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Our previous work demonstrated that basophils regulate a suite of malaria phenotypes, including intestinal mastocytosis and permeability, the immune response to infection, gametocytemia, and parasite transmission to the malaria mosquito Anopheles stephensi. Given that activated basophils are primary sources of the regulatory cytokines IL-4 and IL-13, we sought to examine the contributions of these mediators to basophil-dependent phenotypes in malaria. We generated mice with basophils depleted for IL-4 and IL-13 (baso IL-4/IL-13 (-)) and genotype controls (baso IL-4/IL-13 (+)) by crossing mcpt8-Cre and Il4/Il13fl/fl mice and infected them with Plasmodium yoelii yoelii 17XNL. Conditional deletion was associated with ileal mastocytosis and mast cell (MC) activation, increased intestinal permeability, and increased bacterial 16S levels in blood, but it had no effect on neutrophil activation, parasitemia, or transmission to A. stephensi. Increased intestinal permeability in baso IL-4/IL-13 (-) mice was correlated with elevated plasma eotaxin (CCL11), a potent eosinophil chemoattractant, and increased ileal MCs, proinflammatory IL-17A, and the chemokines MIP-1α (CCL3) and MIP-1β (CCL4). Blood bacterial 16S copies were positively but weakly correlated with plasma proinflammatory cytokines IFN-γ and IL-12p40, suggesting that baso IL-4/IL-13 (-) mice failed to control bacterial translocation into the blood during malaria infection. These observations suggest that basophil-derived IL-4 and IL-13 do not contribute to basophil-dependent regulation of parasite transmission, but these cytokines do orchestrate protection of intestinal barrier integrity after P. yoelii infection. Specifically, basophil-dependent IL-4/IL-13 control MC activation and prevent infection-induced intestinal barrier damage and bacteremia, perhaps via regulation of eosinophils, macrophages, and Th17-mediated inflammation.

Background: The study of rodent malaria parasites has significantly advanced our understanding of malaria parasite biology and host responses to parasite infections. There are four well-characterized rodent malaria parasite species (Plasmodium yoelii, P. chabaudi, P. berghei, and P. vinckei). Each species also has multiple strains that cause different disease phenotypes. P. yoelii nigeriensis N67C and N67, two isogenic parasites, are particularly intriguing as they differ in virulence and incite different immune responses in mice. The genome of the N67 parasite has been assembled recently, but not that of N67C. This study used PacBio HiFi sequencing data to assemble the N67C genome, compared the two genomes, and performed RNA sequencing to identify polymorphisms and differentially expressed genes (DEGs). Results: The assembled N67C parasite genome consisted of 16 scaffolds and three contigs of approximately 22.5 Mb with 100% and 96.6% completeness based on well-characterized single-copy orthologs specific to the Apicomplexa phylum and the Plasmodium genus, respectively. A comparison between the annotated N67C and N67 genomes revealed 133 single nucleotide polymorphisms (SNPs) and 75 indels. Among the polymorphic sites, an S (N67) to N (N67C) amino acid substitution at position 114 (S114N) in the dihydrofolate reductase-thymidylate synthase (DHFR-TS) confers resistance to pyrimethamine in mice. Additionally, 60 differentially expressed single-copy genes (DEGs) were detected after comparing mRNA levels between the two parasites. Starting with the predicted and annotated 5,681 N67C and 5,749 N67 genes, we identified 4,641 orthogroups that included at least one gene from the four P. yoelii parasites (N67, N67C, 17X, and YM), whereas 758 orthogroups showed subspecies or strain-specific patterns. Conclusion: The identification of polymorphic sites between the N67 and N67C genomes, along with the detection of the DEGs, may provide crucial insights into the variations in parasite drug responses and disease severity between these two isogenic parasites. The functional characterization of these genetic differences and candidate genes will deepen our understanding of disease mechanisms and pave the way for developing more effective control measures against malaria. [ABSTRACT FROM AUTHOR]

Malaria, caused by Plasmodium parasites, imposes a significant health burden and live-attenuated parasites are being pursued as vaccines. Here, we report on the creation of a genetically attenuated parasite by the deletion of Plasmodium LINUP, encoding a liver stage nuclear protein. In the rodent parasite Plasmodium yoelii, LINUP expression was restricted to liver stage nuclei after the onset of liver stage schizogony. Compared to wildtype P. yoelii, P. yoelii LINUP gene deletion parasites (linup—) exhibited no phenotype in blood stages and mosquito stages but suffered developmental arrest late in liver stage schizogony with a pronounced defect in exo-erythrocytic merozoite formation. This defect caused severe attenuation of the liver stage-to-blood stage transition and immunization of mice with linup— parasites conferred robust protection against infectious sporozoite challenge. LINUP gene deletion in the human parasite Plasmodium falciparum also caused a severe defect in late liver stage differentiation. Importantly, P. falciparum linup— liver stages completely failed to transition from the liver stage to a viable blood stage infection in a humanized mouse model. These results suggest that P. falciparum LINUP is an ideal target for late liver stage attenuation that can be incorporated into a late liver stage-arresting replication competent whole parasite vaccine. A conserved Plasmodium protein, specific to the liver stage and localized in the nucleus of liver stage schizonts, plays a critical role in liver stage development and differentiation. [ABSTRACT FROM AUTHOR]

The cortical cytoskeleton of subpellicular microtubules (SPMTs) supports the Plasmodium ookinete morphogenesis during mosquito transmission of malaria. SPMTs are hypothesized to function as the cytoskeletal tracks in motor-driven cargo transport for apical organelle and structure assembly in ookinetes. However, the SPMT-based transport motor has not been identified in the Plasmodium. The cytoplasmic dynein is the motor moving towards the minus end of microtubules (MTs) and likely be responsible for cargo transport to the apical part in ookinetes. Here we screen 7 putative dynein heavy chain (DHC) proteins in the P. yoelii and identify DHC3 showing peripheral localization in ookinetes. DHC3 is localized at SPMTs throughout ookinete morphogenesis. We also identify five other dynein subunits localizing at SPMTs. DHC3 disruption impairs ookinete development, shape, and gliding, leading to failure in mosquito infection of Plasmodium. The DHC3-deficient ookinetes display defective formation or localization of apical organelles and structures. Rab11A and Rab11B interact with DHC3 at SPMTs in a DHC3-dependent manner, likely functioning as the receptors for the cargoes driven by SPMT-dynein. Disturbing Rab11A or Rab11B phenocopies DHC3 deficiency in ookinete morphogenesis. Our study reveals an SPMT-based dynein motor driving the transport of Rab11A- and Rab11B-labeled cargoes in the ookinete morphogenesis of Plasmodium. Liu et al. identify a subpellicular microtubule-based dynein motor in ookinetes of Plasmodium. This motor transports the cargos for apical organelle and structure assembly in ookinetes. Dynein-deficient parasites fail ookinete morphogenesis and mosquito transmission of Plasmodium. [ABSTRACT FROM AUTHOR]

Chronic bone loss is an under-recognized complication of malaria, the underlying mechanism of which remains incompletely understood. We have previously shown that persistent accumulation of Plasmodium products in the bone marrow leads to chronic inflammation in osteoblast (OB) and osteoclast (OC) precursors causing bone loss through MyD88, an adaptor molecule for diverse inflammatory signals. However, the specific contribution of MyD88 signaling in OB or OC precursors in malaria-induced bone loss remains elusive. To assess the direct cell-intrinsic role of MyD88 signaling in adult bone metabolism under physiological and infection conditions, we used the Lox-Cre system to specifically deplete MyD88 in the OB or OC lineages. Mice lacking MyD88 primarily in the maturing OBs showed a comparable decrease in trabecular bone density by microcomputed tomography to that of controls after Plasmodium yoelii non-lethal infection. In contrast, mice lacking MyD88 in OC precursors showed significantly less trabecular bone loss than controls, suggesting that malaria-mediated inflammatory mediators are primarily controlled by MyD88 in the OC lineage. Surprisingly, however, depletion of MyD88 in OB, but not in OC, precursors resulted in reduced bone mass with decreased bone formation rates in the trabecular areas of femurs under physiological conditions. Notably, insulin-like growth factor-1, a key molecule for OB differentiation, was significantly lower locally and systemically when MyD88 was depleted in OBs. Thus, our data demonstrate an indispensable intrinsic role for MyD88 signaling in OB differentiation and bone formation, while MyD88 signaling in OC lineages plays a partial role in controlling malaria-induced inflammatory mediators and following bone pathology. These findings may lead to the identification of novel targets for specific intervention of bone pathologies, particularly in malaria-endemic regions. [ABSTRACT FROM AUTHOR]

Abstract Background Malaria is a serious public health concern. Artemisinin and its derivatives are first-line drugs for the treatment of Plasmodium falciparum malaria. In mammals, artemisinin exhibits potent anti-inflammatory and immunoregulatory properties. However, it is unclear whether artemisinin plays a regulatory role in the innate immunity of mosquitoes, thereby affecting the development of Plasmodium in Anopheles when artemisinin and its metabolites enter mosquitoes. This study aims to determine the effect of dihydroartemisinin (DHA), a first-generation semisynthetic derivative of artemisinin, on innate immunity and malaria vector competence of Anopheles stephensi. Methods Anopheles stephensi was fed Plasmodium-infected mice treated with DHA via gavage, Plasmodium-infected blood containing DHA in vitro, or DHA-containing sugar, followed by Plasmodium yoelii infection. The engorged female mosquitoes were separated and dissected 8 and 17 days after infection. Plasmodium oocysts and sporozoites were counted and compared between the control and DHA-treated groups. Additionally, total RNA and proteins were extracted from engorged mosquitoes 24 and 72 h post infection (hpi). Real-time polymerase chain reaction (PCR) and western blotting were performed to detect the transcriptional levels and protein expression of immune molecules in mosquitoes. Finally, the Toll signaling pathway was inhibited via RNA interference and the infection density was analyzed to confirm the role of the Toll signaling pathway in the effect of DHA on the vector competence of mosquitoes. Results DHA treatment via different approaches significantly reduced the number of Plasmodium oocysts and sporozoites in mosquitoes. The transcriptional levels of anti-Plasmodium immune genes (including TEP1, LRIM1, and APL1C), Toll pathway genes (including Tube, MyD88, and Rel1), and the effector defensin 1 were upregulated by DHA treatment at 24 and 72 hpi. TEP1 and Rel1 protein expression was significantly induced under DHA treatment. However, Rel1 knockdown in DHA-treated mosquitoes abrogated DHA-mediated refractoriness to Plasmodium infection. Conclusions DHA treatment effectively inhibited the development of P. yoelii in A. stephensi by upregulating mosquitoes’ Toll signaling pathway, thereby influencing the susceptibility of Anopheles to Plasmodium. Graphical Abstract

Olfactory receptors (Olfr) are G protein-coupled receptors that are normally expressed on olfactory sensory neurons to detect volatile chemicals or odorants. Interestingly, many Olfrs are also expressed in diverse tissues and function in cell-cell recognition, migration, and proliferation as well as immune responses and disease processes. Here, we showed that many Olfr genes were expressed in the mouse spleen, linked to Plasmodium yoelii genetic loci significantly, and/or had genome-wide patterns of LOD scores (GPLSs) similar to those of host Toll-like receptor genes. Expression of specific Olfr genes such as Olfr1386 in HEK293T cells significantly increased luciferase signals driven by IFN-β and NF-κB promoters, with elevated levels of phosphorylated TBK1, IRF3, P38, and JNK. Mice without Olfr1386 were generated using the CRISPR/Cas9 method, and the Olfr1386-/- mice showed significantly lower IFN-α/β levels and longer survival than wild-type (WT) littermates after infection with P. yoelii YM parasites. Inhibition of G protein signaling and P38 activity could affect cyclic AMP-responsive element promoter-driven luciferase signals and IFN-β mRNA levels in HEK293T cells expressing the Olfr1386 gene, respectively. Screening of malaria parasite metabolites identified nicotinamide adenine dinucleotide (NAD) as a potential ligand for Olfr1386, and NAD could stimulate IFN-β responses and phosphorylation of TBK1 and STAT1/2 in RAW264.7 cells. Additionally, parasite RNA (pRNA) could significantly increase Olfr1386 mRNA levels. This study links multiple Olfrs to host immune response pathways, identifies a candidate ligand for Olfr1386, and demonstrates the important roles of Olfr1386 in regulating type I interferon (IFN-I) responses during malaria parasite infections. [ABSTRACT FROM AUTHOR]

Plasmodium sporozoites are the infective forms of the malaria parasite in the mosquito and vertebrate host. Gliding motility allows sporozoites to migrate and invade mosquito salivary glands and mammalian hosts. Motility and invasion are powered by an actin-myosin motor complex linked to the glideosome, which contains glideosome-associated proteins (GAPs), MyoA and the myosin A tailinteracting protein (MTIP). However, the role of several proteins involved in gliding motility remains unknown. We identified that the S14 gene is upregulated in sporozoite from transcriptome data of Plasmodium yoelii and further confirmed its transcription in P. berghei sporozoites using real-time PCR. C-terminal 3×HA-mCherry tagging revealed that S14 is expressed and localized on the inner membrane complex of the sporozoites. We disrupted S14 in P. berghei and demonstrated that it is essential for sporozoite gliding motility, and salivary gland and hepatocyte invasion. The gliding and invasiondeficient S14 knockout sporozoites showed normal expression and organization of inner membrane complex and surface proteins. Taken together, our data show that S14 plays a role in the function of the glideosome and is essential for malaria transmission. [ABSTRACT FROM AUTHOR]

Abstract Background Eukaryotic genes contain introns that are removed by the spliceosomal machinery during mRNA maturation. Introns impose a huge energetic burden on a cell; therefore, they must play an essential role in maintaining genome stability and/or regulating gene expression. Many genes (> 50%) in Plasmodium parasites contain predicted introns, including introns in 5′ and 3′ untranslated regions (UTR). However, the roles of UTR introns in the gene expression of malaria parasites remain unknown. Methods In this study, an episomal dual-luciferase assay was developed to evaluate gene expression driven by promoters with or without a 5′UTR intron from four Plasmodium yoelii genes. To investigate the effect of the 5′UTR intron on endogenous gene expression, the pytctp gene was tagged with 3xHA at the N-terminal of the coding region, and parasites with or without the 5′UTR intron were generated using the CRISPR/Cas9 system. Results We showed that promoters with 5′UTR introns had higher activities in driving gene expression than those without 5′UTR introns. The results were confirmed in recombinant parasites expressing an HA-tagged gene (pytctp) driven by promoter with or without 5′UTR intron. The enhancement of gene expression was intron size dependent, but not the DNA sequence, e.g. the longer the intron, the higher levels of expression. Similar results were observed when a promoter from one strain of P. yoelii was introduced into different parasite strains. Finally, the 5′UTR introns were alternatively spliced in different parasite development stages, suggesting an active mechanism employed by the parasites to regulate gene expression in various developmental stages. Conclusions Plasmodium 5′UTR introns enhance gene expression in a size-dependent manner; the presence of alternatively spliced mRNAs in different parasite developmental stages suggests that alternative slicing of 5′UTR introns is one of the key mechanisms in regulating parasite gene expression and differentiation. Graphical Abstract

Background: Eukaryotic genes contain introns that are removed by the spliceosomal machinery during mRNA maturation. Introns impose a huge energetic burden on a cell; therefore, they must play an essential role in maintaining genome stability and/or regulating gene expression. Many genes (> 50%) in Plasmodium parasites contain predicted introns, including introns in 5′ and 3′ untranslated regions (UTR). However, the roles of UTR introns in the gene expression of malaria parasites remain unknown. Methods: In this study, an episomal dual-luciferase assay was developed to evaluate gene expression driven by promoters with or without a 5′UTR intron from four Plasmodium yoelii genes. To investigate the effect of the 5′UTR intron on endogenous gene expression, the pytctp gene was tagged with 3xHA at the N-terminal of the coding region, and parasites with or without the 5′UTR intron were generated using the CRISPR/Cas9 system. Results: We showed that promoters with 5′UTR introns had higher activities in driving gene expression than those without 5′UTR introns. The results were confirmed in recombinant parasites expressing an HA-tagged gene (pytctp) driven by promoter with or without 5′UTR intron. The enhancement of gene expression was intron size dependent, but not the DNA sequence, e.g. the longer the intron, the higher levels of expression. Similar results were observed when a promoter from one strain of P. yoelii was introduced into different parasite strains. Finally, the 5′UTR introns were alternatively spliced in different parasite development stages, suggesting an active mechanism employed by the parasites to regulate gene expression in various developmental stages. Conclusions: Plasmodium 5′UTR introns enhance gene expression in a size-dependent manner; the presence of alternatively spliced mRNAs in different parasite developmental stages suggests that alternative slicing of 5′UTR introns is one of the key mechanisms in regulating parasite gene expression and differentiation. [ABSTRACT FROM AUTHOR]

Global prevalence of hypertension is on the rise, burdening healthcare, especially in developing countries where infectious diseases, such as malaria, are also rampant. Whether hypertension could predispose or increase susceptibility to malaria, however, has not been extensively explored. Previously, we reported that hypertension is associated with abnormal red blood cell (RBC) physiology and anemia. Since RBC are target host cells for malarial parasite, Plasmodium, we hypothesized that hypertensive patients with abnormal RBC physiology are at greater risk or susceptibility to Plasmodium infection. To test this hypothesis, normotensive (BPN/3J) and hypertensive (BPH/2J) mice were characterized for their RBC physiology and subsequently infected with Plasmodium yoelii (P. yoelii), a murine-specific non-lethal strain. When compared to BPN mice, BPH mice displayed microcytic anemia with RBC highly resistant to osmotic hemolysis. Further, BPH RBC exhibited greater membrane rigidity and an altered lipid composition, as evidenced by higher levels of phospholipids and saturated fatty acid, such as stearate (C18:0), along with lower levels of polyunsaturated fatty acid like arachidonate (C20:4). Moreover, BPH mice had significantly greater circulating Ter119+ CD71+ reticulocytes, or immature RBC, prone to P. yoelii infection. Upon infection with P. yoelii, BPH mice experienced significant body weight loss accompanied by sustained parasitemia, indices of anemia, and substantial increase in systemic pro-inflammatory mediators, compared to BPN mice, indicating that BPH mice were incompetent to clear P. yoelii infection. Collectively, these data demonstrate that aberrant RBC physiology observed in hypertensive BPH mice contributes to an increased susceptibility to P. yoelii infection and malaria-associated pathology. Graphical Abstract [ABSTRACT FROM AUTHOR]

Toll‐like receptors (TLRs) play an important role in inducing innate and acquired immune responses against infection. However, the effect of Toll‐like receptor 7 (TLR7) on follicular helper T (Tfh) cells in mice infected with Plasmodium is still not clear. The results showed that the splenic CD4+CXCR5+PD‐1+ Tfh cells were accumulated after Plasmodium yoelii NSM infection, the content of splenic Tfh cells was correlated to parasitemia and/or the red blood cells (RBCs) counts in the blood. Moreover, the expression of TLR7 was found higher than TLR2, TLR3 and TLR4 in splenic Tfh cells of the WT mice. TLR7 agonist R848 and the lysate of red blood cells of infected mice (iRBCs) could induce the activation and differentiation of splenic Tfh cells. Knockout of TLR7 leads to a decrease in the proportion of Tfh cells, down‐regulated expression of functional molecules CD40L, IFN‐γ, IL‐21 and IL‐10 in Tfh cells; decreased the proportion of plasma cells and antibody production and reduces the expression of STAT3 and Ikzf2 in Tfh cells. Administration of R848 could inhibit parasitemia, enhance splenic Tfh cell activation and increase STAT3 and Ikzf2 expression in Tfh cells. In summary, this study shows that TLR7 could regulate the function of Tfh cells, affecting the immune response in the spleen of Plasmodium yoelii NSM‐infected mice. [ABSTRACT FROM AUTHOR]

The persistent prevalence and dissemination of drug-resistant malaria parasites continue to challenge the progress of malaria eradication efforts. As a result, there is an urgent need to search for and develop innovative therapies. In this study, we screened synthetic 2,5-diphenyloxazole analogs from Oxytropis lanata. Among 48 compounds, 14 potently inhibited the proliferation of P. falciparum strains 3D7 (chloroquine-sensitive) and K1 (multidrug-resistant) in vitro, exhibited IC50 values from 3.38 to 12.65 μM and 1.27–6.19 μM, respectively, and were toxic to human foreskin fibroblasts at 39.53–336.35 μM. Notably, Compounds 31 (2-(2′,3′-dimethoxyphenyl)-5-(2″-hydroxyphenyl)oxazole) and 32 (2-(2′,3′-dimethoxyphenyl)-5-(2″-benzyloxyphenyl)oxazole) exhibited the highest selectivity indices (SIs) against both P. falciparum strains (3D7/K1), with values > 40.20/>126.58 and > 41.27/> 59.06, respectively. In the IC50 speed and stage-specific assays, Compounds 31 and 32 showed slow action, along with distinct effects on the ring and trophozoite stages. Microscopy observations further revealed that both compounds impact the development and delay the progression of the trophozoite and schizont stages in P. falciparum 3D7, especially at concentrations 100 times their IC50 values. In a 72-h in vitro exposure experiment at their respective IC80 in P. falciparum 3D7, significant alterations in parasitemia levels were observed compared to the untreated group. In Compound 31-treated cultures, parasites shrank and were unable to reinvade red blood cells (RBCs) during an extended 144-h incubation period, even after compound removal from the culture. In vivo assessments were conducted on P. yoelii 17XNL-infected mice treated with Compounds 31 and 32 at 20 mg/kg administered once daily for ten days. The treated groups showed statistically significant lower peaks of parasitemia (Compound 31-treated: trial 1 12.7%, trial 2 15.8%; Compound 32-treated: trial 1 12.7%, trial 2 14.0%) compared to the untreated group (trial 1 21.7%, trial 2 28.3%). These results emphasize the potential of further developing 2,5-diphenyloxazoles as promising antimalarial agents.

Mutations in a Plasmodium de-ubiquitinase UBP1 have been linked to antimalarial drug resistance. However, the UBP1-mediated drug-resistant mechanism remains unknown. Through drug selection, genetic mapping, allelic exchange, and functional characterization, here we show that simultaneous mutations of two amino acids (I1560N and P2874T) in the Plasmodium yoelii UBP1 can mediate high-level resistance to mefloquine, lumefantrine, and piperaquine. Mechanistically, the double mutations are shown to impair UBP1 cytoplasmic aggregation and de-ubiquitinating activity, leading to increased ubiquitination levels and altered protein localization, from the parasite digestive vacuole to the plasma membrane, of the P. yoelii multidrug resistance transporter 1 (MDR1). The MDR1 on the plasma membrane enhances the efflux of substrates/drugs out of the parasite cytoplasm to confer multidrug resistance, which can be reversed by inhibition of MDR1 transport. This study reveals a previously unknown drug-resistant mechanism mediated by UBP1 through altered MDR1 localization and substrate transport direction in a mouse model, providing a new malaria treatment strategy. Here, the authors show that two mutations in the Plasmodium de-ubiquitinase UBP1 alter the ubiquitination level, membrane localization, and ligand transport direction of multidrug resistance transporter 1 (MDR1), leading to multiple drug resistances. [ABSTRACT FROM AUTHOR]

Purpose: To investigate the role of nitroquine (CI-679) against artemisinin-resistant Plasmodium falciparum (P. falciparum) C580Y strain. Methods: Antimalarial activity of CI-679 against blood stages in Plasmodium yoelii (P. yoelii) - infected BALB/c mice model was first identified. Thereafter, in vitro assays were performed to investigate the inhibitory activity against blood stages of artemisinin-sensitive P. falciparum 3D7 strain. Finally, the potential effect of CI-679 was also investigated on artemisinin-resistant P. falciparum, which was constructed by introducing C580Y mutation in K13 of the 3D7 using the CRISPR-CAS9 technology. Results: CI-679 significantly suppressed the growth of rodent malaria parasite, P. yoelii BY265, in a dose-dependent manner, and also inhibited the development of the parasites in mice (p < 0.05). Furthermore, CI-679 efficiently inhibited the growth of artemisinin-sensitive P. falciparum 3D7 in vitro, with more sensitivity against late phase of blood stages (p < 0.05). Also, CI-679 suppressed the development of artemisinin-resistant P. falciparum C580Y strain, and the inhibitory effect was comparable to that of artemisinin-sensitive 3D7 strain. Conclusion: CI-679 exhibits potent antimalarial activity against blood stages of P. yoelii BY265 in vivo, and both artemisinin-sensitive P. falciparum 3D7 and artemisinin-resistant P. falciparum C580Y in vitro. Further pharmacokinetic properties, tolerability and safety of the compound need to be investigated to support this claim. [ABSTRACT FROM AUTHOR]

Background The long-acting antimalarial drug piperaquine can be metabolized into the carboxylic acid metabolite (PQM). However, the clinical relevance of PQM remains unclear. Objectives The pharmacodynamics/pharmacokinetics of PQM were studied. Methods The antimalarial activity of PQM was studied in vitro (Plasmodium strains Pf 3D7 and Pf Dd2) and in vivo (murine Plasmodium yoelii). The toxicity of PQM was evaluated in mice, in terms of the general measures of animal well-being, serum biochemical examination and ECG monitoring. The pharmacokinetic profiles of piperaquine and its metabolite PQM were investigated in healthy subjects after recommended oral doses of piperaquine. Results PQM showed no relevant in vitro antimalarial activity (IC50 > 1.0 μM) with no significant toxicity. After recommended oral administration of piperaquine to healthy subjects, the maximum concentration of PQM was less than 30.0 nM, and it did not accumulate after repeated dosing. Conclusions With a low antimalarial potency, PQM should not contribute to the efficacy of piperaquine with clinically acceptable doses. [ABSTRACT FROM AUTHOR]

Background: The rapid reproduction of malaria parasites requires proper iron uptake. However, the process of iron absorption by parasites is rarely studied. Divalent metal transporter (DMT1) is a critical iron transporter responsible for uptaking iron. A homolog of human DMT1 exists in the malaria parasite genome, which in Plasmodium yoelii is hereafter named PyDMT1. Results: PyDMT1 knockout appears to be lethal. Surprisingly, despite dwelling in an iron-rich environment, the parasite cannot afford to lose even partial expression of PyDMT1; PyDMT1 hypomorphs were associated with severe growth defects and quick loss of pathogenicity. Iron supplementation could completely suppress the defect of the PyDMT1 hypomorph during in vitro culturing. Genetic manipulation through host ferritin (Fth1) knockout to increase intracellular iron levels enforced significant growth inhibition in vivo on the normal parasites but not the mutant. In vitro culturing with isolated ferritin knockout mouse erythrocytes completely rescued PyDMT1-hypomorph parasites. Conclusion: A critical iron requirement of malaria parasites at the blood stage as mediated by this newly identified iron importer PyDMT1, and the iron homeostasis in malarial parasites is finely tuned. Tipping the iron balance between the parasite and host will efficiently kill the pathogenicity of the parasite. Lastly, PyDMT1 hypomorph parasites were less sensitive to the action of artemisinin. [ABSTRACT FROM AUTHOR]

• Malaria infection leads to the development of thymic atrophy. • Thymic atrophy does not depend on parasitemia level but on the specific host-parasite interaction. • Thymus atrophy seems to be associated with a corticosterone-induced effect and had no association with elevated serum cytokines levels. • The severity of thymic damage in both malaria models reached the same extent, although it occurs at different stages of infection. The thymus is the anatomical site where T cells undergo a complex process of differentiation, proliferation, selection, and elimination of autorreactive cells which involves molecular signals in different intrathymic environment. However, the immunological functions of the thymus can be compromised upon exposure to different infections, affecting thymocyte populations. In this work, we investigated the impact of malaria parasites on the thymus by using C57BL/6 mice infected with Plasmodium berghei ANKA and Plasmodium yoelii 17XL; these lethal infection models represent the most severe complications, cerebral malaria, and anemia respectively. Data showed a reduction in the thymic weight and cellularity involving different T cell maturation stages, mainly CD4−CD8− and CD4+CD8+ thymocytes, as well as an increased presence of apoptotic cells, leading to significant thymic cortex reduction. Thymus atrophy showed no association with elevated serum cytokines levels, although increased glucocorticoid levels did. The severity of thymic damage in both models reached the same extend although it occurs at different stages of infection, showing that thymic atrophy does not depend on parasitemia level but on the specific host-parasite interaction. [ABSTRACT FROM AUTHOR]

An increase in mast cells (MCs) and MCs mediators has been observed in malaria-associated bacteremia, however, the role of these granulocytes in malarial immunity is poorly understood. Herein, we studied the role of mouse MC protease (Mcpt) 4, an ortholog of human MC chymase, in malaria-induced bacteremia using Mcpt4 knockout (Mcpt4-/-) mice and Mcpt4+/+ C57BL/6J controls, and the non-lethal mouse parasite Plasmodium yoelii yoelii 17XNL. Significantly lower parasitemia was observed in Mcpt4-/- mice compared with Mcpt4+/+ controls by day 10 post infection (PI). Although bacterial 16S DNA levels in blood were not different between groups, increased intestinal permeability to FITC-dextran and altered ileal adherens junction E-cadherin were observed in Mcpt4-/- mice. Relative to infected Mcpt4+/+ mice, ileal MC accumulation in Mcpt4-/- mice occurred two days earlier and IgE levels were higher by days 8-10 PI. Increased levels of circulating myeloperoxidase were observed at 6 and 10 days PI in Mcpt4+/+ but not Mcpt4-/- mice, affirming a role for neutrophil activation that was not predictive of parasitemia or bacterial 16S copies in blood. In contrast, early increased plasma levels of TNF-α, IL-12p40 and IL-3 were observed in Mcpt4-/- mice, while levels of IL-2, IL-10 and MIP1β (CCL4) were increased over the same period in Mcpt4+/+ mice, suggesting that the host response to infection was skewed toward a type-1 immune response in Mcpt4-/- mice and type-2 response in Mcpt4+/+ mice. Spearman analysis revealed an early (day 4 PI) correlation of Mcpt4-/- parasitemia with TNF-α and IFN-γ, inflammatory cytokines known for their roles in pathogen clearance, a pattern that was observed in Mcpt4+/+ mice much later (day 10 PI). Transmission success of P. y. yoelii 17XNL to Anopheles stephensi was significantly higher from infected Mcpt4-/- mice compared with infected Mcpt4+/+ mice, suggesting that Mcpt4 also impacts transmissibility of sexual stage parasites. Together, these results suggest that early MCs activation and release of Mcpt4 suppresses the host immune response to P. y. yoelii 17XNL, perhaps via degradation of TNF-α and promotion of a type-2 immune response that concordantly protects epithelial barrier integrity, while limiting the systemic response to bacteremia and parasite transmissibility.

Malaria poses a serious threat to human health. Existing vector-based interventions have shortcomings, such as environmental pollution, strong resistance to chemical insecticides, and the slow effects of biological insecticides. Therefore, the need to develop novel strategies for controlling malaria, such as reducing mosquito vector competence, is escalating. Human defensin 5 (HD5) has broad-spectrum antimicrobial activity. To determine its effect on Plasmodium development in mosquitoes, HD5 was injected into Anopheles stephensi at various time points. The infection density of Plasmodium yoelii in An. stephensi was substantially reduced by HD5 treatment administered 24 h prior to infection or 6, 12, or 24 h post-infection (hpi). We found that HD5 treatment upregulated the expression of the innate immune effectors TEP1, MyD88, and Rel1 at 24 and 72 hpi. Furthermore, the RNA interference of MyD88, a key upstream molecule in the Toll signaling pathway, decreased the HD5-induced resistance of mosquitoes against Plasmodium infection. These results suggest that HD5 microinjection inhibits the development of malaria parasites in An. stephensi by activating the Toll signaling pathway.

Vaccination strategies in mice inducing high numbers of memory CD8+ T cells specific to a single epitope are able to provide sterilizing protection against infection with Plasmodium sporozoites. We have recently found that Plasmodium-specific CD8+ T cells cluster around sporozoite-infected hepatocytes but whether such clusters are important in elimination of the parasite remains incompletely understood. Here, we used our previously generated data in which we employed intravital microscopy to longitudinally image 32 green fluorescent protein (GFP)-expressing Plasmodium yoelii parasites in livers of mice that had received activated Plasmodium-specific CD8+ T cells after sporozoite infection. We found significant heterogeneity in the dynamics of the normalized GFP signal from the parasites (termed 'vitality index' or VI) that was weakly correlated with the number of T cells near the parasite. We also found that a simple model assuming mass-action, additive killing by T cells well describes the VI dynamics for most parasites and predicts a highly variable killing efficacy by individual T cells. Given our estimated median per capita kill rate of k = 0.031/h we predict that a single T cell is typically incapable of killing a parasite within the 48 h lifespan of the liver stage in mice. Stochastic simulations of T cell clustering and killing of the liver stage also suggested that: (i) three or more T cells per infected hepatocyte are required to ensure sterilizing protection; (ii) both variability in killing efficacy of individual T cells and resistance to killing by individual parasites may contribute to the observed variability in VI decline, and (iii) the stable VI of some clustered parasites cannot be explained by measurement noise. Taken together, our analysis for the first time provides estimates of efficiency at which individual CD8+ T cells eliminate intracellular parasitic infection in vivo. [ABSTRACT FROM AUTHOR]

CD103 is an important marker of tissue-resident memory T cells (TRM) which play important roles in fighting against infection. However, the immunological characteristics of CD103+ T cells are not thoroughly elucidated in the liver of mouse infected with Plasmodium. Six- to eight-week-old C57BL/6 mice were infected with Plasmodium yoelii nigeriensis NSM. Mice were sacrificed on 12–16 days after infection and the livers were picked out. Sections of the livers were stained, and serum aspartate aminotransferase (AST) and alanine transaminase (ALT) levels were measured. Moreover, lymphocytes in the liver were isolated, and the expression of CD103 was determined by using qPCR. The percentage of CD103 on different immune cell populations was dynamically observed by using flow cytometry (FCM). In addition, the phenotype and cytokine production characteristics of CD103+CD8+ Tc cell were analyzed by using flow cytometry, respectively. Erythrocyte stage plasmodium infection could result in severe hepatic damage, a widespread inflammatory response and the decrease of CD103 expression on hepatic immune cells. Only CD8+ Tc and γδT cells expressed higher levels of CD103 in the uninfected state.CD103 expression in CD8+ Tc cells significantly decreased after infection. Compared to that of CD103− CD8+ Tc cells, CD103+ CD8+ Tc cells from the infected mice expressed lower level of CD69, higher level of CD62L, and secreted more IL-4, IL-10, IL-17, and secreted less IFN-γ. CD103+CD8+ Tc cells might mediate the hepatic immune response by secreting IL-4, IL-10, and IL-17 except IFN-γ in the mice infected with the erythrocytic phase plasmodium, which could be involved in the pathogenesis of severe liver damage resulted from the erythrocytic phase plasmodium yoelii nigeriensis NSM infection. [ABSTRACT FROM AUTHOR]

Plasmodium vivax 's biological complexity has restricted in vitro culture development for characterising antigens involved in erythrocyte invasion and their immunological relevance. The murine model is proposed as a suitable alternative in the search for therapeutic candidates since Plasmodium yoelii uses homologous proteins for its invasion. The AMA-1 protein is essential for parasite invasion of erythrocytes as it is considered an important target for infection control. This study has focused on functional Py AMA-1 peptides involved in host-pathogen interaction; the protein is located in regions under negative selection as determined by bioinformatics analysis. It was found that pyama1 has two highly conserved regions amongst species (>70%) under negative selection. Fourteen synthetic peptides spanning both conserved regions were evaluated; 5 Py AMA-1 peptides having high specific binding (HABP) to murine erythrocytes were identified. The parasite's invasion inhibition capability was analysed through in vitro assays, suggesting that peptides 42681 (43-ENTERSIKLINPWDKYMEKY-62), 42903 (206-RYSSNDANNENQPFSFTPEK-225) and 42904 (221-FTPEKIENYKDLSYLTKNLR-240) had greater than 50% inhibition profile and restricted P. yoelii intra-erythrocyte development. This work proposes that the screening of conserved HABPs under negative selective pressure might be good candidates for developing a synthetic anti-malarial vaccine since they share functionally-relevant characteristics, such as interspecies conservation, specific RBC binding profile, invasion and parasite development inhibition capability, and the predicted B-epitopes within were recognised by sera obtained from experimentally-infected mice. • AMA-1 peptides 42681, 42898, 42903, 42904 and 42910 displayed a High Activity Binding (HABPs) to murine erythrocytes. • HABPs 42681, 42903 and 42904 inhibited parasite invasion of erythrocytes in vitro. • HABPs 42681 and 42904 inhibited parasite intra-erythrocyte development. • All HABPs were antigenic. • Peptides 42681, 42903 and 42904 are thus potential candidates for developing a synthetic anti-malarial vaccine. [ABSTRACT FROM AUTHOR]

Plasmodium parasites cause malaria with disease outcomes ranging from mild illness to deadly complications such as severe malarial anemia (SMA), pulmonary edema, acute renal failure, and cerebral malaria. In young children, SMA often requires blood transfusion and is a major cause of hospitalization. Malaria parasite infection leads to the destruction of infected and noninfected erythrocytes as well as dyserythropoiesis; however, the mechanism of dyserythropoiesis accompanied by splenomegaly is not completely understood. Using Plasmodium yoelii yoelii 17XNL as a model, we show that both a defect in erythroblastic island (EBI) macrophages in supporting red blood cell (RBC) maturation and the destruction of reticulocytes/RBCs by the parasites contribute to SMA and splenomegaly. After malaria parasite infection, the destruction of both infected and noninfected RBCs stimulates extramedullary erythropoiesis in mice. The continuous decline of RBCs stimulates active erythropoiesis and drives the expansion of EBIs in the spleen, contributing to splenomegaly. Phagocytosis of malaria parasites by macrophages in the bone marrow and spleen may alter their functional properties and abilities to support erythropoiesis, including reduced expression of the adherence molecule CD169 and inability to support erythroblast differentiation, particularly RBC maturation in vitro and in vivo. Therefore, macrophage dysfunction is a key mechanism contributing to SMA. Mitigating and/or alleviating the inhibition of RBC maturation may provide a treatment strategy for SMA. [ABSTRACT FROM AUTHOR]

Malaria, a life-threatening mosquito-borne disease caused by Plasmodium parasites, continues to pose a significant global health burden. Despite notable progress in combating the disease in recent years, malaria remains prevalent in many regions, particularly in Southeast Asia and most of sub-Saharan Africa, where it claims hundreds of thousands of lives annually. Flavonoids, such as the baicalein class of compounds, are known to have antimalarial properties. In this study, we rationally designed and synthesized a series of baicalein derivatives and identified a lead compound, FNDR-10132, that displayed potent in vitro antimalarial activity against Plasmodium falciparum (P. falciparum), both chloroquine-sensitive (60 nM) and chloroquine-resistant (177 nM) parasites. FNDR-10132 was evaluated for its antimalarial activity in vivo against the chloroquine-resistant strain Plasmodium yoelii N67 in Swiss mice. The oral administration of 100 mg/kg of FNDR-10132 showed 44% parasite suppression on day 4, with a mean survival time of 13.5 ± 2.3 days vs. 8.4 ± 2.3 days of control. Also, FNDR-10132 displayed equivalent activity against the resistant strains of P. falciparum in the 200–300 nM range. This study offers a novel series of antimalarial compounds that could be developed into potent drugs against chloroquine-resistant malarial parasites through further chemistry and DMPK optimization. [ABSTRACT FROM AUTHOR]

Apical membrane antigen 1 (AMA1) is a key malaria vaccine candidate and target of neutralizing antibodies. AMA1 binds to a loop in rhoptry neck protein 2 (RON2L) to form the moving junction during parasite invasion of host cells, and this complex is conserved among apicomplexan parasites. AMA1-RON2L complex immunization achieves higher growth inhibitory activity than AMA1 alone and protects mice against Plasmodium yoelii challenge. Here, three single-component AMA1-RON2L immunogens were designed that retain the structure of the two-component AMA1-RON2L complex: one structure-based design (SBD1) and two insertion fusions. All immunogens elicited high antibody titers with potent growth inhibitory activity, yet these antibodies did not block RON2L binding to AMA1. The SBD1 immunogen induced significantly more potent strain-transcending neutralizing antibody responses against diverse strains of Plasmodium falciparum than AMA1 or AMA1-RON2L complex vaccination. This indicates that SBD1 directs neutralizing antibody responses to strain-transcending epitopes in AMA1 that are independent of RON2L binding. This work underscores the importance of neutralization mechanisms that are distinct from RON2 blockade. The stable single-component SBD1 immunogen elicits potent strain-transcending protection that may drive the development of next-generation vaccines for improved malaria and apicomplexan parasite control. Here the authors use structure-based design to engineer a single component immunogen that mimics the malaria parasite AMA1-RON2 complex required for invasion of host cells, and show that it elicits a potent strain-transcending antibody response in rats. [ABSTRACT FROM AUTHOR]

Background and Purpose: Plasmodium falciparum and P. vivax are responsible for most malaria cases in humans in the African Region and the Americas; these parasites have developed resistance to classic antimalarial drugs. On the other hand, previous investigations of the alkyl-linked bis tetrahydro-(2H)-1,3,5-thiadiazine-2-thione (bis-THTT) derivatives compounds show satisfactory results against protozoan parasites such as Trypanosoma cruzi, Trypanosoma vaginalis, Trypanosoma brucei rhodesiense and Leishmania donovani. Therefore, it is possible to see some effect of bis-THTT derivatives on other protozoan parasites, such as Plasmodium. Experimental Approach: This study aimed to perform an in vivo biological evaluation of bis-THTT (JH1 to JH6) derivatives compounds as possible anti-malaria drugs in BALB/c mice infected with Plasmodium berghei ANKA and Plasmodium yoelii 17XL strains. In this work, we evaluated the compounds as potential antimalarial drugs in BALB/c mice infected with Plasmodium strains. Key Results: For each compound, we assess the percentages of parasitemia by smears from tail blood and the humoral response by indirect ELISA test using each compound as an antigen. We also evaluated the B lymphocyte response and the cytotoxicity of the bis-THTT derivatives compounds with MTT cell proliferation assays. Conclusions: Our results show that the bis-THTT derivatives JH2 and JH4 presented effective parasitemia control in mice infected with P. berghei; JH5 and JH6 compounds have similar infection control results as chloroquine in mice infected P. yoelii strain. The evaluation of bis-THTT derivatives compounds in a model of BALB/c mice infected with P. berghei and P. yoelii allowed us to conclude that some of them have an antimalarial effect; however, none of the tested compounds exceeded the efficiency of chloroquine.

Blood levels of histamine and serotonin (5-HT) are altered in human malaria, and, at these levels, we have shown they have broad, independent effects on Anopheles stephensi following ingestion by this invasive mosquito. Given that histamine and 5-HT are ingested together under natural conditions and that histaminergic and serotonergic signaling are networked in other organisms, we examined effects of combinations of these biogenic amines provisioned to A. stephensi at healthy human levels (high 5-HT, low histamine) or levels associated with severe malaria (low 5-HT, high histamine). Treatments were delivered in water (priming) before feeding A. stephensi on Plasmodium yoelii-infected mice or via artificial blood meal. Relative to effects of histamine and 5-HT alone, effects of biogenic amine combinations were complex. Biogenic amine treatments had the greatest impact on the first oviposition cycle, with high histamine moderating low 5-HT effects in combination. In contrast, clutch sizes were similar across combination and individual treatments. While high histamine alone increased uninfected A. stephensi weekly lifetime blood feeding, neither combination altered this tendency relative to controls. The tendency to re-feed 2 weeks after the first blood meal was altered by combination treatments, but this depended on mode of delivery. For blood delivery, malaria-associated treatments yielded higher percentages of fed females relative to healthy-associated treatments, but the converse was true for priming. Female mosquitoes treated with the malaria-associated combination exhibited enhanced flight behavior and object inspection relative to controls and healthy combination treatment. Mosquitoes primed with the malaria-associated combination exhibited higher mean oocysts and sporozoite infection prevalence relative to the healthy combination, with high histamine having a dominant effect on these patterns. Compared with uninfected A. stephensi, the tendency of infected mosquitoes to take a second blood meal revealed an interaction of biogenic amines with infection. We used a mathematical model to project the impacts of different levels of biogenic amines and associated changes on outbreaks in human populations. While not all outbreak parameters were impacted the same, the sum of effects suggests that histamine and 5-HT alter the likelihood of transmission by mosquitoes that feed on hosts with symptomatic malaria versus a healthy host. [ABSTRACT FROM AUTHOR]

Malaria causes >600 thousand fatalities each year, with most cases attributed to the human-infectious Plasmodium falciparum species. Many rodent-infectious Plasmodium species, like Plasmodium berghei and Plasmodium yoelii, have been used as model species that can expedite studies of this pathogen. P. yoelii is an especially good model for investigating the mosquito and liver stages of development because key attributes closely resemble those of P. falciparum. Because of its importance, in 2002 the 17XNL strain of P. yoelii was the first rodent malaria parasite to be sequenced. Although this was a breakthrough effort, the assembly consisted of >5000 contiguous sequences that adversely impacted the annotated gene models. While other rodent malaria parasite genomes have been sequenced and annotated since then, including the related P. yoelii 17X strain, the 17XNL strain has not. As a result, genomic data for 17X has become the de facto reference genome for the 17XNL strain while leaving open questions surrounding possible differences between the 17XNL and 17X genomes. In this work, we present a high-quality genome assembly for P. yoelii 17XNL using PacBio DNA sequencing. In addition, we use Nanopore and Illumina RNA sequencing of mixed blood stages to create complete gene models that include coding sequences, alternate isoforms, and UTR designations. A comparison of the 17X and this new 17XNL assembly revealed biologically meaningful differences between the strains due to the presence of coding sequence variants. Taken together, our work provides a new genomic framework for studies with this commonly used rodent malaria model species. [ABSTRACT FROM AUTHOR]

Babesia spp. are intraerythrocytic apicomplexans that digest and utilize red blood cells in a similar way to intraerythrocytic Plasmodium spp., but unlike the latter, are not sensitive to artemisinin. A comparison of Babesia and Plasmodium genomes revealed that Babesia genomes, which are smaller than those of Plasmodium, lack numerous genes, and especially haem synthesis-related genes, that are found in the latter. Single-cell sequencing analysis showed that the different treatment groups of Babesia microti with expressed pentose phosphate pathway-related, DNA replication-related, antioxidation-related, glycolysis-related, and glutathione-related genes were not as sensitive to artemether as Plasmodium yoelii 17XNL. In particular, pentose phosphate pathway-related, DNA replication-related, and glutathione-related genes, which were actively expressed in P. yoelii 17XNL, were not actively expressed in B. microti. Supplying iron in vivo can promote the reproduction of B. microti. These results suggest that Babesia spp. lack a similar mechanism to that of malaria parasites through which the haem or iron in hemoglobin is utilized, and that this likely leads to their insensitivity to artemisinin. [ABSTRACT FROM AUTHOR]

Malaria strongly predisposes to bacteremia, which is associated with sequestration of parasitized red blood cells and increased gastrointestinal permeability. The mechanisms underlying this disruption are poorly understood. Here, we evaluated the expression of factors associated with mast cell activation and malaria-associated bacteremia in a rodent model. C57BL/6J mice were infected with Plasmodium yoeliiyoelli 17XNL, and blood and tissues were collected over time to assay for circulating levels of bacterial 16S DNA, IgE, mast cell protease 1 (Mcpt-1) and Mcpt-4, Th1 and Th2 cytokines, and patterns of ileal mastocytosis and intestinal permeability. The anti-inflammatory cytokines (interleukin-4 [IL-4], IL-6, and IL-10) and MCP-1/CCL2 were detected early after P. yoeliiyoelii 17XNL infection. This was followed by the appearance of IL-9 and IL-13, cytokines known for their roles in mast cell activation and growth-enhancing activity as well as IgE production. Later increases in circulating IgE, which can induce mast cell degranulation, as well as Mcpt-1 and Mcpt-4, were observed concurrently with bacteremia and increased intestinal permeability. These results suggest that P. yoeliiyoelii 17XNL infection induces the production of early cytokines that activate mast cells and drive IgE production, followed by elevated IgE, IL-9, and IL-13 that maintain and enhance mast cell activation while disrupting the protease/antiprotease balance in the intestine, contributing to epithelial damage and increased permeability.

Abstract Background The liver is responsible for a range of functions in vertebrates, such as metabolism and immunity. In malaria, the liver plays a crucial role in the interaction between the parasite and host. Although malarial hepatitis is a common clinical complication of severe malaria, other malaria-related liver changes have been overlooked during the blood stage of the parasite life-cycle, in contrast to the many studies that have focused on parasite invasion of and replication in the liver during the hepatic stage of the parasite. Methods A rodent model of malaria was established using Plasmodium yoelii strain 17XL, a lethal strain of rodent malaria, for liver transcriptomic profiling. Results Differentially expressed messenger RNAs were associated with innate and adaptive immune responses, while differentially expressed long noncoding RNAs were enriched in the regulation of metabolism-related pathways, such as lipid metabolism. The coexpression network showed that host genes were related to cellular transport and tissue remodeling. Hub gene analysis of P. yoelii indicated that ubiquitination genes that were coexpressed with the host were evolutionarily conserved. Conclusions Our analysis yielded evidence of activated immune responses, aberrant metabolic processes and tissue remodeling changes in the livers of mice with malaria during the blood stage of the parasite, which provided a systematic outline of liver responses during Plasmodium infection. Graphical Abstract

Sepsis is a common disease in sub-Saharan Africa and Asia, where malaria is also prevalent. To determine whether Plasmodium infection might enhance susceptibility to endotoxin shock, we used a mouse model of lipopolysaccharide (LPS) administration. Our results indicated that Plasmodium yoelii infection in mice strongly enhanced the susceptibility of the host to develop endotoxin shock. This increased susceptibility to endotoxin shock was correlated with a synergistic effect of Plasmodium and LPS on the secretion of Tumor Necrosis Factor (TNF). TNF contributed mostly to lethality after the dual challenge since neutralization with an anti-TNF antibody provided protection from death. Plasmodium infection also induced an enhancement of the serum levels of LPS soluble ligands, sCD14 and Lipopolysaccharide Binding Protein. In this regard, our data confirm that Plasmodium infection can profoundly modify responses to secondary bacteria challenges, resulting in dysregulated cytokine expression and pathological effects. If confirmed in humans, LPS soluble receptors might serve as markers of susceptibility to septic shock. [ABSTRACT FROM AUTHOR]

Introduction: Malaria and Babesiosis are acute zoonotic disease that caused by infection with the parasite in the phylum Apicomplexa. Severe anemia and thrombocytopenia are the most common hematological complication of malaria and babesiosis. However, the mechanisms involved have not been elucidated, and only a few researches focus on the possible role of antierythrocyte and anti-platelet antibodies. Methods: In this study, the Plasmodium yoelii, P. chabaudi, Babesia microti and B. rodhaini infected SCID and ICR mice. The parasitemia, survival rate, platelet count, anti-platelet antibodies, and the level of IFN-g and interleukin (IL) -10 was tested after infection. Furthermore, the P. yoelii, P. chabaudi, B. rodhaini and B. microti infected ICR mice were treated with artesunate and diminaze, the development of the anti-erythrocyte and anti-platelet antibodies in chronic stage were examined. At last, the murine red blood cell and platelet membrane proteins probed with auto-antibodies induced by P. yoelii, P. chabaudi, B. rodhaini, and B. microti infection were characterized by proteomic analysis. Results and discussion: The high anti-platelet and anti-erythrocyte antibodies were detected in ICR mice after P. yoelii, P. chabaudi, B. rodhaini, and B. microti infection. Actin of murine erythrocyte and platelet is a common auto-antigen in Plasmodium and Babesia spp. infected mice. Our findings indicate that anti-erythrocyte and anti-platelet autoantibodies contribute to thrombocytopenia and anemia associated with Plasmodium spp. and Babesia spp. infection. This study will help to understand the mechanisms of malaria and babesiosis-related thrombocytopenia and hemolytic anemia. [ABSTRACT FROM AUTHOR]

Plasmodium malaria parasites use erythrocyte-binding-like (EBL) ligands to invade erythrocytes in their vertebrate host. EBLs are released from micronemes, which are secretory organelles located at the merozoite apical end and bind to erythrocyte surface receptors. Because of their essential nature, EBLs have been studied as vaccine candidates, such as the Plasmodium vivax Duffy binding protein. Previously, we showed through using the rodent malaria parasite Plasmodium yoelii that a single amino acid substitution within the EBL C-terminal Cys-rich domain (region 6) caused mislocalization of this molecule and resulted in alteration of the infection course and virulence between the non-lethal 17X and lethal 17XL strains. In the present study, we generated a panel of transgenic P. yoelii lines in which seven of the eight conserved Cys residues in EBL region 6 were independently substituted to Ala residues to observe the consequence of these substitutions with respect to EBL localization, the infection course, and virulence. Five out of seven transgenic lines showed EBL mislocalizations and higher parasitemias. Among them, three showed increased virulence, whereas the other two did not kill the infected mice. The remaining two transgenic lines showed low parasitemias similar to their parental 17X strain, and their EBL localizations did not change. The results indicate the importance of Cys residues in EBL region 6 for EBL localization, parasite infection course, and virulence and suggest an association between EBL localization and the parasite infection course. [ABSTRACT FROM AUTHOR]

Abstract Background Rapid emergence of Plasmodium resistance to anti-malarial drug mainstays has driven a continual effort to discover novel drugs that target different biochemical pathway (s) during infection. Plasma membrane Calcium + 2 ATPase (PMCA4), a novel plasma membrane protein that regulates Calcium levels in various cells, namely red blood cell (RBC), endothelial cell and platelets, represents a new biochemical pathway that may interfere with susceptibility to malaria and/or severe malaria. Methods This study identified several pharmacological inhibitors of PMCA4, namely ATA and Resveratrol, and tested for their anti-malarial activities in vitro and in vivo using the Plasmodium falciparum 3D7 strain, the Plasmodium berghei ANKA strain, and Plasmodium yoelii 17XL strain as model. Results In vitro propagation of P. falciparum 3D7 strain in the presence of a wide concentration range of the inhibitors revealed that the parasite growth was inhibited in a dose-dependent manner, with IC50s at 634 and 0.231 µM, respectively. Results The results confirmed that both compounds exhibit moderate to potent anti-malarial activities with the strongest parasite growth inhibition shown by resveratrol at 0.231 µM. In vivo models using P. berghei ANKA for experimental cerebral malaria and P. yoelii 17XL for the effect on parasite growth, showed that the highest dose of ATA, 30 mg/kg BW, increased survival of the mice. Likewise, resveratrol inhibited the parasite growth following 4 days intraperitoneal injection at the dose of 100 mg/kg BW. Conclusion The findings indicate that the PMCA4 of the human host may be a potential target for novel anti-malarials, either as single drug or in combination with the currently available effective anti-malarials.

Genetically-growth-attenuated blood-stage parasites were generated in Plasmodium falciparum by targeted deletion of NT1 (Nucleoside Transporter-1) gene, and Pfnt1(-) parasites only grew after providing the culture with supra-physiological concentrations of purines. Genetically-attenuated P. yoelii nt1(-) parasites induced sterile-protection against homologous blood-stage infectious challenge after immunization with single subpatent doses, which remained subpatent even in immune-compromised mice. Here, we showed that immunizations with frozen-stocks of equally-mixed P. berghei and P. yoelii nt1(-) parasites in single subcutaneous doses, which did not lead to patent blood-stage infection, conferred sterile protection against intravenous infectious blood-stage challenge with wild-type parasites of P. berghei ANKA and P. yoelii 17X-NL strains. This data highlights the possibility that a single subcutaneous sub-patent dose of two species of genetically-growth-attenuated parasites, which can protect humans against two Plasmodium spp. infections, could be developed in cultures provided with supra-physiological concentrations of purines, and shipped to endemic areas as frozen-stock doses. [ABSTRACT FROM AUTHOR]

Background: The liver is responsible for a range of functions in vertebrates, such as metabolism and immunity. In malaria, the liver plays a crucial role in the interaction between the parasite and host. Although malarial hepatitis is a common clinical complication of severe malaria, other malaria-related liver changes have been overlooked during the blood stage of the parasite life-cycle, in contrast to the many studies that have focused on parasite invasion of and replication in the liver during the hepatic stage of the parasite. Methods: A rodent model of malaria was established using Plasmodium yoelii strain 17XL, a lethal strain of rodent malaria, for liver transcriptomic profiling. Results: Differentially expressed messenger RNAs were associated with innate and adaptive immune responses, while differentially expressed long noncoding RNAs were enriched in the regulation of metabolism-related pathways, such as lipid metabolism. The coexpression network showed that host genes were related to cellular transport and tissue remodeling. Hub gene analysis of P. yoelii indicated that ubiquitination genes that were coexpressed with the host were evolutionarily conserved. Conclusions: Our analysis yielded evidence of activated immune responses, aberrant metabolic processes and tissue remodeling changes in the livers of mice with malaria during the blood stage of the parasite, which provided a systematic outline of liver responses during Plasmodium infection. [ABSTRACT FROM AUTHOR]

7DW8-5 is a potent glycolipid adjuvant that improves malaria vaccine efficacy in mice by inducing IFN-γ and increasing protective CD8+ T cell responses. The addition of 7DW8-5 was previously shown to improve the efficacy of a CD8+ T cell-mediated heterologous 'prime-and-trap' malaria vaccine against Plasmodium yoelii sporozoite challenge in inbred female mice. Here, we report significant differential sex-specific responses to 7DW8-5 in inbred and outbred mice. Male mice express significantly less IFN-γ and IL-4 compared to females following intravenous 7DW8-5 administration. Additionally, unlike in female mice, 7DW8-5 did not improve the vaccine efficacy against sporozoite challenge in prime-and-trap vaccinated male mice. Our findings highlight the importance of including both female and male sexes in experimental adjuvant studies. [ABSTRACT FROM AUTHOR]

Cerebral malaria (CM), a fatal complication of Plasmodium infection that affects children, especially under the age of five, in sub-Saharan Africa and adults in South-East Asia, results from incompletely understood pathogenetic mechanisms. Increased release of circulating miRNA, proteins, lipids and extracellular vesicles has been found in CM patients and experimental mouse models. We compared lipid profiles derived from the plasma of CBA mice infected with Plasmodium berghei ANKA (PbA), which causes CM, to those from Plasmodium yoelii (Py), which does not. We previously showed that platelet-free plasma (18k fractions enriched from plasma) contains a high number of extracellular vesicles (EVs). Here, we found that this fraction produced at the time of CM differed dramatically from those of non-CM mice, despite identical levels of parasitaemia. Using high-resolution liquid chromatography–mass spectrometry (LCMS), we identified over 300 lipid species within 12 lipid classes. We identified 45 and 75 lipid species, mostly including glycerolipids and phospholipids, with significantly altered concentrations in PbA-infected mice compared to Py-infected and uninfected mice, respectively. Total lysophosphatidylethanolamine (LPE) levels were significantly lower in PbA infection compared to Py infection and controls. These results suggest that experimental CM could be characterised by specific changes in the lipid composition of the 18k fraction containing circulating EVs and can be considered an appropriate model to study the role of lipids in the pathophysiology of CM. [ABSTRACT FROM AUTHOR]

The liver is the first destination of malaria parasites in humans. After reaching the liver by the blood stream, Plasmodium sporozoites cross the liver sinusoid epithelium, enter and exit several hepatocytes, and eventually invade a final hepatocyte host cell. At present, the mechanism of hepatocyte invasion is only partially understood, presenting a key research gap with opportunities for the development of new therapeutics. Recently, human EphA2, a membrane-bound receptor tyrosine kinase, was implicated in hepatocyte infection by the human malaria parasite Plasmodium falciparum and the rodent parasite Plasmodium yoelii, but its role is not known for Plasmodium vivax, a major human parasite whose liver infection poses a specific challenge for malaria treatment and elimination. In this study, the role of EphA2 in P. vivax infection was investigated. It was found that surface expression of several recombinant fragments of EphA2 enhanced the parasite infection rate, thus establishing its role in P. vivax infection. Furthermore, a new permanent cell line (EphA2Extra-HC04) expressing the whole extracellular domain of EphA2 was generated. This cell line supports a higher rate of P. vivax infection and is a valuable tool for P. vivax liver-stage research. [ABSTRACT FROM AUTHOR]

The lipid is a vital metabolic and structural component of the malaria parasite. Malaria parasite-induced liver lipid deposits undergo peroxidation, which ultimately causes tissue damage and histopathological changes, which further lead to many complications. Therefore, it is essential to focus on the factors responsible for this stimulated lipid accumulation during malaria infection. In the present study, we have correlated the significant increase in serum TNF-α and liver triglyceride during Plasmodium yoelii MDR infection in mice. In order to explore the role of TNF-α in inducing lipid accumulation in the liver during malaria infection, we have used a competitive TNF-α inhibitor Etanercept, for the treatment of Plasmodium yoelii MDR (Py MDR) infected mice and found that Etanercept displayed up to a three-fold inhibition of the liver triglyceride level in Py MDR infected mice. These results were also confirmed by triglyceride specific oil red O staining of liver sections. In addition, all the treatment groups also showed inhibition in the level of serum TNF-α and the liver malondialdehyde (MDA), a byproduct of lipid peroxidation. Our study thus concludes that Etanercept significantly reduces Plasmodium-induced liver triglyceride and further saves the host liver from malaria-induced lipid infiltration and liver damage. Therefore, treatment with Etanercept, along with a standard antimalarial, may prove a better therapy for the disease. [ABSTRACT FROM AUTHOR]

Methylene blue (MB) is the oldest synthetic anti-infective. Its high potency against asexual and sexual stages of malaria parasites is well documented. This study aimed to investigate possible additional activities of MB in interfering with parasite transmission and determine target stages in Anopheles vectors and humans. MB's transmission-blocking activity was first evaluated by an ex vivo direct membrane feeding assay (DMFA) using Plasmodium falciparum field isolates. To investigate anti-mosquito stage activity, Plasmodium berghei-infected Anopheles stephensi mosquitoes were fed a second blood meal on mice that had been treated with methylene blue, 3, 6- and 15-days after the initial infectious blood meal. Anti-sporozoite and liver stage activities were evaluated in vitro and in vivo via sporozoite invasion and liver stage development assays, respectively. MB exhibited a robust inhibition of P. falciparum transmission in An. gambiae, even when added shortly before the DMFA but only a moderate effect against P. berghei oocyst development. Exposure of mature P. berghei and P. falciparum sporozoites to MB blocked hepatocyte invasion, yet P. berghei liver stage development was unaffected by MB. Our results indicate previously underappreciated rapid specific activities of methylene blue against Plasmodium transmission stages, preventing the establishment of both mosquito midgut and liver infections as the first essential steps in both hosts. [ABSTRACT FROM AUTHOR]

Abstract Background Medicinal plants have been successfully used as an alternative source of drugs for the treatment of microbial diseases. Finding a novel treatment for malaria is still challenging, and various extracts from different wild desert plants have been reported to have multiple medicinal uses for human public health, this study evaluated the antimalarial efficacy of several Egyptian plant extracts. Methods We assessed the cytotoxic potential of 13 plant extracts and their abilities to inhibit the in vitro growth of Plasmodium falciparum (3D7), and to treat infection with non-lethal Plasmodium yoelii 17XNL in an in vivo malaria model in BALB/c mice. Results In vitro screening identified four promising candidates, Trichodesma africanum, Artemisia judaica, Cleome droserifolia, and Vachellia tortilis, with weak-to-moderate activity against P. falciparum erythrocytic blood stages with mean half-maximal inhibitory concentration 50 (IC50) of 11.7 μg/ml, 20.0 μg/ml, 32.1 μg/ml, and 40.0 μg/ml, respectively. Their selectivity index values were 35.2, 15.8, 11.5, and 13.8, respectively. Among these four candidates, T. africanum crude extract exhibited the highest parasite suppression in a murine malaria model against P. yoelii. Conclusion Our study identified novel natural antimalarial agents of plant origin that have potential for development into therapeutics for treating malaria.

Malaria, caused by Plasmodium parasites, remains a significant global health challenge, particularly in tropical and subtropical regions. At the same time, the prevalence of toxoplasmosis has been reported to be 30% worldwide. Traditional medicines have long played a vital role in discovering and developing novel drugs, and this approach is essential in the face of increasing resistance to current antimalarial and anti- Toxoplasma drugs. In Indonesian traditional medicine, various plants are used for their therapeutic properties. This study focuses on eleven medicinal plants from which nineteen extracts were obtained and screened for their potential medicinal benefits against malaria and toxoplasmosis. The aim of this study was to evaluate the efficacy of extracts from Indonesian medicinal plants to inhibit Plasmodium falciparum , a parasite responsible for malaria, and Toxoplasma gondii , an opportunistic parasite responsible for toxoplasmosis. Nineteen extracts from eleven plants were subjected to in vitro screening against P. falciparum 3D7 (a chloroquine-sensitive strain) and the T. gondii RH strain. In vitro treatments were conducted on P. falciparum 3D7 and K1 (multidrug-resistant strains) using the potent extracts, and in vivo assessments were carried out with mice infected with P. yoelii 17XNL. LCMS analysis was also conducted to identify the main components of the most effective extract. Seven extracts showed significant antiplasmodial activity (>80% inhibition) at a concentration of 100 μg/ml. These extracts were obtained from Dysoxylum parasiticum (Osbeck) Kosterm., Elaeocarpus glaber (Bl.) Bijdr., Eleutherine americana Merr., Kleinhovia hospita L., Peronema canescens Jack, and Plectranthus scutellarioides (L.) R.Br. Notably, the D. parasiticum ethyl acetate extract exhibited high selectivity and efficacy both in vitro and in vivo. Herein, the key active compounds oleamide and erucamide were identified, which had IC 50 values (P. falciparum 3D7/K1) of 17.49/23.63 μM and 32.49/51.59 μM, respectively. The results of this study highlight the antimalarial potential of plant extracts collected from Indonesia. Particularly, extracts from D. parasiticum EtOH and EtOAc stood out for their low toxicity and strong antiplasmodial properties, with the EtOAc extract emerging as a notably promising antimalarial candidate. Key compounds identified within this extract demonstrate the complexity of extracts' action against malaria, potentially targeting both the parasite and the host. This suggests a promising approach for developing new antimalarial strategies that tackle the multifaceted challenges of drug resistance and disease management. Future investigations are necessary to unlock the full therapeutic potential of these extracts. [Display omitted] • This study evaluated nineteen extracts from eleven Indonesian medicinal plants for their effectiveness against P. falciparum. • Seven extracts demonstrated antiplasmodial activity, showing over 80% inhibition at a concentration of 100 μg/ml. • The D. parasiticum EtOAc extract exhibits high selectivity with oleamide and erucamide identified as key active compounds. • These findings underscore the potential of D. parasiticum , as promising sources of malaria treatment. [ABSTRACT FROM AUTHOR]

Abstract Background Regulatory T cells are known to play a key role to counter balance the protective immune response and immune mediated pathology. However, the role of naturally occurring regulatory cells CD4+CD25+Foxp3 + in malaria infection during the disease pathogenesis is controversial. Beside this, ICOS molecule has been shown to be involved in the development and function of regulatory T cell enhance IL-10 production. Therefore, possible involvement of the ICOS dependent regulatory CD4+ICOS+Foxp3 + T cells in resistance/susceptibility during malaria parasite is explored in this study. Methods 5 × 105 red blood cells infected with non-lethal and lethal parasites were inoculated in female Balb/c mice by intra-peritoneal injection. Infected or uninfected mice were sacrificed at early (3rd day post infection) and later stage (10th day post infection) of infection. Harvested cells were analysed by using flow cytometer and serum cytokine by Bioplex assay. Results Thin blood films show that percentages of parasitaemia increases with disease progression in infections with the lethal malaria parasite and mice eventually die by day 14th post-infection. Whereas in case of non-lethal malaria parasite, parasitaemia goes down by 7th day post infection and gets cleared within 13th day. The number of CD4+ ICOS+ T cells increases in lethal infection with disease progression. Surprisingly, in non-lethal parasite, ICOS expression decreases after day 7th post infection as parasitaemia goes down. The frequency of CD4+ICOS+FoxP3+ Tregs was significantly higher in lethal parasitic infection as compared to the non-lethal parasite. The level of IL-12 cytokine was remarkably higher in non-lethal infection compared to the lethal infection. In contrast, the level of IL-10 cytokines was higher in lethal parasite infection compared to the non-lethal parasite. Conclusion Taken together, these data suggest that lethal parasite induce immunosuppressive environment, protecting from host immune responses and help the parasite to survive whereas non-lethal parasite leads to low frequencies of Treg cells seldom impede immune response that allow the parasite to get self-resolved. Graphical Abstract

Disseminated infections with nontyphoidal Salmonella (NTS) are a significant cause of child mortality in sub-Saharan Africa. NTS infection in children is clinically associated with malaria, suggesting that malaria compromises the control of disseminated NTS infection. To study the mechanistic basis for increased NTS susceptibility, we utilized a model of concurrent infection with Salmonella enterica serotype Typhimurium and Plasmodium yoelii nigeriensis (P. yoelii). Underlying malaria blunted monocyte expression of Ly6C, a marker for inflammatory activation, and impaired recruitment of inflammatory cells to the liver. Hepatic mononuclear phagocytes expressed lower levels of inducible nitric oxide synthase, tumor necrosis factor alpha, and granulocyte-macrophage colony-stimulating factor and showed increased levels of production of interleukin-10 and heme oxygenase-1, indicating that the underlying malaria modifies the activation state and inflammatory response of mononuclear phagocytes to NTS. P. yoelii infection also increased intracellular iron levels in liver mononuclear cells, as evidenced by elevated levels of ferritin and by the rescue of an S Typhimurium tonB feoB mutant defective for iron uptake. In addition, concurrent P. yoelii infection partially rescued the systemic colonization defect of an S Typhimurium spiB mutant defective for type III secretion system 2 (T3SS-2), indicating that the ability of phagocytic cells to limit the spread of S Typhimurium is impaired during concurrent P. yoelii infection. These results show that concurrent malaria increases susceptibility to disseminated NTS infection by blunting macrophage bactericidal mechanisms and providing an essential nutrient that enhances bacterial growth.

Abscisic acid (ABA) is an ancient stress hormone and is detectable in a wide variety of organisms where it regulates innate immunity and inflammation. Previously, we showed that oral supplementation with ABA decreased parasitemia in a mouse model of malaria, decreased liver and spleen pathology and reduced parasite transmission to mosquitoes. Here, we report that higher circulating ABA levels were associated with a reduced risk of symptomatic malaria in a cohort of Plasmodium falciparum-infected Ugandan children. To understand possible mechanisms of ABA protection in malaria, we returned to our mouse model to show that ABA effects on Plasmodium yoelii 17XNL infection were accompanied by minimal effects on complete blood count and blood chemistry analytes, suggesting a benefit to host health. In addition, orally delivered ABA induced patterns of gene expression in mouse liver and spleen that suggested enhancement of host anti-parasite defenses. To test these inferences, we utilized passive immunization and knockout mice to demonstrate that ABA supplementation increases circulating levels of protective, parasite-specific IgG and requires caspase-1 to reduce parasitemia. Collectively, ABA induces host responses that ameliorate infection and disease in an animal model and suggest that further studies of ABA in the context of human malaria are warranted.