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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., Competing Interests: Competing interests statement:The authors declare no competing interest.

Rodent malaria parasites have been widely used in all aspects of malaria research to study parasite development within rodent and insect hosts, drug resistance, disease pathogenesis, host immune response, and vaccine efficacy. Rodent malaria parasites were isolated from African thicket rats and initially characterized by scientists at the University of Edinburgh, UK, particularly by Drs. Richard Carter, David Walliker, and colleagues. Through their efforts and elegant work, many rodent malaria parasite species, subspecies, and strains are now available. Because of the ease of maintaining these parasites in laboratory mice, genetic crosses can be performed to map the parasite and host genes contributing to parasite growth and disease severity. Recombinant DNA technologies are now available to manipulate the parasite genomes and to study gene functions efficiently. In this chapter, we provide a brief history of the isolation and species identification of rodent malaria parasites. We also discuss some recent studies to further characterize the different developing stages of the parasites including parasite genomes and chromosomes. Although there are differences between rodent and human malaria parasite infections, the knowledge gained from studies of rodent malaria parasites has contributed greatly to our understanding of and the fight against human malaria., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Genetic mapping has been widely employed to search for genes linked to phenotypes/traits of interest. Because of the ease of maintaining rodent malaria parasites in laboratory mice, many genetic crosses of rodent malaria parasites have been performed to map the parasite genes contributing to malaria parasite development, drug resistance, host immune response, and disease pathogenesis. Drs. Richard Carter, David Walliker, and colleagues at the University of Edinburgh, UK, were the pioneers in developing the systems for genetic mapping of malaria parasite traits, including characterization of genetic markers to follow the inheritance and recombination of parasite chromosomes and performing the first genetic cross using rodent malaria parasites. Additionally, many genetic crosses of inbred mice have been performed to link mouse chromosomal loci to the susceptibility to malaria parasite infections. In this chapter, we review and discuss past and recent advances in genetic marker development, performing genetic crosses, and genetic mapping of both parasite and host genes. Genetic mappings using models of rodent malaria parasites and inbred mice have contributed greatly to our understanding of malaria, including parasite development within their hosts, mechanism of drug resistance, and host-parasite interaction., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Published by Elsevier B.V.)

Background: To understand how Plasmodium falciparum malaria is controlled, it is essential to elucidate the transcriptomic responses of the human host in naturally-exposed populations. Various individual studies of the human transcriptomic responses to naturally transmitted P. falciparum infections have been reported with varying results. Multicohort gene expression analysis by aggregating data from diverse populations into a single analysis will increase the reproducibility and reliability of the results., Methods: In this study, discovery cohorts GSE1124-GPL96, GSE34404, GSE117613, and validation cohort GSE35858 were obtained from the Gene Expression Omnibus. A meta-analysis using data from the multicohort studies was performed to identify the differentially expressed genes (DEGs) between malaria-infected and noninfected individuals using the MetaIntegrator R package. Subsequently, the protein-protein interaction (PPI) networks of the DEGs were constructed using Cytoscape software. Significant modules were selected, and the hub genes were identified using the CytoHubba and MCODE plug-ins. Multicohort WGCNA was conducted to find a correlation between modules and malaria infection. Furthermore, the immune cell profile of the peripheral blood in different groups was identified using ssGSEA., Results: These analyses reveal that neutrophil activation, neutrophil-mediated immunity, and neutrophil degranulation are involved in the human response to natural malaria infection. However, neutrophil cell enrichment and activation were not significantly different between mild malaria and severe malaria groups. Malaria infection also downregulates host genes in ribosome synthesis and protein translation and upregulates host cell division-related genes. Furthermore, immune cell profiling analysis shows that activated dendritic cells and type 2 T helper cells are upregulated, while activated B cells, immature B cells, and monocytes are downregulated in the malaria-infected patients relative to the noninfected individuals. Significantly higher enrichment of activated dendritic cell-related genes and significantly lower enrichment of monocyte-related genes are also observed in the peripheral blood of the severe malaria group than in the mild malaria group., Conclusion: These results reveal important molecular signatures of host responses to malaria infections, providing some bases for developing malaria control strategies and protective vaccines., (© 2022. The Author(s).)

N 6 -Methyladenosine (m 6 A), the most abundant mammalian mRNA modification, has been reported to modulate various viral infections. Although it has been confirmed that RNA modifications can also modulate the replication and development of different parasites, the role of the RNA epitranscriptome in the regulation of host response post parasite infection remains to be elucidated. Here we report host spleen m 6 A epitranscriptome landscapes induced by different strains of the malaria parasite Plasmodium yoelii . We found that malaria parasite infection dramatically changes host spleen m 6 A mRNA modification and gene expression. Additionally, malaria parasite infection reprograms host immune response pathways by regulating the m 6 A modification enzymes. Collectively, our study is the first characterization of host spleen m 6 A methylome triggered by malaria parasite infections, and our data identify m 6 A modifications as significant transcriptome-wide marks during host-parasite interactions. We demonstrate that host mRNA methylation machinery can sense and respond to malaria parasite infections, and provide new insights into epitranscriptomic mechanisms underlying parasite-induced pathogenesis., 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 © 2022 Wang, Wu, Liu, Chen, Pang, Zhou, Xia, Huang, Pan, Su and Wang.)

Cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) play critical roles in the innate immunity against infectious diseases and are required to link pathogen DNA sensing to immune responses. However, the mechanisms by which cGAS-STING-induced cytokines suppress the adaptive immune response against malaria infections remain poorly understood. Here, cGAS-STING signaling is identified to play a detrimental role in regulating anti-malaria immunity. cGAS or STING deficiency in mice markedly prolongs mouse survival during lethal malaria Plasmodium yoelii nigeriensis N67C infections by reducing late interleukin (IL)-6 production. Mechanistically, cGAS/STING recruits myeloid differentiation factor 88 (MyD88) and specifically induces the p38-dependent signaling pathway for late IL-6 production, which, in turn, expands CD11b + Ly6C hi proinflammatory monocytes to inhibit immunity. Moreover, the blockage or ablation of the cGAS-STING-MyD88-p38-IL-6 signaling axis or the depletion of CD11b + Ly6C hi proinflammatory monocytes provides mice a significant survival benefit during N67C and other lethal malaria-strain infections. Taken together, these findings identify a previously unrecognized detrimental role of cGAS-STING-MyD88-p38 axis in infectious diseases through triggering the late IL-6 production and proinflammatory monocyte expansion and provide insight into how targeting the DNA sensing pathway, dysregulated cytokines, and proinflammatory monocytes enhances immunity against infection., (© 2022 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Background: Rodent malaria parasites are important models for studying host-malaria parasite interactions such as host immune response, mechanisms of parasite evasion of host killing, and vaccine development. One of the rodent malaria parasites is Plasmodium yoelii, and multiple P. yoelii strains or subspecies that cause different disease phenotypes have been widely employed in various studies. The genomes and transcriptomes of several P. yoelii strains have been analyzed and annotated, including the lethal strains of P. y. yoelii YM (or 17XL) and non-lethal strains of P. y. yoelii 17XNL/17X. Genomic DNA sequences and cDNA reads from another subspecies P. y. nigeriensis N67 have been reported for studies of genetic polymorphisms and parasite response to drugs, but its genome has not been assembled and annotated., Results: We performed genome sequencing of the N67 parasite using the PacBio long-read sequencing technology, de novo assembled its genome and transcriptome, and predicted 5383 genes with high overall annotation quality. Comparison of the annotated genome of the N67 parasite with those of YM and 17X parasites revealed a set of genes with N67-specific orthology, expansion of gene families, particularly the homologs of the Plasmodium chabaudi erythrocyte membrane antigen, large numbers of SNPs and indels, and proteins predicted to interact with host immune responses based on their functional domains., Conclusions: The genomes of N67 and 17X parasites are highly diverse, having approximately one polymorphic site per 50 base pairs of DNA. The annotated N67 genome and transcriptome provide searchable databases for fast retrieval of genes and proteins, which will greatly facilitate our efforts in studying the parasite biology and gene function and in developing effective control measures against malaria.

Type I interferons (IFN-Is) are important cytokines playing critical roles in various infections, autoimmune diseases, and cancer. Studies have also shown that IFN-Is exhibit 'conflicting' roles in malaria parasite infections. Malaria parasites have a complex life cycle with multiple developing stages in two hosts. Both the liver and blood stages of malaria parasites in a vertebrate host stimulate IFN-I responses. IFN-Is have been shown to inhibit liver and blood stage development, to suppress T cell activation and adaptive immune response, and to promote production of proinflammatory cytokines and chemokines in animal models. Different parasite species or strains trigger distinct IFN-I responses. For example, a Plasmodium yoelii strain can stimulate a strong IFN-I response during early infection, whereas its isogenetic strain does not. Host genetic background also greatly influences IFN-I production during malaria infections. Consequently, the effects of IFN-Is on parasitemia and disease symptoms are highly variable depending on the combination of parasite and host species or strains. Toll-like receptor (TLR) 7, TLR9, melanoma differentiation-associated protein 5 (MDA5), and cyclic GMP-AMP synthase (cGAS) coupled with stimulator of interferon genes (STING) are the major receptors for recognizing parasite nucleic acids (RNA/DNA) to trigger IFN-I responses. IFN-I levels in vivo are tightly regulated, and various novel molecules have been identified to regulate IFN-I responses during malaria infections. Here we review the major findings and progress in ligand recognition, signaling pathways, functions, and regulation of IFN-I responses during malaria infections., 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 © 2020 He, Xia, Tumas, Wu and Su.)

Malaria is the most deadly parasitic disease, affecting hundreds of millions of people worldwide. Malaria parasites have been associated with their hosts for millions of years. During the long history of host-parasite co-evolution, both parasites and hosts have applied pressure on each other through complex host-parasite molecular interactions. Whereas the hosts activate various immune mechanisms to remove parasites during an infection, the parasites attempt to evade host immunity by diversifying their genome and switching expression of targets of the host immune system. Human intervention to control the disease such as antimalarial drugs and vaccination can greatly alter parasite population dynamics and evolution, particularly the massive applications of antimalarial drugs in recent human history. Vaccination is likely the best method to prevent the disease; however, a partially protective vaccine may have unwanted consequences that require further investigation. Studies of host-parasite interactions and co-evolution will provide important information for designing safe and effective vaccines and for preventing drug resistance. In this essay, we will discuss some interesting molecules involved in host-parasite interactions, including important parasite antigens. We also discuss subjects relevant to drug and vaccine development and some approaches for studying host-parasite interactions., (Copyright © 2020 Su, Zhang and Joy.)

Malaria infection induces complex and diverse immune responses. To elucidate the mechanisms underlying host-parasite interaction, we performed a genetic screen during early (24 h) Plasmodium yoelii infection in mice and identified a large number of interacting host and parasite genes/loci after transspecies expression quantitative trait locus (Ts-eQTL) analysis. We next investigated a host E3 ubiquitin ligase gene ( March1 ) that was clustered with interferon (IFN)-stimulated genes (ISGs) based on the similarity of the genome-wide pattern of logarithm of the odds (LOD) scores (GPLS). March1 inhibits MAVS/STING/TRIF-induced type I IFN (IFN-I) signaling in vitro and in vivo. However, in malaria-infected hosts, deficiency of March1 reduces IFN-I production by activating inhibitors such as SOCS1, USP18, and TRIM24 and by altering immune cell populations. March1 deficiency increases CD86 + DC (dendritic cell) populations and levels of IFN-γ and interleukin 10 (IL-10) at day 4 post infection, leading to improved host survival. T cell depletion reduces IFN-γ level and reverse the protective effects of March1 deficiency, which can also be achieved by antibody neutralization of IFN-γ. This study reveals functions of MARCH1 (membrane-associated ring-CH-type finger 1) in innate immune responses and provides potential avenues for activating antimalaria immunity and enhancing vaccine efficacy., Competing Interests: The authors declare no competing interest.

Erythrocyte-binding-like (EBL) proteins are known to play an important role in malaria parasite invasion of red blood cells (RBCs); however, any roles of EBL proteins in regulating host immune responses remain unknown. Here, we show that Plasmodium yoelii EBL (PyEBL) can shape disease severity by modulating the surface structure of infected RBCs (iRBCs) and host immune responses. We identified an amino acid substitution (a change of C to Y at position 741 [C741Y]) in the protein trafficking domain of PyEBL between isogenic P. yoellii nigeriensis strain N67 and N67C parasites that produce different disease phenotypes in C57BL/6 mice. Exchanges of the C741Y alleles altered parasite growth and host survival accordingly. The C741Y substitution also changed protein processing and trafficking in merozoites and in the cytoplasm of iRBCs, reduced PyEBL binding to band 3, increased phosphatidylserine (PS) surface exposure, and elevated the osmotic fragility of iRBCs, but it did not affect invasion of RBCs in vitro Malaria is a deadly parasitic disease that continues to afflict hundreds of millions of people every year. Infections with malaria parasites can be asymptomatic, with mild symptoms, or fatal, depending on a delicate balance of host immune responses. Malaria parasites enter host red blood cells (RBCs) through interactions between parasite ligands and host receptors, such as erythrocyte-binding-like (EBL) proteins and host Duffy antigen receptor for chemokines (DARC). IMPORTANCE Malaria is a deadly parasitic disease that continues to afflict hundreds of millions of people every year. Infections with malaria parasites can be asymptomatic, with mild symptoms, or fatal, depending on a delicate balance of host immune responses. Malaria parasites enter host red blood cells (RBCs) through interactions between parasite ligands and host receptors, such as erythrocyte-binding-like (EBL) proteins and host Duffy antigen receptor for chemokines (DARC). Plasmodium yoelii EBL (PyEBL) is known to play a role in parasite invasion of RBCs. Here, we show that PyEBL also affects disease severity through modulation of host immune responses, particularly type I interferon (IFN-I) signaling. This discovery assigns a new function to PyEBL and provides a mechanism for developing disease control strategies.

Protozoan Plasmodium parasites are the causative agents of malaria, a deadly disease that continues to afflict hundreds of millions of people every year. Infections with malaria parasites can be asymptomatic, with mild or severe symptoms, or fatal, depending on many factors such as parasite virulence and host immune status. Malaria can be treated with various drugs, with artemisinin-based combination therapies (ACTs) being the first-line choice. Recent advances in genetics and genomics of malaria parasites have contributed greatly to our understanding of parasite population dynamics, transmission, drug responses, and pathogenesis. However, knowledge gaps in parasite biology and host-parasite interactions still remain. Parasites resistant to multiple antimalarial drugs have emerged, while advanced clinical trials have shown partial efficacy for one available vaccine. Here we discuss genetic and genomic studies of Plasmodium biology, host-parasite interactions, population structures, mosquito infectivity, antigenic variation, and targets for treatment and immunization. Knowledge from these studies will advance our understanding of malaria pathogenesis, epidemiology, and evolution and will support work to discover and develop new medicines and vaccines., (Copyright © 2019 American Society for Microbiology.)

The inflammasome plays a critical role in inflammation and immune responses against pathogens. However, whether or how inflammasome activation regulates type I interferon (IFN-I) signaling in the context of malaria infection remain unknown. Here we show mice deficient in inflammasome sensors AIM2, NLRP3 or adaptor Caspase-1 produce high levels of IFN-I cytokines and are resistant to lethal Plasmodium yoelii YM infection. Inactivation of inflammasome signaling reduces interleukin (IL)-1β production, but increases IFN-I production. Mechanistically, we show inflammsome activation enhances IL-1β-mediated MyD88-TRAF3-IRF3 signaling and SOCS1 upregulation. However, SOCS1 inhibits MyD88-IRF7-mediated-IFN-I signaling and cytokine production in plasmacytoid dendritic cells. By contrast, ablation of inflammsome components reduces SOCS1 induction, and relieves its inhibition on MyD88-IRF7-dependent-IFN-I signaling, leading to high levels of IFN-α/β production and host survival. Our study identifies a previously unrecognized role of inflammasome activation in the negative regulation of IFN-I signaling pathways and provides potential targets for developing effective malaria vaccines.

Malaria is a disease with diverse symptoms depending on host immune status and pathogenicity of Plasmodium parasites. The continuous parasite growth within a host suggests mechanisms of immune evasion by the parasite and/or immune inhibition in response to infection. To identify pathways commonly inhibited after malaria infection, we infected C57BL/6 mice with four Plasmodium yoelii strains causing different disease phenotypes and 24 progeny of a genetic cross. mRNAs from mouse spleens day 1 and/or day 4 post infection (p.i.) were hybridized to a mouse microarray to identify activated or inhibited pathways, upstream regulators, and host genes playing an important role in malaria infection. Strong interferon responses were observed after infection with the N67 strain, whereas initial inhibition and later activation of hematopoietic pathways were found after infection with 17XNL parasite, showing unique responses to individual parasite strains. Inhibitions of pathways such as Th1 activation, dendritic cell (DC) maturation, and NFAT immune regulation were observed in mice infected with all the parasite strains day 4 p.i., suggesting universally inhibited immune pathways. As a proof of principle, treatment of N67-infected mice with antibodies against T cell receptors OX40 or CD28 to activate the inhibited pathways enhanced host survival. Controlled activation of these pathways may provide important strategies for better disease management and for developing an effective vaccine.

Ookinete gliding motility is essential for penetration of the mosquito midgut wall and transmission of malaria parasites. Cyclic guanosine monophosphate (cGMP) signaling has been implicated in ookinete gliding. However, the upstream mechanism of how the parasites activate cGMP signaling and thus initiate ookinete gliding remains unknown. Using real-time imaging to visualize Plasmodium yoelii guanylate cyclase β (GCβ), we show that cytoplasmic GCβ translocates and polarizes to the parasite plasma membrane at "ookinete extrados site" (OES) during zygote-to-ookinete differentiation. The polarization of enzymatic active GCβ at OES initiates gliding of matured ookinete. Both the P4-ATPase-like domain and guanylate cyclase domain are required for GCβ polarization and ookinete gliding. CDC50A, a co-factor of P4-ATPase, binds to and stabilizes GCβ during ookinete development. Screening of inner membrane complex proteins identifies ISP1 as a key molecule that anchors GCβ/CDC50A complex at the OES of mature ookinetes. This study defines a spatial-temporal mechanism for the initiation of ookinete gliding, where GCβ polarization likely elevates local cGMP levels and activates cGMP-dependent protein kinase signaling., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The RNA-guided endonuclease Cas9 has applied as an efficient gene-editing method in malaria parasite Plasmodium. However, the size (4.2 kb) of the commonly used Cas9 from Streptococcus pyogenes (SpCas9) limits its utility for genome editing in the parasites only introduced with cas9 plasmid. To establish the endogenous and constitutive expression of Cas9 protein in the rodent malaria parasite P. yoelii, we replaced the coding region of an endogenous gene sera1 with the intact SpCas9 coding sequence using the CRISPR/Cas9-mediated genome editing method, generating the cas9-knockin parasite (PyCas9ki) of the rodent malaria parasite P. yoelii. The resulted PyCas9ki parasite displays normal progression during the whole life cycle and possesses the Cas9 protein expression in asexual blood stage. By introducing the plasmid (pYCs) containing only sgRNA and homologous template elements, we successfully achieved both deletion and tagging modifications for different endogenous genes in the genome of PyCas9ki parasite. This cas9-knockin PyCas9ki parasite provides a new platform facilitating gene functions study in the rodent malaria parasite P. yoelii., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Malaria parasites invade red blood cells (RBCs), consume copious amounts of hemoglobin, and severely disrupt iron regulation in humans. Anemia often accompanies malaria disease; however, iron supplementation therapy inexplicably exacerbates malarial infections. Here we found that the iron exporter ferroportin (FPN) was highly abundant in RBCs, and iron supplementation suppressed its activity. Conditional deletion of the Fpn gene in erythroid cells resulted in accumulation of excess intracellular iron, cellular damage, hemolysis, and increased fatality in malaria-infected mice. In humans, a prevalent FPN mutation, Q248H (glutamine to histidine at position 248), prevented hepcidin-induced degradation of FPN and protected against severe malaria disease. FPN Q248H appears to have been positively selected in African populations in response to the impact of malaria disease. Thus, FPN protects RBCs against oxidative stress and malaria infection., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Malaria is a fatal disease that displays a spectrum of symptoms and severity, which are determined by complex host-parasite interactions. It has been difficult to study the effects of parasite strains on disease severity in human infections, but the mechanisms leading to specific disease phenotypes can be investigated using strains of rodent malaria parasites that cause different disease symptoms in inbred mice. Using a unique mouse malaria model, here we investigated the mechanisms of splenic cell death and their relationship to control of parasitemia and host mortality. C57BL/6 mice infected with Plasmodium yoelii nigeriensis N67C display high levels of pro-inflammatory cytokines and chemokines (IL-6, IFN-γ, TNF-α, CXCL1, and CCL2) and extensive splenic damage with dramatic reduction of splenic cell populations. These disease phenotypes were rescued in RAG2 -/- , IFN-γ -/- , or T cell depleted mice, suggesting IFN-γ and T cell mediated disease mechanisms. Additionally, apoptosis was one of the major pathways involved in splenic cell death, which coincides with the peaks of pro-inflammatory cytokines. Our results demonstrate the critical roles of T cells and IFN-γ in mediating splenic cell apoptosis, parasitemia control, and host lethality and thus may provide important insights for preventing/reducing morbidity associated with severe malaria in humans.

Infection of mice with strains of Plasmodium yoelii parasites can result in different pathology, but molecular mechanisms to explain this variation are unclear. Here we show that a P. yoelii gene encoding a HECT-like E3 ubiquitin ligase (Pyheul) influences parasitemia and host mortality. We genetically cross two lethal parasites with distinct disease phenotypes, and identify 43 genetically diverse progeny by typing with microsatellites and 9230 single-nucleotide polymorphisms. A genome-wide quantitative trait loci scan links parasite growth and host mortality to two major loci on chromosomes 1 and 7 with LOD (logarithm of the odds) scores = 6.1 and 8.1, respectively. Allelic exchange of partial sequences of Pyheul in the chromosome 7 locus and modification of the gene expression alter parasite growth and host mortality. This study identifies a gene that may have a function in parasite growth, virulence, and host-parasite interaction, and therefore could be a target for drug or vaccine development.Many strains of Plasmodium differ in virulence, but factors that control these distinctions are not known. Here the authors comparatively map virulence loci using the offspring from a P. yoelii YM and N67 genetic cross, and identify a putative HECT E3 ubiquitin ligase that may explain the variance.

Parasitic protozoan diseases continue to rank among the world's greatest global health problems, which are also common among poor populations. Currently available drugs for treatment present drawbacks, urging the need for more effective, safer, and cheaper drugs. Artemisinin (ART) and its derivatives are some of the most important classes of antimalarial agents originally derived from Artemisia annua L. However, besides the outstanding antimalarial and antischistosomal activities, ART and its derivatives also possess activities against other parasitic protozoa. In this paper we review the activities of ART and its derivatives against protozoan parasites in vitro and in vivo, including Leishmania spp., Trypanosoma spp., Toxoplasma gondii, Neospora caninum, Eimeria tenella, Acanthamoeba castellanii, Naegleria fowleri, Cryptosporidium parvum, Giardia lamblia, and Babesia spp. We conclude that ART and its derivatives may be good alternatives for treating other non-malarial protozoan infections in developing countries, although more studies are necessary before they can be applied clinically., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Type I interferon (IFN) is critical for controlling pathogen infection; however, its regulatory mechanisms in plasmacytoid cells (pDCs) still remain unclear. Here, we have shown that nucleic acid sensors cGAS-, STING-, MDA5-, MAVS-, or transcription factor IRF3-deficient mice produced high amounts of type I IFN-α and IFN-β (IFN-α/β) in the serum and were resistant to lethal plasmodium yoelii YM infection. Robust IFN-α/β production was abolished when gene encoding nucleic acid sensor TLR7, signaling adaptor MyD88, or transcription factor IRF7 was ablated or pDCs were depleted. Further, we identified SOCS1 as a key negative regulator to inhibit MyD88-dependent type I IFN signaling in pDCs. Finally, we have demonstrated that pDCs, cDCs, and macrophages were required for generating IFN-α/β-induced subsequent protective immunity. Thus, our findings have identified a critical regulatory mechanism of type I IFN signaling in pDCs and stage-specific function of immune cells in generating potent immunity against lethal YM infection., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Both type I interferon (IFN-I) and CD40 play a significant role in various infectious diseases, including malaria and autoimmune disorders. CD40 is mostly known to function in adaptive immunity, but previous observations of elevated CD40 levels early after malaria infection of mice led us to investigate its roles in innate IFN-I responses and disease control. Using a Plasmodium yoelii nigeriensis N67 and C57BL/6 mouse model, we showed that infected CD40-/- mice had reduced STING and serum IFN-β levels day-2 post infection, higher day-4 parasitemia, and earlier deaths. CD40 could greatly enhance STING-stimulated luciferase signals driven by the IFN-β promoter in vitro, which was mediated by increased STING protein levels. The ability of CD40 to influence STING expression was confirmed in CD40-/- mice after malaria infection. Substitutions at CD40 TRAF binding domains significantly decreased the IFN-β signals and STING protein level, which was likely mediated by changes in STING ubiquitination and degradation. Increased levels of CD40, STING, and ISRE driven luciferase signal in RAW Lucia were observed after phagocytosis of N67-infected red blood cells (iRBCs), stimulation with parasite DNA/RNA, or with selected TLR ligands [LPS, poly(I:C), and Pam3CSK4]. The results suggest stimulation of CD40 expression by parasite materials through TLR signaling pathways, which was further confirmed in bone marrow derived dendritic cells/macrophages (BMDCs/BMDMs) and splenic DCs from CD40-/-, TLR3-/- TLR4-/-, TRIF-/-, and MyD88-/- mice after iRBC stimulation or parasite infection. Our data connect several signaling pathways consisting of phagocytosis of iRBCs, recognition of parasite DNA/RNA (possibly GPI) by TLRs, elevated levels of CD40 and STING proteins, increased IFN-I production, and longer host survival time. This study reveals previously unrecognized CD40 function in innate IFN-I responses and protective pathways in infections with malaria strains that induce a strong IFN-I response, which may provide important information for better understanding and management of malaria., Competing Interests: The authors have declared that no competing interests exist.

Malaria-associated acute lung injury (ALI) is a frequent complication of severe malaria that is often caused by "excessive" immune responses. To better understand the mechanism of ALI in malaria infection, here we investigated the roles of galectin (Gal)-1, 3, 8, 9 and the receptors of Gal-9 (Tim-3, CD44, CD137, and PDI) in malaria-induced ALI. We injected alpha (α)-lactose into mice-infected with Plasmodium berghei ANKA (PbANKA) to block galectins and found significantly elevated total proteins in bronchoalveolar lavage fluid, higher parasitemia and tissue parasite burden, and increased numbers of CD68(+) alveolar macrophages as well as apoptotic cells in the lungs after blockage. Additionally, mRNA levels of Gal-9, Tim-3, CD44, CD137, and PDI were significantly increased in the lungs at day 5 after infection, and the levels of CD137, IFN-α, IFN-β, IFN-γ, IL-4, and IL-10 in the lungs were also increased after α-lactose treatment. Similarly, the levels of Gal-9, Tim-3, IFN-α, IFN-β, IFN-γ, and IL-10 were all significantly increased in murine peritoneal macrophages co-cultured with PbANKA-infected red blood cells in vitro; but only IFN-α and IFN-β were significantly increased after α-lactose treatment. Our data indicate that Gal-9 interaction with its multiple receptors play an important role in murine malaria-associated ALI.

Invading pathogens trigger specific host responses, an understanding of which might identify genes that function in pathogen recognition and elimination. In this study, we performed trans-species expression quantitative trait locus (ts-eQTL) analysis using genotypes of the Plasmodium yoelii malaria parasite and phenotypes of mouse gene expression. We significantly linked 1,054 host genes to parasite genetic loci (LOD score ≥ 3.0). Using LOD score patterns, which produced results that differed from direct expression-level clustering, we grouped host genes that function in related pathways, allowing functional prediction of unknown genes. As a proof of principle, 14 of 15 randomly selected genes predicted to function in type I interferon (IFN-I) responses were experimentally validated using overexpression, small hairpin RNA knockdown, viral infection, and/or infection of knockout mice. This study demonstrates an effective strategy for studying gene function, establishes a functional gene database, and identifies regulators in IFN-I pathways., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Malaria infection triggers vigorous host immune responses; however, the parasite ligands, host receptors, and the signaling pathways responsible for these reactions remain unknown or controversial. Malaria parasites primarily reside within RBCs, thereby hiding themselves from direct contact and recognition by host immune cells. Host responses to malaria infection are very different from those elicited by bacterial and viral infections and the host receptors recognizing parasite ligands have been elusive. Here we investigated mouse genome-wide transcriptional responses to infections with two strains of Plasmodium yoelii (N67 and N67C) and discovered differences in innate response pathways corresponding to strain-specific disease phenotypes. Using in vitro RNAi-based gene knockdown and KO mice, we demonstrated that a strong type I IFN (IFN-I) response triggered by RNA polymerase III and melanoma differentiation-associated protein 5, not Toll-like receptors (TLRs), binding of parasite DNA/RNA contributed to a decline of parasitemia in N67-infected mice. We showed that conventional dendritic cells were the major sources of early IFN-I, and that surface expression of phosphatidylserine on infected RBCs might promote their phagocytic uptake, leading to the release of parasite ligands and the IFN-I response in N67 infection. In contrast, an elevated inflammatory response mediated by CD14/TLR and p38 signaling played a role in disease severity and early host death in N67C-infected mice. In addition to identifying cytosolic DNA/RNA sensors and signaling pathways previously unrecognized in malaria infection, our study demonstrates the importance of parasite genetic backgrounds in malaria pathology and provides important information for studying human malaria pathogenesis., Competing Interests: The authors declare no conflict of interest.

Plasmodium falciparum malaria, an infectious disease caused by a parasitic protozoan, claims the lives of nearly a million children each year in Africa alone and is a top public health concern. Evidence is accumulating that resistance to artemisinin derivatives, the frontline therapy for the asexual blood stage of the infection, is developing in southeast Asia. Renewed initiatives to eliminate malaria will benefit from an expanded repertoire of antimalarials, including new drugs that kill circulating P. falciparum gametocytes, thereby preventing transmission. Our current understanding of the biology of asexual blood-stage parasites and gametocytes and the ability to culture them in vitro lends optimism that high-throughput screenings of large chemical libraries will produce a new generation of antimalarial drugs. There is also a need for new therapies to reduce the high mortality of severe malaria. An understanding of the pathophysiology of severe disease may identify rational targets for drugs that improve survival.

Malaria is a deadly infectious disease in many tropical and subtropical countries. Previous efforts to eradicate malaria have failed, largely due to the emergence of drug-resistant parasites, insecticide-resistant mosquitoes and, in particular, the lack of drugs or vaccines to block parasite transmission. ATP-binding cassette (ABC) transporters are known to play a role in drug transport, metabolism, and resistance in many organisms, including malaria parasites. To investigate whether a Plasmodium falciparum ABC transporter (Pf14_0244 or PfABCG2) modulates parasite susceptibility to chemical compounds or plays a role in drug resistance, we disrupted the gene encoding PfABCG2, screened the recombinant and the wild-type 3D7 parasites against a library containing 2,816 drugs approved for human or animal use, and identified an antihistamine (ketotifen) that became less active against the PfABCG2-disrupted parasite in culture. In addition to some activity against asexual stages and gametocytes, ketotifen was highly potent in blocking oocyst development of P. falciparum and the rodent parasite Plasmodium yoelii in mosquitoes. Tests of structurally related tricyclic compounds identified additional compounds with similar activities in inhibiting transmission. Additionally, ketotifen appeared to have some activity against relapse of Plasmodium cynomolgi infection in rhesus monkeys. Further clinical evaluation of ketotifen and related compounds, including synthetic new derivatives, in blocking malaria transmission may provide new weapons for the current effort of malaria eradication.

Macrophage migration inhibitory factor (MIF) has been shown to be involved in the pathogenesis of severe malaria. Malaria parasites express an MIF homolog that may play a role in regulating host immune responses, and a recent study showed that overexpression of MIF reduced parasitemia in a mouse malaria model. Another recent study showed migration of monocytes to the spleen contributed to the control of blood stage infection. However, there are few papers describing the effect of MIF on monocyte recruitment/activation during the infection. We generated recombinant Plasmodium yoelii MIF (rPyMIF) and investigated its function on purified mouse CD11b(+) cells in vitro and monocyte responses in vivo. The result shows that rPyMIF protein bound to mouse CD11b(+) cells and inhibited their random migration in vitro. On the other hand, rPyMIF did not induce cytokine release from the cells directly or modulate lipopolysaccharide-induced cytokine release. Mice immunized with rPyMIF showed transient but significantly lower parasitemia than the control mice at day 3 after lethal Py17XL challenge. The total number of CD11b(+) cells in the spleens was significantly higher in rPyMIF-immunized group. Further investigation revealed that there were significantly higher numbers of recruited and activated monocytes in the spleens of rPyMIF immunization group on day 3. These results indicate that PyMIF potentially modulates monocyte recruitment and activation during infection of P. yoelii erythrocytic stages.

Of all the parasitic diseases, malaria is the number one killer. Despite tremendous efforts in disease control and research, nearly a million people, primarily children, still die from the disease each year, partly due to drug resistance and the lack of an effective vaccine. Many parasite antigens have been identified and evaluated for vaccine development; however, none has been approved for human use. Antigenic variation, complex life cycle, and inadequate understanding of the mechanisms of parasite-host interaction and of host immune response all contribute to the lack of an effective vaccine for malaria control. In a recent search of genome-wide polymorphism in Plasmodium falciparum, several molecules were found to be recognized by sera from patients infected with the P. falciparum parasite. Here, we have expressed a 350-amino acid N terminus from one of the homologous candidate antigen genes from the rodent malaria parasite Plasmodium yoelii (Py01157, a putative dentin phosphorin) in bacteria and evaluated the immune response and protection generated after immunization with the recombinant protein. We showed that the recombinant protein was recognized by sera from both mice and humans infected with malaria parasites. Partial protection was observed after challenge with non-lethal P. yoelii 17XNL but not with the lethal P. yoelii 17XL parasite. Further tests using a full-length protein or the conserved C terminus may provide additional information on whether this protein has the potential for being a malaria vaccine.

The Greater Mekong Subregion (GMS), comprised of six countries including Cambodia, China's Yunnan Province, Lao PDR, Myanmar (Burma), Thailand and Vietnam, is one of the most threatening foci of malaria. Since the initiation of the WHO's Mekong Malaria Program a decade ago, malaria situation in the GMS has greatly improved, reflected in the continuous decline in annual malaria incidence and deaths. However, as many nations are moving towards malaria elimination, the GMS nations still face great challenges. Malaria epidemiology in this region exhibits enormous geographical heterogeneity with Myanmar and Cambodia remaining high-burden countries. Within each country, malaria distribution is also patchy, exemplified by 'border malaria' and 'forest malaria' with high transmission occurring along international borders and in forests or forest fringes, respectively. 'Border malaria' is extremely difficult to monitor, and frequent malaria introductions by migratory human populations constitute a major threat to neighboring, malaria-eliminating countries. Therefore, coordination between neighboring countries is essential for malaria elimination from the entire region. In addition to these operational difficulties, malaria control in the GMS also encounters several technological challenges. Contemporary malaria control measures rely heavily on effective chemotherapy and insecticide control of vector mosquitoes. However, the spread of multidrug resistance and potential emergence of artemisinin resistance in Plasmodium falciparum make resistance management a high priority in the GMS. This situation is further worsened by the circulation of counterfeit and substandard artemisinin-related drugs. In most endemic areas of the GMS, P. falciparum and Plasmodium vivax coexist, and in recent malaria control history, P. vivax has demonstrated remarkable resilience to control measures. Deployment of the only registered drug (primaquine) for the radical cure of vivax malaria is severely undermined due to high prevalence of glucose-6-phosphate dehydrogenase deficiency in target human populations. In the GMS, the dramatically different ecologies, diverse vector systems, and insecticide resistance render traditional mosquito control less efficient. Here we attempt to review the changing malaria epidemiology in the GMS, analyze the vector systems and patterns of malaria transmission, and identify the major challenges the malaria control community faces on its way to malaria elimination., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Malaria transmission requires the production of male and female gametocytes in the human host followed by fertilization and sporogonic development in the mosquito midgut. Although essential for the spread of malaria through the population, little is known about the initiation of gametocytogenesis in vitro or in vivo. Using a gametocyte-defective parasite line and genetic complementation, we show that Plasmodium falciparumgametocyte development 1 gene (Pfgdv1), encoding a peri-nuclear protein, is critical for early sexual differentiation. Transcriptional analysis of Pfgdv1 negative and positive parasite lines identified a set of gametocytogenesis early genes (Pfge) that were significantly down-regulated (>10 fold) in the absence of Pfgdv1 and expression was restored after Pfgdv1 complementation. Progressive accumulation of Pfge transcripts during successive rounds of asexual replication in synchronized cultures suggests that gametocytes are induced continuously during asexual growth. Comparison of Pfge gene transcriptional profiles in patient samples divided the genes into two groups differing in their expression in mature circulating gametocytes and providing candidates to evaluate gametocyte induction and maturation separately in vivo. The expression profile of one of the early gametocyte specific genes, Pfge1, correlated significantly with asexual parasitemia, which is consistent with the ongoing induction of gametocytogenesis during asexual growth observed in vitro and reinforces the need for sustained transmission-blocking strategies to eliminate malaria.

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to successful identification of genes or loci underlying various traits in malaria parasites of rodents and humans. The malaria parasite Plasmodium yoelii is one of many malaria species isolated from wild African rodents and has been adapted to grow in laboratories. This species reproduces many of the biologic characteristics of the human malaria parasites; genetic markers such as microsatellite and amplified fragment length polymorphism (AFLP) markers have also been developed for the parasite. Thus, genetic studies in rodent malaria parasites can be performed to complement research on Plasmodium falciparum. Here, we demonstrate the techniques for producing a genetic cross in P. yoelii that were first pioneered by Drs. David Walliker, Richard Carter, and colleagues at the University of Edinburgh. Genetic crosses in P. yoelii and other rodent malaria parasites are conducted by infecting mice Mus musculus with an inoculum containing gametocytes of two genetically distinct clones that differ in phenotypes of interest and by allowing mosquitoes to feed on the infected mice 4 days after infection. The presence of male and female gametocytes in the mouse blood is microscopically confirmed before feeding. Within 48 hrs after feeding, in the midgut of the mosquito, the haploid gametocytes differentiate into male and female gametes, fertilize, and form a diploid zygote (Fig. 1). During development of a zygote into an ookinete, meiosis appears to occur. If the zygote is derived through cross-fertilization between gametes of the two genetically distinct parasites, genetic exchanges (chromosomal reassortment and cross-overs between the non-sister chromatids of a pair of homologous chromosomes; Fig. 2) may occur, resulting in recombination of genetic material at homologous loci. Each zygote undergoes two successive nuclear divisions, leading to four haploid nuclei. An ookinete further develops into an oocyst. Once the oocyst matures, thousands of sporozoites (the progeny of the cross) are formed and released into mosquito hemoceal. Sporozoites are harvested from the salivary glands and injected into a new murine host, where pre-erythrocytic and erythrocytic stage development takes place. Erythrocytic forms are cloned and classified with regard to the characters distinguishing the parental lines prior to genetic linkage mapping. Control infections of individual parental clones are performed in the same way as the production of a genetic cross.

Genetic crosses have been employed to study various traits of rodent malaria parasites and to locate loci that contribute to drug resistance, immune protection, and disease virulence. Compared with human malaria parasites, genetic crossing of rodent malaria parasites is more easily performed; however, genotyping methods using microsatellites (MSs) or large-scale single nucleotide polymorphisms (SNPs) that have been widely used in typing Plasmodium falciparum are not available for rodent malaria species. Here we report a genome-wide search of the Plasmodium yoelii yoelii (P. yoelii) genome for simple sequence repeats (SSRs) and the identification of nearly 600 polymorphic MS markers for typing the genomes of P. yoelii and Plasmodium berghei. The MS markers are randomly distributed across the 14 physical chromosomes assembled from genome sequences of three rodent malaria species, although some variations in the numbers of MS expected according to chromosome size exist. The majority of the MS markers are AT-rich repeats, similar to those found in the P. falciparum genome. The MS markers provide an important resource for genotyping, lay a foundation for developing linkage maps, and will greatly facilitate genetic studies of P. yoelii.

VAR2CSA is the main candidate for a pregnancy malaria vaccine, but vaccine development may be complicated by sequence polymorphism. Here, we obtained partial or full-length var2CSA sequences from 106 parasites and applied novel computational methods and three-dimensional modeling to investigate VAR2CSA geographic variation and selection pressure. Our analysis reveals structural patterns of VAR2CSA sequence variation in which polymorphic sites group into segments of limited diversity. Within these segments, two or three basic types characterize a substantial majority of the parasite samples. Comparison to the primate malaria Plasmodium reichenowi shows that these basic types have ancient origins. Globally, var2CSA genes are comprised of a mosaic of these ancestral polymorphic segments that have recombined extensively between var2CSA alleles. Three-dimensional modeling reveals that polymorphic segments concentrate in flexible loops at characteristic locations in the six VAR2CSA Duffy binding-like (DBL) adhesion domains. Individual DBL domain surfaces have distinct patterns of diversifying selection, suggesting that limited and differing portions of each DBL domain are targeted by host antibody. Since standard phylogenetic tree analysis is inadequate for highly recombining genes like var2CSA, we developed a novel phylogenetic approach that incorporates recombination and tracks new mutations in segment types. In the resulting tree, P. reichenowi is confirmed as an outlier and African and Asian P. falciparum isolates have slightly diverged. These findings validate a new approach to modeling protein evolution in the presence of frequent recombination and provide a clearer understanding of how var gene products function as immunoevasive binding ligands.

Despite significant improvement in the malaria situation of the Greater Mekong Subregion (GMS), malaria control for the region continues to face a multitude of challenges. The extremely patchy malaria distribution, especially along international borders, makes disease surveillance and targeted control difficult. The vector systems are also diverse with dramatic differences in habitat ecology, biting behavior, and vectorial capacity, and there is a lack of effective transmission surveillance and control tools. Finally, in an era of heavy deployment of artemisinin-based combination therapies, the region acts as an epicenter of drug resistance, with the emergence of artemisinin resistant Plasmodium falciparum posing a threat to both regional and global malaria elimination campaigns. This problem is further exacerbated by the circulation of counterfeit and substandard artemisinin drugs. Accordingly, this Southeast Asian Malaria Research Center, consisting of a consortium of US and regional research institutions, has proposed four interlinked projects to address these most urgent problems in malaria control. The aims of these projects will help to substantially improve our understanding of malaria epidemiology, vector systems and their roles in malaria transmission, as well as the mechanisms of drug resistance in parasites. Through the training of next-generation scientists in malaria research, this program will help build up and strengthen regional research infrastructure and capacities, which are essential for sustained malaria control in this region.

Cyclic GMP‐AMP synthase (cGAS) and stimulator of interferon genes (STING) play critical roles in the innate immunity against infectious diseases and are required to link pathogen DNA sensing to immune responses. However, the mechanisms by which cGAS‐STING‐induced cytokines suppress the adaptive immune response against malaria infections remain poorly understood. Here, cGAS‐STING signaling is identified to play a detrimental role in regulating anti‐malaria immunity. cGAS or STING deficiency in mice markedly prolongs mouse survival during lethal malaria Plasmodium yoelii nigeriensis N67C infections by reducing late interleukin (IL)‐6 production. Mechanistically, cGAS/STING recruits myeloid differentiation factor 88 (MyD88) and specifically induces the p38‐dependent signaling pathway for late IL‐6 production, which, in turn, expands CD11b+Ly6Chi proinflammatory monocytes to inhibit immunity. Moreover, the blockage or ablation of the cGAS‐STING‐MyD88‐p38‐IL‐6 signaling axis or the depletion of CD11b+Ly6Chi proinflammatory monocytes provides mice a significant survival benefit during N67C and other lethal malaria‐strain infections. Taken together, these findings identify a previously unrecognized detrimental role of cGAS‐STING‐MyD88‐p38 axis in infectious diseases through triggering the late IL‐6 production and proinflammatory monocyte expansion and provide insight into how targeting the DNA sensing pathway, dysregulated cytokines, and proinflammatory monocytes enhances immunity against infection. [ABSTRACT FROM AUTHOR]