Plasmodium vivax liver stage development and hypnozoite persistence in human liver-chimeric mice.

Plasmodium vivax malaria is characterized by periodic relapses of symptomatic blood stage parasite infections likely initiated by activation of dormant liver stage parasites-hypnozoites. The lack of tractable P. vivax animal models constitutes an obstacle in examining P. vivax liver stage infection and drug efficacy. To overcome this obstacle, we have used human liver-chimeric (huHep) FRG KO mice as a model for P. vivax infection. FRG KO huHep mice support P. vivax sporozoite infection, liver stage development, and hypnozoite formation. We show complete P. vivax liver stage development, including maturation into infectious exo-erythrocytic merozoites as well as the formation and persistence of hypnozoites. Prophylaxis or treatment with the antimalarial primaquine can prevent and eliminate liver stage infection, respectively. Thus, P. vivax-infected FRG KO huHep mice are a model to investigate liver stage development and dormancy and may facilitate the discovery of drugs targeting relapsing malaria.

Cyclic GMP balance is critical for malaria parasite transmission from the mosquito to the mammalian host.

UNLABELLED: Transmission of malaria occurs during Anopheles mosquito vector blood meals, when Plasmodium sporozoites that have invaded the mosquito salivary glands are delivered to the mammalian host. Sporozoites display a unique form of motility that is essential for their movement across cellular host barriers and invasion of hepatocytes. While the molecular machinery powering motility and invasion is increasingly well defined, the signaling events that control these essential parasite activities have not been clearly delineated. Here, we identify a phosphodiesterase (PDEγ) in Plasmodium, a regulator of signaling through cyclic nucleotide second messengers. Reverse transcriptase PCR (RT-PCR) analysis and epitope tagging of endogenous PDEγ detected its expression in blood stages and sporozoites of Plasmodium yoelii. Deletion of PDEγ (pdeγ(-)) rendered sporozoites nonmotile, and they failed to invade the mosquito salivary glands. Consequently, PDEγ deletion completely blocked parasite transmission by mosquito bite. Strikingly, pdeγ(-) sporozoites showed dramatically elevated levels of cyclic GMP (cGMP), indicating that a perturbation in cyclic nucleotide balance is involved in the observed phenotypic defects. Transcriptome sequencing (RNA-Seq) analysis of pdeγ(-) sporozoites revealed reduced transcript abundance of genes that encode key components of the motility and invasion apparatus. Our data reveal a crucial role for PDEγ in maintaining the cyclic nucleotide balance in the malaria parasite sporozoite stage, which in turn is essential for parasite transmission from mosquito to mammal. IMPORTANCE: Malaria is a formidable threat to human health worldwide, and there is an urgent need to identify novel drug targets for this parasitic disease. The parasite is transmitted by mosquito bite, inoculating the host with infectious sporozoite stages. We show that cellular signaling by cyclic nucleotides is critical for transmission of the parasite from the mosquito vector to the mammalian host. Parasite phosphodiesterase γ is essential for maintaining cyclic nucleotide balance, and its deletion blocks transmission of sporozoites. A deeper understanding of the signaling mechanisms involved in transmission might inform the discovery of novel drugs that interrupt this essential step in the parasite life cycle.

SSP3 is a novel Plasmodium yoelii sporozoite surface protein with a role in gliding motility.

Plasmodium sporozoites develop within oocysts in the mosquito midgut wall and then migrate to the salivary glands. After transmission, they embark on a complex journey to the mammalian liver, where they infect hepatocytes. Proteins on the sporozoite surface likely mediate multiple steps of this journey, yet only a few sporozoite surface proteins have been described. Here, we characterize a novel, conserved sporozoite surface protein (SSP3) in the rodent malaria parasite Plasmodium yoelii. SSP3 is a putative type I transmembrane protein unique to Plasmodium. By using epitope tagging and SSP3-specific antibodies in conjunction with immunofluorescence microscopy, we showed that SSP3 is expressed in mosquito midgut oocyst sporozoites, exhibiting an intracellular localization. In sporozoites derived from the mosquito salivary glands, however, SSP3 localized predominantly to the sporozoite surface as determined by immunoelectron microscopy. However, the ectodomain of SSP3 appeared to be inaccessible to antibodies in nonpermeabilized salivary gland sporozoites. Antibody-induced shedding of the major surface protein circumsporozoite protein (CSP) exposed the SSP3 ectodomain to antibodies in some sporozoites. Targeted deletion of SSP3 adversely affected in vitro sporozoite gliding motility, which, surprisingly, impacted neither their cell traversal capacity, host cell invasion in vitro, nor infectivity in vivo. Together, these data reveal a previously unappreciated complexity of the Plasmodium sporozoite surface proteome and the roles of surface proteins in distinct biological activities of sporozoites.

A next-generation genetically attenuated Plasmodium falciparum parasite created by triple gene deletion.

Immunization with live-attenuated Plasmodium sporozoites completely protects against malaria infection. Genetic engineering offers a versatile platform to create live-attenuated sporozoite vaccine candidates. We previously generated a genetically attenuated parasite (GAP) by deleting the P52 and P36 genes in the NF54 wild-type (WT) strain of Plasmodium falciparum (Pf p52(-)/p36(-) GAP). Preclinical assessment of p52(-)/p36(-) GAP in a humanized mouse model indicated an early and severe liver stage growth defect. However, human exposure to >200 Pf p52(-)/p36(-) GAP-infected mosquito bites in a safety trial resulted in peripheral parasitemia in one of six volunteers, revealing that this GAP was incompletely attenuated. We have now created a triple gene deleted GAP by additionally removing the SAP1 gene (Pf p52(-)/p36(-)/sap1(-) GAP) and employed flippase (FLP)/flippase recognition target (FRT) recombination for drug selectable marker cassette removal. This next-generation GAP was indistinguishable from WT parasites in blood stage and mosquito stage development. Using an improved humanized mouse model transplanted with human hepatocytes and human red blood cells, we show that despite a high-dose sporozoite challenge, Pf p52(-)/p36(-)/sap1(-) GAP did not transition to blood stage infection and appeared to be completely attenuated. Thus, clinical testing of Pf p52(-)/p36(-)/sap1(-) GAP assessing safety, immunogenicity, and efficacy against sporozoite challenge is warranted.

A transgenic Plasmodium falciparum NF54 strain that expresses GFP-luciferase throughout the parasite life cycle.

Plasmodium falciparum is the pathogenic agent of the most lethal of human malarias. Transgenic P. falciparum parasites expressing luciferase have been created to study drug interventions of both asexual and sexual blood stages but luciferase-expressing mosquito stage and liver stage parasites have not been created which has prevented the easy quantification of mosquito stage development (e.g. for transmission blocking interventions) and liver stage development (for interventions that prevent infection). To overcome this obstacle, we have created a transgenic P. falciparum NF54 parasite that expresses a GFP-luciferase transgene throughout the life cycle. Luciferase expression is robust and measurable at all life cycle stages, including midgut oocyst, salivary gland sporozoites and liver stages, where in vivo development is easily measurable using humanized mouse infections in conjunction with an in vivo imaging system. This parasite reporter strain will accelerate testing of interventions against pre-erythrocytic life cycle stages.

Metabolomic analysis of patient plasma yields evidence of plant-like α-linolenic acid metabolism in Plasmodium falciparum.

Metabolomics offers a powerful means to investigate human malaria parasite biology and host-parasite interactions at the biochemical level, and to discover novel therapeutic targets and biomarkers of infection. Here, we used an approach based on liquid chromatography and mass spectrometry to perform an untargeted metabolomic analysis of metabolite extracts from Plasmodium falciparum-infected and uninfected patient plasma samples, and from an enriched population of in vitro cultured P. falciparum-infected and uninfected erythrocytes. Statistical modeling robustly segregated infected and uninfected samples based on metabolite species with significantly different abundances. Metabolites of the α-linolenic acid (ALA) pathway, known to exist in plants but not known to exist in P. falciparum until now, were enriched in infected plasma and erythrocyte samples. In vitro labeling with (13)C-ALA showed evidence of plant-like ALA pathway intermediates in P. falciparum. Ortholog searches using ALA pathway enzyme sequences from 8 available plant genomes identified several genes in the P. falciparum genome that were predicted to potentially encode the corresponding enzymes in the hitherto unannotated P. falciparum pathway. These data suggest that our approach can be used to discover novel facets of host/malaria parasite biology in a high-throughput manner.

Evidence that mutant PfCRT facilitates the transmission to mosquitoes of chloroquine-treated Plasmodium gametocytes.

Resistance of the human malarial parasite Plasmodium falciparum to the antimalarial drug chloroquine has rapidly spread from several independent origins and is now widely prevalent throughout the majority of malaria-endemic areas. Field studies have suggested that chloroquine-resistant strains might be more infective to mosquito vectors. To test the hypothesis that the primary chloroquine resistance determinant, mutations in PfCRT, facilitates parasite transmission under drug pressure, we have introduced a mutant or wild-type pfcrt allele into the rodent model malarial parasite Plasmodium berghei. Our results show that mutant PfCRT from the chloroquine-resistant 7G8 strain has no effect on asexual blood stage chloroquine susceptibility in vivo or ex vivo but confers a significant selective advantage in competitive mosquito infections in the presence of this drug, by protecting immature gametocytes from its lethal action. Enhanced infectivity to mosquitoes may have been a key factor driving the worldwide spread of mutant pfcrt.

Metabolomics and malaria biology.

Metabolomics has ushered in a novel and multi-disciplinary realm in biological research. It has provided researchers with a platform to combine powerful biochemical, statistical, computational, and bioinformatics techniques to delve into the mysteries of biology and disease. The application of metabolomics to study malaria parasites represents a major advance in our approach towards gaining a more comprehensive perspective on parasite biology and disease etiology. This review attempts to highlight some of the important aspects of the field of metabolomics, and its ongoing and potential future applications to malaria research.

A non-radioactive DAPI-based high-throughput in vitro assay to assess Plasmodium falciparum responsiveness to antimalarials--increased sensitivity of P. falciparum to chloroquine in Senegal.

The spread of Plasmodium falciparum drug resistance is outpacing new antimalarial development and compromising effective malaria treatment. Combination therapy is widely implemented to prolong the effectiveness of currently approved antimalarials. To maximize utility of available drugs, periodic monitoring of drug efficacy and gathering of accurate information regarding parasite-sensitivity changes are essential. We describe a high-throughput, non-radioactive, field-based assay to evaluate in vitro antimalarial drug sensitivity of P. falciparum isolates from 40 Senegalese patients. Compared with earlier years, we found a significant decrease in chloroquine in vitro and in genotypic resistances (> 50% and > 65%, respectively, in previous studies) with only 23% of isolates showing resistance. This is possibly caused by a withdrawal of chloroquine from Senegal in 2002. We also found a range of artemisinin responses. Prevalence of drug resistance is dynamic and varies by region. Therefore, the implementation of non-radioactive, robust, high-throughput antimalarial sensitivity assays is critical for defining region-specific prophylaxis and treatment guidelines.

Plasmodium falciparum biology: analysis of in vitro versus in vivo growth conditions.

Recent studies of Plasmodium falciparum isolated directly from infected patients indicate that alternative parasite biological states occur in the natural host that are not observed with in vitro cultivated parasites. Variation in host substrates, immune responses and other factors probably induce modifications in parasite biology. These biological states could have important implications for pathogenesis, transmission and therapy. We review the differences between P. falciparum in vitro culture systems and in vivo host environments, as well as evidence that host conditions can alter pathogen biology. For select biological questions, the incorporation of naturally occurring conditions into in vitro experimental manipulation of microbes may provide novel insight into pathogen biology.

The fatty acid biosynthesis enzyme FabI plays a key role in the development of liver-stage malarial parasites.

The fatty acid synthesis type II pathway has received considerable interest as a candidate therapeutic target in Plasmodium falciparum asexual blood-stage infections. This apicoplast-resident pathway, distinct from the mammalian type I process, includes FabI. Here, we report synthetic chemistry and transfection studies concluding that Plasmodium FabI is not the target of the antimalarial activity of triclosan, an inhibitor of bacterial FabI. Disruption of fabI in P. falciparum or the rodent parasite P. berghei does not impede blood-stage growth. In contrast, mosquito-derived, FabI-deficient P. berghei sporozoites are markedly less infective for mice and typically fail to complete liver-stage development in vitro. This defect is characterized by an inability to form intrahepatic merosomes that normally initiate blood-stage infections. These data illuminate key differences between liver- and blood-stage parasites in their requirements for host versus de novo synthesized fatty acids, and create new prospects for stage-specific antimalarial interventions.

Biology of plasmacytoid dendritic cells and natural killer cells in HIV-1 infection.

PURPOSE: This review summarizes recent literature on the biology of dendritic cells and natural killer cells in HIV-1 infection and the importance of crosstalk between them in the development of strong antiviral immunity. RECENT FINDINGS: Type I interferons produced by dendritic cells in response to HIV-1 have been suggested to act as a double-edged sword, stemming HIV-1 replication on the one hand and causing T-cell loss on the other. Recent epidemiologic evidence demonstrates a strong association between the natural killer cell receptor KIR3DS1 (along with its presumed ligand HLA-B Bw4-80I) in the control of HIV-1 replication. SUMMARY: Dendritic and natural killer cells play a central role in the innate immune response to viral infections through both the direct elimination of infected cells and modulation of each other's function.

Chloroquine-resistant isoforms of the Plasmodium falciparum chloroquine resistance transporter acidify lysosomal pH in HEK293 cells more than chloroquine-sensitive isoforms.

The emergence of chloroquine-resistant Plasmodium falciparum malaria imperils the lives of millions of people in Africa, Southeast Asia and South America. Chloroquine resistance is associated with mutations in the P. falciparum chloroquine resistance transporter (PfCRT). We expressed chloroquine-sensitive (HB3) and resistant (Dd2) pfcrt alleles in HEK293 human embryonic kidney cells. PfCRT localized to the lysosomal limiting membrane and was not detected in the plasma membrane. We observed significant acidification of lysosomes containing PfCRT HB3 and Dd2, with Dd2 acidifying significantly more than HB3. A mutant HB3 allele expressing the K76T mutation (earlier found to be key for chloroquine resistance) acidified to the same extent as Dd2, whereas the acidification by a Dd2 allele expressing the T76K "back mutation" was significantly less than Dd2. Thus, the amino acid at position 76 is both an important determinant of chloroquine resistance in parasites and of lysosomal acidification following heterologous expression. PfCRT may be capable of modulating the pH of the parasite digestive vacuole, and thus chloroquine availability. Chloroquine accumulation and glycyl-phenylalanine-2-naphthylamide-induced release of lysosomal Ca(2+) stores were unaffected by PfCRT expression. Cytoplasmic domain mutations did not alter PfCRT sorting to the lysosomal membrane. This heterologous expression system will be useful to characterize PfCRT protein structure and function, and elucidate its molecular role in chloroquine resistance.

PfCRT and the trans-vacuolar proton electrochemical gradient: regulating the access of chloroquine to ferriprotoporphyrin IX.

It is accepted that resistance of Plasmodium falciparum to chloroquine (CQ) is caused primarily by mutations in the pfcrt gene. However, a consensus has not yet been reached on the mechanism by which resistance is achieved. CQ-resistant (CQR) parasite lines accumulate less CQ than do CQ-sensitive (CQS) parasites. The CQR phenotype is complex with a component of reduced energy-dependent CQ uptake and an additional component that resembles energy-dependent CQ efflux. Here we show that the required energy input is in the form of the proton electrochemical gradient across the digestive vacuole (DV) membrane. Collapsing the DV proton gradient (or starving the parasites of glucose) results in similar levels of CQ accumulation in CQS and CQR lines. Under these conditions the accumulation of CQ is stimulated in CQR parasite lines but is reduced in CQS lines. Energy deprivation has no effect on the rate of CQ efflux from CQR lines implying that mutant PfCRT does not function as an efflux pump or active carrier. Using pfcrt-modified parasite lines we show that the entire CQ susceptibility phenotype is switched by the single K76T amino acid change in PfCRT. The efflux of CQ in CQR lines is not directly coupled to the energy supply, consistent with a model in which mutant PfCRT functions as a gated channel or pore, allowing charged CQ species to leak out of the DV.

A critical role for PfCRT K76T in Plasmodium falciparum verapamil-reversible chloroquine resistance.

Chloroquine resistance (CQR) in Plasmodium falciparum is associated with mutations in the digestive vacuole transmembrane protein PfCRT. However, the contribution of individual pfcrt mutations has not been clarified and other genes have been postulated to play a substantial role. Using allelic exchange, we show that removal of the single PfCRT amino-acid change K76T from resistant strains leads to wild-type levels of CQ susceptibility, increased binding of CQ to its target ferriprotoporphyrin IX in the digestive vacuole and loss of verapamil reversibility of CQ and quinine resistance. Our data also indicate that PfCRT mutations preceding residue 76 modulate the degree of verapamil reversibility in CQ-resistant lines. The K76T mutation accounts for earlier observations that CQR can be overcome by subtly altering the CQ side-chain length. Together, these findings establish PfCRT K76T as a critical component of CQR and suggest that CQ access to ferriprotoporphyrin IX is determined by drug-protein interactions involving this mutant residue.

Evidence for a central role for PfCRT in conferring Plasmodium falciparum resistance to diverse antimalarial agents.

Chloroquine resistance in Plasmodium falciparum is primarily conferred by mutations in pfcrt. Parasites resistant to chloroquine can display hypersensitivity to other antimalarials; however, the patterns of crossresistance are complex, and the genetic basis has remained elusive. We show that stepwise selection for resistance to amantadine or halofantrine produced previously unknown pfcrt mutations (including S163R), which were associated with a loss of verapamil-reversible chloroquine resistance. This was accompanied by restoration of efficient chloroquine binding to hematin in these selected lines. This S163R mutation provides insight into a mechanism by which PfCRT could gate the transport of protonated chloroquine through the digestive vacuole membrane. Evidence for the presence of this mutation in a Southeast Asian isolate supports the argument for a broad role for PfCRT in determining levels of susceptibility to structurally diverse antimalarials.

Moe1 and spInt6, the fission yeast homologues of mammalian translation initiation factor 3 subunits p66 (eIF3d) and p48 (eIF3e), respectively, are required for stable association of eIF3 subunits.

The protein encoded by the fission yeast gene, moe1(+) is the homologue of the p66/eIF3d subunit of mammalian translation initiation factor eIF3. In this study, we show that in fission yeast, Moe1 physically associates with eIF3 core subunits as well as with 40 S ribosomal particles as a constituent of the eIF3 protein complex that is similar in size to multisubunit mammalian eIF3. However, strains lacking moe1(+) (Deltamoe1) are viable and show no gross defects in translation initiation, although the rate of translation in the Deltamoe1 cells is about 30-40% slower than wild-type cells. Mutant Deltamoe1 cells are hypersensitive to caffeine and defective in spore formation. These phenotypes of Deltamoe1 cells are similar to those reported previously for deletion of the fission yeast int6(+) gene that encodes the fission yeast homologue of the p48/Int6/eIF3e subunit of mammalian eIF3. Further analysis of eIF3 subunits in Deltamoe1 or Deltaint6 cells shows that in these deletion strains, while all the eIF3 subunits are bound to 40 S particles, dissociation of ribosome-bound eIF3 results in the loss of stable association between the eIF3 subunits. In contrast, eIF3 isolated from ribosomes of wild-type cells are associated with one another in a protein complex. These observations suggest that Moe1 and spInt6 are each required for stable association of eIF3 subunits in fission yeast.