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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]

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]

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. [ABSTRACT FROM AUTHOR]

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]

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]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Parasites, bacteria, and viruses pose serious threats to public health. Many parasite infections, including infections of protozoa and helminths, can inhibit inflammatory responses and impact disease outcomes caused by viral, bacterial, or other parasitic infections. Type I interferon (IFN-I) has been recognized as an essential immune effector in the host defense against various pathogens. In addition, IFN-I responses induced by co-infections with different pathogens may vary according to the host genetic background, immune status, and pathogen burden. However, there is only limited information on the roles of IFN-I in co-infections with parasites and viruses, bacteria, or other parasites. This review summarizes some recent findings on the roles of IFN-I in co-infections with parasites, including Leishmania spp., Plasmodium spp., Eimeria maxima, Heligmosomoides polygyrus, Brugia malayi , or Schistosoma mansoni , and viruses or bacteria and co-infections with different parasites (such as co-infection with Neospora caninum and Toxoplasma gondii , and co-infection with Plasmodium spp. and H. polygyrus). The potential mechanisms of host responses associated with co-infections, which may provide targets for immune intervention and therapies of the co-infections, are also discussed. [ABSTRACT FROM AUTHOR]

Gametocytes differentiation to gametes (gametogenesis) within mosquitos is essential for malaria parasite transmission. Both reduction in temperature and mosquito-derived XA or elevated pH are required for triggering cGMP/PKG dependent gametogenesis. However, the parasite molecule for sensing or transducing these environmental signals to initiate gametogenesis remains unknown. Here we perform a CRISPR/Cas9-based functional screening of 59 membrane proteins expressed in the gametocytes of Plasmodium yoelii and identify that GEP1 is required for XA-stimulated gametogenesis. GEP1 disruption abolishes XA-stimulated cGMP synthesis and the subsequent signaling and cellular events, such as Ca2+ mobilization, gamete formation, and gametes egress out of erythrocytes. GEP1 interacts with GCα, a cGMP synthesizing enzyme in gametocytes. Both GEP1 and GCα are expressed in cytoplasmic puncta of both male and female gametocytes. Depletion of GCα impairs XA-stimulated gametogenesis, mimicking the defect of GEP1 disruption. The identification of GEP1 being essential for gametogenesis provides a potential new target for intervention of parasite transmission. Mosquito-derived xanthurenic acid (XA) is a trigger for gametogenesis of Plasmodium parasites. Here the authors show that the putative amino acid transporter GEP1 is required for XA-stimulated gametogenesis in Plasmodium yoelii and that it interacts with guanylyl cyclase α (GCα), a cGMP synthesizing enzyme in gametocytes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Leucocytozoon parasites infect many species of avian hosts, including domestic chicken, and can inflict heavy economic loss on the poultry industry. Two major species of Leucocytozoon parasites have been reported in China, L. sabrazesi and L. caulleryi, although L. sabrazesi appears to be more widespread than L. caulleryi in southern China. The traditional method for detecting Leucocytozoon infection is microscopic examination of blood smears for the presence of mature gametocytes in circulation, which may miss infections with low parasitemia (gametocytemia) or immature gametocytes. Here we developed a PCR-based method to monitor L. sabrazesi infections at seven sites in four provinces of China after testing two PCR primer pairs based on parasite mitochondrial cytochrome b (cytb) and cytochrome c oxidase III (coxIII) genes. We compared the results of PCR detection with those of microscopic observation. As expected, the PCR assays were more sensitive than microscope examination in detecting L. sabrazesi infection and were able to detect parasite DNA after gametocytes disappeared in the blood stream. Using these methods, we investigated monthly dynamics of L. sabrazesi in chickens from a free-range farm in Xiamen, Fujian province of China, over one year. Our results showed that chickens were infected with L. sabrazesi year-round in southern China. Finally, we tested several compounds for potential treatment of Leucocytozoon infections, including primaquine, ketotifen, clomipramine hydrochloride, desipramine hydrochloride, sulfaquinoxaline, and pyrimethamine. Only primaquine had activity against L. sabrazesi gametocytes. Our results provide important information for controlling parasite transmission in southern China and disease management. [ABSTRACT FROM AUTHOR]

Leucocytozoon parasites infect a large number of avian hosts, including domestic chicken, and cause significant economical loss to the poultry industry. Although the transmission stages of the parasites were observed in avian blood cells more than a century ago, the specific host cell type(s) that the gametocytes infect remain uncertain. Because all the avian blood cells, including red blood cells (RBCs), are nucleated, and the developing parasites dramatically change the morphology of the infected host cells, it has been difficult to identify Leucocytozoon infected host cell(s). Here we use cell-type specific antibodies to investigate the identities of the host cells infected by Leucocytozoon sabrazesi gametocytes. Anti-RBC antibodies stained RBCs membrane strongly, but not the parasite-infected cells, ruling out the possibility of RBCs being the infected host cells. Antibodies recognizing various leukocytes including heterophils, monocytes, lymphocytes, and macrophages did not stain the infected cells either. Antisera raised against a peptide of the parasite cytochrome B (CYTB) stained parasite-infected cells and some leukocytes, particularly cells with a single round nucleus as well as clear/pale cytoplasm suggestive of thrombocytes. Finally, a monoclonal antibody known to specifically bind chicken thrombocytes also stained the infected cells, confirming that L. sabrazesi gametocytes develop within chicken thrombocytes. The identification of L. sabrazesi infected host cell solves a long unresolved puzzle and provides important information for studying parasite invasion of host cells and for developing reagents to interrupt parasite transmission. [ABSTRACT FROM AUTHOR]

Summary 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. [ABSTRACT FROM AUTHOR]

Leucocytozoon parasites infect many species of avian hosts, including domestic chicken, and can inflict heavy economic loss to the poultry industry. Although the prevalence and distribution of two Leucocytozoon species (L. sabrazesi and L. caulleryi) have been reported in China previously, there are many questions related to the parasite infection that remain unanswered, including population diversity and transmission dynamics in domestic chickens. Here we surveyed chicken blood samples from seven sites in four provinces of China to identify Leucocytozoon infection, characterized parasite diversity within individual infected hosts and between sampling sites, and investigated the dynamics of gametocytemia in chickens over time. We found high infection rates in three of the seven sites. Clustering parasite sequences of the mitochondrial cytochrome oxidase III (coxIII) and cytochrome b (cytb) genes showed lack of grouping according to geographic origins and individual hosts carrying large numbers of L. sabrazesi strains. Monitoring gametocytemia in blood samples from infected chickens over time showed ‘relapse’ or persistence of low-level gametocytemia for 4–5 months, which could be explored as an in vivo model for testing drugs against liver stages of Apicomplexan parasites. This study provides important information on population diversity and transmission dynamics of L. sabrazesi and for disease control. [ABSTRACT FROM AUTHOR]

Malaria parasites are transmitted by mosquitoes, and blocking parasite transmission is critical in reducing or eliminating malaria in endemic regions. Here, we report the pharmacological characterization of a new class of malaria transmission-blocking compounds that acts via the inhibition of Plasmodia CDPK4 enzyme. We demonstrate that these compounds achieved selectivity over mammalian kinases by capitalizing on a small serine gatekeeper residue in the active site of the Plasmodium CDPK4 enzyme. To directly confirm the mechanism of action of these compounds, we generated P. falciparum parasites that express a drug-resistant methionine gatekeeper (S147M) CDPK4 mutant. Mutant parasites showed a shift in exflagellation EC50 relative to the wild-type strains in the presence of compound 1294, providing chemical-genetic evidence that CDPK4 is the target of the compound. Pharmacokinetic analyses suggest that coformulation of this transmission-blocking agent with asexual stage antimalarials such as artemisinin combination therapy (ACT) is a promising option for drug delivery that may reduce transmission of malaria including drug-resistant strains. Ongoing studies include refining the compounds to improve efficacy and toxicological properties for efficient blocking of malaria transmission. [ABSTRACT FROM AUTHOR]

Spatial relationships within the eukaryotic nucleus are essential for proper nuclear function. In Plasmodium falciparum, the repositioning of chromosomes has been implicated in the regulation of the expression of genes responsible for antigenic variation, and the formation of a single, peri-nuclear nucleolus results in the clustering of rDNA. Nevertheless, the precise spatial relationships between chromosomes remain poorly understood, because, until recently, techniques with sufficient resolution have been lacking. Here we have used chromosome conformation capture and second-generation sequencing to study changes in chromosome folding and spatial positioning that occur during switches in var gene expression. We have generated maps of chromosomal spatial affinities within the P. falciparum nucleus at 25 Kb resolution, revealing a structured nucleolus, an absence of chromosome territories, and confirming previously identified clustering of heterochromatin foci. We show that switches in var gene expression do not appear to involve interaction with a distant enhancer, but do result in local changes at the active locus. These maps reveal the folding properties of malaria chromosomes, validate known physical associations, and characterize the global landscape of spatial interactions. Collectively, our data provide critical information for a better understanding of gene expression regulation and antigenic variation in malaria parasites. [ABSTRACT FROM AUTHOR]

Cerebral malaria (CM) is a serious and often fatal complication of Plasmodium falciparum infections; however, the precise mechanisms leading to CM is poorly understood. Mouse malaria models have provided insight into the key events in pathogenesis of CM. T-cell immune response is known to play an important role in malaria infection, and members of the T-cell immunoglobulin- and mucin-domain-containing molecule (Tim) family have roles in T-cell-mediated immune responses. Tim-1 and Tim-3 are expressed on terminally differentiated Th2 and Th1 cells, respectively, and participate in the regulation of Th immune response. Until now, the role of Tim family proteins in Plasmodium infection remains unclear. In the present study, the mRNA levels of Tim-1, Tim-3, and some key Th1 and Th2 cytokines in the spleen of Kunming outbred mice infected with Plasmodium berghei ANKA ( PbANKA) were determined using real-time polymerase chain reaction (qRT-PCR). Compared with uninfected controls, Tim-1 expression was significantly decreased in infected mice with CM at day 10 postinfection (p.i.) but significantly increased in infected mice with non-CM at day 22 p.i.; in contrast, Tim-3 expression was significantly increased in infected mice both with CM at day 10 p.i. and with non-CM at day 22 p.i. The expressions of IFN-γ, TNF-α, IL-10, and IL-12 were significantly increased but IL-4 was significantly decreased in infected mice with CM at days 10 p.i., whereas the expressions of IFN-γ, TNF-α, IL-4, IL-10, and TGF-β were significantly increased but IL-12 was significantly decreased in infected mice with non-CM at days 22 p.i. Furthermore, the expression of Tim-1 and Tim-3 could reflect Th2 and Th1 immune response in the spleen of PbANKA-infected mice, respectively. Our data suggest that PbANKA infection could inhibit the differentiation of T lymphocytes toward Th2 cells, promote the Th1 cell differentiation, and induce Th1-biased immune response in the early infective stage, whereas the infection could promote Th2 cell differentiation and induce Th2-biased immune response in the late infective stage. Our data indicate that both Tim-1 and Tim-3 may play a role in the process of PbANKA infection, which may represent a potential therapeutic target. [ABSTRACT FROM AUTHOR]

The recent detection of clinical Artemisinin (ART) resistance manifested as delayed parasite clearance in the Cambodia-Thailand border area raises a serious concern. The mechanism of ART resistance is not clear; but the P. falciparum sarco/endoplasmic reticulum Ca2+-ATPase (PfSERCA or PfATP6) has been speculated to be the target of ARTs and thus a potential marker for ART resistance. Here we amplified and sequenced pfatp6 gene (∼3.6 Kb) in 213 samples collected after 2005 from the Greater Mekong Subregion, where ART drugs have been used extensively in the past. A total of 24 single nucleotide polymorphisms (SNPs), including 8 newly found in this study and 13 nonsynonymous, were identified. However, these mutations were either uncommon or also present in other geographical regions with limited ART use. None of the mutations were suggestive of directional selection by ARTs. We further analyzed pfatp6 from a worldwide collection of 862 P. falciparum isolates in 19 populations from Asia, Africa, South America and Oceania, which include samples from regions prior to and after deployments ART drugs. A total of 71 SNPs were identified, resulting in 106 nucleotide haplotypes. Similarly, many of the mutations were continent-specific and present at frequencies below 5%. The most predominant and perhaps the ancestral haplotype occurred in 441 samples and was present in 16 populations from Asia, Africa, and Oceania. The 3D7 haplotype found in 54 samples was the second most common haplotype and present in nine populations from all four continents. Assessment of the selection strength on pfatp6 in the 19 parasite populations found that pfatp6 in most of these populations was under purifying selection with an average dN/dS ratio of 0.333. Molecular evolution analyses did not detect significant departures from neutrality in pfatp6 for most populations, challenging the suitability of this gene as a marker for monitoring ART resistance. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

BACKGROUND: Although transmission of Plasmodium falciparum (Pf) infection during red blood cell (RBC) transfusion from an infected donor has been well documented, malaria parasites are not known to infect hematopoietic stem cells. We report a case of Pf infection in a patient 11 days after peripheral blood stem cell transplant for sickle cell disease. STUDY DESIGN AND METHODS: Malaria parasites were detected in thick blood smears by Giemsa staining. Pf HRP2 antigen was measured by enzyme-linked immunosorbent assay on whole blood and plasma. Pf DNA was detected in whole blood and stem cell retention samples by real-time polymerase chain reaction using Pf species-specific primers and probes. Genotyping of eight Pf microsatellites was performed on genomic DNA extracted from whole blood. RESULTS: Pf was not detected by molecular, serologic, or parasitologic means in samples from the recipient until Day 11 posttransplant, coincident with the onset of symptoms. In contrast, Pf antigen was retrospectively detected in stored plasma collected 3 months before transplant from the asymptomatic donor. Pf DNA was detected in whole blood from both the donor and the recipient after transplant, and genotyping confirmed shared markers between donor and recipient Pf strains. Lookback analysis of RBC donors was negative for Pf infection. CONCLUSIONS: These findings are consistent with transmission by the stem cell product and have profound implications with respect to the screening of potential stem cell donors and recipients from malaria-endemic regions. [ABSTRACT FROM AUTHOR]

The recent reports of artemisinin (ART) resistance in the Thai-Cambodian border area raise a serious concern on the long-term efficacy of ARTs. To elucidate the resistance mechanisms, we performed in vitro selection with dihydroartemisinin (DHA) and obtained two parasite clones from Dd2 with more than 25-fold decrease in susceptibility to DHA. The DHA-resistant clones were more tolerant of stressful growth conditions and more resistant to several commonly used antimalarial drugs than Dd2. The result is worrisome as many of the drugs are currently used as ART partners in malaria control. This study showed that the DHA resistance is not limited to ring stage, but also occurred in trophozoites and schizonts. Microarray and biochemical analyses revealed pfmdr1 amplification, elevation of the antioxidant defence network, and increased expression of many chaperones in the DHA-resistant parasites. Without drug pressure, the DHA-resistant parasites reverted to sensitivity in approximately 8 weeks, accompanied by de-amplification of pfmdr1 and reduced antioxidant activities. The parallel decrease and increase in pfmdr1 copy number and antioxidant activity and the up and down of DHA sensitivity strongly suggest that pfmdr1 and antioxidant defence play a role in in vitro resistance to DHA, providing potential molecular markers for ART resistance. [ABSTRACT FROM AUTHOR]

Malaria elimination strategies require surveillance of the parasite population for genetic changes that demand a public health response, such as new forms of drug resistance. Here we describe methods for the large-scale analysis of genetic variation in Plasmodium falciparum by deep sequencing of parasite DNA obtained from the blood of patients with malaria, either directly or after short-term culture. Analysis of 86,158 exonic single nucleotide polymorphisms that passed genotyping quality control in 227 samples from Africa, Asia and Oceania provides genome-wide estimates of allele frequency distribution, population structure and linkage disequilibrium. By comparing the genetic diversity of individual infections with that of the local parasite population, we derive a metric of within-host diversity that is related to the level of inbreeding in the population. An open-access web application has been established for the exploration of regional differences in allele frequency and of highly differentiated loci in the P.?falciparum genome. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Drug resistance in malaria parasites is a serious public health burden, and resistance to most of the antimalarial drugs currently in use has been reported. A better understanding of the molecular mechanisms of drug resistance is urgently needed to slow or circumvent the spread of resistance, to allow local treatments to be deployed more effectively to prolong the life span of the current drugs, and to develop new drugs. Although mutations in genes determining resistance to drugs such as chloroquine and the antifolates have been identified, we still do not have a full understanding of the resistance mechanisms, and genes that contribute to resistance to many other drugs remain to be discovered. Genetic mapping is a powerful tool for the identification of mutations conferring drug resistance in malaria parasites because most drug-resistant phenotypes were selected within the past 60 years. High-throughput methods for genotyping large numbers of single nucleotide polymorphisms (SNPs) and microsatellites (MSs) are now available or are being developed, and genome-wide association studies for malaria traits will soon become a reality. Here we discuss strategies and issues related to mapping genes contributing to drug resistance in the human malaria parasite Plasmodium falciparum. [ABSTRACT FROM AUTHOR]

One goal in sequencing the Plasmodium falciparum genome, the agent of the most lethal form of malaria, is to discover vaccine and drug targets. However, identifying those targets in a genome in which ∼60% of genes have unknown functions is an enormous challenge. Because the majority of known malaria antigens and drug-resistant genes are highly polymorphic and under various selective pressures, genome-wide analysis for signatures of selection may lead to discovery of new vaccine and drug candidates. Here we surveyed 3,539 P. falciparum genes (∼65% of the predicted genes) for polymorphisms and identified various highly polymorphic loci and genes, some of which encode new antigens that we confirmed using human immune sera. Our collections of genome-wide SNPs (∼65% nonsynonymous) and polymorphic microsatellites and indels provide a high-resolution map (one marker per ∼4 kb) for mapping parasite traits and studying parasite populations. In addition, we report new antigens, providing urgently needed vaccine candidates for disease control. [ABSTRACT FROM AUTHOR]

Understanding the influences of population structure, selection, and recombination on polymorphism and linkage disequilibrium (LD) is integral to mapping genes contributing to drug resistance or virulence in Plasmodium falciparum. The parasite's short generation time, coupled with a high cross-over rate, can cause rapid LD break-down. However, observations of low genetic variation have led to suggestions of effective clonality: selfing, population admixture, and selection may preserve LD in populations. Indeed, extensive LD surrounding drug-resistant genes has been observed, indicating that recombination and selection play important roles in shaping recent parasite genome evolution. These studies, however, provide only limited information about haplotype variation at local scales. Here we describe the first (to our knowledge) chromosome-wide SNP haplotype and population recombination maps for a global collection of malaria parasites, including the 3D7 isolate, whose genome has been sequenced previously. The parasites are clustered according to continental origin, but alternative groupings were obtained using SNPs at 37 putative transporter genes that are potentially under selection. Geographic isolation and highly variable multiple infection rates are the major factors affecting haplotype structure. Variation in effective recombination rates is high, both among populations and along the chromosome, with recombination hotspots conserved among populations at chromosome ends. This study supports the feasibility of genome-wide association studies in some parasite populations. [ABSTRACT FROM AUTHOR]

Quinine (QN) remains effective against Plasmodium falciparum, but its decreasing efficacy is documented from different continents. Multiple genes are likely to contribute to the evolution of QN resistance. To locate genes contributing to QN response variation, we have searched a P. falciparum genetic cross for quantitative trait loci (QTL). Results identify additive QTL in segments of chromosomes (Chrs) 13, 7 and 5, and pairwise effects from two additional loci of Chrs 9 and 6 that interact, respectively, with the QTL of Chrs 13 and 7. The mapped segments of Chrs 7 and 5 contain pfcrt, the determinant of chloroquine resistance (CQR), and pfmdr1, a gene known to affect QN responses. Association of pfcrt with a QTL of QN resistance supports anecdotal evidence for an evolutionary relationship between CQR and reduced QN sensitivity. The Chr 13 segment contains several candidate genes, one of which ( pfnhe-1) encodes a putative Na+/H+ exchanger. A repeat polymorphism in pfnhe-1 shows significant association with low QN response in a collection of P. falciparum strains from Asia, Africa and Central and South America. Dissection of the genes and modifiers involved in QN response will require experimental strategies that can evaluate multiple genes from different chromosomes in combination. [ABSTRACT FROM AUTHOR]

The malaria parasite lives within erythrocytes and depends on the binding of parasite ligands to host cell surface receptors for invasion. The most virulent human malaria parasite, Plasmodium falciparum, uses multiple ligands, including EBA-175, BAEBL and JESEBL of the Duffy-binding-like (DBL) family of erythrocyte-binding proteins, for invasion of human erythrocytes. Region II of these parasite ligands is the erythrocyte-binding domain. Previously, we had shown that polymorphism in region II of BAEBL leads to different erythrocyte-binding specificities. We have now identified and characterized the binding specificity of six JESERL variants. We sequenced region II of JESEBL from 20 P. falciparum clones collected from various parts of the world where malaria is endemic. We observed eight JESEBL variants that contained amino acid polymorphisms at five positions among all clones. Seven of the eight variants could be connected by a single base change that led to an amino acid change. We investigated the functional significance of these polymorphisms by transiently expressing region II from six of JESEBL variants on the surface of Chinese hamster ovary cells. We observed four erythrocyte-binding patterns to enzyme-treated erythrocytes. Thus, P. falciparum DBL ligands JESEBL and BAEBL can recognize multiple receptors on the erythrocyte surface. In contrast to Plasmodium vivax, which has disappeared from West Africa because of the Duffy-negative blood group, P. falciparum may have been successful in endemic areas because it has mutated the ligands of the DBL family to create multiple pathways of invasion, thus making selection of refractory erythrocytes unlikely. [ABSTRACT FROM AUTHOR]

Summary Mutations and/or overexpression of various transporters are known to confer drug resistance in a variety of organisms. In the malaria parasite Plasmodium falciparum , a homologue of P-glycoprotein, PfMDR1, has been implicated in responses to chloroquine (CQ), quinine (QN) and other drugs, and a putative transporter, PfCRT, was recently demonstrated to be the key molecule in CQ resistance. However, other unknown molecules are probably involved, as different parasite clones carrying the same pfcrt and pfmdr1 alleles show a wide range of quantitative responses to CQ and QN. Such molecules may contribute to increasing incidences of QN treatment failure, the molecular basis of which is not understood. To identify additional genes involved in parasite CQ and QN responses, we assayed the in vitro susceptibilities of 97 culture-adapted cloned isolates to CQ and QN and searched for single nucleotide polymorphisms (SNPs) in DNA encoding 49 putative transporters (total 113 kb) and in 39 housekeeping genes that acted as negative controls. SNPs in 11 of the putative transporter genes, including pfcrt and pfmdr1 , showed significant associations with decreased sensitivity to CQ and/or QN in P. falciparum . Significant linkage disequilibria within and between these genes were also detected, suggesting interactions among the transporter genes. This study provides specific leads for better understanding of complex drug resistances in malaria parasites. [ABSTRACT FROM AUTHOR]

Chloroquine (CQ)-resistant Plasmodium vivax malaria was first reported 12 years ago, nearly 30 years after the recognition of CQ-resistant P. falciparum. Loss of CQ efficacy now poses a severe problem for the prevention and treatment of both diseases. Mutations in a digestive vacuole protein encoded by a 13-exon gene, pfcrt, were shown recently to have a central role in the CQ resistance (CQR) of P. falciparum. Whether mutations in pfcrt orthologues of other Plasmodium species are involved in CQR remains an open question. This report describes pfcrt homologues from P. vivax, P. knowlesi, P. berghei, and Dictyostelium discoideum. Synteny between the P. falciparum and P. vivax genes is demonstrated. However, a survey of patient isolates and monkey-adapted lines has shown no association between in vivo CQR and codon mutations in the P. vivax gene. This is evidence that the molecular events underlying P. vivax CQR differ from those in P. falciparum. [ABSTRACT FROM AUTHOR]

Abstract Microsatellite loci are generally assumed to evolve via a stepwise mutational process and a battery of statistical techniques has been developed in recent years based on this or related mutation models. It is therefore important to investigate the appropriateness of these models in a wide variety of taxa. We used two approaches to examine mutation patterns in the malaria parasite Plasmodium falciparum: (i) we examined sequence variation at 12 tri-nucleotide repeat loci; and (ii) we analysed patterns of repeat structure and heterozygosity at 114 loci using data from 12 laboratory parasite lines. The sequencing study revealed complex patterns of mutation in five of the 12 loci studied. Alleles at two loci contain indels of 24 bp and 57 bp in flanking regions, while in the other three loci, blocks of imperfect microsatellites appear to be duplicated or inserted; these loci essentially consist of minisatellite repeats, with each repeat unit containing four to eight microsatellites. The survey of heterozygosity revealed a positive relationship between repeat number and microsatellite variability for both di- and trinucleotides, indicating a higher mutation rate in loci with longer repeat arrays. Comparisons of levels of variation in different repeat types indicate that the mutation rate of dinucleotide-bearing loci is 1.6–2.1 times faster than trinucleotides, consistent with the lower mean number of repeats in trinucleotide-bearing loci. However, despite the evidence that microsatellite arrays themselves are evolving in a manner consistent with stepwise mutation model in P. falciparum, the high frequency of complex mutations precludes the use of analytical tools based on this mutation model for many microsatellite-bearing loci in this protozoan. The results call into question the generality of models based on stepwise mutation for analysing microsatellite data, but also demonstrate the ease with which loci that violate model assumptions can be detected using minimal sequencing effort. [ABSTRACT FROM AUTHOR]

Autochthonous Plasmodium falciparum malaria (PFM) in Central Europe has been reported repeatedly, transmission of the parasite being attributed to blood transfusion or imported P. falciparum-infected vectors. We report two cases of PFM in German children without travel history to malaria-endemic areas. Both infections occurred during a stay in a hospital where a child from Angola with chronic P. falciparum infection was hospitalized at the time. Known routes of transmission, such as imported mosquitoes or blood transfusion, were very unlikely or could be excluded, whereas evidence was obtained for transmission by the indigenous mosquito species Anopheles plumbeus. [ABSTRACT FROM AUTHOR]

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. The inflammasome is an essential component of inflammatory processes and the host response to infection. Here the authors show that inflammasome activation modulates MyD88-IRF7 type I IFN signalling and anti-malaria immunity. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The author talks about how his laboratory at the U.S. National Institutes of Health (NIH) is developing disease-control measures and medicines to help cure malaria. Topics discussed include the global incidence of malaria, the laboratory's partnership with National Center for Advancing Translational Sciences (NCATS) scientists in identifying potential new medicines to treat the disease, and the development of vaccines to stop malaria infection.

Background: Malaria is still one of the serious public health problems in Grande Comore Island, although the number of annual cases has been greatly reduced in recent years. A better understanding of malaria parasite population diversity and transmission dynamics is critical for assessing the effectiveness of malaria control measures. The objective of this study is to investigate temporal changes in genetic diversity of Plasmodium falciparum populations and multiplicity of infection (MOI) in Grande Comore 10 years after introduction of ACT. Methods: A total of 232 P. falciparum clinical isolates were collected from the Grande Comore Island during two sampling periods (118 for 2006‒2007 group, and 114 for 2013‒2016 group). Parasite isolates were characterized for genetic diversity and complexity of infection by genotyping polymorphic regions in merozoite surface protein gene 1 (msp-1), msp-2, and msp-3 using nested PCR and DNA sequencing. Results: Three msp-1 alleles (K1, MAD20, and RO33), two msp-2 alleles (FC27 and 3D7), and two msp-3 alleles (K1 and 3D7) were detected in parasites of both sampling periods. The RO33 allele of msp-1 (84.8%), 3D7 allele of msp-2 (90.8%), and K1 allele of msp-3 (66.7%) were the predominant allelic types in isolates from 2006–2007 group. In contrast, the RO33 allele of msp-1 (63.4%), FC27 allele of msp-2 (91.1%), and 3D7 allele of msp-3 (53.5%) were the most prevalent among isolates from the 2013–2016 group. Compared with the 2006‒2007 group, polyclonal infection rates of msp-1 (from 76.7 to 29.1%, P < 0.01) and msp-2 (from 62.4 to 28.3%, P < 0.01) allelic types were significantly decreased in those from 2013‒2016 group. Similarly, the MOIs for both msp-1 and msp-2 were higher in P. falciparum isolates in the 2006–2007 group than those in 2013–2016 group (MOI = 3.11 vs 1.63 for msp-1; MOI = 2.75 vs 1.35 for msp-2). DNA sequencing analyses also revealed reduced numbers of distinct sequence variants in the three genes from 2006‒2007 to 2013‒2016: msp-1, from 32 to 23 (about 28% decline); msp-2 from 29 to 21 (about 28% decline), and msp-3 from 11 to 3 (about 72% decline). Conclusions: The present data showed dramatic reduction in genetic diversity and MOI among Grande Comore P. falciparum populations over the course of the study, suggesting a trend of decreasing malaria transmission intensity and genetic diversity in Grande Comore Island. These data provide valuable information for surveillance of P. falciparum infection and for assessing the appropriateness of the current malarial control strategies in the endemic area. [ABSTRACT FROM AUTHOR]