Paul, Aditya S; Egan, Elizabeth S; Duraisingh, Manoj T
Malaria is caused by the infection and proliferation of parasites from the genus Plasmodium in red blood cells (RBCs). A free Plasmodium parasite, or merozoite, released from an infected RBC must invade another RBC host cell to sustain a blood-stage infection. Here, we review recent advances on RBC invasion by Plasmodium merozoites, focusing on specific molecular interactions between host and parasite. Recent work highlights the central role of host-parasite interactions at virtually every stage of RBC invasion by merozoites. Biophysical experiments have for the first time measured the strength of merozoite-RBC attachment during invasion. For P. falciparum, there have been many key insights regarding the invasion ligand PfRh5 in particular, including its influence on host species tropism, a co-crystal structure with its RBC receptor basigin, and its suitability as a vaccine target. For P. vivax, researchers identified the origin and emergence of the parasite from Africa, demonstrating a natural link to the Duffy-negative RBC variant in African populations. For the simian parasite P. knowlesi, zoonotic invasion into human cells is linked to RBC age, which has implications for parasitemia during an infection and thus malaria. New studies of the molecular and cellular mechanisms governing RBC invasion by Plasmodium parasites have shed light on various aspects of parasite biology and host cell tropism, and indicate opportunities for malaria control.
Paul, Aditya S.; Egan, Elizabeth S.; Duraisingh, Manoj T.
Purpose of Review Malaria is caused by the infection and proliferation of parasites from the genus Plasmodium in red blood cells (RBCs). A free Plasmodium parasite, or merozoite, released from an infected RBC must invade another RBC host cell to sustain a blood-stage infection. Here, we review recent advances on RBC invasion by Plasmodium merozoites, focusing on specific molecular interactions between host and parasite. Recent findings Recent work highlights the central role of host-parasite interactions at virtually every stage of RBC invasion by merozoites. Biophysical experiments have for the first time measured the strength of merozoite-RBC attachment during invasion. For P. falciparum, there have been many key insights regarding the invasion ligand PfRh5 in particular, including its influence on host species tropism, a co-crystal structure with its RBC receptor basigin, and its suitability as a vaccine target. For P. vivax, researchers identified the origin and emergence of the parasite from Africa, demonstrating a natural link to the Duffy-negative RBC variant in African populations. For the simian parasite P. knowlesi, zoonotic invasion into human cells is linked to RBC age, which has implications for parasitemia during an infection and thus malaria. Summary New studies of the molecular and cellular mechanisms governing RBC invasion by Plasmodium parasites have shed light on various aspects of parasite biology and host cell tropism; and indicate opportunities for malaria control. PMID:25767956
Kenry; Lim, Ying Bena; Nai, Mui Hoon; Cao, Jianshu; Loh, Kian Ping; Lim, Chwee Teck
The interactions between graphene oxide (GO) and various biological entities have been actively investigated in recent years, resulting in numerous potential bioapplications of these nanomaterials. Despite this, the biological interactions between GO and disease-causing protozoan parasites have not been well elucidated and remain relatively unexplored. Here, we investigate the in vitro interactions between GO nanosheets and a particular species of malaria parasites, Plasmodium falciparum (P. falciparum). We hypothesize that GO nanosheets may exhibit antimalarial characteristic via action mechanisms of physical obstruction of P. falciparum parasites as well as nutrient depletion. To ascertain this, we characterize the physical interactions between GO nanosheets, red blood cells (RBCs), and malarial parasites as well as the adsorption of several biomolecules necessary for parasitic survival and growth on GO nanosheets. Subsequent to establishing the origin of this antimalarial behavior of GO nanosheets, their efficiency in inhibiting parasite invasion is evaluated. We observe that GO nanosheets at various tested concentrations significantly inhibit the invasion of malaria parasites into RBCs. Furthermore, GO nanosheets delay parasite progression from the ring to the trophozoite stage. Overall, this study may further shed light on the graphene-parasite interactions and potentially facilitate the development of nanomaterial-based strategies for combating malaria.
Najer, Adrian; Wu, Dalin; Bieri, Andrej; Brand, Françoise; Palivan, Cornelia G; Beck, Hans-Peter; Meier, Wolfgang
The fight against most infectious diseases, including malaria, is often hampered by the emergence of drug resistance and lack or limited efficacies of vaccines. Therefore, new drugs, vaccines, or other strategies to control these diseases are needed. Here, we present an innovative nanotechnological strategy in which the nanostructure itself represents the active substance with no necessity to release compounds to attain therapeutic effect and which might act in a drug- and vaccine-like dual function. Invasion of Plasmodium falciparum parasites into red blood cells was selected as a biological model for the initial validation of this approach. Stable nanomimics-polymersomes presenting receptors required for parasite attachment to host cells-were designed to efficiently interrupt the life cycle of the parasite by inhibiting invasion. A simple way to build nanomimics without postformation modifications was established. First, a block copolymer of the receptor with a hydrophobic polymer was synthesized and then mixed with a polymersome-forming block copolymer. The resulting nanomimics bound parasite-derived ligands involved in the initial attachment to host cells and they efficiently blocked reinvasion of malaria parasites after their egress from host cells in vitro. They exhibited efficacies of more than 2 orders of magnitude higher than the soluble form of the receptor, which can be explained by multivalent interactions of several receptors on one nanomimic with multiple ligands on the infective parasite. In the future, our strategy might offer interesting treatment options for severe malaria or a way to modulate the immune response.
Dasgupta, Sabyasachi; Auth, Thorsten; Gov, Nir S.; Satchwell, Timothy J.; Hanssen, Eric; Zuccala, Elizabeth S.; Riglar, David T.; Toye, Ashley M.; Betz, Timo; Baum, Jake; Gompper, Gerhard
The blood stage malaria parasite, the merozoite, has a small window of opportunity during which it must successfully target and invade a human erythrocyte. The process of invasion is nonetheless remarkably rapid. To date, mechanistic models of invasion have focused predominantly on the parasite actomyosin motor contribution to the energetics of entry. Here, we have conducted a numerical analysis using dimensions for an archetypal merozoite to predict the respective contributions of the host-parasite interactions to invasion, in particular the role of membrane wrapping. Our theoretical modeling demonstrates that erythrocyte membrane wrapping alone, as a function of merozoite adhesive and shape properties, is sufficient to entirely account for the first key step of the invasion process, that of merozoite reorientation to its apex and tight adhesive linkage between the two cells. Next, parasite-induced reorganization of the erythrocyte cytoskeleton and release of parasite-derived membrane can also account for a considerable energetic portion of actual invasion itself, through membrane wrapping. Thus, contrary to the prevailing dogma, wrapping by the erythrocyte combined with parasite-derived membrane release can markedly reduce the expected contributions of the merozoite actomyosin motor to invasion. We therefore propose that invasion is a balance between parasite and host cell contributions, evolved toward maximal efficient use of biophysical forces between the two cells. PMID:24988340
Moon, Robert W.; Sharaf, Hazem; Hastings, Claire H.; Ho, Yung Shwen; Nair, Mridul B.; Rchiad, Zineb; Knuepfer, Ellen; Mohring, Franziska; Amir, Amirah; Yusuf, Noor A.; Hall, Joanna; Almond, Neil; Lau, Yee Ling; Pain, Arnab; Blackman, Michael J.
The dominant cause of malaria in Malaysia is now Plasmodium knowlesi, a zoonotic parasite of cynomolgus macaque monkeys found throughout South East Asia. Comparative genomic analysis of parasites adapted to in vitro growth in either cynomolgus or human RBCs identified a genomic deletion that includes the gene encoding normocyte-binding protein Xa (NBPXa) in parasites growing in cynomolgus RBCs but not in human RBCs. Experimental deletion of the NBPXa gene in parasites adapted to growth in human RBCs (which retain the ability to grow in cynomolgus RBCs) restricted them to cynomolgus RBCs, demonstrating that this gene is selectively required for parasite multiplication and growth in human RBCs. NBPXa-null parasites could bind to human RBCs, but invasion of these cells was severely impaired. Therefore, NBPXa is identified as a key mediator of P. knowlesi human infection and may be a target for vaccine development against this emerging pathogen. PMID:27303038
Moon, Robert W; Sharaf, Hazem; Hastings, Claire H; Ho, Yung Shwen; Nair, Mridul B; Rchiad, Zineb; Knuepfer, Ellen; Ramaprasad, Abhinay; Mohring, Franziska; Amir, Amirah; Yusuf, Noor A; Hall, Joanna; Almond, Neil; Lau, Yee Ling; Pain, Arnab; Blackman, Michael J; Holder, Anthony A
The dominant cause of malaria in Malaysia is now Plasmodium knowlesi, a zoonotic parasite of cynomolgus macaque monkeys found throughout South East Asia. Comparative genomic analysis of parasites adapted to in vitro growth in either cynomolgus or human RBCs identified a genomic deletion that includes the gene encoding normocyte-binding protein Xa (NBPXa) in parasites growing in cynomolgus RBCs but not in human RBCs. Experimental deletion of the NBPXa gene in parasites adapted to growth in human RBCs (which retain the ability to grow in cynomolgus RBCs) restricted them to cynomolgus RBCs, demonstrating that this gene is selectively required for parasite multiplication and growth in human RBCs. NBPXa-null parasites could bind to human RBCs, but invasion of these cells was severely impaired. Therefore, NBPXa is identified as a key mediator of P. knowlesi human infection and may be a target for vaccine development against this emerging pathogen.
Yahata, Kazuhide; Treeck, Moritz; Culleton, Richard; Gilberger, Tim-Wolf; Kaneko, Osamu
In order to propagate within the mammalian host, malaria parasites must invade red blood cells (RBCs). This process offers a window of opportunity in which to target the parasite with drugs or vaccines. However, most of the studies relating to RBC invasion have analyzed the molecular interactions of parasite proteins with host cells under static conditions, and the dynamics of these interactions remain largely unstudied. Time-lapse imaging of RBC invasion is a powerful technique to investigate cell invasion and has been reported for Plasmodium knowlesi and Plasmodium falciparum. However, experimental modification of genetic loci is laborious and time consuming for these species. We have established a system of time-lapse imaging for the rodent malaria parasite Plasmodium yoelii, for which modification of genetic loci is quicker and simpler. We compared the kinetics of RBC invasion by P. yoelii with that of P. falciparum and found that the overall kinetics during invasion were similar, with some exceptions. The most striking of these differences is that, following egress from the RBC, the shape of P. yoelii merozoites gradually changes from flat elongated ovals to spherical bodies, a process taking about 60 sec. During this period merozoites were able to attach to and deform the RBC membrane, but were not able to reorient and invade. We propose that this morphological change of P. yoelii merozoites may be related to the secretion or activation of invasion-related proteins. Thus the P. yoelii merozoite appears to be an excellent model to analyze the molecular dynamics of RBC invasion, particularly during the morphological transition phase, which could serve as an expanded window that cannot be observed in P. falciparum. PMID:23227208
Biryukov, Sergei; Angov, Evelina; Landmesser, Mary E; Spring, Michele D; Ockenhouse, Christian F; Stoute, José A
Plasmodium falciparum malaria is a deadly pathogen. The invasion of red blood cells (RBCs) by merozoites is a target for vaccine development. Although anti-merozoite antibodies can block invasion in vitro, there is no efficacy in vivo. To explain this discrepancy we hypothesized that complement activation could enhance RBC invasion by binding to the complement receptor 1 (CR1). Here we show that a monoclonal antibody directed against the merozoite and human polyclonal IgG from merozoite vaccine recipients enhanced RBC invasion in a complement-dependent manner and that soluble CR1 inhibited this enhancement. Sialic acid-independent strains, that presumably are able to bind to CR1 via a native ligand, showed less complement-dependent enhancement of RBC invasion than sialic acid-dependent strains that do not utilize native CR1 ligands. Confocal fluorescent microscopy revealed that complement-dependent invasion resulted in aggregation of CR1 at the RBC surface in contact with the merozoite. Finally, total anti-P. berghei IgG enhanced parasite growth and C3 deficiency decreased parasite growth in mice. These results demonstrate, contrary to current views, that complement activation in conjunction with antibodies can paradoxically aid parasites invade RBCs and should be considered in future design and testing of merozoite vaccines. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.
Marzal, Alfonso; Ricklefs, Robert E.; Valkiūnas, Gediminas; Albayrak, Tamer; Arriero, Elena; Bonneaud, Camille; Czirják, Gábor A.; Ewen, John; Hellgren, Olof; Hořáková, Dita; Iezhova, Tatjana A.; Jensen, Henrik; Križanauskienė, Asta; Lima, Marcos R.; de Lope, Florentino; Magnussen, Eyðfinn; Martin, Lynn B.; Møller, Anders P.; Palinauskas, Vaidas; Pap, Péter L.; Pérez-Tris, Javier; Sehgal, Ravinder N. M.; Soler, Manuel; Szöllősi, Eszter; Westerdahl, Helena; Zetindjiev, Pavel; Bensch, Staffan
Invasive species can displace natives, and thus identifying the traits that make aliens successful is crucial for predicting and preventing biodiversity loss. Pathogens may play an important role in the invasive process, facilitating colonization of their hosts in new continents and islands. According to the Novel Weapon Hypothesis, colonizers may out-compete local native species by bringing with them novel pathogens to which native species are not adapted. In contrast, the Enemy Release Hypothesis suggests that flourishing colonizers are successful because they have left their pathogens behind. To assess the role of avian malaria and related haemosporidian parasites in the global spread of a common invasive bird, we examined the prevalence and genetic diversity of haemosporidian parasites (order Haemosporida, genera Plasmodium and Haemoproteus) infecting house sparrows (Passer domesticus). We sampled house sparrows (N = 1820) from 58 locations on 6 continents. All the samples were tested using PCR-based methods; blood films from the PCR-positive birds were examined microscopically to identify parasite species. The results show that haemosporidian parasites in the house sparrows' native range are replaced by species from local host-generalist parasite fauna in the alien environments of North and South America. Furthermore, sparrows in colonized regions displayed a lower diversity and prevalence of parasite infections. Because the house sparrow lost its native parasites when colonizing the American continents, the release from these natural enemies may have facilitated its invasion in the last two centuries. Our findings therefore reject the Novel Weapon Hypothesis and are concordant with the Enemy Release Hypothesis. PMID:21779353
Bosch, Jürgen; Turley, Stewart; Roach, Claudia M.; Daly, Thomas M.; Bergman, Lawrence W.; Hol, Wim G. J.
The Myosin A-tail Interacting Protein (MTIP) of the malaria parasite links the actomyosin motor of the host cell invasion machinery to its inner membrane complex. We report here that at neutral pH Plasmodium falciparum MTIP in complex with Myosin A adopts a compact conformation, with its two domains completely surrounding the Myosin A-tail helix, dramatically different from previously observed extended MTIP structures. Crystallographic and mutagenesis studies show that H810 and K813 of Myosin A are key players in the formation of the compact MTIP:Myosin A complex. Only the unprotonated state of Myosin A-H810 is compatible with the compact complex. Most surprisingly, every side chain atom of Myosin A-K813 is engaged in contacts with MTIP. While this side chain was previously considered to prevent a compact conformation of MTIP with Myosin A, it actually appears to be essential for the formation of the compact complex. The hydrophobic pockets and adaptability seen in the available series of MTIP structures bodes well for the discovery of inhibitors of cell invasion by malaria parasites. PMID:17628590
Martínez-de la Puente, Josué; Muñoz, Joaquín; Capelli, Gioia; Montarsi, Fabrizio; Soriguer, Ramón; Arnoldi, Daniele; Rizzoli, Annapaola; Figuerola, Jordi
The invasive Asian tiger mosquito Aedes albopictus has dramatically expanded its distribution range, being catalogued as one of the world's 100 worst invasive alien species. As vectors of pathogens, Ae. albopictus may create novel epidemiological scenarios in the invaded areas. Here, the frequency of encounters of Ae. albopictus with the avian malaria parasite Plasmodium and the related Haemoproteus was studied in an area with established populations in northeastern Italy and compared with those from four native mosquito species, Anopheles maculipennis s.l., Culex hortensis, Culex pipiens, and Ochlerotatus caspius. The abdomens of mosquitoes with a recent blood meal were used to identify both the blood meal source and the parasites harboured. Aedes albopictus had a clear antropophilic behaviour while An. maculipennis and Oc. caspius fed mainly on non-human mammals. Birds were the most common hosts of Cx. pipiens and reptiles of Cx. hortensis. Parasites were isolated from three mosquito species, with Cx. pipiens (30%) showing the highest parasite prevalence followed by Cx. hortensis (9%) and Ae. albopictus (5%). These results are the first identifying the avian malaria parasites harboured by mosquitoes in Italy and represent the first evidence supporting that, although Ae. albopictus could be involved in the transmission of avian malaria parasites, the risk of avian malaria parasite spread by this invasive mosquito in Europe would be minimal.
Benzaquen-Geffin, R; Milner, Y; Ginsburg, H
Extraction by boiling of the buffy coat of human blood yields a protein solution which inhibits the propagation of the human malaria parasite Plasmodium falciparum in culture with a 50% inhibitory dose of 105 micrograms of protein per ml. The inhibitory activity is associated exclusively with the lymphocytes and affects solely the invasion of erythrocytes by free merozoites. Boiled extracts of isolated lymphocytes had a 50% inhibitory dose of 22 micrograms/ml. Fractionation of surface-labeled or pronase-treated lymphocytes revealed that the antimalarial lymphocyte factor is associated with the intracellular aspect of the membrane fraction and is probably not involved in the host defense system against malaria. Further purification by salt extraction, ion-exchange chromatography, molecular gel filtration, and electroelution from lithium dodecyl sulfate-polyacrylamide gels resulted in 300- to 550-fold purification, i.e., a 50% inhibitory dose of 40 to 70 ng/ml. All inhibitory fractions contained a 48-kilodalton polypeptide which eluted from a gel filtration column as a 400-kilodalton species, implying multimeric association. Some 6,000 molecules of the 48-kilodalton polypeptide bind with high affinity to one merozoite, the free form of the parasite. The Kd of 0.1 to 0.5 nM for the binding of the 48-kilodalton polypeptide correlated well with the 50% inhibitory dose of 0.3 to 0.4 nM obtained with purified active antimalarial lymphocyte factor. We therefore suggest that the 48-kilodalton polypeptide partially purified from lymphocyte membranes is the antimalarial lymphocyte factor and that it exerts its inhibitory activity by binding to merozoites, thereby preventing their invasion into erythrocytes. The antimalarial lymphocyte factor or a polypeptide sequence thereof could serve for further probing of invasion at the molecular level.
Benzaquen-Geffin, R; Milner, Y; Ginsburg, H
Extraction by boiling of the buffy coat of human blood yields a protein solution which inhibits the propagation of the human malaria parasite Plasmodium falciparum in culture with a 50% inhibitory dose of 105 micrograms of protein per ml. The inhibitory activity is associated exclusively with the lymphocytes and affects solely the invasion of erythrocytes by free merozoites. Boiled extracts of isolated lymphocytes had a 50% inhibitory dose of 22 micrograms/ml. Fractionation of surface-labeled or pronase-treated lymphocytes revealed that the antimalarial lymphocyte factor is associated with the intracellular aspect of the membrane fraction and is probably not involved in the host defense system against malaria. Further purification by salt extraction, ion-exchange chromatography, molecular gel filtration, and electroelution from lithium dodecyl sulfate-polyacrylamide gels resulted in 300- to 550-fold purification, i.e., a 50% inhibitory dose of 40 to 70 ng/ml. All inhibitory fractions contained a 48-kilodalton polypeptide which eluted from a gel filtration column as a 400-kilodalton species, implying multimeric association. Some 6,000 molecules of the 48-kilodalton polypeptide bind with high affinity to one merozoite, the free form of the parasite. The Kd of 0.1 to 0.5 nM for the binding of the 48-kilodalton polypeptide correlated well with the 50% inhibitory dose of 0.3 to 0.4 nM obtained with purified active antimalarial lymphocyte factor. We therefore suggest that the 48-kilodalton polypeptide partially purified from lymphocyte membranes is the antimalarial lymphocyte factor and that it exerts its inhibitory activity by binding to merozoites, thereby preventing their invasion into erythrocytes. The antimalarial lymphocyte factor or a polypeptide sequence thereof could serve for further probing of invasion at the molecular level. Images PMID:3542831
Baldwin, Michael R.; Li, Xuerong; Hanada, Toshihiko; Liu, Shih-Chun
Plasmodium falciparum invasion of human red blood cells (RBCs) is an intricate process requiring a number of distinct ligand-receptor interactions at the merozoite-erythrocyte interface. Merozoite surface protein 1 (MSP1), a highly abundant ligand coating the merozoite surface in all species of malaria parasites, is essential for RBC invasion and considered a leading candidate for inclusion in a multiple-subunit vaccine against malaria. Our previous studies identified an interaction between the carboxyl-terminus of MSP1 and RBC band 3. Here, by employing phage display technology, we report a novel interaction between the amino-terminus of MSP1 and RBC glycophorin A (GPA). Mapping of the binding domains established a direct interaction between malaria MSP1 and human GPA within a region of MSP1 known to potently inhibit P falciparum invasion of human RBCs. Furthermore, a genetically modified mouse model lacking the GPA– band 3 complex in RBCs is completely resistant to malaria infection in vivo. These findings suggest an essential role of the MSP1-GPA–band 3 complex during the initial adhesion phase of malaria parasite invasion of RBCs. PMID:25778531
Baldwin, Michael R; Li, Xuerong; Hanada, Toshihiko; Liu, Shih-Chun; Chishti, Athar H
Plasmodium falciparum invasion of human red blood cells (RBCs) is an intricate process requiring a number of distinct ligand-receptor interactions at the merozoite-erythrocyte interface. Merozoite surface protein 1 (MSP1), a highly abundant ligand coating the merozoite surface in all species of malaria parasites, is essential for RBC invasion and considered a leading candidate for inclusion in a multiple-subunit vaccine against malaria. Our previous studies identified an interaction between the carboxyl-terminus of MSP1 and RBC band 3. Here, by employing phage display technology, we report a novel interaction between the amino-terminus of MSP1 and RBC glycophorin A (GPA). Mapping of the binding domains established a direct interaction between malaria MSP1 and human GPA within a region of MSP1 known to potently inhibit P falciparum invasion of human RBCs. Furthermore, a genetically modified mouse model lacking the GPA- band 3 complex in RBCs is completely resistant to malaria infection in vivo. These findings suggest an essential role of the MSP1-GPA-band 3 complex during the initial adhesion phase of malaria parasite invasion of RBCs.
Zuccala, Elizabeth S.; Gout, Alexander M.; Dekiwadia, Chaitali; Marapana, Danushka S.; Angrisano, Fiona; Turnbull, Lynne; Riglar, David T.; Rogers, Kelly L.; Whitchurch, Cynthia B.; Ralph, Stuart A.; Speed, Terence P.; Baum, Jake
Host cell infection by apicomplexan parasites plays an essential role in lifecycle progression for these obligate intracellular pathogens. For most species, including the etiological agents of malaria and toxoplasmosis, infection requires active host-cell invasion dependent on formation of a tight junction – the organising interface between parasite and host cell during entry. Formation of this structure is not, however, shared across all Apicomplexa or indeed all parasite lifecycle stages. Here, using an in silico integrative genomic search and endogenous gene-tagging strategy, we sought to characterise proteins that function specifically during junction-dependent invasion, a class of proteins we term invasins to distinguish them from adhesins that function in species specific host-cell recognition. High-definition imaging of tagged Plasmodium falciparum invasins localised proteins to multiple cellular compartments of the blood stage merozoite. This includes several that localise to distinct subcompartments within the rhoptries. While originating from the same organelle, however, each has very different dynamics during invasion. Apical Sushi Protein and Rhoptry Neck protein 2 release early, following the junction, whilst a novel rhoptry protein PFF0645c releases only after invasion is complete. This supports the idea that organisation of proteins within a secretory organelle determines the order and destination of protein secretion and provides a localisation-based classification strategy for predicting invasin function during apicomplexan parasite invasion. PMID:23049965
White, Nicholas J
Following anti-malarial drug treatment asexual malaria parasite killing and clearance appear to be first order processes. Damaged malaria parasites in circulating erythrocytes are removed from the circulation mainly by the spleen. Splenic clearance functions increase markedly in acute malaria. Either the entire infected erythrocytes are removed because of their reduced deformability or increased antibody binding or, for the artemisinins which act on young ring stage parasites, splenic pitting of drug-damaged parasites is an important mechanism of clearance. The once-infected erythrocytes returned to the circulation have shortened survival. This contributes to post-artesunate haemolysis that may follow recovery in non-immune hyperparasitaemic patients. As the parasites mature Plasmodium vivax-infected erythrocytes become more deformable, whereas Plasmodium falciparum-infected erythrocytes become less deformable, but they escape splenic filtration by sequestering in venules and capillaries. Sequestered parasites are killed in situ by anti-malarial drugs and then disintegrate to be cleared by phagocytic leukocytes. After treatment with artemisinin derivatives some asexual parasites become temporarily dormant within their infected erythrocytes, and these may regrow after anti-malarial drug concentrations decline. Artemisinin resistance in P. falciparum reflects reduced ring stage susceptibility and manifests as slow parasite clearance. This is best assessed from the slope of the log-linear phase of parasitaemia reduction and is commonly measured as a parasite clearance half-life. Pharmacokinetic-pharmacodynamic modelling of anti-malarial drug effects on parasite clearance has proved useful in predicting therapeutic responses and in dose-optimization.
Huang, Hong Ming; Bauer, Denis C.; Lelliott, Patrick M.; Greth, Andreas; McMorran, Brendan J.; Foote, Simon J.; Burgio, Gaetan
Genetic defects in various red blood cell (RBC) cytoskeletal proteins have been long associated with changes in susceptibility towards malaria infection. In particular, while ankyrin (Ank-1) mutations account for approximately 50% of hereditary spherocytosis (HS) cases, an association with malaria is not well-established, and conflicting evidence has been reported. We describe a novel N-ethyl-N-nitrosourea (ENU)-induced ankyrin mutation MRI61689 that gives rise to two different ankyrin transcripts: one with an introduced splice acceptor site resulting a frameshift, the other with a skipped exon. Ank-1(MRI61689/+) mice exhibit an HS-like phenotype including reduction in mean corpuscular volume (MCV), increased osmotic fragility and reduced RBC deformability. They were also found to be resistant to rodent malaria Plasmodium chabaudi infection. Parasites in Ank-1(MRI61689/+) erythrocytes grew normally, but red cells showed resistance to merozoite invasion. Uninfected Ank-1(MRI61689/+) erythrocytes were also more likely to be cleared from circulation during infection; the “bystander effect”. This increased clearance is a novel resistance mechanism which was not observed in previous ankyrin mouse models. We propose that this bystander effect is due to reduced deformability of Ank-1(MRI61689/+) erythrocytes. This paper highlights the complex roles ankyrin plays in mediating malaria resistance. PMID:27848995
Ito, Daisuke; Schureck, Marc A; Desai, Sanjay A
Malaria parasites evade immune detection by growth and replication within erythrocytes. After erythrocyte invasion, the intracellular pathogen must increase host cell uptake of nutrients from plasma. Here, we report that the parasite-encoded RhopH complex contributes to both invasion and channel-mediated nutrient uptake. As rhoph2 and rhoph3 gene knockouts were not viable in the human P. falciparum pathogen, we used conditional knockdowns to determine that the encoded proteins are essential and to identify their stage-specific functions. We exclude presumed roles for RhopH2 and CLAG3 in erythrocyte invasion but implicate a RhopH3 contribution either through ligand-receptor interactions or subsequent parasite internalization. These proteins then traffic via an export translocon to the host membrane, where they form a nutrient channel. Knockdown of either RhopH2 or RhopH3 disrupts the entire complex, interfering with organellar targeting and subsequent trafficking. Therapies targeting this complex should attack the pathogen at two critical points in its cycle. DOI: http://dx.doi.org/10.7554/eLife.23485.001 PMID:28221136
Mueller, Ann-Kristin; Kohlhepp, Florian; Hammerschmidt, Christiane; Michel, Kristin
The interplay between vector and pathogen is essential for vector-borne disease transmission. Dissecting the molecular basis of refractoriness of some vectors may pave the way to novel disease control mechanisms. A pathogen often needs to overcome several physical barriers, such as the peritrophic matrix, midgut epithelium and salivary glands. Additionally, the arthropod vector elicites immune responses that can severely limit transmission success. One important step in the transmission of most vector-borne diseases is the entry of the disease agent into the salivary glands of its arthropod vector. The salivary glands of blood-feeding arthropods produce a complex mixture of molecules that facilitate blood feeding by inhibition of the host haemostasis, inflammation and immune reactions. Pathogen entry into salivary glands is a receptor-mediated process, which requires molecules on the surface of the pathogen and salivary gland. In most cases, the nature of these molecules remains unknown. Recent advances in our understanding of malaria parasite entry into mosquito salivary glands strongly suggests that specific carbohydrate molecules on the salivary gland surface function as docking receptors for malaria parasites. PMID:20621627
Olshina, Maya A; Angrisano, Fiona; Marapana, Danushka S; Riglar, David T; Bane, Kartik; Wong, Wilson; Catimel, Bruno; Yin, Meng-Xin; Holmes, Andrew B; Frischknecht, Friedrich; Kovar, David R; Baum, Jake
Gliding motility in Plasmodium parasites, the aetiological agents of malaria disease, is mediated by an actomyosin motor anchored in the outer pellicle of the motile cell. Effective motility is dependent on a parasite myosin motor and turnover of dynamic parasite actin filaments. To date, however, the basis for directional motility is not known. Whilst myosin is very likely orientated as a result of its anchorage within the parasite, how actin filaments are orientated to facilitate directional force generation remains unexplained. In addition, recent evidence has questioned the linkage between actin filaments and secreted surface antigens leaving the way by which motor force is transmitted to the extracellular milieu unknown. Malaria parasites possess a markedly reduced repertoire of actin regulators, among which few are predicted to interact with filamentous (F)-actin directly. One of these, PF3D7_1251200, shows strong homology to the coronin family of actin-filament binding proteins, herein referred to as PfCoronin. Here the N terminal beta propeller domain of PfCoronin (PfCor-N) was expressed to assess its ability to bind and bundle pre-formed actin filaments by sedimentation assay, total internal reflection fluorescence (TIRF) microscopy and confocal imaging as well as to explore its ability to bind phospholipids. In parallel a tagged PfCoronin line in Plasmodium falciparum was generated to determine the cellular localization of the protein during asexual parasite development and blood-stage merozoite invasion. A combination of biochemical approaches demonstrated that the N-terminal beta-propeller domain of PfCoronin is capable of binding F-actin and facilitating formation of parallel filament bundles. In parasites, PfCoronin is expressed late in the asexual lifecycle and localizes to the pellicle region of invasive merozoites before and during erythrocyte entry. PfCoronin also associates strongly with membranes within the cell, likely mediated by interactions
Singh, Shailja; Alam, M. Mahmood; Pal-Bhowmick, Ipsita; Brzostowski, Joseph A.; Chitnis, Chetan E.
The invasion of erythrocytes by Plasmodium merozoites requires specific interactions between host receptors and parasite ligands. Parasite proteins that bind erythrocyte receptors during invasion are localized in apical organelles called micronemes and rhoptries. The regulated secretion of microneme and rhoptry proteins to the merozoite surface to enable receptor binding is a critical step in the invasion process. The sequence of these secretion events and the external signals that trigger release are not known. We have used time-lapse video microscopy to study changes in intracellular calcium levels in Plasmodium falciparum merozoites during erythrocyte invasion. In addition, we have developed flow cytometry based methods to measure relative levels of cytosolic calcium and study surface expression of apical organelle proteins in P. falciparum merozoites in response to different external signals. We demonstrate that exposure of P. falciparum merozoites to low potassium ion concentrations as found in blood plasma leads to a rise in cytosolic calcium levels through a phospholipase C mediated pathway. Rise in cytosolic calcium triggers secretion of microneme proteins such as the 175 kD erythrocyte binding antigen (EBA175) and apical membrane antigen-1 (AMA-1) to the merozoite surface. Subsequently, interaction of EBA175 with glycophorin A (glyA), its receptor on erythrocytes, restores basal cytosolic calcium levels and triggers release of rhoptry proteins. Our results identify for the first time the external signals responsible for the sequential release of microneme and rhoptry proteins during erythrocyte invasion and provide a starting point for the dissection of signal transduction pathways involved in regulated exocytosis of these key apical organelles. Signaling pathway components involved in apical organelle discharge may serve as novel targets for drug development since inhibition of microneme and rhoptry secretion can block invasion and limit blood
Harris, Karen S.; Casey, Joanne L.; Coley, Andrew M.; Karas, John A.; Sabo, Jennifer K.; Tan, Yen Yee; Dolezal, Olan; Norton, Raymond S.; Hughes, Andrew B.; Scanlon, Denis; Foley, Michael
Apical membrane antigen 1 (AMA1) of the malaria parasite Plasmodium falciparum has been implicated in the invasion of host erythrocytes and is an important vaccine candidate. We have previously described a 20-residue peptide, R1, that binds to AMA1 and subsequently blocks parasite invasion. Because this peptide appears to target a site critical for AMA1 function, it represents an important lead compound for anti-malarial drug development. However, the effectiveness of this peptide inhibitor was limited to a subset of parasite isolates, indicating a requirement for broader strain specificity. Furthermore, a barrier to the utility of any peptide as a potential therapeutic is its susceptibility to rapid proteolytic degradation. In this study, we sought to improve the proteolytic stability and AMA1 binding properties of the R1 peptide by systematic methylation of backbone amides (N-methylation). The inclusion of a single N-methyl group in the R1 peptide backbone dramatically increased AMA1 affinity, bioactivity, and proteolytic stability without introducing global structural alterations. In addition, N-methylation of multiple R1 residues further improved these properties. Therefore, we have shown that modifications to a biologically active peptide can dramatically enhance activity. This approach could be applied to many lead peptides or peptide therapeutics to simultaneously optimize a number of parameters. PMID:19164290
Pinzón, Carlos Giovanni; Curtidor, Hernando; García, Jeison; Vanegas, Magnolia; Vizcaíno, Carolina; Patarroyo, Manuel A; Patarroyo, Manuel E
In this study, we synthesized the complete sequence of the CLAG-9 protein as 67 20-mer-long non-overlapped peptides and assessed their ability to bind to erythrocytes in receptor-ligand assays. Twenty CLAG-9 peptides were found to have specific high-affinity binding ability to erythrocytes (thereby named as HABPs), with nanomolar dissociation constants. CLAG-9 HABPs interacted with different erythrocyte surface receptors having apparent molecular weights of 85, 63 and 34 kDa. CLAG-9 HABPs binding was also affected by pre-treatment of RBCs with enzymes and inhibited erythrocyte invasion in vitro by up to 72% at 200 microM. These results suggest that some protein fragments of CLAG-9 may be part of the molecular machinery used by malaria parasites to invade erythrocytes, hence supporting their study as possible vaccine candidates. Copyright 2010 Elsevier Ltd. All rights reserved.
Templeton, Thomas J.; Asada, Masahito; Jiratanh, Montakan; Ishikawa, Sohta A.; Tiawsirisup, Sonthaya; Sivakumar, Thillaiampalam; Namangala, Boniface; Takeda, Mika; Mohkaew, Kingdao; Ngamjituea, Supawan; Inoue, Noboru; Sugimoto, Chihiro; Inagaki, Yuji; Suzuki, Yasuhiko; Yokoyama, Naoaki; Kaewthamasorn, Morakot; Kaneko, Osamu
Haemosporida parasites of even-toed ungulates are diverse and globally distributed, but since their discovery in 1913 their characterization has relied exclusively on microscopy-based descriptions. In order to bring molecular approaches to bear on the identity and evolutionary relationships of ungulate malaria parasites, we conducted Plasmodium cytb-specific nested PCR surveys using blood from water buffalo in Vietnam and Thailand, and goats in Zambia. We found that Plasmodium is readily detectable from water buffalo in these countries, indicating that buffalo Plasmodium is distributed in a wider region than India, which is the only area in which buffalo Plasmodium has been reported. Two types (I and II) of Plasmodium sequences were identified from water buffalo and a third type (III) was isolated from goat. Morphology of the parasite was confirmed in Giemsa-reagent stained blood smears for the Type I sample. Complete mitochondrial DNA sequences were isolated and used to infer a phylogeny in which ungulate malaria parasites form a monophyletic clade within the Haemosporida, and branch prior to the clade containing bird, lizard and other mammalian Plasmodium. Thus it is likely that host switching of Plasmodium from birds to mammals occurred multiple times, with a switch to ungulates independently from other mammalian Plasmodium. PMID:26996979
immunity (23, 24) and its relevance to human malaria (25). 4. The effect of the B- thalassemia mutation on ralaria-infectcd mice arid the role of the spleen...detected. Thus, Pc96 shares a cross-reactive epitope with these three primate malaria antigens. 4. Effect of B- thalassemia on malaria-infected mice and...B- thalassemia against malaria, rodent malaria parasites were studied in C57BL/6J mice with B- thalassemia , in mice in which the thalassemia had been
Malaria is a disease caused by parasites that are carried by mosquitoes. Once in the bloodstream, the parasite inhabits the red blood cell (RBC). This picture shows purple-stained malaria parasites inside red blood cells.
Orjuela-Sánchez, Pamela; Brandi, Michelle C; Ferreira, Marcelo U
Microsatellites have been increasingly used to investigate the population structure of malaria parasites, to map genetic loci contributing to phenotypes such as drug resistance and virulence in laboratory crosses and genome-wide association studies and to distinguish between treatment failures and new infections in clinical trials. Here, we provide optimized protocols for genotyping highly polymorphic microsatellites sampled from across the genomes of the human malaria parasites Plasmodium falciparum and P. vivax that have been extensively used in research laboratories worldwide.
Torchin, M.E.; Lafferty, K.D.; Kuris, A.M.
Introduced marine species are a major environmental and economic problem. The rate of these biological invasions has substantially increased in recent years due to the globalization of the world's economies. The damage caused by invasive species is often a result of the higher densities and larger sizes they attain compared to where they are native. A prominent hypothesis explaining the success of introduced species is that they are relatively free of the effects of natural enemies. Most notably, they may encounter fewer parasites in their introduced range compared to their native range. Parasites are ubiquitous and pervasive in marine systems, yet their role in marine invasions is relatively unexplored. Although data on parasites of marine organisms exist, the extent to which parasites can mediate marine invasions, or the extent to which invasive parasites and pathogens are responsible for infecting or potentially decimating native marine species have not been examined. In this review, we present a theoretical framework to model invasion success and examine the evidence for a relationship between parasite presence and the success of introduced marine species. For this, we compare the prevalence and species richness of parasites in several introduced populations of marine species with populations where they are native. We also discuss the potential impacts of introduced marine parasites on native ecosystems.
Sibley, L. D.
Intracellular parasites use various strategies to invade cells and to subvert cellular signaling pathways and, thus, to gain a foothold against host defenses. Efficient cell entry, ability to exploit intracellular niches, and persistence make these parasites treacherous pathogens. Most intracellular parasites gain entry via host-mediated processes, but apicomplexans use a system of adhesion-based motility called ``gliding'' to actively penetrate host cells. Actin polymerization-dependent motility facilitates parasite migration across cellular barriers, enables dissemination within tissues, and powers invasion of host cells. Efficient invasion has brought widespread success to this group, which includes Toxoplasma, Plasmodium, and Cryptosporidium.
Beier, J C
Mosquitoes of the genus Anopheles transmit malaria parasites to humans. Anopheles mosquito species vary in their vector potential because of environmental conditions and factors affecting their abundance, blood-feeding behavior, survival, and ability to support malaria parasite development. In the complex life cycle of the parasite in female mosquitoes, a process termed sporogony, mosquitoes acquire gametocyte-stage parasites from blood-feeding on an infected host. The parasites carry out fertilization in the midgut, transform to ookinetes, then oocysts, which produce sporozoites. Sporozoites invade the salivary glands and are transmitted when the mosquito feeds on another host. Most individual mosquitoes that ingest gametocytes do not support development to the sporozoite stage. Bottle-necks occur at every stage of the cycle in the mosquito. Powerful new techniques and approaches exist for evaluating malaria parasite development and for identifying mechanisms regulating malaria parasite-vector interactions. This review focuses on those interactions that are important for the development of new approaches for evaluating and blocking transmission in nature.
Summary Despite decades of research, we still know little about the mechanics of Plasmodium host cell invasion. Fundamentally, while the essential or non‐essential nature of different parasite proteins is becoming clearer, their actual function and how each comes together to govern invasion are poorly understood. Furthermore, in recent years an emerging world view is shifting focus away from the parasite actin–myosin motor being the sole force responsible for entry to an appreciation of host cell dynamics and forces and their contribution to the process. In this review, we discuss merozoite invasion of the erythrocyte, focusing on the complex set of pre‐invasion events and how these might prime the red cell to facilitate invasion. While traditionally parasite interactions at this stage have been viewed simplistically as mediating adhesion only, recent work makes it apparent that by interacting with a number of host receptors and signalling pathways, combined with secretion of parasite‐derived lipid material, that the merozoite may initiate cytoskeletal re‐arrangements and biophysical changes in the erythrocyte that greatly reduce energy barriers for entry. Seen in this light Plasmodium invasion may well turn out to be a balance between host and parasite forces, much like that of other pathogen infection mechanisms. PMID:26663815
Vaughan, Ashley M; Kappe, Stefan H I
In their infection cycle, malaria parasites undergo replication and population expansions within the vertebrate host and the mosquito vector. Host infection initiates with sporozoite invasion of hepatocytes, followed by a dramatic parasite amplification event during liver stage parasite growth and replication within hepatocytes. Each liver stage forms up to 90,000 exoerythrocytic merozoites, which are in turn capable of initiating a blood stage infection. Liver stages not only exploit host hepatocyte resources for nutritional needs but also endeavor to prevent hepatocyte cell death and detection by the host's immune system. Research over the past decade has identified numerous parasite factors that play a critical role during liver infection and has started to delineate a complex web of parasite-host interactions that sustain successful parasite colonization of the mammalian host. Targeting the parasites' obligatory infection of the liver as a gateway to the blood, with drugs and vaccines, constitutes the most effective strategy for malaria eradication, as it would prevent clinical disease and onward transmission of the parasite.
Malaria is a disease caused by parasites. This picture shows dark orange-stained malaria parasites inside red blood cells (a) and outside the cells (b). Note the large cells that look like targets; ...
Favuzza, Paola; Guffart, Elena; Tamborrini, Marco; Scherer, Bianca; Dreyer, Anita M; Rufer, Arne C; Erny, Johannes; Hoernschemeyer, Joerg; Thoma, Ralf; Schmid, Georg; Gsell, Bernard; Lamelas, Araceli; Benz, Joerg; Joseph, Catherine; Matile, Hugues; Pluschke, Gerd; Rudolph, Markus G
Invasion of erythrocytes by Plasmodial merozoites is a composite process involving the interplay of several proteins. Among them, the Plasmodium falciparum Cysteine-Rich Protective Antigen (PfCyRPA) is a crucial component of a ternary complex, including Reticulocyte binding-like Homologous protein 5 (PfRH5) and the RH5-interacting protein (PfRipr), essential for erythrocyte invasion. Here we present the crystal structure of PfCyRPA and of its complex with the antigen-binding fragment of a parasite growth inhibitory antibody. While PfCyRPA adopts a 6-bladed β-propeller structure with similarity to the classic sialidase fold, it possesses no sialidase activity, indicating that it fulfills a non-enzymatic function. Characterization of the epitope recognized by protective antibodies will facilitate design of peptidomimetics that focus vaccine responses on protective epitopes. Both in vitro and in vivo anti-PfCyRPA and anti-PfRH5 antibodies showed more potent parasite growth inhibitory activity in combination than on their own, supporting a combined delivery of PfCyRPA and PfRH5 in vaccines.
Favuzza, Paola; Guffart, Elena; Tamborrini, Marco; Scherer, Bianca; Dreyer, Anita M; Rufer, Arne C; Erny, Johannes; Hoernschemeyer, Joerg; Thoma, Ralf; Schmid, Georg; Gsell, Bernard; Lamelas, Araceli; Benz, Joerg; Joseph, Catherine; Matile, Hugues; Pluschke, Gerd; Rudolph, Markus G
Invasion of erythrocytes by Plasmodial merozoites is a composite process involving the interplay of several proteins. Among them, the Plasmodium falciparum Cysteine-Rich Protective Antigen (PfCyRPA) is a crucial component of a ternary complex, including Reticulocyte binding-like Homologous protein 5 (PfRH5) and the RH5-interacting protein (PfRipr), essential for erythrocyte invasion. Here, we present the crystal structures of PfCyRPA and its complex with the antigen-binding fragment of a parasite growth inhibitory antibody. PfCyRPA adopts a 6-bladed β-propeller structure with similarity to the classic sialidase fold, but it has no sialidase activity and fulfills a purely non-enzymatic function. Characterization of the epitope recognized by protective antibodies may facilitate design of peptidomimetics to focus vaccine responses on protective epitopes. Both in vitro and in vivo anti-PfCyRPA and anti-PfRH5 antibodies showed more potent parasite growth inhibitory activity in combination than on their own, supporting a combined delivery of PfCyRPA and PfRH5 in vaccines. DOI: http://dx.doi.org/10.7554/eLife.20383.001 PMID:28195038
Moriyama, Yoshinori; Hayashi, Mitsuko; Yatsushiro, Shouki; Yamamoto, Akitsugu
The malaria parasite is a unicellular protozoan parasite of the genus Plasmodium that causes one of the most serious infectious diseases for human beings. Like other protozoa, the malaria parasite possesses acidic organelles, which may play an essential role(s) in energy acquisition, resistance to antimalarial agents, and vesicular trafficking. Recent evidence has indicated that two types of vacuolar proton pumps, vacuolar H+-ATPase and vacuolar H+-pyrophosphatase, are responsible for their acidification. In this mini-review, we discuss the recent progress on vacuolar proton pumps in the malaria parasite.
Walker, Dawn M; Oghumu, Steve; Gupta, Gaurav; McGwire, Bradford S; Drew, Mark E; Satoskar, Abhay R
Numerous disease-causing parasites must invade host cells in order to prosper. Collectively, such pathogens are responsible for a staggering amount of human sickness and death throughout the world. Leishmaniasis, Chagas disease, toxoplasmosis, and malaria are neglected diseases and therefore are linked to socio-economical and geographical factors, affecting well-over half the world's population. Such obligate intracellular parasites have co-evolved with humans to establish a complexity of specific molecular parasite-host cell interactions, forming the basis of the parasite's cellular tropism. They make use of such interactions to invade host cells as a means to migrate through various tissues, to evade the host immune system, and to undergo intracellular replication. These cellular migration and invasion events are absolutely essential for the completion of the lifecycles of these parasites and lead to their for disease pathogenesis. This review is an overview of the molecular mechanisms of protozoan parasite invasion of host cells and discussion of therapeutic strategies, which could be developed by targeting these invasion pathways. Specifically, we focus on four species of protozoan parasites Leishmania, Trypanosoma cruzi, Plasmodium, and Toxoplasma, which are responsible for significant morbidity and mortality.
Liu, Weimin; Li, Yingying; Shaw, Katharina S.; Learn, Gerald H.; Plenderleith, Lindsey J.; Malenke, Jordan A.; Sundararaman, Sesh A.; Ramirez, Miguel A.; Crystal, Patricia A.; Smith, Andrew G.; Bibollet-Ruche, Frederic; Ayouba, Ahidjo; Locatelli, Sabrina; Esteban, Amandine; Mouacha, Fatima; Guichet, Emilande; Butel, Christelle; Ahuka-Mundeke, Steve; Inogwabini, Bila-Isia; Ndjango, Jean-Bosco N.; Speede, Sheri; Sanz, Crickette M.; Morgan, David B.; Gonder, Mary K.; Kranzusch, Philip J.; Walsh, Peter D.; Georgiev, Alexander V.; Muller, Martin N.; Piel, Alex K.; Stewart, Fiona A.; Wilson, Michael L.; Pusey, Anne E.; Cui, Liwang; Wang, Zenglei; Färnert, Anna; Sutherland, Colin J.; Nolder, Debbie; Hart, John A.; Hart, Terese B.; Bertolani, Paco; Gillis, Amethyst; LeBreton, Matthew; Tafon, Babila; Kiyang, John; Djoko, Cyrille F.; Schneider, Bradley S.; Wolfe, Nathan D.; Mpoudi-Ngole, Eitel; Delaporte, Eric; Carter, Richard; Culleton, Richard L.; Shaw, George M.; Rayner, Julian C.; Peeters, Martine; Hahn, Beatrice H.; Sharp, Paul M.
Plasmodium vivax is the leading cause of human malaria in Asia and Latin America but is absent from most of central Africa due to the near fixation of a mutation that inhibits the expression of its receptor, the Duffy antigen, on human erythrocytes. The emergence of this protective allele is not understood because P. vivax is believed to have originated in Asia. Here we show, using a non-invasive approach, that wild chimpanzees and gorillas throughout central Africa are endemically infected with parasites that are closely related to human P. vivax. Sequence analyses reveal that ape parasites lack host specificity and are much more diverse than human parasites, which form a monophyletic lineage within the ape parasite radiation. These findings indicate that human P. vivax is of African origin and likely selected for the Duffy-negative mutation. All extant human P. vivax parasites are derived from a single ancestor that escaped out of Africa. PMID:24557500
Quartan malaria; Falciparum malaria; Biduoterian fever; Blackwater fever; Tertian malaria; Plasmodium ... Malaria is caused by a parasite that is passed to humans by the bite of infected Anopheles ...
Rénia, Laurent; Goh, Yun Shan
Parasites of the genus Plasmodium have a complex life cycle. They alternate between their final mosquito host and their intermediate hosts. The parasite can be either extra- or intracellular, depending on the stage of development. By modifying their shape, motility, and metabolic requirements, the parasite adapts to the different environments in their different hosts. The parasite has evolved to escape the multiple immune mechanisms in the host that try to block parasite development at the different stages of their development. In this article, we describe the mechanisms reported thus far that allow the Plasmodium parasite to evade innate and adaptive immune responses. PMID:27872623
Gomes, Pollyanna S.; Bhardwaj, Jyoti; Rivera-Correa, Juan; Freire-De-Lima, Celio G.; Morrot, Alexandre
Malaria is one of the most life-threatening infectious diseases worldwide. Immunity to malaria is slow and short-lived despite the repeated parasite exposure in endemic areas. Malaria parasites have evolved refined machinery to evade the immune system based on a range of genetic changes that include allelic variation, biomolecular exposure of proteins, and intracellular replication. All of these features increase the probability of survival in both mosquitoes and the vertebrate host. Plasmodium species escape from the first immunological trap in its invertebrate vector host, the Anopheles mosquitoes. The parasites have to pass through various immunological barriers within the mosquito such as anti-microbial molecules and the mosquito microbiota in order to achieve successful transmission to the vertebrate host. Within these hosts, Plasmodium species employ various immune evasion strategies during different life cycle stages. Parasite persistence against the vertebrate immune response depends on the balance among virulence factors, pathology, metabolic cost of the host immune response, and the parasites ability to evade the immune response. In this review we discuss the strategies that Plasmodium parasites use to avoid the vertebrate host immune system and how they promote successful infection and transmission. PMID:27799922
Gomes, Pollyanna S; Bhardwaj, Jyoti; Rivera-Correa, Juan; Freire-De-Lima, Celio G; Morrot, Alexandre
Malaria is one of the most life-threatening infectious diseases worldwide. Immunity to malaria is slow and short-lived despite the repeated parasite exposure in endemic areas. Malaria parasites have evolved refined machinery to evade the immune system based on a range of genetic changes that include allelic variation, biomolecular exposure of proteins, and intracellular replication. All of these features increase the probability of survival in both mosquitoes and the vertebrate host. Plasmodium species escape from the first immunological trap in its invertebrate vector host, the Anopheles mosquitoes. The parasites have to pass through various immunological barriers within the mosquito such as anti-microbial molecules and the mosquito microbiota in order to achieve successful transmission to the vertebrate host. Within these hosts, Plasmodium species employ various immune evasion strategies during different life cycle stages. Parasite persistence against the vertebrate immune response depends on the balance among virulence factors, pathology, metabolic cost of the host immune response, and the parasites ability to evade the immune response. In this review we discuss the strategies that Plasmodium parasites use to avoid the vertebrate host immune system and how they promote successful infection and transmission.
Huber, M; Cabib, E; Miller, L H
Malaria parasites (ookinetes) appear to digest the peritrophic membrane in the mosquito midgut during penetration. Previous studies demonstrated that lectins specific for N-acetylglucosamine bind to the peritrophic membrane and proposed that the membrane contains chitin [Rudin, W. & Hecker, H. (1989) Parasitol. Res. 75, 268-279]. In the present study, we show that the peritrophic membrane is digested by Serratia marcescens chitinase (EC 126.96.36.199), leading to the release of N-acetylglucosamine and fragmentation of the membrane. We also report the presence of a malaria parasite chitinase that digests 4-methylumbelliferyl chitotriose. The enzyme is not detectable until 15 hr after zygote formation, the time required for maturation of the parasite from a zygote to an ookinete, the invasive form of the parasite. At 20 hr, the enzyme begins to appear in the culture supernatant. The chitinase extracted from the parasite and found in the culture supernatant consists of a major band and two minor bands of activity on native polyacrylamide gel electrophoresis. The presence of chitin in the peritrophic membrane, the disruption of the peritrophic membrane during invasion, and the presence of chitinase in ookinetes suggest that the chitinase in ookinetes is used in the penetration of the peritrophic membrane. Images PMID:2011589
Däbritz, Jan; Schneider, Markward; Just-Nuebling, Gudrun; Groll, Andreas H
Malaria is the most important parasitic infection in people, affecting 5-10% of the world's population with more than two million deaths a year. Whereas invasive bacterial infections are not uncommon during severe Plasmodium falciparum malaria, only a few cases of opportunistic fungal infections have been reported. Here, we present a fatal case of disseminated hyalohyphomycosis associated with acute P. falciparum malaria in a non-immune traveller, review the cases reported in the literature and discuss the theoretical foundations for the increased susceptibility of non-immune individuals with severe P. falciparum malaria to opportunistic fungal infections. Apart from the availability of free iron as sequelae of massive haemolysis, tissue damage, acidosis and measures of advanced life support, patients with complicated P. falciparum malaria also are profoundly immunosuppressed by the organism's interaction with innate and adaptive host immune mechanisms.
Bennink, Sandra; Kiesow, Meike J.
Summary The mosquito midgut stages of malaria parasites are crucial for establishing an infection in the insect vector and to thus ensure further spread of the pathogen. Parasite development in the midgut starts with the activation of the intraerythrocytic gametocytes immediately after take‐up and ends with traversal of the midgut epithelium by the invasive ookinetes less than 24 h later. During this time period, the plasmodia undergo two processes of stage conversion, from gametocytes to gametes and from zygotes to ookinetes, both accompanied by dramatic morphological changes. Further, gamete formation requires parasite egress from the enveloping erythrocytes, rendering them vulnerable to the aggressive factors of the insect gut, like components of the human blood meal. The mosquito midgut stages of malaria parasites are unprecedented objects to study a variety of cell biological aspects, including signal perception, cell conversion, parasite/host co‐adaptation and immune evasion. This review highlights recent insights into the molecules involved in gametocyte activation and gamete formation as well as in zygote‐to‐ookinete conversion and ookinete midgut exit; it further discusses factors that can harm the extracellular midgut stages as well as the measures of the parasites to protect themselves from any damage. PMID:27111866
Carlton, Jane M.; Adams, John H.; Silva, Joana C.; Bidwell, Shelby L.; Lorenzi, Hernan; Caler, Elisabet; Crabtree, Jonathan; Angiuoli, Samuel V.; Merino, Emilio F.; Amedeo, Paolo; Cheng, Qin; Coulson, Richard M. R.; Crabb, Brendan S.; del Portillo, Hernando A.; Essien, Kobby; Feldblyum, Tamara V.; Fernandez-Becerra, Carmen; Gilson, Paul R.; Gueye, Amy H.; Guo, Xiang; Kang’a, Simon; Kooij, Taco W. A.; Korsinczky, Michael; Meyer, Esmeralda V.-S.; Nene, Vish; Paulsen, Ian; White, Owen; Ralph, Stuart A.; Ren, Qinghu; Sargeant, Tobias J.; Salzberg, Steven L.; Stoeckert, Christian J.; Sullivan, Steven A.; Yamamoto, Marcio Massao; Hoffman, Stephen L.; Wortman, Jennifer R.; Gardner, Malcolm J.; Galinski, Mary R.; Barnwell, John W.; Fraser-Liggett, Claire M.
The human malaria parasite Plasmodium vivax is responsible for 25-40% of the ~515 million annual cases of malaria worldwide. Although seldom fatal, the parasite elicits severe and incapacitating clinical symptoms and often relapses months after a primary infection has cleared. Despite its importance as a major human pathogen, P. vivax is little studied because it cannot be propagated in the laboratory except in non-human primates. We determined the genome sequence of P. vivax in order to shed light on its distinctive biologic features, and as a means to drive development of new drugs and vaccines. Here we describe the synteny and isochore structure of P. vivax chromosomes, and show that the parasite resembles other malaria parasites in gene content and metabolic potential, but possesses novel gene families and potential alternate invasion pathways not recognized previously. Completion of the P. vivax genome provides the scientific community with a valuable resource that can be used to advance scientific investigation into this neglected species. PMID:18843361
Garcia, C R; Ann, S E; Tavares, E S; Dluzewski, A R; Mason, W T; Paiva, F B
Calcium uptake by permeabilized P. chabaudi malaria parasites was measured at the trophozoite stage to assess calcium accumulation by the parasite organelles. As determined with 45Ca2+, the total calcium in the parasite was found to be 11 pmoles/10(7) cells. When the K+/H+ uncoupling agent, nigericin was present, this level fell to 6.5 pmoles/10(7) cells. A similar regulatory mechanism operates in P. falciparum, since addition of nigericin to intact parasites in calcium free-medium resulted in a transient elevation of free calcium in the parasite cytosol, as judged by fluorescent imaging of single cells loaded with the calcium indicator fluo-3,AM. 7-Chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl) and monensin, inhibitors of H+ ATPases and K+/H+ ionophore respectively, induced calcium elevation in fluo-3, AM-labeled intact P. chabaudi parasites. We conclude that malaria parasites utilize acidic intracellular compartments to regulate their cytosolic free calcium concentration.
Ord, Rosalynn L; Rodriguez, Marilis; Lobo, Cheryl A
With drug resistance to available therapeutics continuing to develop against Plasmodium falciparum malaria, the development of an effective vaccine candidate remains a major research goal. Successful interruption of invasion of parasites into erythrocytes during the blood stage of infection will prevent the severe clinical symptoms and complications associated with malaria. Previously studied blood stage antigens have highlighted the hurdles that are inherent to this life-cycle stage, namely that highly immunogenic antigens are also globally diverse, resulting in protection only against the vaccine strain, or that naturally acquired immunity to blood stage antigens do not always correlate with actual protection. The blood stage antigen reticulocyte binding homolog RH5 is essential for parasite viability, has globally limited diversity, and is associated with protection from disease. Here we summarize available information on this invasion ligand and recent findings that highlight its candidacy for inclusion in a blood-stage malaria vaccine. PMID:25844685
Klug, Dennis; Frischknecht, Friedrich
Malaria is transmitted when an infected Anopheles mosquito deposits Plasmodium sporozoites in the skin during a bite. Sporozoites are formed within oocysts at the mosquito midgut wall and are released into the hemolymph, from where they invade the salivary glands and are subsequently transmitted to the vertebrate host. We found that a thrombospondin-repeat containing sporozoite-specific protein named thrombospondin-releated protein 1 (TRP1) is important for oocyst egress and salivary gland invasion, and hence for the transmission of malaria. We imaged the release of sporozoites from oocysts in situ, which was preceded by active motility. Parasites lacking TRP1 failed to migrate within oocysts and did not egress, suggesting that TRP1 is a vital component of the events that precede intra-oocyst motility and subsequently sporozoite egress and salivary gland invasion. DOI: http://dx.doi.org/10.7554/eLife.19157.001 PMID:28115054
James, A A
Genetic approaches to controlling the transmission of mosquito-borne diseases are being developed to augment the available chemical control practices and environmental manipulation methods. Much progress has been made in laboratory-based research that seeks to develop antipathogen or antivector effector genes and methods for genetically manipulating host vector strains. Research is summarized here in the development of a malaria-resistant phenotype using as a model system the avian parasite, Plasmodium gallinaceum, and the mosquito, Aedes aegypti. Robust transformation technology based on a number of transposable elements, the identification of promoter regions derived from endogenous mosquito genes, and the development of single-chain antibodies as effector genes have made it possible to produce malaria-resistant mosquitoes. Future challenges include discovery of methods for spreading antiparasite genes through mosquito populations, determining the threshold levels below which parasite intensities of infection must be held, and defining the circumstances in which a genetic control strategy would be employed in the field.
Hayakawa, Toshiyuki; Culleton, Richard; Otani, Hiroto; Horii, Toshihiro; Tanabe, Kazuyuki
Malaria parasites (genus Plasmodium) infect all classes of terrestrial vertebrates and display host specificity in their infections. It is therefore assumed that malaria parasites coevolved intimately with their hosts. Here, we propose a novel scenario of malaria parasite-host coevolution. A phylogenetic tree constructed using the malaria parasite mitochondrial genome reveals that the extant primate, rodent, bird, and reptile parasite lineages rapidly diverged from a common ancestor during an evolutionary short time period. This rapid diversification occurred long after the establishment of the primate, rodent, bird, and reptile host lineages, which implies that host-switch events contributed to the rapid diversification of extant malaria parasite lineages. Interestingly, the rapid diversification coincides with the radiation of the mammalian genera, suggesting that adaptive radiation to new mammalian hosts triggered the rapid diversification of extant malaria parasite lineages.
Mudeppa, Devaraja G.; Kumar, Shiva; Kokkonda, Sreekanth; White, John; Rathod, Pradipsinh K.
Historically, type II topoisomerases have yielded clinically useful drugs for the treatment of bacterial infections and cancer, but the corresponding enzymes from malaria parasites remain understudied. This is due to the general challenges of producing malaria proteins in functional forms in heterologous expression systems. Here, we express full-length Plasmodium falciparum topoisomerase II (PfTopoII) in a wheat germ cell-free transcription-translation system. Functional activity of soluble PfTopoII from the translation lysates was confirmed through both a plasmid relaxation and a DNA decatenation activity that was dependent on magnesium and ATP. To facilitate future drug discovery, a convenient and sensitive fluorescence assay was established to follow DNA decatenation, and a stable, truncated PfTopoII was engineered for high level enzyme production. PfTopoII was purified using a DNA affinity column. Existing TopoII inhibitors previously developed for other non-malaria indications inhibited PfTopoII, as well as malaria parasites in culture at submicromolar concentrations. Even before optimization, inhibitors of bacterial gyrase, GSK299423, ciprofloxacin, and etoposide exhibited 15-, 57-, and 3-fold selectivity for the malarial enzyme over human TopoII. Finally, it was possible to use the purified PfTopoII to dissect the different modes by which these varying classes of TopoII inhibitors could trap partially processed DNA. The present biochemical advancements will allow high throughput chemical screening of compound libraries and lead optimization to develop new lines of antimalarials. PMID:26055707
De Niz, Mariana; Ullrich, Ann-Katrin; Heiber, Arlett; Blancke Soares, Alexandra; Pick, Christian; Lyck, Ruth; Keller, Derya; Kaiser, Gesine; Prado, Monica; Flemming, Sven; del Portillo, Hernando; Janse, Chris J.; Heussler, Volker; Spielmann, Tobias
Sequestration of red blood cells infected with the human malaria parasite Plasmodium falciparum in organs such as the brain is considered important for pathogenicity. A similar phenomenon has been observed in mouse models of malaria, using the rodent parasite Plasmodium berghei, but it is unclear whether the P. falciparum proteins known to be involved in this process are conserved in the rodent parasite. Here we identify the P. berghei orthologues of two such key factors of P. falciparum, SBP1 and MAHRP1. Red blood cells infected with P. berghei parasites lacking SBP1 or MAHRP1a fail to bind the endothelial receptor CD36 and show reduced sequestration and virulence in mice. Complementation of the mutant P. berghei parasites with the respective P. falciparum SBP1 and MAHRP1 orthologues restores sequestration and virulence. These findings reveal evolutionary conservation of the machinery underlying sequestration of divergent malaria parasites and support the notion that the P. berghei rodent model is an adequate tool for research on malaria virulence. PMID:27225796
De Niz, Mariana; Ullrich, Ann-Katrin; Heiber, Arlett; Blancke Soares, Alexandra; Pick, Christian; Lyck, Ruth; Keller, Derya; Kaiser, Gesine; Prado, Monica; Flemming, Sven; Del Portillo, Hernando; Janse, Chris J; Heussler, Volker; Spielmann, Tobias
Sequestration of red blood cells infected with the human malaria parasite Plasmodium falciparum in organs such as the brain is considered important for pathogenicity. A similar phenomenon has been observed in mouse models of malaria, using the rodent parasite Plasmodium berghei, but it is unclear whether the P. falciparum proteins known to be involved in this process are conserved in the rodent parasite. Here we identify the P. berghei orthologues of two such key factors of P. falciparum, SBP1 and MAHRP1. Red blood cells infected with P. berghei parasites lacking SBP1 or MAHRP1a fail to bind the endothelial receptor CD36 and show reduced sequestration and virulence in mice. Complementation of the mutant P. berghei parasites with the respective P. falciparum SBP1 and MAHRP1 orthologues restores sequestration and virulence. These findings reveal evolutionary conservation of the machinery underlying sequestration of divergent malaria parasites and support the notion that the P. berghei rodent model is an adequate tool for research on malaria virulence.
Angrisano, Fiona; Delves, Michael J.; Zuccala, Elizabeth S.; Turnbull, Lynne; Dekiwadia, Chaitali; Olshina, Maya A.; Marapana, Danushka S.; Wong, Wilson; Mollard, Vanessa; Bradin, Clare H.; Tonkin, Christopher J.; Gunning, Peter W.; Ralph, Stuart A.; Whitchurch, Cynthia B.; Sinden, Robert E.; Cowman, Alan F.; McFadden, Geoffrey I.; Baum, Jake
Actin dynamics have been implicated in a variety of developmental processes during the malaria parasite lifecycle. Parasite motility, in particular, is thought to critically depend on an actomyosin motor located in the outer pellicle of the parasite cell. Efforts to understand the diverse roles actin plays have, however, been hampered by an inability to detect microfilaments under native conditions. To visualise the spatial dynamics of actin we generated a parasite-specific actin antibody that shows preferential recognition of filamentous actin and applied this tool to different lifecycle stages (merozoites, sporozoites and ookinetes) of the human and mouse malaria parasite species Plasmodium falciparum and P. berghei along with tachyzoites from the related apicomplexan parasite Toxoplasma gondii. Actin filament distribution was found associated with three core compartments: the nuclear periphery, pellicular membranes of motile or invasive parasite forms and in a ring-like distribution at the tight junction during merozoite invasion of erythrocytes in both human and mouse malaria parasites. Localisation at the nuclear periphery is consistent with an emerging role of actin in facilitating parasite gene regulation. During invasion, we show that the actin ring at the parasite-host cell tight junction is dependent on dynamic filament turnover. Super-resolution imaging places this ring posterior to, and not concentric with, the junction marker rhoptry neck protein 4. This implies motor force relies on the engagement of dynamic microfilaments at zones of traction, though not necessarily directly through receptor-ligand interactions at sites of adhesion during invasion. Combined, these observations extend current understanding of the diverse roles actin plays in malaria parasite development and apicomplexan cell motility, in particular refining understanding on the linkage of the internal parasite gliding motor with the extra-cellular milieu. PMID:22389687
Some hours after invading the erythrocytes of its human host, the malaria parasite Plasmodium falciparum induces an increase in the permeability of the erythrocyte membrane to monovalent ions. The resulting net influx of Na(+) and net efflux of K(+), down their respective concentration gradients, converts the erythrocyte cytosol from an initially high-K(+), low-Na(+) solution to a high-Na(+), low-K(+) solution. The intraerythrocytic parasite itself exerts tight control over its internal Na(+), K(+), Cl(-), and Ca(2+) concentrations and its intracellular pH through the combined actions of a range of membrane transport proteins. The molecular mechanisms underpinning ion regulation in the parasite are receiving increasing attention, not least because PfATP4, a P-type ATPase postulated to be involved in Na(+) regulation, has emerged as a potential antimalarial drug target, susceptible to inhibition by a wide range of chemically unrelated compounds.
Román, Gustavo C
Neurologists should be aware of parasitic diseases occurring in travelers and recent migrants because the world has become a global village as a result of tourism and immigration. Global warming is changing the distribution of diseases formerly confined to the tropics. The two most common parasitic diseases of the nervous system are Plasmodium falciparum malaria presenting as a febrile encephalopathy with normal CSF and neurocysticercosis causing seizures with focal MRI lesions or with intracranial hypertension. Numerous parasites may cause larva migrans with eosinophilic meningitis. Spinal cord involvement is the signature presentation of schistosomiasis. Trypanosoma cruzi, the agent of Chagas disease in the Americas, may cause myocardiopathy and embolic stroke. Sleeping sickness remains the most common manifestation of African trypanosomiasis. These conditions are challenging to diagnose unless a history of travel is elicited. Prospective travelers should be advised of preventive measures to avoid potentially severe infections of the nervous system.
Kehr, Sebastian; Jortzik, Esther; Delahunty, Claire; Yates, John R.; Rahlfs, Stefan
Abstract Aims: Protein S-glutathionylation is a widely distributed post-translational modification of thiol groups with glutathione that can function as a redox-sensitive switch to mediate redox regulation and signal transduction. The malaria parasite Plasmodium falciparum is exposed to intense oxidative stress and possesses the enzymatic system required to regulate protein S-glutathionylation, but despite its potential importance, protein S-glutathionylation has not yet been studied in malaria parasites. In this work we applied a method based on enzymatic deglutathionylation, affinity purification of biotin-maleimide-tagged proteins, and proteomic analyses to characterize the Plasmodium glutathionylome. Results: We identified 493 targets of protein S-glutathionylation in Plasmodium. Functional profiles revealed that the targets are components of central metabolic pathways, such as nitrogen compound metabolism and protein metabolism. Fifteen identified proteins with important functions in metabolic pathways (thioredoxin reductase, thioredoxin, thioredoxin peroxidase 1, glutathione reductase, glutathione S-transferase, plasmoredoxin, mitochondrial dihydrolipoamide dehydrogenase, glutamate dehydrogenase 1, glyoxalase I and II, ornithine δ-aminotransferase, lactate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase [GAPDH], pyruvate kinase [PK], and phosphoglycerate mutase) were further analyzed to study their ability to form mixed disulfides with glutathione. We demonstrate that P. falciparum GAPDH, PK, and ornithine δ-aminotransferase are reversibly inhibited by S-glutathionylation. Further, we provide evidence that not only P. falciparum glutaredoxin 1, but also thioredoxin 1 and plasmoredoxin are able to efficiently catalyze protein deglutathionylation. Innovation: We used an affinity-purification based proteomic approach to characterize the Plasmodium glutathionylome. Conclusion: Our results indicate a wide regulative use of S-glutathionylation in the
Arrighi, Romanico B G; Lycett, Gareth; Mahairaki, Vassiliki; Siden-Kiamos, Inga; Louis, Christos
During the invasion of the mosquito midgut epithelium, Plasmodium ookinetes come to rest on the basal lamina, where they transform into the sporozoite-producing oocysts. Laminin, one of the basal lamina's major components, has previously been shown to bind several surface proteins of Plasmodium ookinetes. Here, using the recently developed RNAi technique in mosquitoes, we used a specific dsRNA construct targeted against the LANB2 gene (laminin gamma1) of Anopheles gambiae to reduce its mRNA levels, leading to a substantial reduction in the number of successfully developed oocysts in the mosquito midgut. Moreover, this molecular relationship is corroborated by the intimate association of developing P. berghei parasites and laminin in the gut, as observed using confocal microscopy. Our data support the notion of laminin playing a functional role in the development of the malaria parasite within the mosquito midgut.
Grimberg, Brian T.
Historically, examinations of the inhibition of malaria parasite growth/invasion, whether using drugs or antibodies, have relied on the use of microscopy or radioactive hypoxanthine uptake. These are considered gold standards for measuring the effectiveness of antimalarial treatments, however, these methods have well known shortcomings. With the advent of flow cytometry coupled with the use of fluorescent DNA stains allowed for increased speed, reproducibility, and qualitative estimates of the effectiveness of antibodies and drugs to limit malaria parasite growth which addresses the challenges of traditional techniques. Because materials and machines available to research facilities are so varied, different methods have been developed to investigate malaria parasites by flow cytometry. This review is intended to serve as a reference guide for advanced users and importantly, as a primer for new users, to support expanded use and improvements to malaria flow cytometry, particularly in endemic countries. PMID:21296083
Singh, B; Cox-Singh, J
Malaysia is a developing country with a range of parasitic infections. Indeed, soil-transmitted helminths and malaria parasites continue to have a significant impact on public health in Malaysia. In this article, the prevalence and distribution of these parasites, the problems associated with parasitic infections, the control measures taken to deal with these parasites and implications for the future will be discussed.
Smith, Ryan C; Vega-Rodríguez, Joel; Jacobs-Lorena, Marcelo
Nearly one million people are killed every year by the malaria parasite Plasmodium. Although the disease-causing forms of the parasite exist only in the human blood, mosquitoes of the genus Anopheles are the obligate vector for transmission. Here, we review the parasite life cycle in the vector and highlight the human and mosquito contributions that limit malaria parasite development in the mosquito host. We address parasite killing in its mosquito host and bottlenecks in parasite numbers that might guide intervention strategies to prevent transmission. PMID:25185005
Greischar, Megan A.; Mideo, Nicole; Read, Andrew F.; Bjørnstad, Ottar N.
Many microparasites infect new hosts with specialized life stages, requiring a subset of the parasite population to forgo proliferation and develop into transmission forms. Transmission stage production influences infectivity, host exploitation, and the impact of medical interventions like drug treatment. Predicting how parasites will respond to public health efforts on both epidemiological and evolutionary timescales requires understanding transmission strategies. These strategies can rarely be observed directly and must typically be inferred from infection dynamics. Using malaria as a case study, we test previously described methods for inferring transmission stage investment against simulated data generated with a model of within-host infection dynamics, where the true transmission investment is known. We show that existing methods are inadequate and potentially very misleading. The key difficulty lies in separating transmission stages produced by different generations of parasites. We develop a new approach that performs much better on simulated data. Applying this approach to real data from mice infected with a single Plasmodium chabaudi strain, we estimate that transmission investment varies from zero to 20%, with evidence for variable investment over time in some hosts, but not others. These patterns suggest that, even in experimental infections where host genetics and other environmental factors are controlled, parasites may exhibit remarkably different patterns of transmission investment. PMID:26890485
Greischar, Megan A; Mideo, Nicole; Read, Andrew F; Bjørnstad, Ottar N
Many microparasites infect new hosts with specialized life stages, requiring a subset of the parasite population to forgo proliferation and develop into transmission forms. Transmission stage production influences infectivity, host exploitation, and the impact of medical interventions like drug treatment. Predicting how parasites will respond to public health efforts on both epidemiological and evolutionary timescales requires understanding transmission strategies. These strategies can rarely be observed directly and must typically be inferred from infection dynamics. Using malaria as a case study, we test previously described methods for inferring transmission stage investment against simulated data generated with a model of within-host infection dynamics, where the true transmission investment is known. We show that existing methods are inadequate and potentially very misleading. The key difficulty lies in separating transmission stages produced by different generations of parasites. We develop a new approach that performs much better on simulated data. Applying this approach to real data from mice infected with a single Plasmodium chabaudi strain, we estimate that transmission investment varies from zero to 20%, with evidence for variable investment over time in some hosts, but not others. These patterns suggest that, even in experimental infections where host genetics and other environmental factors are controlled, parasites may exhibit remarkably different patterns of transmission investment.
AD-A279 410 GRANT NO: DAMN17-89-Z-9003 TITLE: MOLECULAR GENETIC ANALYSIS OF PARASITE SURVIVAL IN R. E&LEZjpAIM MALARIA PRINCIPAL INVESTIGATOR... Analysis of Parasite Survival Grant No. in P. Falciparum Malaria DAMDi 7-89- Z-9003 -6. AUTHOR(S) Jeffrey V. Ravetch, M.D., Ph.D. 7. PERFORMING...consequences of genetic variation for parasite survival. Genetic polymorphisms in PRfalciparum were initially detected by pulsed-field gel analysis of intact
Yusuf, Noor A.; Green, Judith L.; Wall, Richard J.; Knuepfer, Ellen; Moon, Robert W.; Schulte-Huxel, Christina; Stanway, Rebecca R.; Martin, Stephen R.; Howell, Steven A.; Douse, Christopher H.; Cota, Ernesto; Tate, Edward W.; Tewari, Rita; Holder, Anthony A.
Myosin B (MyoB) is one of the two short class XIV myosins encoded in the Plasmodium genome. Class XIV myosins are characterized by a catalytic “head,” a modified “neck,” and the absence of a “tail” region. Myosin A (MyoA), the other class XIV myosin in Plasmodium, has been established as a component of the glideosome complex important in motility and cell invasion, but MyoB is not well characterized. We analyzed the properties of MyoB using three parasite species as follows: Plasmodium falciparum, Plasmodium berghei, and Plasmodium knowlesi. MyoB is expressed in all invasive stages (merozoites, ookinetes, and sporozoites) of the life cycle, and the protein is found in a discrete apical location in these polarized cells. In P. falciparum, MyoB is synthesized very late in schizogony/merogony, and its location in merozoites is distinct from, and anterior to, that of a range of known proteins present in the rhoptries, rhoptry neck or micronemes. Unlike MyoA, MyoB is not associated with glideosome complex proteins, including the MyoA light chain, myosin A tail domain-interacting protein (MTIP). A unique MyoB light chain (MLC-B) was identified that contains a calmodulin-like domain at the C terminus and an extended N-terminal region. MLC-B localizes to the same extreme apical pole in the cell as MyoB, and the two proteins form a complex. We propose that MLC-B is a MyoB-specific light chain, and for the short class XIV myosins that lack a tail region, the atypical myosin light chains may fulfill that role. PMID:25802338
Woehlbier, Ute; Epp, Christian; Kauth, Christian W.; Lutz, Rolf; Long, Carole A.; Coulibaly, Boubacar; Kouyaté, Bocar; Arevalo-Herrera, Myriam; Herrera, Sócrates; Bujard, Hermann
The 190-kDa merozoite surface protein 1 (MSP-1) of Plasmodium falciparum, an essential component in the parasite's life cycle, is a primary candidate for a malaria vaccine. Rabbit antibodies elicited by the heterologously produced MSP-1 processing products p83, p30, p38, and p42, derived from strain 3D7, were analyzed for the potential to inhibit in vitro erythrocyte invasion by the parasite and parasite growth. Our data show that (i) epitopes recognized by antibodies, which inhibit parasite replication, are distributed throughout the entire MSP-1 molecule; (ii) when combined, antibodies specific for different regions of MSP-1 inhibit in a strictly additive manner; (iii) anti-MSP-1 antibodies interfere with erythrocyte invasion as well as with the intraerythrocytic growth of the parasite; and (iv) antibodies raised against MSP-1 of strain 3D7 strongly cross-inhibit replication of the heterologous strain FCB-1. Accordingly, anti-MSP-1 antibodies appear to be capable of interfering with parasite multiplication at more than one level. Since the overall immunogenicity profile of MSP-1 in rabbits closely resembles that found in sera of Aotus monkeys immunized with parasite-derived MSP-1 and of humans semi-immune to malaria from whom highly inhibiting antigen-specific antibodies were recovered, we consider the findings reported here to be relevant for the development of MSP-1-based vaccines against malaria. PMID:16428781
Xangsayarath, Phonepadith; Tang, Jianxia; Yahata, Kazuhide; Zoungrana, Augustin; Mitaka, Hayato; Acharjee, Arita; Datta, Partha P.; Hunt, Paul; Carter, Richard; Kaneko, Osamu; Mustonen, Ville; Pain, Arnab
Identifying the genetic determinants of phenotypes that impact disease severity is of fundamental importance for the design of new interventions against malaria. Here we present a rapid genome-wide approach capable of identifying multiple genetic drivers of medically relevant phenotypes within malaria parasites via a single experiment at single gene or allele resolution. In a proof of principle study, we found that a previously undescribed single nucleotide polymorphism in the binding domain of the erythrocyte binding like protein (EBL) conferred a dramatic change in red blood cell invasion in mutant rodent malaria parasites Plasmodium yoelii. In the same experiment, we implicated merozoite surface protein 1 (MSP1) and other polymorphic proteins, as the major targets of strain-specific immunity. Using allelic replacement, we provide functional validation of the substitution in the EBL gene controlling the growth rate in the blood stages of the parasites. PMID:28704525
Najer, Adrian; Thamboo, Sagana; Palivan, Cornelia G; Beck, Hans-Peter; Meier, Wolfgang
Malaria is an infectious disease that needs to be addressed using innovative approaches to counteract spread of drug resistance and to establish or optimize vaccination strategies. With our approach, we aim for a dual action with drug- and 'vaccine-like' activity against malaria. By inhibiting entry of malaria parasites into host red blood cells (RBCs) - using polymer vesicle-based (polymersome) nanomimics of RBC membranes - the life cycle of the parasite is interrupted and the exposed parasites are accessible to the host immune system. Here, we describe how host cell-sized RBC membrane mimics, formed with the same block copolymers as nanomimics, also bind the corresponding malaria parasite ligand and whole malaria parasites, similar to nanomimics. This was demonstrated using fluorescence imaging techniques and confirms the suitability of giant polymersomes (GUVs) as simple mimics for RBC membranes.
Fried, Michal; Hixson, Kim K.; Anderson, Lori; Ogata, Yuko; Mutabingwa, Theonest K.; Duffy, Patrick E.
Malaria proteins expressed on the surface of Plasmodium falciparum infected erythrocytes (IE) mediate adhesion and are targeted by protective immune responses. During pregnancy, IE sequester in the placenta. Placental IE bind to the molecule chondroitin sulfate A (CSA) and preferentially transcribe the gene that encodes VAR2CSA, a member of the PfEMP1 variant surface antigen family. Over successive pregnancies women develop specific immunity to CSA-binding IE and antibodies to VAR2CSA. We used tandem mass spectrometry together with accurate mass and time tag technology to study IE membrane fractions of placental parasites. VAR2CSA peptides were detected in placental IE and in IE from children, but the MC variant of VAR2CSA was specifically associated with placental IE. We identified six conserved hypothetical proteins with putative TM or signal peptides that were exclusively expressed by the placental IE, and 11 such proteins that were significantly more abundant in placental IE. One of these hypothetical proteins, PFI1785w, is a 42kDa molecule detected by Western blot in parasites infecting pregnant women but not those infecting children.
Taylor, Terrie E; Fu, Wenjiang J; Carr, Richard A; Whitten, Richard O; Mueller, Jeffrey S; Fosiko, Nedson G; Lewallen, Susan; Liomba, N George; Molyneux, Malcolm E; Mueller, Jeffrey G
To study the pathogenesis of fatal cerebral malaria, we conducted autopsies in 31 children with this clinical diagnosis. We found that 23% of the children had actually died from other causes. The remaining patients had parasites sequestered in cerebral capillaries, and 75% of those had additional intra- and perivascular pathology. Retinopathy was the only clinical sign distinguishing malarial from nonmalarial coma. These data have implications for treating malaria patients, designing clinical trials and assessing malaria-specific disease associations.
Deroost, Katrien; Pham, Thao-Thy; Opdenakker, Ghislain; Van den Steen, Philippe E
Coevolution of humans and malaria parasites has generated an intricate balance between the immune system of the host and virulence factors of the parasite, equilibrating maximal parasite transmission with limited host damage. Focusing on the blood stage of the disease, we discuss how the balance between anti-parasite immunity versus immunomodulatory and evasion mechanisms of the parasite may result in parasite clearance or chronic infection without major symptoms, whereas imbalances characterized by excessive parasite growth, exaggerated immune reactions or a combination of both cause severe pathology and death, which is detrimental for both parasite and host. A thorough understanding of the immunological balance of malaria and its relation to other physiological balances in the body is of crucial importance for developing effective interventions to reduce malaria-related morbidity and to diminish fatal outcomes due to severe complications. Therefore, we discuss in this review the detailed mechanisms of anti-malarial immunity, parasite virulence factors including immune evasion mechanisms and pathogenesis. Furthermore, we propose a comprehensive classification of malaria complications according to the different types of imbalances.
Leffler, Ellen M; Band, Gavin; Busby, George B J; Kivinen, Katja; Le, Quang Si; Clarke, Geraldine M; Bojang, Kalifa A; Conway, David J; Jallow, Muminatou; Sisay-Joof, Fatoumatta; Bougouma, Edith C; Mangano, Valentina D; Modiano, David; Sirima, Sodiomon B; Achidi, Eric; Apinjoh, Tobias O; Marsh, Kevin; Ndila, Carolyne M; Peshu, Norbert; Williams, Thomas N; Drakeley, Chris; Manjurano, Alphaxard; Reyburn, Hugh; Riley, Eleanor; Kachala, David; Molyneux, Malcolm; Nyirongo, Vysaul; Taylor, Terrie; Thornton, Nicole; Tilley, Louise; Grimsley, Shane; Drury, Eleanor; Stalker, Jim; Cornelius, Victoria; Hubbart, Christina; Jeffreys, Anna E; Rowlands, Kate; Rockett, Kirk A; Spencer, Chris C A; Kwiatkowski, Dominic P
The malaria parasite Plasmodium falciparum invades human red blood cells by a series of interactions between host and parasite surface proteins. By analyzing genome sequence data from human populations, including 1269 individuals from sub-Saharan Africa, we identify a diverse array of large copy-number variants affecting the host invasion receptor genes GYPA and GYPB We find that a nearby association with severe malaria is explained by a complex structural rearrangement involving the loss of GYPB and gain of two GYPB-A hybrid genes, which encode a serologically distinct blood group antigen known as Dantu. This variant reduces the risk of severe malaria by 40% and has recently increased in frequency in parts of Kenya, yet it appears to be absent from west Africa. These findings link structural variation of red blood cell invasion receptors with natural resistance to severe malaria. Copyright © 2017, American Association for the Advancement of Science.
Okoye, Vincent C. N.; Bennett, Vann
Human erythrocyte band 3, a major membrane-spanning protein, was purified and incorporated into liposomes. These liposomes, at nanomolar concentrations of protein, inhibited invasion of human erythrocytes in vitro by the malaria parasite Plasmodium falciparum. Liposomes containing human band 3 were ten times more effective in inhibiting invasion than those with pig band 3 and six times more effective than liposomes containing human erythrocyte glycophorin. Liposomes alone or liposomes containing erythrocyte glycolipids did not inhibit invasion. These results suggest that band 3 participates in the invasion process in a step involving a specific, high-affinity interaction between band 3 and some component of the parasite.
Rosenthal, Philip J
Controlling the spread of antimalarial drug resistance, especially resistance of Plasmodium falciparum to artemisinin-based combination therapies, is a high priority. Available data indicate that, as with other microorganisms, the spread of drug-resistant malaria parasites is limited by fitness costs that frequently accompany resistance. Resistance-mediating polymorphisms in malaria parasites have been identified in putative drug transporters and in target enzymes. The impacts of these polymorphisms on parasite fitness have been characterized in vitro and in animal models. Additional insights have come from analyses of samples from clinical studies, both evaluating parasites under different selective pressures and determining the clinical consequences of infection with different parasites. With some exceptions, resistance-mediating polymorphisms lead to malaria parasites that, compared with wild type, grow less well in culture and in animals, and are replaced by wild type when drug pressure diminishes in the clinical setting. In some cases, the fitness costs of resistance may be offset by compensatory mutations that increase virulence or changes that enhance malaria transmission. However, not enough is known about effects of resistance mediators on parasite fitness. A better appreciation of the costs of fitness-mediating mutations will facilitate the development of optimal guidelines for the treatment and prevention of malaria.
Introduced species disrupt native communities and biodiversity worldwide. Parasitic infections (and at times, their absence) are thought to be a key component in the success and impact of biological invasions by plants and animals. They can facilitate or limit invasions, and positively or negatively...
Zheng, Hong; Tan, Zhangping
Malaria is a mosquito-borne infectious disease of humans. It begins with a bite from an infected female Anopheles mosquito and leads to the development of the pre-erythrocytic and blood stages. Blood-stage infection is the exclusive cause of clinical symptoms of malaria. In contrast, the pre-erythrocytic stage is clinically asymptomatic and could be an excellent target for preventive therapies. Although the robust host immune responses limit the development of the liver stage, malaria parasites have also evolved strategies to suppress host defenses at the pre-erythrocytic stage. This paper reviews the immune evasion strategies of malaria parasites at the pre-erythrocytic stage, which could provide us with potential targets to design prophylactic strategies against malaria. PMID:24891764
Das, D K; Mukherjee, R; Chakraborty, C
Malaria, being an epidemic disease, demands its rapid and accurate diagnosis for proper intervention. Microscopic image-based characterization of erythrocytes plays an integral role in screening of malaria parasites. In practice, microscopic evaluation of blood smear image is the gold standard for malaria diagnosis; where the pathologist visually examines the stained slide under the light microscope. This visual inspection is subjective, error-prone and time consuming. In order to address such issues, computational microscopic imaging methods have been given importance in recent times in the field of digital pathology. Recently, such quantitative microscopic techniques have rapidly evolved for abnormal erythrocyte detection, segmentation and semi/fully automated classification by minimizing such diagnostic errors for computerized malaria detection. The aim of this paper is to present a review on enhancement, segmentation, microscopic feature extraction and computer-aided classification for malaria parasite detection. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.
Flueck, Christian; Baker, David A
Blood-stage malaria parasites evade the immune system by switching the protein exposed at the surface of the infected erythrocyte. A small proportion of these parasites commits to sexual development to mediate mosquito transmission. Two studies in this issue (Brancucci et al., 2014; Coleman et al., 2014) shed light on shared epigenetic machinery underlying both of these events.
Duraisingh, Manoj T; Lodish, Harvey F
Sickle cell hemoglobin conveys resistance to malaria. In this issue of Cell Host & Microbe, LaMonte et al. (2012) demonstrate a surprising mechanism for this innate immunity. A microRNA enriched in sickle red blood cells is translocated into the parasite, incorporated covalently into P. falciparum mRNAs and inhibits parasite growth.
Rahlfs, Stefan; Przyborski, Jude M.; Becker, Katja
Malaria, caused by the apicomplexan parasite Plasmodium, still represents a major threat to human health and welfare and leads to about one million human deaths annually. Plasmodium is a rapidly multiplying unicellular organism undergoing a complex developmental cycle in man and mosquito – a life style that requires rapid adaptation to various environments. In order to deal with high fluxes of reactive oxygen species and maintain redox regulatory processes and pathogenicity, Plasmodium depends upon an adequate redox balance. By systematically studying the subcellular localization of the major antioxidant and redox regulatory proteins, we obtained the first complete map of redox compartmentation in Plasmodium falciparum. We demonstrate the targeting of two plasmodial peroxiredoxins and a putative glyoxalase system to the apicoplast, a non-photosynthetic plastid. We furthermore obtained a complete picture of the compartmentation of thioredoxin- and glutaredoxin-like proteins. Notably, for the two major antioxidant redox-enzymes – glutathione reductase and thioredoxin reductase – Plasmodium makes use of alternative-translation-initiation (ATI) to achieve differential targeting. Dual localization of proteins effected by ATI is likely to occur also in other Apicomplexa and might open new avenues for therapeutic intervention. PMID:21203490
Carruthers, Vern B.
ABSTRACT Toxoplasma gondii and its Plasmodium kin share a well-conserved invasion process, including sequential secretion of adhesive molecules for host cell attachment and invasion. However, only a few orthologs have been shown to be important for efficient invasion by both genera. Bioinformatic screening to uncover potential new players in invasion identified a previously unrecognized T. gondii ortholog of Plasmodium glycosylphosphatidylinositol-anchored micronemal antigen (TgGAMA). We show that TgGAMA localizes to the micronemes and is processed into several proteolytic products within the parasite prior to secretion onto the parasite surface during invasion. TgGAMA from parasite lysate bound to several different host cell types in vitro, suggesting a role in parasite attachment. Consistent with this function, tetracycline-regulatable TgGAMA and TgGAMA knockout strains showed significant reductions in host cell invasion at the attachment step, with no defects in any of the other stages of the parasite lytic cycle. Together, the results of this work reveal a new conserved component of the adhesive repertoire of apicomplexan parasites. IMPORTANCE Toxoplasma gondii is a successful human pathogen in the same phylum as malaria-causing Plasmodium parasites. Invasion of a host cell is an essential process that begins with secretion of adhesive proteins onto the parasite surface for attachment and subsequent penetration of the host cell. Conserved invasion proteins likely play roles that were maintained through the divergence of these parasites. Here, we identify a new conserved invasion protein called glycosylphosphatidylinositol-anchored micronemal antigen (GAMA). Tachyzoites lacking TgGAMA were partially impaired in parasite attachment and invasion of host cells, yielding the first genetic evidence of a specific role in parasite entry into host cells. These findings widen our appreciation of the repertoire of conserved proteins that apicomplexan parasites employ for
Kono, Maya; Heincke, Dorothee; Wilcke, Louisa; Wong, Tatianna Wai Ying; Bruns, Caroline; Herrmann, Susann; Spielmann, Tobias; Gilberger, Tim W.
ABSTRACT The intraerythrocytic developmental cycle of Plasmodium falciparum is completed with the release of up to 32 invasive daughter cells, the merozoites, into the blood stream. Before release, the final step of merozoite development is the assembly of the cortical pellicle, a multi-layered membrane structure. This unique apicomplexan feature includes the inner membrane complex (IMC) and the parasite's plasma membrane. A dynamic ring structure, referred to as the basal complex, is part of the IMC and helps to divide organelles and abscises in the maturing daughter cells. Here, we analyze the dynamics of the basal complex of P. falciparum. We report on a novel transmembrane protein of the basal complex termed BTP1, which is specific to the genus Plasmodium. It colocalizes with the known basal complex marker protein MORN1 and shows distinct dynamics as well as localization when compared to other IMC proteins during schizogony. Using a parasite plasma membrane marker cell line, we correlate dynamics of the basal complex with the acquisition of the maternal membrane. We show that plasma membrane invagination and IMC propagation are interlinked during the final steps of cell division. PMID:26763910
Frischknecht, Friedrich; Baldacci, Patricia; Martin, Béatrice; Zimmer, Christophe; Thiberge, Sabine; Olivo-Marin, Jean-Christophe; Shorte, Spencer L; Ménard, Robert
Malaria is contracted when Plasmodium sporozoites are inoculated into the vertebrate host during the blood meal of a mosquito. In infected mosquitoes, sporozoites are present in large numbers in the secretory cavities of the salivary glands at the most distal site of the salivary system. However, how sporozoites move through the salivary system of the mosquito, both in resting and feeding mosquitoes, is unknown. Here, we observed fluorescent Plasmodium berghei sporozoites within live Anopheles stephensi mosquitoes and their salivary glands and ducts. We show that sporozoites move in the mosquito by gliding, a type of motility associated with their capacity to invade host cells. Unlike in vitro, sporozoite gliding inside salivary cavities and ducts is modulated in speed and motion pattern. Imaging of sporozoite discharge through the proboscis of salivating mosquitoes indicates that sporozoites need to locomote from cavities into ducts to be ejected and that their progression inside ducts favours their early ejection. These observations suggest that sporozoite gliding allows not only for cell invasion but also for parasite locomotion in host tissues, and that it may control parasite transmission.
Duval, Linda; Nerrienet, Eric; Rousset, Dominique; Sadeuh Mba, Serge Alain; Houze, Sandrine; Fourment, Mathieu; Le Bras, Jacques; Robert, Vincent; Ariey, Frederic
Since the 1970's, the diversity of Plasmodium parasites in African great apes has been neglected. Surprisingly, P. reichenowi, a chimpanzee parasite, is the only such parasite to have been molecularly characterized. This parasite is closely phylogenetically related to P. falciparum, the principal cause of the greatest malaria burden in humans. Studies of malaria parasites from anthropoid primates may provide relevant phylogenetic information, improving our understanding of the origin and evolutionary history of human malaria species. In this study, we screened 130 DNA samples from chimpanzees (Pan troglodytes) and gorillas (Gorilla gorilla) from Cameroon for Plasmodium infection, using cytochrome b molecular tools. Two chimpanzees from the subspecies Pan t. troglodytes presented single infections with Plasmodium strains molecularly related to the human malaria parasite P. ovale. These chimpanzee parasites and 13 human strains of P. ovale originated from a various sites in Africa and Asia were characterized using cytochrome b and cytochrome c oxidase 1 mitochondrial partial genes and nuclear ldh partial gene. Consistent with previous findings, two genetically distinct types of P. ovale, classical and variant, were observed in the human population from a variety of geographical locations. One chimpanzee Plasmodium strain was genetically identical, on all three markers tested, to variant P. ovale type. The other chimpanzee Plasmodium strain was different from P. ovale strains isolated from humans. This study provides the first evidence of possibility of natural cross-species exchange of P. ovale between humans and chimpanzees of the subspecies Pan t. troglodytes.
Duval, Linda; Nerrienet, Eric; Rousset, Dominique; Sadeuh Mba, Serge Alain; Houze, Sandrine; Fourment, Mathieu; Le Bras, Jacques; Robert, Vincent; Ariey, Frederic
Since the 1970's, the diversity of Plasmodium parasites in African great apes has been neglected. Surprisingly, P. reichenowi, a chimpanzee parasite, is the only such parasite to have been molecularly characterized. This parasite is closely phylogenetically related to P. falciparum, the principal cause of the greatest malaria burden in humans. Studies of malaria parasites from anthropoid primates may provide relevant phylogenetic information, improving our understanding of the origin and evolutionary history of human malaria species. In this study, we screened 130 DNA samples from chimpanzees (Pan troglodytes) and gorillas (Gorilla gorilla) from Cameroon for Plasmodium infection, using cytochrome b molecular tools. Two chimpanzees from the subspecies Pan t. troglodytes presented single infections with Plasmodium strains molecularly related to the human malaria parasite P. ovale. These chimpanzee parasites and 13 human strains of P. ovale originated from a various sites in Africa and Asia were characterized using cytochrome b and cytochrome c oxidase 1 mitochondrial partial genes and nuclear ldh partial gene. Consistent with previous findings, two genetically distinct types of P. ovale, classical and variant, were observed in the human population from a variety of geographical locations. One chimpanzee Plasmodium strain was genetically identical, on all three markers tested, to variant P. ovale type. The other chimpanzee Plasmodium strain was different from P. ovale strains isolated from humans. This study provides the first evidence of possibility of natural cross-species exchange of P. ovale between humans and chimpanzees of the subspecies Pan t. troglodytes. PMID:19436742
SILVA, JOANA C.; EGAN, AMY; FRIEDMAN, ROBERT; MUNRO, JAMES B.; CARLTON, JANE M.; HUGHES, AUSTIN L.
SUMMARY Objective The evolutionary history of human malaria parasites (genus Plasmodium) has long been a subject of speculation and controversy. The complete genome sequences of the two most widespread human malaria parasites, P. falciparum and P. vivax, and of the monkey parasite P. knowlesi are now available, together with the draft genomes of the chimpanzee parasite P. reichenowi, three rodent parasites, P. yoelii yoelli, P. berghei and P. chabaudi chabaudi, and one avian parasite, P. gallinaceum. Methods We present here an analysis of 45 orthologous gene sequences across the eight species that resolves the relationships of major Plasmodium lineages, and provides the first comprehensive dating of the age of those groups. Results Our analyses support the hypothesis that the last common ancestor of P. falciparum and the chimpanzee parasite P. reichenowi occurred around the time of the human-chimpanzee divergence. P. falciparum infections of African apes are most likely derived from humans and not the other way around. On the other hand, P. vivax, split from the monkey parasite P. knowlesi in the much more distant past, during the time that encompasses the separation of the Great Apes and Old World Monkeys. Conclusion The results support an ancient association between malaria parasites and their primate hosts, including humans. PMID:21118608
Douglas, Ross G; Amino, Rogerio; Sinnis, Photini; Frischknecht, Freddy
Malaria parasites undergo a complex life cycle between their hosts and vectors. During this cycle the parasites invade different types of cells, migrate across barriers, and transfer from one host to another. Recent literature hints at a misunderstanding of the difference between active, parasite-driven migration and passive, circulation-driven movement of the parasite or parasite-infected cells in the various bodily fluids of mosquito and mammalian hosts. Because both active migration and passive transport could be targeted in different ways to interfere with the parasite, a distinction between the two ways the parasite uses to get from one location to another is essential. We discuss the two types of motion needed for parasite dissemination and elaborate on how they could be targeted by future vaccines or drugs.
Feis, Marieke E; Goedknegt, M Anouk; Thieltges, David W; Buschbaum, Christian; Wegner, K Mathias
Host-parasite coevolution has rarely been observed in natural systems. Its study often relies on microparasitic infections introducing a potential bias in the estimation of the evolutionary change of host and parasite traits. Using biological invasions as a tool to study host-parasite coevolution in nature can overcome these biases. We demonstrate this with a cross-infection experiment in the invasive macroparasite Mytilicola intestinalis and its bivalve host, the blue mussel Mytilus edulis. The invasion history of the parasite is well known for the southeastern North Sea and is characterised by two separate invasion fronts that reached opposite ends of the Wadden Sea (i.e. Texel, The Netherlands and Sylt, Germany) in a similar time frame. The species' natural history thus makes this invasion an ideal natural experiment to study host-parasite coevolution in nature. We infected hosts from Texel, Sylt and Kiel (Baltic Sea, where the parasite is absent) with parasites from Texel and Sylt, to form sympatric, allopatric and naïve infestation combinations, respectively. We measured infection rate, host condition and parasite growth to show that sympatric host-parasite combinations diverged in terms of pre- and post-infection traits within <100 generations since their introduction. Texel parasites were more infective and more efficient at exploiting the host's resources. Hosts on Texel, on the other hand, evolved resistance to infection, whereas hosts on Sylt may have evolved tolerance. This illustrates that different coevolutionary trajectories can evolve along separate invasion fronts of the parasite, highlighting the use of biological invasions in studies of host-parasite coevolution in nature.
Liew, Kingsley J L; Hu, Guangan; Bozdech, Zbynek; Peter, Preiser R
In recent years a number of genome sequences for different plasmodium species have become available. This has allowed the identification of numerous conserved genes across the different species and has significantly enhanced our understanding of parasite biology. In contrast little is known about species specific differences between the different genomes partly due to the lower sequence coverage and therefore relatively poor annotation of some of the draft genomes particularly the rodent malarias parasite species. To improve the current annotation and gene identification status of the draft genomes of P. berghei, P. chabaudi and P. yoelii, we performed genome-wide comparisons between these three species. Through analyses via comparative genome hybridizations using a newly designed pan-rodent array as well as in depth bioinformatics analysis, we were able to improve on the coverage of the draft rodent parasite genomes by detecting orthologous genes between these related rodent parasite species. More than 1,000 orthologs for P. yoelii were now newly associated with a P. falciparum gene. In addition to extending the current core gene set for all plasmodium species this analysis also for the first time identifies a relatively small number of genes that are unique to the primate malaria parasites while a larger gene set is uniquely conserved amongst the rodent malaria parasites. These findings allow a more thorough investigation of the genes that are important for host specificity in malaria.
Videvall, Elin; Cornwallis, Charlie K; Ahrén, Dag; Palinauskas, Vaidas; Valkiūnas, Gediminas; Hellgren, Olof
Malaria parasites (Plasmodium spp.) include some of the world's most widespread and virulent pathogens. Our knowledge of the molecular mechanisms these parasites use to invade and exploit their hosts other than in mice and primates is, however, extremely limited. It is therefore imperative to characterize transcriptome-wide gene expression from nonmodel malaria parasites and how this varies across individual hosts. Here, we used high-throughput Illumina RNA sequencing on blood from wild-caught Eurasian siskins experimentally infected with a clonal strain of the avian malaria parasite Plasmodium ashfordi (lineage GRW2). Using a bioinformatic multistep approach to filter out host transcripts, we successfully assembled the blood-stage transcriptome of P. ashfordi. A total of 11 954 expressed transcripts were identified, and 7860 were annotated with protein information. We quantified gene expression levels of all parasite transcripts across three hosts during two infection stages - peak and decreasing parasitemia. Interestingly, parasites from the same host displayed remarkably similar expression profiles during different infection stages, but showed large differences across hosts, indicating that P. ashfordi may adjust its gene expression to specific host individuals. We further show that the majority of transcripts are most similar to the human parasite Plasmodium falciparum, and a large number of red blood cell invasion genes were discovered, suggesting evolutionary conserved invasion strategies between mammalian and avian Plasmodium. The transcriptome of P. ashfordi and its host-specific gene expression advances our understanding of Plasmodium plasticity and is a valuable resource as it allows for further studies analysing gene evolution and comparisons of parasite gene expression. © 2017 John Wiley & Sons Ltd.
Conway, David J
More human death and disease is caused by malaria parasites than by all other eukaryotic pathogens combined. As early as the sequencing of the first human genome, malaria parasite genomics was prioritized to fuel the discovery of vaccine candidate antigens. This stimulated increased research on malaria, generating new understanding of the cellular and molecular mechanisms of infection and immunity. This review of recent developments illustrates how new approaches in parasite genomics, and increasingly large amounts of data from population studies, are helping to identify antigens that are promising lead targets. Although these results have been encouraging, effective discovery and characterization need to be coupled with more innovation and funding to translate findings into newly designed vaccine products for clinical trials.
Khan, Shahid M; Janse, Chris J; Kappe, Stefan H I; Mikolajczak, Sebastian A
Vaccination with live-attenuated Plasmodium sporozoites that arrest in the liver can completely protect against a malaria infection both in animal models and in humans; this has provided the conceptual basis for the most promising, but also challenging, approach to develop an efficacious malaria vaccine. Advances in genetic manipulation of Plasmodium in conjunction with improved genomic and biological information has enabled new approaches to design genetically attenuated parasites (GAPs). In this review we discuss the principles in discovery and development of GAPs in preclinical models that are important in selecting GAP parasites for first-in-human clinical studies. Finally, we highlight the challenges in manufacture, formulation and delivery of a live-attenuated whole parasite malaria vaccine, as well as the further refinements that may be implemented in the next generation GAP vaccines.
Salman, Ahmed M; Mogollon, Catherin Marin; Lin, Jing-Wen; van Pul, Fiona J A; Janse, Chris J; Khan, Shahid M
We describe methods for the rapid generation of transgenic rodent Plasmodium berghei (Pb) parasites that express human malaria parasite (HMP) proteins, using the recently developed GIMO-based transfection methodology. Three different genetic modifications are described resulting in three types of transgenic parasites. (1) Additional Gene (AG) mutants. In these mutants the HMP gene is introduced as an "additional gene" into a silent/neutral locus of the Pb genome under the control of either a constitutive or stage-specific Pb promoter. This method uses the GIMO-transfection protocol and AG mutants are generated by replacing the positive-negative selection marker (SM) hdhfr::yfcu cassette in a neutral locus of a standard GIMO mother line with the HMP gene expression cassette, resulting in SM free transgenic parasites. (2) Double-step Replacement (DsR) mutants. In these mutants the coding sequence (CDS) of the Pb gene is replaced with the CDS of the HMP ortholog in a two-step GIMO-transfection procedure. This process involves first the replacement of the Pb CDS with the hdhfr::yfcu SM, followed by insertion of the HMP ortholog at the same locus thereby replacing hdhfr::yfcu with the HMP CDS. These steps use the GIMO-transfection protocol, which exploits both positive selection for Pb orthologous gene-deletion and negative selection for HMP gene-insertion, resulting in SM free transgenic parasites. (3) Double-step Insertion (DsI) mutants. When a Pb gene is essential for blood stage development the DsR strategy is not possible. In these mutants the HMP expression cassette is first introduced into the neutral locus in a standard GIMO mother line as described for AG mutants but under the control elements of the Pb orthologous gene; subsequently, the Pb ortholog CDS is targeted for deletion through replacement of the Pb CDS with the hdhfr::yfcu SM, resulting in transgenic parasites with a new GIMO locus permissive for additional gene-insertion modifications.The different
Farrow, Rachel E; Green, Judith; Katsimitsoulia, Zoe; Taylor, William R; Holder, Anthony A; Molloy, Justin E
Plasmodium falciparum is the most virulent causative agent of malaria in man accounting for 80% of all malarial infections and 90% of the one million annual deaths attributed to malaria. P. falciparum is a unicellular, Apicomplexan parasite, that spends part of its lifecycle in the mosquito and part in man and it has evolved a special form of motility that enables it to burrow into animal cells, a process termed "host cell invasion". The acute, life threatening, phase of malarial infection arises when the merozoite form of the parasite undergoes multiple cycles of red blood cell invasion and rapid proliferation. Here, we discuss the molecular machinery that enables malarial parasites to invade red blood cells and we focus particularly on the ATP-driven acto-myosin motor that powers invasion.
Tangchaikeeree, Tienrat; Jangpatarapongsa, Kulachart; Polpanich, Duangporn; Thiramanas, Raweewan; Pornjarone, Atcharavalai; Udnaen, Somkiat; Udomsangpetch, Rachanee; Tangboriboonrat, Pramuan
The simple and less expensive technique based on magnetic nanoparticles (MNPs) was developed for separation of malaria parasites containing specific antigens. The carboxylated MNPs were chemically bound with anti-P. falciparum IgG antibodies (Ab-MNPs) purified from the plasma of malaria patients and then used for removal of P. falciparum malaria-infected erythrocytes from other non-infected blood cells in malaria culture at a given percent parasitemia. The results from optical microscope showed that all blood stages parasites, i.e., ring, trophozoite and schizont, could be separated from other blood components with high purity (> or = 95%) and yield of 33.5% (the early stages of ring and trophozoite:the schizont stage were 1:1.34). Highly specific interaction between Ab-MNPs and the P. falciparum malaria infected erythrocytes was confirmed by scanning electron microscope. When compared to the centrifugation with Percoll gradient and depletion by sorbitol lysis which are specific to the mature and the ring stages, respectively, our technique would be more useful for production of high quality of parasites to use in malaria pathogenesis or immunological studies, and in detection techniques.
Beeson, James G.; Drew, Damien R.; Boyle, Michelle J.; Feng, Gaoqian; Fowkes, Freya J.I.; Richards, Jack S.
Malaria accounts for an enormous burden of disease globally, with Plasmodium falciparum accounting for the majority of malaria, and P. vivax being a second important cause, especially in Asia, the Americas and the Pacific. During infection with Plasmodium spp., the merozoite form of the parasite invades red blood cells and replicates inside them. It is during the blood-stage of infection that malaria disease occurs and, therefore, understanding merozoite invasion, host immune responses to merozoite surface antigens, and targeting merozoite surface proteins and invasion ligands by novel vaccines and therapeutics have been important areas of research. Merozoite invasion involves multiple interactions and events, and substantial processing of merozoite surface proteins occurs before, during and after invasion. The merozoite surface is highly complex, presenting a multitude of antigens to the immune system. This complexity has proved challenging to our efforts to understand merozoite invasion and malaria immunity, and to developing merozoite antigens as malaria vaccines. In recent years, there has been major progress in this field, and several merozoite surface proteins show strong potential as malaria vaccines. Our current knowledge on this topic is reviewed, highlighting recent advances and research priorities. PMID:26833236
Beeson, James G; Drew, Damien R; Boyle, Michelle J; Feng, Gaoqian; Fowkes, Freya J I; Richards, Jack S
Malaria accounts for an enormous burden of disease globally, with Plasmodium falciparum accounting for the majority of malaria, and P. vivax being a second important cause, especially in Asia, the Americas and the Pacific. During infection with Plasmodium spp., the merozoite form of the parasite invades red blood cells and replicates inside them. It is during the blood-stage of infection that malaria disease occurs and, therefore, understanding merozoite invasion, host immune responses to merozoite surface antigens, and targeting merozoite surface proteins and invasion ligands by novel vaccines and therapeutics have been important areas of research. Merozoite invasion involves multiple interactions and events, and substantial processing of merozoite surface proteins occurs before, during and after invasion. The merozoite surface is highly complex, presenting a multitude of antigens to the immune system. This complexity has proved challenging to our efforts to understand merozoite invasion and malaria immunity, and to developing merozoite antigens as malaria vaccines. In recent years, there has been major progress in this field, and several merozoite surface proteins show strong potential as malaria vaccines. Our current knowledge on this topic is reviewed, highlighting recent advances and research priorities. © FEMS 2016.
Ricklefs, Robert E; Outlaw, Diana C; Svensson-Coelho, Maria; Medeiros, Matthew C I; Ellis, Vincenzo A; Latta, Steven
The malaria parasites (Apicomplexa: Haemosporida) of birds are believed to have diversified across the avian host phylogeny well after the origin of most major host lineages. Although many symbionts with direct transmission codiversify with their hosts, mechanisms of species formation in vector-borne parasites, including the role of host shifting, are poorly understood. Here, we examine the hosts of sister lineages in a phylogeny of 181 putative species of malaria parasites of New World terrestrial birds to determine the role of shifts between host taxa in the formation of new parasite species. We find that host shifting, often across host genera and families, is the rule. Sympatric speciation by host shifting would require local reproductive isolation as a prerequisite to divergent selection, but this mechanism is not supported by the generalized host-biting behavior of most vectors of avian malaria parasites. Instead, the geographic distribution of individual parasite lineages in diverse hosts suggests that species formation is predominantly allopatric and involves host expansion followed by local host-pathogen coevolution and secondary sympatry, resulting in local shifting of parasite lineages across hosts.
Ricklefs, Robert E.; Outlaw, Diana C.; Svensson-Coelho, Maria; Medeiros, Matthew C. I.; Ellis, Vincenzo A.; Latta, Steven
The malaria parasites (Apicomplexa: Haemosporida) of birds are believed to have diversified across the avian host phylogeny well after the origin of most major host lineages. Although many symbionts with direct transmission codiversify with their hosts, mechanisms of species formation in vector-borne parasites, including the role of host shifting, are poorly understood. Here, we examine the hosts of sister lineages in a phylogeny of 181 putative species of malaria parasites of New World terrestrial birds to determine the role of shifts between host taxa in the formation of new parasite species. We find that host shifting, often across host genera and families, is the rule. Sympatric speciation by host shifting would require local reproductive isolation as a prerequisite to divergent selection, but this mechanism is not supported by the generalized host-biting behavior of most vectors of avian malaria parasites. Instead, the geographic distribution of individual parasite lineages in diverse hosts suggests that species formation is predominantly allopatric and involves host expansion followed by local host–pathogen coevolution and secondary sympatry, resulting in local shifting of parasite lineages across hosts. PMID:25271324
Malaria parasites are continually evolving to evade the immune system and human attempts to control the disease. To eliminate malaria from regions where it is deeply entrenched we need ways of monitoring what is going on in the parasite population, detecting problematic changes as soon as they arise, and executing a prompt and effective response based on a deep understanding of this natural evolutionary process. Powerful new tools to address this problem are emerging from the fast-growing field of genomic epidemiology, driven by new sequencing technologies and computational methods that allow parasite genome variation to be studied in much greater detail and in many more samples than was previously considered possible. These new tools will provide a deep understanding of what is going on in the parasite population, generating actionable knowledge for strategic planning of control interventions, for monitoring their effects and steering them for greatest impact, and for raising the alert if things start to go wrong.
Mikolajczak, Sebastian A; Kappe, Stefan H
All mammalian malaria parasite species have an initial tissue stage in liver cells. The liver stage produces new parasite forms that can enter and live inside red blood cells. Accordingly, the first place of residence provides parasites with a radically different cellular and molecular environment from their subsequent red blood cell home. Liver stages have remained refractory to reveal their secrets, yet the last few years have seen several advances in elucidating their biology. This review looks at the more recent findings concerning the liver stage-host hepatocyte association, some of which may become powerful weapons in the prevention of malaria infection. We also outline areas of liver stage research and technological development that provide promising foci to accelerate a better understanding of this most elusive of the parasites many life cycle stages.
Malaria parasites are continually evolving to evade the immune system and human attempts to control the disease. To eliminate malaria from regions where it is deeply entrenched we need ways of monitoring what is going on in the parasite population, detecting problematic changes as soon as they arise, and executing a prompt and effective response based on a deep understanding of this natural evolutionary process. Powerful new tools to address this problem are emerging from the fast-growing field of genomic epidemiology, driven by new sequencing technologies and computational methods that allow parasite genome variation to be studied in much greater detail and in many more samples than was previously considered possible. These new tools will provide a deep understanding of what is going on in the parasite population, generating actionable knowledge for strategic planning of control interventions, for monitoring their effects and steering them for greatest impact, and for raising the alert if things start to go wrong. PMID:25733556
Desai, Sanjay A.
Malaria parasites increase erythrocyte permeability to diverse solutes including anions, some cations, and organic solutes, as characterized with several independent methods. Over the last decade, patch-clamp studies have determined that the permeability results from one or more ion channels on the infected erythrocyte host membrane. However, the biological role(s) served by these channels, if any, remain controversial. Recent studies implicate the plasmodial surface anion channel (PSAC) and a role in parasite nutrient acquisition. A debated alternative role in remodeling host ion composition for the benefit of the parasite appears to be nonessential. Because both channel activity and the associated clag3 genes are strictly conserved in malaria parasites, channel-mediated permeability is an attractive target for development of new therapies. PMID:24507014
Lakshmanan, Viswanathan; Fishbaugher, Matthew E.; Morrison, Bob; Baldwin, Michael; Macarulay, Michael; Vaughan, Ashley M.; Mikolajczak, Sebastian A.
ABSTRACT 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. PMID:25784701
Douglass, Alyse N; Kain, Heather S; Abdullahi, Marian; Arang, Nadia; Austin, Laura S; Mikolajczak, Sebastian A; Billman, Zachary P; Hume, Jen C C; Murphy, Sean C; Kappe, Stefan H I; Kaushansky, Alexis
Eliminating malaria parasites during the asymptomatic but obligate liver stages (LSs) of infection would stop disease and subsequent transmission. Unfortunately, only a single licensed drug that targets all LSs, Primaquine, is available. Targeting host proteins might significantly expand the repertoire of prophylactic drugs against malaria. Here, we demonstrate that both Bcl-2 inhibitors and P53 agonists dramatically reduce LS burden in a mouse malaria model in vitro and in vivo by altering the activity of key hepatocyte factors on which the parasite relies. Bcl-2 inhibitors act primarily by inducing apoptosis in infected hepatocytes, whereas P53 agonists eliminate parasites in an apoptosis-independent fashion. In combination, Bcl-2 inhibitors and P53 agonists act synergistically to delay, and in some cases completely prevent, the onset of blood stage disease. Both families of drugs are highly effective at doses that do not cause substantial hepatocyte cell death in vitro or liver damage in vivo. P53 agonists and Bcl-2 inhibitors were also effective when administered to humanized mice infected with Plasmodium falciparum. Our data demonstrate that host-based prophylaxis could be developed into an effective intervention strategy that eliminates LS parasites before the onset of clinical disease and thus opens a new avenue to prevent malaria. PMID:25648263
Adegoke, S A; Oyelami, O A; Olatunya, O S; Adeyemi, L A
One hundred and twenty children with acute uncomplicated malaria who were managed at the children's outpatient department of the Wesley Guild Hospital, Ilesa (a unit of Obafemi Awolowo University Teaching Hospitals' Complex, Ile-Ife, Osun state, Nigeria) were recruited into the study to determine the effects of lime juice on malaria parasite clearance. These children were randomized into treatment with World Health Organization recommended antimalarials (artemisinin combination therapy, ACT) either alone or with lime juice. Nine of them were lost to follow-up, four were in the group that were managed with ACT and lime, and five in the group that were managed on ACT alone. The average (SD) time to achieve >75% reduction in parasite load was significantly lower in patients on ACT and lime; 30.5 ± 2.4 h against 38.6 ± 3.3 h for those on ACT alone (p < 0.001). Also, while a significantly higher proportion of children on antimalarial drugs and lime juice achieved complete parasite clearance by 72 h of therapy (p = 0.007), ten (18.2%) patients without lime had early treatment failure (p = 0.003). There were no side effects with the use of lime juice. It may therefore be inferred, from this preliminary work, that lime juice when used with the appropriate antimalarial may enhance malaria parasite clearance especially in those with uncomplicated malaria.
Ginsburg, Hagai; Abdel-Haleem, Alyaa M
Malaria Parasite Metabolic Pathways (MPMP) is the website for the functional genomics of intraerythrocytic Plasmodium falciparum. All the published information about targeted chemical compounds has now been added. Users can find the drug target and publication details linked to a drug database for further information about the medicinal properties of each compound.
Otto, Thomas D; Rayner, Julian C; Böhme, Ulrike; Pain, Arnab; Spottiswoode, Natasha; Sanders, Mandy; Quail, Michael; Ollomo, Benjamin; Renaud, François; Thomas, Alan W; Prugnolle, Franck; Conway, David J; Newbold, Chris; Berriman, Matthew
Plasmodium falciparum causes most human malaria deaths, having prehistorically evolved from parasites of African Great Apes. Here we explore the genomic basis of P. falciparum adaptation to human hosts by fully sequencing the genome of the closely related chimpanzee parasite species P. reichenowi, and obtaining partial sequence data from a more distantly related chimpanzee parasite (P. gaboni). The close relationship between P. reichenowi and P. falciparum is emphasized by almost complete conservation of genomic synteny, but against this strikingly conserved background we observe major differences at loci involved in erythrocyte invasion. The organization of most virulence-associated multigene families, including the hypervariable var genes, is broadly conserved, but P. falciparum has a smaller subset of rif and stevor genes whose products are expressed on the infected erythrocyte surface. Genome-wide analysis identifies other loci under recent positive selection, but a limited number of changes at the host-parasite interface may have mediated host switching.
Crick, Alex J; Theron, Michel; Tiffert, Teresa; Lew, Virgilio L; Cicuta, Pietro; Rayner, Julian C
Erythrocyte invasion by Plasmodium falciparum merozoites is an essential step for parasite survival and hence the pathogenesis of malaria. Invasion has been studied intensively, but our cellular understanding has been limited by the fact that it occurs very rapidly: invasion is generally complete within 1 min, and shortly thereafter the merozoites, at least in in vitro culture, lose their invasive capacity. The rapid nature of the process, and hence the narrow time window in which measurements can be taken, have limited the tools available to quantitate invasion. Here we employ optical tweezers to study individual invasion events for what we believe is the first time, showing that newly released P. falciparum merozoites, delivered via optical tweezers to a target erythrocyte, retain their ability to invade. Even spent merozoites, which had lost the ability to invade, retain the ability to adhere to erythrocytes, and furthermore can still induce transient local membrane deformations in the erythrocyte membrane. We use this technology to measure the strength of the adhesive force between merozoites and erythrocytes, and to probe the cellular mode of action of known invasion inhibitory treatments. These data add to our understanding of the erythrocyte-merozoite interactions that occur during invasion, and demonstrate the power of optical tweezers technologies in unraveling the blood-stage biology of malaria.
Crick, Alex J.; Theron, Michel; Tiffert, Teresa; Lew, Virgilio L.; Cicuta, Pietro; Rayner, Julian C.
Erythrocyte invasion by Plasmodium falciparum merozoites is an essential step for parasite survival and hence the pathogenesis of malaria. Invasion has been studied intensively, but our cellular understanding has been limited by the fact that it occurs very rapidly: invasion is generally complete within 1 min, and shortly thereafter the merozoites, at least in in vitro culture, lose their invasive capacity. The rapid nature of the process, and hence the narrow time window in which measurements can be taken, have limited the tools available to quantitate invasion. Here we employ optical tweezers to study individual invasion events for what we believe is the first time, showing that newly released P. falciparum merozoites, delivered via optical tweezers to a target erythrocyte, retain their ability to invade. Even spent merozoites, which had lost the ability to invade, retain the ability to adhere to erythrocytes, and furthermore can still induce transient local membrane deformations in the erythrocyte membrane. We use this technology to measure the strength of the adhesive force between merozoites and erythrocytes, and to probe the cellular mode of action of known invasion inhibitory treatments. These data add to our understanding of the erythrocyte-merozoite interactions that occur during invasion, and demonstrate the power of optical tweezers technologies in unraveling the blood-stage biology of malaria. PMID:25140419
Malaria is a serious disease caused by a parasite. You get it when an infected mosquito bites you. Malaria is a major cause of death worldwide, but ... at risk. There are four different types of malaria caused by four related parasites. The most deadly ...
Imwong, Mallika; Stepniewska, Kasia; Tripura, Rupam; Peto, Thomas J.; Lwin, Khin Maung; Vihokhern, Benchawan; Wongsaen, Klanarong; von Seidlein, Lorenz; Dhorda, Mehul; Snounou, Georges; Keereecharoen, Lilly; Singhasivanon, Pratap; Sirithiranont, Pasathorn; Chalk, Jem; Nguon, Chea; Day, Nicholas P. J.; Nosten, Francois; Dondorp, Arjen; White, Nicholas J.
Background. Asymptomatic parasitemia is common even in areas of low seasonal malaria transmission, but the true proportion of the population infected has not been estimated previously because of the limited sensitivity of available detection methods. Methods. Cross-sectional malaria surveys were conducted in areas of low seasonal transmission along the border between eastern Myanmar and northwestern Thailand and in western Cambodia. DNA was quantitated by an ultrasensitive polymerase chain reaction (uPCR) assay (limit of accurate detection, 22 parasites/mL) to characterize parasite density distributions for Plasmodium falciparum and Plasmodium vivax, and the proportions of undetected infections were imputed. Results. The prevalence of asymptomatic malaria as determined by uPCR was 27.5% (1303 of 4740 people tested). Both P. vivax and P. falciparum density distributions were unimodal and log normal, with modal values well within the quantifiable range. The estimated proportions of all parasitemic individuals identified by uPCR were >70% among individuals infected with P. falciparum and >85% among those infected with P. vivax. Overall, 83% of infections were predicted to be P. vivax infections, 13% were predicted to be P. falciparum infections, and 4% were predicted to be mixed infections. Geometric mean parasite densities were similar; 5601 P. vivax parasites/mL and 5158 P. falciparum parasites/mL. Conclusions. This uPCR method identified most infected individuals in malaria-endemic areas. Malaria parasitemia persists in humans at levels that optimize the probability of generating transmissible gametocyte densities without causing illness. PMID:26681777
Angus, B J; Chotivanich, K; Udomsangpetch, R; White, N J
During acute falciparum malaria infection, red blood cells (RBC) containing abundant ring-infected erythrocyte surface antigen (Pf 155 or RESA), but no intracellular parasites, are present in the circulation. These RESA-positive parasite negative RBC are not seen in parasite cultures in vitro. This indicates that in acute falciparum malaria there is active removal of intraerythrocytic parasites by a host mechanism in vivo (probably the spleen) without destruction of the parasitized RBC. This may explain the observed disparity between the drop in hematocrit and decrease in parasite count in some hyperparasitemic patients. The fate of these "once-parasitized" RBC in vivo is not known.
Lelliott, Patrick M; Lampkin, Shelley; McMorran, Brendan J; Foote, Simon J; Burgio, Gaetan
Malaria treatments are becoming less effective due to the rapid spread of drug resistant parasites. Increased understanding of the host/parasite interaction is crucial in order to develop treatments that will be less prone to resistance. Parasite invasion of the red blood cell (RBC) is a critical aspect of the parasite life cycle and is, therefore, a promising target for the development of malaria treatments. Assays for analysing parasite invasion in vitro have been developed, but no equivalent assays exist for in vivo studies. This article describes a novel flow cytometric in vivo parasite invasion assay. Experiments were conducted with mice infected with erythrocytic stages of Plasmodium chabaudi adami strain DS. Exogenously labelled blood cells were transfused into infected mice at schizogony, and collected blood samples stained and analysed using flow cytometry to specifically detect and measure proportions of labelled RBC containing newly invaded parasites. A combination of antibodies (CD45 and CD71) and fluorescent dyes, Hoechst (DNA) and JC-1 (mitochondrial membrane potential), were used to differentiate parasitized RBCs from uninfected cells, RBCs containing Howell-Jolly bodies, leukocytes and RBC progenitors. Blood cells were treated ex vivo with proteases to examine the effects on in vivo parasite invasion. The staining and flow cytometry analysis method was accurate in determining the parasitaemia down to 0.013% with the limit of detection at 0.007%. Transfused labelled blood supported normal rates of parasite invasion. Protease-treated red cells resulted in 35% decrease in the rate of parasite invasion within 30 minutes of introduction into the bloodstream of infected mice. The invasion assay presented here is a versatile method for the study of in vivo red cell invasion efficiency of Plasmodium parasites in mice, and allows direct comparison of invasion in red cells derived from two different populations. The method also serves as an accurate
Patel, Kashyap; Batty, Kevin T; Moore, Brioni R; Gibbons, Peter L; Bulitta, Jürgen B; Kirkpatrick, Carl M
Murine models are used to study erythrocytic stages of malaria infection, because parasite morphology and development are comparable to those in human malaria infections. Mechanism-based pharmacokinetic-pharmacodynamic (PK-PD) models for antimalarials are scarce, despite their potential to optimize antimalarial combination therapy. The aim of this study was to develop a mechanism-based growth model (MBGM) for Plasmodium berghei and then characterize the parasiticidal effect of dihydroartemisinin (DHA) in murine malaria (MBGM-PK-PD). Stage-specific (ring, early trophozoite, late trophozoite, and schizont) parasite density data from Swiss mice inoculated with Plasmodium berghei were used for model development in S-ADAPT. A single dose of intraperitoneal DHA (10 to 100 mg/kg) or vehicle was administered 56 h postinoculation. The MBGM explicitly reflected all four erythrocytic stages of the 24-hour P. berghei life cycle. Merozoite invasion of erythrocytes was described by a first-order process that declined with increasing parasitemia. An efflux pathway with subsequent return was additionally required to describe the schizont data, thus representing parasite sequestration or trapping in the microvasculature, with a return to circulation. A 1-compartment model with zero-order absorption described the PK of DHA, with an estimated clearance and distribution volume of 1.95 liters h(-1) and 0.851 liter, respectively. Parasite killing was described by a turnover model, with DHA inhibiting the production of physiological intermediates (IC(50), 1.46 ng/ml). Overall, the MBGM-PK-PD described the rise in parasitemia, the nadir following DHA dosing, and subsequent parasite resurgence. This novel model is a promising tool for studying malaria infections, identifying the stage specificity of antimalarials, and providing insight into antimalarial treatment strategies.
The capacity to migrate is fundamental to multicellular and single-celled life. Apicomplexan parasites, an ancient protozoan clade that includes malaria parasites (Plasmodium) and Toxoplasma, achieve remarkable speeds of directional cell movement. This rapidity is achieved via a divergent actomyosin motor system, housed within a narrow compartment that lies underneath the length of the parasite plasma membrane. How this motor functions at a mechanistic level during motility and host cell invasion is a matter of debate. Here, we integrate old and new insights toward refining the current model for the function of this motor with the aim of revitalizing interest in the mechanics of how these deadly pathogens move. PMID:27573462
Tomavo, Stanislas; Slomianny, Christian; Meissner, Markus; Carruthers, Vern B
Toxoplasma (toxoplasmosis) and Plasmodium (malaria) use unique secretory organelles for migration, cell invasion, manipulation of host cell functions, and cell egress. In particular, the apical secretory micronemes and rhoptries of apicomplexan parasites are essential for successful host infection. New findings reveal that the contents of these organelles, which are transported through the endoplasmic reticulum (ER) and Golgi, also require the parasite endosome-like system to access their respective organelles. In this review, we discuss recent findings that demonstrate that these parasites reduced their endosomal system and modified classical regulators of this pathway for the biogenesis of apical organelles.
Zheng Zhang; Ong, L L Sharon; Kong Fang; Matthew, Athul; Dauwels, Justin; Ming Dao; Asada, Harry
This paper presents a method to detect unlabeled malaria parasites in red blood cells. The current "gold standard" for malaria diagnosis is microscopic examination of thick blood smear, a time consuming process requiring extensive training. Our goal is to develop an automate process to identify malaria infected red blood cells. Major issues in automated analysis of microscopy images of unstained blood smears include overlapping cells and oddly shaped cells. Our approach creates robust templates to detect infected and uninfected red cells. Histogram of Oriented Gradients (HOGs) features are extracted from templates and used to train a classifier offline. Next, the ViolaJones object detection framework is applied to detect infected and uninfected red cells and the image background. Results show our approach out-performs classification approaches with PCA features by 50% and cell detection algorithms applying Hough transforms by 24%. Majority of related work are designed to automatically detect stained parasites in blood smears where the cells are fixed. Although it is more challenging to design algorithms for unstained parasites, our methods will allow analysis of parasite progression in live cells under different drug treatments.
Alcantara, Laura M; Kim, Junwon; Moraes, Carolina B; Franco, Caio H; Franzoi, Kathrin D; Lee, Sukjun; Freitas-Junior, Lucio H; Ayong, Lawrence S
Members of the ATP-binding cassette (ABC)-type transporter superfamily have been implicated in multidrug resistance in malaria, and various mechanistic models have been postulated to explain their interaction with diverse antimalarial drugs. To gain insight into the pharmacological benefits of inhibiting ABC-type transporters in malaria chemotherapy, we investigated the in vitro chemosensitization potential of various P-glycoprotein inhibitors. A fluorescent chloroquine derivative was synthesized and used to assess the efflux dynamics of chloroquine in MDR and wild type Plasmodium falciparum parasites. This novel BODIPY-based probe accumulated in the digestive vacuole (DV) of CQ-sensitive parasites but less so in MDR cells. Pre-exposure of the MDR parasites to non-cytocidal concentrations of unlabeled chloroquine resulted in a diffused cytoplasmic retention of the probe whereas a similar treatment with the CQR-reversing agent, chlorpheniramine, resulted in DV accumulation. A diffused cytoplasmic distribution of the probe was also obtained following treatment with the P-gp specific inhibitors zosuquidar and tariquidar, whereas treatments with the tyrosine kinase inhibitors gefitinib or imatinib produced a partial accumulation within the DV. Isobologram analyses of the interactions between these inhibitors and the antimalarial drugs chloroquine, mefloquine, and artemisinin revealed distinct patterns of drug synergism, additivity and antagonism. Taken together, the data indicate that competitive tyrosine kinase and noncompetitive P-glycoprotein ATPase-specific inhibitors represent two new classes of chemosensitizing agents in malaria parasites, but caution against the indiscriminate use of these agents in antimalarial drug combinations.
Zerka, Agata; Kaczmarek, Radosław; Jaśkiewicz, Ewa
Malaria is caused by infection with protozoan parasites belonging to the genus Plasmodium, which have arguably exerted the greatest selection pressure on humans in the history of our species. Besides humans, different Plasmodium parasites infect a wide range of animal hosts, from marine invertebrates to primates. On the other hand, individual Plasmodium species show high host specificity. The extraordinary evolution of Plasmodium probably began when a free-living red algae turned parasitic, and culminated with its ability to thrive inside a human red blood cell. Studies on the African apes generated new data on the evolution of malaria parasites in general and the deadliest human-specific species, Plasmodium falciparum, in particular. Initially, it was hypothesized that P. falciparum descended from the chimpanzee malaria parasite P. reichenowi, after the human and the chimp lineage diverged about 6 million years ago. However, a recently identified new species infecting gorillas, unexpectedly showed similarity to P. falciparum and was therefore named P. praefalciparum. That finding spurred an alternative hypothesis, which proposes that P. falciparum descended from its gorilla rather than chimp counterpart. In addition, the gorilla-to-human host shift may have occurred more recently (about 10 thousand years ago) than the theoretical P. falciparum-P. reichenowi split. One of the key aims of the studies on Plasmodium evolution is to elucidate the mechanisms that allow the incessant host shifting and retaining the host specificity, especially in the case of human-specific species. Thorough understanding of these phenomena will be necessary to design effective malaria treatment and prevention strategies.
Divis, Paul C.; Siner, Angela; Zainudin, Ramlah; Wong, Ing Tien; Lu, Chan Woon; Singh-Khaira, Sarina K.; Millar, Scott B.; Lynch, Sean; Willmann, Matthias; Singh, Balbir; Krishna, Sanjeev; Cox-Singh, Janet
Emerging pathogens undermine initiatives to control the global health impact of infectious diseases. Zoonotic malaria is no exception. Plasmodium knowlesi, a malaria parasite of Southeast Asian macaques, has entered the human population. P. knowlesi, like Plasmodium falciparum, can reach high parasitaemia in human infections, and the World Health Organization guidelines for severe malaria list hyperparasitaemia among the measures of severe malaria in both infections. Not all patients with P. knowlesi infections develop hyperparasitaemia, and it is important to determine why. Between isolate variability in erythrocyte invasion, efficiency seems key. Here we investigate the idea that particular alleles of two P. knowlesi erythrocyte invasion genes, P. knowlesi normocyte binding protein Pknbpxa and Pknbpxb, influence parasitaemia and human disease progression. Pknbpxa and Pknbpxb reference DNA sequences were generated from five geographically and temporally distinct P. knowlesi patient isolates. Polymorphic regions of each gene (approximately 800 bp) were identified by haplotyping 147 patient isolates at each locus. Parasitaemia in the study cohort was associated with markers of disease severity including liver and renal dysfunction, haemoglobin, platelets and lactate, (r = ≥0.34, p = <0.0001 for all). Seventy-five and 51 Pknbpxa and Pknbpxb haplotypes were resolved in 138 (94%) and 134 (92%) patient isolates respectively. The haplotypes formed twelve Pknbpxa and two Pknbpxb allelic groups. Patients infected with parasites with particular Pknbpxa and Pknbpxb alleles within the groups had significantly higher parasitaemia and other markers of disease severity. Our study strongly suggests that P. knowlesi invasion gene variants contribute to parasite virulence. We focused on two invasion genes, and we anticipate that additional virulent loci will be identified in pathogen genome-wide studies. The multiple sustained entries of this diverse pathogen into the
Ahmed, Atique M; Pinheiro, Miguel M; Divis, Paul C; Siner, Angela; Zainudin, Ramlah; Wong, Ing Tien; Lu, Chan Woon; Singh-Khaira, Sarina K; Millar, Scott B; Lynch, Sean; Willmann, Matthias; Singh, Balbir; Krishna, Sanjeev; Cox-Singh, Janet
Emerging pathogens undermine initiatives to control the global health impact of infectious diseases. Zoonotic malaria is no exception. Plasmodium knowlesi, a malaria parasite of Southeast Asian macaques, has entered the human population. P. knowlesi, like Plasmodium falciparum, can reach high parasitaemia in human infections, and the World Health Organization guidelines for severe malaria list hyperparasitaemia among the measures of severe malaria in both infections. Not all patients with P. knowlesi infections develop hyperparasitaemia, and it is important to determine why. Between isolate variability in erythrocyte invasion, efficiency seems key. Here we investigate the idea that particular alleles of two P. knowlesi erythrocyte invasion genes, P. knowlesi normocyte binding protein Pknbpxa and Pknbpxb, influence parasitaemia and human disease progression. Pknbpxa and Pknbpxb reference DNA sequences were generated from five geographically and temporally distinct P. knowlesi patient isolates. Polymorphic regions of each gene (approximately 800 bp) were identified by haplotyping 147 patient isolates at each locus. Parasitaemia in the study cohort was associated with markers of disease severity including liver and renal dysfunction, haemoglobin, platelets and lactate, (r = ≥ 0.34, p = <0.0001 for all). Seventy-five and 51 Pknbpxa and Pknbpxb haplotypes were resolved in 138 (94%) and 134 (92%) patient isolates respectively. The haplotypes formed twelve Pknbpxa and two Pknbpxb allelic groups. Patients infected with parasites with particular Pknbpxa and Pknbpxb alleles within the groups had significantly higher parasitaemia and other markers of disease severity. Our study strongly suggests that P. knowlesi invasion gene variants contribute to parasite virulence. We focused on two invasion genes, and we anticipate that additional virulent loci will be identified in pathogen genome-wide studies. The multiple sustained entries of this diverse pathogen into the human
Moon, Seunghyun; Lee, Sukjun; Kim, Heechang; Freitas-Junior, Lucio H; Kang, Myungjoo; Ayong, Lawrence; Hansen, Michael A E
With more than 40% of the world's population at risk, 200-300 million infections each year, and an estimated 1.2 million deaths annually, malaria remains one of the most important public health problems of mankind today. With the propensity of malaria parasites to rapidly develop resistance to newly developed therapies, and the recent failures of artemisinin-based drugs in Southeast Asia, there is an urgent need for new antimalarial compounds with novel mechanisms of action to be developed against multidrug resistant malaria. We present here a novel image analysis algorithm for the quantitative detection and classification of Plasmodium lifecycle stages in culture as well as discriminating between viable and dead parasites in drug-treated samples. This new algorithm reliably estimates the number of red blood cells (isolated or clustered) per fluorescence image field, and accurately identifies parasitized erythrocytes on the basis of high intensity DAPI-stained parasite nuclei spots and Mitotracker-stained mitochondrial in viable parasites. We validated the performance of the algorithm by manual counting of the infected and non-infected red blood cells in multiple image fields, and the quantitative analyses of the different parasite stages (early rings, rings, trophozoites, schizonts) at various time-point post-merozoite invasion, in tightly synchronized cultures. Additionally, the developed algorithm provided parasitological effective concentration 50 (EC50) values for both chloroquine and artemisinin, that were similar to known growth inhibitory EC50 values for these compounds as determined using conventional SYBR Green I and lactate dehydrogenase-based assays.
Gerald, Noel; Mahajan, Babita; Kumar, Sanjai
Malaria is caused by intraerythrocytic protozoan parasites belonging to Plasmodium spp. (phylum Apicomplexa) that produce significant morbidity and mortality, mostly in developing countries. Plasmodium parasites have a complex life cycle that includes multiple stages in anopheline mosquito vectors and vertebrate hosts. During the life cycle, the parasites undergo several cycles of extreme population growth within a brief span, and this is critical for their continued transmission and a contributing factor for their pathogenesis in the host. As with other eukaryotes, successful mitosis is an essential requirement for Plasmodium reproduction; however, some aspects of Plasmodium mitosis are quite distinct and not fully understood. In this review, we will discuss the current understanding of the architecture and key events of mitosis in Plasmodium falciparum and related parasites and compare them with the traditional mitotic events described for other eukaryotes. PMID:21317311
Lehane, Adele M; Ridgway, Melanie C; Baker, Eileen; Kirk, Kiaran
The antimalarial spiroindolones disrupt Plasmodium falciparum Na(+) regulation and induce an alkalinization of the parasite cytosol. It has been proposed that they do so by inhibiting PfATP4, a parasite plasma membrane P-type ATPase postulated to export Na(+) and import H(+) equivalents. Here, we screened the 400 antiplasmodial compounds of the open access 'Malaria Box' for their effects on parasite ion regulation. Twenty eight compounds affected parasite Na(+) and pH regulation in a manner consistent with PfATP4 inhibition. Six of these, with chemically diverse structures, were selected for further analysis. All six showed reduced antiplasmodial activity against spiroindolone-resistant parasites carrying mutations in pfatp4. We exposed parasites to incrementally increasing concentrations of two of the six compounds and in both cases obtained resistant parasites with mutations in pfatp4. The finding that diverse chemotypes have an apparently similar mechanism of action indicates that PfATP4 may be a significant Achilles' heel for the parasite. © 2014 John Wiley & Sons Ltd.
Sims, Paul F G; Hyde, John E
The lethal species of malaria parasite, Plasmodium falciparum, continues to exact a huge toll of mortality and morbidity, particularly in sub-Saharan Africa. Completion of the genome sequence of this organism and advances in proteomics and mass spectrometry have opened up unprecedented opportunities for understanding the complex biology of this parasite and how it responds to drug challenge and other interventions. This review describes recent progress that has been made in applying proteomics technology to this important pathogen and provides a look forward to likely future developments.
Goedknegt, M. Anouk; Feis, Marieke E.; Wegner, K. Mathias; Luttikhuizen, Pieternella C.; Buschbaum, Christian; Camphuysen, Kees (C. J.); van der Meer, Jaap; Thieltges, David W.
Worldwide, marine and coastal ecosystems are heavily invaded by introduced species and the potential role of parasites in the success and impact of marine invasions has been increasingly recognized. In this review, we link recent theoretical developments in invasion ecology with empirical studies from marine ecosystems in order to provide a conceptual framework for studying the role of parasites and their hosts in marine invasions. Based on an extensive literature search, we identified six mechanisms in which invaders directly or indirectly affect parasite and host populations and communities: I) invaders can lose some or all of their parasites during the invasion process (parasite release or reduction), often causing a competitive advantage over native species; II) invaders can also act as a host for native parasites, which may indirectly amplify the parasite load of native hosts (parasite spillback); III) invaders can also be parasites themselves and be introduced without needing co-introduction of the host (introduction of free-living infective stages); IV) alternatively, parasites may be introduced together with their hosts (parasite co-introduction with host); V) consequently, these co-introduced parasites can sometimes also infect native hosts (parasite spillover); and VI) invasive species may be neither a host nor a parasite, but nevertheless affect native parasite host interactions by interfering with parasite transmission (transmission interference). We discuss the ecological and evolutionary implications of each of these mechanisms and generally note several substantial effects on natural communities and ecosystems via i) mass mortalities of native populations creating strong selection gradients, ii) indirect changes in species interactions within communities and iii) trophic cascading and knock-on effects in food webs that may affect ecosystem function and services. Our review demonstrates a wide range of ecological and evolutionary implications of
Hammami, Imen; Nuel, Grégory; Garcia, André
Malaria is a global health problem responsible for nearly one million deaths every year around 85% of which concern children younger than five years old in Sub-Saharan Africa. In addition, around 300 million clinical cases are declared every year. The level of infection, expressed as parasite density, is classically defined as the number of asexual parasites relative to a microliter of blood. Microscopy of Giemsa-stained thick blood films is the gold standard for parasite enumeration. Parasite density estimation methods usually involve threshold values; either the number of white blood cells counted or the number of high power fields read. However, the statistical properties of parasite density estimators generated by these methods have largely been overlooked. Here, we studied the statistical properties (mean error, coefficient of variation, false negative rates) of parasite density estimators of commonly used threshold-based counting techniques depending on variable threshold values. We also assessed the influence of the thresholds on the cost-effectiveness of parasite density estimation methods. In addition, we gave more insights on the behavior of measurement errors according to varying threshold values, and on what should be the optimal threshold values that minimize this variability.
Kim, Charles C; Wilson, Emily B; DeRisi, Joseph L
Malaria parasites generate free haem upon catabolism of host haemoglobin during their intraerythrocytic growth cycle. In order to minimize oxidative toxicity of the ferric iron, the free haem molecules are polymerized into the biomineral beta-haematin (commonly referred to as haemozoin). Haemozoin crystals are paramagnetic, and this property can be exploited for the purification of late stage parasites as they contain larger haemozoin crystals than early stage parasites and uninfected cells. Commercially available magnets that were originally developed for the purpose of antibody-mediated cell purification are widely used for this purpose. As these methods are not necessarily optimized for parasite purification, the relationship between magnetic field strength and the quantity and quality of yield during parasite purification was explored. Inexpensive rare-earth neodymium magnets with commercially available disposable columns were employed to explore the relationship between magnetic field strength and recovery of free haemozoin and infected erythrocytes (iRBCs). Yields of free haemozoin increased nearly linearly with increasing magnetic field strength to the strongest fields tested (8,500 Gauss). Stronger magnetic fields also improved the recovery of iRBCs with no detrimental effects on parasite viability. An in-house constructed magnetic stand, built for $75 in materials, produced superior results when compared with much more expensive commercial products. Existing protocols for the magnetic purification of free haemozoin and iRBCs result in sub-optimal yields. Inexpensive high-strength neodymium magnets offer a better option, resulting in higher yields with no detrimental effects on parasite viability.
Matsuoka, Hiroyuki; Tomita, Hiroyuki; Hattori, Ryuta; Arai, Meiji; Hirai, Makoto
We produced a transgenic rodent malaria parasite (Plasmodium berghei) that contained the luciferase gene under a promoter region of elongation factor-1α. These transgenic (TG) parasites expressed luciferase in all stages of their life cycle, as previously reported. However, we were the first to succeed in observing sporozoites as a mass in mouse skin following their deposition by the probing of infective mosquitoes. Our transgenic parasites may have emitted stronger bioluminescence than previous TG parasites. The estimated number of injected sporozoites by mosquitoes was between 34 and 775 (median 80). Since luciferase activity diminished immediately after the death of the parasites, luciferase activity could be an indicator of the existence of live parasites. Our results indicated that sporozoites survived at the probed site for more than 42 hours. We also detected sporozoites in the liver within 15 min of the intravenous injection. Bioluminescence was not observed in the lung, kidney or spleen. We confirmed the observation that the liver was the first organ in which malaria parasites entered and increased in number.
Poulin, Benoit; Patzewitz, Eva-Maria; Brady, Declan; Silvie, Olivier; Wright, Megan H.; Ferguson, David J. P.; Wall, Richard J.; Whipple, Sarah; Guttery, David S.; Tate, Edward W.; Wickstead, Bill; Holder, Anthony A.; Tewari, Rita
Summary The phylum Apicomplexa comprises over 5000 intracellular protozoan parasites, including Plasmodium and Toxoplasma, that are clinically important pathogens affecting humans and livestock. Malaria parasites belonging to the genus Plasmodium possess a pellicle comprised of a plasmalemma and inner membrane complex (IMC), which is implicated in parasite motility and invasion. Using live cell imaging and reverse genetics in the rodent malaria model P. berghei, we localise two unique IMC sub-compartment proteins (ISPs) and examine their role in defining apical polarity during zygote (ookinete) development. We show that these proteins localise to the anterior apical end of the parasite where IMC organisation is initiated, and are expressed at all developmental stages, especially those that are invasive. Both ISP proteins are N-myristoylated, phosphorylated and membrane-bound. Gene disruption studies suggest that ISP1 is likely essential for parasite development, whereas ISP3 is not. However, an absence of ISP3 alters the apical localisation of ISP1 in all invasive stages including ookinetes and sporozoites, suggesting a coordinated function for these proteins in the organisation of apical polarity in the parasite. PMID:24244852
Haldar, K; Samuel, B U; Mohandas, N; Harrison, T; Hiller, N L
Studies in the past year displaced long-standing dogmas and provided many new molecular insights into how proteins and solutes move between the erythrocyte plasma membrane and the malarial vacuole. Highlights include a demonstration that (1) detergent-resistant membrane (DRM) rafts exist in the red cell membrane and their resident proteins are detected as rafts in the plasmodial vacuole, (2) a voltage-gated channel in the infected red cell membrane mediates uptake of extracellular nutrient solutes, and (3) intraerythrocytic membranes transport a parasite-encoded adherence antigen to the red cell surface.
Dankwa, Selasi; Lim, Caeul; Bei, Amy K; Jiang, Rays H Y; Abshire, James R; Patel, Saurabh D; Goldberg, Jonathan M; Moreno, Yovany; Kono, Maya; Niles, Jacquin C; Duraisingh, Manoj T
Plasmodium knowlesi is a zoonotic parasite transmitted from macaques causing malaria in humans in Southeast Asia. Plasmodium parasites bind to red blood cell (RBC) surface receptors, many of which are sialylated. While macaques synthesize the sialic acid variant N-glycolylneuraminic acid (Neu5Gc), humans cannot because of a mutation in the enzyme CMAH that converts N-acetylneuraminic acid (Neu5Ac) to Neu5Gc. Here we reconstitute CMAH in human RBCs for the reintroduction of Neu5Gc, which results in enhancement of P. knowlesi invasion. We show that two P. knowlesi invasion ligands, PkDBPβ and PkDBPγ, bind specifically to Neu5Gc-containing receptors. A human-adapted P. knowlesi line invades human RBCs independently of Neu5Gc, with duplication of the sialic acid-independent invasion ligand, PkDBPα and loss of PkDBPγ. Our results suggest that absence of Neu5Gc on human RBCs limits P. knowlesi invasion, but that parasites may evolve to invade human RBCs through the use of sialic acid-independent pathways.
Dankwa, Selasi; Lim, Caeul; Bei, Amy K.; Jiang, Rays H. Y.; Abshire, James R.; Patel, Saurabh D.; Goldberg, Jonathan M.; Moreno, Yovany; Kono, Maya; Niles, Jacquin C.; Duraisingh, Manoj T.
Plasmodium knowlesi is a zoonotic parasite transmitted from macaques causing malaria in humans in Southeast Asia. Plasmodium parasites bind to red blood cell (RBC) surface receptors, many of which are sialylated. While macaques synthesize the sialic acid variant N-glycolylneuraminic acid (Neu5Gc), humans cannot because of a mutation in the enzyme CMAH that converts N-acetylneuraminic acid (Neu5Ac) to Neu5Gc. Here we reconstitute CMAH in human RBCs for the reintroduction of Neu5Gc, which results in enhancement of P. knowlesi invasion. We show that two P. knowlesi invasion ligands, PkDBPβ and PkDBPγ, bind specifically to Neu5Gc-containing receptors. A human-adapted P. knowlesi line invades human RBCs independently of Neu5Gc, with duplication of the sialic acid-independent invasion ligand, PkDBPα and loss of PkDBPγ. Our results suggest that absence of Neu5Gc on human RBCs limits P. knowlesi invasion, but that parasites may evolve to invade human RBCs through the use of sialic acid-independent pathways. PMID:27041489
Guerra, Carlos A; Hay, Simon I; Lucioparedes, Lorena S; Gikandi, Priscilla W; Tatem, Andrew J; Noor, Abdisalan M; Snow, Robert W
Background Open access to databases of information generated by the research community can synergize individual efforts and are epitomized by the genome mapping projects. Open source models for outputs of scientific research funded by tax-payers and charities are becoming the norm. This has yet to be extended to malaria epidemiology and control. Methods The exhaustive searches and assembly process for a global database of malaria parasite prevalence as part of the Malaria Atlas Project (MAP) are described. The different data sources visited and how productive these were in terms of availability of parasite rate (PR) data are presented, followed by a description of the methods used to assemble a relational database and an associated geographic information system. The challenges facing spatial data assembly from varied sources are described in an effort to help inform similar future applications. Results At the time of writing, the MAP database held 3,351 spatially independent PR estimates from community surveys conducted since 1985. These include 3,036 Plasmodium falciparum and 1,347 Plasmodium vivax estimates in 74 countries derived from 671 primary sources. More than half of these data represent malaria prevalence after the year 2000. Conclusion This database will help refine maps of the global spatial limits of malaria and be the foundation for the development of global malaria endemicity models as part of MAP. A widespread application of these maps is envisaged. The data compiled and the products generated by MAP are planned to be released in June 2009 to facilitate a more informed approach to global malaria control. PMID:17306022
Yiangou, Loukia; Montandon, Ruddy; Modrzynska, Katarzyna; Rosen, Barry; Bushell, Wendy; Hale, Christine; Billker, Oliver; Rayner, Julian C.
The clinical complications of malaria are caused by the parasite expansion in the blood. Invasion of erythrocytes is a complex process that depends on multiple receptor-ligand interactions. Identification of host receptors is paramount for fighting the disease as it could reveal new intervention targets, but the enucleated nature of erythrocytes makes genetic approaches impossible and many receptors remain unknown. Host-parasite interactions evolve rapidly and are therefore likely to be species-specific. As a results, understanding of invasion receptors outside the major human pathogen Plasmodium falciparum is very limited. Here we use mouse embryonic stem cells (mESCs) that can be genetically engineered and differentiated into erythrocytes to identify receptors for the rodent malaria parasite Plasmodium berghei. Two proteins previously implicated in human malaria infection: glycophorin C (GYPC) and Band-3 (Slc4a1) were deleted in mESCs to generate stable cell lines, which were differentiated towards erythropoiesis. In vitro infection assays revealed that while deletion of Band-3 has no effect, absence of GYPC results in a dramatic decrease in invasion, demonstrating the crucial role of this protein for P. berghei infection. This stem cell approach offers the possibility of targeting genes that may be essential and therefore difficult to disrupt in whole organisms and has the potential to be applied to a variety of parasites in diverse host cell types. PMID:27362409
Heck, J E
Human malaria is caused by four species of the genus plasmodium. The sexual stage of the parasite occurs in the mosquito and asexual reproduction occurs in man. Symptoms of fever, chills, headache, and myalgia result from the invasion and rupture of erythrocytes. Merozoites are released from erythrocytes and invade other cells, thus propagating the infection. The most vulnerable hosts are nonimmune travelers, young children living in the tropics, and pregnant women. P. falciparum causes the most severe infections because it infects RBCs of all ages and has the propensity to develop resistance to antimalarials. Rapid diagnosis can be made with a malarial smear, and treatment should be initiated promptly. In some regions (Mexico, Central America except Panama, and North Africa) chloroquine phosphate is effective therapy. In subsaharan Africa, South America, and Southeast Asia, chloroquine resistance has become widespread, and other antimalarials are necessary. The primary care physician should have a high index of suspicion for malaria in the traveler returning from the tropics. Malaria should also be suspected in the febrile transfusion recipient and newborns of mothers with malaria.
Katakai, Yuko; Komaki-Yasuda, Kanako; Tangpukdee, Noppadon; Wilairatana, Polrat; Krudsood, Srivicha; Kano, Shigeyuki
The NOW® Malaria Test, an immunochromatographic test (ICT), was evaluated to determine its ability to quantitatively detect malaria parasites using 100 blood samples from Thailand, including 50 Plasmodium falciparum (Pf) infections and 50 P. vivax (Pv) infections. Intensities of the thickness of the visible bands of the positive ICT were compared with the parasite densities. In cases of Pf infection, the intensities of both HRP-2 bands (T1 bands: Pf specific bands) and aldolase bands (T2 bands: pan-Plasmodium bands) correlated with the parasite densities. The intensities of T2 bands in Pf positive samples showed better correlation with the parasite densities than the T1 bands. In the cases of Pv infection, the intensities of T2 bands were also well correlated with parasite density. These results suggest that the ICT is useful not only for rapid detection of malaria parasites but also for estimating parasite density. PMID:22438699
Katakai, Yuko; Komaki-Yasuda, Kanako; Tangpukdee, Noppadon; Wilairatana, Polrat; Krudsood, Srivicha; Kano, Shigeyuki
The NOW® Malaria Test, an immunochromatographic test (ICT), was evaluated to determine its ability to quantitatively detect malaria parasites using 100 blood samples from Thailand, including 50 Plasmodium falciparum (Pf) infections and 50 P. vivax (Pv) infections. Intensities of the thickness of the visible bands of the positive ICT were compared with the parasite densities. In cases of Pf infection, the intensities of both HRP-2 bands (T1 bands: Pf specific bands) and aldolase bands (T2 bands: pan-Plasmodium bands) correlated with the parasite densities. The intensities of T2 bands in Pf positive samples showed better correlation with the parasite densities than the T1 bands. In the cases of Pv infection, the intensities of T2 bands were also well correlated with parasite density. These results suggest that the ICT is useful not only for rapid detection of malaria parasites but also for estimating parasite density.
Kishore, Sandeep P; Stiller, John W; Deitsch, Kirk W
The acquisition of complex transcriptional regulatory abilities and epigenetic machinery facilitated the transition of the ancestor of apicomplexans from a free-living organism to an obligate parasite. The ability to control sophisticated gene expression patterns enabled these ancient organisms to evolve several differentiated forms, invade multiple hosts and evade host immunity. How these abilities were acquired remains an outstanding question in protistan biology. In this work, we study SET domain bearing genes that are implicated in mediating immune evasion, invasion and cytoadhesion pathways of modern apicomplexans, including malaria parasites. We provide the first conclusive evidence of a horizontal gene transfer of a Histone H4 Lysine 20 (H4K20) modifier, Set8, from an animal host to the ancestor of apicomplexans. Set8 is known to contribute to the coordinated expression of genes involved in immune evasion in modern apicomplexans. We also show the likely transfer of a H3K36 methyltransferase (Ashr3 from plants), possibly derived from algal endosymbionts. These transfers appear to date to the transition from free-living organisms to parasitism and coincide with the proposed horizontal acquisition of cytoadhesion domains, the O-glycosyltransferase that modifies these domains, and the primary family of transcription factors found in apicomplexan parasites. Notably, phylogenetic support for these conclusions is robust and the genes clearly are dissimilar to SET sequences found in the closely related parasite Perkinsus marinus, and in ciliates, the nearest free-living organisms with complete genome sequences available. Animal and plant sources of epigenetic machinery provide new insights into the evolution of parasitism in apicomplexans. Along with the horizontal transfer of cytoadhesive domains, O-linked glycosylation and key transcription factors, the acquisition of SET domain methyltransferases marks a key transitional event in the evolution to parasitism in
Background The acquisition of complex transcriptional regulatory abilities and epigenetic machinery facilitated the transition of the ancestor of apicomplexans from a free-living organism to an obligate parasite. The ability to control sophisticated gene expression patterns enabled these ancient organisms to evolve several differentiated forms, invade multiple hosts and evade host immunity. How these abilities were acquired remains an outstanding question in protistan biology. Results In this work, we study SET domain bearing genes that are implicated in mediating immune evasion, invasion and cytoadhesion pathways of modern apicomplexans, including malaria parasites. We provide the first conclusive evidence of a horizontal gene transfer of a Histone H4 Lysine 20 (H4K20) modifier, Set8, from an animal host to the ancestor of apicomplexans. Set8 is known to contribute to the coordinated expression of genes involved in immune evasion in modern apicomplexans. We also show the likely transfer of a H3K36 methyltransferase (Ashr3 from plants), possibly derived from algal endosymbionts. These transfers appear to date to the transition from free-living organisms to parasitism and coincide with the proposed horizontal acquisition of cytoadhesion domains, the O-glycosyltransferase that modifies these domains, and the primary family of transcription factors found in apicomplexan parasites. Notably, phylogenetic support for these conclusions is robust and the genes clearly are dissimilar to SET sequences found in the closely related parasite Perkinsus marinus, and in ciliates, the nearest free-living organisms with complete genome sequences available. Conclusions Animal and plant sources of epigenetic machinery provide new insights into the evolution of parasitism in apicomplexans. Along with the horizontal transfer of cytoadhesive domains, O-linked glycosylation and key transcription factors, the acquisition of SET domain methyltransferases marks a key transitional event in
Alleva, L M; Kirk, K
The regulation of intracellular Ca(2+) in the intraerythrocytic form of the human malaria parasite, Plasmodium falciparum, was investigated using parasites 'isolated' from their host cells by saponin-permeabilisation of the erythrocyte membrane. The isolated parasites maintained tight control over their resting cytosolic Ca(2+) concentration which ranged from approximately 100 nM in the absence of extracellular Ca(2+) to approximately 700 nM in the presence of 1 mM extracellular Ca(2+). The parasite has two functionally discrete intracellular Ca(2+) stores. One is an 'endoplasmic reticulum (ER)-like' store, the other an 'acidic store'. The ER-like store was discharged by cyclopiazonic acid (CPA), an inhibitor of sarco/endoplasmic reticulum Ca(2+)-ATPases (SERCAs) of animal and plant cells, but not by thapsigargin (TG), a more specific inhibitor of SERCAs of animal cells. The acidic store was discharged by nigericin and by NH(4)(+). The amount of Ca(2+) in the ER-like store increased with increasing extracellular Ca(2+) concentration, whereas the amount of Ca(2+) in the acidic store did not. Ca(2+) released from the ER-like store by CPA was cleared from the parasite cytosol by uptake into the acidic store (over a range of extracellular Ca(2+) concentrations), consistent with the acidic store serving as a Ca(2+) reservoir within the intracellular parasite.
Russell, Bruce M; Cooke, Brian M
Our current understanding of how malaria parasites remodel their host red blood cells (RBCs) and ultimately cause disease is largely based on studies of Plasmodium falciparum. In this review, we expand our knowledge to include what is currently known about pathophysiological changes to RBCs that are infected by non-falciparum malaria parasites. We highlight the potential folly of making generalizations about the rheology of malaria infection, and emphasize the need for more systematic studies into the erythrocytic biology of non-falciparum malaria parasites. We propose that a better understanding of the mechanisms that underlie the changes to RBCs induced by malaria parasites other than P. falciparum may be highly informative for the development of therapeutics that specifically disrupt the altered rheological profile of RBCs infected with either sexual- or asexual-stage parasites, resulting in drugs that block transmission, reduce disease severity, and help delay the onset of resistance to current and future anti-malaria drugs.
Jones, Catherine M; Brown, Mark J F; Ings, Thomas
Biological invasions are facilitated by the global transportation of species and climate change. Given that invasions may cause ecological and economic damage and pose a major threat to biodiversity, understanding the mechanisms behind invasion success is essential. Both the release of non-native populations from natural enemies, such as parasites, and the genetic diversity of these populations may play key roles in their invasion success. We investigated the roles of parasite communities, through enemy release and parasite acquisition, and genetic diversity in the invasion success of the non-native bumblebee, Bombus hypnorum, in the United Kingdom. The invasive B. hypnorum had higher parasite prevalence than most, or all native congeners for two high-impact parasites, probably due to higher susceptibility and parasite acquisition. Consequently parasites had a higher impact on B. hypnorum queens’ survival and colony-founding success than on native species. Bombus hypnorum also had lower functional genetic diversity at the sex-determining locus than native species. Higher parasite prevalence and lower genetic diversity have not prevented the rapid invasion of the United Kingdom by B. hypnorum. These data may inform our understanding of similar invasions by commercial bumblebees around the world. This study suggests that concerns about parasite impacts on the small founding populations common to re-introduction and translocation programs may be less important than currently believed. PMID:24749545
Treeck, Moritz; Struck, Nicole S; Haase, Silvia; Langer, Christine; Herrmann, Susann; Healer, Julie; Cowman, Alan F; Gilberger, Tim W
The proliferation of the malaria parasite Plasmodium falciparum within the human host is dependent upon invasion of erythrocytes. This process is accomplished by the merozoite, a highly specialized form of the parasite. Secretory organelles including micronemes and rhoptries play a pivotal role in the invasion process by storing and releasing parasite proteins. The mechanism of protein sorting to these compartments is unclear. Using a transgenic approach we show that trafficking of the most abundant micronemal proteins (members of the EBL-family: EBA-175, EBA-140/BAEBL, and EBA-181/JSEBL) is independent of their cytoplasmic and transmembrane domains, respectively. To identify the minimal sequence requirements for microneme trafficking, we generated parasites expressing EBA-GFP chimeric proteins and analyzed their distribution within the infected erythrocyte. This revealed that: (i) a conserved cysteine-rich region in the ectodomain is necessary for protein trafficking to the micronemes and (ii) correct sorting is dependent on accurate timing of expression.
Kobylinski, Kevin C.; Alout, Haoues; Foy, Brian D.; Clements, Archie; Adisakwattana, Poom; Swierczewski, Brett E.; Richardson, Jason H.
Recently there have been calls for the eradication of malaria and the elimination of soil-transmitted helminths (STHs). Malaria and STHs overlap in distribution, and STH infections are associated with increased risk for malaria. Indeed, there is evidence that suggests that STH infection may facilitate malaria transmission. Malaria and STH coinfection may exacerbate anemia, especially in pregnant women, leading to worsened child development and more adverse pregnancy outcomes than these diseases would cause on their own. Ivermectin mass drug administration (MDA) to humans for malaria parasite transmission suppression is being investigated as a potential malaria elimination tool. Adding albendazole to ivermectin MDAs would maximize effects against STHs. A proactive, integrated control platform that targets malaria and STHs would be extremely cost-effective and simultaneously reduce human suffering caused by multiple diseases. This paper outlines the benefits of adding albendazole to ivermectin MDAs for malaria parasite transmission suppression. PMID:25070998
Gardner, Malcolm J; Hall, Neil; Fung, Eula; White, Owen; Berriman, Matthew; Hyman, Richard W; Carlton, Jane M; Pain, Arnab; Nelson, Karen E; Bowman, Sharen; Paulsen, Ian T; James, Keith; Eisen, Jonathan A; Rutherford, Kim; Salzberg, Steven L; Craig, Alister; Kyes, Sue; Chan, Man-Suen; Nene, Vishvanath; Shallom, Shamira J; Suh, Bernard; Peterson, Jeremy; Angiuoli, Sam; Pertea, Mihaela; Allen, Jonathan; Selengut, Jeremy; Haft, Daniel; Mather, Michael W; Vaidya, Akhil B; Martin, David M A; Fairlamb, Alan H; Fraunholz, Martin J; Roos, David S; Ralph, Stuart A; McFadden, Geoffrey I; Cummings, Leda M; Subramanian, G Mani; Mungall, Chris; Venter, J Craig; Carucci, Daniel J; Hoffman, Stephen L; Newbold, Chris; Davis, Ronald W; Fraser, Claire M; Barrell, Bart
The parasite Plasmodium falciparum is responsible for hundreds of millions of cases of malaria, and kills more than one million African children annually. Here we report an analysis of the genome sequence of P. falciparum clone 3D7. The 23-megabase nuclear genome consists of 14 chromosomes, encodes about 5,300 genes, and is the most (A + T)-rich genome sequenced to date. Genes involved in antigenic variation are concentrated in the subtelomeric regions of the chromosomes. Compared to the genomes of free-living eukaryotic microbes, the genome of this intracellular parasite encodes fewer enzymes and transporters, but a large proportion of genes are devoted to immune evasion and host-parasite interactions. Many nuclear-encoded proteins are targeted to the apicoplast, an organelle involved in fatty-acid and isoprenoid metabolism. The genome sequence provides the foundation for future studies of this organism, and is being exploited in the search for new drugs and vaccines to fight malaria.
Lucchi, Naomi W.; Gaye, Marie; Diallo, Mammadou Alpha; Goldman, Ira F.; Ljolje, Dragan; Deme, Awa Bineta; Badiane, Aida; Ndiaye, Yaye Die; Barnwell, John W.; Udhayakumar, Venkatachalam; Ndiaye, Daouda
Isothermal nucleic acid amplification assays such as the loop mediated isothermal amplification (LAMP), are well suited for field use as they do not require thermal cyclers to amplify the DNA. To further facilitate the use of LAMP assays in remote settings, simpler sample preparation methods and lyophilized reagents are required. The performance of a commercial malaria LAMP assay (Illumigene Malaria LAMP) was evaluated using two sample preparation workflows (simple filtration prep (SFP)) and gravity-driven filtration prep (GFP)) and pre-dispensed lyophilized reagents. Laboratory and clinical samples were tested in a field laboratory in Senegal and the results independently confirmed in a reference laboratory in the U.S.A. The Illumigene Malaria LAMP assay was easily implemented in the clinical laboratory and gave similar results to a real-time PCR reference test with limits of detection of ≤2.0 parasites/μl depending on the sample preparation method used. This assay reliably detected Plasmodium sp. parasites in a simple low-tech format, providing a much needed alternative to the more complex molecular tests for malaria diagnosis. PMID:27827432
Park, Se Eun; Pak, Gi Deok; Aaby, Peter; Adu-Sarkodie, Yaw; Ali, Mohammad; Aseffa, Abraham; Biggs, Holly M; Bjerregaard-Andersen, Morten; Breiman, Robert F; Crump, John A; Cruz Espinoza, Ligia Maria; Eltayeb, Muna Ahmed; Gasmelseed, Nagla; Hertz, Julian T; Im, Justin; Jaeger, Anna; Parfait Kabore, Leon; von Kalckreuth, Vera; Keddy, Karen H; Konings, Frank; Krumkamp, Ralf; MacLennan, Calman A; Meyer, Christian G; Montgomery, Joel M; Ahmet Niang, Aissatou; Nichols, Chelsea; Olack, Beatrice; Panzner, Ursula; Park, Jin Kyung; Rabezanahary, Henintsoa; Rakotozandrindrainy, Raphaël; Sampo, Emmanuel; Sarpong, Nimako; Schütt-Gerowitt, Heidi; Sooka, Arvinda; Soura, Abdramane Bassiahi; Sow, Amy Gassama; Tall, Adama; Teferi, Mekonnen; Yeshitela, Biruk; May, Jürgen; Wierzba, Thomas F; Clemens, John D; Baker, Stephen; Marks, Florian
Country-specific studies in Africa have indicated that Plasmodium falciparum is associated with invasive nontyphoidal Salmonella (iNTS) disease. We conducted a multicenter study in 13 sites in Burkina Faso, Ethiopia, Ghana, Guinea-Bissau, Kenya, Madagascar, Senegal, South Africa, Sudan, and Tanzania to investigate the relationship between the occurrence of iNTS disease, other systemic bacterial infections, and malaria. Febrile patients received a blood culture and a malaria test. Isolated bacteria underwent antimicrobial susceptibility testing, and the association between iNTS disease and malaria was assessed. A positive correlation between frequency proportions of malaria and iNTS was observed (P = .01; r = 0.70). Areas with higher burden of malaria exhibited higher odds of iNTS disease compared to other bacterial infections (odds ratio [OR], 4.89; 95% CI, 1.61-14.90; P = .005) than areas with lower malaria burden. Malaria parasite positivity was associated with iNTS disease (OR, 2.44; P = .031) and gram-positive bacteremias, particularly Staphylococcus aureus, exhibited a high proportion of coinfection with Plasmodium malaria. Salmonella Typhimurium and Salmonella Enteritidis were the predominant NTS serovars (53/73; 73%). Both moderate (OR, 6.05; P = .0001) and severe (OR, 14.62; P < .0001) anemia were associated with iNTS disease. A positive correlation between iNTS disease and malaria endemicity, and the association between Plasmodium parasite positivity and iNTS disease across sub-Saharan Africa, indicates the necessity to consider iNTS as a major cause of febrile illness in malaria-holoendemic areas. Prevention of iNTS disease through iNTS vaccines for areas of high malaria endemicity, targeting high-risk groups for Plasmodium parasitic infection, should be considered. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail firstname.lastname@example.org.
Rutledge, Gavin G.; Böhme, Ulrike; Sanders, Mandy; Reid, Adam J.; Cotton, James A.; Maiga-Ascofare, Oumou; Djimdé, Abdoulaye A.; Apinjoh, Tobias O.; Amenga-Etego, Lucas; Manske, Magnus; Barnwell, John W.; Renaud, François; Ollomo, Benjamin; Prugnolle, Franck; Anstey, Nicholas M.; Auburn, Sarah; Price, Ric N.; McCarthy, James S.; Kwiatkowski, Dominic P.; Newbold, Chris I.; Berriman, Matthew; Otto, Thomas D.
Elucidation of the evolutionary history and interrelatedness of Plasmodium species that infect humans has been hampered by a lack of genetic information for three human-infective species: P. malariae and two P. ovale species (P. o. curtisi and P. o. wallikeri)1. These species are prevalent across most regions in which malaria is endemic2,3 and are often undetectable by light microscopy4, rendering their study in human populations difficult5. The exact evolutionary relationship of these species to the other human-infective species has been contested6,7. Using a new reference genome for P. malariae and a manually curated draft P. o. curtisi genome, we are now able to accurately place these species within the Plasmodium phylogeny. Sequencing of a P. malariae relative that infects chimpanzees reveals similar signatures of selection in the P. malariae lineage to another Plasmodium lineage shown to be capable of colonization of both human and chimpanzee hosts. Molecular dating suggests that these host adaptations occurred over similar evolutionary timescales. In addition to the core genome that is conserved between species, differences in gene content can be linked to their specific biology. The genome suggests that P. malariae expresses a family of heterodimeric proteins on its surface that have structural similarities to a protein crucial for invasion of red blood cells. The data presented here provide insight into the evolution of the Plasmodium genus as a whole. PMID:28117441
Rutledge, Gavin G; Böhme, Ulrike; Sanders, Mandy; Reid, Adam J; Cotton, James A; Maiga-Ascofare, Oumou; Djimdé, Abdoulaye A; Apinjoh, Tobias O; Amenga-Etego, Lucas; Manske, Magnus; Barnwell, John W; Renaud, François; Ollomo, Benjamin; Prugnolle, Franck; Anstey, Nicholas M; Auburn, Sarah; Price, Ric N; McCarthy, James S; Kwiatkowski, Dominic P; Newbold, Chris I; Berriman, Matthew; Otto, Thomas D
Elucidation of the evolutionary history and interrelatedness of Plasmodium species that infect humans has been hampered by a lack of genetic information for three human-infective species: P. malariae and two P. ovale species (P. o. curtisi and P. o. wallikeri). These species are prevalent across most regions in which malaria is endemic and are often undetectable by light microscopy, rendering their study in human populations difficult. The exact evolutionary relationship of these species to the other human-infective species has been contested. Using a new reference genome for P. malariae and a manually curated draft P. o. curtisi genome, we are now able to accurately place these species within the Plasmodium phylogeny. Sequencing of a P. malariae relative that infects chimpanzees reveals similar signatures of selection in the P. malariae lineage to another Plasmodium lineage shown to be capable of colonization of both human and chimpanzee hosts. Molecular dating suggests that these host adaptations occurred over similar evolutionary timescales. In addition to the core genome that is conserved between species, differences in gene content can be linked to their specific biology. The genome suggests that P. malariae expresses a family of heterodimeric proteins on its surface that have structural similarities to a protein crucial for invasion of red blood cells. The data presented here provide insight into the evolution of the Plasmodium genus as a whole.
Sigala, Paul A; Crowley, Jan R; Henderson, Jeffrey P; Goldberg, Daniel E
Heme metabolism is central to blood-stage infection by the malaria parasite Plasmodium falciparum. Parasites retain a heme biosynthesis pathway but do not require its activity during infection of heme-rich erythrocytes, where they can scavenge host heme to meet metabolic needs. Nevertheless, heme biosynthesis in parasite-infected erythrocytes can be potently stimulated by exogenous 5-aminolevulinic acid (ALA), resulting in accumulation of the phototoxic intermediate protoporphyrin IX (PPIX). Here we use photodynamic imaging, mass spectrometry, parasite gene disruption, and chemical probes to reveal that vestigial host enzymes in the cytoplasm of Plasmodium-infected erythrocytes contribute to ALA-stimulated heme biosynthesis and that ALA uptake depends on parasite-established permeability pathways. We show that PPIX accumulation in infected erythrocytes can be harnessed for antimalarial chemotherapy using luminol-based chemiluminescence and combinatorial stimulation by low-dose artemisinin to photoactivate PPIX to produce cytotoxic reactive oxygen. This photodynamic strategy has the advantage of exploiting host enzymes refractory to resistance-conferring mutations. DOI: http://dx.doi.org/10.7554/eLife.09143.001 PMID:26173178
Pollitt, Laura C; Churcher, Thomas S; Dawes, Emma J; Khan, Shahid M; Sajid, Mohammed; Basáñez, María-Gloria; Colegrave, Nick; Reece, Sarah E
The utility of using evolutionary and ecological frameworks to understand the dynamics of infectious diseases is gaining increasing recognition. However, integrating evolutionary ecology and infectious disease epidemiology is challenging because within-host dynamics can have counterintuitive consequences for between-host transmission, especially for vector-borne parasites. A major obstacle to linking within- and between-host processes is that the drivers of the relationships between the density, virulence, and fitness of parasites are poorly understood. By experimentally manipulating the intensity of rodent malaria (Plasmodium berghei) infections in Anopheles stephensi mosquitoes under different environmental conditions, we show that parasites experience substantial density-dependent fitness costs because crowding reduces both parasite proliferation and vector survival. We then use our data to predict how interactions between parasite density and vector environmental conditions shape within-vector processes and onward disease transmission. Our model predicts that density-dependent processes can have substantial and unexpected effects on the transmission potential of vector-borne disease, which should be considered in the development and evaluation of transmission-blocking interventions. PMID:23789029
Liu, Xuewu; Huang, Yuxiao; Liang, Jiao; Zhang, Shuai; Li, Yinghui; Wang, Jun; Shen, Yan; Xu, Zhikai; Zhao, Ya
The invasion of red blood cells (RBCs) by malarial parasites is an essential step in the life cycle of Plasmodium falciparum. Human-parasite surface protein interactions play a critical role in this process. Although several interactions between human and parasite proteins have been discovered, the mechanism related to invasion remains poorly understood because numerous human-parasite protein interactions have not yet been identified. High-throughput screening experiments are not feasible for malarial parasites due to difficulty in expressing the parasite proteins. Here, we performed computational prediction of the PPIs involved in malaria parasite invasion to elucidate the mechanism by which invasion occurs. In this study, an expectation maximization algorithm was used to estimate the probabilities of domain-domain interactions (DDIs). Estimates of DDI probabilities were then used to infer PPI probabilities. We found that our prediction performance was better than that based on the information of D. melanogaster alone when information related to the six species was used. Prediction performance was assessed using protein interaction data from S. cerevisiae, indicating that the predicted results were reliable. We then used the estimates of DDI probabilities to infer interactions between 490 parasite and 3,787 human membrane proteins. A small-scale dataset was used to illustrate the usability of our method in predicting interactions between human and parasite proteins. The positive predictive value (PPV) was lower than that observed in S. cerevisiae. We integrated gene expression data to improve prediction accuracy and to reduce false positives. We identified 80 membrane proteins highly expressed in the schizont stage by fast Fourier transform method. Approximately 221 erythrocyte membrane proteins were identified using published mass spectral datasets. A network consisting of 205 interactions was predicted. Results of network analysis suggest that SNARE proteins of
Spielmann, Tobias; Gardiner, Donald L; Beck, Hans-Peter; Trenholme, Katharine R; Kemp, David J
The parasite-host cell interface is a key compartment of vacuolated intracellular pathogens but little is known about its molecular composition and architecture. We used in vivo cross-linking to analyse the parasite-host cell interface of asexual stages of the most virulent human malaria parasite Plasmodium falciparum. We show that the integral membrane protein members of the early transcribed membrane protein (ETRAMP) family and exported protein 1 (EXP-1), which are components of the parasite-host cell interface, form complexes of oligomeric arrays in this compartment. The most notable feature is that each ETRAMP member and EXP-1 define separate arrays, demonstrating that the protein distribution in this membrane is non-random. Each of three recombinant ETRAMPs readily oligomerized in bacterial membranes, confirming that these arrays can form independently of other Plasmodium proteins. We propose that the malaria parasite-host cell interface contains patches of integral membrane proteins forming a mosaic of different microdomains in this membrane.
Bhargav, Saligram Prabhakar; Vahokoski, Juha; Kumpula, Esa-Pekka; Kursula, Inari
Malaria is a devastating disease caused by apicomplexan parasites of the genus Plasmodium that use a divergent actin-powered molecular motor for motility and invasion. Plasmodium actin differs from canonical actins in sequence, structure and function. Here, the purification, crystallization and secondary-structure analysis of the two Plasmodium actin isoforms are presented. The recombinant parasite actins were folded and could be purified to homogeneity. Plasmodium actins I and II were crystallized in complex with the gelsolin G1 domain; the crystals diffracted to resolutions of 1.19 and 2.2 Å and belonged to space groups P212121 and P21, respectively, each with one complex in the asymmetric unit. PMID:24100575
Staines, Henry M.; Alkhalil, Abdulnaser; Allen, Richard J.; De Jonge, Hugo R.; Derbyshire, Elvira; Egée, Stéphane; Ginsburg, Hagai; Hill, David A.; Huber, Stephan M.; Kirk, Kiaran; Lang, Florian; Lisk, Godfrey; Oteng, Eugene; Pillai, Ajay D.; Rayavara, Kempaiah; Rouhani, Sherin; Saliba, Kevin J.; Shen, Crystal; Solomon, Tsione; Thomas, Serge L. Y.; Verloo, Patrick; Desai, Sanjay A.
The altered permeability characteristics of erythrocytes infected with malaria parasites have been a source of interest for over 30 years. Recent electrophysiological studies have provided strong evidence that these changes reflect transmembrane transport through ion channels in the host erythrocyte plasma membrane. However, conflicting results and differing interpretations of the data have led to confusion in this field. In an effort to unravel these issues, the groups involved recently came together for a week of discussion and experimentation. In this article, the various models for altered transport are reviewed, together with the areas of consensus in the field and those that require a better understanding. PMID:17292372
Background Malaria parasites generate free haem upon catabolism of host haemoglobin during their intraerythrocytic growth cycle. In order to minimize oxidative toxicity of the ferric iron, the free haem molecules are polymerized into the biomineral beta-haematin (commonly referred to as haemozoin). Haemozoin crystals are paramagnetic, and this property can be exploited for the purification of late stage parasites as they contain larger haemozoin crystals than early stage parasites and uninfected cells. Commercially available magnets that were originally developed for the purpose of antibody-mediated cell purification are widely used for this purpose. As these methods are not necessarily optimized for parasite purification, the relationship between magnetic field strength and the quantity and quality of yield during parasite purification was explored. Methods Inexpensive rare-earth neodymium magnets with commercially available disposable columns were employed to explore the relationship between magnetic field strength and recovery of free haemozoin and infected erythrocytes (iRBCs). Results Yields of free haemozoin increased nearly linearly with increasing magnetic field strength to the strongest fields tested (8,500 Gauss). Stronger magnetic fields also improved the recovery of iRBCs with no detrimental effects on parasite viability. An in-house constructed magnetic stand, built for $75 in materials, produced superior results when compared with much more expensive commercial products. Conclusions Existing protocols for the magnetic purification of free haemozoin and iRBCs result in sub-optimal yields. Inexpensive high-strength neodymium magnets offer a better option, resulting in higher yields with no detrimental effects on parasite viability. PMID:20074366
Malaria is a vector borne disease widely occurring at equatorial region. Even after decades of campaigning of malaria control, still today it is high mortality causing disease due to improper and late diagnosis. To prevent number of people getting affected by malaria, the diagnosis should be in early stage and accurate. This paper presents an automatic method for diagnosis of malaria parasite in the blood images. Image processing techniques are used for diagnosis of malaria parasite and to detect their stages. The diagnosis of parasite stages is done using features like statistical features and textural features of malaria parasite in blood images. This paper gives a comparison of the textural based features individually used and used in group together. The comparison is made by considering the accuracy, sensitivity, and specificity of the features for the same images in database. PMID:27247560
Singh, Upasana Shyamsunder; Siwal, Nisha; Pande, Veena
India is highly endemic to malaria with prevalence of all five species of human malaria parasites of Plasmodium genus. India is set for malaria elimination by 2030. Since cases of mixed Plasmodium species infections remain usually undetected but cause huge disease burden, in order to understand the distributional prevalence of both monospecies infections and mixed species infections in India, we collated published data on the differential infection incidences of the five different malaria parasites based on PCR diagnostic assay. About 11% of total cases were due to mixed species infection. Among several interesting observations on both single and mixed parasitic infections, incidences of Plasmodium falciparum monoinfection were found to be significantly higher than P. vivax monoinfection. Also, P. malariae seems to be emerging as a potential malaria threat in India. Putting all the facts together, it appears that the dream of achieving malaria elimination in India will not be completely successful without dealing with mixed species infection. PMID:28900620
Larremore, Daniel B.; Sundararaman, Sesh A.; Liu, Weimin; Proto, William R.; Clauset, Aaron; Loy, Dorothy E.; Speede, Sheri; Plenderleith, Lindsey J.; Sharp, Paul M.; Hahn, Beatrice H.; Rayner, Julian C.; Buckee, Caroline O.
Antigens encoded by the var gene family are major virulence factors of the human malaria parasite Plasmodium falciparum, exhibiting enormous intra- and interstrain diversity. Here we use network analysis to show that var architecture and mosaicism are conserved at multiple levels across the Laverania subgenus, based on var-like sequences from eight single-species and three multi-species Plasmodium infections of wild-living or sanctuary African apes. Using select whole-genome amplification, we also find evidence of multi-domain var structure and synteny in Plasmodium gaboni, one of the ape Laverania species most distantly related to P. falciparum, as well as a new class of Duffy-binding-like domains. These findings indicate that the modular genetic architecture and sequence diversity underlying var-mediated host-parasite interactions evolved before the radiation of the Laverania subgenus, long before the emergence of P. falciparum. PMID:26456841
Liu, Weimin; Li, Yingying; Learn, Gerald H.; Rudicell, Rebecca S.; Robertson, Joel D.; Keele, Brandon F.; Ndjango, Jean-Bosco N.; Sanz, Crickette M.; Morgan, David B.; Locatelli, Sabrina; Gonder, Mary K.; Kranzusch, Philip J.; Walsh, Peter D.; Delaporte, Eric; Mpoudi-Ngole, Eitel; Georgiev, Alexander V.; Muller, Martin N.; Shaw, George M.; Peeters, Martine; Sharp, Paul M.; Rayner, Julian C.; Hahn, Beatrice H.
Plasmodium falciparum is the most prevalent and lethal of the malaria parasites infecting humans, yet the origin and evolutionary history of this important pathogen remain controversial. Here, we developed a novel polymerase chain reaction based single genome amplification strategy to identify and characterize Plasmodium spp. DNA sequences in fecal samples of wild-living apes. Among nearly 3,000 specimens collected from field sites throughout central Africa, we found Plasmodium infection in chimpanzees (Pan troglodytes) and western gorillas (Gorilla gorilla), but not in eastern gorillas (Gorilla beringei) or bonobos (Pan paniscus). Ape plasmodial infections were highly prevalent, widely distributed, and almost always comprised of mixed parasite species. Analysis of more than 1,100 mitochondrial, apicoplast and nuclear gene sequences from chimpanzees and gorillas revealed that 99% grouped within one of six host-specific lineages representing distinct Plasmodium species within the subgenus Laverania. One of these from western gorillas was comprised of parasites that were nearly identical to P. falciparum. In phylogenetic analyses of full-length mitochondrial sequences, human P. falciparum formed a monophyletic lineage within the gorilla parasite radiation. These findings indicate that P. falciparum is of gorilla and not of chimpanzee, bonobo or ancient human origin. PMID:20864995
Kümpornsin, Krittikorn; Modchang, Charin; Heinberg, Adina; Ekland, Eric H.; Jirawatcharadech, Piyaporn; Chobson, Pornpimol; Suwanakitti, Nattida; Chaotheing, Sastra; Wilairat, Prapon; Deitsch, Kirk W.; Kamchonwongpaisan, Sumalee; Fidock, David A.; Kirkman, Laura A.; Yuthavong, Yongyuth; Chookajorn, Thanat
Biological robustness allows mutations to accumulate while maintaining functional phenotypes. Despite its crucial role in evolutionary processes, the mechanistic details of how robustness originates remain elusive. Using an evolutionary trajectory analysis approach, we demonstrate how robustness evolved in malaria parasites under selective pressure from an antimalarial drug inhibiting the folate synthesis pathway. A series of four nonsynonymous amino acid substitutions at the targeted enzyme, dihydrofolate reductase (DHFR), render the parasites highly resistant to the antifolate drug pyrimethamine. Nevertheless, the stepwise gain of these four dhfr mutations results in tradeoffs between pyrimethamine resistance and parasite fitness. Here, we report the epistatic interaction between dhfr mutations and amplification of the gene encoding the first upstream enzyme in the folate pathway, GTP cyclohydrolase I (GCH1). gch1 amplification confers low level pyrimethamine resistance and would thus be selected for by pyrimethamine treatment. Interestingly, the gch1 amplification can then be co-opted by the parasites because it reduces the cost of acquiring drug-resistant dhfr mutations downstream in the same metabolic pathway. The compensation of compromised fitness by extra GCH1 is an example of how robustness can evolve in a system and thus expand the accessibility of evolutionary trajectories leading toward highly resistant alleles. The evolution of robustness during the gain of drug-resistant mutations has broad implications for both the development of new drugs and molecular surveillance for resistance to existing drugs. PMID:24739308
Pollitt, Laura C.; Mideo, Nicole; Drew, Damien R.; Schneider, Petra; Colegrave, Nick; Reece, Sarah E.
All organisms must trade off resource allocation between different life processes that determine their survival and reproduction. Malaria parasites replicate asexually in the host but must produce sexual stages to transmit between hosts. Because different specialized stages are required for these functions, the division of resources between these life-history components is a key problem for natural selection to solve. Despite the medical and economic importance of these parasites, their reproductive strategies remain poorly understood and often seem counterintuitive. Here, we tested recent theory predicting that in-host competition shapes how parasites trade off investment in in-host replication relative to between-host transmission. We demonstrate, across several genotypes, that Plasmodium chabaudi parasites detect the presence of competing genotypes and facultatively respond by reducing their investment in sexual stages in the manner predicted to maximize their competitive ability. Furthermore, we show that genotypes adjust their allocation to sexual stages in line with the availability of exploitable red blood cell resources. Our findings are predicted by evolutionary theory developed to explain life-history trade-offs in more traditionally studied multicellular taxa and suggest that the answer to the long-standing question of why so few transmission stages are produced is that in most natural infections heavy investment in reproduction may compromise in-host survival. PMID:21460544
Corey, Victoria C; Lukens, Amanda K; Istvan, Eva S; Lee, Marcus C S; Franco, Virginia; Magistrado, Pamela; Coburn-Flynn, Olivia; Sakata-Kato, Tomoyo; Fuchs, Olivia; Gnädig, Nina F; Goldgof, Greg; Linares, Maria; Gomez-Lorenzo, Maria G; De Cózar, Cristina; Lafuente-Monasterio, Maria Jose; Prats, Sara; Meister, Stephan; Tanaseichuk, Olga; Wree, Melanie; Zhou, Yingyao; Willis, Paul A; Gamo, Francisco-Javier; Goldberg, Daniel E; Fidock, David A; Wirth, Dyann F; Winzeler, Elizabeth A
Microbial resistance to chemotherapy has caused countless deaths where malaria is endemic. Chemotherapy may fail either due to pre-existing resistance or evolution of drug-resistant parasites. Here we use a diverse set of antimalarial compounds to investigate the acquisition of drug resistance and the degree of cross-resistance against common resistance alleles. We assess cross-resistance using a set of 15 parasite lines carrying resistance-conferring alleles in pfatp4, cytochrome bc1, pfcarl, pfdhod, pfcrt, pfmdr, pfdhfr, cytoplasmic prolyl t-RNA synthetase or hsp90. Subsequently, we assess whether resistant parasites can be obtained after several rounds of drug selection. Twenty-three of the 48 in vitro selections result in resistant parasites, with time to resistance onset ranging from 15 to 300 days. Our data indicate that pre-existing resistance may not be a major hurdle for novel-target antimalarial candidates, and focusing our attention on fast-killing compounds may result in a slower onset of clinical resistance.
Corey, Victoria C.; Lukens, Amanda K.; Istvan, Eva S.; Lee, Marcus C. S.; Franco, Virginia; Magistrado, Pamela; Coburn-Flynn, Olivia; Sakata-Kato, Tomoyo; Fuchs, Olivia; Gnädig, Nina F.; Goldgof, Greg; Linares, Maria; Gomez-Lorenzo, Maria G.; De Cózar, Cristina; Lafuente-Monasterio, Maria Jose; Prats, Sara; Meister, Stephan; Tanaseichuk, Olga; Wree, Melanie; Zhou, Yingyao; Willis, Paul A.; Gamo, Francisco-Javier; Goldberg, Daniel E.; Fidock, David A.; Wirth, Dyann F.; Winzeler, Elizabeth A.
Microbial resistance to chemotherapy has caused countless deaths where malaria is endemic. Chemotherapy may fail either due to pre-existing resistance or evolution of drug-resistant parasites. Here we use a diverse set of antimalarial compounds to investigate the acquisition of drug resistance and the degree of cross-resistance against common resistance alleles. We assess cross-resistance using a set of 15 parasite lines carrying resistance-conferring alleles in pfatp4, cytochrome bc1, pfcarl, pfdhod, pfcrt, pfmdr, pfdhfr, cytoplasmic prolyl t-RNA synthetase or hsp90. Subsequently, we assess whether resistant parasites can be obtained after several rounds of drug selection. Twenty-three of the 48 in vitro selections result in resistant parasites, with time to resistance onset ranging from 15 to 300 days. Our data indicate that pre-existing resistance may not be a major hurdle for novel-target antimalarial candidates, and focusing our attention on fast-killing compounds may result in a slower onset of clinical resistance. PMID:27301419
Kümpornsin, Krittikorn; Modchang, Charin; Heinberg, Adina; Ekland, Eric H; Jirawatcharadech, Piyaporn; Chobson, Pornpimol; Suwanakitti, Nattida; Chaotheing, Sastra; Wilairat, Prapon; Deitsch, Kirk W; Kamchonwongpaisan, Sumalee; Fidock, David A; Kirkman, Laura A; Yuthavong, Yongyuth; Chookajorn, Thanat
Biological robustness allows mutations to accumulate while maintaining functional phenotypes. Despite its crucial role in evolutionary processes, the mechanistic details of how robustness originates remain elusive. Using an evolutionary trajectory analysis approach, we demonstrate how robustness evolved in malaria parasites under selective pressure from an antimalarial drug inhibiting the folate synthesis pathway. A series of four nonsynonymous amino acid substitutions at the targeted enzyme, dihydrofolate reductase (DHFR), render the parasites highly resistant to the antifolate drug pyrimethamine. Nevertheless, the stepwise gain of these four dhfr mutations results in tradeoffs between pyrimethamine resistance and parasite fitness. Here, we report the epistatic interaction between dhfr mutations and amplification of the gene encoding the first upstream enzyme in the folate pathway, GTP cyclohydrolase I (GCH1). gch1 amplification confers low level pyrimethamine resistance and would thus be selected for by pyrimethamine treatment. Interestingly, the gch1 amplification can then be co-opted by the parasites because it reduces the cost of acquiring drug-resistant dhfr mutations downstream in the same metabolic pathway. The compensation of compromised fitness by extra GCH1 is an example of how robustness can evolve in a system and thus expand the accessibility of evolutionary trajectories leading toward highly resistant alleles. The evolution of robustness during the gain of drug-resistant mutations has broad implications for both the development of new drugs and molecular surveillance for resistance to existing drugs.
Matsubara, Ryuma; Aonuma, Hiroka; Kojima, Mikiko; Tahara, Michiru; Andrabi, Syed Bilal Ahmad; Sakakibara, Hitoshi; Nagamune, Kisaburo
The apicomplexan parasite Toxoplasma gondii produces the plant hormone abscisic acid, but it is unclear if phytohormones are produced by the malaria parasite Plasmodium spp., the most important parasite of this phylum. Here, we report detection of salicylic acid, an immune-related phytohormone of land plants, in P. berghei ANKA and T. gondii cell lysates. However, addition of salicylic acid to P. falciparum and T. gondii culture had no effect. We transfected P. falciparum 3D7 with the nahG gene, which encodes a salicylic acid-degrading enzyme isolated from plant-infecting Pseudomonas sp., and established a salicylic acid-deficient mutant. The mutant had a significantly decreased concentration of parasite-synthesized prostaglandin E2, which potentially modulates host immunity as an adaptive evolution of Plasmodium spp. To investigate the function of salicylic acid and prostaglandin E2 on host immunity, we established P. berghei ANKA mutants expressing nahG. C57BL/6 mice infected with nahG transfectants developed enhanced cerebral malaria, as assessed by Evans blue leakage and brain histological observation. The nahG-transfectant also significantly increased the mortality rate of mice. Prostaglandin E2 reduced the brain symptoms by induction of T helper-2 cytokines. As expected, T helper-1 cytokines including interferon-γ and interleukin-2 were significantly elevated by infection with the nahG transfectant. Thus, salicylic acid of Plasmodium spp. may be a new pathogenic factor of this threatening parasite and may modulate immune function via parasite-produced prostaglandin E2. PMID:26466097
Matsubara, Ryuma; Aonuma, Hiroka; Kojima, Mikiko; Tahara, Michiru; Andrabi, Syed Bilal Ahmad; Sakakibara, Hitoshi; Nagamune, Kisaburo
The apicomplexan parasite Toxoplasma gondii produces the plant hormone abscisic acid, but it is unclear if phytohormones are produced by the malaria parasite Plasmodium spp., the most important parasite of this phylum. Here, we report detection of salicylic acid, an immune-related phytohormone of land plants, in P. berghei ANKA and T. gondii cell lysates. However, addition of salicylic acid to P. falciparum and T. gondii culture had no effect. We transfected P. falciparum 3D7 with the nahG gene, which encodes a salicylic acid-degrading enzyme isolated from plant-infecting Pseudomonas sp., and established a salicylic acid-deficient mutant. The mutant had a significantly decreased concentration of parasite-synthesized prostaglandin E2, which potentially modulates host immunity as an adaptive evolution of Plasmodium spp. To investigate the function of salicylic acid and prostaglandin E2 on host immunity, we established P. berghei ANKA mutants expressing nahG. C57BL/6 mice infected with nahG transfectants developed enhanced cerebral malaria, as assessed by Evans blue leakage and brain histological observation. The nahG-transfectant also significantly increased the mortality rate of mice. Prostaglandin E2 reduced the brain symptoms by induction of T helper-2 cytokines. As expected, T helper-1 cytokines including interferon-γ and interleukin-2 were significantly elevated by infection with the nahG transfectant. Thus, salicylic acid of Plasmodium spp. may be a new pathogenic factor of this threatening parasite and may modulate immune function via parasite-produced prostaglandin E2.
Tuteja, Renu; Ansari, Abulaish; Chauhan, Virander Singh
Transcription is a process by which the genetic information stored in DNA is converted into mRNA by enzymes known as RNA polymerase. Bacteria use only one RNA polymerase to transcribe all of its genes while eukaryotes contain three RNA polymerases to transcribe the variety of eukaryotic genes. RNA polymerase also requires other factors/proteins to produce the transcript. These factors generally termed as transcription factors (TFs) are either associated directly with RNA polymerase or add in building the actual transcription apparatus. TFs are the most common tools that our cells use to control gene expression. Plasmodium falciparum is responsible for causing the most lethal form of malaria in humans. It shows most of its characteristics common to eukaryotic transcription but it is assumed that mechanisms of transcriptional control in P. falciparum somehow differ from those of other eukaryotes. In this article we describe the studies on the main TFs such as myb protein, high mobility group protein and ApiA2 family proteins from malaria parasite. These studies show that these TFs are slowly emerging to have defined roles in the regulation of gene expression in the parasite.
Hellmann, Janina Kristin; Münter, Sylvia; Kudryashev, Mikhail; Schulz, Simon; Heiss, Kirsten; Müller, Ann-Kristin; Matuschewski, Kai; Spatz, Joachim P.; Schwarz, Ulrich S.; Frischknecht, Friedrich
Migrating cells are guided in complex environments mainly by chemotaxis or structural cues presented by the surrounding tissue. During transmission of malaria, parasite motility in the skin is important for Plasmodium sporozoites to reach the blood circulation. Here we show that sporozoite migration varies in different skin environments the parasite encounters at the arbitrary sites of the mosquito bite. In order to systematically examine how sporozoite migration depends on the structure of the environment, we studied it in micro-fabricated obstacle arrays. The trajectories observed in vivo and in vitro closely resemble each other suggesting that structural constraints can be sufficient to guide Plasmodium sporozoites in complex environments. Sporozoite speed in different environments is optimized for migration and correlates with persistence length and dispersal. However, this correlation breaks down in mutant sporozoites that show adhesion impairment due to the lack of TRAP-like protein (TLP) on their surfaces. This may explain their delay in infecting the host. The flexibility of sporozoite adaption to different environments and a favorable speed for optimal dispersal ensures efficient host switching during malaria transmission. PMID:21698220
A-Elgayoum, Salwa M E; El-Rayah, El-Amin; Giha, Hayder A
Invasive procedures for diagnostic or therapeutic purposes bear a relative risk of transmission of serious blood-borne infectious disease. In this study, a noninvasive approach to malaria diagnosis using polymerase chain reaction (PCR) for the detection of parasite DNA in saliva, buccal mucosa and urine (alternative samples) was examined. Saliva, buccal mucosa and urine samples were collected simultaneously with blood samples from 93 patients with microscopically confirmed Plasmodium falciparum infection. Species-specific primers detected the parasite DNA only in blood samples. However, when the PCR analysis was repeated using MSP1 and MSP2 primers in a subgroup of 21 complete sets of samples, the parasite DNA was detected in all except 3 samples, which were found to be negative with the MSP2 primers. Parasite density, body temperature or patient age did not influence the PCR results. In conclusion, P. falciparum parasite DNA was detected equally in saliva, buccal mucosa and urine of malaria patients, regardless of their ages, body temperatures or parasite density. Surprisingly, the parasite DNA was not amplified by species-specific primers in the alternative samples whereas it was in the blood samples.
Nkuo-Akenji, Theresa; Ntonifor, Nelson N; Ndukum, Maze B; Abongwa, Edith L; Nkwescheu, Armand; Anong, Damain N; Songmbe, Michael; Boyo, Michael G; Ndamukong, Kenneth N; Titanji, Vincent P K
The impact of some environmental factors on malaria parasite prevalence was investigated in rural Bolifamba, Cameroon. The study population comprised 1454 subjects aged 0 - 65 years. Malaria parasite prevalence was higher in the rainy (50.1 %) than in the dry season (44.2 %) with a significant difference (P = 0.001) in mean parasite density between seasons. Individuals < 15 years old had significantly higher malaria parasite prevalence (55.5 %) than those > 15 years (37.4 %). Malaria parasite prevalence (P = 0.001) and parasite density (P = 0.03) were higher in the individuals of wooden plank houses than those of cement brick houses. Inhabitants of houses surrounded by bushes or garbage heaps and swamps or stagnant water showed higher malaria parasite prevalence and densities compared with those from cleaner surroundings. Anopheles gambiae (63.8 %) and A. funestus (32.8 %) were associated with perennial transmission of malaria. Our data indicates that poor environmental sanitation and housing conditions may be significant risk factors for malaria parasite burden in Bolifamba.
Cheeseman, Ian H.; Miller, Becky; Tan, John C.; Tan, Asako; Nair, Shalini; Nkhoma, Standwell C.; De Donato, Marcos; Rodulfo, Hectorina; Dondorp, Arjen; Branch, Oralee H.; Mesia, Lastenia Ruiz; Newton, Paul; Mayxay, Mayfong; Amambua-Ngwa, Alfred; Conway, David J.; Nosten, François; Ferdig, Michael T.; Anderson, Tim J. C.
If copy number variants (CNVs) are predominantly deleterious, we would expect them to be more efficiently purged from populations with a large effective population size (Ne) than from populations with a small Ne. Malaria parasites (Plasmodium falciparum) provide an excellent organism to examine this prediction, because this protozoan shows a broad spectrum of population structures within a single species, with large, stable, outbred populations in Africa, small unstable inbred populations in South America and with intermediate population characteristics in South East Asia. We characterized 122 single-clone parasites, without prior laboratory culture, from malaria-infected patients in seven countries in Africa, South East Asia and South America using a high-density single-nucleotide polymorphism/CNV microarray. We scored 134 high-confidence CNVs across the parasite exome, including 33 deletions and 102 amplifications, which ranged in size from <500 bp to 59 kb, as well as 10,107 flanking, biallelic single-nucleotide polymorphisms. Overall, CNVs were rare, small, and skewed toward low frequency variants, consistent with the deleterious model. Relative to African and South East Asian populations, CNVs were significantly more common in South America, showed significantly less skew in allele frequencies, and were significantly larger. On this background of low frequency CNV, we also identified several high-frequency CNVs under putative positive selection using an FST outlier analysis. These included known adaptive CNVs containing rh2b and pfmdr1, and several other CNVs (e.g., DNA helicase and three conserved proteins) that require further investigation. Our data are consistent with a significant impact of genetic structure on CNV burden in an important human pathogen. PMID:26613787
Cheeseman, Ian H; Miller, Becky; Tan, John C; Tan, Asako; Nair, Shalini; Nkhoma, Standwell C; De Donato, Marcos; Rodulfo, Hectorina; Dondorp, Arjen; Branch, Oralee H; Mesia, Lastenia Ruiz; Newton, Paul; Mayxay, Mayfong; Amambua-Ngwa, Alfred; Conway, David J; Nosten, François; Ferdig, Michael T; Anderson, Tim J C
If copy number variants (CNVs) are predominantly deleterious, we would expect them to be more efficiently purged from populations with a large effective population size (Ne) than from populations with a small Ne. Malaria parasites (Plasmodium falciparum) provide an excellent organism to examine this prediction, because this protozoan shows a broad spectrum of population structures within a single species, with large, stable, outbred populations in Africa, small unstable inbred populations in South America and with intermediate population characteristics in South East Asia. We characterized 122 single-clone parasites, without prior laboratory culture, from malaria-infected patients in seven countries in Africa, South East Asia and South America using a high-density single-nucleotide polymorphism/CNV microarray. We scored 134 high-confidence CNVs across the parasite exome, including 33 deletions and 102 amplifications, which ranged in size from <500 bp to 59 kb, as well as 10,107 flanking, biallelic single-nucleotide polymorphisms. Overall, CNVs were rare, small, and skewed toward low frequency variants, consistent with the deleterious model. Relative to African and South East Asian populations, CNVs were significantly more common in South America, showed significantly less skew in allele frequencies, and were significantly larger. On this background of low frequency CNV, we also identified several high-frequency CNVs under putative positive selection using an FST outlier analysis. These included known adaptive CNVs containing rh2b and pfmdr1, and several other CNVs (e.g., DNA helicase and three conserved proteins) that require further investigation. Our data are consistent with a significant impact of genetic structure on CNV burden in an important human pathogen.
Wetzel, Johanna; Herrmann, Susann; Swapna, Lakshmipuram Seshadri; Prusty, Dhaneswar; John Peter, Arun T; Kono, Maya; Saini, Sidharth; Nellimarla, Srinivas; Wong, Tatianna Wai Ying; Wilcke, Louisa; Ramsay, Olivia; Cabrera, Ana; Biller, Laura; Heincke, Dorothee; Mossman, Karen; Spielmann, Tobias; Ungermann, Christian; Parkinson, John; Gilberger, Tim W
To survive and persist within its human host, the malaria parasite Plasmodium falciparum utilizes a battery of lineage-specific innovations to invade and multiply in human erythrocytes. With central roles in invasion and cytokinesis, the inner membrane complex, a Golgi-derived double membrane structure underlying the plasma membrane of the parasite, represents a unique and unifying structure characteristic to all organisms belonging to a large phylogenetic group called Alveolata. More than 30 structurally and phylogenetically distinct proteins are embedded in the IMC, where a portion of these proteins displays N-terminal acylation motifs. Although N-terminal myristoylation is catalyzed co-translationally within the cytoplasm of the parasite, palmitoylation takes place at membranes and is mediated by palmitoyl acyltransferases (PATs). Here, we identify a PAT (PfDHHC1) that is exclusively localized to the IMC. Systematic phylogenetic analysis of the alveolate PAT family reveals PfDHHC1 to be a member of a highly conserved, apicomplexan-specific clade of PATs. We show that during schizogony this enzyme has an identical distribution like two dual-acylated, IMC-localized proteins (PfISP1 and PfISP3). We used these proteins to probe into specific sequence requirements for IMC-specific membrane recruitment and their interaction with differentially localized PATs of the parasite.
Paul, Rick E L; Nu, Van Anh Ton; Krettli, Antoniana U; Brey, Paul T
The role of species interactions in structuring parasite communities remains controversial. Here, we show that interspecific competition between two avian malaria parasite species, Plasmodium gallinaceum and P. juxtanucleare, occurs as a result of interference during parasite fertilization within the bloodmeal of the mosquito. The significant reduction in the transmission success of P. gallinaceum to mosquitoes, due to the co-infecting P. juxtanucleare, is predicted to have compromised its colonization of regions occupied by P. juxtanucleare and, thus, may have contributed to the restricted global distribution of P. gallinaceum. Such interspecies interactions may occur between human malaria parasites and, thus, impact upon parasite species epidemiology, especially in regions of seasonal transmission. PMID:12573069
Haeberlein, Simone; Chevalley-Maurel, Séverine; Ozir-Fazalalikhan, Arifa; Koppejan, Hester; Winkel, Beatrice M F; Ramesar, Jai; Khan, Shahid M; Sauerwein, Robert W; Roestenberg, Meta; Janse, Chris J; Smits, Hermelijn H; Franke-Fayard, Blandine
In humans and murine models of malaria, intradermal immunization (ID-I) with genetically attenuated sporozoites that arrest in liver induces lower protective immunity than intravenous immunization (IV-I). It is unclear whether this difference is caused by fewer sporozoites migrating into the liver or by suboptimal hepatic and injection site-dependent immune responses. We therefore developed a Plasmodium yoelii immunization/boost/challenge model to examine parasite liver loads as well as hepatic and lymph node immune responses in protected and unprotected ID-I and IV-I animals. Despite introducing the same numbers of genetically attenuated parasites in the liver, ID-I resulted in lower sterile protection (53-68%) than IV-I (93-95%). Unprotected mice developed less sporozoite-specific CD8(+) and CD4(+) effector T-cell responses than protected mice. After immunization, ID-I mice showed more interleukin-10-producing B and T cells in livers and skin-draining lymph nodes, but fewer hepatic CD8 memory T cells and CD8(+) dendritic cells compared to IV-I mice. Our results indicate that the lower protection efficacy obtained by intradermal sporozoite administration is not linked to low hepatic parasite numbers as presumed before, but correlates with a shift towards regulatory immune responses. Overcoming these immune suppressive responses is important not only for live-attenuated malaria vaccines but also for other live vaccines administered in the skin.
Murphy, Sean C.; Fernandez-Pol, Sebastian; Chung, Paul H.; Prasanna Murthy, S. N.; Milne, Stephen B.; Salomao, Marcela; Brown, H. Alex; Lomasney, Jon W.; Mohandas, Narla
Studies of detergent-resistant membrane (DRM) rafts in mature erythrocytes have facilitated identification of proteins that regulate formation of endovacuolar structures such as the parasitophorous vacuolar membrane (PVM) induced by the malaria parasite Plasmodium falciparum. However, analyses of raft lipids have remained elusive because detergents interfere with lipid detection. Here, we use primaquine to perturb the erythrocyte membrane and induce detergent-free buoyant vesicles, which are enriched in cholesterol and major raft proteins flotillin and stomatin and contain low levels of cytoskeleton, all characteristics of raft microdomains. Lipid mass spectrometry revealed that phosphatidylethanolamine and phosphatidylglycerol are depleted in endovesicles while phosphoinositides are highly enriched, suggesting raft-based endovesiculation can be achieved by simple (non–receptor-mediated) mechanical perturbation of the erythrocyte plasma membrane and results in sorting of inner leaflet phospholipids. Live-cell imaging of lipid-specific protein probes showed that phosphatidylinositol (4,5) bisphosphate (PIP2) is highly concentrated in primaquine-induced vesicles, confirming that it is an erythrocyte raft lipid. However, the malarial PVM lacks PIP2, although another raft lipid, phosphatidylserine, is readily detected. Thus, different remodeling/sorting of cytoplasmic raft phospholipids may occur in distinct endovacuoles. Importantly, erythrocyte raft lipids recruited to the invasion junction by mechanical stimulation may be remodeled by the malaria parasite to establish blood-stage infection. PMID:17526861
Sarduy, Emir Salas; de los A. Chávez Planes, María
Papain-like cysteine proteases of malaria parasites are considered important chemotherapeutic targets or valuable models for the evaluation of drug candidates. Consequently, many of these enzymes have been cloned and expressed in Escherichia coli for their biochemical characterization. However, their expression has been problematic, showing low yield and leading to the formation of insoluble aggregates. Given that highly-productive expression systems are required for the high-throughput evaluation of inhibitors, we analyzed the existing expression systems to identify the causes of such apparent issues. We found that significant divergences in codon and nucleotide composition from host genes are the most probable cause of expression failure, and propose several strategies to overcome these limitations. Finally we predict that yeast hosts Saccharomyces cerevisiae and Pichia pastoris may be better suited than E. coli for the efficient expression of plasmodial genes, presumably leading to soluble and active products reproducing structural and functional characteristics of the natural enzymes. PMID:23018863
Suh, Kathryn N.; Kain, Kevin C.; Keystone, Jay S.
Malaria is a parasitic infection of global importance. Although relatively uncommon in developed countries, where the disease occurs mainly in travellers who have returned from endemic regions, it remains one of the most prevalent infections of humans worldwide. In endemic regions, malaria is a significant cause of morbidity and mortality and creates enormous social and economic burdens. Current efforts to control malaria focus on reducing attributable morbidity and mortality. Targeted chemoprophylaxis and use of insecticide-treated bed nets have been successful in some endemic areas. For travellers to malaria-endemic regions, personal protective measures and appropriate chemoprophylaxis can significantly reduce the risk of infection. Prompt evaluation of the febrile traveller, a high degree of suspicion of malaria, rapid and accurate diagnosis, and appropriate antimalarial therapy are essential in order to optimize clinical outcomes of infected patients. Additional approaches to malaria control, including genetic manipulation of mosquitoes and malaria vaccines, are areas of ongoing research. PMID:15159369
Prajapati, Surendra Kumar; Singh, Om Prakash
The immune evasion gene family of malaria parasites encodes variant surface proteins that are expressed at the surface of infected erythrocytes and help the parasite in evading the host immune response by means of antigenic variation. The identification of Plasmodium vivax vir orthologous immune evasion gene family from primate malaria parasites would provide new insight into the evolution of virulence and pathogenesis. Three vir subfamilies viz. vir-B, vir-D and vir-G were successfully PCR amplified from primate malaria parasites, cloned and sequenced. DNA sequence analysis confirmed orthologues of vir-D subfamily in Plasmodium cynomolgi, Plasmodium simium, Plasmodium simiovale and Plasmodium fieldi. The identified vir-D orthologues are 1-9 distinct members of the immune evasion gene family which have 68-83% sequence identity with vir-D and 71.2-98.5% sequence identity within the members identified from primate malaria parasites. The absence of other vir subfamilies among primate malaria parasites reflects the limitations in the experimental approach. This study clearly identified the presence of vir-D like sequences in four species of Plasmodium infecting primates that would be useful in understanding the evolution of virulence in malaria parasites.
Tsai, Meng-Hsiun; Yu, Shyr-Shen; Chan, Yung-Kuan; Jen, Chun-Chu
In this study, an automatic malaria parasite detector is proposed to perceive the malaria-infected erythrocytes in a blood smear image and to separate parasites from the infected erythrocytes. The detector hence can verify whether a patient is infected with malaria. It could more objectively and efficiently help a doctor in diagnosing malaria. The experimental results show that the proposed method can provide impressive performance in segmenting the malaria-infected erythrocytes and the parasites from a blood smear image taken under a microscope. This paper also presents a weighted Sobel operation to compute the image gradient. The experimental results demonstrates that the weighted Sobel operation can provide more clear-cut and thinner object contours in object segmentation.
Mangal, Praveen; Mittal, Shilpa; Kachhawa, Kamal; Agrawal, Divya; Rath, Bhabagrahi; Kumar, Sanjay
Malaria remains a major health hazard in the modern world, particularly in developing countries. In Plasmodium falciparum malaria, there is a direct correlation between asexual erythrocytic stage parasite density and disease severity. Accordingly, the correlations between parasite density and various clinical presentations, severity, and outcome were examined in falciparum malaria in India. The study was conducted in a tertiary health-care center in North India. Of 100 cases of falciparum malaria, 65 patients were male and 35 were female. A total of 54 patients were in the uncomplicated group and 46 patients were in the complicated malaria group. Fever, anemia, icterus, splenomegaly, hepatomegaly, and hepatosplenomegaly were common clinical findings. All clinical findings were significantly more common in the complicated malaria group and patients with a high parasite density than in the uncomplicated group and those with a low parasite density. All patients in the uncomplicated malaria group had a parasite density of <5% while most patients in the complicated malaria group had a parasite density of >5%, and the difference between groups was statistically significant. The incidence of cerebral malaria was significantly higher in cases with a high parasite density; 58.33% mortality was observed in these cases. Cerebral malaria and hyperbilirubinemia was the most frequently encountered combination of complications. In P. falciparum malaria, parasite density was associated with complications and poor clinical outcomes. These results may inform treatment decisions and suggest that a threshold parasite density of 5% is informative.
Kristensson, Krister; Masocha, Willias; Bentivoglio, Marina
Invasion of the central nervous system (CNS) is a most devastating complication of a parasitic infection. Several physical and immunological barriers provide obstacles to such an invasion. In this broad overview focus is given to the physical barriers to neuroinvasion of parasites provided at the portal of entry of the parasites, i.e., the skin and epithelial cells of the gastrointestinal tract, and between the blood and the brain parenchyma, i.e., the blood-brain barrier (BBB). A description is given on how human pathogenic parasites can reach the CNS via the bloodstream either as free-living or extracellular parasites, by embolization of eggs, or within red or white blood cells when adapted to intracellular life. Molecular mechanisms are discussed by which parasites can interact with or pass across the BBB. The possible targeting of the circumventricular organs by parasites, as well as the parasites' direct entry to the brain from the nasal cavity through the olfactory nerve pathway, is also highlighted. Finally, examples are given which illustrate different mechanisms by which parasites can cause dysfunction or damage in the CNS related to toxic effects of parasite-derived molecules or to immune responses to the infection.
Díez-Padrisa, Núria; Bassat, Quique; Machevo, Sonia; Quintó, Llorenç; Morais, Luis; Nhampossa, Tacilta; O'Callaghan-Gordo, Cristina; Torres, Antoni; Alonso, Pedro L.; Roca, Anna
Background Pneumonia is the major cause of mortality and morbidity in children worldwide. Procalcitonin (PCT) and C-reactive protein (CRP) are used in developed countries to differentiate between viral and bacterial causes of pneumonia. Validity of these markers needs to be further explored in Africa. Methodology and Principal Findings We assessed the utility of PCT and CRP to differentiate viral from invasive bacterial pneumonia in children <5 years hospitalized with clinical severe pneumonia (CSP) in rural Mozambique, a malaria-endemic area with high HIV prevalence. Prognostic capacity of these markers was also evaluated. Out of 835 children with CSP, 87 fulfilled definition of viral pneumonia and 89 of invasive bacterial pneumonia. In absence of malaria parasites, levels of PCT and CRP were lower in the viral group when compared to the invasive bacterial one (PCT: median = 0.21 versus 8.31 ng/ml, p<0.001; CRP: 18.3 vs. 185.35 mg/l, p<0.001). However, in presence of malaria parasites distribution between clinical groups overlapped (PCT: median = 23.1 vs. 21.75 ng/ml, p = 0.825; CRP: median = 96.8 vs. 217.4 mg/l, p = 0.052). None of the two markers could predict mortality. Conclusions Presence of malaria parasites should be taken into consideration, either for clinical or epidemiological purposes, if using PCT or CRP to differentiate viral from invasive bacterial pneumonia in malaria-endemic areas. PMID:20976241
Ding, Hui; Li, Dongmei
As a parasitic protozoan, Plasmodium falciparum (P. falciparum) can cause malaria. The mitochondrial proteins of malaria parasite play important roles in the discovery of anti-malarial drug targets. Thus, accurate identification of mitochondrial proteins of malaria parasite is a key step for understanding their functions and finding potential drug targets. In this work, we developed a sequence-based method to identify the mitochondrial proteins of malaria parasite. At first, we extended adjoining dipeptide composition to g-gap dipeptide composition for discretely formulating the protein sequences. Subsequently, the analysis of variance (ANOVA) combined with incremental feature selection (IFS) was used to pick out the optimal features. Finally, the jackknife cross-validation was used to evaluate the performance of the proposed model. Evaluation results showed that the maximum accuracy of 97.1% could be achieved by using 101 optimal 5-gap dipeptides. The comparison with previous methods demonstrated that our method was accurate and efficient.
Fitchett, Joseph R; Cooke, Mary K
Genetic engineering provides an ingenious method of attenuating Plasmodium falciparum parasites for next generation vaccines. A novel approach stimulates new optimism in the struggle to eliminate the burden of malaria.
This Memorandum presents current knowledge concerning the epidemiology of drug resistance of malaria parasites and outlines 33 research proposals which could lead to a better understanding of that epidemiology and to a better management of the problem. PMID:3325184
Garcia-Longoria, Luz; Møller, Anders P; Balbontín, Javier; de Lope, Florentino; Marzal, Alfonso
Escape behaviour is the behaviour that birds and other animals display when already caught by a predator. An individual exhibiting higher intensity of such anti-predator behaviour could have greater probabilities of escape from predators. Parasites are known to affect different aspects of host behaviour to increase their own fitness. Vector-transmitted parasites such as malaria parasites should gain by manipulating their hosts to enhance the probability of transmission. Several studies have shown that malaria parasites can manipulate their vectors leading to increased transmission success. However, little is known about whether malaria parasites can manipulate escape behaviour of their avian hosts thereby increasing the spread of the parasite. Here we used an experimental approach to explore if Plasmodium relictum can manipulate the escape behaviour of one of its most common avian hosts, the house sparrow Passer domesticus. We experimentally tested whether malaria parasites manipulate the escape behaviour of their avian host. We showed a decrease in the intensity of biting and tonic immobility after removal of infection with anti-malaria medication compared to pre-experimental behaviour. These outcomes suggest that infected sparrows performed more intense escape behaviour, which would increase the likelihood of individuals escaping from predators, but also benefit the parasite by increasing its transmission opportunities.
Gazarini, Marcos L; Thomas, Andrew P; Pozzan, Tullio; Garcia, Célia R S
Malaria parasites, Plasmodia, spend most of their asexual life cycle within red blood cells, where they proliferate and mature. The erythrocyte cytoplasm has very low [Ca2+] (<100 nM), which is very different from the extracellular environment encountered by most eukaryotic cells. The absence of extracellular Ca2+ is usually incompatible with normal cell functions and survival. In the present work, we have tested the possibility that Plasmodia overcome the limitation posed by the erythrocyte intracellular environment through the maintenance of a high [Ca2+] within the parasitophorous vacuole (PV), the compartment formed during invasion and within which the parasites grow and divide. Thus, Plasmodia were allowed to invade erythrocytes in the presence of Ca2+ indicator dyes. This allowed selective loading of the Ca2+ probes within the PV. The [Ca2+] within this compartment was found to be approximately 40 microM, i.e., high enough to be compatible with a normal loading of the Plasmodia intracellular Ca2+ stores, a prerequisite for the use of a Ca2+-based signaling mechanism. We also show that reduction of extracellular [Ca2+] results in a slow depletion of the [Ca2+] within the PV. A transient drop of [Ca2+] in the PV for a period as short as 2 h affects the maturation process of the parasites within the erythrocytes, with a major reduction 48 h later in the percentage of schizonts, the form that re-invades the red blood cells.
Taylor, Steve M.; Fairhurst, Rick M.
Purpose of review Multiple red cell variants are known to confer protection from malaria. Here we review advances in identifying new variants that modulate malaria risk and in defining molecular mechanisms that mediate malaria protection. Recent findings New red cell variants, including an innate variant in the red cell’s major Ca2+ pump and the acquired state of iron deficiency, have been associated with protection from clinical falciparum malaria. The hemoglobin (Hb) mutants HbC and HbS – known to protect carriers from severe falciparum malaria – enhance parasite passage to mosquitoes and may promote malaria transmission. At the molecular level, substantial advances have been made in understanding the impact of HbS and HbC upon the interactions between host microRNAs and Plasmodium falciparum protein translation; remodeling of red cell cytoskeletal components and transport of parasite proteins to the red cell surface; and chronic activation of the human innate immune system which induces tolerance to blood-stage parasites. Several polymorphisms have now been associated with protection from clinical vivax malaria or reduced P. vivax density, including Southeast Asian ovalocytosis and two common forms of glucose-6-phosphate dehydrogenase deficiency. Summary Red cell variants that modulate malaria risk can serve as models to identify clinically relevant mechanisms of pathogenesis, and thus define parasite and host targets for next-generation therapies. PMID:24675047
Yashima, Akihito; Mizuno, Masashi; Yuzawa, Yukio; Shimada, Koki; Suzuki, Norihiko; Tawada, Hideo; Sato, Waichi; Tsuboi, Naotake; Maruyama, Shoichi; Ito, Yasuhiko; Matsuo, Seiichi; Ohno, Tamio
Malaria is an important tropical disease and has remained a serious health problem in many countries. One of the critical complications of malarial infection is renal injury, such as acute renal failure and chronic glomerulopathy. Few animal models of nephropathy related to malarial infection have been reported. Therefore, we developed and investigated a novel malarial nephropathy model in mice infected by murine malaria parasites. NC mice and C57BL/6J mice were infected with Ttwo different murine malaria parasites, Plasmodium (P.) chabaudi AS and P. yoelii 17X. After the infection, renal pathology and blood and urinary biochemistry were analyzed. NC mice infected by the murine malaria parasite P. chabaudi AS, but not P. yoelii 17X, developed mesangial proliferative glomerulonephritis with endothelial damage, and decreased serum albumin concentration and increased proteinuria. These pathological changes were accompanied by deposition of immunoglobulin G and complement component 3, mainly in the mesangium until day 4 and in the mesangium and glomerular capillaries from day 8. On day 21, renal pathology developed to focal segmental sclerosis according to light microscopy. In C57BL/6J mice, renal injuries were not observed from either parasite infection. The clinical and pathological features of P. chabaudi AS infection in NC mice might be similar to quartan malarial nephropathy resulting from human malaria parasite P. malariae infection. The NC mouse model might therefore be useful in analyzing the underlying mechanisms and developing therapeutic approaches to malaria-related nephropathy.
Absalon, Sabrina; Robbins, Jonathan A.; Dvorin, Jeffrey D.
Blood-stage replication of the human malaria parasite Plasmodium falciparum occurs via schizogony, wherein daughter parasites are formed by a specialized cytokinesis known as segmentation. Here we identify a parasite protein, which we name P. falciparum Merozoite Organizing Protein (PfMOP), as essential for cytokinesis of blood-stage parasites. We show that, following PfMOP knockdown, parasites undergo incomplete segmentation resulting in a residual agglomerate of partially divided cells. While organelles develop normally, the structural scaffold of daughter parasites, the inner membrane complex (IMC), fails to form in this agglomerate causing flawed segmentation. In PfMOP-deficient gametocytes, the IMC formation defect causes maturation arrest with aberrant morphology and death. Our results provide insight into the mechanisms of replication and maturation of malaria parasites. PMID:27121004
Imwong, Mallika; Woodrow, Charles J; Hendriksen, Ilse C E; Veenemans, Jacobien; Verhoef, Hans; Faiz, M Abul; Mohanty, Sanjib; Mishra, Saroj; Mtove, George; Gesase, Samwel; Seni, Amir; Chhaganlal, Kajal D; Day, Nicholas P J; Dondorp, Arjen M; White, Nicholas J
In malaria-endemic areas, Plasmodium falciparum parasitemia is common in apparently healthy children and severe malaria is commonly misdiagnosed in patients with incidental parasitemia. We assessed whether the plasma Plasmodium falciparum DNA concentration is a useful datum for distinguishing uncomplicated from severe malaria in African children and Asian adults. P. falciparum DNA concentrations were measured by real-time polymerase chain reaction (PCR) in 224 African children (111 with uncomplicated malaria and 113 with severe malaria) and 211 Asian adults (100 with uncomplicated malaria and 111 with severe malaria) presenting with acute falciparum malaria. The diagnostic accuracy of plasma P. falciparum DNA concentrations in identifying severe malaria was 0.834 for children and 0.788 for adults, similar to that of plasma P. falciparum HRP2 levels and substantially superior to that of parasite densities (P < .0001). The diagnostic accuracy of plasma P. falciparum DNA concentrations plus plasma P. falciparum HRP2 concentrations was significantly greater than that of plasma P. falciparum HRP2 concentrations alone (0.904 for children [P = .004] and 0.847 for adults [P = .003]). Quantitative real-time PCR measurement of parasite DNA in plasma is a useful method for diagnosing severe falciparum malaria on fresh or archived plasma samples.
Eastman, Richard T; Pattaradilokrat, Sittiporn; Raj, Dipak K; Dixit, Saurabh; Deng, Bingbing; Miura, Kazutoyo; Yuan, Jing; Tanaka, Takeshi Q; Johnson, Ronald L; Jiang, Hongying; Huang, Ruili; Williamson, Kim C; Lambert, Lynn E; Long, Carole; Austin, Christopher P; Wu, Yimin; Su, Xin-Zhuan
Malaria is a deadly infectious disease in many tropical and subtropical countries. Previous efforts to eradicate malaria have failed, largely due to the emergence of drug-resistant parasites, insecticide-resistant mosquitoes and, in particular, the lack of drugs or vaccines to block parasite transmission. ATP-binding cassette (ABC) transporters are known to play a role in drug transport, metabolism, and resistance in many organisms, including malaria parasites. To investigate whether a Plasmodium falciparum ABC transporter (Pf14_0244 or PfABCG2) modulates parasite susceptibility to chemical compounds or plays a role in drug resistance, we disrupted the gene encoding PfABCG2, screened the recombinant and the wild-type 3D7 parasites against a library containing 2,816 drugs approved for human or animal use, and identified an antihistamine (ketotifen) that became less active against the PfABCG2-disrupted parasite in culture. In addition to some activity against asexual stages and gametocytes, ketotifen was highly potent in blocking oocyst development of P. falciparum and the rodent parasite Plasmodium yoelii in mosquitoes. Tests of structurally related tricyclic compounds identified additional compounds with similar activities in inhibiting transmission. Additionally, ketotifen appeared to have some activity against relapse of Plasmodium cynomolgi infection in rhesus monkeys. Further clinical evaluation of ketotifen and related compounds, including synthetic new derivatives, in blocking malaria transmission may provide new weapons for the current effort of malaria eradication.
Annam, Vamseedhar; Mohan, Chakkirala Nalini; Mrinalini, Vazhayil Ramunny
Malaria is a commonest mosquito-borne infectious disease worldwide. Early identification and management of malaria prevents complications and mortality. Identification of the malaria mainly relies on detection of the parasite on blood smears. The present study was conducted to compare Toluidine blue method with Leishman method for detection of malaria parasite and also to study the efficacy and advantages of using Toluidine blue method. In 540 consecutive patients with clinical suspicion of malaria, peripheral smears were prepared. Smears were processed for both conventional Leishman method and Toluidine blue method simultaneously. The significance of Toluidine blue method over Leishman method was analyzed using Chi-square (χ(2)) test. Out of 540 smears, 28.3% (153/540) were positive for malaria parasite on conventional Leishman method, while the smear positivity was more by Toluidine blue method to 33.3% (180/540) [P value < 0.01]. The remaining 66.67% (360/540) were negative by both Toluidine blue method and conventional Leishman method. The Toluidine blue method is simple, rapid, inexpensive, and easily available. The implementation of Toluidine blue method clearly improves microscopic detection of malaria parasite and can be a useful contribution to routine hematology even at rural health sectors.
Marrama, Laurence; Konate, Lassana; Phimpraphi, Waraphon; Sokhna, Cheikh; Tall, Adama; Diène Sarr, Fatoumata; Peerapittayamongkol, Chayanon; Louicharoen, Chalisa; Schneider, Bradley S.; Levescot, Anaïs; Talman, Arthur; Casademont, Isabelle; Menard, Didier; Trape, Jean-François; Rogier, Christophe; Kaewkunwal, Jaranit; Sura, Thanyachai; Nuchprayoon, Issarang; Ariey, Frederic; Baril, Laurence; Singhasivanon, Pratap; Mercereau-Puijalon, Odile; Paul, Rick
Background Studies on human genetic factors associated with malaria have hitherto concentrated on their role in susceptibility to and protection from disease. In contrast, virtually no attention has been paid to the role of human genetics in eliciting the production of parasite transmission stages, the gametocytes, and thus enhancing the spread of disease. Methods and Findings We analysed four longitudinal family-based cohort studies from Senegal and Thailand followed for 2–8 years and evaluated the relative impact of the human genetic and non-genetic factors on gametocyte production in infections of Plasmodium falciparum or P. vivax. Prevalence and density of gametocyte carriage were evaluated in asymptomatic and symptomatic infections by examination of Giemsa-stained blood smears and/or RT-PCR (for falciparum in one site). A significant human genetic contribution was found to be associated with gametocyte prevalence in asymptomatic P. falciparum infections. By contrast, there was no heritability associated with the production of gametocytes for P. falciparum or P. vivax symptomatic infections. Sickle cell mutation, HbS, was associated with increased gametocyte prevalence but its contribution was small. Conclusions The existence of a significant human genetic contribution to gametocyte prevalence in asymptomatic infections suggests that candidate gene and genome wide association approaches may be usefully applied to explore the underlying human genetics. Prospective epidemiological studies will provide an opportunity to generate novel and perhaps more epidemiologically pertinent gametocyte data with which similar analyses can be performed and the role of human genetics in parasite transmission ascertained. PMID:20613877
Yu, Xinran; Korkmaz, Turgay; Lilburn, Timothy G; Cai, Hong; Gu, Jianying; Wang, Yufeng
Annotating and understanding the function of proteins and other elements in a genome can be difficult in the absence of a well-studied and evolutionarily close relative. The causative agent of malaria, one of the oldest and most deadly global infectious diseases, is a good example of this problem. The burden of malaria is huge and there is a pressing need for new, more effective antimalarial strategies. However, techniques such as homology-dependent annotation transfer are severely impaired in this parasite because there are no well-understood close relatives. To circumvent this approach we developed a network-based method that uses a heavy path network-mining algorithm. We uncovered the protein-protein associations that are implicated in important cellular processes including genome integrity, DNA repair, transcriptional regulation, invasion, and pathogenesis, thus demonstrating the utility of this method. The URL of the source code for super-sequence mining method is http://www.cs.utsa.edu/~korkmaz/research/heavy-path-mining/.
Argy, Nicolas; Kendjo, Eric; Augé-Courtoi, Claire; Cojean, Sandrine; Clain, Jérôme; Houzé, Pascal; Thellier, Marc; Hubert, Veronique; Deloron, Philippe; Houzé, Sandrine
Imported malaria in France is characterized by various clinical manifestations observed in a heterogeneous population of patients such as travelers/expatriates and African migrants. In this population, host factors and parasite biomass associated with severe imported malaria are poorly known. From data collected by the Centre National de Référence du Paludisme, we identified epidemiological, demographic and biological features including parasite biomass and anti-plasmodial antibody levels (negative, positive and strongly positive serology) associated with different disease severity groups (very severe, moderately severe, and uncomplicated malaria) in 3 epidemiological groups (travelers/expatriates, first- and second-generation migrants). Age, ethnicity, absence of prior infection with P. falciparum, antibody levels, plasma PfHRP2 levels, total and circulating parasite biomass were related to severe malaria onset. Sequestered parasite biomass tended to be increased in very severe malaria, and was strongly correlated to the antibody level of the host. Prior exposure to P. falciparum is associated with high anti-plasmodial antibody levels which influence clinical presentation of imported malaria and its correlated circulating and sequestered parasite burden.
Kendjo, Eric; Augé-Courtoi, Claire; Cojean, Sandrine; Clain, Jérôme; Houzé, Pascal; Thellier, Marc; Hubert, Veronique; Deloron, Philippe; Houzé, Sandrine
Objectives Imported malaria in France is characterized by various clinical manifestations observed in a heterogeneous population of patients such as travelers/expatriates and African migrants. In this population, host factors and parasite biomass associated with severe imported malaria are poorly known. Methods From data collected by the Centre National de Référence du Paludisme, we identified epidemiological, demographic and biological features including parasite biomass and anti-plasmodial antibody levels (negative, positive and strongly positive serology) associated with different disease severity groups (very severe, moderately severe, and uncomplicated malaria) in 3 epidemiological groups (travelers/expatriates, first- and second-generation migrants). Results Age, ethnicity, absence of prior infection with P. falciparum, antibody levels, plasma PfHRP2 levels, total and circulating parasite biomass were related to severe malaria onset. Sequestered parasite biomass tended to be increased in very severe malaria, and was strongly correlated to the antibody level of the host. Conclusions Prior exposure to P. falciparum is associated with high anti-plasmodial antibody levels which influence clinical presentation of imported malaria and its correlated circulating and sequestered parasite burden. PMID:28410415
Escalating drug resistance in malaria parasites and lack of vaccine entails the discovery of novel drug targets and inhibitor molecules. The multi-component protein translation machinery is a rich source of such drug targets. Malaria parasites contain three translational compartments: the cytoplasm, apicoplast and mitochondrion, of which the latter two are of the prokaryotic type. Recent explorations by many groups into the malaria parasite protein translation enzymes, aminoacyl-tRNA synthetases (aaRSs), have yielded many promising inhibitors. The understanding of the biology of this unique set of 36 enzymes has become much clearer in recent times. Current review discusses the advances made in understanding of crucial aaRSs from Plasmodium and also the specific inhibitors found against malaria aaRSs.
Rogers, M. John; Cundliffe, Eric; McCutchan, Thomas F.
The antibiotic micrococcin is a potent growth inhibitor of the human malaria parasite Plasmodium falciparum, with a 50% inhibitory concentration of 35 nM. This is comparable to or less than the corresponding levels of commonly used antimalarial drugs. Micrococcin, like thiostrepton, putatively targets protein synthesis in the plastid-like organelle of the parasite. PMID:9517961
Rogers, M J; Cundliffe, E; McCutchan, T F
The antibiotic micrococcin is a potent growth inhibitor of the human malaria parasite Plasmodium falciparum, with a 50% inhibitory concentration of 35 nM. This is comparable to or less than the corresponding levels of commonly used antimalarial drugs. Micrococcin, like thiostrepton, putatively targets protein synthesis in the plastid-like organelle of the parasite.
Hayakawa, Eri H; Matsuoka, Hiroyuki
Scanning electron microscopy (SEM) is a powerful tool used to investigate object surfaces and has been widely applied in both material science and biology. With respect to the study of malaria, SEM revealed that erythrocytes infected with Plasmodium falciparum, a human parasite, display 'knob-like' structures on their surface comprising parasitized proteins. However, detailed methodology for SEM studies of malaria parasites is lacking in the literature making such studies challenging. Here, we provide a step-by-step guide to preparing Plasmodium-infected erythrocytes from two mouse strains for SEM analysis with minimal structural deterioration. We tested three species of murine malaria parasites, P. berghei, P. yoelii, and P. chabaudi, as well as non-parasitized human erythrocytes and P. falciparum-infected erythrocytes for comparisons. Our data demonstrated that the surface structures of parasitized erythrocytes between the three species of murine parasites in the two different strains of mice were indistinguishable and no surface alterations were observed in P. falciparum-erythrocytes. Our SEM observations contribute towards an understanding of the molecular mechanisms of parasite maturation in the erythrocyte cytoplasm and, along with future studies using our detailed methodology, may help to gain insight into the clinical phenomena of human malaria. Copyright © 2016 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.
Bensch, Staffan; Canbäck, Björn; DeBarry, Jeremy D.; Johansson, Tomas; Hellgren, Olof; Kissinger, Jessica C.; Palinauskas, Vaidas; Videvall, Elin; Valkiūnas, Gediminas
The phylogenetic relationships among hemosporidian parasites, including the origin of Plasmodium falciparum, the most virulent malaria parasite of humans, have been heavily debated for decades. Studies based on multiple-gene sequences have helped settle many of these controversial phylogenetic issues. However, denser taxon sampling and genome-wide analyses are needed to confidently resolve the evolutionay relationships among hemosporidian parasites. Genome sequences of several Plasmodium parasites are available but only for species infecting primates and rodents. To root the phylogenetic tree of Plasmodium, genomic data from related parasites of birds or reptiles are required. Here, we use a novel approach to isolate parasite DNA from microgametes and describe the first genome of a bird parasite in the sister genus to Plasmodium, Haemoproteus tartakovskyi. Similar to Plasmodium parasites, H. tartakovskyi has a small genome (23.2 Mb, 5,990 genes) and a GC content (25.4%) closer to P. falciparum (19.3%) than to Plasmodium vivax (42.3%). Combined with novel transcriptome sequences of the bird parasite Plasmodium ashfordi, our phylogenomic analyses of 1,302 orthologous genes demonstrate that mammalian-infecting malaria parasites are monophyletic, thus rejecting the repeatedly proposed hypothesis that the ancestor of Laverania parasites originated from a secondary host shift from birds to humans. Genes and genomic features previously found to be shared between P. falciparum and bird malaria parasites, but absent in other mammal malaria parasites, are therefore signatures of maintained ancestral states. We foresee that the genome of H. tartakovskyi will open new directions for comparative evolutionary analyses of malarial adaptive traits. PMID:27190205
Kurth, Florian; Develoux, Michel; Mechain, Matthieu; Clerinx, Jan; Antinori, Spinello; Gjørup, Ida E; Gascon, Joaquím; Mørch, Kristine; Nicastri, Emanuele; Ramharter, Michael; Bartoloni, Alessandro; Visser, Leo; Rolling, Thierry; Zanger, Philipp; Calleri, Guido; Salas-Coronas, Joaquín; Nielsen, Henrik; Just-Nübling, Gudrun; Neumayr, Andreas; Hachfeld, Anna; Schmid, Matthias L; Antonini, Pietro; Pongratz, Peter; Kern, Peter; Saraiva da Cunha, José; Soriano-Arandes, Antoni; Schunk, Mirjam; Suttorp, Norbert; Hatz, Christoph; Zoller, Thomas
Intravenous artesunate improves survival in severe malaria, but clinical trial data from nonendemic countries are scarce. The TropNet severe malaria database was analyzed to compare outcomes of artesunate vs quinine treatment. Artesunate reduced parasite clearance time and duration of intensive care unit and hospital treatment in European patients with imported severe malaria.
Milner, Danny A; Lee, Jonathan J; Frantzreb, Charles; Whitten, Richard O; Kamiza, Steve; Carr, Richard A; Pradham, Alana; Factor, Rachel E; Playforth, Krupa; Liomba, George; Dzamalala, Charles; Seydel, Karl B; Molyneux, Malcolm E; Taylor, Terrie E
Children in sub-Saharan Africa continue to acquire and die from cerebral malaria, despite efforts to control or eliminate the causative agent, Plasmodium falciparum. We present a quantitative histopathological assessment of the sequestration of parasitized erythrocytes in multiple organs obtained during a prospective series of 103 autopsies performed between 1996 and 2010 in Blantyre, Malawi, on pediatric patients who died from cerebral malaria and controls. After the brain, sequestration of parasites was most intense in the gastrointestinal tract, both in patients with cerebral malaria and those with parasitemia in other organs. Within cases of histologically defined cerebral malaria, which includes phenotypes termed "sequestration only" (CM1) and "sequestration with extravascular pathology" (CM2), CM1 was associated with large parasite numbers in the spleen and CM2 with intense parasite sequestration in the skin. A striking histological finding overall was the marked sequestration of parasitized erythrocytes across most organs in patients with fatal cerebral malaria, supporting the hypothesis that the disease is, in part, a result of a high level of total-body parasite sequestration. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: email@example.com.
Rosenthal, P J
The effects of peptide proteinase inhibitors on globin hydrolysis by cultured malaria parasites were studied. All of the four cysteine proteinase inhibitors evaluated blocked globin hydrolysis, as documented by the development of a morphological abnormality in which parasite food vacuoles filled with undegraded globin and by SDS-PAGE showing that the cysteine proteinase inhibitor-treated parasites accumulated large quantities of globin. The aspartic proteinase inhibitor pepstatin did not block globin hydrolysis by cultured parasites. None of seven antimalarial drugs tested elicited the food vacuole abnormality caused by cysteine proteinase inhibitors, indicating that this morphological alteration was not simply a sign of nonspecific parasite toxicity. Our results indicate that a trophozoite cysteine proteinase is required for initial cleavages of globin by intact malaria parasites.
There has been considerable progress in the biological characterization of malaria parasites in the past few years. Physiological parameters such as host adaptation, virulence, exoerythrocytic development, in vitro growth of erythrocytic stages, and drug sensitivity are of particular importance to epidemiologists. Advances in enzyme analysis, 2-dimensional protein electrophoresis, and nucleic acid analysis have produced several new techniques that can be applied to the malaria parasite. Similarly, antigenic characterization is expected to progress as a result of technical improvements. Many of the biological parameters are needed for the study of parasite genetics, a field which has expanded greatly through the development of cloning techniques. The latter also hold interest for the production, and the future use in research, of biologically well characterized standard clones. In this connexion, the cryopreservation and banking of malaria parasites deserve attention, in order to ensure the supply of well defined, viable isolates and clones to interested research workers.
Lindner, Scott E.; Swearingen, Kristian E.; Harupa, Anke; Vaughan, Ashley M.; Sinnis, Photini; Moritz, Robert L.; Kappe, Stefan H. I.
Malaria infections of mammals are initiated by the transmission of Plasmodium salivary gland sporozoites during an Anopheles mosquito vector bite. Sporozoites make their way through the skin and eventually to the liver, where they infect hepatocytes. Blocking this initial stage of infection is a promising malaria vaccine strategy. Therefore, comprehensively elucidating the protein composition of sporozoites will be invaluable in identifying novel targets for blocking infection. Previous efforts to identify the proteins expressed in Plasmodium mosquito stages were hampered by the technical difficulty of separating the parasite from its vector; without effective purifications, the large majority of proteins identified were of vector origin. Here we describe the proteomic profiling of highly purified salivary gland sporozoites from two Plasmodium species: human-infective Plasmodium falciparum and rodent-infective Plasmodium yoelii. The combination of improved sample purification and high mass accuracy mass spectrometry has facilitated the most complete proteome coverage to date for a pre-erythrocytic stage of the parasite. A total of 1991 P. falciparum sporozoite proteins and 1876 P. yoelii sporozoite proteins were identified, with >86% identified with high sequence coverage. The proteomic data were used to confirm the presence of components of three features critical for sporozoite infection of the mammalian host: the sporozoite motility and invasion apparatus (glideosome), sporozoite signaling pathways, and the contents of the apical secretory organelles. Furthermore, chemical labeling and identification of proteins on live sporozoites revealed previously uncharacterized complexity of the putative sporozoite surface-exposed proteome. Taken together, the data constitute the most comprehensive analysis to date of the protein expression of salivary gland sporozoites and reveal novel potential surface-exposed proteins that might be valuable targets for antibody blockage
Dunn, Alison M; Hatcher, Melanie J
Species distributions are changing at an unprecedented rate owing to human activity. We examine how two key processes of redistribution - biological invasion and disease emergence - are interlinked. There are many parallels between invasion and emergence processes, and invasions can drive the spread of new diseases to wildlife. We examine the potential impacts of invasion and disease emergence, and discuss how these threats can be countered, focusing on biosecurity. In contrast with international policy on emerging diseases of humans and managed species, policy on invasive species and parasites of wildlife is fragmented, and the lack of international cooperation encourages individual parties to minimize their input into control. We call for international policy that acknowledges the strong links between emerging diseases and invasion risk.
Sibley, L. David
Summary Intracellular parasitism has arisen only a few times during the long ancestry of protozoan parasites including in diverse groups such as microsporidians, kinetoplastids, and apicomplexans. Strategies used to gain entry differ widely from injection (e.g. microsporidians), active penetration of the host cell (e.g. Toxoplasma), recruitment of lysosomes to a plasma membrane wound (e.g. Trypanosoma cruzi), to host cell-mediated phagocytosis (e.g. Leishmania). The resulting range of intracellular niches is equally diverse ranging from cytosolic (e.g. T. cruzi) to residing within a nonfusigenic vacuole (e.g. Toxoplasma, Encephalitizoon) or a modified phagolysosome (e.g. Leishmania). These lifestyle choices influence access to nutrients, interaction with host cell signaling pathways, and detection by pathogen recognition systems. As such, intracellular life requires a repertoire of adaptations to assure entry-exit from the cell, as well as to thwart innate immune mechanisms and prevent clearance. Elucidating these pathways at the cellular and molecular level may identify key steps that can be targeted to reduce parasite survival or augment immunological responses and thereby prevent disease. PMID:21349087
Diagne, Christophe; Ribas, Alexis; Charbonnel, Nathalie; Dalecky, Ambroise; Tatard, Caroline; Gauthier, Philippe; Haukisalmi, Voitto; Fossati-Gaschignard, Odile; Bâ, Khalilou; Kane, Mamadou; Niang, Youssoupha; Diallo, Mamoudou; Sow, Aliou; Piry, Sylvain; Sembène, Mbacké; Brouat, Carine
Understanding why some exotic species become widespread and abundant in their colonised range is a fundamental issue that still needs to be addressed. Among many hypotheses, newly established host populations may benefit from a parasite loss ("enemy release" hypothesis) through impoverishment of their original parasite communities or reduced infection levels. Moreover, the fitness of competing native hosts may be negatively affected by the acquisition of exotic taxa from invaders ("parasite spillover") and/or by an increased transmission risk of native parasites due to their amplification by invaders ("parasite spillback"). We focused on gastrointestinal helminth communities to determine whether these predictions could explain the ongoing invasion success of the commensal house mouse (Mus musculus domesticus) and black rat (Rattus rattus), as well as the associated decrease in native Mastomys spp., in Senegal. For both invasive species, our results were consistent with the predictions of the enemy release hypothesis. A decrease in overall gastrointestinal helminth prevalence and infracommunity species richness was observed along the invasion gradients as well as lower specific prevalence/abundance (Aspiculuris tetraptera in Mus musculus domesticus, Hymenolepis diminuta in Rattus rattus) on the invasion fronts. Conversely, we did not find strong evidence of GIH spillover or spillback in invasion fronts, where native and invasive rodents co-occurred. Further experimental research is needed to determine whether and how the loss of gastrointestinal helminths and reduced infection levels along invasion routes may result in any advantageous effects on invader fitness and competitive advantage.
Phillips, Margaret A; Burrows, Jeremy N; Manyando, Christine; van Huijsduijnen, Rob Hooft; Van Voorhis, Wesley C; Wells, Timothy N C
Malaria is caused in humans by five species of single-celled eukaryotic Plasmodium parasites (mainly Plasmodium falciparum and Plasmodium vivax) that are transmitted by the bite of Anopheles spp. mosquitoes. Malaria remains one of the most serious infectious diseases; it threatens nearly half of the world's population and led to hundreds of thousands of deaths in 2015, predominantly among children in Africa. Malaria is managed through a combination of vector control approaches (such as insecticide spraying and the use of insecticide-treated bed nets) and drugs for both treatment and prevention. The widespread use of artemisinin-based combination therapies has contributed to substantial declines in the number of malaria-related deaths; however, the emergence of drug resistance threatens to reverse this progress. Advances in our understanding of the underlying molecular basis of pathogenesis have fuelled the development of new diagnostics, drugs and insecticides. Several new combination therapies are in clinical development that have efficacy against drug-resistant parasites and the potential to be used in single-dose regimens to improve compliance. This ambitious programme to eliminate malaria also includes new approaches that could yield malaria vaccines or novel vector control strategies. However, despite these achievements, a well-coordinated global effort on multiple fronts is needed if malaria elimination is to be achieved.
Tran, Tuan M.; Ongoiba, Aissata; Coursen, Jill; Crosnier, Cecile; Diouf, Ababacar; Huang, Chiung-Yu; Li, Shanping; Doumbo, Safiatou; Doumtabe, Didier; Kone, Younoussou; Bathily, Aboudramane; Dia, Seydou; Niangaly, Moussa; Dara, Charles; Sangala, Jules; Miller, Louis H.; Doumbo, Ogobara K.; Kayentao, Kassoum; Long, Carole A.; Miura, Kazutoyo; Wright, Gavin J.; Traore, Boubacar; Crompton, Peter D.
Background. Plasmodium falciparum reticulocyte-binding protein homologue 5 (PfRH5) is a blood-stage parasite protein essential for host erythrocyte invasion. PfRH5-specific antibodies raised in animals inhibit parasite growth in vitro, but the relevance of naturally acquired PfRH5-specific antibodies in humans is unclear. Methods. We assessed pre–malaria season PfRH5-specific immunoglobulin G (IgG) levels in 357 Malian children and adults who were uninfected with Plasmodium. Subsequent P. falciparum infections were detected by polymerase chain reaction every 2 weeks and malaria episodes by weekly physical examination and self-referral for 7 months. The primary outcome was time between the first P. falciparum infection and the first febrile malaria episode. PfRH5-specific IgG was assayed for parasite growth-inhibitory activity. Results. The presence of PfRH5-specific IgG at enrollment was associated with a longer time between the first blood-stage infection and the first malaria episode (PfRH5-seropositive median: 71 days, PfRH5-seronegative median: 18 days; P = .001). This association remained significant after adjustment for age and other factors associated with malaria risk/exposure (hazard ratio, .62; P = .02). Concentrated PfRH5-specific IgG purified from Malians inhibited P. falciparum growth in vitro. Conclusions. Naturally acquired PfRH5-specific IgG inhibits parasite growth in vitro and predicts protection from malaria. These findings strongly support efforts to develop PfRH5 as an urgently needed blood-stage malaria vaccine. Clinical Trials Registration NCT01322581. PMID:24133188
Marchesini, N; Luo, S; Rodrigues, C O; Moreno, S N; Docampo, R
Plasmodium berghei trophozoites were loaded with the fluorescent calcium indicator, fura-2 acetoxymethyl ester, to measure their intracellular Ca(2+) concentration ([Ca(2+)](i)). [Ca(2+)](i) was increased in the presence of the sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase inhibitor, thapsigargin. Trophozoites also possess a significant amount of Ca(2+) stored in an acidic compartment. This was indicated by: (1) the increase in [Ca(2+)](i) induced by bafilomycin A(1), nigericin, monensin, or the weak base, NH(4)Cl, in the nominal absence of extracellular Ca(2+), and (2) the effect of ionomycin, which cannot take Ca(2+) out of acidic organelles and was more effective after alkalinization of this compartment by addition of bafilomycin A(1), nigericin, monensin, or NH(4)Cl. Inorganic PP(i) promoted the acidification of a subcellular compartment in cell homogenates of trophozoites. The proton gradient driven by PP(i) collapsed by addition of the K(+)/H(+) exchanger, nigericin, and eliminated by the PP(i) analogue, aminomethylenediphosphonate (AMDP). Both PP(i) hydrolysis and proton transport were dependent upon K(+), and Na(+) caused partial inhibition of these activities. PP(i) hydrolysis was sensitive in a dose-dependent manner to AMDP, imidodiphosphate, sodium fluoride, dicyclohexylcarbodi-imide and to the thiol reagent, N-ethylmaleimide. Immunofluorescence microscopy using antibodies raised against conserved peptide sequences of a plant vacuolar pyrophosphatase (V-H(+)-PPase) suggested that the proton pyrophosphatase is located in intracellular vacuoles and the plasma membrane of trophozoites. AMDP caused an increase in [Ca(2+)](i) in the nominal absence of extracellular Ca(2+). Ionomycin was more effective in releasing Ca(2+) from this acidic intracellular compartment after treatment of the cells with AMDP. Taken together, these results suggest the presence in malaria parasites of acidocalcisomes with similar characteristics to those described in
Marchesini, N; Luo, S; Rodrigues, C O; Moreno, S N; Docampo, R
Plasmodium berghei trophozoites were loaded with the fluorescent calcium indicator, fura-2 acetoxymethyl ester, to measure their intracellular Ca(2+) concentration ([Ca(2+)](i)). [Ca(2+)](i) was increased in the presence of the sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase inhibitor, thapsigargin. Trophozoites also possess a significant amount of Ca(2+) stored in an acidic compartment. This was indicated by: (1) the increase in [Ca(2+)](i) induced by bafilomycin A(1), nigericin, monensin, or the weak base, NH(4)Cl, in the nominal absence of extracellular Ca(2+), and (2) the effect of ionomycin, which cannot take Ca(2+) out of acidic organelles and was more effective after alkalinization of this compartment by addition of bafilomycin A(1), nigericin, monensin, or NH(4)Cl. Inorganic PP(i) promoted the acidification of a subcellular compartment in cell homogenates of trophozoites. The proton gradient driven by PP(i) collapsed by addition of the K(+)/H(+) exchanger, nigericin, and eliminated by the PP(i) analogue, aminomethylenediphosphonate (AMDP). Both PP(i) hydrolysis and proton transport were dependent upon K(+), and Na(+) caused partial inhibition of these activities. PP(i) hydrolysis was sensitive in a dose-dependent manner to AMDP, imidodiphosphate, sodium fluoride, dicyclohexylcarbodi-imide and to the thiol reagent, N-ethylmaleimide. Immunofluorescence microscopy using antibodies raised against conserved peptide sequences of a plant vacuolar pyrophosphatase (V-H(+)-PPase) suggested that the proton pyrophosphatase is located in intracellular vacuoles and the plasma membrane of trophozoites. AMDP caused an increase in [Ca(2+)](i) in the nominal absence of extracellular Ca(2+). Ionomycin was more effective in releasing Ca(2+) from this acidic intracellular compartment after treatment of the cells with AMDP. Taken together, these results suggest the presence in malaria parasites of acidocalcisomes with similar characteristics to those described in
Keitany, Gladys J; Sack, Brandon; Smithers, Hannah; Chen, Lin; Jang, Ihn K; Sebastian, Leslie; Gupta, Megha; Sather, D Noah; Vignali, Marissa; Vaughan, Ashley M; Kappe, Stefan H I; Wang, Ruobing
Understanding protective immunity to malaria is essential for the design of an effective vaccine to prevent the large number of infections and deaths caused by this parasitic disease. To date, whole-parasite immunization with attenuated parasites is the most effective method to confer sterile protection against malaria infection in clinical trials. Mouse model studies have highlighted the essential role that CD8(+) T cells play in protection against preerythrocytic stages of malaria; however, there is mounting evidence that antibodies are also important in these stages. Here, we show that experimental immunization of mice with Plasmodium yoelii fabb/f(-) (Pyfabb/f(-)), a genetically attenuated rodent malaria parasite that arrests late in the liver stage, induced functional antibodies that inhibited hepatocyte invasion in vitro and reduced liver-stage burden in vivo. These antibodies were sufficient to induce sterile protection from challenge by P. yoelii sporozoites in the absence of T cells in 50% of mice when sporozoites were administered by mosquito bite but not when they were administered by intravenous injection. Moreover, among mice challenged by mosquito bite, a higher proportion of BALB/c mice than C57BL/6 mice developed sterile protection (62.5% and 37.5%, respectively). Analysis of the antibody isotypes induced by immunization with Pyfabb/f(-) showed that, overall, BALB/c mice developed an IgG1-biased response, whereas C57BL/6 mice developed an IgG2b/c-biased response. Our data demonstrate for the first time that antibodies induced by experimental immunization of mice with a genetically attenuated rodent parasite play a protective role during the preerythrocytic stages of malaria. Furthermore, they highlight the importance of considering both the route of challenge and the genetic background of the mouse strains used when interpreting vaccine efficacy studies in animal models of malaria infection. Copyright © 2014, American Society for Microbiology. All
Mooney, J P; Butler, B P; Lokken, K L; Xavier, M N; Chau, J Y; Schaltenberg, N; Dandekar, S; George, M D; Santos, R L; Luckhart, S; Tsolis, R M
Coinfection can markedly alter the response to a pathogen, thereby changing its clinical presentation. For example, non-typhoidal Salmonella (NTS) serotypes are associated with gastroenteritis in immunocompetent individuals. In contrast, individuals with severe pediatric malaria can develop bacteremic infections with NTS, during which symptoms of gastroenteritis are commonly absent. Here we report that, in both a ligated ileal loop model and a mouse colitis model, malaria parasites caused a global suppression of gut inflammatory responses and blunted the neutrophil influx that is characteristic of NTS infection. Further, malaria parasite infection led to increased recovery of Salmonella enterica serotype Typhimurium from the draining mesenteric lymph node (MLN) of mice. In the mouse colitis model, blunted intestinal inflammation during NTS infection was independent of anemia but instead required parasite-induced synthesis of interleukin (IL)-10. Blocking of IL-10 in coinfected mice reduced dissemination of S. Typhimurium to the MLN, suggesting that induction of IL-10 contributes to development of disseminated infection. Thus IL-10 produced during the immune response to malaria in this model contributes to suppression of mucosal inflammatory responses to invasive NTS, which may contribute to differences in the clinical presentation of NTS infection in the setting of malaria.
Zhang, Yao; Huang, Changjin; Kim, Sangtae; Golkaram, Mahdi; Dixon, Matthew W A; Tilley, Leann; Li, Ju; Zhang, Sulin; Suresh, Subra
During its asexual development within the red blood cell (RBC), Plasmodium falciparum (Pf), the most virulent human malaria parasite, exports proteins that modify the host RBC membrane. The attendant increase in cell stiffness and cytoadherence leads to sequestration of infected RBCs in microvasculature, which enables the parasite to evade the spleen, and leads to organ dysfunction in severe cases of malaria. Despite progress in understanding malaria pathogenesis, the molecular mechanisms responsible for the dramatic loss of deformability of Pf-infected RBCs have remained elusive. By recourse to a coarse-grained (CG) model that captures the molecular structures of Pf-infected RBC membrane, here we show that nanoscale surface protrusions, known as "knobs," introduce multiple stiffening mechanisms through composite strengthening, strain hardening, and knob density-dependent vertical coupling. On one hand, the knobs act as structural strengtheners for the spectrin network; on the other, the presence of knobs results in strain inhomogeneity in the spectrin network with elevated shear strain in the knob-free regions, which, given its strain-hardening property, effectively stiffens the network. From the trophozoite to the schizont stage that ensues within 24-48 h of parasite invasion into the RBC, the rise in the knob density results in the increased number of vertical constraints between the spectrin network and the lipid bilayer, which further stiffens the membrane. The shear moduli of Pf-infected RBCs predicted by the CG model at different stages of parasite maturation are in agreement with experimental results. In addition to providing a fundamental understanding of the stiffening mechanisms of Pf-infected RBCs, our simulation results suggest potential targets for antimalarial therapies.
Akompong, Thomas; Ghori, Nafisa; Haldar, Kasturi
The human malaria parasite Plasmodium falciparum digests hemoglobin and polymerizes the released free heme into hemozoin. This activity occurs in an acidic organelle called the food vacuole and is essential for survival of the parasite in erythrocytes. Since acidic conditions are known to enhance the auto-oxidation of hemoglobin, we investigated whether hemoglobin ingested by the parasite was oxidized and whether the oxidation process could be a target for chemotherapy against malaria. We released parasites from their host cells and separately analyzed hemoglobin ingested by the parasites from that remaining in the erythrocytes. Isolated parasites contained elevated amounts (38.5% ± 3.5%) of oxidized hemoglobin (methemoglobin) compared to levels (0.8% ± 0.2%) found in normal, uninfected erythrocytes. Further, treatment of infected cells with the reducing agent riboflavin for 24 h decreased the parasite methemoglobin level by 55%. It also inhibited hemozoin production by 50% and decreased the average size of the food vacuole by 47%. Administration of riboflavin for 48 h resulted in a 65% decrease in food vacuole size and inhibited asexual parasite growth in cultures. High doses of riboflavin are used clinically to treat congenital methemoglobinemia without any adverse side effects. This activity, in conjunction with its impressive antimalarial activity, makes riboflavin attractive as a safe and inexpensive drug for treating malaria caused by P. falciparum. PMID:10602728
Background Malaria is a major health and socio-economical problem in tropical and sub-tropical areas of the world. Several methodologies have been used to assess parasite viability during the adaption of field strains to culture or the assessment of drug potential, but these are in general not able to provide an accurate real-time assessment of whether parasites are alive or dead. Methods Different commercial dyes and kits were assessed for their potential to allow for the real-time detection of whether a blood stage malaria parasite is dead or alive. Results Here, a methodology is presented based on the potential-sensitive mitochondrial probe JC-1, which allows for the real-time visualization of live (red staining) and/or dead (absence of red staining) blood stage parasites in vitro and ex vivo. This method is applicable across malaria parasite species and strains and allows to visualize all parasite blood stages including gametocytes. Further, this methodology has been assessed also for use in drug sensitivity testing. Conclusions The JC-1 staining approach is a versatile methodology that can be used to assess parasite viability during the adaptation of field samples to culture and during drug treatment. It was found to hold promise in the assessment of drugs expected to lead to delayed death phenotypes and it currently being evaluated as a method for the assessment of parasite viability during the adaptation of patient-derived Plasmodium vivax to long-term in vitro culture. PMID:23758788
Lutz, Holly L; Patterson, Bruce D; Kerbis Peterhans, Julian C; Stanley, William T; Webala, Paul W; Gnoske, Thomas P; Hackett, Shannon J; Stanhope, Michael J
Phylogenies of parasites provide hypotheses on the history of their movements between hosts, leading to important insights regarding the processes of host switching that underlie modern-day epidemics. Haemosporidian (malaria) parasites lack a well resolved phylogeny, which has impeded the study of evolutionary processes associated with host-switching in this group. Here we present a novel phylogenetic hypothesis that suggests bats served as the ancestral hosts of malaria parasites in primates and rodents. Expanding upon current taxon sampling of Afrotropical bat and bird parasites, we find strong support for all major nodes in the haemosporidian tree using both Bayesian and maximum likelihood approaches. Our analyses support a single transition of haemosporidian parasites from saurian to chiropteran hosts, and do not support a monophyletic relationship between Plasmodium parasites of birds and mammals. We find, for the first time, that Hepatocystis and Plasmodium parasites of mammals represent reciprocally monophyletic evolutionary lineages. These results highlight the importance of broad taxonomic sampling when analyzing phylogenetic relationships, and have important implications for our understanding of key host switching events in the history of malaria parasite evolution. Copyright © 2016 Elsevier Inc. All rights reserved.
Espinosa, Diego A; Radtke, Andrea J; Zavala, Fidel
Rodent transgenic parasites are useful tools for the preclinical evaluation of malaria vaccines. Over the last decade, several studies have reported the development of transgenic rodent parasites expressing P. falciparum antigens for the assessment of vaccine-induced immune responses, which traditionally have been limited to in vitro assays. However, the genetic manipulation of rodent Plasmodium species can have detrimental effects on the parasite's infectivity and development. In this chapter, we present a few guidelines for designing transfection plasmids, which should improve transfection efficiency and facilitate the generation of functional transgenic parasite strains. In addition, we provide a transfection protocol for the development of transgenic P. berghei parasites as well as practical methods to assess the viability and infectivity of these newly generated strains throughout different stages of their life cycle. These techniques should allow researchers to develop novel rodent malaria parasites expressing antigens from human malaria species and to determine whether these transgenic strains are fully infectious and thus represent stringent platforms for the in vivo evaluation of malaria vaccine candidates.
Giddam, Ashwini Kumar; Reiman, Jennifer M; Zaman, Mehfuz; Skwarczynski, Mariusz; Toth, Istvan; Good, Michael F
Although attenuated malaria parasitized red blood cells (pRBCs) are promising vaccine candidates, their application in humans may be restricted for ethical and regulatory reasons. Therefore, we developed an organic microparticle-based delivery platform as a whole parasite malaria-antigen carrier to mimic pRBCs. Killed blood stage parasites were encapsulated within liposomes that are targeted to antigen presenting cells (APCs). Mannosylated lipid core peptides (MLCPs) were used as targeting ligands for the liposome-encapsulated parasite antigens. MLCP-liposomes, but not unmannosylated liposomes, were taken-up efficiently by APCs which then significantly upregulated expression of MHC-ll and costimulatory molecules, CD80 and CD86. Two such vaccines using rodent model systems were constructed - one with Plasmodium chabaudi and the other with P. yoelii. MLCP-liposome vaccines were able to control the parasite burden and extended the survival of mice. Thus, we have demonstrated an alternative delivery system to attenuated pRBCs with similar vaccine efficacy and added clinical advantages. Such liposomes are promising candidates for a human malaria vaccine. Attenuated whole parasite-based vaccines, by incorporating all parasite antigens, are very promising candidates, but issues relating to production, storage and safety concerns are significantly slowing their development. We therefore developed a semi-synthetic whole parasite malaria vaccine that is easily manufactured and stored. Two such prototype vaccines (a P. chabaudi and a P. yoelii vaccine) have been constructed. They are non-infectious, highly immunogenic and give good protection profiles. This semi-synthetic delivery platform is an exciting strategy to accelerate the development of a licensed malaria vaccine. Moreover, this strategy can be potentially applied to a wide range of pathogens. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
Barata, Lídia; Houzé, Pascal; Boutbibe, Khadija; Zanghi, Gigliola; Franetich, Jean-François; Mazier, Dominique; Clain, Jérôme
The interaction between atovaquone and proguanil has never been studied against liver stage malaria, which is the main target of this drug combination when used for chemoprevention. Using human hepatocytes lacking cytochrome P450 activity, and thus avoiding proguanil metabolizing into potent cycloguanil, we show in vitro that the atovaquone-proguanil combination synergistically inhibits the growth of rodent Plasmodium yoelii parasites. These results provide a pharmacological basis for the high efficacy of atovaquone-proguanil used as malaria chemoprevention.
Barata, Lídia; Houzé, Pascal; Boutbibe, Khadija; Zanghi, Gigliola; Franetich, Jean-François
The interaction between atovaquone and proguanil has never been studied against liver stage malaria, which is the main target of this drug combination when used for chemoprevention. Using human hepatocytes lacking cytochrome P450 activity, and thus avoiding proguanil metabolizing into potent cycloguanil, we show in vitro that the atovaquone-proguanil combination synergistically inhibits the growth of rodent Plasmodium yoelii parasites. These results provide a pharmacological basis for the high efficacy of atovaquone-proguanil used as malaria chemoprevention. PMID:26926628
Vaughan, Ashley M; Kappe, Stefan H I; Ploss, Alexander; Mikolajczak, Sebastian A
Malaria is a disease caused by infection with Plasmodium parasites that are transmitted by mosquito bite. Five different species of Plasmodium infect humans with severe disease, but human malaria is primarily caused by Plasmodium falciparum. The burden of malaria on the developing world is enormous, and a fully protective vaccine is still elusive. One of the biggest challenges in the quest for the development of new antimalarial drugs and vaccines is the lack of accessible animal models to study P. falciparum infection because the parasite is restricted to the great apes and human hosts. Here, we review the current state of research in this field and provide an outlook of the development of humanized small animal models to study P. falciparum infection that will accelerate fundamental research into human parasite biology and could accelerate drug and vaccine design in the future.
Cunnington, Aubrey J.; Riley, Eleanor M.; Walther, Michael
Severe malaria defines individuals at increased risk of death from their infection. Proposed pathogenic mechanisms include parasite sequestration, inflammation, and endothelial dysfunction. Severe malaria is not a single entity, manifesting with distinct syndromes such as severe anemia, severe respiratory distress or coma, each characterized by differences in epidemiology, underlying biology, and risk of death. The relative contribution of the various pathogenic mechanisms may differ between syndromes, and this is supported by accumulating evidence, which challenges sequestration as the initiating event. Here we propose that high parasite biomass is the common initiating feature, but subtle variations in the interaction between the host and parasite exist, and understanding these differences may be crucial to improve outcomes in patients with severe malaria. PMID:24210256
Eckerle, Isabella; Ebinger, Damaris; Gotthardt, Daniel; Eberhardt, Ralf; Schnabel, Philipp A; Stremmel, Wolfgang; Junghanss, Thomas; Eisenbach, Christoph
Invasive fungal infection is rarely reported in association with malaria, even though malaria-associated inhibition of phagocyte function is a well-known condition. Invasive aspergillosis is frequently found in severely immuno-compromised patients but not in healthy individuals. Here, a case of pulmonary invasive aspergillosis in a previously healthy patient with severe P. falciparum malaria is presented, who was successfully treated with voriconazol and caspofungin. This is the first survival of malaria-associated invasive aspergillosis. PMID:19619319
Zhu, Feng; Liu, Taiping; Zhao, Chenhao; Lu, Xiao; Zhang, Jian; Xu, Wenyue
As a malaria transmission-blocking vaccine alone does not confer a direct benefit to the recipient, it is necessary to develop a vaccine that not only blocks malaria transmission but also protects vaccinated individuals. In this study we observed that a whole-killed blood-stage vaccine (WKV) not only conferred protection against the blood-stage challenge but also markedly inhibited the transmission of different strains of the malaria parasite. Although the parasitemia is much lower in WKV-immunized mice challenged with malaria parasites, the gametocytemia is comparable between control and immunized mice during the early stages of infection. The depletion of CD4(+) T cells prior to the adoptive transfer of parasites into WKV-immunized mice has no effect on the development of the malaria parasite in the mosquito, but the adoptive transfer of the serum from the immunized mice into the parasite-inoculated mice remarkably suppresses the development of malaria parasites in mosquitoes. Furthermore, immunized mice challenged with the malaria parasite generate higher levels of parasite-specific Abs and the inflammatory cytokines MCP-1 and IFN-γ. However, the adoptive transfer of parasite-specific IgG or the depletion of MCP-1, but not IFN-γ, to some extent is closely associated with the suppression of malaria parasite development in mosquitoes. These data strongly suggest that WKV-induced immune responses confer protection against the mosquito stage, which is largely dependent on malaria parasite-specific Abs and MCP-1. This finding sheds new light on blocking malaria transmission through the immunization of individuals with the WKV. Copyright © 2016 by The American Association of Immunologists, Inc.
Kute, Vivek B; Goswami, Jitendra G; Vanikar, Aruna V; Shah, Pankaj R; Gumber, Manoj R; Patel, Himanshu V; Kanodia, Kamal V; Trivedi, Hargovind L
Severe and complicated malaria is usually caused by Plasmodium falciparum malaria (PF) but it has been increasingly observed that Plasmodium vivax malaria (PV), which was otherwise considered to be benign malaria, with a low case-fatality ratio, can also occasionally result in severe disease as with PF malaria. There is an urgent need to re-examine the clinical spectrum and burden of PV so that adequate control measures can be implemented against this emerging but neglected disease. We report a case of severe PV malaria with multi-organ dysfunction. Patients exhibited acute kidney injury, severe anemia/thrombocytopenia, jaundice, hypoglycemia, hyponatremia, and pulmonary edema. Peripheral blood microscopy by trained and expert pathologist and rapid diagnostic test showed the presence of PV and absence of PF. The patient recovered completely with anti-malarial drugs, supportive measures, and hemodialysis.Recent microrheologic research that analyzed malaria severity in PV clearly demonstrated enhanced aggregation, erythrocyte clumping, and reduced deformability affecting microcirculation. Our case report highlights the fact that PV malaria is benign by name but not always by nature. PV can lead to unusual and potentially life-threatening complications. Further large-scale multi-centric studies are needed to define this less known entity.
Elliott, J L; Saliba, K J; Kirk, K
The mature, intraerythrocytic form of the human malaria parasite, Plasmodium falciparum, is reliant on glycolysis for its energetic requirements. It produces large quantities of lactic acid, which have to be removed from the parasite's cytosol to maintain the cell's integrity and metabolic viability. Here we show that the monocarboxylates lactate and pyruvate are both transported across the parasite's plasma membrane via a H(+)/monocarboxylate symport process that is saturable and inhibited by the bioflavonoid phloretin. The results provide direct evidence for the presence at the parasite surface of a H(+)-coupled monocarboxylate transporter with features in common with members of the MCT (monocarboxylate transporter) family of higher eukaryotes. PMID:11311136
Hikosaka, Kenji; Hirai, Makoto; Komatsuya, Keisuke; Ono, Yasuo; Kita, Kiyoshi
The intraerythrocytic form of the human malaria parasite Plasmodium falciparum relies on glycolysis for its energy requirements. In glycolysis, lactate is an end product. It is therefore known that lactate accumulates in in vitro culture; however, its influence on parasite growth remains unknown. Here we investigated the effect of lactate on the development of P. falciparum during in vitro culture under lactate supplementation in detail. Results revealed that lactate retarded parasite development and reduced the number of merozoites in the schizont stage. These findings suggest that lactate has the potential to affect parasite development.
Davis, T M; Singh, B; Sheridan, G
A 29-year-old woman with ovale malaria (most likely contracted, together with asymptomatic schistosomiasis, in East Africa two years previously) had fever, nausea and confusion, jaundice, anaemia, thrombocytopenia, hyponatraemia and hypokalaemia. She was initially diagnosed with and treated for blood-smear-positive vivax malaria. Because of the unusual clinical presentation, blood was analysed by a malaria species-specific nested polymerase chain reaction (PCR) assay which identified Plasmodium ovale as the only infecting species. This case illustrates (i) that a detailed travel history remains a vital part of clinical assessment, (ii) ovale malaria can have an exceptionally long incubation period and features of a moderately severe acute infection, and (iii) PCR assay may prove a valuable adjunct to blood film examination in the diagnosis and speciation of malaria.
Artemisinin-based combination therapies (ACTs) are currently the first-line drugs for treating uncomplicated falciparum malaria, the most deadly of the human malarias. Malaria parasite clearance rates estimated from patients' blood following ACT treatment have been widely adopted as a measure of drug effectiveness and as surveillance tools for detecting the presence of potential artemisinin resistance. This metric has not been investigated in detail, nor have its properties or potential shortcomings been identified. Herein, the pharmacology of drug treatment, parasite biology, and human immunity are combined to investigate the dynamics of parasite clearance following ACT. This approach parsimoniously recovers the principal clinical features and dynamics of clearance. Human immunity is the primary determinant of clearance rates, unless or until artemisinin killing has fallen to near-ineffective levels. Clearance rates are therefore highly insensitive metrics for surveillance that may lead to overconfidence, as even quite substantial reductions in drug sensitivity may not be detected as lower clearance rates. Equally serious is the use of clearance rates to quantify the impact of ACT regimen changes, as this strategy will plausibly miss even very substantial increases in drug effectiveness. In particular, the malaria community may be missing the opportunity to dramatically increase ACT effectiveness through regimen changes, particularly through a switch to twice-daily regimens and/or increases in artemisinin dosing levels. The malaria community therefore appears overreliant on a single metric of drug effectiveness, the parasite clearance rate, that has significant and serious shortcomings. PMID:26239987
Lalremruata, Albert; Magris, Magda; Vivas-Martínez, Sarai; Koehler, Maike; Esen, Meral; Kempaiah, Prakasha; Jeyaraj, Sankarganesh; Perkins, Douglas Jay; Mordmüller, Benjamin; Metzger, Wolfram G
The quartan malaria parasite Plasmodium malariae is the widest spread and best adapted human malaria parasite. The simian Plasmodium brasilianum causes quartan fever in New World monkeys and resembles P. malariae morphologically. Since the genetics of the two parasites are nearly identical, differing only in a range of mutations expected within a species, it has long been speculated that the two are the same. However, no naturally acquired infection with parasites termed as P. brasilianum has been found in humans until now. We investigated malaria cases from remote Yanomami indigenous communities of the Venezuelan Amazon and analyzed the genes coding for the circumsporozoite protein (CSP) and the small subunit of ribosomes (18S) by species-specific PCR and capillary based-DNA sequencing. Based on 18S rRNA gene sequencing, we identified 12 patients harboring malaria parasites which were 100% identical with P. brasilianum isolated from the monkey, Alouatta seniculus. Translated amino acid sequences of the CS protein gene showed identical immunodominant repeat units between quartan malaria parasites isolated from both humans and monkeys. This study reports, for the first time, naturally acquired infections in humans with parasites termed as P. brasilianum. We conclude that quartan malaria parasites are easily exchanged between humans and monkeys in Latin America. We hypothesize a lack of host specificity in mammalian hosts and consider quartan malaria to be a true anthropozoonosis. Since the name P. brasilianum suggests a malaria species distinct from P. malariae, we propose that P. brasilianum should have a nomenclatorial revision in case further research confirms our findings. The expansive reservoir of mammalian hosts discriminates quartan malaria from other Plasmodium spp. and requires particular research efforts.
Lalremruata, Albert; Magris, Magda; Vivas-Martínez, Sarai; Koehler, Maike; Esen, Meral; Kempaiah, Prakasha; Jeyaraj, Sankarganesh; Perkins, Douglas Jay; Mordmüller, Benjamin; Metzger, Wolfram G.
Background The quartan malaria parasite Plasmodium malariae is the widest spread and best adapted human malaria parasite. The simian Plasmodium brasilianum causes quartan fever in New World monkeys and resembles P. malariae morphologically. Since the genetics of the two parasites are nearly identical, differing only in a range of mutations expected within a species, it has long been speculated that the two are the same. However, no naturally acquired infection with parasites termed as P. brasilianum has been found in humans until now. Methods We investigated malaria cases from remote Yanomami indigenous communities of the Venezuelan Amazon and analyzed the genes coding for the circumsporozoite protein (CSP) and the small subunit of ribosomes (18S) by species-specific PCR and capillary based-DNA sequencing. Findings Based on 18S rRNA gene sequencing, we identified 12 patients harboring malaria parasites which were 100% identical with P. brasilianum isolated from the monkey, Alouatta seniculus. Translated amino acid sequences of the CS protein gene showed identical immunodominant repeat units between quartan malaria parasites isolated from both humans and monkeys. Interpretation This study reports, for the first time, naturally acquired infections in humans with parasites termed as P. brasilianum. We conclude that quartan malaria parasites are easily exchanged between humans and monkeys in Latin America. We hypothesize a lack of host specificity in mammalian hosts and consider quartan malaria to be a true anthropozoonosis. Since the name P. brasilianum suggests a malaria species distinct from P. malariae, we propose that P. brasilianum should have a nomenclatorial revision in case further research confirms our findings. The expansive reservoir of mammalian hosts discriminates quartan malaria from other Plasmodium spp. and requires particular research efforts. PMID:26501116
McMorrow, M. L.; Aidoo, M.; Kachur, S. P.
Rapid diagnostic tests (RDTs) for malaria have improved the availability of parasite-based diagnosis throughout the malaria-endemic world. Accurate malaria diagnosis is essential for malaria case management, surveillance, and elimination. RDTs are inexpensive, simple to perform, and provide results in 15–20 min. Despite high sensitivity and specificity for Plasmodium falciparum infections, RDTs have several limitations that may reduce their utility in low-transmission settings: they do not reliably detect low-density parasitaemia (≤200 parasites/μL), many are less sensitive for Plasmodium vivax infections, and their ability to detect Plasmodium ovale and Plasmodium malariae is unknown. Therefore, in elimination settings, alternative tools with higher sensitivity for low-density infections (e.g. nucleic acid-based tests) are required to complement field diagnostics, and new highly sensitive and specific field-appropriate tests must be developed to ensure accurate diagnosis of symptomatic and asymptomatic carriers. As malaria transmission declines, the proportion of low-density infections among symptomatic and asymptomatic persons is likely to increase, which may limit the utility of RDTs. Monitoring malaria in elimination settings will probably depend on the use of more than one diagnostic tool in clinical-care and surveillance activities, and the combination of tools utilized will need to be informed by regular monitoring of test performance through effective quality assurance. PMID:21910780
Background Erythrocyte invasion by Plasmodium falciparum is a complex process that involves two families; Erythrocyte Binding-Like (EBL) and the Reticulocyte Binding-Like (PfRh) proteins. Antibodies that inhibit merozoite attachment and invasion are believed to be important in mediating naturally acquired immunity and immunity generated by parasite blood stage vaccine candidates. The hypotheses tested in this study were 1) that antibody responses against specific P. falciparum invasion ligands (EBL and PfRh) differ between symptomatic and asymptomatic individuals living in the low-transmission region of the Peruvian Amazon and 2), such antibody responses might have an association, either direct or indirect, with clinical immunity observed in asymptomatically parasitaemic individuals. Methods ELISA was used to assess antibody responses (IgG, IgG1 and IgG3) against recombinant P. falciparum invasion ligands of the EBL (EBA-175, EBA-181, EBA-140) and PfRh families (PfRh1, PfRh2a, PfRh2b, PfRh4 and PfRh5) in 45 individuals infected with P. falciparum from Peruvian Amazon. Individuals were classified as having symptomatic malaria (N=37) or asymptomatic infection (N=8). Results Antibody responses against both EBL and PfRh family proteins were significantly higher in asymptomatic compared to symptomatic individuals, demonstrating an association with clinical immunity. Significant differences in the total IgG responses were observed with EBA-175, EBA-181, PfRh2b, and MSP119 (as a control). IgG1 responses against EBA-181, PfRh2a and PfRh2b were significantly higher in the asymptomatic individuals. Total IgG antibody responses against PfRh1, PfRh2a, PfRh2b, PfRh5, EBA-175, EBA-181 and MSP119 proteins were negatively correlated with level of parasitaemia. IgG1 responses against EBA-181, PfRh2a and PfRh2b and IgG3 response for PfRh2a were also negatively correlated with parasitaemia. Conclusions These data suggest that falciparum malaria patients who develop clinical immunity
Khan, Shahid M; Kroeze, Hans; Franke-Fayard, Blandine; Janse, Chris J
Genetically modified Plasmodium parasites are central gene function reagents in malaria research. The Rodent Malaria genetically modified DataBase (RMgmDB) ( www.pberghei.eu ) is a manually curated Web - based repository that contains information on genetically modified rodent malaria parasites. It provides easy and rapid access to information on the genotype and phenotype of genetically modified mutant and reporter parasites. Here, we provide guidelines for generating and describing rodent malaria parasite mutants. Standardization in describing mutant genotypes and phenotypes is important not only to enhance publication quality but also to facilitate cross-linking and mining data from multiple sources, and should permit information derived from mutant parasites to be used in integrative system biology approaches. We also provide guidelines on how to submit information to RMgmDB on non-published mutants, mutants that do not exhibit a clear phenotype, as well as negative attempts to disrupt/mutate genes. Such information helps to prevent unnecessary duplication of experiments in different laboratories, and can provide indirect evidence that these genes are essential for blood-stage development.
Chakarov, Nayden; Linke, Burkhard; Boerner, Martina; Goesmann, Alexander; Krüger, Oliver; Hoffman, Joseph I
Parasite transmission strategies strongly impact host-parasite co-evolution and virulence. However, studies of vector-borne parasites such as avian malaria have neglected the potential effects of host relatedness on the exchange of parasites. To test whether extended parental care in the presence of vectors increases the probability of transmission from parents to offspring, we used high-throughput sequencing to develop microsatellites for malaria-like Leucocytozoon parasites of a wild raptor population. We show that host siblings carry genetically more similar parasites than unrelated chicks both within and across years. Moreover, chicks of mothers of the same plumage morph carried more similar parasites than nestlings whose mothers were of different morphs, consistent with matrilineal transmission of morph-specific parasite strains. Ours is the first evidence of an association between host relatedness and parasite genetic similarity, consistent with vector-mediated parent-to-offspring transmission. The conditions for such 'quasi-vertical' transmission may be common and could suppress the evolution of pathogen virulence.
Hallett, Rachel L; Sutherland, Colin J; Alexander, Neal; Ord, Rosalynn; Jawara, Musa; Drakeley, Chris J; Pinder, Margaret; Walraven, Gijs; Targett, Geoffrey A T; Alloueche, Ali
Malaria parasites carrying genes conferring resistance to antimalarials are thought to have a selective advantage which leads to higher rates of transmissibility from the drug-treated host. This is a likely mechanism for the increasing prevalence of parasites with resistance to chloroquine (CQ) and sulfadoxine-pyrimethamine in sub-Saharan Africa. Combination therapy is the key strategy being implemented to reduce the impact of resistance, but its effect on the transmission of genetically resistant parasites from treated patients to mosquito vectors has not been measured directly. In a trial comparing CQ monotherapy to the combination CQ plus artesunate (AS) in Gambian children with uncomplicated falciparum malaria, we measured transmissibility by feeding Anopheles gambiae mosquitoes with blood from 43 gametocyte-positive patients through a membrane. In the CQ-treated group, gametocytes from patients carrying parasites with the CQ resistance-associated allele pfcrt-76T prior to treatment produced infected mosquitoes with 38 times higher Plasmodium falciparum oocyst burdens than mosquitoes fed on gametocytes from patients infected with sensitive parasites (P < 0.001). Gametocytes from parasites carrying the resistance-associated allele pfmdr1-86Y produced 14-fold higher oocyst burdens than gametocytes from patients infected with sensitive parasites (P = 0.011). However, parasites carrying either of these resistance-associated alleles pretreatment were not associated with higher mosquito oocyst burdens in the CQ-AS-treated group. Thus, combination therapy overcomes the transmission advantage enjoyed by drug-resistant parasites.
Osgood, S M; Schall, J J
Sex ratio theory posits that the adaptive proportion of male to female gametocytes of a malaria parasite within the vertebrate host depends on the degree of inbreeding within the vector. Gametocyte sex ratio could be phenotypically flexible, being altered based on the infection's clonal diversity, and thus likely inbreeding. This idea was tested by manipulating the clonal diversity of infections of Plasmodium mexicanum in its lizard host, Sceloporus occidentalis. Naive lizards were inoculated with infected blood from a single donor or 3 pooled donors. Donors varied in their gametocyte sex ratios (17-46%, male), and sex ratio theory allowed estimation of the clonal diversity within donor and recipient infections. Phenotypic plasticity would produce a correlation between donor and recipient infections for infections initiated from a single donor, and a less female-biased gametocyte sex ratio in recipients that received a mixed blood inoculum (with predicted higher clonal diversity) than recipients receiving blood from a single donor. Neither pattern was observed. Gametocyte sex ratio of most infections ranged from 35 to 42% male, expected if clonal diversity was high for all infections. Alternative explanations are suggested for the observed variation of gametocyte sex ratio among P. mexicanum infections.
Hallée, Stéphanie; Richard, Dave
Invasion of a red blood cell by Plasmodium falciparum merozoites is an essential step in the malaria lifecycle. Several of the proteins involved in this process are stored in the apical complex of the merozoite, a structure containing secretory organelles that are released at specific times during invasion. The molecular players involved in erythrocyte invasion thus represent potential key targets for both therapeutic and vaccine-based strategies to block parasite development. In our quest to identify and characterize new effectors of invasion, we investigated the P. falciparum homologue of a P. berghei protein putatively localized to the rhoptries, the Putative rhoptry protein 2 (PbPRP2). We show that in P. falciparum, the protein colocalizes extensively with the Golgi apparatus across the asexual erythrocytic cycle. Furthermore, imaging of merozoites caught at different times during invasion show that PfPRP2 is not secreted during the process instead staying associated with the Golgi apparatus. Our evidence therefore suggests that PfPRP2 is a Golgi protein and that it is likely not a direct effector in the process of merozoite invasion.
Hallée, Stéphanie; Richard, Dave
Invasion of a red blood cell by Plasmodium falciparum merozoites is an essential step in the malaria lifecycle. Several of the proteins involved in this process are stored in the apical complex of the merozoite, a structure containing secretory organelles that are released at specific times during invasion. The molecular players involved in erythrocyte invasion thus represent potential key targets for both therapeutic and vaccine-based strategies to block parasite development. In our quest to identify and characterize new effectors of invasion, we investigated the P. falciparum homologue of a P. berghei protein putatively localized to the rhoptries, the Putative rhoptry protein 2 (PbPRP2). We show that in P. falciparum, the protein colocalizes extensively with the Golgi apparatus across the asexual erythrocytic cycle. Furthermore, imaging of merozoites caught at different times during invasion show that PfPRP2 is not secreted during the process instead staying associated with the Golgi apparatus. Our evidence therefore suggests that PfPRP2 is a Golgi protein and that it is likely not a direct effector in the process of merozoite invasion. PMID:26375591
Kapishnikov, S; Leiserowitz, L; Yang, Y; Cloetens, P; Pereiro, E; Awamu Ndonglack, F; Matuschewski, K; Als-Nielsen, J
Red blood cells infected by the malaria parasite Plasmodium falciparum are correlatively imaged by tomography using soft X-rays as well as by scanning hard nano-X-ray beam to obtain fluorescence maps of various elements such as S and Fe. In this way one can deduce the amount of Fe bound either in hemoglobin or in hemozoin crystals in the digestive vacuole of the malaria parasite as well as determine the hemoglobin concentrations in the cytosols of the red blood cell and of the parasite. Fluorescence map of K shows that in the parasite's schizont stage the K concentration in the red blood cell cytosol is diminished by a factor of seven relative to a pristine red blood cell but the total amount of K in the infected red blood cell is the same as in the pristine red blood cell.
Malleret, Benoît; Claser, Carla; Ong, Alice Soh Meoy; Suwanarusk, Rossarin; Sriprawat, Kanlaya; Howland, Shanshan Wu; Russell, Bruce; Nosten, Francois; Rénia, Laurent
Microscopic examination of Giemsa-stained thin blood smears remains the gold standard method used to quantify and stage malaria parasites. However, this technique is tedious, and requires trained microscopists. We have developed a fast and simple flow cytometry method to quantify and stage, various malaria parasites in red blood cells in whole blood or in vitro cultured Plasmodium falciparum. The parasites were stained with dihydroethidium and Hoechst 33342 or SYBR Green I and leukocytes were identified with an antibody against CD45. Depending on the DNA stains used, samples were analyzed using different models of flow cytometers. This protocol, which does not require any washing steps, allows infected red blood cells to be distinguished from leukocytes, as well as allowing non-infected reticulocytes and normocytes to be identified. It also allows assessing the proportion of parasites at different developmental stages. Lastly, we demonstrate how this technique can be applied to antimalarial drug testing.
Arisue, Nobuko; Hashimoto, Tetsuo; Mitsui, Hideya; Palacpac, Nirianne M Q; Kaneko, Akira; Kawai, Satoru; Hasegawa, Masami; Tanabe, Kazuyuki; Horii, Toshihiro
Apicoplast, a nonphotosynthetic plastid derived from secondary symbiotic origin, is essential for the survival of malaria parasites of the genus Plasmodium. Elucidation of the evolution of the apicoplast genome in Plasmodium species is important to better understand the functions of the organelle. However, the complete apicoplast genome is available for only the most virulent human malaria parasite, Plasmodium falciparum. Here, we obtained the near-complete apicoplast genome sequences from eight Plasmodium species that infect a wide variety of vertebrate hosts and performed structural and phylogenetic analyses. We found that gene repertoire, gene arrangement, and other structural attributes were highly conserved. Phylogenetic reconstruction using 30 protein-coding genes of the apicoplast genome inferred, for the first time, a close relationship between P. ovale and rodent parasites. This close relatedness was robustly supported using multiple evolutionary assumptions and models. The finding suggests that an ancestral host switch occurred between rodent and human Plasmodium parasites.
Kaddumukasa, Mark; Lwanira, Catherine; Lugaajju, Allan; Katabira, Elly; Persson, Kristina E. M.; Wahlgren, Mats; Kironde, Fred
Introduction There is no approved vaccine for malaria, and precisely how human antibody responses to malaria parasite components and potential vaccine molecules are developed and maintained remains poorly defined. In this study, antibody anamnestic or memory response elicited by a single episode of P. falciparum infection was investigated. Methods This study involved 362 malaria patients aged between 6 months to 60 years, of whom 19% were early-diagnosed people living with HIV/AIDS (PLWHA). On the day malaria was diagnosed and 42 days later, blood specimens were collected. Parasite density, CD4+ cells, and antibodies specific to synthetic peptides representing antigenic regions of the P. falciparum proteins GLURP, MSP3 and HRPII were measured. Results On the day of malaria diagnosis, Immunoglobulin (IgG) antibodies against GLURP, MSP3 and HRP II peptides were present in the blood of 75%, 41% and 60% of patients, respectively. 42 days later, the majority of patients had boosted their serum IgG antibody more than 1.2 fold. The increase in level of IgG antibody against the peptides was not affected by parasite density at diagnosis. The median CD4+ cell counts of PLWHAs and HIV negative individuals were not statistically different, and median post-infection increases in anti-peptide IgG were similar in both groups of patients. Conclusion In the majority (70%) of individuals, an infection of P. falciparum elicits at least 20% increase in level of anti-parasite IgG. This boost in anti-P. falciparum IgG is not affected by parasite density on the day of malaria diagnosis, or by HIV status. PMID:25906165
Zhang, Hong-Wei; Li, San-Jin; Hu, Tao; Yu, Yong-Min; Yang, Cheng-Yun; Zhou, Rui-Min; Liu, Ying; Tang, Jing; Wang, Jing-Jing; Wang, Xiu-Yun; Sun, Yong-Xiang; Feng, Zhan-Chun; Xu, Bian-Li
The spleen plays a pivotal role in the rapid clearance of parasitized red blood cells in patients with falciparum malaria after artemisinin treatment. Prolonged parasite clearance can be found in patients who have had a splenectomy, or those with hemoglobin abnormalities and/or reduced immunity, which are all distinguishable from artemisinin resistance. This paper reports on a case of prolonged parasite clearance in a Chinese splenectomized patient with falciparum malaria imported from Nigeria. A 35-year-old Chinese male suffered 2 days of febrile illness after returning to Zhumadian city of Henan province from Nigeria on October 1, 2014. The main symptoms were febrile, including the highest axillary temperature of 40 °C, headache, and chills. A peripheral blood smear showed parasitemia (53 913 asexual parasites/μl) of Plasmodium falciparum. The patient had not used any chemoprophylaxis against malaria in Nigeria when he worked there as a construction worker between 2009 and 2014. The patient had three episodes of malaria in Nigeria and had a splenectomy due to a traffic accident 8 years ago from the time he was admitted to hospital. The patient was orally administrated a total of 320 mg/2.56 g dihydroartemisinin-piperaquine for 2 days and intravenously administrated a total of 3 000 mg artesunate for 18 days. The axillary temperature of the patient ranged between 37.0 and 37.7 °C from Day 0 to Day 3, and blood microscopy revealed falciparum malaria parasitemia (26 674 asexual parasites/μl) on Day 3. The patient was afebrile on Day 4, falciparum malaria parasitemia was continuously present and then gradually decreased on the next days, and was negative on Day 21. The patient was cured and left hospital on Day 24 after no plasmodium falciparum was found in the blood on Day 21 to Day 23. No mutation was found in the K13 propeller gene when compared with the PF3D7_1343700 K13 propeller gene reference sequence. This is the first reported case in China of
Barber, Bridget E.; William, Timothy; Grigg, Matthew J.; Parameswaran, Uma; Piera, Kim A.; Price, Ric N.; Yeo, Tsin W.; Anstey, Nicholas M.
Plasmodium vivax can cause severe malaria, however its pathogenesis is poorly understood. In contrast to P. falciparum, circulating vivax parasitemia is low, with minimal apparent sequestration in endothelium-lined microvasculature, and pathogenesis thought unrelated to parasite biomass. However, the relationships between vivax disease-severity and total parasite biomass, endothelial autocrine activation and microvascular dysfunction are unknown. We measured circulating parasitemia and markers of total parasite biomass (plasma parasite lactate dehydrogenase [pLDH] and PvLDH) in adults with severe (n = 9) and non-severe (n = 53) vivax malaria, and examined relationships with disease-severity, endothelial activation, and microvascular function. Healthy controls and adults with non-severe and severe falciparum malaria were enrolled for comparison. Median peripheral parasitemia, PvLDH and pLDH were 2.4-fold, 3.7-fold and 6.9-fold higher in severe compared to non-severe vivax malaria (p = 0.02, p = 0.02 and p = 0.015, respectively), suggesting that, as in falciparum malaria, peripheral P. vivax parasitemia underestimates total parasite biomass, particularly in severe disease. P. vivax schizonts were under-represented in peripheral blood. Severe vivax malaria was associated with increased angiopoietin-2 and impaired microvascular reactivity. Peripheral vivax parasitemia correlated with endothelial activation (angiopoietin-2, von-Willebrand-Factor [VWF], E-selectin), whereas markers of total vivax biomass correlated only with systemic inflammation (IL-6, IL-10). Activity of the VWF-cleaving-protease, ADAMTS13, was deficient in proportion to endothelial activation, IL-6, thrombocytopenia and vivax disease-severity, and associated with impaired microvascular reactivity in severe disease. Impaired microvascular reactivity correlated with lactate in severe vivax malaria. Findings suggest that tissue accumulation of P. vivax may occur, with the hidden
Bell, Andrew S; Huijben, Silvie; Paaijmans, Krijn P; Sim, Derek G; Chan, Brian H K; Nelson, William A; Read, Andrew F
The evolution of drug resistant Plasmodium parasites is a major challenge to effective malaria control. In theory, competitive interactions between sensitive parasites and resistant parasites within infections are a major determinant of the rate at which parasite evolution undermines drug efficacy. Competitive suppression of resistant parasites in untreated hosts slows the spread of resistance; competitive release following treatment enhances it. Here we report that for the murine model Plasmodium chabaudi, co-infection with drug-sensitive parasites can prevent the transmission of initially rare resistant parasites to mosquitoes. Removal of drug-sensitive parasites following chemotherapy enabled resistant parasites to transmit to mosquitoes as successfully as sensitive parasites in the absence of treatment. We also show that the genetic composition of gametocyte populations in host venous blood accurately reflects the genetic composition of gametocytes taken up by mosquitoes. Our data demonstrate that, at least for this mouse model, aggressive chemotherapy leads to very effective transmission of highly resistant parasites that are present in an infection, the very parasites which undermine the long term efficacy of front-line drugs.
Cervantes, Serena; Bunnik, Evelien M; Saraf, Anita; Conner, Christopher M; Escalante, Aster; Sardiu, Mihaela E; Ponts, Nadia; Prudhomme, Jacques; Florens, Laurence; Le Roch, Karine G
Autophagy is a catabolic pathway typically induced by nutrient starvation to recycle amino acids, but can also function in removing damaged organelles. In addition, this pathway plays a key role in eukaryotic development. To date, not much is known about the role of autophagy in apicomplexan parasites and more specifically in the human malaria parasite Plasmodium falciparum. Comparative genomic analysis has uncovered some, but not all, orthologs of autophagy-related (ATG) genes in the malaria parasite genome. Here, using a genome-wide in silico analysis, we confirmed that ATG genes whose products are required for vesicle expansion and completion are present, while genes involved in induction of autophagy and cargo packaging are mostly absent. We subsequently focused on the molecular and cellular function of P. falciparum ATG8 (PfATG8), an autophagosome membrane marker and key component of the autophagy pathway, throughout the parasite asexual and sexual erythrocytic stages. In this context, we showed that PfATG8 has a distinct and atypical role in parasite development. PfATG8 localized in the apicoplast and in vesicles throughout the cytosol during parasite development. Immunofluorescence assays of PfATG8 in apicoplast-minus parasites suggest that PfATG8 is involved in apicoplast biogenesis. Furthermore, treatment of parasite cultures with bafilomycin A 1 and chloroquine, both lysosomotropic agents that inhibit autophagosome and lysosome fusion, resulted in dramatic morphological changes of the apicoplast, and parasite death. Furthermore, deep proteomic analysis of components associated with PfATG8 indicated that it may possibly be involved in ribophagy and piecemeal microautophagy of the nucleus. Collectively, our data revealed the importance and specificity of the autophagy pathway in the malaria parasite and offer potential novel therapeutic strategies.
Ch'ng, Jun-Hong; Yeo, Su-Ping; Shyong-Wei Tan, Kevin
The protozoan pathogens responsible for malaria are from the Plasmodium genus, with Plasmodium falciparum and Plasmodium vivax accounting for almost all clinical infections. With recent estimates of mortality exceeding 800,000 annually, malaria continues to take a terrible toll on lives and the early promises of medicine to eradicate the disease have yet to approach realization, in part due to the spread of drug resistant parasites. Recent reports of artemisinin-resistance have prompted renewed efforts to identify novel therapeutic options, and one such pathway being considered for antimalarial exploit is the parasite's programmed cell death (PCD) pathway. In this mini-review, we will discuss the roles of the plasmodium mitochondria in cell death and as a target of antimalarial compounds, taking into account recent data suggesting that PCD pathways involving the mitochondria may be attractive antimalarial targets.
Pakpour, Nazzy; Cheung, Kong Wai; Luckhart, Shirley
More than half of the world's population is at risk of malaria and simultaneously, many malaria-endemic regions are facing dramatic increases in the prevalence of type 2 diabetes. Studies in murine malaria models have examined the impact of malaria infection on type 2 diabetes pathology, it remains unclear how this chronic metabolic disorder impacts the transmission of malaria. In this report, the ability type 2 diabetic rodents infected with malaria to transmit parasites to Anopheles stephensi mosquitoes is quantified. The infection prevalence and intensity of An. stephensi mosquitoes that fed upon control or type 2 diabetic C57BL/6 db/db mice infected with either lethal Plasmodium berghei NK65 or non-lethal Plasmodium yoelii 17XNL murine malaria strains were determined. Daily parasitaemias were also recorded. A higher percentage of mosquitoes (87.5 vs 61.5 % for P. yoelii and 76.9 vs 50 % for P. berghei) became infected following blood feeding on Plasmodium-infected type 2 diabetic mice compared to mosquitoes that fed on infected control animals, despite no significant differences in circulating gametocyte levels. These results suggest that type 2 diabetic mice infected with malaria are more efficient at infecting mosquitoes, raising the question of whether a similar synergy exists in humans.
Gabrieli, Paolo; Buckee, Caroline O.; Catteruccia, Flaminia
The control of mosquito populations with insecticide treated bed nets and indoor residual sprays remains the cornerstone of malaria reduction and elimination programs. In light of widespread insecticide resistance in mosquitoes, however, alternative strategies for reducing transmission by the mosquito vector are urgently needed, including the identification of safe compounds that affect vectorial capacity via mechanisms that differ from fast-acting insecticides. Here, we show that compounds targeting steroid hormone signaling disrupt multiple biological processes that are key to the ability of mosquitoes to transmit malaria. When an agonist of the steroid hormone 20-hydroxyecdysone (20E) is applied to Anopheles gambiae females, which are the dominant malaria mosquito vector in Sub Saharan Africa, it substantially shortens lifespan, prevents insemination and egg production, and significantly blocks Plasmodium falciparum development, three components that are crucial to malaria transmission. Modeling the impact of these effects on Anopheles population dynamics and Plasmodium transmission predicts that disrupting steroid hormone signaling using 20E agonists would affect malaria transmission to a similar extent as insecticides. Manipulating 20E pathways therefore provides a powerful new approach to tackle malaria transmission by the mosquito vector, particularly in areas affected by the spread of insecticide resistance. PMID:27977810
Pospich, Sabrina; Kumpula, Esa-Pekka; von der Ecken, Julian; Vahokoski, Juha; Kursula, Inari; Raunser, Stefan
During their life cycle, apicomplexan parasites, such as the malaria parasite Plasmodium falciparum, use actomyosin-driven gliding motility to move and invade host cells. For this process, actin filament length and stability are temporally and spatially controlled. In contrast to canonical actin, P. falciparum actin 1 (PfAct1) does not readily polymerize into long, stable filaments. The structural basis of filament instability, which plays a pivotal role in host cell invasion, and thus infectivity, is poorly understood, largely because high-resolution structures of PfAct1 filaments were missing. Here, we report the near-atomic structure of jasplakinolide (JAS)-stabilized PfAct1 filaments determined by electron cryomicroscopy. The general filament architecture is similar to that of mammalian F-actin. The high resolution of the structure allowed us to identify small but important differences at inter- and intrastrand contact sites, explaining the inherent instability of apicomplexan actin filaments. JAS binds at regular intervals inside the filament to three adjacent actin subunits, reinforcing filament stability by hydrophobic interactions. Our study reveals the high-resolution structure of a small molecule bound to F-actin, highlighting the potential of electron cryomicroscopy for structure-based drug design. Furthermore, our work serves as a strong foundation for understanding the structural design and evolution of actin filaments and their function in motility and host cell invasion of apicomplexan parasites.
Kaushansky, A; Metzger, P G; Douglass, A N; Mikolajczak, S A; Lakshmanan, V; Kain, H S; Kappe, S HI
Intracellular eukaryotic parasites and their host cells constitute complex, coevolved cellular interaction systems that frequently cause disease. Among them, Plasmodium parasites cause a significant health burden in humans, killing up to one million people annually. To succeed in the mammalian host after transmission by mosquitoes, Plasmodium parasites must complete intracellular replication within hepatocytes and then release new infectious forms into the blood. Using Plasmodium yoelii rodent malaria parasites, we show that some liver stage (LS)-infected hepatocytes undergo apoptosis without external triggers, but the majority of infected cells do not, and can also resist Fas-mediated apoptosis. In contrast, apoptosis is dramatically increased in hepatocytes infected with attenuated parasites. Furthermore, we find that blocking total or mitochondria-initiated host cell apoptosis increases LS parasite burden in mice, suggesting that an anti-apoptotic host environment fosters parasite survival. Strikingly, although LS infection confers strong resistance to extrinsic host hepatocyte apoptosis, infected hepatocytes lose their ability to resist apoptosis when anti-apoptotic mitochondrial proteins are inhibited. This is demonstrated by our finding that B-cell lymphoma 2 family inhibitors preferentially induce apoptosis in LS-infected hepatocytes and significantly reduce LS parasite burden in mice. Thus, targeting critical points of susceptibility in the LS-infected host cell might provide new avenues for malaria prophylaxis. PMID:23928701
Sánchez-Arcila, Juan Camilo; Perce-da-Silva, Daiana de Souza; Vasconcelos, Mariana Pinheiro Alves; Rodrigues-da-Silva, Rodrigo Nunes; Pereira, Virginia Araujo; Aprígio, Cesarino Junior Lima; Lima, Cleoni Alves Mendes; Fonseca e Fonseca, Bruna de Paula; Banic, Dalma Maria; Lima-Junior, Josué da Costa; Oliveira-Ferreira, Joseli
In Brazil, malaria is prevalent in the Amazon region and these regions coincide with high prevalence of intestinal parasites but few studies explore the interaction between malaria and other parasites. Therefore, the present study evaluates changes in cytokine, chemokine, C-reactive protein, and nitric oxide (NO) concentrations in 264 individuals, comparing plasma from infected individuals with concurrent malaria and intestinal parasites to individuals with either malaria infection alone and uninfected. In the studied population 24% of the individuals were infected with Plasmodium and 18% coinfected with intestinal parasites. Protozoan parasites comprised the bulk of the intestinal parasites infections and subjects infected with intestinal parasites were more likely to have malaria. The use of principal component analysis and cluster analysis associated increased levels of IL-6, TNF-α, IL-10, and CRP and low levels of IL-17A predominantly with individuals with malaria alone and coinfected individuals. In contrast, low levels of almost all inflammatory mediators were associated predominantly with individuals uninfected while increased levels of IL-17A were associated predominantly with individuals with intestinal parasites only. In conclusion, our data suggest that, in our population, the infection with intestinal parasites (mainly protozoan) does not modify the pattern of cytokine production in individuals infected with P. falciparum and P. vivax.
Perce-da-Silva, Daiana de Souza; Lima-Junior, Josué da Costa
In Brazil, malaria is prevalent in the Amazon region and these regions coincide with high prevalence of intestinal parasites but few studies explore the interaction between malaria and other parasites. Therefore, the present study evaluates changes in cytokine, chemokine, C-reactive protein, and nitric oxide (NO) concentrations in 264 individuals, comparing plasma from infected individuals with concurrent malaria and intestinal parasites to individuals with either malaria infection alone and uninfected. In the studied population 24% of the individuals were infected with Plasmodium and 18% coinfected with intestinal parasites. Protozoan parasites comprised the bulk of the intestinal parasites infections and subjects infected with intestinal parasites were more likely to have malaria. The use of principal component analysis and cluster analysis associated increased levels of IL-6, TNF-α, IL-10, and CRP and low levels of IL-17A predominantly with individuals with malaria alone and coinfected individuals. In contrast, low levels of almost all inflammatory mediators were associated predominantly with individuals uninfected while increased levels of IL-17A were associated predominantly with individuals with intestinal parasites only. In conclusion, our data suggest that, in our population, the infection with intestinal parasites (mainly protozoan) does not modify the pattern of cytokine production in individuals infected with P. falciparum and P. vivax. PMID:25309052
Background In malaria endemic areas, individuals are frequently asymptomatic and may be undetected by conventional microscopy or newer, rapid diagnostic tests. Molecular techniques allow a more accurate assessment of this asymptomatic parasite burden, the extent of which is important for malaria control. This study examines the relative prevalence of sub-microscopic level parasite carriage and clonal complexity of infections (multiplicity of infection) over a range of endemicities in a region of north-eastern Tanzania where altitude is an established proxy of malaria transmission. The PCR prevalence was then compared against other measures of transmission intensity collected in the same area. Methods This study used 1,121 blood samples collected from a previously conducted cross-sectional malario-metric survey during the short rainy season in 2001 from 13 villages (three at < 600 m, four at 600-1,200 m and six at > 1,200 m in altitude above sea level). Samples were analysed by PCR for carriage of parasites and multiplicity of infection. These data were compared with other measures of transmission intensity collected from the same area. Results Parasite prevalence was 34.7% by PCR and 13.6% by microscopy; a 2.5-fold difference in line with other recent observations. This fold difference was relatively consistent at the different altitude bands despite a marked decrease in parasite prevalence with altitude: < 600 m 70.9 vs 28.6, 600-1,200 m 35.5 vs 9.9, > 1,200 m 15.8 vs 5.9. The difference between parasite prevalence by PCR was 3.2 in individuals aged between 15 and 45 years (34.5 vs 10.9) compared with 2.5 in those aged 1-5 (34.0 vs 13.5) though this was not statistically significant. Multiplicity of infection (MOI) ranged from 1.2 to 3.7 and was positively associated with parasite prevalence assessed by both PCR and microscopy. There was no association of MOI and age. Village level PCR parasite prevalence was strongly correlated with altitude, sero-conversion rate
Lambrechts, Louis; Chavatte, Jean-Marc; Snounou, Georges; Koella, Jacob C
The genetic basis of a host's resistance to parasites has important epidemiological and evolutionary consequences. Understanding this genetic basis can be complicated by non-genetic factors, such as environmental quality, which may influence the expression of genetic resistance and profoundly alter patterns of disease and the host's response to selection. In particular, understanding the environmental influence on the genetic resistance of mosquitoes to malaria gives valuable knowledge concerning the use of malaria-resistant transgenic mosquitoes as a measure of malaria control. We made a step towards this understanding by challenging eight isofemale lines of the malaria vector Anopheles stephensi with the rodent malaria parasite Plasmodium yoelii yoelii and by feeding the mosquitoes with different concentrations of glucose. The isofemale lines differed in infection loads (the numbers of oocysts), corroborating earlier studies showing a genetic basis of resistance. In contrast, the proportion of infected mosquitoes did not differ among lines, suggesting that the genetic component underlying infection load differs from the genetic component underlying infection rate. In addition, the mean infection load and, in particular, its heritable variation in mosquitoes depended on the concentration of glucose, which suggests that the environment affects the expression and the evolution of the mosquitoes' resistance in nature. We found no evidence of genotype-by-environment interactions, i.e. the lines responded similarly to environmental variation. Overall, these results indicate that environmental variation can significantly reduce the importance of genes in determining the resistance of mosquitoes to malaria infection. PMID:16777744
Carucci, D J
Infection with any of the four species of Plasmodium single cell parasites that infects humans causes the clinical disease, malaria. Of these, it is Plasmodium falciparum that is responsible for the majority of the 1.5-2.3 million deaths due to this disease each year. Worldwide there are between 300-500 million cases of malaria annually. To date there is no licensed vaccine and resistance to most of the available drugs used to prevent and/or treat malaria is spreading. There is therefore an urgent need to develop new and effective drugs and vaccines against this devastating parasite. We have outlined a strategy using a combination of DNA-based vaccines and the data derived from the soon-to-be completed P. falciparum genome and the genomes of other species of Plasmodium to develop new vaccines against malaria. Much of the technology that we are developing for vaccine target identification is directly applicable to the identification of potential targets for drug discovery. The publicly available genome sequence data also provides a means for researchers whose focus may not be primarily malaria to leverage their research on cancer, yeast biology and other research areas to the biological problems of malaria.
Abkallo, Hussein M; Liu, Weimin; Hokama, Sarina; Ferreira, Pedro E; Nakazawa, Shusuke; Maeno, Yoshimasa; Quang, Nguyen T; Kobayashi, Nobuyuki; Kaneko, Osamu; Huffman, Michael A; Kawai, Satoru; Marchand, Ron P; Carter, Richard; Hahn, Beatrice H; Culleton, Richard
Following the bite of an infective mosquito, malaria parasites first invade the liver where they develop and replicate for a number of days before being released into the bloodstream where they invade red blood cells and cause disease. The biology of the liver stages of malaria parasites is relatively poorly understood due to the inaccessibility of the parasites to sampling during this phase of their life cycle. Here we report the detection in blood and faecal samples of malaria parasite DNA throughout their development in the livers of mice and before the parasites begin their growth in the blood circulation. It is shown that parasite DNA derived from pre-erythrocytic stage parasites reaches the faeces via the bile. We then show that different primate malaria species can be detected by PCR in blood and faecal samples from naturally infected captive macaque monkeys. These results demonstrate that pre-erythrocytic parasites can be detected and quantified in experimentally infected animals. Furthermore, these results have important implications for both molecular epidemiology and phylogenetics of malaria parasites. In the former case, individuals who are malaria parasite negative by microscopy, but PCR positive for parasite DNA in their blood, are considered to be "sub-microscopic" blood stage parasite carriers. We now propose that PCR positivity is not necessarily an indicator of the presence of blood stage parasites, as the DNA could derive from pre-erythrocytic parasites. Similarly, in the case of molecular phylogenetics based on DNA sequences alone, we argue that DNA amplified from blood or faeces does not necessarily come from a parasite species that infects the red blood cells of that particular host.
Njunda, Anna Longdoh; Fon, Shuri Ghasarah; Assob, Jules Clement Nguedia; Nsagha, Dickson Shey; Kwenti, Tayong Dizzle Bita; Kwenti, Tebit Emmanuel
The purpose of this study was to determine the prevalence of coinfection with malaria and intestinal parasites, as well as to determine its association with anaemia in children aged 10 years and below in Muyuka, Cameroon. This was a cross-sectional study. Participants were febrile children who were admitted to the Muyuka district hospital between April and October 2012. Blood and stool samples were collected from those participants who gave consent to take part in the study. Haemoglobin concentration (Hb) and complete blood count (CBC) were performed using an automated haematology analyser (Mindray®, BC-2800). Giemsa-stained blood film was examined to detect malaria parasites, while the formol-ether concentration technique was used to detect intestinal parasitic infections (IPIs). The Pearson's chi-square, Student's T-test and correlation analysis were all performed as part of the statistical analyses. Four hundred and eleven (411) children successfully took part in this study. The prevalence of malaria, IPIs, malaria and IPI coinfection, and anaemia observed were 98.5 %, 11.9 %, 11.9 % and 44.8 %, respectively. Anaemia and IPIs were significantly associated with age; anaemia was more prevalent in children under five years of age (p = 0.000), whereas IPIs were more prevalent in children aged between five and 10 years (p = 0.006). The parasite species isolated included Ascaris lumbricoides (36 [73.5 %]), Entamoeba histolytica/dispar (9 [18.4 %]) and hookworm (4 [8.2 %]). The mean Hb observed was 10.64 g/dl (±1.82). A significant negative correlation was observed between malaria parasite density and Hb. There was no significant difference in the prevalence of anaemia among children infected with malaria, IPIs, or malaria and IPI coinfection, or among non-infected children. Similarly, the mean Hb did not differ among infected and non-infected children. This study showed that malaria and IPIs still constitute a major public health problem in the study area despite a
Martinsen, Ellen S.; McInerney, Nancy; Brightman, Heidi; Ferebee, Ken; Walsh, Tim; McShea, William J.; Forrester, Tavis D.; Ware, Lisa; Joyner, Priscilla H.; Perkins, Susan L.; Latch, Emily K.; Yabsley, Michael J.; Schall, Joseph J.; Fleischer, Robert C.
Malaria parasites of the genus Plasmodium are diverse in mammal hosts, infecting five mammalian orders in the Old World, but were long considered absent from the diverse deer family (Cervidae) and from New World mammals. There was a description of a Plasmodium parasite infecting a single splenectomized white-tailed deer (WTD; Odocoileus virginianus) in 1967 but none have been reported since, which has proven a challenge to our understanding of malaria parasite biogeography. Using both microscopy and polymerase chain reaction, we screened a large sample of native and captive ungulate species from across the United States for malaria parasites. We found a surprisingly high prevalence (up to 25%) and extremely low parasitemia of Plasmodium parasites in WTD throughout the eastern United States. We did not detect infections in the other ungulate species nor in western WTD. We also isolated the parasites from the mosquito Anopheles punctipennis. Morphologically, the parasites resemble the parasite described in 1967, Plasmodium odocoilei. Our analysis of the cytochrome b gene revealed two divergent Plasmodium clades in WTD representative of species that likely diverged 2.3 to 6 million years ago, concurrent with the arrival of the WTD ancestor into North America across Beringia. Multigene phylogenetic analysis placed these clades within the larger malaria parasite clade. We document Plasmodium parasites to be common in WTD, endemic to the New World, and as the only known malaria parasites from deer (Cervidae). These findings reshape our knowledge of the phylogeography of the malaria parasites and suggest that other mammal taxa may harbor infection by endemic and occult malaria parasites. PMID:26989785
Martinsen, Ellen S; McInerney, Nancy; Brightman, Heidi; Ferebee, Ken; Walsh, Tim; McShea, William J; Forrester, Tavis D; Ware, Lisa; Joyner, Priscilla H; Perkins, Susan L; Latch, Emily K; Yabsley, Michael J; Schall, Joseph J; Fleischer, Robert C
Malaria parasites of the genus Plasmodium are diverse in mammal hosts, infecting five mammalian orders in the Old World, but were long considered absent from the diverse deer family (Cervidae) and from New World mammals. There was a description of a Plasmodium parasite infecting a single splenectomized white-tailed deer (WTD; Odocoileus virginianus) in 1967 but none have been reported since, which has proven a challenge to our understanding of malaria parasite biogeography. Using both microscopy and polymerase chain reaction, we screened a large sample of native and captive ungulate species from across the United States for malaria parasites. We found a surprisingly high prevalence (up to 25%) and extremely low parasitemia of Plasmodium parasites in WTD throughout the eastern United States. We did not detect infections in the other ungulate species nor in western WTD. We also isolated the parasites from the mosquito Anopheles punctipennis. Morphologically, the parasites resemble the parasite described in 1967, Plasmodium odocoilei. Our analysis of the cytochrome b gene revealed two divergent Plasmodium clades in WTD representative of species that likely diverged 2.3 to 6 million years ago, concurrent with the arrival of the WTD ancestor into North America across Beringia. Multigene phylogenetic analysis placed these clades within the larger malaria parasite clade. We document Plasmodium parasites to be common in WTD, endemic to the New World, and as the only known malaria parasites from deer (Cervidae). These findings reshape our knowledge of the phylogeography of the malaria parasites and suggest that other mammal taxa may harbor infection by endemic and occult malaria parasites.
Dhiman, Sunil; Bhola, Rakesh Kumar; Goswami, Diganta; Rabha, Bipul; Kumar, Dinesh; Baruah, Indra; Singh, Lokendra
This study was carried out to determine the human host preference and presence of Plasmodium parasite in field collected Anopheles mosquitoes among four villages around a military cantonment located in malaria endemic Sonitpur district of Assam, India. Encountered malaria vector mosquitoes were identified and tested for host preference and Plasmodium presence using PCR method. Human host preference was detected using simple PCR, whereas vectorial status for Plasmodium parasite was confirmed using first round PCR with genus specific primers and thereafter nested PCR with three Plasmodium species specific primers. Out of 1874 blood fed vector mosquitoes collected, 187 (10%) were processed for PCR, which revealed that 40·6% had fed on human blood; 9·2% of human blood fed mosquito were harbouring Plasmodium parasites, 71·4% of which were confirmed to Plasmodium falciparum. In addition to An. minimus, An. annularis and An. culicifacies were also found positive for malaria parasites. The present study exhibits the human feeding tendency of Anopheles vectors highlighting their malaria parasite transmission potential. The present study may serve as a model for understanding the human host preference of malaria vectors and detection of malaria parasite inside the anopheline vector mosquitoes in order to update their vectorial status for estimating the possible role of these mosquitoes in malaria transmission. The study has used PCR method and suggests that PCR-based method should be used in this entire malarious region to correctly report the vectorial position of different malaria vectors.
In humans, infections contribute highly to mortality and morbidity rates worldwide. Malaria tropica is one of the major infectious diseases globally and is caused by the protozoan parasite Plasmodium falciparum. Plasmodia have accompanied human beings since the emergence of humankind. Due to its pathogenicity, malaria is a powerful selective force on the human genome. Genetic epidemiology approaches such as family and twin studies, candidate gene studies, and disease-association studies have identified a number of genes that mediate relative protection against the severest forms of the disease. New molecular approaches, including genome-wide association studies, have recently been performed to expand our knowledge on the functional effect of human variation in malaria. For the future, a systematic determination of gene-dosage effects and expression profiles of protective genes might unveil the functional impact of structural alterations in these genes on either side of the host-parasite interaction. PMID:19725943
Urbán, Patricia; Ranucci, Elisabetta; Fernàndez-Busquets, Xavier
Malaria is arguably one of the main medical concerns worldwide because of the numbers of people affected, the severity of the disease and the complexity of the life cycle of its causative agent, the protist Plasmodium spp. With the advent of nanoscience, renewed hopes have appeared of finally obtaining the long sought-after magic bullet against malaria in the form of a nanovector for the targeted delivery of antimalarial compounds exclusively to Plasmodium-infected cells, thus increasing drug efficacy and minimizing the induction of resistance to newly developed therapeutic agents. Polyamidoamine-derived nanovectors combine into a single chemical structure drug encapsulating capacity, antimalarial activity, low unspecific toxicity, specific targeting to Plasmodium, optimal in vivo activity and affordable synthesis cost. After having shown their efficacy in targeting drugs to intraerythrocytic parasites, now polyamidoamines face the challenge of spearheading a new generation of nanocarriers aiming at the malaria parasite stages in the mosquito vector.
Orbán, Ágnes; Butykai, Ádám; Molnár, András; Pröhle, Zsófia; Fülöp, Gergö; Zelles, Tivadar; Forsyth, Wasan; Hill, Danika; Müller, Ivo; Schofield, Louis; Rebelo, Maria; Hänscheid, Thomas; Karl, Stephan; Kézsmárki, István
Improving the efficiency of malaria diagnosis is one of the main goals of current malaria research. We have recently developed a magneto-optical (MO) method which allows high-sensitivity detection of malaria pigment (hemozoin crystals) in blood via the magnetically induced rotational motion of the hemozoin crystals. Here, we evaluate this MO technique for the detection of Plasmodium falciparum in infected erythrocytes using in-vitro parasite cultures covering the entire intraerythrocytic life cycle. Our novel method detected parasite densities as low as ∼40 parasites per microliter of blood (0.0008% parasitemia) at the ring stage and less than 10 parasites/µL (0.0002% parasitemia) in the case of the later stages. These limits of detection, corresponding to approximately 20 pg/µL of hemozoin produced by the parasites, exceed that of rapid diagnostic tests and compete with the threshold achievable by light microscopic observation of blood smears. The MO diagnosis requires no special training of the operator or specific reagents for parasite detection, except for an inexpensive lysis solution to release intracellular hemozoin. The devices can be designed to a portable format for clinical and in-field tests. Besides testing its diagnostic performance, we also applied the MO technique to investigate the change in hemozoin concentration during parasite maturation. Our preliminary data indicate that this method may offer an efficient tool to determine the amount of hemozoin produced by the different parasite stages in synchronized cultures. Hence, it could eventually be used for testing the susceptibility of parasites to antimalarial drugs. PMID:24824542
Klein, Eili Y; Smith, David L; Boni, Maciej F; Laxminarayan, Ramanan
Mutations in Plasmodium falciparum that confer resistance to first-line antimalarial drugs have spread throughout the world from a few independent foci, all located in areas that were likely characterized by low or unstable malaria transmission. One of the striking differences between areas of low or unstable malaria transmission and hyperendemic areas is the difference in the size of the population of immune individuals. However, epidemiological models of malaria transmission have generally ignored the role of immune individuals in transmission, assuming that they do not affect the fitness of the parasite. This model reconsiders the role of immunity in the dynamics of malaria transmission and its impact on the evolution of antimalarial drug resistance under the assumption that immune individuals are infectious. The model is constructed as a two-stage susceptible-infected-susceptible (SIS) model of malaria transmission that assumes that individuals build up clinical immunity over a period of years. This immunity reduces the frequency and severity of clinical symptoms, and thus their use of drugs. It also reduces an individual's level of infectiousness, but does not impact the likelihood of becoming infected. Simulations found that with the introduction of resistance into a population, clinical immunity can significantly alter the fitness of the resistant parasite, and thereby impact the ability of the resistant parasite to spread from an initial host by reducing the effective reproductive number of the resistant parasite as transmission intensity increases. At high transmission levels, despite a higher basic reproductive number, R0, the effective reproductive number of the resistant parasite may fall below the reproductive number of the sensitive parasite. These results suggest that high-levels of clinical immunity create a natural ecological refuge for drug-sensitive parasites. This provides an epidemiological rationale for historical patterns of resistance
Gupta, Himanshu; Srivastava, Shikha; Chaudhari, Sima; Vasudevan, Thanvanthri G; Hande, Manjunath H; D'souza, Sydney C; Umakanth, Shashikiran; Satyamoorthy, Kapaettu
For the effective control of malaria, development of sensitive, accurate and rapid tool to diagnose and manage the disease is essential. In humans subjects, the severe form of malaria is caused by Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) and there is need to identify these parasites in acute, chronic and latent (during and post-infection) stages of the disease. In this study, we report a species specific and sensitive diagnostic method for the detection of Pf and Pv in humans. First, we identified intra and intergenic multiloci short stretch of 152 (PfMLS152) and 110 (PvMLS110) nucleotides which is present up to 44 and 34 times in the genomes of Pf and Pv respectively. We developed the single-step amplification-based method using isolated DNA or from lysed red blood cells for the detection of the two malaria parasites. The limit of detection of real-time polymerase chain reaction based assays were 0.1copyof parasite/μl for PfMLS152 and PvMLS110 target sequences. Next, we have tested 250 clinically suspected cases of malaria to validate the method. Sensitivity and specificity for both targets were 100% compared to the quantitative buffy coat microscopy analysis and real-time PCR (Pf-chloroquine resistance transporter (PfCRT) and Pv-lactate dehydrogenase (PvLDH)) based assays. The sensitivity of microscopy and real-time PCR (PfCRT and PvLDH primers) assays were 80.63%; 95%CI 75.22%-85.31%; p<0.05 and 97.61%; 95%CI 94.50%-99.21%; p<0.05 in detecting malaria infection respectively when compared to PfMLS152 and PvMLS110 targets to identify malaria infection in patients. These improved assays may have potential applications in evaluating malaria in asymptomatic patients, treatment, blood donors and in vaccine studies.
A new lizard malaria parasite Plasmodium intabazwe n. sp. (Apicomplexa: Haemospororida: Plasmodiidae) in the Afromontane Pseudocordylus melanotus (Sauria: Cordylidae) with a review of African saurian malaria parasites.
van As, Johann; Cook, Courtney A; Netherlands, Edward C; Smit, Nico J
Saurian malaria parasites are diverse apicomplexan blood parasites including the family Plasmodiidae Mesnil, 1903, and have been studied since the early 1900s. Currently, at least 27 species of Plasmodium are recorded in African lizards, and to date only two species, Plasmodium zonuriae (Pienaar, 1962) and Plasmodium cordyli Telford, 1987, have been reported from the African endemic family Cordylidae. This paper presents a description of a new malaria parasite in a cordylid lizard and provides a phylogenetic hypothesis for saurian Plasmodium species from South Africa. Furthermore, it provides a tabular review of the Plasmodium species that to date have been formally described infecting species of African lizards. Blood samples were collected from 77 specimens of Pseudocordylus melanotus (A. Smith, 1838) from Platberg reserve in the Eastern Free State, and two specimens of Cordylus vittifer (Reichenow, 1887) from the Roodewalshoek conservancy in Mpumalanga (South Africa). Blood smears were Giemsa-stained, screened for haematozoa, specifically saurian malaria parasites, parasite stages were photographed and measured. A small volume was also preserved for TEM studies. Plasmodium and Haemoproteus primer sets, with a nested-polymerase chain reaction (PCR) protocol, were employed to target a fragment of the cytochrome-b (cyt-b) gene region. Resulting sequences of the saurian Plasmodium species' isolates were compared with each other and to other known Plasmodium spp. sequences in the GenBank database. The presence of P. zonuriae in both specimens of the type lizard host C. vittifer was confirmed using morphological characteristics, which subsequently allowed for the species' molecular characterisation. Of the 77 P. melanotus, 44 were parasitised by a Plasmodium species, which when compared morphologically to other African saurian Plasmodium spp. and molecularly to P. zonuriae, supported its description as a new species Plasmodium intabazwe n. sp. This is the first
Ramaprasad, Abhinay; Pain, Arnab; Ravasi, Timothy
Malaria, caused by the protozoan parasite Plasmodium falciparum, affects around 225 million people yearly and a huge international effort is directed towards combating this grave threat to world health and economic development. Considerable advances have been made in malaria research triggered by the sequencing of its genome in 2002, followed by several high-throughput studies defining the malaria transcriptome and proteome. A protein-protein interaction (PPI) network seeks to trace the dynamic interactions between proteins, thereby elucidating their local and global functional relationships. Experimentally derived PPI network from high-throughput methods such as yeast two hybrid (Y2H) screens are inherently noisy, but combining these independent datasets by computational methods tends to give a greater accuracy and coverage. This review aims to discuss the computational approaches used till date to construct a malaria protein interaction network and to catalog the functional predictions and biological inferences made from analysis of the PPI network.
Drake, John M
The enemy-release hypothesis for biological invasions supposes that invasive species may be more successful in their introduced ranges than in their native ranges owing to the absence of coevolved natural enemies. Recent studies supporting this hypothesis have found that introduced plants and animals are less parasitized in their introduced ranges than in their native ranges. Expanding on this theory, I hypothesize that the role of enemy release may differ among the introduction, establishment and spread phases of an invasion. I present a simple model indicating that parasite release is unlikely to greatly affect the chance of establishment in populations with and without an immune subpopulation. The specific numerical relationship between the number of individuals introduced and the chance of establishment depends on a relationship between virulence, here conceptualized as the chance for the extinction of a lineage, and the fraction of the population infected at introduction. These results support the idea of a 'filter effect' in which different biological processes regulate the different phases of an invasion. PMID:14667361
Zhang, Cui; Xiao, Bo; Jiang, Yuanyuan; Zhao, Yihua; Li, Zhenkui; Gao, Han; Ling, Yuan; Wei, Jun; Li, Shaoneng; Lu, Mingke; Yuan, Jing
ABSTRACT Malaria parasites are unicellular organisms residing inside the red blood cells, and current methods for editing the parasite genes have been inefficient. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats and Cas9 endonuclease-mediated genome editing) system is a new powerful technique for genome editing and has been widely employed to study gene function in various organisms. However, whether this technique can be applied to modify the genomes of malaria parasites has not been determined. In this paper, we demonstrated that Cas9 is able to introduce site-specific DNA double-strand breaks in the Plasmodium yoelii genome that can be repaired through homologous recombination. By supplying engineered homologous repair templates, we generated targeted deletion, reporter knock-in, and nucleotide replacement in multiple parasite genes, achieving up to 100% efficiency in gene deletion and 22 to 45% efficiencies in knock-in and allelic replacement. Our results establish methodologies for introducing desired modifications in the P. yoelii genome with high efficiency and accuracy, which will greatly improve our ability to study gene function of malaria parasites. PMID:24987097
Bob, Ndeye Sakha; Diop, Bernard Marcel; Renaud, Francois; Marrama, Laurence; Durand, Patrick; Tall, Adama; Ka, Boubacar; Ekala, Marie Therese; Bouchier, Christiane; Mercereau-Puijalon, Odile; Jambou, Ronan
Background Transmission of malaria in West African urban areas is low and healthcare facilities are well organized. However, malaria mortality remains high. We conducted a survey in Dakar with the general objective to establish who died from severe malaria (SM) in urban areas (particularly looking at the age-groups) and to compare parasite isolates associated with mild or severe malaria. Methodology/Principal Findings The current study included mild- (MM) and severe malaria (SM) cases, treated in dispensaries (n = 2977) and hospitals (n = 104), We analysed Pfdhfr/Pfcrt-exon2 and nine microsatellite loci in 102 matched cases of SM and MM. Half of the malaria cases recorded at the dispensaries and 87% of SM cases referred to hospitals, occurred in adults, although adults only accounted for 26% of all dispensary consultations. This suggests that, in urban settings, whatever the reason for this adult over-representation, health-workers are forced to take care of increasing numbers of malaria cases among adults. Inappropriate self treatment and mutations in genes associated with drug resistance were found associated with SM in adults. SM was also associated with a specific pool of isolates highly polymorphic and different from those associated with MM. Conclusion In this urban setting, adults currently represent one of the major groups of patients attending dispensaries for malaria treatment. For these patients, despite the low level of transmission, SM was associated with a specific and highly polymorphic pool of parasites which may have been selected by inappropriate treatment. PMID:20352101
Background Malaria case management is a key strategy for malaria control. Effective coverage of parasite-based malaria diagnosis (PMD) remains limited in malaria endemic countries. This study assessed the health system's capacity to absorb PMD at primary health care facilities in Uganda. Methods In a cross sectional survey, using multi-stage cluster sampling, lower level health facilities (LLHF) in 11 districts in Uganda were assessed for 1) tools, 2) skills, 3) staff and infrastructure, and 4) structures, systems and roles necessary for the implementing of PMD. Results Tools for PMD (microscopy and/or RDTs) were available at 30 (24%) of the 125 LLHF. All LLHF had patient registers and 15% had functional in-patient facilities. Three months’ long stock-out periods were reported for oral and parenteral quinine at 39% and 47% of LLHF respectively. Out of 131 health workers interviewed, 86 (66%) were nursing assistants; 56 (43%) had received on-job training on malaria case management and 47 (36%) had adequate knowledge in malaria case management. Overall, only 18% (131/730) Ministry of Health approved staff positions were filled by qualified personnel and 12% were recruited or transferred within six months preceding the survey. Of 186 patients that received referrals from LLHF, 130(70%) had received pre-referral anti-malarial drugs, none received pre-referral rectal artesunate and 35% had been referred due to poor response to antimalarial drugs. Conclusion Primary health care facilities had inadequate human and infrastructural capacity to effectively implement universal parasite-based malaria diagnosis. The priority capacity building needs identified were: 1) recruitment and retention of qualified staff, 2) comprehensive training of health workers in fever management, 3) malaria diagnosis quality control systems and 4) strengthening of supply chain, stock management and referral systems. PMID:22920954
Kyabayinze, Daniel J; Achan, Jane; Nakanjako, Damalie; Mpeka, Betty; Mawejje, Henry; Mugizi, Rukaaka; Kalyango, Joan N; D'Alessandro, Umberto; Talisuna, Ambrose; Jean-Pierre, Van geertruyden
Malaria case management is a key strategy for malaria control. Effective coverage of parasite-based malaria diagnosis (PMD) remains limited in malaria endemic countries. This study assessed the health system's capacity to absorb PMD at primary health care facilities in Uganda. In a cross sectional survey, using multi-stage cluster sampling, lower level health facilities (LLHF) in 11 districts in Uganda were assessed for 1) tools, 2) skills, 3) staff and infrastructure, and 4) structures, systems and roles necessary for the implementing of PMD. Tools for PMD (microscopy and/or RDTs) were available at 30 (24%) of the 125 LLHF. All LLHF had patient registers and 15% had functional in-patient facilities. Three months' long stock-out periods were reported for oral and parenteral quinine at 39% and 47% of LLHF respectively. Out of 131 health workers interviewed, 86 (66%) were nursing assistants; 56 (43%) had received on-job training on malaria case management and 47 (36%) had adequate knowledge in malaria case management. Overall, only 18% (131/730) Ministry of Health approved staff positions were filled by qualified personnel and 12% were recruited or transferred within six months preceding the survey. Of 186 patients that received referrals from LLHF, 130(70%) had received pre-referral anti-malarial drugs, none received pre-referral rectal artesunate and 35% had been referred due to poor response to antimalarial drugs. Primary health care facilities had inadequate human and infrastructural capacity to effectively implement universal parasite-based malaria diagnosis. The priority capacity building needs identified were: 1) recruitment and retention of qualified staff, 2) comprehensive training of health workers in fever management, 3) malaria diagnosis quality control systems and 4) strengthening of supply chain, stock management and referral systems.
Arranz, Asier; Frean, John
Background There are 600,000 new malaria cases daily worldwide. The gold standard for estimating the parasite burden and the corresponding severity of the disease consists in manually counting the number of parasites in blood smears through a microscope, a process that can take more than 20 minutes of an expert microscopist’s time. Objective This research tests the feasibility of a crowdsourced approach to malaria image analysis. In particular, we investigated whether anonymous volunteers with no prior experience would be able to count malaria parasites in digitized images of thick blood smears by playing a Web-based game. Methods The experimental system consisted of a Web-based game where online volunteers were tasked with detecting parasites in digitized blood sample images coupled with a decision algorithm that combined the analyses from several players to produce an improved collective detection outcome. Data were collected through the MalariaSpot website. Random images of thick blood films containing Plasmodium falciparum at medium to low parasitemias, acquired by conventional optical microscopy, were presented to players. In the game, players had to find and tag as many parasites as possible in 1 minute. In the event that players found all the parasites present in the image, they were presented with a new image. In order to combine the choices of different players into a single crowd decision, we implemented an image processing pipeline and a quorum algorithm that judged a parasite tagged when a group of players agreed on its position. Results Over 1 month, anonymous players from 95 countries played more than 12,000 games and generated a database of more than 270,000 clicks on the test images. Results revealed that combining 22 games from nonexpert players achieved a parasite counting accuracy higher than 99%. This performance could be obtained also by combining 13 games from players trained for 1 minute. Exhaustive computations measured the parasite
Beigier-Bompadre, Macarena; Becker, Martina; Kroczek, Richard A.; Kaufmann, Stefan H. E.; Matuschewski, Kai
ABSTRACT Protective immunity against preerythrocytic malaria parasite infection is difficult to achieve. Intracellular Plasmodium parasites likely minimize antigen presentation by surface-expressed major histocompatibility complex class I (MHC-I) molecules on infected cells, yet they actively remodel their host cells by export of parasite factors. Whether exported liver-stage proteins constitute better candidates for MHC-I antigen presentation to CD8+ T lymphocytes remains unknown. Here, we systematically characterized the contribution of protein export to the magnitude of antigen-specific T-cell responses against Plasmodium berghei liver-stage parasites in C57BL/6 mice. We generated transgenic sporozoites that secrete a truncated ovalbumin (OVA) surrogate antigen only in the presence of an amino-terminal protein export element. Immunization with live attenuated transgenic sporozoites revealed that antigen export was not critical for CD8+ T-cell priming but enhanced CD8+ T-cell proliferation in the liver. Upon transfer of antigen-specific CD8+ T cells, liver-stage parasites secreting the target protein were eliminated more efficiently. We conclude that Plasmodium parasites strictly control protein export during liver infection to minimize immune recognition. Strategies that enhance the discharge of parasite proteins into infected hepatocytes could improve the efficacy of candidate preerythrocytic malaria vaccines. PMID:25073641
Lungato, Lisandro; Gazarini, Marcos L; Paredes-Gamero, Edgar J; Tufik, Sergio; D'Almeida, Vânia
Parasitic diseases like malaria are a major public health problem in many countries and disrupted sleep patterns are an increasingly common part of modern life. The aim of this study was to assess the effects of paradoxical sleep deprivation (PSD) and sleep rebound (RB) on malarial parasite infection in mice. After PSD, one group was immediately infected with parasites (PSD). The two other PSD rebound groups were allowed to sleep normally for either 24 h (24 h RB) or 48 h (48 h RB). After the recovery periods, mice were inoculated with parasites. The PSD group was the most affected by parasites presenting the higher death rate (0.02), higher number of infected cells (p < 0.01), and decrease in body weight (p < 0.04) compared to control and 48 h RB groups. The 24 h RB group was also different from control group in survival (p < 0.03), number of infected cells (p < 0.05) and body weight (p < 0.04). After 48 hours of sleep rebound animals were allowed to restore their response to parasitic infection similar to normal sleep animals. These results suggest that PSD is damaging to the immune system and leads to an increased infection severity of malaria parasites; only 48 hours of recovery sleep was sufficient to return the mice infection response to baseline values.
Apoptosis is a precisely regulated process of cell death which occurs widely in multicellular organisms and is essential for normal development and immune defences. In recent years, interest has grown in the occurrence of apoptosis in unicellular organisms. In particular, as apoptosis has been reported in a wide range of species, including protozoan malaria parasites and trypanosomes, it may provide a novel target for intervention. However, it is important to understand when and why parasites employ an apoptosis strategy before the likely long- and short-term success of such an intervention can be evaluated. The occurrence of apoptosis in unicellular parasites provides a challenge for evolutionary theory to explain as organisms are expected to have evolved to maximise their own proliferation, not death. One possible explanation is that protozoan parasites undergo apoptosis in order to gain a group benefit from controlling their density as this prevents premature vector mortality. However, experimental manipulations to examine the ultimate causes behind apoptosis in parasites are lacking. In this review, we focus on malaria parasites to outline how an evolutionary framework can help make predictions about the ecological circumstances under which apoptosis could evolve. We then highlight the ecological considerations that should be taken into account when designing evolutionary experiments involving markers of cell death, and we call for collaboration between researchers in different fields to identify and develop appropriate markers in reference to parasite ecology and to resolve debates on terminology. PMID:21080937
Beatty, Wandy; Angel, Ross; Slebodnick, Carla; Andersen, John; Kumar, Sanjai; Rathore, Dharmendar
When malaria parasites infect host red blood cells (RBC) and proteolyze hemoglobin, a unique, albeit poorly understood parasite-specific mechanism, detoxifies released heme into hemozoin (Hz). Here, we report the identification and characterization of a novel Plasmodium Heme Detoxification Protein (HDP) that is extremely potent in converting heme into Hz. HDP is functionally conserved across Plasmodium genus and its gene locus could not be disrupted. Once expressed, the parasite utilizes a circuitous “Outbound–Inbound” trafficking route by initially secreting HDP into the cytosol of infected RBC. A subsequent endocytosis of host cytosol (and hemoglobin) delivers HDP to the food vacuole (FV), the site of Hz formation. As Hz formation is critical for survival, involvement of HDP in this process suggests that it could be a malaria drug target. PMID:18437218
Dalton, John P; Demanga, Corine G; Reiling, Sarah J; Wunderlich, Juliane; Eng, Jenny W L; Rohrbach, Petra
We describe methods for the large-scale in vitro culturing of synchronous and asynchronous blood-stage Plasmodium falciparum parasites in sterile disposable plastic bioreactors controlled by wave-induced motion (wave bioreactor). These cultures perform better than static flask cultures in terms of preserving parasite cell cycle synchronicity and reducing the number of multiple-infected erythrocytes. The straight-forward methods described here will facilitate the large scale production of malaria parasites for antigen and organelle isolation and characterisation, for the high throughput screening of compound libraries with whole cells or extracts, and the development of live- or whole-cell malaria vaccines under good manufacturing practice compliant standards. Crown Copyright Â© 2012. Published by Elsevier Ltd. All rights reserved.
Ghansah, Anita; Amenga-Etego, Lucas; Amambua-Ngwa, Alfred; Andagalu, Ben; Apinjoh, Tobias; Bouyou-Akotet, Marielle; Cornelius, Victoria; Golassa, Lemu; Andrianaranjaka, Voahangy Hanitriniaina; Ishengoma, Deus; Johnson, Kimberly; Kamau, Edwin; Maïga-Ascofaré, Oumou; Mumba, Dieudonne; Tindana, Paulina; Tshefu-Kitoto, Antoinette; Randrianarivelojosia, Milijaona; William, Yavo; Kwiatkowski, Dominic P; Djimde, Abdoulaye A
The African continent continues to bear the greatest burden of malaria and the greatest diversity of parasites, mosquito vectors, and human victims. The evolutionary plasticity of malaria parasites and their vectors is a major obstacle to eliminating the disease. Of current concern is the recently reported emergence of resistance to the front-line drug, artemisinin, in South-East Asia in Plasmodium falciparum, which calls for preemptive surveillance of the African parasite population for genetic markers of emerging drug resistance. Here we describe the Plasmodium Diversity Network Africa (PDNA), which has been established across 11 countries in sub-Saharan Africa to ensure that African scientists are enabled to work together and to play a key role in the global effort for tracking and responding to this public health threat.
Hopp, Ann-Katrin; Saenger, Mélanie; Soichot, Julien; Scholze, Heidi; Boch, Jens; Blandin, Stéphanie A.; Marois, Eric
Mosquitoes genetically engineered to be resistant to Plasmodium parasites represent a promising novel approach in the fight against malaria. The insect immune system itself is a source of anti-parasitic genes potentially exploitable for transgenic designs. The Anopheles gambiae thioester containing protein 1 (TEP1) is a potent anti-parasitic protein. TEP1 is secreted and circulates in the mosquito hemolymph, where its activated cleaved form binds and eliminates malaria parasites. Here we investigated whether TEP1 can be used to create malaria resistant mosquitoes. Using a GFP reporter transgene, we determined that the fat body is the main site of TEP1 expression. We generated transgenic mosquitoes that express TEP1r, a potent refractory allele of TEP1, in the fat body and examined the activity of the transgenic protein in wild-type or TEP1 mutant genetic backgrounds. Transgenic TEP1r rescued loss-of-function mutations, but did not increase parasite resistance in the presence of a wild-type susceptible allele. Consistent with previous reports, TEP1 protein expressed from the transgene in the fat body was taken up by hemocytes upon a challenge with injected bacteria. Furthermore, although maturation of transgenic TEP1 into the cleaved form was impaired in one of the TEP1 mutant lines, it was still sufficient to reduce parasite numbers and induce parasite melanization. We also report here the first use of Transcription Activator Like Effectors (TALEs) in Anopheles gambiae to stimulate expression of endogenous TEP1. We found that artificial elevation of TEP1 expression remains moderate in vivo and that enhancement of endogenous TEP1 expression did not result in increased resistance to Plasmodium. Taken together, our results reveal the difficulty of artificially influencing TEP1-mediated Plasmodium resistance, and contribute to further our understanding of the molecular mechanisms underlying mosquito resistance to Plasmodium parasites. PMID:28095489
Background Reliable methods to preserve mosquito vectors for malaria studies are necessary for detecting Plasmodium parasites. In field settings, however, maintaining a cold chain of storage from the time of collection until laboratory processing, or accessing other reliable means of sample preservation is often logistically impractical or cost prohibitive. As the Plasmodium infection rate of Anopheles mosquitoes is a central component of the entomological inoculation rate and other indicators of transmission intensity, storage conditions that affect pathogen detection may bias malaria surveillance indicators. This study investigated the effect of storage time and temperature on the ability to detect Plasmodium parasites in desiccated Anopheles mosquitoes by real-time polymerase chain reaction (PCR). Methods Laboratory-infected Anopheles stephensi mosquitoes were chloroform-killed and stored over desiccant for 0, 1, 3, and 6 months while being held at four different temperatures: 28, 37, -20 and -80°C. The detection of Plasmodium DNA was evaluated by real-time PCR amplification of a 111 base pair region of block 4 of the merozoite surface protein. Results Varying the storage time and temperature of desiccated mosquitoes did not impact the sensitivity of parasite detection. A two-way factorial analysis of variance suggested that storage time and temperature were not associated with a loss in the ability to detect parasites. Storage of samples at 28°C resulted in a significant increase in the ability to detect parasite DNA, though no other positive associations were observed between the experimental storage treatments and PCR amplification. Conclusions Cold chain maintenance of desiccated mosquito samples is not necessary for real-time PCR detection of parasite DNA. Though field-collected mosquitoes may be subjected to variable conditions prior to molecular processing, the storage of samples over an inexpensive and logistically accessible desiccant will likely
DeWitt CC, Quino -Ascurra HA, Kester KE, Kain KC, Walsh DS, Ballou WR, Gasser RA, 2001. Malaria rapid diagnostic devices: performance characteristics of...Wongsrichanalai C, Magill AJ, Craig LG, Sirichais- inthop J, Bautista CT, Miller RS, Ockenhouse CF, Kester KE, Aronson NE, Andersen EM, Quino
In 1988, the number of malaria cases reported was 134.2 thousand (Taiwan Province not included). Comparing with 210.6 thousand cases reported in 1987, a decrease of 36.3% was noted. Based on data reported by each county in the country, about 950.7 million people in 2,541 counties/cities were living in areas where malaria incidence was less than 0.1 per 1,000 (including originally malaria-free areas as well as areas free from the disease); 96.6 million people in 207 counties/cities with an incidence of 0.1-1.0 per 1,000; 30.4 million in 79 counties/cities with an incidence of 1.1-10.0 per 1000, and 0.4 million in 4 counties with an incidence of above 10.0 per 1,000. While the incidence decreased markedly in most endemic areas, it fluctuated in the southern part of China due to the increase in the floating population, shortage of insecticides, and natural calamities in some areas. In Hainan province the incidence increased by 21.1% over 1987; increase in incidence and focal outbreaks also occurred in some areas of Yunnan, Guangdong, Guangxi, Guizhou provinces/autonomous region. In the provinces of Anhui, Jiangsu, Henan and Jiangxi, the major endemic areas in central China, the incidence decreased by 43.6-71.4% over last year, there were 57 thousand cases, accounting for 42.6% of the total cases recorded in the country. Downward trends of incidence continued in other provinces, autonomous regions and municipalities. The distribution of falciparum malaria was confined to 63 counties of 6 provinces/autonomous region, while non-indigenous falciparum malaria cases were found in 88 counties of 8 provinces due to the population movements.(ABSTRACT TRUNCATED AT 250 WORDS)
Kirk, K; Wong, H Y; Elford, B C; Newbold, C I; Ellory, J C
Human erythrocytes infected in vitro with the malaria parasite Plasmodium falciparum showed a markedly increased rate of choline influx compared with normal cells. Choline transport into uninfected cells (cultured in parallel with infected cells) obeyed Michaelis-Menten kinetics (Km approximately 11 microM). In malaria-parasite-infected cells there was an additional choline-transport component which failed to saturate at extracellular concentrations of up to 500 microM. This component was less sensitive than the endogenous transporter to inhibition by the Cinchona bark alkaloids quinine, quinidine, cinchonine and cinchonidine, but showed a much greater sensitivity than the native system to inhibition by piperine. The sensitivity of the induced choline transport to these reagents was similar to that of the malaria-induced (ouabain- and bumetanide-resistant) Rb(+)-transport pathway; however, the relative magnitudes of the piperine-sensitive choline and Rb+ fluxes in malaria-parasite-infected cells varied between cultures. This suggests either that the enhanced transport of the two cations was via functionally distinct (albeit pharmacologically similar) pathways, or that the transport was mediated by a pathway with variable substrate selectivity. PMID:1898345
Costa, Daniela Camargos; Madureira, Ana Paula; Amaral, Lara Cotta; Sanchez, Bruno Antônio Marinho; Gomes, Luciano Teixeira; Fontes, Cor Jésus Fernandes; Limongi, Jean Ezequiel; Brito, Cristiana Ferreira Alves de; Carvalho, Luzia Helena
The polymerase chain reaction (PCR)-based methods for the diagnosis of malaria infection are expected to accurately identify submicroscopic parasite carriers. Although a significant number of PCR protocols have been described, few studies have addressed the performance of PCR amplification in cases of field samples with submicroscopic malaria infection. Here, the reproducibility of two well-established PCR protocols (nested-PCR and real-time PCR for the Plasmodium 18 small subunit rRNA gene) were evaluated in a panel of 34 blood field samples from individuals that are potential reservoirs of malaria infection, but were negative for malaria by optical microscopy. Regardless of the PCR protocol, a large variation between the PCR replicates was observed, leading to alternating positive and negative results in 38% (13 out of 34) of the samples. These findings were quite different from those obtained from the microscopy-positive patients or the unexposed individuals; the diagnosis of these individuals could be confirmed based on the high reproducibility and specificity of the PCR-based protocols. The limitation of PCR amplification was restricted to the field samples with very low levels of parasitaemia because titrations of the DNA templates were able to detect < 3 parasites/µL in the blood. In conclusion, conventional PCR protocols require careful interpretation in cases of submicroscopic malaria infection, as inconsistent and false-negative results can occur.
Costa, Daniela Camargos; Madureira, Ana Paula; Amaral, Lara Cotta; Sanchez, Bruno Antônio Marinho; Gomes, Luciano Teixeira; Fontes, Cor Jésus Fernandes; Limongi, Jean Ezequiel; de Brito, Cristiana Ferreira Alves; Carvalho, Luzia Helena
The polymerase chain reaction (PCR)-based methods for the diagnosis of malaria infection are expected to accurately identify submicroscopic parasite carriers. Although a significant number of PCR protocols have been described, few studies have addressed the performance of PCR amplification in cases of field samples with submicroscopic malaria infection. Here, the reproducibility of two well-established PCR protocols (nested-PCR and real-time PCR for the Plasmodium 18 small subunit rRNA gene) were evaluated in a panel of 34 blood field samples from individuals that are potential reservoirs of malaria infection, but were negative for malaria by optical microscopy. Regardless of the PCR protocol, a large variation between the PCR replicates was observed, leading to alternating positive and negative results in 38% (13 out of 34) of the samples. These findings were quite different from those obtained from the microscopy-positive patients or the unexposed individuals; the diagnosis of these individuals could be confirmed based on the high reproducibility and specificity of the PCR-based protocols. The limitation of PCR amplification was restricted to the field samples with very low levels of parasitaemia because titrations of the DNA templates were able to detect < 3 parasites/µL in the blood. In conclusion, conventional PCR protocols require careful interpretation in cases of submicroscopic malaria infection, as inconsistent and false-negative results can occur. PMID:24626306
Cai, Shengxin; Risinger, April L; Nair, Shalini; Peng, Jiangnan; Anderson, Timothy J C; Du, Lin; Powell, Douglas R; Mooberry, Susan L; Cichewicz, Robert H
Some of the most valuable antimalarial compounds, including quinine and artemisinin, originated from plants. While these drugs have served important roles over many years for the treatment of malaria, drug resistance has become a widespread problem. Therefore, a critical need exists to identify new compounds that have efficacy against drug-resistant malaria strains. In the current study, extracts prepared from plants readily obtained from local sources were screened for activity against Plasmodium falciparum. Bioassay-guided fractionation was used to identify 18 compounds from five plant species. These compounds included eight lupane triterpenes (1-8), four kaempferol 3-O-rhamnosides (10-13), four kaempferol 3-O-glucosides (14-17), and the known compounds amentoflavone and knipholone. These compounds were tested for their efficacy against multi-drug-resistant malaria parasites and counterscreened against HeLa cells to measure their antimalarial selectivity. Most notably, one of the new lupane triterpenes (3) isolated from the supercritical extract of Buxus sempervirens, the common boxwood, showed activity against both drug-sensitive and -resistant malaria strains at a concentration that was 75-fold more selective for the drug-resistant malaria parasites as compared to HeLa cells. This study demonstrates that new antimalarial compounds with efficacy against drug-resistant strains can be identified from native and introduced plant species in the United States, which traditionally have received scant investigation compared to more heavily explored tropical and semitropical botanical resources from around the world.
Background To achieve the goal of malaria elimination in low transmission areas such as in Cambodia, new, inexpensive, high-throughput diagnostic tools for identifying very low parasite densities in asymptomatic carriers are required. This will enable a switch from passive to active malaria case detection in the field. Methods DNA extraction and real-time PCR assays were implemented in an “in-house” designed mobile laboratory allowing implementation of a robust, sensitive and rapid malaria diagnostic strategy in the field. This tool was employed in a survey organized in the context of the MalaResT project (NCT01663831). Results The real-time PCR screening and species identification assays were performed in the mobile laboratory between October and November 2012, in Rattanakiri Province, to screen approximately 5,000 individuals in less than four weeks and treat parasite carriers within 24–48 hours after sample collection. An average of 240 clinical samples (and 40 quality control samples) was tested every day, six/seven days per week. Some 97.7% of the results were available <24 hours after the collection. A total of 4.9% were positive for malaria. Plasmodium vivax was present in 61.1% of the positive samples, Plasmodium falciparum in 45.9%, Plasmodium malariae in 7.0% and Plasmodium ovale in 2.0%. Conclusions The operational success of this diagnostic set-up proved that molecular testing and subsequent treatment is logistically achievable in field settings. This will allow the detection of clusters of asymptomatic carriers and to provide useful epidemiological information. Fast results will be of great help for staff in the field to track and treat asymptomatic parasitaemic cases. The concept of the mobile laboratory could be extended to other countries for the molecular detection of malaria or other pathogens, or to culture vivax parasites, which does not support long-time delay between sample collection and culture. PMID:24206649
Hastings, Ian M; Kay, Katherine; Hodel, Eva Maria
Artemisinin-based combination therapies (ACTs) are currently the first-line drugs for treating uncomplicated falciparum malaria, the most deadly of the human malarias. Malaria parasite clearance rates estimated from patients' blood following ACT treatment have been widely adopted as a measure of drug effectiveness and as surveillance tools for detecting the presence of potential artemisinin resistance. This metric has not been investigated in detail, nor have its properties or potential shortcomings been identified. Herein, the pharmacology of drug treatment, parasite biology, and human immunity are combined to investigate the dynamics of parasite clearance following ACT. This approach parsimoniously recovers the principal clinical features and dynamics of clearance. Human immunity is the primary determinant of clearance rates, unless or until artemisinin killing has fallen to near-ineffective levels. Clearance rates are therefore highly insensitive metrics for surveillance that may lead to overconfidence, as even quite substantial reductions in drug sensitivity may not be detected as lower clearance rates. Equally serious is the use of clearance rates to quantify the impact of ACT regimen changes, as this strategy will plausibly miss even very substantial increases in drug effectiveness. In particular, the malaria community may be missing the opportunity to dramatically increase ACT effectiveness through regimen changes, particularly through a switch to twice-daily regimens and/or increases in artemisinin dosing levels. The malaria community therefore appears overreliant on a single metric of drug effectiveness, the parasite clearance rate, that has significant and serious shortcomings. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
Rode, Nicolas O; Lievens, Eva J P; Segard, Adeline; Flaven, Elodie; Jabbour-Zahab, Roula; Lenormand, Thomas
We investigated the host specificity of two cryptic microsporidian species (Anostracospora rigaudi and Enterocytospora artemiae) infecting invasive (Artemia franciscana) and native (Artemia parthenogenetica) hosts in sympatry. Anostracospora rigaudi was on average four times more prevalent in the native host, whereas E. artemiae was three times more prevalent in the invasive host. Infection with An. rigaudi strongly reduced female reproduction in both host species, whereas infection with E. artemiae had weaker effects on female reproduction. We contrasted microsporidian prevalence in native A. franciscana populations (New World) and in both invaded and non-invaded Artemia populations (Old World). At a community level, microsporidian prevalence was twice as high in native compared with invasive hosts, due to the contrasting host-specificity of An. rigaudi and E. artemiae. At a higher biogeographical level, microsporidian prevalence in A. franciscana did not differ between the invaded populations and the native populations used for the introduction. Although E. artemiae was the only species found both in New and Old World populations, no evidence of its co-introduction with the invasive host was found in our experimental and phylogeographic tests. These results suggest that the success of A. franciscana invasion is probably due to a lower susceptibility to virulent microsporidian parasites rather than to decreased microsporidian prevalence compared with A. parthenogenetica or to lower microsporidian virulence in introduced areas. Published by Elsevier Ltd.
Angrisano, Fiona; Tan, Yan-Hong; Sturm, Angelika; McFadden, Geoffrey I; Baum, Jake
Vector-borne diseases constitute an enormous burden on public health across the world. However, despite the importance of interactions between infectious pathogens and their respective vector for disease transmission, the biology of the pathogen in the insect is often less well understood than the forms that cause human infections. Even with the global impact of Plasmodium parasites, the causative agents of malarial disease, no vaccine exists to prevent infection and resistance to all frontline drugs is emerging. Malaria parasite migration through the mosquito host constitutes a major population bottleneck of the lifecycle and therefore represents a powerful, although as yet relatively untapped, target for therapeutic intervention. The understanding of parasite-mosquito interactions has increased in recent years with developments in genome-wide approaches, genomics and proteomics. Each development has shed significant light on the biology of the malaria parasite during the mosquito phase of the lifecycle. Less well understood, however, is the process of midgut colonisation and oocyst formation, the precursor to parasite re-infection from the next mosquito bite. Here, we review the current understanding of cellular and molecular events underlying midgut colonisation centred on the role of the motile ookinete. Further insight into the major interactions between the parasite and the mosquito will help support the broader goal to identify targets for transmission-blocking therapies against malarial disease. Copyright © 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.
Rahbari, Mahsa; Bogeski, Ivan
Hydrogen peroxide is an important antimicrobial agent but is also crucially involved in redox signaling and pathogen-host cell interactions. As a basis for systematically investigating intracellular H2O2 dynamics and regulation in living malaria parasites, we established the genetically encoded fluorescent H2O2 sensors roGFP2-Orp1 and HyPer-3 in Plasmodium falciparum. Both ratiometric redox probes as well as the pH control SypHer were expressed in the cytosol of blood-stage parasites. Both redox sensors showed reproducible sensitivity towards H2O2 in the lower micromolar range in vitro and in the parasites. Due to the pH sensitivity of HyPer-3, we used parasites expressing roGFP2-Orp1 for evaluation of short-, medium-, and long-term effects of antimalarial drugs on H2O2 levels and detoxification in Plasmodium. None of the quinolines or artemisinins tested had detectable direct effects on the H2O2 homeostasis at pharmacologically relevant concentrations. However, pre-treatment of the cells with antimalarial drugs or heat shock led to a higher tolerance towards exogenous H2O2. The systematic evaluation and comparison of the two genetically encoded cytosolic H2O2 probes in malaria parasites provides a basis for studying parasite-host cell interactions or drug effects with spatio-temporal resolution while preserving cell integrity. PMID:28369083
Huijben, Silvie; Nelson, William A; Wargo, Andrew R; Sim, Derek G; Drew, Damien R; Read, Andrew F
A major determinant of the rate at which drug-resistant malaria parasites spread through a population is the ecology of resistant and sensitive parasites sharing the same host. Drug treatment can significantly alter this ecology by removing the drug-sensitive parasites, leading to competitive release of resistant parasites. Here, we test the hypothesis that the spread of resistance can be slowed by reducing drug treatment and hence restricting competitive release. Using the rodent malaria model Plasmodium chabaudi, we found that low-dose chemotherapy did reduce competitive release. A higher drug dose regimen exerted stronger positive selection on resistant parasites for no detectable clinical gain. We estimated instantaneous selection coefficients throughout the course of replicate infections to analyze the temporal pattern of the strength and direction of within-host selection. The strength of selection on resistance varied through the course of infections, even in untreated infections, but increased immediately following drug treatment, particularly in the high-dose groups. Resistance remained under positive selection for much longer than expected from the half life of the drug. Although there are many differences between mice and people, our data do raise the question whether the aggressive treatment regimens aimed at complete parasite clearance are the best resistance-management strategies for humans.
Schaer, Juliane; Perkins, Susan L; Decher, Jan; Leendertz, Fabian H; Fahr, Jakob; Weber, Natalie; Matuschewski, Kai
As the only volant mammals, bats are captivating for their high taxonomic diversity, for their vital roles in ecosystems--particularly as pollinators and insectivores--and, more recently, for their important roles in the maintenance and transmission of zoonotic viral diseases. Genome sequences have identified evidence for a striking expansion of and positive selection in gene families associated with immunity. Bats have also been known to be hosts of malaria parasites for over a century, and as hosts, they possess perhaps the most phylogenetically diverse set of hemosporidian genera and species. To provide a molecular framework for the study of these parasites, we surveyed bats in three remote areas of the Upper Guinean forest ecosystem. We detected four distinct genera of hemosporidian parasites: Plasmodium, Polychromophilus, Nycteria, and Hepatocystis. Intriguingly, the two species of Plasmodium in bats fall within the clade of rodent malaria parasites, indicative of multiple host switches across mammalian orders. We show that Nycteria species form a very distinct phylogenetic group and that Hepatocystis parasites display an unusually high diversity and prevalence in epauletted fruit bats. The diversity and high prevalence of novel lineages of chiropteran hemosporidians underscore the exceptional position of bats among all other mammalian hosts of hemosporidian parasites and support hypotheses of pathogen tolerance consistent with the exceptional immunology of bats.
Schaer, Juliane; Perkins, Susan L.; Decher, Jan; Leendertz, Fabian H.; Fahr, Jakob; Weber, Natalie; Matuschewski, Kai
As the only volant mammals, bats are captivating for their high taxonomic diversity, for their vital roles in ecosystems—particularly as pollinators and insectivores—and, more recently, for their important roles in the maintenance and transmission of zoonotic viral diseases. Genome sequences have identified evidence for a striking expansion of and positive selection in gene families associated with immunity. Bats have also been known to be hosts of malaria parasites for over a century, and as hosts, they possess perhaps the most phylogenetically diverse set of hemosporidian genera and species. To provide a molecular framework for the study of these parasites, we surveyed bats in three remote areas of the Upper Guinean forest ecosystem. We detected four distinct genera of hemosporidian parasites: Plasmodium, Polychromophilus, Nycteria, and Hepatocystis. Intriguingly, the two species of Plasmodium in bats fall within the clade of rodent malaria parasites, indicative of multiple host switches across mammalian orders. We show that Nycteria species form a very distinct phylogenetic group and that Hepatocystis parasites display an unusually high diversity and prevalence in epauletted fruit bats. The diversity and high prevalence of novel lineages of chiropteran hemosporidians underscore the exceptional position of bats among all other mammalian hosts of hemosporidian parasites and support hypotheses of pathogen tolerance consistent with the exceptional immunology of bats. PMID:24101466
Mahajan, Sumit S; Gut, Jiri; Rosenthal, Philip J; Renslo, Adam R
Background The malaria parasites Plasmodium falciparum and Plasmodium vivax generate significant concentrations of free unbound ferrous iron heme as a side product of hemoglobin degradation. The presence of these chemically reactive forms of iron, rare in healthy cells, presents an opportunity for parasite-selective drug delivery. Accordingly, our group is developing technologies for the targeted delivery of therapeutics to the intra-erythrocytic malaria parasite. These so-called ‘fragmenting hybrids’ employ a 1,2,4-trioxolane ring system as an iron(II)-sensing ‘trigger’ moiety and a ‘traceless’ retro-Michael linker to which a variety of partner drug species may be attached. After ferrous iron-promoted activation in the parasite, the partner drug is released via a β-elimination reaction. Methods In this report, we describe three orthogonal experimental approaches that were explored in order to generate in vitro proof-of-concept for ferrous iron-dependent drug delivery from a prototypical fragmenting hybrid. Conclusion Studies of two fragmenting hybrids by orthogonal approaches confirm that a partner drug species can be delivered to live P. falciparum parasites. A key advantage of this approach is the potential to mask a partner drug’s intrinsic bioactivity prior to release in the parasite. PMID:23234548
Ademolue, Temitope W; Aniweh, Yaw; Kusi, Kwadwo A; Awandare, Gordon A
In individuals living in malaria-endemic regions, parasitaemia thresholds for the onset of clinical symptoms vary with transmission intensity. The mechanisms that mediate this relationship are however, unclear. Since inflammatory responses to parasite infection contribute to the clinical manifestation of malaria, this study investigated inflammatory cytokine responses in children with malaria from areas of different transmission intensities (ranging from low to high). Blood samples were obtained from children confirmed with malaria at community hospitals in three areas with differing transmission intensities. Cytokine levels were assessed using the Luminex(®)-based magnetic bead array system, and levels were compared across sites using appropriate statistical tests. The relative contributions of age, gender, parasitaemia and transmission intensity on cytokine levels were investigated using multivariate regression analysis. Parasite density increased with increasing transmission intensity in children presenting to hospital with symptomatic malaria, indicating that the parasitaemia threshold for clinical malaria increases with increasing transmission intensity. Furthermore, levels of pro-inflammatory cytokines, including tumour necrosis factor alpha (TNF-α), interferon-gamma (IFN-γ), interleukin (IL)-1β, IL-2, IL-6, IL-8, and IL-12, decreased with increasing transmission intensity, and correlated significantly with parasitaemia levels in the low transmission area but not in high transmission areas. Similarly, levels of anti-inflammatory cytokines, including IL-4, IL-7, IL-10 and IL-13, decreased with increasing transmission intensity, with IL-10 showing strong correlation with parasitaemia levels in the low transmission area. Multiple linear regression analyses revealed that transmission intensity was a stronger predictor of cytokine levels than age, gender and parasitaemia. Taken together, the data demonstrate a strong relationship between the prevailing
Hanssen, Eric; Knoechel, Christian; Klonis, Nectarios; Abu-Bakar, Nurhidanatasha; Deed, Samantha; LeGros, Mark; Larabell, Carolyn; Tilley, Leann
Cryo transmission x-ray microscopy in the “water window” of photon energies has recently been introduced as a method that exploits the natural contrast of biological samples. We have used cryo tomographic x-ray imaging of the intraerythrocytic malaria parasite, Plasmodium falciparum, to undertake a survey of the cellular features of this important human pathogen. We examined whole hydrated cells at different stages of growth and defined some of the structures with different x-ray density, including the parasite nucleus, cytoplasm, digestive vacuole and the hemoglobin degradation product, hemozoin. As the parasite develops from an early cup-shaped morphology to a more rounded shape, puncta of hemozoin are formed; these coalesce in the mature trophozoite into a central compartment. In some trophozoite stage parasites we observed invaginations of the parasite surface and, using a selective permeabilization process, showed that these remain connected to the RBC cytoplasm. Some of these invaginations have large openings consistent with phagocytic structures and we observed independent endocytic vesicles in the parasite cytoplasm which appear to play a role in hemoglobin uptake. In schizont stage parasites staggered mitosis was observed and x-ray-dense lipid-rich structures were evident at their apical ends of the developing daughter cells. Treatment of parasites with the antimalarial drug artemisinin appears to affect parasite development and their ability to produce the hemoglobin breakdown product, hemozoin. PMID:20826218
Rupp, Ingrid; Sologub, Ludmilla; Williamson, Kim C; Scheuermayer, Matthias; Reininger, Luc; Doerig, Christian; Eksi, Saliha; Kombila, Davy U; Frank, Matthias; Pradel, Gabriele
Physical contact is important for the interaction between animal cells, but it can represent a major challenge for protists like malaria parasites. Recently, novel filamentous cell-cell contacts have been identified in different types of eukaryotic cells and termed nanotubes due to their morphological appearance. Nanotubes represent small dynamic membranous extensions that consist of F-actin and are considered an ancient feature evolved by eukaryotic cells to establish contact for communication. We here describe similar tubular structures in the malaria pathogen Plasmodium falciparum, which emerge from the surfaces of the forming gametes upon gametocyte activation in the mosquito midgut. The filaments can exhibit a length of > 100 μm and contain the F-actin isoform actin 2. They actively form within a few minutes after gametocyte activation and persist until the zygote transforms into the ookinete. The filaments originate from the parasite plasma membrane, are close ended and express adhesion proteins on their surfaces that are typically found in gametes, like Pfs230, Pfs48/45 or Pfs25, but not the zygote surface protein Pfs28. We show that these tubular structures represent long-distance cell-to-cell connections between sexual stage parasites and demonstrate that they meet the characteristics of nanotubes. We propose that malaria parasites utilize these adhesive “nanotubes” in order to facilitate intercellular contact between gametes during reproduction in the mosquito midgut. PMID:21173797
Sanderson, Theo; Rayner, Julian C.
Two decades after the first Plasmodium transfection, attempts have been made to disrupt more than 3,151 genes in malaria parasites, across five Plasmodium species. While results from rodent malaria transfections have been curated and systematised, empowering large-scale analysis, phenotypic data from human malaria parasite transfections currently exists as individual reports scattered across a the literature. To facilitate systematic analysis of published experimental genetic data across Plasmodium species, we have built PhenoPlasm ( http://www.phenoplasm.org), a database of phenotypes generated by transfection experiments in all Plasmodium parasites. The site provides a simple interface linking citation-backed Plasmodium reverse-genetic phenotypes to gene IDs. The database has been populated with phenotypic data on 367 P. falciparum genes, curated from 176 individual publications, as well as existing data on rodent Plasmodium species from RMgmDB and PlasmoGEM. This is the first time that all available data on P. falciparum transfection experiments has been brought together in a single place. These data are presented using ortholog mapping to allow a researcher interested in a gene in one species to see results across other Plasmodium species. The collaborative nature of the database enables any researcher to add new phenotypes as they are discovered. As an example of database utility, we use the currently available datasets to identify RAP (RNA-binding domain abundant in Apicomplexa)-domain containing proteins as crucial to parasite survival. PMID:28748223
Rupp, Ingrid; Sologub, Ludmilla; Williamson, Kim C; Scheuermayer, Matthias; Reininger, Luc; Doerig, Christian; Eksi, Saliha; Kombila, Davy U; Frank, Matthias; Pradel, Gabriele
Physical contact is important for the interaction between animal cells, but it can represent a major challenge for protists like malaria parasites. Recently, novel filamentous cell-cell contacts have been identified in different types of eukaryotic cells and termed nanotubes due to their morphological appearance. Nanotubes represent small dynamic membranous extensions that consist of F-actin and are considered an ancient feature evolved by eukaryotic cells to establish contact for communication. We here describe similar tubular structures in the malaria pathogen Plasmodium falciparum, which emerge from the surfaces of the forming gametes upon gametocyte activation in the mosquito midgut. The filaments can exhibit a length of > 100 μm and contain the F-actin isoform actin 2. They actively form within a few minutes after gametocyte activation and persist until the zygote transforms into the ookinete. The filaments originate from the parasite plasma membrane, are close ended and express adhesion proteins on their surfaces that are typically found in gametes, like Pfs230, Pfs48/45 or Pfs25, but not the zygote surface protein Pfs28. We show that these tubular structures represent long-distance cell-to-cell connections between sexual stage parasites and demonstrate that they meet the characteristics of nanotubes. We propose that malaria parasites utilize these adhesive "nanotubes" in order to facilitate intercellular contact between gametes during reproduction in the mosquito midgut.
Verhulst, Niels O.; Smallegange, Renate C.; Takken, Willem
Malaria is caused by Plasmodium parasites which are transmitted by mosquitoes. Until recently, human malaria was considered to be caused by human-specific Plasmodium species. Studies on Plasmodium parasites in non-human primates (NHPs), however, have identified parasite species in gorillas and chimpanzees that are closely related to human Plasmodium species. Moreover, P. knowlesi, long known as a parasite of monkeys, frequently infects humans. The requirements for such a cross-species exchange and especially the role of mosquitoes in this process are discussed, as the latter may act as bridge vectors of Plasmodium species between different primates. Little is known about the mosquito species that would bite both humans and NHPs and if so, whether humans and NHPs share the same Plasmodium vectors. To understand the vector-host interactions that can lead to an increased Plasmodium transmission between species, studies are required that reveal the nature of these interactions. Studying the potential role of NHPs as a Plasmodium reservoir for humans will contribute to the ongoing efforts of human malaria elimination, and will help to focus on critical areas that should be considered in achieving this goal. PMID:22701434
Miller, Jessica L; Sack, Brandon K; Baldwin, Michael; Vaughan, Ashley M; Kappe, Stefan H I
Mosquito-transmitted malaria parasites infect hepatocytes and asymptomatically replicate as liver stages. Using RNA sequencing, we show that a rodent malaria liver-stage infection stimulates a robust innate immune response including type I interferon (IFN) and IFNγ pathways. Liver-stage infection is suppressed by these infection-engendered innate responses. This suppression was abrogated in mice deficient in IFNγ, the type I IFN α/β receptor (IFNAR), and interferon regulatory factor 3. Natural killer and CD49b(+)CD3(+) natural killer T (NKT) cells increased in the liver after a primary infection, and CD1d-restricted NKT cells, which secrete IFNγ, were critical in reducing liver-stage burden of a secondary infection. Lack of IFNAR signaling abrogated the increase in NKT cell numbers in the liver, showing a link between type I IFN signaling, cell recruitment, and subsequent parasite elimination. Our findings demonstrate innate immune sensing of malaria parasite liver-stage infection and that the ensuing innate responses can eliminate the parasite.
Pinto, M J; Rodrigues, S R; Desouza, R; Verenkar, M P
A rapid test for diagnosis of malaria based on acridine orange staining of centrifuged blood samples in a microhematocrit tube (QBC) was compared with thick and thin peripheral blood smears in 2274 samples. Malaria was diagnosed in 239 (10.5%) patients by Leishman's staining technique and QBC method. The QBC method allowed detection of an additional 89 (3.9%) cases. Thus the prevalence rate of malaria during the study was 14.4%. In 1946 patients who were negative by the QBC technique, the Leishman's stained smears did not provide any help in malaria diagnosis. Analysis of the relative quantity of parasites in the specimens, in the QBC method, revealed that 80 out of 89 QBC positive but smear negative cases, had a very low parasite number (less than 10 parasites per QBC field). Although QBC method was superior to the smear for malarial parasite detection, species identification was not possible in 26 (7.9%) cases by this technique. In 95.7% (n = 314) QBC positive cases, the buffy coat in the QBC tube appeared pigmented (gray to black). The colour of the buffy coat was therefore considered by us as a predictor of positivity and could be taken as an indicator for a careful and more prolonged search for the parasites. Thus, the QBC technique has its advantages in terms of speed, sensitivity and ease, especially in an endemic area as ours, where the level of parasitaemia is low and more than 70 to 80 smears need to be examined per day. However, the age old Romanowsky stains still appear superior for species identification.
Madabushi, Amrita; Chakraborty, Sibani; Fisher, S. Zoë; Clemente, José C.; Yowell, Charles; Agbandje-McKenna, Mavis; Dame, John B.; Dunn, Ben M.; McKenna, Robert
Plasmepsin 4 from the malarial parasite P. malariae has been crystallized in complex with a small molecular inhibitor. Preliminary X-ray analysis of the diffraction data collected at 3.3 Å resolution is reported.
Ramiro, Ricardo S; Alpedrinha, João; Carter, Lucy; Gardner, Andy; Reece, Sarah E
Malaria parasites must undergo a round of sexual reproduction in the blood meal of a mosquito vector to be transmitted between hosts. Developing a transmission-blocking intervention to prevent parasites from mating is a major goal of biomedicine, but its effectiveness could be compromised if parasites can compensate by simply adjusting their sex allocation strategies. Recently, the application of evolutionary theory for sex allocation has been supported by experiments demonstrating that malaria parasites adjust their sex ratios in response to infection genetic diversity, precisely as predicted. Theory also predicts that parasites should adjust sex allocation in response to host immunity. Whilst data are supportive, the assumptions underlying this prediction - that host immune responses have differential effects on the mating ability of males and females - have not yet been tested. Here, we combine experimental work with theoretical models in order to investigate whether the development and fertility of male and female parasites is affected by innate immune factors and develop new theory to predict how parasites' sex allocation strategies should evolve in response to the observed effects. Specifically, we demonstrate that reactive nitrogen species impair gametogenesis of males only, but reduce the fertility of both male and female gametes. In contrast, tumour necrosis factor-α does not influence gametogenesis in either sex but impairs zygote development. Therefore, our experiments demonstrate that immune factors have complex effects on each sex, ranging from reducing the ability of gametocytes to develop into gametes, to affecting the viability of offspring. We incorporate these results into theory to predict how the evolutionary trajectories of parasite sex ratio strategies are shaped by sex differences in gamete production, fertility and offspring development. We show that medical interventions targeting offspring development are more likely to be 'evolution
Morand, Serge; Bordes, Frédéric; Chen, Hsuan-Wien; Claude, Julien; Cosson, Jean-François; Galan, Maxime; Czirják, Gábor Á; Greenwood, Alex D; Latinne, Alice; Michaux, Johan; Ribas, Alexis
We summarize the current knowledge on parasitism-related invasion processes of the globally invasive Rattus lineages, originating from Asia, and how these invasions have impacted the local epidemiology of rodent-borne diseases. Parasites play an important role in the invasion processes and successes of their hosts through multiple biological mechanisms such as "parasite release," "immunocompetence advantage," "biotic resistance" and "novel weapon." Parasites may also greatly increase the impact of invasions by spillover of parasites and other pathogens, introduced with invasive hosts, into new hosts, potentially leading to novel emerging diseases. Another potential impact is the ability of the invader to amplify local parasites by spillback. In both cases, local fauna and humans may be exposed to new health risks, which may decrease biodiversity and potentially cause increases in human morbidity and mortality. Here we review the current knowledge on these processes and propose some research priorities.
Brazier, Andrew J.; Avril, Marion; Bernabeu, Maria; Benjamin, Maxwell
ABSTRACT Plasmodium falciparum, the most deadly of the human malaria parasites, is a member of the Laverania subgenus that also infects African Great Apes. The virulence of P. falciparum is related to cytoadhesion of infected erythrocytes in microvasculature, but the origin of dangerous parasite adhesion traits is poorly understood. To investigate the evolutionary history of the P. falciparum cytoadhesion pathogenicity determinant, we studied adhesion domains from the chimpanzee malaria parasite P. reichenowi. We demonstrate that the P. reichenowi var gene repertoire encodes cysteine-rich interdomain region (CIDR) domains which bind human CD36 and endothelial protein C receptor (EPCR) with the same levels of affinity and at binding sites similar to those bound by P. falciparum. Moreover, P. reichenowi domains interfere with the protective function of the activated protein C-EPCR pathway on endothelial cells, a presumptive virulence trait in humans. These findings provide evidence for ancient evolutionary origins of two key cytoadhesion properties of P. falciparum that contribute to human infection and pathogenicity. IMPORTANCE Cytoadhesion of P. falciparum-infected erythrocytes in the microcirculation is a major virulence determinant. P. falciparum is descended from a subgenus of parasites that also infect chimpanzees and gorillas and exhibits strict host species specificity. Despite their high genetic similarity to P. falciparum, it is unknown whether ape parasites encode adhesion properties similar to those of P. falciparum or are as virulent in their natural hosts. Consequently, it has been unclear when virulent adhesion traits arose in P. falciparum and how long they have been present in the parasite population. It is also unknown whether cytoadhesive interactions pose a barrier to cross-species transmission. We show that parasite domains from the chimpanzee malaria parasite P. reichenowi bind human receptors with specificity similar to that of P
Winograd, E.; Greenan, J.R.T.; Sherman, I.W.
Erythrocytes infected with a knobby variant of Plasmodium falciparum selectively bind IgG autoantibodies in normal human serum. Quantification of membrane-bound IgG, by use of /sup 125/I-labeled protein A, revealed that erythrocytes infected with the knobby variant bound 30 times more protein A than did noninfected erythrocytes; infection with a knobless variant resulted in less than a 2-fold difference compared with noninfected erythrocytes. IgG binding to knobby erythrocytes appeared to be related to parasite development, since binding of /sup 125/I-labeled protein A to cells bearing young trophozoites (less than 20 hr after parasite invasion) was similar to binding to uninfected erythrocytes. By immunoelectron microscopy, the membrane-bound IgG on erythrocytes infected with the knobby variant was found to be preferentially associated with the protuberances (knobs) of the plasma membrane. The removal of aged or senescent erythrocytes from the peripheral circulation is reported to involve the binding of specific antibodies to an antigen (senescent antigen) related to the major erythrocyte membrane protein band 3. Since affinity-purified autoantibodies against band 3 specifically bound to the plasma membrane of erythrocytes infected with the knobby variant of P. falciparum, it is clear that the malaria parasite induces expression of senescent antigen.
Blanquart, Samuel; Gascuel, Olivier
Plasmodium falciparum is responsible for the most acute form of human malaria. Most recent studies demonstrate that it belongs to a monophyletic lineage specialized in the infection of great ape hosts. Several other Plasmodium species cause human malaria. They all belong to another distinct lineage of parasites which infect a wider range of primate species. All known mammalian malaria parasites appear to be monophyletic. Their clade includes the two previous distinct lineages of parasites of primates and great apes, one lineage of rodent parasites, and presumably Hepatocystis species. Plasmodium falciparum and great ape parasites are commonly thought to be the sister-group of all other mammal-infecting malaria parasites. However, some studies supported contradictory origins and found parasites of great apes to be closer to those of rodents, or to those of other primates. To distinguish between these mutually exclusive hypotheses on the origin of Plasmodium falciparum and its great ape infecting relatives, we performed a comprehensive phylogenetic analysis based on a data set of three mitochondrial genes from 33 to 84 malaria parasites. We showed that malarial mitochondrial genes have evolved slowly and are compositionally homogeneous. We estimated their phylogenetic relationships using Bayesian and maximum-likelihood methods. Inferred trees were checked for their robustness to the (i) site selection, (ii) assumptions of various probabilistic models, and (iii) taxon sampling. Our results robustly support a common ancestry of rodent parasites and Plasmodium falciparum's relatives infecting great apes. Our results refute the most common view of the origin of great ape malaria parasites, and instead demonstrate the robustness of a less well-established phylogenetic hypothesis, under which Plasmodium falciparum and its relatives infecting great apes are closely related to rodent parasites. This study sheds light on the evolutionary history of Plasmodium falciparum, a
Matz, Joachim M; Kooij, Taco W A
Plasmodium berghei was identified as a parasite of thicket rats (Grammomys dolichurus) and Anopheles dureni mosquitoes in African highland forests. Successful adaptation to a range of rodent and mosquito species established P. berghei as a malaria model parasite. The introduction of stable transfection technology, permitted classical reverse genetics strategies and thus systematic functional profiling of the gene repertoire. In the past 10 years following the publication of the P. berghei genome sequence, many new tools for experimental genetics approaches have been developed and existing ones have been improved. The infection of mice is the principal limitation towards a genome-wide repository of mutant parasite lines. In the past few years, there have been some promising and most welcome developments that allow rapid selection and isolation of recombinant parasites while simultaneously minimising animal usage. Here, we provide an overview of all the currently available tools and methods.
Curtidor, Hernando; Patiño, Liliana C; Arévalo-Pinzón, Gabriela; Vanegas, Magnolia; Patarroyo, Manuel E; Patarroyo, Manuel A
Plasmodium falciparum malaria parasite invasion of erythrocytes is an essential step in host infection and the proteins involved in such invasion are the main target in developing an antimalarial vaccine. Secretory organelle-derived proteins (micronemal AMA1 protein and the RON2, 4, and 5 rhoptry neck proteins) have been recently described as components of moving junction complex formation allowing merozoites to move into a newly created parasitophorous vacuole. This study led to identifying RON5 regions involved in binding to human erythrocytes by using a highly robust, sensitive and specific receptor-ligand interaction assay; it is further shown that the RON5 protein remains highly conserved throughout different parasite strains. It is shown that the binding peptide-erythrocyte interaction is saturable and sensitive to chymotrypsin and trypsin. Invasion inhibition assays using erythrocyte binding peptides showed that the RON5-erythrocyte interaction could be critical for merozoite invasion of erythrocytes. This work provides evidence (for the first time) suggesting a fundamental role for RON5 in erythrocyte invasion. Copyright © 2013 Elsevier Inc. All rights reserved.
Borrmann, Steffen; Matuschewski, Kai
Plasmodium undergoes an obligate liver phase before the onset of malaria, which is caused exclusively by cyclic propagation of the parasite inside erythrocytes. The diagnostically inaccessible and clinically silent pre-erythrocytic expansion phase is a promising target for inducing sterilizing immunity against reinfections. Recent studies in rodent and human malaria models called attention to the induction of potent protective immunity by administration of anti-malarial drugs during sporozoite exposure. Here, we review the concept of drug-mediated pathogen arrest as a natural immunization strategy. This previously unrecognized immunological benefit might also open new opportunities for population-wide presumptive drug administration as an adjunct malaria control tool. Copyright © 2011 Elsevier Ltd. All rights reserved.
Mita, Toshihiro; Tachibana, Shin-Ichiro; Hashimoto, Muneaki; Hirai, Makoto
Although artemisinin combination therapies have been deployed as a first-line treatment for uncomplicated malaria in almost all endemic countries, artemisinin-resistant parasites have emerged and have gradually spread across the Greater Mekong subregions. There is growing concern that the resistant parasites may migrate to or emerge indigenously in sub-Saharan Africa, which might provoke a global increase in malaria-associated morbidity and mortality. Therefore, development of molecular markers that enable identification of artemisinin resistance with high sensitivity is urgently required to combat this issue. In 2014, a potential artemisinin-resistance responsible gene, Plasmodium falciparum kelch13, was discovered. Here, we review the genetic features of P. falciparum kelch13 and discuss its related resistant mechanisms and potential as a molecular marker.
Park, Yongkeun; Diez-Silva, Monica; Fu, Dan; Popescu, Gabriel; Choi, Wonshik; Barman, Ishan; Suresh, Subra; Feld, Michael S.
We present the light scattering of individual Plasmodium falciparum-parasitized human red blood cells (Pf-RBCs), and demonstrate progressive alterations to the scattering signal arising from the development of malaria-inducing parasites. By selectively imaging the electric fields using quantitative phase microscopy and a Fourier transform light scattering technique, we calculate the light scattering maps of individual Pf-RBCs. We show that the onset and progression of pathological states of the Pf-RBCs can be clearly identified by the static scattering maps. Progressive changes to the biophysical properties of the Pf-RBC membrane are captured from dynamic light scattering.
Peterson, Tina M L; Luckhart, Shirley
Malaria parasite infection in anopheline mosquitoes induces nitrosative and oxidative stresses that limit parasite development, but also damage mosquito tissues in proximity to the response. Based on these observations, we proposed that cellular defenses in the mosquito may be induced to minimize self-damage. Specifically, we hypothesized that peroxiredoxins (Prxs), enzymes known to detoxify reactive oxygen species (ROS) and reactive nitrogen oxide species (RNOS), protect mosquito cells. We identified an Anopheles stephensi 2-Cys Prx ortholog of Drosophila melanogaster Prx-4783, which protects fly cells against oxidative stresses. To assess function, AsPrx-4783 was overexpressed in D. melanogaster S2 and in A. stephensi (MSQ43) cells and silenced in MSQ43 cells with RNA interference before treatment with various ROS and RNOS. Our data revealed that AsPrx-4783 and DmPrx-4783 differ in host cell protection and that AsPrx-4783 protects A. stephensi cells against stresses that are relevant to malaria parasite infection in vivo, namely nitric oxide (NO), hydrogen peroxide, nitroxyl, and peroxynitrite. Further, AsPrx-4783 expression is induced in the mosquito midgut by parasite infection at times associated with peak nitrosative and oxidative stresses. Hence, whereas the NO-mediated defense response is toxic to both host and parasite, AsPrx-4783 may shift the balance in favor of the mosquito.
Birget, Philip L G; Repton, Charlotte; O'Donnell, Aidan J; Schneider, Petra; Reece, Sarah E
The trade-off between survival and reproduction is fundamental in the life history of all sexually reproducing organisms. This includes malaria parasites, which rely on asexually replicating stages for within-host survival and on sexually reproducing stages (gametocytes) for between-host transmission. The proportion of asexual stages that form gametocytes (reproductive effort) varies during infections-i.e. is phenotypically plastic-in response to changes in a number of within-host factors, including anaemia. However, how the density and age structure of red blood cell (RBC) resources shape plasticity in reproductive effort and impacts upon parasite fitness is controversial. Here, we examine how and why the rodent malaria parasite Plasmodium chabaudi alters its reproductive effort in response to experimental perturbations of the density and age structure of RBCs. We show that all four of the genotypes studied increase reproductive effort when the proportion of RBCs that are immature is elevated during host anaemia, and that the responses of the genotypes differ. We propose that anaemia (counterintuitively) generates a resource-rich environment in which parasites can afford to allocate more energy to reproduction (i.e. transmission) and that anaemia also exposes genetic variation to selection. From an applied perspective, adaptive plasticity in parasite reproductive effort could explain the maintenance of genetic variation for virulence and why anaemia is often observed as a risk factor for transmission in human infections. © 2017 The Authors.
Peterson, Tina M.L.; Luckhart, Shirley
Malaria parasite infection in anopheline mosquitoes induces nitrosative and oxidative stresses that limit parasite development, but also damage mosquito tissues in proximity to the response. Based on these observations, we proposed that cellular defenses in the mosquito may be induced to minimize self-damage. Specifically, we hypothesized that peroxiredoxins (Prxs), enzymes known to detoxify reactive oxygen species (ROS) and reactive nitrogen oxide species (RNOS), protect mosquito cells. We identified an Anopheles stephensi 2-Cys Prx ortholog of Drosophila melanogaster Prx-4783, which protects fly cells against oxidative stresses. To assess function, AsPrx-4783 was overexpressed in D. melanogaster (S2) and in A. stephensi (MSQ43) cells and silenced in MSQ43 cells with RNA interference before treatment with various ROS and RNOS. Our data revealed that AsPrx-4783 and DmPrx-4783 differ in host cell protection and that AsPrx-4783 protects A. stephensi cells against stresses that are relevant to malaria parasite infection in vivo, namely nitric oxide (NO), hydrogen peroxide, nitroxyl, and peroxynitrite. Further, AsPrx-4783 expression is induced in the mosquito midgut by parasite infection at times associated with peak nitrosative and oxidative stresses. Hence, whereas the NO-mediated defense response is toxic to both host and parasite, AsPrx-4783 may shift the balance in favor of the mosquito. PMID:16540402
Thomas, James A.; Collins, Christine R.; Das, Sujaan; Hackett, Fiona; Graindorge, Arnault; Bell, Donald; Deu, Edgar; Blackman, Michael J.
Malaria is caused by an obligate intracellular protozoan parasite that replicates within and destroys erythrocytes. Asexual blood stages of the causative agent of the most virulent form of human malaria, Plasmodium falciparum, can be cultivated indefinitely in vitro in human erythrocytes, facilitating experimental analysis of parasite cell biology, biochemistry and genetics. However, efforts to improve understanding of the basic biology of this important pathogen and to develop urgently required new antimalarial drugs and vaccines, suffer from a paucity of basic research tools. This includes a simple means of quantifying the effects of drugs, antibodies and gene modifications on parasite fitness and replication rates. Here we describe the development and validation of an extremely simple, robust plaque assay that can be used to visualise parasite replication and resulting host erythrocyte destruction at the level of clonal parasite populations. We demonstrate applications of the plaque assay by using it for the phenotypic characterisation of two P. falciparum conditional mutants displaying reduced fitness in vitro. PMID:27332706
Soares, Letícia; Marra, Peter; Gray, Lindsey; Ricklefs, Robert E
Island populations are vulnerable to introduced pathogens, as evidenced by extinction or population decline of several endemic Hawaiian birds caused by the malaria parasite, Plasmodium relictum (order Haemosporida). We analyzed blood samples from 363 birds caught near Guantánamo Bay, Cuba, for the presence of haemosporidian infections. We characterized parasite lineages by determining nucleotide variation of the parasite's mitochondrial cyt b gene. Fifty-nine individuals were infected, and we identified 7 lineages of haemosporidian parasites. Fifty individuals were infected by 6 Haemoproteus sp. lineages, including a newly characterized lineage of Haem. (Parahaemoproteus) sp. CUH01. Nine individuals carried the P. relictum lineage GRW4, including 5 endemic Cuban Grassquits (Tiaris canorus) and 1 migratory Cape May Warbler (Setophaga tigrina). A sequence of the merozoite surface protein gene from one Cuban Grassquit infected with GRW4 matched that of the Hawaiian haplotype Pr9. Our results indicate that resident and migratory Cuban birds are infected with a malaria lineage that has severely affected populations of several endemic Hawaiian birds. We suggest GRW4 may be associated with the lack of several bird species on Cuba that are ubiquitous elsewhere in the West Indies. From the standpoint of avian conservation in the Caribbean Basin, it will be important to determine the distribution of haemosporidian parasites, especially P. relictum GRW4, in Cuba as well as the pathogenicity of this lineage in species that occur and are absent from Cuba. © 2017 Society for Conservation Biology.
Malaria is one of the most serious infectious diseases at the beginning of the twenty-first century. Various membrane proteins are present in Plasmodium falciparum, the principal malaria pathogen. Among them, P. falciparum reticulocyte-binding protein homolog 5 (PfRh5) is indispensable for erythrocyte invasion, and has become a promising vaccine target. Basigin (CD147, EMMPRIN) has been identified as the erythrocyte receptor of PfRh5, and shown to be essential for the invasion of multiple strains of the pathogen. Fundamental information on basigin is fully described, including structure as a member of the immunoglobulin superfamily and function based on its interactions with external molecules and with proteins within the same membrane. The involvement of basigin in many diseases such as cancer and inflammatory diseases is also described, the implication being that anti-basigin therapy might be helpful to treat certain illnesses. Finally, PfRh5 as a vaccine candidate is covered, and its interaction with basigin is evaluated. The identification of basigin, a well-characterized membrane protein, as a receptor essential for malaria infection will contribute significantly to prevention and treatment of malaria. As an example, anti-basigin therapy can be considered an alternative approach to the treatment of drug-resistant malaria.
Schall, J J
The negative consequences of parasitic infection (virulence) were examined for two lizard malaria parasite-host associations: Plasmodium agamae and P. giganteum, parasites of the rainbow lizard, Agama agama, in Sierra Leone, West Africa; and P. mexicanum in the western fence lizard, Sceloporus occidentalis, in northern California. These malaria species vary greatly in their reproductive characteristics: P. agamae produces only 8 merozoites per schizont, P. giganteum yields over 100, and P. mexicanum an intermediate number. All three parasites appear to have had an ancient association with their host. In fence lizards, infection with malaria is associated with increased numbers of immature erythrocytes, decreased haemoglobin levels, decreased maximal oxygen consumption, and decreased running stamina. Not affected were numbers of erythrocytes, resting metabolic rate, and sprint running speed which is supported by anaerobic means in lizards. Infected male fence lizards had smaller testes, stored less fat in preparation for winter dormancy, were more often socially submissive and, unexpectedly, were more extravagantly coloured on the ventral surface (a sexually dimorphic trait) than non-infected males. Females also stored less fat and produced smaller clutches of eggs, a directly observed reduction in fitness. Infected fence lizards do not develop behavioural fevers. P. mexicanum appears to have broad thermal buffering abilities and thermal tolerance; the parasite's population growth was unaffected by experimental alterations in the lizard's body temperature. The data are less complete for A. agama, but infected lizards suffered similar haematological and physiological effects. Infected animals may be socially submissive because they appear to gather less insect prey, possibly a result of being forced into inferior territories. Infection does not reduce clutch size in rainbow lizards, but may lengthen the time between clutches. These results are compared with
Martin, Rowena E; Henry, Roselani I; Abbey, Janice L; Clements, John D; Kirk, Kiaran
Background The uptake of nutrients, expulsion of metabolic wastes and maintenance of ion homeostasis by the intraerythrocytic malaria parasite is mediated by membrane transport proteins. Proteins of this type are also implicated in the phenomenon of antimalarial drug resistance. However, the initial annotation of the genome of the human malaria parasite Plasmodium falciparum identified only a limited number of transporters, and no channels. In this study we have used a combination of bioinformatic approaches to identify and attribute putative functions to transporters and channels encoded by the malaria parasite, as well as comparing expression patterns for a subset of these. Results A computer program that searches a genome database on the basis of the hydropathy plots of the corresponding proteins was used to identify more than 100 transport proteins encoded by P. falciparum. These include all the transporters previously annotated as such, as well as a similar number of candidate transport proteins that had escaped detection. Detailed sequence analysis enabled the assignment of putative substrate specificities and/or transport mechanisms to all those putative transport proteins previously without. The newly-identified transport proteins include candidate transporters for a range of organic and inorganic nutrients (including sugars, amino acids, nucleosides and vitamins), and several putative ion channels. The stage-dependent expression of RNAs for 34 candidate transport proteins of particular interest are compared. Conclusion The malaria parasite possesses substantially more membrane transport proteins than was originally thought, and the analyses presented here provide a range of novel insights into the physiology of this important human pathogen. PMID:15774027
Yu, Fu-Dong; Yang, Shao-You; Li, Yuan-Yuan; Hu, Wei
Malaria continues to be one of the most severe global infectious diseases, as a major threat to human health and economic development. Network-based biological analysis is a promising approach to uncover key genes and biological processes from a network viewpoint, which could not be recognized from individual gene-based signatures. We integrated gene co-expression profile with protein-protein interaction and transcriptional regulation information to construct a comprehensive gene co-expression network of Plasmodium falciparum. Based on this network, we identified 10 core modules by using ICE (Iterative Clique Enumeration) algorithm, which were essential for malaria parasite development in intraerythrocytic developmental cycle (IDC) stages. In each module, all genes were highly correlated probably due to co-regulation or formation of a protein complex. Some of these genes were recognized to be differentially coexpressed among three close-by IDC stages. The gene of prpf8 (PFD0265w) encoding pre-mRNA processing splicing factor 8 product was identified as DCGs (differentially co-expressed genes) among IDC stages, although this gene function was seldom reported in previous researches. Integrating the species-specific gene prediction and differential co-expression gene detection, we found some modules could perform species-specific functions according to some of genes in these modules were species-specific genes, like the module 10. Furthermore, in order to reveal the underlying mechanisms of the erythrocyte invasion by P. falciparum, Steiner Tree algorithm was employed to identify the invasion subnetwork from our gene co-expression network. The subnetwork-based analysis indicated that some important Plasmodium parasite specific genes could corporate with each other and be co-regulated during the parasite invasion process, which including a head-to-head gene pair of PfRH2a (PF13_0198) and PfRH2b (MAL13P1.176). This study based on gene co-expression network could shed new
Khoury, David S.; Cromer, Deborah; Best, Shannon E.; James, Kylie R.; Sebina, Ismail; Haque, Ashraful; Davenport, Miles P.
The best correlate of malaria severity in human Plasmodium falciparum (Pf) infection is the total parasite load. Pf-infected humans could control parasite loads by two mechanisms, either decreasing parasite multiplication, or increasing parasite clearance. However, few studies have directly measured these two mechanisms in vivo. Here, we have directly quantified host clearance of parasites during Plasmodium infection in mice. We transferred labelled red blood cells (RBCs) from Plasmodium infected donors into uninfected and infected recipients, and tracked the fate of donor parasites by frequent blood sampling. We then applied age-based mathematical models to characterise parasite clearance in the recipient mice. Our analyses revealed an increased clearance of parasites in infected animals, particularly parasites of a younger developmental stage. However, the major decrease in parasite multiplication in infected mice was not mediated by increased clearance alone, but was accompanied by a significant reduction in the susceptibility of RBCs to parasitisation.
Hughes, Austin L; Verra, Federica
Phylogenetic analyses of the mitochondrial cytochrome b (cytb), apicoplast caseinolytic protease C (clpC), and 18S rRNA sequences of Plasmodium isolates from chimpanzees along with those of the virulent human malaria parasite P. falciparum showed that the common chimpanzee (Pan troglodytes) malaria parasites, assigned by Rich et al. (2009) to P. reichenowi, constitute a paraphyletic assemblage. The assumption that P. falciparum diverged from P. reichenowi as recently as 5000-50,000 years ago would require a rate of synonymous substitution/site/year in cytb and clpC on the order of 10(-5)-10(-6), several orders of magnitude higher than any known from eukaryotic organelle genomes, and would imply an unrealistically recent timing of the most recent common ancestor of P. falciparum mitochondrial genomes. The available data are thus most consistent with the hypothesis that P. reichenowi (in the strict sense) and P. falciparum co-speciated with their hosts about 5-7 million years ago.
Lin, Lee C.; Rovie-Ryan, Jeffrine J.; Kadir, Khamisah A.; Anderios, Fread; Hisam, Shamilah; Sharma, Reuben S.K.; Singh, Balbir; Conway, David J.
Multilocus microsatellite genotyping of Plasmodium knowlesi isolates previously indicated 2 divergent parasite subpopulations in humans on the island of Borneo, each associated with a different macaque reservoir host species. Geographic divergence was also apparent, and independent sequence data have indicated particularly deep divergence between parasites from mainland Southeast Asia and Borneo. To resolve the overall population structure, multilocus microsatellite genotyping was conducted on a new sample of 182 P. knowlesi infections (obtained from 134 humans and 48 wild macaques) from diverse areas of Malaysia, first analyzed separately and then in combination with previous data. All analyses confirmed 2 divergent clusters of human cases in Malaysian Borneo, associated with long-tailed macaques and pig-tailed macaques, and a third cluster in humans and most macaques in peninsular Malaysia. High levels of pairwise divergence between each of these sympatric and allopatric subpopulations have implications for the epidemiology and control of this zoonotic species. PMID:28322705
M, Eze Evelyn; Ezeiruaku, F C; Ukaji, D C
This study examined the experiential relationship between the parasite density and haematological parameters in male patients with Plasmodium falciparum infection in Port Harcourt, Nigeria reporting to malaria clinics. A total of one hundred and thirty-six (136) male patients were recruited. QBC haematological analysis, QBC malaria parasite specie identification and quantification and thin blood film for differential leucocytes count was used. The mean values of the haematological parameters in each quartile of parasite densities were determined using Microsoft Excel statistical package. Regression analysis was employed to model the experiential relationship between parasite density and haematological parameters. All regression relationships were tested and the relationship with the highest coefficient of determination (R2) was accepted as the valid relationship. The relationships tested included linear, polynomial, exponential, logarithmic and power relationships. The X- axis of the regression graphs stand for the parasite density while Y-axis stands for the respective haematological parameters Neutrophil count had a negative exponential relationship with the parasite density and is related to the parasite density by a polynomial equation model: ynm = -7E-07x2 - 0.0003x + 56.685.The coefficient of determination (R2) was 0.6140. This means that the rate of change of the parasitemia will depend on the initial value of the neutrophil. As the neutrophil increases, the parasitemia will tend to decrease in a double, triple and quadruple manner. The relationship between lymphocyte count, monocyte count and eosinophil count and parasite density was logarithmic and expressed by the following linear equation models: ylm = -2.371ln(x) + 37.296, ymm = 0.6965ln(x) + 5.7692 and yem = 0.9334ln(x) + 4.1718 in the same order. Their respective high coefficients of determination (R2) were 0.8027, 0.8867 and 0.9553. This logarithmic relationship means that each doubling of
M., Eze Evelyn; Ezeiruaku, F. C.; Ukaji, D. C.
This study examined the experiential relationship between the parasite density and haematological parameters in male patients with Plasmodium falciparum infection in Port Harcourt, Nigeria reporting to malaria clinics. A total of one hundred and thirty-six (136) male patients were recruited. QBC haematological analysis, QBC malaria parasite specie identification and quantification and thin blood film for differential leucocytes count was used. The mean values of the haematological parameters in each quartile of parasite densities were determined using Microsoft Excel statistical package. Regression analysis was employed to model the experiential relationship between parasite density and haematological parameters. All regression relationships were tested and the relationship with the highest coefficient of determination (R2) was accepted as the valid relationship. The relationships tested included linear, polynomial, exponential, logarithmic and power relationships. The X- axis of the regression graphs stand for the parasite density while Y-axis stands for the respective haematological parameters Neutrophil count had a negative exponential relationship with the parasite density and is related to the parasite density by a polynomial equation model: ynm = -7E-07x2 - 0.0003x + 56.685. The coefficient of determination (R2) was 0.6140. This means that the rate of change of the parasitemia will depend on the initial value of the neutrophil. As the neutrophil increases, the parasitemia will tend to decrease in a double, triple and quadruple manner. The relationship between lymphocyte count, monocyte count and eosinophil count and parasite density was logarithmic and expressed by the following linear equation models: ylm = -2.371ln(x) + 37.296, ymm = 0.6965ln(x) + 5.7692 and yem = 0.9334ln(x) + 4.1718 in the same order. Their respective high coefficients of determination (R2) were 0.8027, 0.8867 and 0.9553. This logarithmic relationship means that each doubling of monocyte
Egan, Timothy J; Combrinck, Jill M; Egan, Joanne; Hearne, Giovanni R; Marques, Helder M; Ntenteni, Skhumbuzo; Sewell, B Trevor; Smith, Peter J; Taylor, Dale; van Schalkwyk, Donelly A; Walden, Jason C
Chemical analysis has shown that Plasmodium falciparum trophozoites contain 61+/-2% of the iron within parasitized erythrocytes, of which 92+/-6% is located within the food vacuole. Of this, 88+/-9% is in the form of haemozoin. (57)Fe-Mössbauer spectroscopy shows that haemozoin is the only detectable iron species in trophozoites. Electron spectroscopic imaging confirms this conclusion. PMID:12033986
Kim, Jong-Dae; Nam, Kyeong-Min; Park, Chan-Young; Kim, Yu-Seop; Song, Hye-Jeong
Malaria must be diagnosed quickly and accurately at the initial infection stage and treated early to cure it properly. The malaria diagnosis method using a microscope requires much labor and time of a skilled expert and the diagnosis results vary greatly between individual diagnosticians. Therefore, to be able to measure the malaria parasite infection quickly and accurately, studies have been conducted for automated classification techniques using various parameters. In this study, by measuring classification technique performance according to changes of two parameters, the parameter values were determined that best distinguish normal from plasmodium-infected red blood cells. To reduce the stain deviation of the acquired images, a principal component analysis (PCA) grayscale conversion method was used, and as parameters, we used a malaria infected area and a threshold value used in binarization. The parameter values with the best classification performance were determined by selecting the value (72) corresponding to the lowest error rate on the basis of cell threshold value 128 for the malaria threshold value for detecting plasmodium-infected red blood cells.
Fricke, Jennifer M; Vardo-Zalik, Anne M; Schall, Jos J
Gene flow, and resulting degree of genetic differentiation among populations, will shape geographic genetic patterns and possibly local adaptation of parasites and their hosts. Some studies of Plasmodium falciparum in humans show substantial differentiation of the parasite in locations separated by only a few kilometers, a paradoxical finding for a parasite in a large, mobile host. We examined genetic differentiation of the malaria parasite Plasmodium mexicanum, and its lizard host, Sceloporus occidentalis, at 8 sites in northern California, with the use of variable microsatellite markers for both species. These lizards are small and highly territorial, so we expected local genetic differentiation of both parasite and lizard. Populations of P. mexicanum were found to be differentiated by analysis of 5 markers (F(st) values >0.05-0.10) over distances as short as 230-400 m, and greatly differentiated (F(st) values >0.25) for sites separated by approximately 10 km. In contrast, the lizard host had no, or very low, levels of differentiation for 3 markers, even for sites >40 km distant. Thus, gene flow for the lizard was great, but despite the mobility of the vertebrate host, the parasite was locally genetically distinct. This discrepancy could result if infected lizards move little, but their noninfected relatives were more mobile. Previous studies on the virulence of P. mexicanum for fence lizards support this hypothesis. However, changing prevalence of the parasite, without changes in density of the lizard, could also result in this pattern.
Spillman, Natalie J; Allen, Richard J W; Kirk, Kiaran
The intraerythrocytic malaria parasite, Plasmodium falciparum maintains an intracellular pH (pH(i)) of around 7.3. If subjected to an experimentally imposed acidification the parasite extrudes H(+), thereby undergoing a pH(i) recovery. In a recent study, Bennett et al. [Bennett TN, Patel J, Ferdig MT, Roepe PD. P. falciparum Na(+)/H(+) exchanger activity and quinine resistance. Mol Biochem Parasitol 2007;153:48-58] used the H(+) ionophore nigericin, in conjunction with an acidic medium, to acidify the parasite cytosol, and then used bovine serum albumin (BSA) to scavenge the nigericin from the parasite membrane. The ensuing Na(+)-dependent pH(i) recovery, seen following an increase in the extracellular pH, was attributed to a plasma membrane Na(+)/H(+) exchanger. This is at odds with previous reports that the primary H(+) extrusion mechanism in the parasite is a plasma membrane V-type H(+)-ATPase. Here we present evidence that the Na(+)-dependent efflux of H(+) from parasites acidified using nigericin/BSA is attributable to Na(+)/H(+) exchange via residual nigericin remaining in the parasite plasma membrane, rather than to endogenous transporter activity.
Huijben, Silvie; Chan, Brian H K; Read, Andrew F
Resistant malaria parasites are frequently found in mixed infections with drug-sensitive parasites. Particularly early in the evolutionary process, the frequency of these resistant mutants can be extremely low and below the level of molecular detection. We tested whether the rarity of resistance in infections impacted the health outcomes of treatment failure and the potential for onward transmission of resistance. Mixed infections of different ratios of resistant and susceptible Plasmodium chabaudi parasites were inoculated in laboratory mice and dynamics tracked during the course of infection using highly sensitive genotype-specific quantitative polymerase chain reaction (qPCR). Frequencies of resistant parasites ranged from 10% to 0.003% at the onset of treatment. We found that the rarer the resistant parasites were, the lower the likelihood of their onward transmission, but the worse the treatment failure was in terms of parasite numbers and disease severity. Strikingly, drug resistant parasites had the biggest impact on health outcomes when they were too rare to be detected by any molecular methods currently available for field samples. Indeed, in the field, these treatment failures would not even have been attributed to resistance.
Lutz, Holly L; Marra, Nicholas J; Grewe, Felix; Carlson, Jenny S; Palinauskas, Vaidas; Valkiūnas, Gediminas; Stanhope, Michael J
Acquiring genomic material from avian malaria parasites for genome sequencing has proven problematic due to the nucleation of avian erythrocytes, which produces a large ratio of host to parasite DNA (∼1 million to 1 bp). We tested the ability of laser capture microdissection microscopy to isolate parasite cells from individual avian erythrocytes for four avian Plasmodium species, and subsequently applied whole genome amplification and Illumina sequencing methods to Plasmodium relictum (lineage pSGS1) to produce sequence reads of the P. relictum genome. We assembled ∼335 kbp of parasite DNA from this species, but were unable to completely avoid contamination by host DNA and other sources. However, it is clear that laser capture microdissection holds promise for the isolation of genomic material from haemosporidian parasites in intracellular life stages. In particular, laser capture microdissection may prove useful for isolating individual parasite species from co-infected hosts. Although not explicitly tested in this study, laser capture microdissection may also have important applications for isolation of rare parasite lineages and museum specimens for which no fresh material exists.
Bernabeu, Maria; Danziger, Samuel A.; Avril, Marion; Vaz, Marina; Babar, Prasad H.; Brazier, Andrew J.; Herricks, Thurston; Maki, Jennifer N.; Pereira, Ligia; Mascarenhas, Anjali; Gomes, Edwin; Chery, Laura; Aitchison, John D.; Rathod, Pradipsinh K.; Smith, Joseph D.
The interplay between cellular and molecular determinants that lead to severe malaria in adults is unexplored. Here, we analyzed parasite virulence factors in an infected adult population in India and investigated whether severe malaria isolates impair endothelial protein C receptor (EPCR), a protein involved in coagulation and endothelial barrier permeability. Severe malaria isolates overexpressed specific members of the Plasmodium falciparum var gene/PfEMP1 (P. falciparum erythrocyte membrane protein 1) family that bind EPCR, including DC8 var genes that have previously been linked to severe pediatric malaria. Machine learning analysis revealed that DC6- and DC8-encoding var transcripts in combination with high parasite biomass were the strongest indicators of patient hospitalization and disease severity. We found that DC8 CIDRα1 domains from severe malaria isolates had substantial differences in EPCR binding affinity and blockade activity for its ligand activated protein C. Additionally, even a low level of inhibition exhibited by domains from two cerebral malaria isolates was sufficient to interfere with activated protein C-barrier protective activities in human brain endothelial cells. Our findings demonstrate an interplay between parasite biomass and specific PfEMP1 adhesion types in the development of adult severe malaria, and indicate that low impairment of EPCR function may contribute to parasite virulence. PMID:27185931
Kafsack, Björn F C; Rovira-Graells, Núria; Clark, Taane G; Bancells, Cristina; Crowley, Valerie M; Campino, Susana G; Williams, April E; Drought, Laura G; Kwiatkowski, Dominic P; Baker, David A; Cortés, Alfred; Llinás, Manuel
The life cycles of many parasites involve transitions between disparate host species, requiring these parasites to go through multiple developmental stages adapted to each of these specialized niches. Transmission of malaria parasites (Plasmodium spp.) from humans to the mosquito vector requires differentiation from asexual stages replicating within red blood cells into non-dividing male and female gametocytes. Although gametocytes were first described in 1880, our understanding of the molecular mechanisms involved in commitment to gametocyte formation is extremely limited, and disrupting this critical developmental transition remains a long-standing goal. Here we show that expression levels of the DNA-binding protein PfAP2-G correlate strongly with levels of gametocyte formation. Using independent forward and reverse genetics approaches, we demonstrate that PfAP2-G function is essential for parasite sexual differentiation. By combining genome-wide PfAP2-G cognate motif occurrence with global transcriptional changes resulting from PfAP2-G ablation, we identify early gametocyte genes as probable targets of PfAP2-G and show that their regulation by PfAP2-G is critical for their wild-type level expression. In the asexual blood-stage parasites pfap2-g appears to be among a set of epigenetically silenced loci prone to spontaneous activation. Stochastic activation presents a simple mechanism for a low baseline of gametocyte production. Overall, these findings identify PfAP2-G as a master regulator of sexual-stage development in malaria parasites and mark the first discovery of a transcriptional switch controlling a differentiation decision in protozoan parasites.
Background Frozen blood clots remaining after serum collection is an often disregarded source of host and pathogen DNA due to troublesome handling and suboptimal outcome. Methods High-speed shaking of clot samples in a cell disruptor manufactured for homogenization of tissue and faecal specimens was evaluated for processing frozen blood clots for DNA extraction. The method was compared to two commercial clot protocols based on a chemical kit and centrifugation through a plastic sieve, followed by the same DNA extraction protocol. Blood clots with different levels of parasitaemia (1-1,000 p/μl) were prepared from parasite cultures to assess sensitivity of PCR detection. In addition, clots retrieved from serum samples collected within two epidemiological studies in Kenya (n = 630) were processed by high speed shaking and analysed by PCR for detection of malaria parasites and the human α-thalassaemia gene. Results High speed shaking succeeded in fully dispersing the clots and the method generated the highest DNA yield. The level of PCR detection of P. falciparum parasites and the human thalassaemia gene was the same as samples optimally collected with an anticoagulant. The commercial clot protocol and centrifugation through a sieve failed to fully dissolve the clots and resulted in lower sensitivity of PCR detection. Conclusions High speed shaking was a simple and efficacious method for homogenizing frozen blood clots before DNA purification and resulted in PCR templates of high quality both from humans and malaria parasites. This novel method enables genetic studies from stored blood clots. PMID:21824391
Butzloff, Sabine; Groves, Matthew R; Wrenger, Carsten; Müller, Ingrid B
The malaria parasite Plasmodium falciparum proliferates within human erythrocytes and is thereby exposed to a variety of reactive oxygen species (ROS) such as hydrogen peroxide, hydroxyl radical, superoxide anion, and highly reactive singlet oxygen ((1)O(2)). While most ROS are already well studied in the malaria parasite, singlet oxygen has been neglected to date. In this study we visualized the generation of (1)O(2) by live cell fluorescence microscopy using 3-(p-aminophenyl) fluorescein as an indicator dye. While (1) O(2) is found restrictively in the parasite, its amount varies during erythrocytic schizogony. Since the photosensitizer cercosporin generates defined amounts of (1)O(2) we have established a new cytometric method that allows the stage specific quantification of (1)O(2). Therefore, the parasites were first classified into three main stages according to their respective pixel-area of 200-600 pixels for rings, 700-1,200 pixels for trophozoites and 1,400-2,500 pixels for schizonts. Interestingly the highest mean concentration of endogenous (1)O(2) of 0.34 nM is found in the trophozoites stage, followed by 0.20 nM (ring stage) and 0.10 nM (schizont stage) suggesting that (1)O(2) derives predominantly from the digestion of hemoglobin.
Pollitt, Laura C.; Bram, Joshua T.; Blanford, Simon; Jones, Matthew J.; Read, Andrew F.
Very little is known about how vector-borne pathogens interact within their vector and how this impacts transmission. Here we show that mosquitoes can accumulate mixed strain malaria infections after feeding on multiple hosts. We found that parasites have a greater chance of establishing and reach higher densities if another strain is already present in a mosquito. Mixed infections contained more parasites but these larger populations did not have a detectable impact on vector survival. Together these results suggest that mosquitoes taking multiple infective bites may disproportionally contribute to malaria transmission. This will increase rates of mixed infections in vertebrate hosts, with implications for the evolution of parasite virulence and the spread of drug-resistant strains. Moreover, control measures that reduce parasite prevalence in vertebrate hosts will reduce the likelihood of mosquitoes taking multiple infective feeds, and thus disproportionally reduce transmission. More generally, our study shows that the types of strain interactions detected in vertebrate hosts cannot necessarily be extrapolated to vectors. PMID:26181518
Pollitt, Laura C; Bram, Joshua T; Blanford, Simon; Jones, Matthew J; Read, Andrew F
Very little is known about how vector-borne pathogens interact within their vector and how this impacts transmission. Here we show that mosquitoes can accumulate mixed strain malaria infections after feeding on multiple hosts. We found that parasites have a greater chance of establishing and reach higher densities if another strain is already present in a mosquito. Mixed infections contained more parasites but these larger populations did not have a detectable impact on vector survival. Together these results suggest that mosquitoes taking multiple infective bites may disproportionally contribute to malaria transmission. This will increase rates of mixed infections in vertebrate hosts, with implications for the evolution of parasite virulence and the spread of drug-resistant strains. Moreover, control measures that reduce parasite prevalence in vertebrate hosts will reduce the likelihood of mosquitoes taking multiple infective feeds, and thus disproportionally reduce transmission. More generally, our study shows that the types of strain interactions detected in vertebrate hosts cannot necessarily be extrapolated to vectors.
Nguitragool, Wang; Rayavara, Kempaiah; Desai, Sanjay A.
The plasmodial surface anion channel mediates uptake of nutrients and other solutes into erythrocytes infected with malaria parasites. The clag3 genes of P. falciparum determine this channel’s activity in human malaria, but how the encoded proteins contribute to transport is unknown. Here, we used proteases to examine the channel’s composition and function. While proteases with distinct specificities all cleaved within an extracellular domain of CLAG3, they produced differing degrees of transport inhibition. Chymotrypsin-induced inhibition depended on parasite genotype, with channels induced by the HB3 parasite affected to a greater extent than those of the Dd2 clone. Inheritance of functional proteolysis in the HB3×Dd2 genetic cross, DNA transfection, and gene silencing experiments all pointed to the clag3 genes, providing independent evidence for a role of these genes. Protease protection assays with a Dd2-specific inhibitor and site-directed mutagenesis revealed that a variant L1115F residue on a CLAG3 extracellular loop contributes to inhibitor binding and accounts for differences in functional proteolysis. These findings indicate that surface-exposed CLAG3 is the relevant pool of this protein for channel function. They also suggest structural models for how exposed CLAG3 domains contribute to pore formation and parasite nutrient uptake. PMID:24699906
Zheng, Hong; Tan, Zhangping; Zhou, TaoLi; Zhu, Feng; Ding, Yan; Liu, Taiping; Wu, Yuzhang; Xu, Wenyue
TLRs (Toll-like receptors) play an important role in the initiation of innate immune responses against invading microorganisms. Although several TLRs have been reported to be involved in the innate immune response against the blood-stage of malaria parasites, the role of TLRs in the development of the pre-erythrocytic stage is still largely unknown. Here, we found that sporozoite and its lysate could significantly activate the TLR2, and induce macrophages to release proinflammatory cytokines, including IL-6, MCP-1 and TNF-α, in a TLR2-dependent manner. Further studies showed that sporozoite and its lysate could be recognized by either TLR2 homodimers or TLR2/1 and TLR2/6 heterodimers, implicating the complexity of TLR2 agonist in sporozoite. Interestingly, the TLR2 signaling can significantly suppress the development of the pre-erythrocytic stage of Plasmodium yoelii, as both liver parasite load and subsequent parasitemia were significantly elevated in both TLR2- and MyD88-deficient mice. Additionally, the observed higher level of parasite burden in TLR2−/− mice was found to be closely associated with a reduction in proinflammatory cytokines in the liver. Therefore, we provide the first evidence that sporozoites can activate the TLR2 signaling, which in turn significantly inhibits the intrahepatic parasites. This may provide us with novel clues to design preventive anti-malaria therapies. PMID:26667391
Lee, Andrew H; Symington, Lorraine S; Fidock, David A
Research into the complex genetic underpinnings of the malaria parasite Plasmodium falciparum is entering a new era with the arrival of site-specific genome engineering. Previously restricted only to model systems but now expanded to most laboratory organisms, and even to humans for experimental gene therapy studies, this technology allows researchers to rapidly generate previously unattainable genetic modifications. This technological advance is dependent on DNA double-strand break repair (DSBR), specifically homologous recombination in the case of Plasmodium. Our understanding of DSBR in malaria parasites, however, is based largely on assumptions and knowledge taken from other model systems, which do not always hold true in Plasmodium. Here we describe the causes of double-strand breaks, the mechanisms of DSBR, and the differences between model systems and P. falciparum. These mechanisms drive basic parasite functions, such as meiosis, antigen diversification, and copy number variation, and allow the parasite to continually evolve in the contexts of host immune pressure and drug selection. Finally, we discuss the new technologies that leverage DSBR mechanisms to accelerate genetic investigations into this global infectious pathogen.
Lee, Andrew H.; Symington, Lorraine S.
SUMMARY Research into the complex genetic underpinnings of the malaria parasite Plasmodium falciparum is entering a new era with the arrival of site-specific genome engineering. Previously restricted only to model systems but now expanded to most laboratory organisms, and even to humans for experimental gene therapy studies, this technology allows researchers to rapidly generate previously unattainable genetic modifications. This technological advance is dependent on DNA double-strand break repair (DSBR), specifically homologous recombination in the case of Plasmodium. Our understanding of DSBR in malaria parasites, however, is based largely on assumptions and knowledge taken from other model systems, which do not always hold true in Plasmodium. Here we describe the causes of double-strand breaks, the mechanisms of DSBR, and the differences between model systems and P. falciparum. These mechanisms drive basic parasite functions, such as meiosis, antigen diversification, and copy number variation, and allow the parasite to continually evolve in the contexts of host immune pressure and drug selection. Finally, we discuss the new technologies that leverage DSBR mechanisms to accelerate genetic investigations into this global infectious pathogen. PMID:25184562
Aminake, Makoah Nigel; Arndt, Hans-Dieter; Pradel, Gabriele
The ubiquitin/proteasome system serves as a regulated protein degradation pathway in eukaryotes, and is involved in many cellular processes featuring high protein turnover rates, such as cell cycle control, stress response and signal transduction. In malaria parasites, protein quality control is potentially important because of the high replication rate and the rapid transformations of the parasite during life cycle progression. The proteasome is the core of the degradation pathway, and is a major proteolytic complex responsible for the degradation and recycling of non-functional ubiquitinated proteins. Annotation of the genome for Plasmodium falciparum, the causative agent of malaria tropica, revealed proteins with similarity to human 26S proteasome subunits. In addition, a bacterial ClpQ/hslV threonine peptidase-like protein was identified. In recent years several independent studies indicated an essential function of the parasite proteasome for the liver, blood and transmission stages. In this review, we compile evidence for protein recycling in Plasmodium parasites and discuss the role of the 26S proteasome as a prospective multi-stage target for antimalarial drug discovery programs. PMID:24533266
Claessens, Antoine; Affara, Muna; Assefa, Samuel A.; Kwiatkowski, Dominic P.; Conway, David J.
Cultured human pathogens may differ significantly from source populations. To investigate the genetic basis of laboratory adaptation in malaria parasites, clinical Plasmodium falciparum isolates were sampled from patients and cultured in vitro for up to three months. Genome sequence analysis was performed on multiple culture time point samples from six monoclonal isolates, and single nucleotide polymorphism (SNP) variants emerging over time were detected. Out of a total of five positively selected SNPs, four represented nonsense mutations resulting in stop codons, three of these in a single ApiAP2 transcription factor gene, and one in SRPK1. To survey further for nonsense mutants associated with culture, genome sequences of eleven long-term laboratory-adapted parasite strains were examined, revealing four independently acquired nonsense mutations in two other ApiAP2 genes, and five in Epac. No mutants of these genes exist in a large database of parasite sequences from uncultured clinical samples. This implicates putative master regulator genes in which multiple independent stop codon mutations have convergently led to culture adaptation, affecting most laboratory lines of P. falciparum. Understanding the adaptive processes should guide development of experimental models, which could include targeted gene disruption to adapt fastidious malaria parasite species to culture. PMID:28117431
Kaushansky, Alexis; Mikolajczak, Sebastian A.; Vignali, Marissa; Kappe, Stefan H.I.
Forty percent of people worldwide are at risk of malaria infection, and despite control efforts it remains the most deadly parasitic disease. Unfortunately, rapid discovery and development of new interventions for malaria are hindered by the lack of small animal models that support the complex life cycles of the main parasite species infecting humans. Such tools must accommodate human parasite tropism for human tissue. Mouse models with human tissue developed to date have already enhanced our knowledge of human parasites, and are useful tools for assessing anti-parasitic interventions. Although these systems are imperfect, their continued refinement will likely broaden their utility. Some of the malaria parasite’s interactions with human hepatocytes and human erythrocytes can already be modeled with available humanized mouse systems. However, interactions with other relevant human tissues such as the skin and immune system, as well as most transitions between life cycle stages in vivo will require refinement of existing humanized mouse models. Here, we review the recent successes achieved in modeling human malaria parasite biology in humanized mice, and discuss how these models have potential to become an valuable part of the toolbox used for understanding the biology of, and development of interventions to, malaria. PMID:24506682
Blackburn, Tim M; Ewen, John G
We provide an overview of the current state of knowledge of parasites in biological invasions by alien species. Parasites have frequently been invoked as drivers of invasions, but have received less attention as invasion passengers. The evidence to date that parasites drive invasions by hosts is weak: while there is abundant evidence that parasites have effects in the context of alien invasions, there is little evidence to suggest that parasites have differential effects on alien species that succeed versus fail in the invasion process. Particular case studies are suggestive but not yet informative about general effects. What evidence there is for parasites as aliens suggests that the same kind of factors determine their success as for non-parasites. Thus, availability is likely to be an important determinant of the probability of translocation. Establishment and spread are likely to depend on propagule pressure and on the environment being suitable (all necessary hosts and vectors are present); the likelihood of both of these dependencies being favourable will be affected by traits relating to parasite life history and demography. The added complication for the success of parasites as aliens is that often this will depend on the success of their hosts. We discuss how these conclusions help us to understand the likely effects of parasites on the success of establishing host populations (alien or native).
Duarte, Neuza; Ramesar, Jai; Avramut, M. Cristina; Koster, Abraham J.; Dessens, Johannes T.; Frischknecht, Friedrich; Chevalley-Maurel, Séverine; Janse, Chris J.; Franke-Fayard, Blandine; Mair, Gunnar R.
Transmission of the malaria parasite from the mammalian host to the mosquito vector requires the formation of adequately adapted parasite forms and stage-specific organelles. Here we show that formation of the crystalloid—a unique and short-lived organelle of the Plasmodium ookinete and oocyst stage required for sporogony—is dependent on the precisely timed expression of the S-acyl-transferase DHHC10. DHHC10, translationally repressed in female Plasmodium berghei gametocytes, is activated translationally during ookinete formation, where the protein is essential for the formation of the crystalloid, the correct targeting of crystalloid-resident protein LAP2, and malaria parasite transmission. PMID:27303037
Davenport, Gregory C.; Hittner, James B.; Otieno, Vincent; ...
Bmore » acteremia and malaria coinfection is a common and life-threatening condition in children residing in sub-Saharan Africa. We previously showed that coinfection with Gram negative (G[−]) entericacilli and Plasmodium falciparum ( Pf [+]) was associated with reduced high-density parasitemia (HDP, >10,000 parasites/ μ L), enhanced respiratory distress, and severe anemia. Since inflammatory mediators are largely unexplored in such coinfections, circulating cytokines were determined in four groups of children ( n = 206 , aged <3 yrs): healthy; Pf [+] alone; G[−] coinfected; and G[+] coinfected. Staphylococcus aureus and non-Typhi Salmonella were the most frequently isolated G[+] and G[−] organisms, respectively. Coinfected children, particularly those with G[−] pathogens, had lower parasite burden (peripheral and geometric mean parasitemia and HDP). In addition, both coinfected groups had increased IL-4, IL-5, IL-7, IL-12, IL-15, IL-17, IFN- γ , and IFN- α and decreased TNF- α relative to malaria alone. Children with G[−] coinfection had higher IL-1 β and IL-1Ra and lower IL-10 than the Pf [+] group and higher IFN- γ than the G[+] group. To determine how the immune response to malaria regulates parasitemia, cytokine production was investigated with a multiple mediation model. Cytokines with the greatest mediational impact on parasitemia were IL-4, IL-10, IL-12, and IFN- γ . Results here suggest that enhanced immune activation, especially in G[−] coinfected children, acts to reduce malaria parasite burden.« less
Verma, Ruchi; Varshney, Grish C; Raghava, G P S
The rate of human death due to malaria is increasing day-by-day. Thus the malaria causing parasite Plasmodium falciparum (PF) remains the cause of concern. With the wealth of data now available, it is imperative to understand protein localization in order to gain deeper insight into their functional roles. In this manuscript, an attempt has been made to develop prediction method for the localization of mitochondrial proteins. In this study, we describe a method for predicting mitochondrial proteins of malaria parasite using machine-learning technique. All models were trained and tested on 175 proteins (40 mitochondrial and 135 non-mitochondrial proteins) and evaluated using five-fold cross validation. We developed a Support Vector Machine (SVM) model for predicting mitochondrial proteins of P. falciparum, using amino acids and dipeptides composition and achieved maximum MCC 0.38 and 0.51, respectively. In this study, split amino acid composition (SAAC) is used where composition of N-termini, C-termini, and rest of protein is computed separately. The performance of SVM model improved significantly from MCC 0.38 to 0.73 when SAAC instead of simple amino acid composition was used as input. In addition, SVM model has been developed using composition of PSSM profile with MCC 0.75 and accuracy 91.38%. We achieved maximum MCC 0.81 with accuracy 92% using a hybrid model, which combines PSSM profile and SAAC. When evaluated on an independent dataset our method performs better than existing methods. A web server PFMpred has been developed for predicting mitochondrial proteins of malaria parasites ( http://www.imtech.res.in/raghava/pfmpred/).
Davenport, Gregory C.; Mukundan, Harshini; Fenimore, Paul W.; Hengartner, Nicolas W.; McMahon, Benjamin H.; Ong'echa, John M.
Bacteremia and malaria coinfection is a common and life-threatening condition in children residing in sub-Saharan Africa. We previously showed that coinfection with Gram negative (G[−]) enteric Bacilli and Plasmodium falciparum (Pf[+]) was associated with reduced high-density parasitemia (HDP, >10,000 parasites/μL), enhanced respiratory distress, and severe anemia. Since inflammatory mediators are largely unexplored in such coinfections, circulating cytokines were determined in four groups of children (n = 206, aged <3 yrs): healthy; Pf[+] alone; G[−] coinfected; and G[+] coinfected. Staphylococcus aureus and non-Typhi Salmonella were the most frequently isolated G[+] and G[−] organisms, respectively. Coinfected children, particularly those with G[−] pathogens, had lower parasite burden (peripheral and geometric mean parasitemia and HDP). In addition, both coinfected groups had increased IL-4, IL-5, IL-7, IL-12, IL-15, IL-17, IFN-γ, and IFN-α and decreased TNF-α relative to malaria alone. Children with G[−] coinfection had higher IL-1β and IL-1Ra and lower IL-10 than the Pf[+] group and higher IFN-γ than the G[+] group. To determine how the immune response to malaria regulates parasitemia, cytokine production was investigated with a multiple mediation model. Cytokines with the greatest mediational impact on parasitemia were IL-4, IL-10, IL-12, and IFN-γ. Results here suggest that enhanced immune activation, especially in G[−] coinfected children, acts to reduce malaria parasite burden. PMID:27418744
Tonkin, Michelle L; Crawford, Joanna; Lebrun, Maryse L; Boulanger, Martin J
Host cell invasion by the obligate intracellular apicomplexan parasites, including Plasmodium (malaria) and Toxoplasma (toxoplasmosis), requires a step-wise mechanism unique among known host–pathogen interactions. A key step is the formation of the moving junction (MJ) complex, a circumferential constriction between the apical tip of the parasite and the host cell membrane that traverses in a posterior direction to enclose the parasite in a protective vacuole essential for intracellular survival. The leading model of MJ assembly proposes that Rhoptry Neck Protein 2 (RON2) is secreted into the host cell and integrated into the membrane where it serves as the receptor for apical membrane antigen 1 (AMA1) on the parasite surface. We have previously demonstrated that the AMA1-RON2 interaction is an effective target for inhibiting apicomplexan invasion. To better understand the AMA1-dependant molecular recognition events that promote invasion, including the significant AMA1-RON2 interaction, we present the structural characterization of AMA1 from the apicomplexan parasites Babesia divergens (BdAMA1) and Neospora caninum (NcAMA1) by X-ray crystallography. These studies offer intriguing structural insight into the RON2-binding surface groove in the AMA1 apical domain, which shows clear evidence for receptor–ligand co-evolution, and the hyper variability of the membrane proximal domain, which in Plasmodium is responsible for direct binding to erythrocytes. By incorporating the structural analysis of BdAMA1 and NcAMA1 with existing AMA1 structures and complexes we were able to define conserved pockets in the AMA1 apical groove that could be targeted for the design of broadly reactive therapeutics. PMID:23169033
Ch’ng, Jun-Hong; Ursing, Johan; Tan, Kevin Shyong-Wei
The antimalarial drug chloroquine (CQ) has been sidelined in the fight against falciparum malaria due to wide-spread CQ resistance. Replacement drugs like sulfadoxine, pyrimethamine and mefloquine have also since been surpassed with the evolution of multi-drug resistant parasites. Even the currently recommended artemisinin-based combination therapies show signs of compromise due to the recent spread of artemisinin delayed-clearance parasites. Though there have been promising breakthroughs in the pursuit of new effective antimalarials, the development and strategic deployment of such novel chemical entities takes time. We therefore argue that there is a crucial need to re-examine the usefulness of ‘outdated’ drugs like chloroquine, and explore if they might be effective alternative therapies in the interim. We suggest that a novel parasite cell death (pCD) pathway may be exploited through the reformulation of CQ to address this need.
Soni, Rani; Sharma, Drista; Rai, Praveen; Sharma, Bhaskar; Bhatt, Tarun K.
Irrespective of various efforts, malaria persist the most debilitating effect in terms of morbidity and mortality. Moreover, the existing drugs are also vulnerable to the emergence of drug resistance. To explore the potential targets for designing the most effective antimalarial therapies, it is required to focus on the facts of biochemical mechanism underlying the process of parasite survival and disease pathogenesis. This review is intended to bring out the existing knowledge about the functions and components of the major signaling pathways such as kinase signaling, calcium signaling, and cyclic nucleotide-based signaling, serving the various aspects of the parasitic asexual stage and highlighted the Toll-like receptors, glycosylphosphatidylinositol-mediated signaling, and molecular events in cytoadhesion, which elicit the host immune response. This discussion will facilitate a look over essential components for parasite survival and disease progression to be implemented in discovery of novel antimalarial drugs and vaccines. PMID:28400771
Soni, Rani; Sharma, Drista; Rai, Praveen; Sharma, Bhaskar; Bhatt, Tarun K
Irrespective of various efforts, malaria persist the most debilitating effect in terms of morbidity and mortality. Moreover, the existing drugs are also vulnerable to the emergence of drug resistance. To explore the potential targets for designing the most effective antimalarial therapies, it is required to focus on the facts of biochemical mechanism underlying the process of parasite survival and disease pathogenesis. This review is intended to bring out the existing knowledge about the functions and components of the major signaling pathways such as kinase signaling, calcium signaling, and cyclic nucleotide-based signaling, serving the various aspects of the parasitic asexual stage and highlighted the Toll-like receptors, glycosylphosphatidylinositol-mediated signaling, and molecular events in cytoadhesion, which elicit the host immune response. This discussion will facilitate a look over essential components for parasite survival and disease progression to be implemented in discovery of novel antimalarial drugs and vaccines.
Jasinskiene, Nijole; Coleman, Judy; Ashikyan, Aurora; Salampessy, Michael; Marinotti, Osvaldo; James, Anthony A
Genetic strategies for controlling malaria transmission based on engineering pathogen resistance in Anopheles mosquitoes are being tested in a number of animal models. A key component is the effector molecule and the efficiency with which it reduces parasite transmission. Single-chain antibodies (scFvs) that bind the circumsporozoite protein of the avian parasite, Plasmodium gallinaceum, can reduce mean intensities of sporozoite infection of salivary glands by two to four orders of magnitude in transgenic Aedes aegypti. Significantly, mosquitoes with as few as 20 sporozoites in their salivary glands are infectious for a vertebrate host, Gallus gallus. Although scFvs hold promise as effector molecules, they will have to reduce mean intensities of infection to zero to prevent parasite transmission and disease. We conclude that similar endpoints must be reached with human pathogens if we are to expect an effect on disease transmission.
Walker, Melanie; Kublin, James G.; Zunt, Joseph R.
Immunosuppression associated with HIV infection or following transplantation increases susceptibility to central nervous system (CNS) infections. Because of increasing international travel, parasites that were previously limited to tropical regions pose an increasing infectious threat to populations at risk for acquiring opportunistic infection, especially people with HIV infection or individuals who have received a solid organ or bone marrow transplant. Although long-term immunosuppression caused by medications such as prednisone likely also increases the risk for acquiring infection and for developing CNS manifestations, little published information is available to support this hypothesis. In an earlier article published in Clinical Infectious Diseases, we described the neurologic manifestations of some of the more common parasitic CNS infections. This review will discuss the presentation, diagnosis, and treatment of the following additional parasitic CNS infections: malaria, microsporidiosis, leishmaniasis, and African trypanosomiasis. PMID:16323101
Walker, Melanie; Kublin, James G; Zunt, Joseph R
Immunosuppression associated with HIV infection or following transplantation increases susceptibility to central nervous system (CNS) infections. Because of increasing international travel, parasites that were previously limited to tropical regions pose an increasing infectious threat to populations at risk for acquiring opportunistic infection, especially people with HIV infection or individuals who have received a solid organ or bone marrow transplant. Although long-term immunosuppression caused by medications such as prednisone likely also increases the risk for acquiring infection and for developing CNS manifestations, little published information is available to support this hypothesis. In an earlier article published in Clinical Infectious Diseases, we described the neurologic manifestations of some of the more common parasitic CNS infections. This review will discuss the presentation, diagnosis, and treatment of the following additional parasitic CNS infections: malaria, microsporidiosis, leishmaniasis, and African trypanosomiasis.
Bour, Tania; Mahmoudi, Nassira; Kapps, Delphine; Thiberge, Sabine; Bargieri, Daniel; Ménard, Robert; Frugier, Magali
The malaria-causing Plasmodium parasites are transmitted to vertebrates by mosquitoes. To support their growth and replication, these intracellular parasites, which belong to the phylum Apicomplexa, have developed mechanisms to exploit their hosts. These mechanisms include expropriation of small metabolites from infected host cells, such as purine nucleotides and amino acids. Heretofore, no evidence suggested that transfer RNAs (tRNAs) could also be exploited. We identified an unusual gene in Apicomplexa with a coding sequence for membrane-docking and structure-specific tRNA binding. This Apicomplexa protein-designated tRip (tRNA import protein)-is anchored to the parasite plasma membrane and directs import of exogenous tRNAs. In the absence of tRip, the fitness of the parasite stage that multiplies in the blood is significantly reduced, indicating that the parasite may need host tRNAs to sustain its own translation and/or as regulatory RNAs. Plasmodium is thus the first example, to our knowledge, of a cell importing exogenous tRNAs, suggesting a remarkable adaptation of this parasite to extend its reach into host cell biology.
Bour, Tania; Mahmoudi, Nassira; Kapps, Delphine; Thiberge, Sabine; Bargieri, Daniel; Ménard, Robert; Frugier, Magali
The malaria-causing Plasmodium parasites are transmitted to vertebrates by mosquitoes. To support their growth and replication, these intracellular parasites, which belong to the phylum Apicomplexa, have developed mechanisms to exploit their hosts. These mechanisms include expropriation of small metabolites from infected host cells, such as purine nucleotides and amino acids. Heretofore, no evidence suggested that transfer RNAs (tRNAs) could also be exploited. We identified an unusual gene in Apicomplexa with a coding sequence for membrane-docking and structure-specific tRNA binding. This Apicomplexa protein—designated tRip (tRNA import protein)—is anchored to the parasite plasma membrane and directs import of exogenous tRNAs. In the absence of tRip, the fitness of the parasite stage that multiplies in the blood is significantly reduced, indicating that the parasite may need host tRNAs to sustain its own translation and/or as regulatory RNAs. Plasmodium is thus the first example, to our knowledge, of a cell importing exogenous tRNAs, suggesting a remarkable adaptation of this parasite to extend its reach into host cell biology. PMID:27071116
Mukhin, Andrey; Palinauskas, Vaidas; Platonova, Elena; Kobylkov, Dmitry; Vakoliuk, Irina; Valkiūnas, Gediminas
Avian malaria parasites (Haemosporida, Plasmodium) are of cosmopolitan distribution, and they have a significant impact on vertebrate host fitness. Experimental studies show that high parasitemia often develops during primary malaria infections. However, field studies only occasionally reveal high parasitemia in free-living birds sampled using the traditional methods of mist-netting or trapping, and light chronic infections predominate. The reason for this discrepancy between field observation and experimental data remains insufficiently understood. Since mist-netting is a passive capture method, two main parameters determine its success in sampling infected birds in wildlife, i. e. the presence of parasitized birds at a study site and their mobility. In other words, the trapping probability depends on the survival rate of birds and their locomotor activity during infection. Here we test (1) the mortality rate of wild birds infected with Plasmodium relictum (the lineage pSGS1), (2) the changes in their behaviour during presence of an aerial predator, and (3) the changes in their locomotor activity at the stage of high primary parasitemia.We show that some behavioural features which might affect a bird's survival during a predator attack (time of reaction, speed of flush flight and take off angle) did not change significantly during primary infection. However, the locomotor activity of infected birds was almost halved compared to control (non-infected) birds during the peak of parasitemia. We report (1) the markedly reduced mobility and (2) the 20% mortality rate caused by P. relictum and conclude that these factors are responsible for the underrepresentation of birds in mist nets and traps during the stage of high primary parasitemia in wildlife. This study indicates that the widespread parasite, P. relictum (pSGS1) influences the behaviour of birds during primary parasitemia. Experimental studies combined with field observations are needed to better understand the
Mukhin, Andrey; Palinauskas, Vaidas; Platonova, Elena; Kobylkov, Dmitry; Vakoliuk, Irina; Valkiūnas, Gediminas
Avian malaria parasites (Haemosporida, Plasmodium) are of cosmopolitan distribution, and they have a significant impact on vertebrate host fitness. Experimental studies show that high parasitemia often develops during primary malaria infections. However, field studies only occasionally reveal high parasitemia in free-living birds sampled using the traditional methods of mist-netting or trapping, and light chronic infections predominate. The reason for this discrepancy between field observation and experimental data remains insufficiently understood. Since mist-netting is a passive capture method, two main parameters determine its success in sampling infected birds in wildlife, i. e. the presence of parasitized birds at a study site and their mobility. In other words, the trapping probability depends on the survival rate of birds and their locomotor activity during infection. Here we test (1) the mortality rate of wild birds infected with Plasmodium relictum (the lineage pSGS1), (2) the changes in their behaviour during presence of an aerial predator, and (3) the changes in their locomotor activity at the stage of high primary parasitemia.We show that some behavioural features which might affect a bird's survival during a predator attack (time of reaction, speed of flush flight and take off angle) did not change significantly during primary infection. However, the locomotor activity of infected birds was almost halved compared to control (non-infected) birds during the peak of parasitemia. We report (1) the markedly reduced mobility and (2) the 20% mortality rate caused by P. relictum and conclude that these factors are responsible for the underrepresentation of birds in mist nets and traps during the stage of high primary parasitemia in wildlife. This study indicates that the widespread parasite, P. relictum (pSGS1) influences the behaviour of birds during primary parasitemia. Experimental studies combined with field observations are needed to better understand the
Thavayogarajah, Thuvaraka; Gangopadhyay, Preetish; Rahlfs, Stefan; Becker, Katja; Lingelbach, Klaus; Przyborski, Jude M.; Holder, Anthony A.
Plasmodium falciparum invades human red blood cells, residing in a parasitophorous vacuole (PV), with a parasitophorous vacuole membrane (PVM) separating the PV from the host cell cytoplasm. Here we have investigated the role of N-myristoylation and two other N-terminal motifs, a cysteine potential S-palmitoylation site and a stretch of basic residues, as the driving force for protein targeting to the parasite plasma membrane (PPM) and subsequent translocation across this membrane. Plasmodium falciparum adenylate kinase 2 (Pf AK2) contains these three motifs, and was previously proposed to be targeted beyond the parasite to the PVM, despite the absence of a signal peptide for entry into the classical secretory pathway. Biochemical and microscopy analyses of PfAK2 variants tagged with green fluorescent protein (GFP) showed that these three motifs are involved in targeting the protein to the PPM and translocation across the PPM to the PV. It was shown that the N-terminal 37 amino acids of PfAK2 alone are sufficient to target and translocate GFP across the PPM. As a control we examined the N-myristoylated P. falciparum ADP-ribosylation factor 1 (PfARF1). PfARF1 was found to co-localise with a Golgi marker. To determine whether or not the putative palmitoylation and the cluster of lysine residues from the N-terminus of PfAK2 would modulate the subcellular localization of PfARF1, a chimeric fusion protein containing the N-terminus of PfARF1 and the two additional PfAK2 motifs was analysed. This chimeric protein was targeted to the PPM, but not translocated across the membrane into the PV, indicating that other features of the N-terminus of PfAK2 also play a role in the secretion process. PMID:25909331
Dende, Chaitanya; Meena, Jairam; Nagarajan, Perumal; Panda, Amulya K; Rangarajan, Pundi N; Padmanaban, Govindarajan
Malaria afflicts around 200 million people annually, with a mortality number close to 600,000. The mortality rate in Human Cerebral Malaria (HCM) is unacceptably high (15-20%), despite the availability of artemisinin-based therapy. An effective adjunct therapy is urgently needed. Experimental Cerebral Malaria (ECM) in mice manifests many of the neurological features of HCM. Migration of T cells and parasite-infected RBCs (pRBCs) into the brain are both necessary to precipitate the disease. We have been able to simultaneously target both these parameters of ECM. Curcumin alone was able to reverse all the parameters investigated in this study that govern inflammatory responses, CD8(+) T cell and pRBC sequestration into the brain and blood brain barrier (BBB) breakdown. But the animals eventually died of anemia due to parasite build-up in blood. However, arteether-curcumin (AC) combination therapy even after the onset of symptoms provided complete cure. AC treatment is a promising therapeutic option for HCM.
Jongwutiwes, Somchai; Putaporntip, Chaturong; Hughes, Austin L.
A number of cell surface antigens of the infective stages of malaria parasites (genus Plasmodium) have been proposed as vaccine candidates, but high levels of polymorphism at the loci encoding these antigens are problematic for vaccine effectiveness. In order to test for the effects of anti-malarial control measures (including drugs and vector control) on polymorphism at antigen-encoding loci, we analyzed sequences of four antigen-encoding loci from P. vivax and two from P. falciparum collected in 2006–2007 from two areas of Thailand: (1) the NW, where malaria cases have remained high until recently; and (2) the South, where control measures have caused a dramatic decline in numbers of cases since 1990. Polymorphism in non-repeat regions of antigen-encoding loci was dramatically reduced in the South compared to the NW. These results suggest a two-pronged strategy for malaria eradication: (1) strenuous non-vaccine control measures that will cause a severe population bottleneck in the parasite; and (2) a subsequent local vaccine focused on one or a few locally occurring alleles at antigen-encoding loci. PMID:20199765
Bushman, Mary; Morton, Lindsay; Duah, Nancy; Quashie, Neils; Abuaku, Benjamin; Koram, Kwadwo A; Dimbu, Pedro Rafael; Plucinski, Mateusz; Gutman, Julie; Lyaruu, Peter; Kachur, S Patrick; de Roode, Jacobus C; Udhayakumar, Venkatachalam
Infections with the malaria parasite Plasmodium falciparum typically comprise multiple strains, especially in high-transmission areas where infectious mosquito bites occur frequently. However, little is known about the dynamics of mixed-strain infections, particularly whether strains sharing a host compete or grow independently. Competition between drug-sensitive and drug-resistant strains, if it occurs, could be a crucial determinant of the spread of resistance. We analysed 1341 P. falciparum infections in children from Angola, Ghana and Tanzania and found compelling evidence for competition in mixed-strain infections: overall parasite density did not increase with additional strains, and densities of individual chloroquine-sensitive (CQS) and chloroquine-resistant (CQR) strains were reduced in the presence of competitors. We also found that CQR strains exhibited low densities compared with CQS strains (in the absence of chloroquine), which may underlie observed declines of chloroquine resistance in many countries following retirement of chloroquine as a first-line therapy. Our observations support a key role for within-host competition in the evolution of drug-resistant malaria. Malaria control and resistance-management efforts in high-transmission regions may be significantly aided or hindered by the effects of competition in mixed-strain infections. Consideration of within-host dynamics may spur development of novel strategies to minimize resistance while maximizing the benefits of control measures. © 2016 The Author(s).
Bertin, Gwladys I.; Sabbagh, Audrey; Argy, Nicolas; Salnot, Virginie; Ezinmegnon, Sem; Agbota, Gino; Ladipo, Yélé; Alao, Jules M.; Sagbo, Gratien; Guillonneau, François; Deloron, Philippe
Plasmodium falciparum is responsible of severe malaria, including cerebral malaria (CM). During its intra-erythrocytic maturation, parasite-derived proteins are expressed, exported and presented at the infected erythrocyte membrane. To identify new CM-specific parasite membrane proteins, we conducted a mass spectrometry-based proteomic study and compared the protein expression profiles between 9 CM and 10 uncomplicated malaria (UM) samples. Among the 1097 Plasmodium proteins identified, we focused on the 499 membrane-associated and hypothetical proteins for comparative analysis. Filter-based feature selection methods combined with supervised data analysis identified a subset of 29 proteins distinguishing CM and UM samples with high classification accuracy. A hierarchical clustering analysis of these 29 proteins based on the similarity of their expression profiles revealed two clusters of 15 and 14 proteins, respectively under- and over-expressed in CM. Among the over-expressed proteins, the MESA protein is expressed at the erythrocyte membrane, involved in proteins trafficking and in the export of variant surface antigens (VSAs), but without antigenic function. Antigen 332 protein is exported at the erythrocyte, also involved in protein trafficking and in VSAs export, and exposed to the immune system. Our proteomics data demonstrate an association of selected proteins in the pathophysiology of CM. PMID:27245217
Dende, Chaitanya; Meena, Jairam; Nagarajan, Perumal; Panda, Amulya K.; Rangarajan, Pundi N.; Padmanaban, Govindarajan
Malaria afflicts around 200 million people annually, with a mortality number close to 600,000. The mortality rate in Human Cerebral Malaria (HCM) is unacceptably high (15–20%), despite the availability of artemisinin-based therapy. An effective adjunct therapy is urgently needed. Experimental Cerebral Malaria (ECM) in mice manifests many of the neurological features of HCM. Migration of T cells and parasite-infected RBCs (pRBCs) into the brain are both necessary to precipitate the disease. We have been able to simultaneously target both these parameters of ECM. Curcumin alone was able to reverse all the parameters investigated in this study that govern inflammatory responses, CD8+ T cell and pRBC sequestration into the brain and blood brain barrier (BBB) breakdown. But the animals eventually died of anemia due to parasite build-up in blood. However, arteether-curcumin (AC) combination therapy even after the onset of symptoms provided complete cure. AC treatment is a promising therapeutic option for HCM. PMID:26227888
Fuchs, Silke; Behrends, Volker; Bundy, Jacob G; Crisanti, Andrea; Nolan, Tony
The blood meal of the female malaria mosquito is a pre-requisite to egg production and also represents the transmission route for the malaria parasite. The proper and rapid assimilation of proteins and nutrients in the blood meal creates a significant metabolic challenge for the mosquito. To better understand this process we generated a global profile of metabolite changes in response to blood meal of Anopheles gambiae, using Gas Chromatography-Mass Spectrometry (GC-MS). To disrupt a key pathway of amino acid metabolism we silenced the gene phenylalanine hydroxylase (PAH) involved in the conversion of the amino acid phenylalanine into tyrosine. We observed increased levels of phenylalanine and the potentially toxic metabolites phenylpyruvate and phenyllactate as well as a reduction in the amount of tyrosine available for melanin synthesis. This in turn resulted in a significant impairment of the melanotic encapsulation response against the rodent malaria parasite Plasmodium berghei. Furthermore silencing of PAH resulted in a significant impairment of mosquito fertility associated with reduction of laid eggs, retarded vitellogenesis and impaired melanisation of the chorion. Carbidopa, an inhibitor of the downstream enzyme DOPA decarboxylase that coverts DOPA into dopamine, produced similar effects on egg melanization and hatching rate suggesting that egg chorion maturation is mainly regulated via dopamine. This study sheds new light on the role of amino acid metabolism in regulating reproduction and immunity.
Honma, Hajime; Hirai, Makoto; Nakamura, Shota; Hakimi, Hassan; Kawazu, Shin-Ichiro; Palacpac, Nirianne M Q; Hisaeda, Hajime; Matsuoka, Hiroyuki; Kawai, Satoru; Endo, Hiroyoshi; Yasunaga, Teruo; Ohashi, Jun; Mita, Toshihiro; Horii, Toshihiro; Furusawa, Mitsuru; Tanabe, Kazuyuki
Plasmodium falciparum malaria imposes a serious public health concern throughout the tropics. Although genetic tools are principally important to fully investigate malaria parasites, currently available forward and reverse tools are fairly limited. It is expected that parasites with a high mutation rate can readily acquire novel phenotypes/traits; however, they remain an untapped tool for malaria biology. Here, we generated a mutator malaria parasite (hereinafter called a 'malaria mutator'), using site-directed mutagenesis and gene transfection techniques. A mutator Plasmodium berghei line with a defective proofreading 3' → 5' exonuclease activity in DNA polymerase δ (referred to as PbMut) and a control P. berghei line with wild-type DNA polymerase δ (referred to as PbCtl) were maintained by weekly passage in ddY mice for 122 weeks. High-throughput genome sequencing analysis revealed that two PbMut lines had 175-178 mutations and a 86- to 90-fold higher mutation rate than that of a PbCtl line. PbMut, PbCtl, and their parent strain, PbWT, showed similar course of infection. Interestingly, PbMut lost the ability to form gametocytes during serial passages. We believe that the malaria mutator system could provide a novel and useful tool to investigate malaria biology.
Rajakaruna, Rupika S; Alifrangis, Michael; Amerasinghe, Priyanie H; Konradsen, Flemming
With the dramatic drop in the transmission of malaria in Sri Lanka in recent years, the country entered the malaria pre-elimination stage in 2008. Assessing the community prevalence of hidden malaria parasites following several years of extremely low transmission is central to the process of complete elimination. The existence of a parasite reservoir in a population free from clinical manifestations, would influence the strategy for surveillance and control towards complete elimination. The prevalence of hidden parasite reservoirs in two historically malaria endemic districts, Anuradhapura and Kurunegala, previously considered as high malaria transmission areas in Sri Lanka, where peaks of transmission follow the rainy seasons was assessed. Blood samples of non-febrile individuals aged five to 55 years were collected from randomly selected areas in the two districts at community level and a questionnaire was used to collect demographic information and movement of the participants. A simple, highly sensitive nested PCR was carried out to detect both Plasmodium falciparum and Plasmodium vivax, simultaneously. In total, 3,023 individuals from 101 villages participated from both districts comprising mostly adults between the ages 19-55 years. Out of these, only about 1.4% of them (n = 19) could recall having had malaria during the past five years. Analysis of a subset of samples (n = 1322) from the two districts using PCR showed that none of the participants had hidden parasites. A reservoir of hidden parasites is unlikely to be a major concern or a barrier to the ongoing malaria elimination efforts in Sri Lanka. However, as very low numbers of indigenous cases are still recorded, an island-wide assessment and in particular, continued alertness and follow up action are still needed. The findings of this study indicate that any future assessments should be based on an adaptive sampling approach, involving prompt sampling of all subjects within a specified radius
Chen, Lili; He, Zhengxiang; Qin, Li; Li, Qinyan; Shi, Xibao; Zhao, Siting; Chen, Ling; Zhong, Nanshan; Chen, Xiaoping
Lung cancer is the most common malignancy in humans and its high fatality means that no effective treatment is available. Developing new therapeutic strategies for lung cancer is urgently needed. Malaria has been reported to stimulate host immune responses, which are believed to be efficacious for combating some clinical cancers. This study is aimed to provide evidence that malaria parasite infection is therapeutic for lung cancer. Antitumor effect of malaria infection was examined in both subcutaneously and intravenously implanted murine Lewis lung cancer (LLC) model. The results showed that malaria infection inhibited LLC growth and metastasis and prolonged the survival of tumor-bearing mice. Histological analysis of tumors from mice infected with malaria revealed that angiogenesis was inhibited, which correlated with increased terminal deoxynucleotidyl transferase-mediated (TUNEL) staining and decreased Ki-67 expression in tumors. Through natural killer (NK) cell cytotoxicity activity, cytokine assays, enzyme-linked immunospot assay, lymphocyte proliferation, and flow cytometry, we demonstrated that malaria infection provided anti-tumor effects by inducing both a potent anti-tumor innate immune response, including the secretion of IFN-γ and TNF-α and the activation of NK cells as well as adaptive anti-tumor immunity with increasing tumor-specific T-cell proliferation and cytolytic activity of CD8(+) T cells. Notably, tumor-bearing mice infected with the parasite developed long-lasting and effective tumor-specific immunity. Consequently, we found that malaria parasite infection could enhance the immune response of lung cancer DNA vaccine pcDNA3.1-hMUC1 and the combination produced a synergistic antitumor effect. Malaria infection significantly suppresses LLC growth via induction of innate and adaptive antitumor responses in a mouse model. These data suggest that the malaria parasite may provide a novel strategy or therapeutic vaccine vector for anti-lung cancer
Chen, Lili; He, Zhengxiang; Qin, Li; Li, Qinyan; Shi, Xibao; Zhao, Siting; Chen, Ling; Zhong, Nanshan; Chen, Xiaoping
Background Lung cancer is the most common malignancy in humans and its high fatality means that no effective treatment is available. Developing new therapeutic strategies for lung cancer is urgently needed. Malaria has been reported to stimulate host immune responses, which are believed to be efficacious for combating some clinical cancers. This study is aimed to provide evidence that malaria parasite infection is therapeutic for lung cancer. Methodology/Principal Findings Antitumor effect of malaria infection was examined in both subcutaneously and intravenously implanted murine Lewis lung cancer (LLC) model. The results showed that malaria infection inhibited LLC growth and metastasis and prolonged the survival of tumor-bearing mice. Histological analysis of tumors from mice infected with malaria revealed that angiogenesis was inhibited, which correlated with increased terminal deoxynucleotidyl transferase-mediated (TUNEL) staining and decreased Ki-67 expression in tumors. Through natural killer (NK) cell cytotoxicity activity, cytokine assays, enzyme-linked immunospot assay, lymphocyte proliferation, and flow cytometry, we demonstrated that malaria infection provided anti-tumor effects by inducing both a potent anti-tumor innate immune response, including the secretion of IFN-γ and TNF-α and the activation of NK cells as well as adaptive anti-tumor immunity with increasing tumor-specific T-cell proliferation and cytolytic activity of CD8+ T cells. Notably, tumor-bearing mice infected with the parasite developed long-lasting and effective tumor-specific immunity. Consequently, we found that malaria parasite infection could enhance the immune response of lung cancer DNA vaccine pcDNA3.1-hMUC1 and the combination produced a synergistic antitumor effect. Conclusions/Significance Malaria infection significantly suppresses LLC growth via induction of innate and adaptive antitumor responses in a mouse model. These data suggest that the malaria parasite may provide a
Kwenti, Tebit E.; Nkume, Franklin A.; Tanjeko, Ajime T.; Kwenti, Tayong D. B.
Background The interaction between intestinal parasites and malaria is still not clear. Data in published literature are conflicting. We studied the effect of intestinal parasitic infection (IPI) on the clinical outcome of malaria in coinfected children. Methods In a cross sectional study performed between October 2014 and September 2015, children infected with malaria, as demonstrated by the presence of asexual parasites in Giemsa stained blood films, were enrolled. Stool samples were obtained from participants and subjected to the formol-ether concentration technique for the detection of intestinal parasites. The Complete blood count was performed using an automated haematology analyser (Mindray, BC-2800). The risk ratio, Pearson’s chi-square and the student T test were all performed as part of the statistical analyses. Statistical significance was set at p < 0.05. Results In all, 405 children successfully took part in the study. The children were between 1 week and 120 months of age (mean ± SD = 41.5 ± 33.5). Coinfection with intestinal parasites was observed in 11.6%. The rate of severe malaria (SM) attack in this study was 10.9%. SM was not observed to be associated with age (p = 0.377) or gender (p = 0.387), meanwhile coinfection with intestinal parasites was associated with age (p = 0.003). Among SM cases, IPI prevalence was higher in children with mild (WHO group 3) severe malaria (p = 0.027). Overall, IPI was not observed to be associated with SM (p = 0.656) or malaria parasite density (p = 0.185) or haemoglobin concentration (p = 0.205). The main clinical features of SM observed were hyperpyrexia (68.2%), severe malarial anaemia (61.4%), and multiple convulsion (52.3%). Conclusion IPI was not observed to be associated with the severity of malaria, the malaria parasite density, and the haemoglobin concentration in coinfected children in Cameroon. The clinical outcome of malaria in children coinfected with intestinal parasites may depend on the
Ingasia, Luicer A; Cheruiyot, Jelagat; Okoth, Sheila Akinyi; Andagalu, Ben; Kamau, Edwin
Transmission intensity, movement of human and vector hosts, biogeographical features, and malaria control measures are some of the important factors that determine Plasmodium falciparum parasite genetic variability and population structure. Kenya has different malaria ecologies which might require different disease intervention methods. Refined parasite population genetic studies are critical for informing malaria control and elimination strategies. This study describes the genetic diversity and population structure of P. falciparum parasites from the different malaria ecological zones in Kenya. Twelve multi-locus microsatellite (MS) loci previously described were genotyped in 225 P. falciparum isolates collected between 2012 and 2013 from five sites; three in lowland endemic regions (Kisumu, Kombewa, and Malindi) and two in highland, epidemic regions (Kisii and Kericho). Parasites from the lowland endemic and highland epidemic regions of western Kenya had high genetic diversity compared to coastal lowland endemic region of Kenya [Malindi]. The Kenyan parasites had a mean genetic differentiation index (FST) of 0.072 (p=0.011). The multi-locus genetic analysis of the 12 MS revealed all the parasites had unique haplotypes. Significant linkage disequilibrium (LD) was observed in all the five parasite populations. Kisumu had the most significant index of association values (0.16; p<0.0001) whereas Kisii had the least significant index of association values (0.03; p<0.0001). Our data suggest high genetic diversity in Kenyan parasite population with the exception of parasite from Malindi where malaria has been on the decline. The presence of significant LD suggests that there is occurrence of inbreeding in the parasite population. Parasite populations from Kisii showed the strongest evidence for epidemic population structure whereas the rest of the regions showed panmixia. Defining the genetic diversity of the parasites in different ecological regions of Kenya after
Alout, Haoues; Djègbè, Innocent; Chandre, Fabrice; Djogbénou, Luc Salako; Dabiré, Roch Kounbobr; Corbel, Vincent; Cohuet, Anna
Currently, there is a strong trend towards increasing insecticide-based vector control coverage in malaria endemic countries. The ecological consequence of insecticide applications has been mainly studied regarding the selection of resistance mechanisms; however, little is known about their impact on vector competence in mosquitoes responsible for malaria transmission. As they have limited toxicity to mosquitoes owing to the selection of resistance mechanisms, insecticides may also interact with pathogens developing in mosquitoes. In this study, we explored the impact of insecticide exposure on Plasmodium falciparum development in insecticide-resistant colonies of Anopheles gambiae s.s., homozygous for the ace-1 G119S mutation (Acerkis) or the kdr L1014F mutation (Kdrkis). Exposure to bendiocarb insecticide reduced the prevalence and intensity of P. falciparum oocysts developing in the infected midgut of the Acerkis strain, whereas exposure to dichlorodiphenyltrichloroethane reduced only the prevalence of P. falciparum infection in the Kdrkis strain. Thus, insecticide resistance leads to a selective pressure of insecticides on Plasmodium parasites, providing, to our knowledge, the first evidence of genotype by environment interactions on vector competence in a natural Anopheles–Plasmodium combination. Insecticide applications would affect the transmission of malaria in spite of resistance and would reduce to some degree the impact of insecticide resistance on malaria control interventions. PMID:24850924
Turner, Louise; Lavstsen, Thomas; Berger, Sanne S.; Wang, Christian W.; Petersen, Jens E.V.; Avril, Marion; Brazier, Andrew J.; Freeth, Jim; Jespersen, Jakob S.; Nielsen, Morten A.; Magistrado, Pamela; Lusingu, John; Smith, Joseph D.; Higgins, Matthew K.; Theander, Thor G.
Sequestration of Plasmodium falciparum-infected erythrocytes in host blood vessels is a key triggering event in the pathogenesis of severe childhood malaria, which is responsible for about one million deaths every year1. Sequestration is mediated by specific interactions between members of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family and receptors on the endothelial lining2. Severe malaria is associated with expression of specific PfEMP1 subtypes containing domain cassettes (DC) 8 and 133, but the endothelial receptor for parasites expressing these proteins was unknown4,5. Here, we identify endothelial protein C receptor (EPCR), which mediates cytoprotective effects of activated protein C6, as the endothelial receptor for DC8 and DC13 PfEMP1. We show that EPCR binding is mediated through the N-terminal cysteine-rich interdomain region (CIDRα1) of DC8 and group A PfEMP1 subfamilies and that CIDRα1 interferes with protein C binding to EPCR. This PfEMP1 adhesive property links P. falciparum cytoadhesion to a host receptor involved in anticoagulation and endothelial cytoprotective pathways and has implications for understanding malaria pathology and the development of new malaria interventions. PMID:23739325
Proellocks, Nicholas I; Coppel, Ross L; Mohandas, Narla; Cooke, Brian M
Malaria, caused by Plasmodium spp., continues to be a major threat to human health and a significant cause of socioeconomic hardship in many countries. Almost half of the world's population live in malaria-endemic regions and many of them suffer one or more, often life-threatening episodes of malaria every year, the symptoms of which are attributable to replication of the parasite within red blood cells (RBCs). In the case of Plasmodium falciparum, the species responsible for most malaria-related deaths, parasite replication within RBCs is accompanied by striking alterations to the morphological, biochemical and biophysical properties of the host cell that are essential for the parasites' survival. To achieve this, the parasite establishes a unique and extensive protein export network in the infected RBC, dedicating at least 6% of its genome to the process. Understanding the full gamut of proteins involved in this process and the mechanisms by which P. falciparum alters the structure and function of RBCs is important both for a more complete understanding of the pathogenesis of malaria and for development of new therapeutic strategies to prevent or treat this devastating disease. This review focuses on what is currently known about exported parasite proteins, their interactions with the RBC and their likely pathophysiological consequences.
Plasmodium falciparum 19-kilodalton merozoite surface protein 1 (MSP1)-specific antibodies that interfere with parasite growth in vitro can inhibit MSP1 processing, merozoite invasion, and intracellular parasite development.
Moss, David K; Remarque, Edmond J; Faber, Bart W; Cavanagh, David R; Arnot, David E; Thomas, Alan W; Holder, Anthony A
Merozoite surface protein 1 (MSP1) is a target for malaria vaccine development. Antibodies to the 19-kDa carboxy-terminal region referred to as MSP1(19) inhibit erythrocyte invasion and parasite growth, with some MSP1-specific antibodies shown to inhibit the proteolytic processing of MSP1 that occurs at invasion. We investigated a series of antibodies purified from rabbits immunized with MSP1(19) and AMA1 recombinant proteins for their ability to inhibit parasite growth, initially looking at MSP1 processing. Although significant inhibition of processing was mediated by several of the antibody samples, there was no clear relationship with overall growth inhibition by the same antibodies. However, no antibody samples inhibited processing but not invasion, suggesting that inhibition of MSP1 processing contributes to but is not the only mechanism of antibody-mediated inhibition of invasion and growth. Examining other mechanisms by which MSP1-specific antibodies inhibit parasite growth, we show that MSP1(19)-specific antibodies are taken up into invaded erythrocytes, where they persist for significant periods and result in delayed intracellular parasite development. This delay may result from antibody interference with coalescence of MSP1(19)-containing vesicles with the food vacuole. Antibodies raised against a modified recombinant MSP1(19) sequence were more efficient at delaying intracellular growth than those to the wild-type protein. We propose that antibodies specific for MSP1(19) can mediate inhibition of parasite growth by at least three mechanisms: inhibition of MSP1 processing, direct inhibition of invasion, and inhibition of parasite development following invasion. The balance between mechanisms may be modulated by modifying the immunogen used to induce the antibodies.
Elsworth, Brendan; Matthews, Kathryn; Nie, Catherine Q; Kalanon, Ming; Charnaud, Sarah C; Sanders, Paul R; Chisholm, Scott A; Counihan, Natalie A; Shaw, Philip J; Pino, Paco; Chan, Jo-Anne; Azevedo, Mauro F; Rogerson, Stephen J; Beeson, James G; Crabb, Brendan S; Gilson, Paul R; de Koning-Ward, Tania F
During the blood stages of malaria, several hundred parasite-encoded proteins are exported beyond the double-membrane barrier that separates the parasite from the host cell cytosol. These proteins have a variety of roles that are essential to virulence or parasite growth. There is keen interest in understanding how proteins are exported and whether common machineries are involved in trafficking the different classes of exported proteins. One potential trafficking machine is a protein complex known as the Plasmodium translocon of exported proteins (PTEX). Although PTEX has been linked to the export of one class of exported proteins, there has been no direct evidence for its role and scope in protein translocation. Here we show, through the generation of two parasite lines defective for essential PTEX components (HSP101 or PTEX150), and analysis of a line lacking the non-essential component TRX2 (ref. 12), greatly reduced trafficking of all classes of exported proteins beyond the double membrane barrier enveloping the parasite. This includes proteins containing the PEXEL motif (RxLxE/Q/D) and PEXEL-negative exported proteins (PNEPs). Moreover, the export of proteins destined for expression on the infected erythrocyte surface, including the major virulence factor PfEMP1 in Plasmodium falciparum, was significantly reduced in PTEX knockdown parasites. PTEX function was also essential for blood-stage growth, because even a modest knockdown of PTEX components had a strong effect on the parasite's capacity to complete the erythrocytic cycle both in vitro and in vivo. Hence, as the only known nexus for protein export in Plasmodium parasites, and an essential enzymic machine, PTEX is a prime drug target.
Ewen, John G; Bensch, Staffan; Blackburn, Tim M; Bonneaud, Camille; Brown, Ruth; Cassey, Phillip; Clarke, Rohan H; Pérez-Tris, Javier
Knowledge of the processes favouring the establishment of exotic parasites is poor. Herein, we test the characteristics of successful exotic parasites that have co-established in the remote island archipelago of New Zealand, due to the introduction of numerous avian host species. Our results show that avian malaria parasites (AM; parasites of the genus Plasmodium) that successfully invaded are more globally generalist (both geographically widespread and with a broad taxonomic range of hosts) than AM parasites not co-introduced to New Zealand. Furthermore, the successful AM parasites are presently more prevalent in their native range than AM parasites found in the same native range but not co-introduced to New Zealand. This has resulted in an increased number and greater taxonomic diversity of AM parasites now in New Zealand.
Hansen, Finn K.; Sumanadasa, Subathdrage D. M.; Stenzel, Katharina; Duffy, Sandra; Meister, Stephan; Marek, Linda; Schmetter, Rebekka; Kuna, Krystina; Hamacher, Alexandra; Mordmüller, Benjamin; Kassack, Matthias U.; Winzeler, Elizabeth A.; Avery, Vicky M.; Andrews, Katherine T.; Kurz, Thomas
In this work we investigated the antiplasmodial activity of a series of HDAC inhibitors containing an alkoxyamide connecting-unit linker region. HDAC inhibitor 1a (LMK235), previously shown to be a novel and specific inhibitor of human HDAC4 and 5, was used as a starting point to rapidly construct a mini-library of HDAC inhibitors using a straightforward solid-phase supported synthesis. Several of these novel HDAC inhibitors were found to have potent in vitro activity against asexual stage P. falciparum malaria parasites. Representative compounds were shown to hyperacetylate P. falciparum histones and to inhibit deacetylase activity of recombinant PfHDAC1 and P. falciparum nuclear extracts. All compounds were also screened in vitro for activity against P. berghei exo-erythrocytic stages and selected compounds were further tested against late stage (IV and V) P. falciparum gametocytes. Of note, some compounds showed nanomolar activity against all three life cycle stages tested (asexual, exo-erythrocytic and gametocyte stages) and several compounds displayed significantly increased parasite selectivity compared to the reference HDAC inhibitor suberoylanilide hydroxamic acid (SAHA). These data suggest that it may be possible to develop HDAC inhibitors that target multiple malaria parasite life cycle stages. PMID:24904967
Motz, Victoria L; Lewis, William D; Vardo-Zalik, Anne M
Plasmodium mexicanum is a malaria parasite that naturally infects the western fence lizard, Sceloporus occidentalis , in northern California. We set out to determine whether lizards naturally infected with this malaria parasite have different leukocyte profiles, indicating an immune response to infection. We used 29 naturally infected western fence lizards paired with uninfected lizards based on sex, snout-to-vent length, tail status, and the presence-absence of ectoparasites such as ticks and mites, as well as the presence-absence of another hemoparasite, Schellackia occidentalis. Complete white blood cell (WBC) counts were conducted on blood smears stained with Giemsa, and the proportion of granulocytes per microliter of blood was estimated using the Avian Leukopet method. The abundance of each WBC class (lymphocytes, monocytes, heterophils, eosinophils, and basophils) in infected and uninfected lizards was compared to determine whether leukocyte densities varied with infection status. We found that the numbers of WBCs and lymphocytes per microliter of blood significantly differed (P < 0.05) between the 2 groups for females but not for males, whereas parasitemia was significantly correlated with lymphocyte counts for males, but not for females. This study supports the theory that infection with P. mexicanum stimulates the lizard's immune response to increase the levels of circulating WBCs, but what effect this has on the biology of the parasite remains unclear.
Mok, Sachel; Ashley, Elizabeth A; Ferreira, Pedro E; Zhu, Lei; Lin, Zhaoting; Yeo, Tomas; Chotivanich, Kesinee; Imwong, Mallika; Pukrittayakamee, Sasithon; Dhorda, Mehul; Nguon, Chea; Lim, Pharath; Amaratunga, Chanaki; Suon, Seila; Hien, Tran Tinh; Htut, Ye; Faiz, M Abul; Onyamboko, Marie A; Mayxay, Mayfong; Newton, Paul N; Tripura, Rupam; Woodrow, Charles J; Miotto, Olivo; Kwiatkowski, Dominic P; Nosten, François; Day, Nicholas P J; Preiser, Peter R; White, Nicholas J; Dondorp, Arjen M; Fairhurst, Rick M; Bozdech, Zbynek
Artemisinin resistance in Plasmodium falciparum threatens global efforts to control and eliminate malaria. Polymorphisms in the kelch domain-carrying protein K13 are associated with artemisinin resistance, but the underlying molecular mechanisms are unknown. We analyzed the in vivo transcriptomes of 1043 P. falciparum isolates from patients with acute malaria and found that artemisinin resistance is associated with increased expression of unfolded protein response (UPR) pathways involving the major PROSC and TRiC chaperone complexes. Artemisinin-resistant parasites also exhibit decelerated progression through the first part of the asexual intraerythrocytic development cycle. These findings suggest that artemisinin-resistant parasites remain in a state of decelerated development at the young ring stage, whereas their up-regulated UPR pathways mitigate protein damage caused by artemisinin. The expression profiles of UPR-related genes also associate with the geographical origin of parasite isolates, further suggesting their role in emerging artemisinin resistance in the Greater Mekong Subregion. Copyright © 2015, American Association for the Advancement of Science.
Hosseini, S Majid; Feng, James J
The pathogenesis of malaria is largely due to stiffening of the infected red blood cells (RBCs). Contemporary understanding ascribes the loss of RBC deformability to a 10-fold increase in membrane stiffness caused by extra cross-linking in the spectrin network. Local measurements by micropipette aspiration, however, have reported only an increase of ∼3-fold in the shear modulus. We believe the discrepancy stems from the rigid parasite particles inside infected cells, and have carried out numerical simulations to demonstrate this mechanism. The cell membrane is represented by a set of discrete particles connected by linearly elastic springs. The cytosol is modeled as a homogeneous Newtonian fluid, and discretized by particles as in standard smoothed particle hydrodynamics. The malaria parasite is modeled as an aggregate of particles constrained to rigid-body motion. We simulate RBC stretching tests by optical tweezers in three dimensions. The results demonstrate that the presence of a sizeable parasite greatly reduces the ability of RBCs to deform under stretching. With the solid inclusion, the observed loss of deformability can be predicted quantitatively using the local membrane elasticity measured by micropipettes.
Perkins, S L
Species of malaria parasite (phylum Apicomplexa: genus Plasmodium) have traditionally been described using the similarity species concept (based primarily on differences in morphological or life-history characteristics). The biological species concept (reproductive isolation) and phylogenetic species concept (based on monophyly) have not been used before in defining species of Plasmodium. Plasmodium azurophilum, described from Anolis lizards in the eastern Caribbean, is actually a two-species cryptic complex. The parasites were studied from eight islands, from Puerto Rico in the north to Grenada in the south. Morphology of the two species is very similar (differences are indistinguishable to the eye), but one infects only erythrocytes and the other only white blood cells. Molecular data for the cytochrome b gene reveal that the two forms are reproductively isolated; distinct haplotypes are present on each island and are never shared between the erythrocyte-infecting and leucocyte-infecting species. Each forms a monophyletic lineage indicating that they diverged before becoming established in the anoles of the eastern Caribbean. This comparison of the similarity, biological and phylogenetic species concepts for malaria parasites reveals the limited value of using only similarity measures in defining protozoan species. PMID:11413654
Chanturiya, Alexandr N.; Glushakova, Svetlana; Yin, Dan; Zimmerberg, Joshua
Digestion of red blood cell (RBC) hemoglobin by the malaria parasite results in the formation of paramagnetic hemazoin crystals inside the parasite body. A number of reports suggest that magnetic field interaction with hamazoin crystals significantly reduces the number of infected cells in culture, and thus magnetic field can be used to combat malaria. We studies the effects of magnetic filed on the Plasmodium falciparum asexual life cycle inside RBCs under various experimental conditions. No effect was found during prolonged exposure of infected RBCs to constant magnetic fields up to 6000 Gauss. Infected RBCs were also exposed, under temperature-controlled conditions, to oscillating magnetic fields with frequencies in the range of 500-20000 kHz, and field strength 30-600 Gauss. This exposure often changed the proportion of different parasite stages in treated culture compared to controls. However, no significant effect on parasitemia was observed in treated cultures. This result indicates that the magnetic field effect on Plasmodium falciparum is negligible, or that hypothetical negative and positive effects on different stages within one 48-hour compensate each other.
Loop-mediated isothermal amplification (LAMP) is an innovative molecular technique that has been validated for point-of-care testing to diagnose malaria. Molecular detection and tracking of anti-malarial drug resistance is mainly based on highly sophisticated, costly and time-consuming techniques. With the validation of resistance-associated gene mutations in malaria parasites, there is a need to develop rapid, easy-to-use molecular tests for anti-malarial drug resistance genotyping. LAMP could be further developed as a point-of-care test to rapidly detect anti-malarial drug resistance-associated molecular markers, thereby help detecting and monitoring drug resistance in surveillance studies. PMID:24934581
Kenthirapalan, Sanketha; Waters, Andrew P.; Matuschewski, Kai; Kooij, Taco W. A.
Assigning function to orphan membrane transport proteins and prioritizing candidates for detailed biochemical characterization remain fundamental challenges and are particularly important for medically relevant pathogens, such as malaria parasites. Here we present a comprehensive genetic analysis of 35 orphan transport proteins of Plasmodium berghei during its life cycle in mice and Anopheles mosquitoes. Six genes, including four candidate aminophospholipid transporters, are refractory to gene deletion, indicative of essential functions. We generate and phenotypically characterize 29 mutant strains with deletions of individual transporter genes. Whereas seven genes appear to be dispensable under the experimental conditions tested, deletion of any of the 22 other genes leads to specific defects in life cycle progression in vivo and/or host transition. Our study provides growing support for a potential link between heavy metal homeostasis and host switching and reveals potential targets for rational design of new intervention strategies against malaria. PMID:26796412
Barber, Bridget E; Grigg, Matthew J; William, Timothy; Piera, Kim A; Boyle, Michelle J; Yeo, Tsin W; Anstey, Nicholas M
In populations pauci-immune to malaria, risk of severe malaria increases with age. This is particularly apparent in Plasmodium knowlesi malaria. However, pathophysiological mechanisms underlying knowlesi malaria, and of the age-related increase in risk of severe malaria in general, are poorly understood. In Malaysian patients aged ≥12 years with severe (n = 47) and nonsevere (n = 99) knowlesi malaria, severe (n = 21) and nonsevere (n = 109) falciparum malaria, and healthy controls (n = 50), we measured parasite biomass, systemic inflammation (interleukin 6 [IL-6]), endothelial activation (angiopoietin-2), and microvascular function, and evaluated the effects of age. Plasmodium knowlesi parasitemia correlated with age (Spearman's correlation coefficient [rs] = 0.36; P < .0001). In knowlesi malaria, IL-6, angiopoietin-2, and microvascular dysfunction were increased in severe compared to nonsevere disease, and all correlated with age, independent of parasitemia. In falciparum malaria, angiopoietin-2 increased with age, independent of parasite biomass (histidine-rich protein 2 [HRP2]). Independent risk factors for severe malaria included parasitemia and angiopoietin-2 in knowlesi malaria, and HRP2, angiopoietin-2, and microvascular dysfunction in falciparum malaria. Parasite biomass, endothelial activation, and microvascular dysfunction are associated with severe disease in knowlesi malaria and likely contribute to pathogenesis. The association of each of these processes with aging may account for the greater severity of malaria observed in older adults in low-endemic regions.
Background Plasmodium parasites are causative agents of malaria which affects >500 million people and claims ~2 million lives annually. The completion of Plasmodium genome sequencing and availability of PlasmoDB database has provided a platform for systematic study of parasite genome. Aminoacyl-tRNA synthetases (aaRSs) are pivotal enzymes for protein translation and other vital cellular processes. We report an extensive analysis of the Plasmodium falciparum genome to identify and classify aaRSs in this organism. Results Using various computational and bioinformatics tools, we have identified 37 aaRSs in P. falciparum. Our key observations are: (i) fraction of proteome dedicated to aaRSs in P. falciparum is very high compared to many other organisms; (ii) 23 out of 37 Pf-aaRS sequences contain signal peptides possibly directing them to different cellular organelles; (iii) expression profiles of Pf-aaRSs vary considerably at various life cycle stages of the parasite; (iv) several PfaaRSs posses very unusual domain architectures; (v) phylogenetic analyses reveal evolutionary relatedness of several parasite aaRSs to bacterial and plants aaRSs; (vi) three dimensional structural modelling has provided insights which could be exploited in inhibitor discovery against parasite aaRSs. Conclusion We have identified 37 Pf-aaRSs based on our bioinformatics analysis. Our data reveal several unique attributes in this protein family. We have annotated all 37 Pf-aaRSs based on predicted localization, phylogenetics, domain architectures and their overall protein expression profiles. The sets of distinct features elaborated in this work will provide a platform for experimental dissection of this family of enzymes, possibly for the discovery of novel drugs against malaria. PMID:20042123
Liu, Huaie; Feng, Guohua; Zeng, Weilin; Li, Xiaomei; Bai, Yao; Deng, Shuang; Ruan, Yonghua; Morris, James; Li, Siman; Yang, Zhaoqing; Cui, Liwang
The conventional method of estimating parasite densities employ an assumption of 8000 white blood cells (WBCs)/μl. However, due to leucopenia in malaria patients, this number appears to overestimate parasite densities. In this study, we assessed the accuracy of parasite density estimated using this assumed WBC count in eastern Myanmar, where Plasmodium vivax has become increasingly prevalent. From 256 patients with uncomplicated P. vivax malaria, we estimated parasite density and counted WBCs by using an automated blood cell counter. It was found that WBC counts were not significantly different between patients of different gender, axillary temperature, and body mass index levels, whereas they were significantly different between age groups of patients and the time points of measurement. The median parasite densities calculated with the actual WBC counts (1903/μl) and the assumed WBC count of 8000/μl (2570/μl) were significantly different. We demonstrated that using the assumed WBC count of 8000 cells/μl to estimate parasite densities of P. vivax malaria patients in this area would lead to an overestimation. For P. vivax patients aged five years and older, an assumed WBC count of 5500/μl best estimated parasite densities. This study provides more realistic assumed WBC counts for estimating parasite densities in P. vivax patients from low-endemicity areas of Southeast Asia.
Krücken, Jürgen; Mehnert, Liv I; Dkhil, Mohamed A; El-Khadragy, Manal; Benten, W Peter M; Mossmann, Horst; Wunderlich, Frank
It is currently accepted that malaria-parasitized red blood cells (pRBC) are eliminated, like senescent erythrocytes, phagocytically by macrophages in the red pulp of the spleen. Here, however, we show that self-healing Plasmodium chabaudi malaria activates spleen closure in C57BL/6 mice. Confocal laser scanning microscopy revealed that spleen closing was manifested by elimination of entry into the red pulp of 3-microm polystyrol particles, pRBC, and nonparasitized red blood cells but not of bovine serum albumin. This spleen closure did not reflect a reduction in the number of phagocytic cells, as shown by flow cytometry, whereas marginal zone macrophages (MZM) were lost and red pulp macrophages entered the white pulp. Splenic trapping of pBRC was strongly reduced in the absence of MZM and marginal metallophilic macrophages (MMM), as it is in noninfected mice with a disrupted lymphotoxin beta receptor (LTbetaR(-/-)), and it was still significantly reduced when the number of MZM and MMM was diminished, as in tumor necrosis factor alpha-deficient (TNF-alpha(-/-)) mice. Moreover, mice deficient in TNF-alpha, tumor necrosis factor receptor I (TNFRI(-/-)), and LTbetaR exhibited progressive impairment in malaria-induced spleen closing. Treatment of C57BL/6 mice with TNF-alpha induced loss of MZM and spleen closing by about 20%. Our data indicate that TNF/TNFRI signaling is involved in regulating malaria-induced spleen closure, which is maximal during crisis, when parasitemia declines more than 100-fold. Consequently, the vast majority of pRBC cannot be destroyed by the spleen during crisis, suggesting that the known sophisticated sequestration system of Plasmodium parasites did not evolve to avoid splenic clearance.
Krungkrai, Sudaratana R; Krungkrai, Jerapan
Plasmodium falciparum (P. falciparum) is responsible for the majority of life-threatening cases of human malaria, causing 1.5-2.7 million annual deaths. The global emergence of drug-resistant malaria parasites necessitates identification and characterization of novel drug targets and their potential inhibitors. We identified the carbonic anhydrase (CA) genes in P. falciparum. The pfCA gene encodes anα-carbonic anhydrase, a Zn2+-metalloenzme, possessing catalytic properties distinct from that of the human host CA enzyme. The amino acid sequence of the pfCA enzyme is different from the analogous protozoan and human enzymes. A library of aromatic/heterocyclic sulfonamides possessing a large diversity of scaffolds were found to be very good inhibitors for the malarial enzyme at moderate-low micromolar and submicromolar inhibitions. The structure of the groups substituting the aromatic-ureido- or aromatic-azomethine fragment of the molecule and the length of the parent sulfonamide were critical parameters for the inhibitory properties of the sulfonamides. One derivative, that is, 4- (3, 4-dichlorophenylureido)thioureido-benzenesulfonamide (compound 10) was the most effective in vitro Plasmodium falciparum CA inhibitor, and was also the most effective antimalarial compound on the in vitro P. falciparum growth inhibition. The compound 10 was also effective in vivo antimalarial agent in mice infected with Plasmodium berghei, an animal model of drug testing for human malaria infection. It is therefore concluded that the sulphonamide inhibitors targeting the parasite CA may have potential for the development of novel therapies against human malaria. PMID:23569766
Kumari, Amrita; Gakhar, S K; Hooda, Vikas
Several studies have been made to study the effect of antisera raised against different tissues (hemolymh, ovary, midgut and salivary glands) on the fecundity and malaria parasite development in the different species of mosquitoes but there are no reports on the antisera raised against the hemolymph of Anopheles culicifacies, the principal malaria vector in India accounting for 65% of malaria cases. Hence, an attempt was made to study the same and evaluate its impact on malaria parasite development. Polyclonal and multifactorial antibodies were produced in rabbits against heterogenous mixture of hemolymph proteins. Antibodies against hemolymph proteins were screened for their potential to influence reproductive performance of mosquitoes. Antibody titer in rabbit serum was determined by ELISA and putative candidate antigens were identified in the hemolymph of An. culicifacies by western blotting. Cross reactivity amongst various tissues vis-a-vis hemolymph protein was also identified. In addition, a significant reduction in oocyst development was also observed in An. culicifacies mosquitoes that ingested antihemolymph antibodies along with Plasmodium vivax. The maximum reduction in fecundity (57%) was observed during fourth week, after the last booster and number of oocyts per infected mosquito reduced by 73.35% in the group of mosquitoes that ingested antihemolymph antibodies along with the infected blood meal respectively. However, the ingestion of antibodies against hemolymph proteins did not have significant influence on hatchability. Antisera raised against hemolymph proteins of An. culicifacies recognized 11 polypeptides by western blotting. During the present study, 11 putative candidate antigens were identified in the hemolymph of An. culicifacies, against which antibodies produced significantly reduced the fecundity by 57%. In addition, a significant reduction in oocyst development was also observed in An. culicifacies that ingested antihemolymph antibodies
Svensson-Coelho, Maria; Ellis, Vincenzo A; Loiselle, Bette A; Blake, John G; Ricklefs, Robert E
How specialization of consumers with respect to resources varies with respect to latitude is poorly understood. Coexistence of many species in the tropics might be possible only if specialization also increases. Alternatively, lower average abundance of more diverse biotic resources in the tropics might force consumers to become more generalized foragers. We examine levels of reciprocal specialization in an antagonistic system-avian malaria-to determine whether the number of host species used and/or parasite lineages harbored differ between a temperate and a tropical assemblage. We evaluate the results of network analysis, which can incorporate both bird and parasite perspectives on specialization in one quantitative index, in comparison to null models. Specialization was significantly greater in both sample sites than predicted from null models. We found evidence for lower per-host species parasite diversity in temperate compared to tropical birds. However, specialization did not differ between the tropical and temperate sites from the parasite perspective. We supplemented the network analysis with estimates of specialization that incorporate phylogenetic relationships of associates and found no differences between sites. Thus, our analyses indicate that specialization within an antagonistic host-parasite (resource-consumer) system varies little between tropical and temperate localities.
Peterson, Tina M.L.; Gow, Andrew J.; Luckhart, Shirley
Malaria parasite infection in anopheline mosquitoes is limited by inflammatory levels of nitric oxide metabolites. To assess the mechanisms of parasite stasis or toxicity, we investigated the biochemistry of these metabolites within the blood-filled mosquito midgut. Our data indicate that nitrates, but not nitrites, are elevated in the Plasmodium-infected midgut. Although levels of S-nitrosothiols do not change with infection, blood proteins are S-nitrosylated after ingestion by the mosquito. In addition, photolyzable nitric oxide, which can be attributed to metal nitrosyls, is elevated following infection and, based on the abundance of hemoglobin, likely includes heme iron nitrosyl. The persistance of oxyhemoglobin throughout blood digestion and changes in hemoglobin conformation in response to infection suggest that hemoglobin catalyzes the synthesis of nitric oxide metabolites in a reducing environment. Provision of urate, a potent reductant and scavenger of oxidants and nitrating agents, as a dietary supplement to mosquitoes increased parasite infection levels relative to allantoin-fed controls, suggesting that nitrosative and/or oxidative stresses negatively impact developing parasites. Collectively, our results reveal a unique role for nitric oxide in an oxyhemoglobin-rich environment. In contrast to facilitating oxygen delivery by hemoglobin in the mammalian vasculature, nitric oxide synthesis in the blood-filled mosquito midgut drives the formation of toxic metabolites that limit parasite development. PMID:17157200
van Santen, Susanne; de Mast, Quirijn; Swinkels, Dorine W.; van der Ven, André J.A.M.
Epidemiological studies have demonstrated an association between malaria and invasive non-typhoid Salmonella (NTS) infections, especially in children. We explore the role of iron as a possible co-factor in this association. Malarial disease, among others, is associated with enhanced erythrophagocytosis and inflammation, which increases the iron content of macrophages and thereby also the survival of Salmonellae spp within macrophages. Whether iron supplementation programs augment the risk of invasive NTS infections in malaria endemic regions is an important global health issue that still needs to be determined. PMID:23601932
Glenn, Matthew P; Chang, Sung-Youn; Hornéy, Carrie; Rivas, Kasey; Yokoyama, Kohei; Pusateri, Erin E; Fletcher, Steven; Cummings, Christopher G; Buckner, Frederick S; Pendyala, Prakash R; Chakrabarti, Debopam; Sebti, Saïd M; Gelb, Michael; Van Voorhis, Wesley C; Hamilton, Andrew D
Third world nations require immediate access to inexpensive therapeutics to counter the high mortality inflicted by malaria. Here, we report a new class of antimalarial protein farnesyltransferase (PFT) inhibitors, designed with specific emphasis on simple molecular architecture, to facilitate easy access to therapies based on this recently validated antimalarial target. This novel series of compounds represents the first Plasmodium falciparum selective PFT inhibitors reported (up to 145-fold selectivity), with lead inhibitors displaying excellent in vitro activity (IC(50) < 1 nM) and toxicity to cultured parasites at low concentrations (ED(50) < 100 nM). Initial studies of absorption, metabolism, and oral bioavailability are reported.
Glenn, Matthew P.; Chang, Sung-Youn; Hornéy, Carrie; Rivas, Kasey; Yokoyama, Kohei; Pusateri, Erin E.; Fletcher, Steven; Cummings, Christopher G.; Buckner, Frederick S.; Pendyala, Prakash R.; Chakrabarti, Debopam; Sebti, Saïd M.; Gelb, Michael; Van Voorhis, Wesley C.; Hamilton, Andrew D.
Third world nations require immediate access to inexpensive therapeutics to counter the high mortality inflicted by malaria. Here, we report a new class of antimalarial protein farnesyltransferase (PFT) inhibitors, designed with specific emphasis on simple molecular architecture, to facilitate easy access to therapies based on this recently validated antimalarial target. This novel series of compounds represents the first Plasmodium falciparum selective PFT inhibitors reported (up to 145-fold selectivity), with lead inhibitors displaying excellent in vitro activity (IC50 < 1 nM) and toxicity to cultured parasites at low concentrations (ED50 < 100 nM). Initial studies of absorption, metabolism, and oral bioavailability are reported. PMID:16970397
Church, James; Maitland, Kathryn
Severe malaria remains a major cause of pediatric hospital admission across Africa. Invasive bacterial infection (IBI) is a recognized complication of Plasmodium falciparum malaria, resulting in a substantially worse outcome. Whether a biological relationship exists between malaria infection and IBI susceptibility remains unclear. We, therefore, examined the extent, nature and evidence of this association. We conducted a systematic search in August 2012 of three major scientific databases, PubMed, Embase and Africa Wide Information, for articles describing bacterial infection among children with P. falciparum malaria using the search string '(malaria OR plasmodium) AND (bacteria OR bacterial OR bacteremia OR bacteraemia OR sepsis OR septicaemia OR septicemia).' Eligiblity criteria also included studies of children hospitalized with malaria or outpatient attendances in sub-Saharan Africa. A total of 25 studies across 11 African countries fulfilled our criteria. They comprised twenty cohort analyses, two randomized controlled trials and three prospective epidemiological studies. In the meta-analysis of 7,208 children with severe malaria the mean prevalence of IBI was 6.4% (95% confidence interval (CI) 5.81 to 6.98%). In a further meta-analysis of 20,889 children hospitalised with all-severity malaria and 27,641 children with non-malarial febrile illness the mean prevalence of IBI was 5.58 (95% CI 5.5 to 5.66%) in children with malaria and 7.77% (95% CI 7.72 to 7.83%) in non-malaria illness. Ten studies reported mortality stratified by IBI. Case fatality was higher at 81 of 336, 24.1% (95% CI 18.9 to 29.4) in children with malaria/IBI co-infection compared to 585 of 5,760, 10.2% (95% CI 9.3 to 10.98) with malaria alone. Enteric gram-negative organisms were over-represented in malaria cases, non-typhoidal Salmonellae being the most commonest isolate. There was weak evidence indicating IBI was more common in the severe anemia manifestation of severe malaria. The
Background Severe malaria remains a major cause of pediatric hospital admission across Africa. Invasive bacterial infection (IBI) is a recognized complication of Plasmodium falciparum malaria, resulting in a substantially worse outcome. Whether a biological relationship exists between malaria infection and IBI susceptibility remains unclear. We, therefore, examined the extent, nature and evidence of this association. Methods We conducted a systematic search in August 2012 of three major scientific databases, PubMed, Embase and Africa Wide Information, for articles describing bacterial infection among children with P. falciparum malaria using the search string ‘(malaria OR plasmodium) AND (bacteria OR bacterial OR bacteremia OR bacteraemia OR sepsis OR septicaemia OR septicemia).’ Eligiblity criteria also included studies of children hospitalized with malaria or outpatient attendances in sub-Saharan Africa. Results A total of 25 studies across 11 African countries fulfilled our criteria. They comprised twenty cohort analyses, two randomized controlled trials and three prospective epidemiological studies. In the meta-analysis of 7,208 children with severe malaria the mean prevalence of IBI was 6.4% (95% confidence interval (CI) 5.81 to 6.98%). In a further meta-analysis of 20,889 children hospitalised with all-severity malaria and 27,641 children with non-malarial febrile illness the mean prevalence of IBI was 5.58 (95% CI 5.5 to 5.66%) in children with malaria and 7.77% (95% CI 7.72 to 7.83%) in non-malaria illness. Ten studies reported mortality stratified by IBI. Case fatality was higher at 81 of 336, 24.1% (95% CI 18.9 to 29.4) in children with malaria/IBI co-infection compared to 585 of 5,760, 10.2% (95% CI 9.3 to 10.98) with malaria alone. Enteric gram-negative organisms were over-represented in malaria cases, non-typhoidal Salmonellae being the most commonest isolate. There was weak evidence indicating IBI was more common in the severe anemia manifestation
Squire, D S; Asmah, R H; Brown, C A; Adjei, D N; Obeng-Nkrumah, N; Ayeh-Kumi, P F
Malaria is hyper-endemic in Ghana. Haematological alterations in the disease pathology may offer complimentary criteria to improve clinical and microscopy diagnosis. Our primary outcome was to evaluate haematological parameters in children with Plasmodium falciparum infections and report their predictive risk and diagnostic performance for malaria infections in Ghana. Haematological data, including thin and thick blood films were examined for children less than 12 years of age in a multicenter-based active case finding approach. Haematological changes were common in P. falciparum infected children and more pronounced in severe malaria cases. More so, a unit increase in parasiteamia increased the odds for severe malaria infection by 93 % [OR, 95 % CI: 1.93 (1.28-2.91); P value = 0.02]. In multivariate regression, low haemoglobin was a significant haematological change in predicting P. falciparum infections [OR, 95 % CI: 3.20 (1.26-7.09); P value = 0.001]. Low haemoglobin levels <11 g/dl was the most reliable indicator for P. falciparum infections [with a sensitivity of (64 %), specificity (71 %), positive predictive value (83 %) and likelihood ratio (2.2)]-even when evaluated in combination with leucocytosis, lymphocytopaenia and high neutrophil counts >7,500 µL. In malaria endemic settings, low haemoglobin concentration (<11 g/dl) in children with febrile illness should prompt a more diligent search for the malarial parasite to limit the misuse and abuse of anti-malarial drugs.
Lee, Andrew H; Fidock, David A
Malaria control efforts have been continuously stymied by drug-resistant strains of Plasmodium falciparum, which typically originate in Southeast Asia prior to spreading into high-transmission settings in Africa. One earlier proposed explanation for Southeast Asia being a hotbed of resistance has been the hypermutability or "Accelerated Resistance to Multiple Drugs" (ARMD) phenotype, whereby multidrug-resistant Southeast Asian parasites were reported to exhibit 1,000-fold higher rates of resistance to unrelated antimalarial agents when compared to drug-sensitive parasites. However, three recent studies do not recapitulate this hypermutability phenotype. Intriguingly, genome sequencing of recently derived multidrug-resistant Cambodian isolates has identified a high proportion of DNA repair gene mutations in multidrug-resistant parasites, suggesting their potential role in shaping local parasite evolution. By adapting fluctuation assays for use in P. falciparum, we have examined the in vitro mutation rates of five recent Cambodian isolates and three reference laboratory strains. For these studies we also generated a knockout parasite line lacking the DNA repair factor Exonuclease I. In these assays, parasites were typed for their ability to acquire resistance to KAE609, currently in advanced clinical trials, yielding 13 novel mutations in the Na+/H+-ATPase PfATP4, the primary resistance determinant. We observed no evidence of hypermutability. Instead, we found evidence of a mild mutator (up to a 3.4-fold increase in mutation rate) phenotype in two artemisinin-resistant Cambodian isolates, which carry DNA repair gene mutations. We observed that one such mutation in the Mismatch Repair protein Mlh1 contributes to the mild mutator phenotype when modeled in yeast (scmlh1-P157S). Compared to basal rates of mutation, a mild mutator phenotype may provide a greater overall benefit for parasites in Southeast Asia in terms of generating drug resistance without incurring
Henry, Roselani I.; Cobbold, Simon A.; Allen, Richard J. W.; Khan, Asif; Hayward, Rhys; Lehane, Adele M.; Bray, Patrick G.; Howitt, Susan M.; Biagini, Giancarlo A.; Saliba, Kevin J.; Kirk, Kiaran
The intraerythrocytic malaria parasite exerts tight control over its ionic composition. In this study, a combination of fluorescent ion indicators and 36Cl− flux measurements was used to investigate the transport of Cl− and the Cl−-dependent transport of “H+-equivalents” in mature (trophozoite stage) parasites, isolated from their host erythrocytes. Removal of extracellular Cl−, resulting in an outward [Cl−] gradient, gave rise to a cytosolic alkalinization (i.e. a net efflux of H+-equivalents). This was reversed on restoration of extracellular Cl−. The flux of H+-equivalents was inhibited by 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid and, when measured in ATP-depleted parasites, showed a pronounced dependence on the pH of the parasite cytosol; the flux was low at cytosolic pH values < 7.2 but increased steeply with cytosolic pH at values > 7.2. 36Cl− influx measurements revealed the presence of a Cl− uptake mechanism with characteristics similar to those of the Cl−-dependent H+-equivalent flux. The intracellular concentration of Cl− in the parasite was estimated to be ∼48 mm in situ. The data are consistent with the intraerythrocytic parasite having in its plasma membrane a 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid-sensitive transporter that, under physiological conditions, imports Cl− together with H+-equivalents, resulting in an intracellular Cl− concentration well above that which would occur if Cl− ions were distributed passively in accordance with the parasite's large, inwardly negative membrane potential. PMID:20332090
Gisselberg, Jolyn E.; Dellibovi-Ragheb, Teegan A.; Matthews, Krista A.; Bosch, Gundula; Prigge, Sean T.
The apicoplast organelle of the malaria parasite Plasmodium falciparum contains metabolic pathways critical for liver-stage and blood-stage development. During the blood stages, parasites lacking an apicoplast can grow in the presence of isopentenyl pyrophosphate (IPP), demonstrating that isoprenoids are the only metabolites produced in the apicoplast which are needed outside of the organelle. Two of the isoprenoid biosynthesis enzymes are predicted to rely on iron-sulfur (FeS) cluster cofactors, however, little is known about FeS cluster synthesis in the parasite or the roles that FeS cluster proteins play in parasite biology. We investigated two putative FeS cluster synthesis pathways (Isc and Suf) focusing on the initial step of sulfur acquisition. In other eukaryotes, these proteins can be located in multiple subcellular compartments, raising the possibility of cross-talk between the pathways or redundant functions. In P. falciparum, SufS and its partner SufE were found exclusively the apicoplast and SufS was shown to have cysteine desulfurase activity in a complementation assay. IscS and its effector Isd11 were solely mitochondrial, suggesting that the Isc pathway cannot contribute to apicoplast FeS cluster synthesis. The Suf pathway was disrupted with a dominant negative mutant resulting in parasites that were only viable when supplemented with IPP. These parasites lacked the apicoplast organelle and its organellar genome – a phenotype not observed when isoprenoid biosynthesis was specifically inhibited with fosmidomycin. Taken together, these results demonstrate that the Suf pathway is essential for parasite survival and has a fundamental role in maintaining the apicoplast organelle in addition to any role in isoprenoid biosynthesis. PMID:24086138
Adjalley, Sophie H; Chabbert, Christophe D; Klaus, Bernd; Pelechano, Vicent; Steinmetz, Lars M
A comprehensive map of transcription start sites (TSSs) across the highly AT-rich genome of P. falciparum would aid progress toward deciphering the molecular mechanisms that underlie the timely regulation of gene expression in this malaria parasite. Using high-throughput sequencing technologies, we generated a comprehensive atlas of transcription initiation events at single-nucleotide resolution during the parasite intra-erythrocytic developmental cycle. This detailed analysis of TSS usage enabled us to define architectural features of plasmodial promoters. We demonstrate that TSS selection and strength are constrained by local nucleotide composition. Furthermore, we provide evidence for coordinate and stage-specific TSS usage from distinct sites within the same transcription unit, thereby producing transcript isoforms, a subset of which are developmentally regulated. This work offers a framework for further investigations into the interactions between genomic sequences and regulatory factors governing the complex transcriptional program of this major human pathogen.
Yoshida, Satoko; Cui, Songkui; Ichihashi, Yasunori; Shirasu, Ken
Parasitic plants thrive by infecting other plants. Flowering plants evolved parasitism independently at least 12 times, in all cases developing a unique multicellular organ called the haustorium that forms upon detection of haustorium-inducing factors derived from the host plant. This organ penetrates into the host stem or root and connects to its vasculature, allowing exchange of materials such as water, nutrients, proteins, nucleotides, pathogens, and retrotransposons between the host and the parasite. In this review, we focus on the formation and function of the haustorium in parasitic plants, with a specific emphasis on recent advances in molecular studies of root parasites in the Orobanchaceae and stem parasites in the Convolvulaceae.
Li, Junmin; Jin, Zexin; Song, Wenjing
Field studies have shown that native, parasitic plants grow vigorously on invasive plants and can cause more damage to invasive plants than native plants. However, no empirical test has been conducted and the mechanism is still unknown. We conducted a completely randomized greenhouse experiment using 3 congeneric pairs of exotic, invasive and native, non-invasive herbaceous plant species to quantify the damage caused by parasitic plants to hosts and its correlation with the hosts' growth rate and resource use efficiency. The biomass of the parasitic plants on exotic, invasive hosts was significantly higher than on congeneric native, non-invasive hosts. Parasites caused more damage to exotic, invasive hosts than to congeneric, native, non-invasive hosts. The damage caused by parasites to hosts was significantly positively correlated with the biomass of parasitic plants. The damage of parasites to hosts was significantly positively correlated with the relative growth rate and the resource use efficiency of its host plants. It may be the mechanism by which parasitic plants grow more vigorously on invasive hosts and cause more damage to exotic, invasive hosts than to native, non-invasive hosts. These results suggest a potential biological control effect of native, parasitic plants on invasive species by reducing the dominance of invasive species in the invaded community.
Li, Junmin; Jin, Zexin; Song, Wenjing
Field studies have shown that native, parasitic plants grow vigorously on invasive plants and can cause more damage to invasive plants than native plants. However, no empirical test has been conducted and the mechanism is still unknown. We conducted a completely randomized greenhouse experiment using 3 congeneric pairs of exotic, invasive and native, non-invasive herbaceous plant species to quantify the damage caused by parasitic plants to hosts and its correlation with the hosts' growth rate and resource use efficiency. The biomass of the parasitic plants on exotic, invasive hosts was significantly higher than on congeneric native, non-invasive hosts. Parasites caused more damage to exotic, invasive hosts than to congeneric, native, non-invasive hosts. The damage caused by parasites to hosts was significantly positively correlated with the biomass of parasitic plants. The damage of parasites to hosts was significantly positively correlated with the relative growth rate and the resource use efficiency of its host plants. It may be the mechanism by which parasitic plants grow more vigorously on invasive hosts and cause more damage to exotic, invasive hosts than to native, non-invasive hosts. These results suggest a potential biological control effect of native, parasitic plants on invasive species by reducing the dominance of invasive species in the invaded community. PMID:22493703
Ferrer, E S; García-Navas, V; Sanz, J J; Ortego, J
Understanding the importance of host genetic diversity for coping with parasites and infectious diseases is a long-standing goal in evolutionary biology. Here, we study the association between probability of infection by avian malaria (Plasmodium relictum) and individual genetic diversity in three blue tit (Cyanistes caeruleus) populations that strongly differ in prevalence of this parasite. For this purpose, we screened avian malaria infections and genotyped 789 blue tits across 26 microsatellite markers. We used two different arrays of markers: 14 loci classified as neutral and 12 loci classified as putatively functional. We found a significant relationship between probability of infection and host genetic diversity estimated at the subset of neutral markers that was not explained by strong local effects and did not differ among the studied populations. This relationship was not linear, and probability of infection increased up to values of homozygosity by locus (HL) around 0.15, reached a plateau at values of HL from 0.15 to 0.40 and finally declined among a small proportion of highly homozygous individuals (HL > 0.4). We did not find evidence for significant identity disequilibrium, which may have resulted from a low variance of inbreeding in the study populations and/or the small power of our set of markers to detect it. A combination of subtle positive and negative local effects and/or a saturation threshold in the association between probability of infection and host genetic diversity in combination with increased resistance to parasites in highly homozygous individuals may explain the observed negative quadratic relationship. Overall, our study highlights that parasites play an important role in shaping host genetic variation and suggests that the use of large sets of neutral markers may be more appropriate for the study of heterozygosity-fitness correlations.
Stanisic, Danielle I.; Gerrard, John; Fink, James; Griffin, Paul M.; Liu, Xue Q.; Sundac, Lana; Sekuloski, Silvana; Rodriguez, Ingrid B.; Pingnet, Jolien; Yang, Yuedong; Zhou, Yaoqi; Trenholme, Katharine R.; Wang, Claire Y. T.; Hackett, Hazel; Chan, Jo-Anne A.; Langer, Christine; Hanssen, Eric; Hoffman, Stephen L.; Beeson, James G.; McCarthy, James S.
Plasmodium falciparum is the most virulent human malaria parasite because of its ability to cytoadhere in the microvasculature. Nonhuman primate studies demonstrated relationships among knob expression, cytoadherence, and infectivity. This has not been examined in humans. Cultured clinical-grade P. falciparum parasites (NF54, 7G8, and 3D7B) and ex vivo-derived cell banks were characterized. Knob and knob-associated histidine-rich protein expression, CD36 adhesion, and antibody recognition of parasitized erythrocytes (PEs) were evaluated. Parasites from the cell banks were administered to malaria-naive human volunteers to explore infectivity. For the NF54 and 3D7B cell banks, blood was collected from the study participants for in vitro characterization. All parasites were infective in vivo. However, infectivity of NF54 was dramatically reduced. In vitro characterization revealed that unlike other cell bank parasites, NF54 PEs lacked knobs and did not cytoadhere. Recognition of NF54 PEs by immune sera was observed, suggesting P. falciparum erythrocyte membrane protein 1 expression. Subsequent recovery of knob expression and CD36-mediated adhesion were observed in PEs derived from participants infected with NF54. Knobless cell bank parasites have a dramatic reduction in infectivity and the ability to adhere to CD36. Subsequent infection of malaria-naive volunteers restored knob expression and CD36-mediated cytoadherence, thereby showing that the human environment can modulate virulence. PMID:27382019
Lettoof, Damian C.; Greenlees, Matthew J.; Stockwell, Michelle; Shine, Richard
One of the most devastating impacts of an invasive species is the introduction of novel parasites or diseases to native fauna. Invasive cane toads (Rhinella marina) in Australia contain several types of parasites, raising concern that the toads may increase rates of parasitism in local anuran species. We sampled cane toads and sympatric native frogs (Limnodynastes peronii, Litoria latopalmata, and Litoria nasuta) at the southern invasion front of cane toads in north-eastern New South Wales (NSW). We dissected and swabbed these anurans to score the presence and abundance of nematodes (Rhabdias lungworms, and gastric encysting nematodes), myxozoans, and chytrid fungus. To determine if cane toad invasion influences rates of parasitism in native frogs, we compared the prevalence and intensity of parasites in frogs from areas with toads, to frogs from areas without toads. Contrary to the situation on the (rapidly-expanding) tropical invasion front, cane toads on the slowly-expanding southern front were heavily infected with rhabditoid lungworms. Toads also contained gastric-encysting nematodes, and one toad was infected by chytrid fungus, but we did not find myxozoans in any toads. All parasite groups were recorded in native frogs, but were less common in areas invaded by toads than in nearby yet to be invaded areas. Contrary to our predictions, toad invasion was associated with a reduced parasite burden in native frogs. Thus, cane toads do not appear to transfer novel parasites to native frog populations, or act as a reservoir for native parasites to ‘spill-back’ into native frogs. Instead, cane toads may reduce frog-parasite numbers by taking up native parasites that are then killed by the toad’s immune defences. PMID:24533330
Frame, I J; Deniskin, Roman; Arora, Avish; Akabas, Myles H
Infection with Plasmodium species parasites causes malaria. Plasmodium parasites are purine auxotrophs. In all life cycle stages, they require purines for RNA and DNA synthesis and other cellular metabolic processes. Purines are imported from the host erythrocyte by equilibrative nucleoside transporters (ENTs). They are processed via purine salvage pathway enzymes to form the required purine nucleotides. The Plasmodium falciparum genome encodes four putative ENTs (PfENT1-4). Genetic, biochemical, and physiologic evidence suggest that PfENT1 is the primary purine transporter supplying the purine salvage pathway. Protein mass spectrometry shows that PfENT1 is expressed in all parasite stages. PfENT1 knockout parasites are not viable in culture at purine concentrations found in human blood (<10 μM). Thus, PfENT1 is a potential target for novel antimalarial drugs, but no PfENT1 inhibitors have been identified to test the hypothesis. Identifying inhibitors of PfENT1 is an essential step to validate PfENT1 as a potential antimalarial drug target.
Koepfli, Cristian; Nguitragool, Wang; Hofmann, Natalie E.; Robinson, Leanne J.; Ome-Kaius, Maria; Sattabongkot, Jetsumon; Felger, Ingrid; Mueller, Ivo
Accurate quantification of parasite density in the human host is essential for understanding the biology and pathology of malaria. Semi-quantitative molecular methods are widely applied, but the need for an external standard curve makes it difficult to compare parasite density estimates across studies. Droplet digital PCR (ddPCR) allows direct quantification without the need for a standard curve. ddPCR was used to diagnose and quantify P. falciparum and P. vivax in clinical patients as well as in asymptomatic samples. ddPCR yielded highly reproducible measurements across the range of parasite densities observed in humans, and showed higher sensitivity than qPCR to diagnose P. falciparum, and equal sensitivity for P. vivax. Correspondence in quantification was very high (>0.95) between qPCR and ddPCR. Quantification between technical replicates by ddPCR differed 1.5–1.7-fold, compared to 2.4–6.2-fold by qPCR. ddPCR facilitates parasite quantification for studies where absolute densities are required, and will increase comparability of results reported from different laboratories. PMID:27982132
Babiker, Hamza A; Hastings, Ian M; Swedberg, Göte
Malaria, a leading parasitic disease, inflicts an enormous toll on human lives and is caused by protozoal parasites belonging to the genus Plasmodium. Antimalarial drugs targeting essential biochemical processes in the parasite are the primary resources for management and control. However, the parasite has established mutations, substantially reducing the efficacy of these drugs. First-line therapy is faced the with the consistent evolution of drug-resistant genotypes carrying these mutations. However, drug-resistant genotypes are likely to be less fit than the wild-type, suggesting that they might disappear by reducing the volume of drug pressure. A substantial body of epidemiological evidence confirmed that the frequency of resistant genotypes wanes when active drug selection declines. Drug selection on the parasite genome that removes genetic variation in the vicinity of drug-resistant genes (hitch-hiking) is common among resistant parasites in the field. This can further disadvantage drug-resistant strains and limit their variability in the face of a mounting immune response. Attempts to provide unequivocal evidence for the fitness cost of drug resistance have monitored the outcomes of laboratory competition experiments of deliberate mixtures of sensitive and resistant strains, in the absence of drug pressure, using isogenic clones produced either by drug selection or gene manipulation. Some of these experiments provided inconclusive results, but they all suggested reduced fitness of drug-resistant clones in the absence of drug pressure. In addition, biochemical analyses provided clearer information demonstrating that the mutation of some antimalarial-targeted enzymes lowers their activity compared with the wild-type enzyme. Here, we review current evidences for the disadvantage of drug-resistance mutations, and discuss some strategies of drug deployment to maximize the cost of resistance and limit its spread.
Moreira, Cristina K.; Naissant, Bernina; Coppi, Alida; Bennett, Brandy L.; Aime, Elena; Franke-Fayard, Blandine; Janse, Chris J.; Coppens, Isabelle; Sinnis, Photini; Templeton, Thomas J.
The phist gene family has members identified across the Plasmodium genus, defined by the presence of a domain of roughly 150 amino acids having conserved aromatic residues and an all alpha-helical structure. The family is highly amplified in P. falciparum, with 65 predicted genes in the genome of the 3D7 isolate. In contrast, in the rodent malaria parasite P. berghei 3 genes are identified, one of which is an apparent pseudogene. Transcripts of the P. berghei phist genes are predominant in schizonts, whereas in P. falciparum transcript profiles span different asexual blood stages and gametocytes. We pursued targeted disruption of P. berghei phist genes in order to characterize a simplistic model for the expanded phist gene repertoire in P. falciparum. Unsuccessful attempts to disrupt P. berghei PBANKA_114540 suggest that this phist gene is essential, while knockout of phist PBANKA_122900 shows an apparent normal progression and non-essential function throughout the life cycle. Epitope-tagging of P. falciparum and P. berghei phist genes confirmed protein export to the erythrocyte cytoplasm and localization with a punctate pattern. Three P. berghei PEXEL/HT-positive exported proteins exhibit at least partial co-localization, in support of a common vesicular compartment in the cytoplasm of erythrocytes infected with rodent malaria parasites. PMID:27022937
Abdul-Ghani, Rashad; Al-Mekhlafi, Abdulsalam M; Alabsi, Mogeeb S
Microbial applications in malaria transmission control have drawn global attention. Mosquito midgut microbiota can modulate vector immunity and block Plasmodium development. Paratransgenic manipulation of bacterial symbionts and Wolbachia can affect reproductive characteristics of mosquitoes. Bacillus-based biolarvicides can control mosquito larvae in different breeding habitats, but their effectiveness differs according to the type of formulation applied, and the physical and ecological conditions of the environment. Entomopathogenic fungi show promise as effective and evolution-proof agents against adult mosquitoes. In addition, transgenic fungi can express anti-plasmodial effector molecules that can target the parasite inside its vector. Despite showing effectiveness in domestic environments as well as against insecticide-resistant mosquitoes, claims towards their deployability in the field and their possible use in integrated vector management programmes have yet to be investigated. Viral pathogens show efficacy in the interruption of sporogonic development of the parasite, and protozoal pathogens exert direct pathogenic potential on larvae and adults with substantial effects on mosquito longevity and fecundity. However, the technology required for their isolation and maintenance impedes their field application. Many agents show promising findings; however, the question remains about the epidemiologic reality of these approaches because even thos