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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]

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]

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]

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]

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]

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]