aimed at evaluating the antimalarial activity of the aqueous extract of A. pirottae against chloroquine sensitive P. berghei in mice., Materials and Methods: The extract was obtained by macerating the latex of A. pirottae with distilled water. To determine its antiplasmodial activity, a 4-day suppressive model was used by dividing 40 mice into five groups of 8 mice each and given 200, 400 & 600mg/kg of the extract, the standard drug (chloroquine 25mg/kg) and the vehicle (distilled water). Then parasite suppression by the extract, survival time and prevention of loss of body weight, rectal temperature and packed cell volume were assessed. All data were presented as the Mean ± SEM (Standard Error of the Mean) and analyzed using IBM SPSS version 20., Results: The extract showed moderate antimalarial activity by significantly (p < 0.001) suppressing parasitemia at all dose levels with maximum parasitemia suppression of 47.0% and significantly (p < 0.01) increasing survival time. Furthermore, 400 mg/kg and 600 mg/kg doses showed significant (p < 0.01) prevention of loss in body weight, rectal temperature and packed cell volume., Conclusion: Based to the results of this study, A. pirottae is endowed with a moderate antimalarial activity that is in agreement with the traditional claim of A. pirottae, hence may be used as a basis for further studies to be conducted on antimalarial activity of the plant., Competing Interests: Declaration of competing interest No competing interests between authors., (Copyright © 2020 Elsevier B.V. All rights reserved.)

G-strand binding protein 2 (GBP2) is a Ser/Arg-rich (SR) protein involved in mRNA surveillance and nuclear mRNA quality control in yeast. However, the roles of GBP2 in virulence and sexual development in Plasmodium parasites are unclear, although GBP2 is involved in the asexual development of Plasmodium berghei, the rodent malaria parasite. In this study, we investigated the role of GBP2 in virulence and sexual development of P. berghei using gbp2-deleted P. berghei (Δgbp2 parasites). Then, to identify factors affected by gbp2 deletion, we performed a comparative proteomic analysis of the Δgbp2 parasites. We found that GBP2 was not associated with the development of experimental cerebral malaria during infection with P. berghei, but asexual development of the parasite was delayed with deletion of gbp2. However, the development of P. berghei gametocytes was significantly reduced with deletion of gbp2. Comparative proteomic analysis revealed that the levels of adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP), and hypoxanthine-guanine phosphoribosyltransferase (HGPRT) in Δgbp2 parasites were significantly higher than those in wild-type (WT) parasites, suggesting that biosynthesis of purine nucleotides may be involved in function of GBP2. Therefore, we investigated the effect of purine starvation on the sexual development and proteome. In nt1-deleted P. berghei (Δnt1 parasites), the production of male and female gametocytes was significantly reduced compared to that in WT parasites. Moreover, we found that protein levels of GBP2 in Δnt1 parasites were markedly lower than in WT parasites. These findings suggest that GBP2 is primarily involved in the sexual development of malaria parasites, and its function may be suppressed by purine starvation., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Due to the lack of efficiency to control malaria elicited by sub-unit vaccine preparations, vaccination with live-attenuated Plasmodium parasite as reported 70 years ago with irradiated sporozoites regained recently a significant interest. The complex life cycle of the parasite and the different stages of development between mammal host and anopheles do not help to propose an easy vaccine strategy. In order to achieve a complete long-lasting protection against Plasmodium infection and disease, we considered a genetically attenuated blood stage parasite in the hmgb2 gene coding for the high-mobility-group-box 2 (HMGB2). This Plasmodium protein belongs to the HMGB family and hold as the mammal proteins, a double life since it acts first as a nuclear factor involved in chromatin remodelling and transcription regulation and second, when secreted as an active pro-inflammatory alarmin protein. Even though the number of reports on whole living attenuated blood stage parasites is limited when compared to attenuated sporozoites, the results reported with Plasmodium KO parasites are very encouraging. In this report, we present a novel strategy based on pre-immunization with Δhmgb2PbNK65 parasitized red blood cells that confer long-lasting protection in a murine experimental cerebral malaria model against two highly pathogenic homologous and heterologous parasites., Competing Interests: The authors have declared that no competing interests exist.

Upon entering its mammalian host, the malaria parasite productively invades two distinct cell types, that is, hepatocytes and erythrocytes during which several adhesins/invasins are thought to be involved. Many surface-located proteins containing thrombospondin Type I repeat (TSR) which help establish host-parasite molecular crosstalk have been shown to be essential for mammalian infection. Previous reports indicated that antibodies produced against Plasmodium falciparum secreted protein with altered thrombospondin repeat (SPATR) block hepatocyte invasion by sporozoites but no genetic evidence of its contribution to invasion has been reported. After failing to generate Spatr knockout in Plasmodium berghei blood stages, a conditional mutagenesis system was employed. Here, we show that SPATR plays an essential role during parasite's blood stages. Mutant salivary gland sporozoites exhibit normal motility, hepatocyte invasion, liver stage development and rupture of the parasitophorous vacuole membrane resulting in merosome formation. But these mutant hepatic merozoites failed to establish a blood stage infection in vivo. We provide direct evidence that SPATR is not required for hepatocyte invasion but plays an essential role during the blood stages of P. berghei., (© 2019 John Wiley & Sons Ltd.)

Background: Malaria disease commences when blood-stage parasites, called merozoites, invade human erythrocytes. Whilst the process of invasion is traditionally seen as being entirely merozoite-driven, emerging data suggests erythrocyte biophysical properties markedly influence invasion. Cholesterol is a major determinant of cell membrane biophysical properties demanding its interrogation as a potential mediator of resistance to merozoite invasion of the erythrocyte., Methods: Biophysical measurements of erythrocyte deformability by flicker spectroscopy were used to assess changes in erythrocyte bending modulus on forced integration of cholesterol and how these artificial changes affect invasion by human Plasmodium falciparum merozoites. To validate these observations in a natural context, either murine Plasmodium berghei or human Plasmodium falciparum merozoites were tested for their ability to invade erythrocytes from a hypercholesterolaemic mouse model or human clinical erythrocyte samples deriving from patients with a range of serum cholesterol concentrations, respectively., Results: Erythrocyte bending modulus (a measure of deformability) was shown to be markedly affected by artificial modulation of cholesterol content and negatively correlated with merozoite invasion efficiency. In an in vitro infection context, however, erythrocytes taken from hypercholesterolaemic mice or from human clinical samples with varying serum cholesterol levels showed little difference in their susceptibility to merozoite invasion. Explaining this, membrane cholesterol levels in both mouse and human hypercholesterolaemia erythrocytes were subsequently found to be no different from matched normal serum controls., Conclusions: Based on these observations, serum cholesterol does not appear to impact on erythrocyte susceptibility to merozoite entry. Indeed, no relationship between serum cholesterol and cholesterol content of the erythrocyte is apparent. This work, nonetheless, suggests that native polymorphisms which do affect membrane lipid composition would be expected to affect parasite entry. This supports investigation of erythrocyte biophysical properties in endemic settings, which may yet identify naturally protective lipid-related polymorphisms.

Plasmodium gene functions in mosquito and liver stages remain poorly characterized due to limitations in the throughput of phenotyping at these stages. To fill this gap, we followed more than 1,300 barcoded P. berghei mutants through the life cycle. We discover 461 genes required for efficient parasite transmission to mosquitoes through the liver stage and back into the bloodstream of mice. We analyze the screen in the context of genomic, transcriptomic, and metabolomic data by building a thermodynamic model of P. berghei liver-stage metabolism, which shows a major reprogramming of parasite metabolism to achieve rapid growth in the liver. We identify seven metabolic subsystems that become essential at the liver stages compared with asexual blood stages: type II fatty acid synthesis and elongation (FAE), tricarboxylic acid, amino sugar, heme, lipoate, and shikimate metabolism. Selected predictions from the model are individually validated in single mutants to provide future targets for drug development., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Malaria is a pathogenic disease in mammal species and typically causes destruction of red blood cells (RBCs). The malaria-infected RBCs undergoes alterations in morphology and its rheological properties, and the altered rheological properties of RBCs have a significant impact on disease pathophysiology. In this study, we investigated detailed topological and biomechanical properties of RBCs infected with malaria Plasmodium berghei ANKA using atomic force microscopy. Mouse (BALB/c) RBCs were obtained on Days 4, 10, and 14 after infection. We found that malaria-infected RBCs changed significantly in shape. The RBCs maintained a biconcave disk shape until Day 4 after infection and then became lopsided on Day 7 after infection. The central region of RBCs began to swell beginning on Day 10 after infection. More schizont stages were present on Days 10 and 14 compared with on Day 4. The malaria-infected RBCs also showed changes in mechanical properties and the cytoskeleton. The stiffness of infected RBCs increased 4.4-4.6-fold and their cytoskeletal F-actin level increased 18.99-67.85% compared with the control cells. The increase in F-actin depending on infection time was in good agreement with the increased stiffness of infected RBCs. Because more schizont stages were found at a late period of infection at Days 10 and 14, the significant changes in biomechanical properties might contribute to the destruction of RBCs, possibly resulting in the release of merozoites into the blood circulation., (© 2019 Wiley Periodicals, Inc.)

Intracellular Plasmodium parasites develop inside a parasitophorous vacuole (PV), a specialised compartment enclosed by a membrane (PVM) that contains proteins of both host and parasite origin. Although exported protein 1 (EXP1) is one of the earliest described parasitic PVM proteins, its function throughout the Plasmodium life cycle remains insufficiently understood. Here, we show that whereas the N-terminus of Plasmodium berghei EXP1 (PbEXP1) is essential for parasite survival in the blood, parasites lacking PbEXP1's entire C-terminal (CT) domain replicate normally in the blood but cause less severe pathology than their wild-type counterparts. Moreover, truncation of PbEXP1's CT domain not only impairs parasite development in the mosquito but also abrogates PbEXP1 localization to the PVM of intrahepatic parasites, severely limiting their replication and preventing their egress into the blood. Our findings highlight the importance of EXP1 during the Plasmodium life cycle and identify this protein as a promising target for antiplasmodial intervention., (© 2019 The Authors. Cellular Microbiology published by John Wiley & Sons Ltd.)

Background: The complex life cycle of malaria parasites requires well-orchestrated stage specific gene expression. In the vertebrate host the parasites grow and multiply by schizogony in two different environments: within erythrocytes and within hepatocytes. Whereas erythrocytic parasites are well-studied in this respect, relatively little is known about the exo-erythrocytic stages., Methods: In an attempt to fill this gap, genome wide RNA-seq analyses of various exo-erythrocytic stages of Plasmodium berghei including sporozoites, samples from a time-course of liver stage development and detached cells were performed. These latter contain infectious merozoites and represent the final step in exo-erythrocytic development., Results: The analysis represents the complete transcriptome of the entire life cycle of P. berghei parasites with temporal detailed analysis of the liver stage allowing comparison of gene expression across the progression of the life cycle. These RNA-seq data from different developmental stages were used to cluster genes with similar expression profiles, in order to infer their functions. A comparison with published data from other parasite stages confirmed stage-specific gene expression and revealed numerous genes that are expressed differentially in blood and exo-erythrocytic stages. One of the most exo-erythrocytic stage-specific genes was PBANKA&#95;1003900, which has previously been annotated as a "gametocyte specific protein". The promoter of this gene drove high GFP expression in exo-erythrocytic stages, confirming its expression profile seen by RNA-seq., Conclusions: The comparative analysis of the genome wide mRNA expression profiles of erythrocytic and different exo-erythrocytic stages could be used to improve the understanding of gene regulation in Plasmodium parasites and can be used to model exo-erythrocytic stage metabolic networks toward the identification of differences in metabolic processes during schizogony in erythrocytes and hepatocytes.

The pre-erythrocytic liver stage of the malaria parasite, comprising sporozoites and the liver stages into which they develop, remains one of the least understood parts of the lifecycle, in part owing to the low numbers of parasites. Nonetheless, it is recognized as an important target for antimalarial drugs and vaccines. Here we provide the first proteomic analysis of merosomes, which define the final phase of the liver stage and are responsible for initiating the blood stage of infection. We identify a total of 1879 parasite proteins, and a core set of 1188 proteins quantitatively detected in every biological replicate, providing an extensive picture of the protein repertoire of this stage. This unique data set will allow us to explore key questions about the biology of merosomes and hepatic merozoites.

Plasmodium sporozoites are infective forms of the parasite to mammalian hepatocytes. Sporozoite surface or secreted proteins likely play an important role in recognition, invasion and successful establishment of hepatocyte infection. By approaches of reverse genetics, we report the functional analysis of two Plasmodium berghei (Pb) sporozoite specific genes- PbS10 and PbS23/SSP3 that encode for proteins with a putative signal peptide. The expression of both genes was high in oocyst and salivary gland sporozoite stages as compared to other life cycle stages and PbS23/SSP3 protein was detected in salivary gland sporozoites. Both mutants were indistinguishable to wild-type parasites with regard to asexual growth in RBC, ability to complete sexual reproduction and form sporozoites in vector host. While the sporozoite stage of both mutants were able to glide and invade hepatocytes normally in vitro and in vivo, PbS10 mutants suffered growth attenuation at an early stage while PbS23/SSP3 mutants manifested defect during late exo-erythrocytic form maturation. Interestingly, both mutants gave rare breakthrough infections, suggesting that while both were critical for liver stage development, their depletion did not completely abrogate blood stage infection. These findings have important implications for weakening sporozoites by multiple gene attenuation towards the generation of a safe whole organism vaccine., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The malaria parasite sporozoite sequentially invades mosquito salivary glands and mammalian hepatocytes; and is the Plasmodium lifecycle infective form mediating parasite transmission by the mosquito vector. The identification of several sporozoite-specific secretory proteins involved in invasion has revealed that sporozoite motility and specific recognition of target cells are crucial for transmission. It has also been demonstrated that some components of the invasion machinery are conserved between erythrocytic asexual and transmission stage parasites. The application of a sporozoite stage-specific gene knockdown system in the rodent malaria parasite, Plasmodium berghei, enables us to investigate the roles of such proteins previously intractable to study due to their essentiality for asexual intraerythrocytic stage development, the stage at which transgenic parasites are derived. Here, we focused on the rhoptry neck protein 11 (RON11) that contains multiple transmembrane domains and putative calcium-binding EF-hand domains. PbRON11 is localised to rhoptry organelles in both merozoites and sporozoites. To repress PbRON11 expression exclusively in sporozoites, we produced transgenic parasites using a promoter-swapping strategy. PbRON11-repressed sporozoites showed significant reduction in attachment and motility in vitro, and consequently failed to efficiently invade salivary glands. PbRON11 was also determined to be essential for sporozoite infection of the liver, the first step during transmission to the vertebrate host. RON11 is demonstrated to be crucial for sporozoite invasion of both target host cells - mosquito salivary glands and mammalian hepatocytes - via involvement in sporozoite motility., (Copyright © 2019 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The essential and distinct functions of Protein Phosphatase type 1 (PP1) catalytic subunit in eukaryotes are exclusively achieved through its interaction with a myriad of regulatory partners. In this work, we report the molecular and functional characterization of Gametocyte EXported Protein 15 (GEXP15), a Plasmodium specific protein, as a regulator of PP1. In vitro interaction studies demonstrated that GEXP15 physically interacts with PP1 through the RVxF binding motif in P. berghei. Functional assays showed that GEXP15 was able to increase PP1 activity and the mutation of the RVxF motif completely abolished this regulation. Immunoprecipitation assays of tagged GEXP15 or PP1 in P. berghei followed by immunoblot or mass spectrometry analyses confirmed their interaction and showed that they are present both in schizont and gametocyte stages in shared protein complexes involved in the spliceosome and proteasome pathways and known to play essential role in parasite development. Phenotypic analysis of viable GEXP15 deficient P. berghei blood parasites showed that they were unable to develop lethal infection in BALB/c mice or to establish experimental cerebral malaria in C57BL/6 mice. Further, although deficient parasites produced gametocytes they did not produce any oocysts/sporozoites indicating a high fitness cost in the mosquito. Global proteomic and phosphoproteomic analyses of GEXP15 deficient schizonts revealed a profound defect with a significant decrease in the abundance and an impact on phosphorylation status of proteins involved in regulation of gene expression or invasion. Moreover, depletion of GEXP15 seemed to impact mainly the abundance of some specific proteins of female gametocytes. Our study provides the first insight into the contribution of a PP1 regulator to Plasmodium virulence and suggests that GEXP15 affects both the asexual and sexual life cycle., Competing Interests: The authors have declared that no competing interests exist.

The Plasmodium subtilisin-like serine protease SUB1 is expressed in hepatic and both asexual and sexual blood parasite stages. SUB1 is required for egress of invasive forms of the parasite from both erythrocytes and hepatocytes, but its subcellular localisation, function, and potential substrates in the sexual stages are unknown. Here, we have characterised the expression profile and subcellular localisation of SUB1 in Plasmodium berghei sexual stages. We show that the protease is selectively expressed in mature male gametocytes and localises to secretory organelles known to be involved in gamete egress, called male osmiophilic bodies. We have investigated PbSUB1 function in the sexual stages by generating P. berghei transgenic lines deficient in PbSUB1 expression or enzyme activity in gametocytes. Our results demonstrate that PbSUB1 plays a role in male gamete egress. We also show for the first time that the PbSUB1 substrate PbSERA3 is expressed in gametocytes and processed by PbSUB1 upon gametocyte activation. Taken together, our results strongly suggest that PbSUB1 is not only a promising drug target for asexual stages but could also be an attractive malaria transmission-blocking target., (© 2019 The Authors Cellular Microbiology Published by John Wiley & Sons Ltd.)

Plasmodium parasites must export proteins into their erythrocytic host to survive. Exported proteins must cross the parasite plasma membrane (PPM) and the parasitophorous vacuolar membrane (PVM) encasing the parasite to access the host cell. Crossing the PVM requires protein unfolding and passage through a translocon, the Plasmodium translocon of exported proteins (PTEX). In this study, we provide the first direct evidence that heat shock protein 101 (HSP101), a core component of PTEX, unfolds proteins for translocation across the PVM by creating transgenic Plasmodium parasites in which the unfoldase and translocation functions of HSP101 have become uncoupled. Strikingly, while these parasites could export native proteins, they were unable to translocate soluble, tightly folded reporter proteins bearing the Plasmodium export element (PEXEL) across the PVM into host erythrocytes under the same conditions. In contrast, an identical PEXEL reporter protein but harboring a transmembrane domain could be exported, suggesting that a prior unfolding step occurs at the PPM. Together, these results demonstrate that the export of parasite proteins is dependent on how these proteins are presented to the secretory pathway before they reach PTEX as well as their folded status. Accordingly, only tightly folded soluble proteins secreted into the vacuolar space and not proteins containing transmembrane domains or the majority of erythrocyte-stage exported proteins have an absolute requirement for the full unfoldase activity of HSP101 to be exported. IMPORTANCE The Plasmodium parasites that cause malaria export hundreds of proteins into their host red blood cell (RBC). These exported proteins drastically alter the structural and functional properties of the RBC and play critical roles in parasite virulence and survival. To access the RBC cytoplasm, parasite proteins must pass through the Plasmodium translocon of exported proteins (PTEX) located at the membrane interfacing the parasite and host cell. Our data provide evidence that HSP101, a component of PTEX, serves to unfold protein cargo requiring translocation. We also reveal that addition of a transmembrane domain to soluble cargo influences its ability to be translocated by parasites in which the HSP101 motor and unfolding activities have become uncoupled. Therefore, we propose that proteins with transmembrane domains use an alternative unfolding pathway prior to PTEX to facilitate export., (Copyright © 2019 Matthews et al.)

Plasmodium sporozoites invade hepatocytes to initiate infection in the mammalian host. In the infected hepatocytes, sporozoites undergo rapid expansion and differentiation, resulting in the formation and release of thousands of invasive merozoites into the bloodstream. Both sporozoites and merozoites invade their host cells by activation of a signaling cascade followed by discharge of micronemal content. cAMP-dependent protein kinase catalytic subunit (PKAc)-mediated signaling plays an important role in merozoite invasion of erythrocytes, but its role during other stages of the parasite remains unknown. Becaused of the essentiality of PKAc in blood stages, we generated conditional mutants of PKAc by disrupting the gene in Plasmodium berghei sporozoites. The mutant salivary gland sporozoites were able to glide, invaded hepatocytes, and matured into hepatic merozoites which were released successfully from merosome, however failed to initiate blood stage infection when inoculated into mice. Our results demonstrate that malaria parasite complete preerythrocytic stages development without PKAc, raising the possibility that the PKAc independent signaling operates in preerythrocytic stages of P. berghei ., (© 2019 Choudhary et al.)

Atovaquone-proguanil (Malarone®) is used for malaria prophylaxis and treatment. While the cytochrome bc1-inhibitor atovaquone has potent activity, proguanil's action is attributed to its cyclization-metabolite, cycloguanil. Evidence suggests that proguanil has limited intrinsic activity, associated with mitochondrial-function. Here we demonstrate that proguanil, and cyclization-blocked analogue tBuPG, have potent, but slow-acting, in vitro anti-plasmodial activity. Activity is folate-metabolism and isoprenoid biosynthesis-independent. In yeast dihydroorotate dehydrogenase-expressing parasites, proguanil and tBuPG slow-action remains, while bc1-inhibitor activity switches from comparatively fast to slow-acting. Like proguanil, tBuPG has activity against P. berghei liver-stage parasites. Both analogues act synergistically with bc1-inhibitors against blood-stages in vitro, however cycloguanil antagonizes activity. Together, these data suggest that proguanil is a potent slow-acting anti-plasmodial agent, that bc1 is essential to parasite survival independent of dihydroorotate dehydrogenase-activity, that Malarone® is a triple-drug combination that includes antagonistic partners and that a cyclization-blocked proguanil may be a superior combination partner for bc1-inhibitors in vivo., Competing Interests: The authors declare no competing interests.

Ferlins mediate calcium-dependent vesicular fusion. Although conserved throughout eukaryotic evolution, their function in unicellular organisms including apicomplexan parasites is largely unknown. Here, we define a crucial role for a ferlin-like protein (FLP) in host-to-vector transmission of the rodent malaria parasite Plasmodium berghei. Infection of the mosquito vectors requires the formation of free gametes and their fertilisation in the mosquito midgut. Mature gametes will only emerge upon secretion of factors that stimulate the disruption of the red blood cell membrane and the parasitophorous vacuole membrane. Genetic depletion of FLP in sexual stages leads to a complete life cycle arrest in the mosquito. Although mature gametes form normally, mutants lacking FLP remain trapped in the red blood cell. The egress defect is rescued by detergent-mediated membrane lysis. In agreement with ferlin vesicular localisation, HA-tagged FLP labels intracellular speckles, which relocalise to the cell periphery during gamete maturation. Our data define FLP as a novel critical factor for Plasmodium fertilisation and transmission and suggest an evolutionarily conserved example of ferlin-mediated exocytosis., (© 2018 John Wiley & Sons Ltd.)

Malaria remains a major public health problem worldwide, and it is responsible for high rates of morbidity and mortality. Resistance to current antimalarial drugs has been identified, and new drugs are urgently needed. In this study, we designed and synthesized seventeen novel quinolines based on the structures of mefloquine ((2,8-bis(trifluoromethyl)quinolin-4-yl)(piperidin-2-yl)methanol) and amodiaquine (4-((7-chloroquinolin-4-yl)amino)-2-((diethylamino)methyl)phenol) using ring bioisosteric replacement and molecular hybridization of the functional groups. The compounds were evaluated in vitro against Plasmodium falciparum and in vivo in mice infected with P. berghei. All derivatives presented anti-P. falciparum activity with IC 50 values ranging from 0.083 to 33.0 µM. The compound with the best anti-P. falciparum activity was N-(5-methyl-4H-1,2,4-triazol-3-yl)-2,8-bis(trifluoromethyl)quinolin-4-amine (12) which showed an IC 50 of 0.083 µM. The three most active compounds were selected for antimalarial activity tests against P. berghei-infected mice. Compound 12 was the most active on the 5th day after infection, reducing parasitemia by 66%, which is consistent with its in vitro activity. This is an important result as 12, a simpler molecule than mefloquine, does not contain the stereogenic center, and consequently, its synthesis in the laboratory is easier and less expensive. This system proved promising for the design of potential antimalarial compounds., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Plasmodium sp. are obligate intracellular parasites that derive most of their nutrients from their host meaning the metabolic circuitry of both are intricately linked. We employed untargeted, global mass spectrometry to identify metabolites present in the culture supernatants of P. falciparum-infected red blood cells synchronized at ring, trophozoite and schizont developmental stages. This revealed a temporal regulation in release of a distinct set of metabolites compared with supernatants of non-infected red blood cells. Of the distinct metabolites we identified pipecolic acid to be abundantly present in parasite lysate, infected red blood cells and infected culture supernatant. Further, we performed targeted metabolomics to quantify pipecolic acid concentrations in both the supernatants of red blood cells infected with P. falciparum, as well as in the plasma and infected RBCs of P. berghei-infected mice. Measurable and significant hyperpipecolatemia suggest that pipecolic acid has the potential to be a diagnostic marker for malaria.

Background: Medicinal plant research may contribute to develop new pharmacological control tools for vector borne diseases, such as malaria., Methods: The effects of methanol extracts (ME) obtained from seed kernel of ripe and unripe Azadirachta indica fruits were studied on erythrocytic proliferation of the rodent malaria parasite Plasmodium berghei strain ANKA and on mice pro-inflammatory response, as evaluated by measuring the matrix-metalloproteinase-9 (MMP-9) and tumour necrosis factor (TNF) plasma levels, in two mouse strains (C57BL/6 and BALB/c) which are considered as prototypical of Th1 and Th2 immune response, respectively., Results: ME obtained from seed kernel of unripe Azadirachta indica fruits decreased by about 30% the proportion of erythrocytes infected with the malaria parasite in C57BL/6 mice in the 4 days suppressive test. In this treatment group, MMP-9 and TNF levels were notably higher than those measured in the same mouse strain treated with the anti-malarial drug artesunate, Azadirachta indica kernel extracts from ripe fruits or solvent. In BALB/c mice, treatment with kernel extracts did not influence parasitaemia. MMP-9 and TNF levels measured in this mouse strain were notably lower than those recorded in C57BL/6 mice and did not vary among treatment groups., Conclusions: The effects of the ME on the parasite-host interactions appeared to be mouse strain-dependent, but also related to the ripening stage of the neem fruits, as only the unripe fruit seed kernel extracts displayed appreciable bioactivity.

During Plasmodium malaria parasite infection in a human, the intraerythrocytic stages lead to the clinical manifestations of the disease, especially hypoglycemia. Hypoglycemia is a recognized feature of severe malaria and linked with a high risk of mortality for children. Hence, the present study aimed to investigate the protective effect of T. crispa stem extract on hypoglycemia induced by P. berghei infection tested with a mouse model. ICR mice were inoculated with 1 × 10 7 parasitized erythrocytes of P. berghei ANKA (PbANKA) by intraperitoneal injection and given 50, 100, and 200 mg/kg of ethanolic extract for 4-consecutive days. The results showed that T. crispa stem extract exerted a protective effect (100%) on hypoglycemia induced by PbANKA infection at doses of 100 and 200 mg/kg. A significantly (p < .05) prolonged mean survival time (28.0 ± 1.9 days) of the extract treated mice was also observed. Additionally, no effect on blood glucose levels was seen in normal mice treated with all doses of extract. It can be concluded that T. crispa stem extract may have beneficial properties in protecting against hypoglycemia, and in increasing survival time during malaria infection., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Bioactive components of Ganoderma lucidum has recently gained intense research attention due to their acclaimed nutritional and medicinal properties. Thus, the terpenoid extract from the fruit bodies of G. lucidum (GT) was evaluated for activity against Plasmodium berghei in mice in two separate experiments. In addition, the effects of the extract on erythrocyte and hepatic lipids as well as liver HMG-CoA reductase activity before and after the treatments were also assessed. Mice with established infection were administered 100 and 250 mg/kg/day GT alone and in combination with chloroquine (CQ), in either case two separate controls designated: CQ (30 mg/kg chloroquine) and INF-CTR (1 mL DMSO) were also included. Treatment was administered orally for 12 days and parasitemia determined every three days. Percentage survival was significantly increased to 87% from 66% due to combination of GT100 with CQ compared to GT100 alone and to 75% from 62% when GT250 was administered with CQ compared to GT250 alone. Erythrocyte triglycerides, total cholesterol (TC), LDL and phospholipids contents were significantly lower in GT + CQ-treated mice compared to CQ alone and INF-CTR. Similarly, hepatic TC and phospholipid levels were significantly lower in the GT + CQ-treated mice compared to CQ alone and INF-CTR and HMG-CoA reductase activity in the liver was significantly inhibited due to administration of GT + CQ. Data from this study suggest that the anti-plasmodial action of GT could involve mechanisms associated with its hypolipidemic activity. It was also demonstrated that chloroquine, when administered in combination with GT, potentiates its curative effect in P. berghei-infected mice.

It is known that premature elimination of non-parasitized RBCs (nRBCs) plays an important role in the pathogenesis of malarial anemia, in which suicidal death process (eryptosis) of nRBCs has been suggested to be involved. To check this possibility, we investigate eryptosis during infection of P. berghei ANKA in Wistar rats, a malaria experimental model that, similar to human malaria, the infection courses with low parasitemia and acute anemia. As expected, P. berghei ANKA infection was marked by low parasite burdens that reached a mean peak of 3% between days six and nine post-infection and solved spontaneously. A significant reduction of the hemoglobin levels (~ 30%) was also observed on days subsequent to the peak of parasitemia, persisting until day 16 post-infection. In eryptosis assays, it was observed a significant increase in the levels of PS-exposing nRBC, which coincided with the reduction of hemoglobin levels and was positively related to anemia. In addition to PS externalization, eryptosis of nRBC induced by P. berghei infection was characterized by cytoplasm calcium influx, but not caspases activity. These results confirm our previous studies evidencing a pro-eryptotic effect of malaria infection on nRBCs and show that a caspase-independent eryptotic process is implicated in anemia induced by P. berghei ANKA infection in Wistar rats.

The rate at which Plasmodium falciparum is developing resistance to clinically used antimalarial drugs is alarming. Therefore, there is a compelling need to develop an efficient drug delivery system to improve the efficacy of existing antimalarial agents and circumvent drug resistance. Here, we report the antibacterial drug doxycycline (DOXY) in liposomal formulations exhibits enhanced antiplasmodial activity against blood stage forms of P. falciparum (3D7) in culture and established Plasmodium berghei NK-65 infection in murine model. Parasite killing on blood stage forms in culture was determined by a radiolabeled [ 3 H] hypoxanthine incorporation assay and infected erythrocytes stained with Giemsa were counted using microscopy in vivo. The 50% inhibitory concentration (IC 50 ) of DOXY-stearylamine liposome (IC 50 0.36 μM) and DOXY-SPC:Chol-liposome (IC 50 0.85 μM) exhibited marked growth inhibition of parasites compared with free DOXY (IC 50 14 μM), with minimal toxicity to normal erythrocytes. Administration of polyethylene glycol distearoyl phosphatidylethanolamine-methoxy-polyethylene glycol2000 (DSPE-mPEG-2000) coated liposomes loaded with DOXY at 2.5 mg/kg per day resulted in efficacious killing of blood parasites with improved survival in mice relative to the free drug in both chloroquine sensitive and resistant strains of P. berghei infection. This is the first report to demonstrate that DOXY in liposomal system has immense chemotherapeutic potential against plasmodial infections at lower dosages.

Plasmodium sporozoites deposited in the skin following a mosquito bite must migrate and invade blood vessels to complete their development in the liver. Once in the bloodstream, sporozoites arrest in the liver sinusoids, but the molecular determinants that mediate this specific homing are not yet genetically defined. Here we investigate the involvement of the thrombospondin-related sporozoite protein (TRSP) in this process using knockout Plasmodium berghei parasites and in vivo bioluminescence imaging in mice. Resorting to a homing assay, trsp knockout sporozoites were found to arrest in the liver similar to control parasites. Moreover, we found no defects in the establishment of infection in mice following inoculation of trsp knockout sporozoites via intravenous and cutaneous injection or mosquito bite. Accordingly, mutant sporozoites were also able to successfully invade hepatocytes in vitro. Altogether, these results suggest TRSP may have a redundant role in the completion of the pre-erythrocytic phase of the malaria parasite. Nonetheless, identifying molecules with paramount roles in this phase could aid in the search for new antigens needed for the design of a protective vaccine against malaria.

Many people are affected by Malaria around the world, and the parasite is developing resistance against available drugs. Currently, isoquine and N-tert-butyl isoquine are some of the most promising antimalarial candidates that have already reached Phase I and II clinical trials, respectively. Nevertheless, pharmacodynamic studies have demonstrated that isoquine is highly sensitive to form O-glucuronide metabolite, which may affect its accumulation in tissues. To avoid the O-glucuronide formation and its negative influence in the accumulation process, a series of novel five dehydroxy isoquine derivatives were designed and prepared herein as potential antimalarial agents. By a simple three-step procedure, five dehydroxy isoquines were prepared and subsequently examined on the inhibition of haemozoin formation, the main target of the 4-aminoquinolines. Four derivatives displayed significant inhibitory activities at low IC 50 values from 1.66 to 1.86 μM comparable to CQ. On the basis of the results, these four compounds were subsequently tested against Plasmodium berghei ANKA model in mice, showing to be as active as CQ with significant curative responses and parasitemia suppression in mice infected. On the other hand, these four compounds showed an acceptable non specific cytotoxicity on murine peritoneal macrophague and human erythrocyte cells. Thus, the presented data indicate that the dehydroxy isoquines 4b, 4c and 4e constitute promising cost-effective leads for the development of new antiplasmodial targeted at blood-stage malaria parasites., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Malaria parasites alternate between intracellular and extracellular stages and successful egress from the host cell is crucial for continuation of the life cycle. We investigated egress of Plasmodium berghei gametocytes, an essential process taking place within a few minutes after uptake of a blood meal by the mosquito. Egress entails the rupture of two membranes surrounding the parasite: the parasitophorous vacuole membrane (PVM), and the red blood cell membrane (RBCM). High-speed video microscopy of 56 events revealed that egress in both genders comprises four well-defined phases, although each event is slightly different. The first phase is swelling of the host cell, followed by rupture and immediate vesiculation of the PVM. These vesicles are extruded through a single stabilized pore of the RBCM, and the latter is subsequently vesiculated releasing the free gametes. The time from PVM vesiculation to completion of egress varies between events. These observations were supported by immunofluorescence microscopy using antibodies against proteins of the RBCM and PVM. The combined results reveal dynamic re-organization of the membranes and the cortical cytoskeleton of the erythrocyte during egress.

Plasmodium berghei (Pb) XAT, a rodent malaria parasite, is an irradiation-attenuated variant derived from the lethal strain Pb NK65. Differences in genome sequence, protein structure and function between Pb XAT and Pb NK65 are currently unknown. In this study, to investigate genetic alterations in Pb XAT, we performed comparative genomics and proteomics analyses of nonlethal and lethal strains of Pb. We found mutations, such as a deletion mutation in rhoptry-associated protein (rap) 1, and deletion of rap2/3 and skeleton-binding protein 1 (sbp1), in Pb XAT. RAP1 is required for targeting of RAP2 to the rhoptries. However, the contribution of RAP2/3 to the lethality of Plasmodium is unclear. Therefore, we generated RAP1- and RAP2/3-deficient mutants of Pb ANKA, a reference strain of P. berghei. Furthermore, we investigated the effect of RAP1 and RAP2/3 deficiency on the outcome of infection. The parasitemia in mice infected with RAP1-deficient parasites was increased compared to that in control parasite-infected mice during the early phase of infection. However, mice infected with RAP1-deficient parasites survived longer than did control parasite-infected mice. Moreover, mice infected with RAP2/3-deficient parasites showed low levels of parasitemia and ultimately recovered from the infection The aim of this study was to investigate the effect of RAP2/3 expression on the outcome of infection with Pb XAT using a RAP2/3-expressing Pb XAT. Results showed that complementation of RAP2/3 expression in Pb XAT partially restored virulence. Our findings suggest that RAP1 and RAP2/3 contribute to virulence and a decrease in their expression explains the loss of virulence of the Pb XAT strain., (Copyright © 2018 Elsevier Inc. All rights reserved.)