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Sample records for haemosporidian parasites plasmodium

  1. Haemosporidian Parasites of Antelopes and Other Vertebrates from Gabon, Central Africa.

    PubMed

    Boundenga, Larson; Makanga, Boris; Ollomo, Benjamin; Gilabert, Aude; Rougeron, Virginie; Mve-Ondo, Bertrand; Arnathau, Céline; Durand, Patrick; Moukodoum, Nancy Diamella; Okouga, Alain-Prince; Delicat-Loembet, Lucresse; Yacka-Mouele, Lauriane; Rahola, Nil; Leroy, Eric; Ba, Cheikh Tidiane; Renaud, Francois; Prugnolle, Franck; Paupy, Christophe

    2016-01-01

    Re-examination, using molecular tools, of the diversity of haemosporidian parasites (among which the agents of human malaria are the best known) has generally led to rearrangements of traditional classifications. In this study, we explored the diversity of haemosporidian parasites infecting vertebrate species (particularly mammals, birds and reptiles) living in the forests of Gabon (Central Africa), by analyzing a collection of 492 bushmeat samples. We found that samples from five mammalian species (four duiker and one pangolin species), one bird and one turtle species were infected by haemosporidian parasites. In duikers (from which most of the infected specimens were obtained), we demonstrated the existence of at least two distinct parasite lineages related to Polychromophilus species (i.e., bat haemosporidian parasites) and to sauropsid Plasmodium (from birds and lizards). Molecular screening of sylvatic mosquitoes captured during a longitudinal survey revealed the presence of these haemosporidian parasite lineages also in several Anopheles species, suggesting a potential role in their transmission. Our results show that, differently from what was previously thought, several independent clades of haemosporidian parasites (family Plasmodiidae) infect mammals and are transmitted by anopheline mosquitoes. PMID:26863304

  2. Haemosporidian Parasites of Antelopes and Other Vertebrates from Gabon, Central Africa

    PubMed Central

    Ollomo, Benjamin; Gilabert, Aude; Rougeron, Virginie; Mve-Ondo, Bertrand; Arnathau, Céline; Durand, Patrick; Moukodoum, Nancy Diamella; Okouga, Alain-Prince; Delicat-Loembet, Lucresse; Yacka-Mouele, Lauriane; Rahola, Nil; Leroy, Eric; BA, Cheikh Tidiane; Renaud, Francois; Prugnolle, Franck; Paupy, Christophe

    2016-01-01

    Re-examination, using molecular tools, of the diversity of haemosporidian parasites (among which the agents of human malaria are the best known) has generally led to rearrangements of traditional classifications. In this study, we explored the diversity of haemosporidian parasites infecting vertebrate species (particularly mammals, birds and reptiles) living in the forests of Gabon (Central Africa), by analyzing a collection of 492 bushmeat samples. We found that samples from five mammalian species (four duiker and one pangolin species), one bird and one turtle species were infected by haemosporidian parasites. In duikers (from which most of the infected specimens were obtained), we demonstrated the existence of at least two distinct parasite lineages related to Polychromophilus species (i.e., bat haemosporidian parasites) and to sauropsid Plasmodium (from birds and lizards). Molecular screening of sylvatic mosquitoes captured during a longitudinal survey revealed the presence of these haemosporidian parasite lineages also in several Anopheles species, suggesting a potential role in their transmission. Our results show that, differently from what was previously thought, several independent clades of haemosporidian parasites (family Plasmodiidae) infect mammals and are transmitted by anopheline mosquitoes. PMID:26863304

  3. Haemosporidian blood parasites in European birds of prey and owls.

    PubMed

    Krone, O; Waldenstrm, J; Valki?nas, G; Lessow, O; Mller, K; Iezhova, T A; Fickel, J; Bensch, S

    2008-06-01

    Avian blood parasites have been intensively studied using morphological methods with limited information on their host specificity and species taxonomic status. Now the analysis of gene sequences, especially the mitochondrial cytochrome b gene of the avian haemosporidian species of Haemoproteus, Plasmodium, and Leucocytozoon, offers a new tool to review the parasite specificity and status. By comparing morphological and genetic techniques, we observed nearly the same overall prevalence of haemosporidian parasites by microscopy (19.8%) and polymerase chain reaction (PCR) (21.8%) analyses. However, in contrast to the single valid Leucocytozoon species (L. toddi) in the Falconiformes we detected 4 clearly distinctive strains by PCR screening. In the Strigiformes, where the only valid Leucocytozoon species is L. danilewskyi, we detected 3 genetically different strains of Leucocytozoon spp. Two strains of Haemoproteus spp. were detected in the birds of prey and owls examined, whereas the strain found in the tawny owl belonged to the morphospecies Haemoproteus noctuae. Three Plasmodium spp. strains that had already been found in Passeriformes were also detected in the birds of prey and owls examined here, supporting previous findings indicating a broad and nonspecific host spectrum bridging different bird orders. PMID:18605786

  4. Avian haemosporidian parasites infection in wild birds inhabiting Minami-daito Island of the Northwest Pacific, Japan.

    PubMed

    Murata, Koichi; Nii, Ryosuke; Yui, Saori; Sasaki, Emi; Ishikawa, Satoshi; Sato, Yukita; Matsui, Shin; Horie, Sayaka; Akatani, Kana; Takagi, Masaoki; Sawabe, Kyoko; Tsuda, Yoshio

    2008-05-01

    Haemosporidian parasites infection among wild birds inhabiting Minami-daito Island was studied. Blood films from 183 birds representing 4 species of 4 families were examined microscopically. Avian haemosporidian parasites were detected in 3 species with an overall prevalence of 59.6%. None of the 30 Daito scops owls (Otus scops interpositus) examined were infected. Either Haemoproteus sp. or Plasmodium sp. infection was found in 14 of 31 (45.2%) Borodino islands white-eyes (Zosterops japonicus daitoensis). Plasmodium spp. were found in 94 of 102 (92.2%) bull-headed shrikes (Lanius bucephalus) and 1 of 20 (5%) tree sparrows (Passer montanus). PMID:18525175

  5. Local host specialization, host-switching, and dispersal shape the regional distributions of avian haemosporidian parasites

    PubMed Central

    Ellis, Vincenzo A.; Collins, Michael D.; Medeiros, Matthew C. I.; Sari, Eloisa H. R.; Coffey, Elyse D.; Dickerson, Rebecca C.; Lugarini, Camile; Stratford, Jeffrey A.; Henry, Donata R.; Merrill, Loren; Matthews, Alix E.; Hanson, Alison A.; Roberts, Jackson R.; Joyce, Michael; Kunkel, Melanie R.; Ricklefs, Robert E.

    2015-01-01

    The drivers of regional parasite distributions are poorly understood, especially in comparison with those of free-living species. For vector-transmitted parasites, in particular, distributions might be influenced by host-switching and by parasite dispersal with primary hosts and vectors. We surveyed haemosporidian blood parasites (Plasmodium and Haemoproteus) of small land birds in eastern North America to characterize a regional parasite community. Distributions of parasite populations generally reflected distributions of their hosts across the region. However, when the interdependence between hosts and parasites was controlled statistically, local host assemblages were related to regional climatic gradients, but parasite assemblages were not. Moreover, because parasite assemblage similarity does not decrease with distance when controlling for host assemblages and climate, parasites evidently disperse readily within the distributions of their hosts. The degree of specialization on hosts varied in some parasite lineages over short periods and small geographic distances independently of the diversity of available hosts and potentially competing parasite lineages. Nonrandom spatial turnover was apparent in parasite lineages infecting one host species that was well-sampled within a single year across its range, plausibly reflecting localized adaptations of hosts and parasites. Overall, populations of avian hosts generally determine the geographic distributions of haemosporidian parasites. However, parasites are not dispersal-limited within their host distributions, and they may switch hosts readily. PMID:26305975

  6. Local host specialization, host-switching, and dispersal shape the regional distributions of avian haemosporidian parasites.

    PubMed

    Ellis, Vincenzo A; Collins, Michael D; Medeiros, Matthew C I; Sari, Eloisa H R; Coffey, Elyse D; Dickerson, Rebecca C; Lugarini, Camile; Stratford, Jeffrey A; Henry, Donata R; Merrill, Loren; Matthews, Alix E; Hanson, Alison A; Roberts, Jackson R; Joyce, Michael; Kunkel, Melanie R; Ricklefs, Robert E

    2015-09-01

    The drivers of regional parasite distributions are poorly understood, especially in comparison with those of free-living species. For vector-transmitted parasites, in particular, distributions might be influenced by host-switching and by parasite dispersal with primary hosts and vectors. We surveyed haemosporidian blood parasites (Plasmodium and Haemoproteus) of small land birds in eastern North America to characterize a regional parasite community. Distributions of parasite populations generally reflected distributions of their hosts across the region. However, when the interdependence between hosts and parasites was controlled statistically, local host assemblages were related to regional climatic gradients, but parasite assemblages were not. Moreover, because parasite assemblage similarity does not decrease with distance when controlling for host assemblages and climate, parasites evidently disperse readily within the distributions of their hosts. The degree of specialization on hosts varied in some parasite lineages over short periods and small geographic distances independently of the diversity of available hosts and potentially competing parasite lineages. Nonrandom spatial turnover was apparent in parasite lineages infecting one host species that was well-sampled within a single year across its range, plausibly reflecting localized adaptations of hosts and parasites. Overall, populations of avian hosts generally determine the geographic distributions of haemosporidian parasites. However, parasites are not dispersal-limited within their host distributions, and they may switch hosts readily. PMID:26305975

  7. Parasite prevalence corresponds to host life history in a diverse assemblage of afrotropical birds and haemosporidian parasites.

    PubMed

    Lutz, Holly L; Hochachka, Wesley M; Engel, Joshua I; Bell, Jeffrey A; Tkach, Vasyl V; Bates, John M; Hackett, Shannon J; Weckstein, Jason D

    2015-01-01

    Avian host life history traits have been hypothesized to predict rates of infection by haemosporidian parasites. Using molecular techniques, we tested this hypothesis for parasites from three haemosporidian genera (Plasmodium, Haemoproteus, and Leucocytozoon) collected from a diverse sampling of birds in northern Malawi. We found that host life history traits were significantly associated with parasitism rates by all three parasite genera. Nest type and nest location predicted infection probability for all three parasite genera, whereas flocking behavior is an important predictor of Plasmodium and Haemoproteus infection and habitat is an important predictor of Leucocytozoon infection. Parasite prevalence was 79.1% across all individuals sampled, higher than that reported for comparable studies from any other region of the world. Parasite diversity was also exceptionally high, with 248 parasite cytochrome b lineages identified from 152 host species. A large proportion of Plasmodium, Haemoproteus, and Leucocytozoon parasite DNA sequences identified in this study represent new, previously undocumented lineages (n = 201; 81% of total identified) based on BLAST queries against the avian malaria database, MalAvi. PMID:25853491

  8. Parasite Prevalence Corresponds to Host Life History in a Diverse Assemblage of Afrotropical Birds and Haemosporidian Parasites

    PubMed Central

    Lutz, Holly L.; Hochachka, Wesley M.; Engel, Joshua I.; Bell, Jeffrey A.; Tkach, Vasyl V.; Bates, John M.; Hackett, Shannon J.; Weckstein, Jason D.

    2015-01-01

    Avian host life history traits have been hypothesized to predict rates of infection by haemosporidian parasites. Using molecular techniques, we tested this hypothesis for parasites from three haemosporidian genera (Plasmodium, Haemoproteus, and Leucocytozoon) collected from a diverse sampling of birds in northern Malawi. We found that host life history traits were significantly associated with parasitism rates by all three parasite genera. Nest type and nest location predicted infection probability for all three parasite genera, whereas flocking behavior is an important predictor of Plasmodium and Haemoproteus infection and habitat is an important predictor of Leucocytozoon infection. Parasite prevalence was 79.1% across all individuals sampled, higher than that reported for comparable studies from any other region of the world. Parasite diversity was also exceptionally high, with 248 parasite cytochrome b lineages identified from 152 host species. A large proportion of Plasmodium, Haemoproteus, and Leucocytozoon parasite DNA sequences identified in this study represent new, previously undocumented lineages (n = 201; 81% of total identified) based on BLAST queries against the avian malaria database, MalAvi. PMID:25853491

  9. Avian haemosporidian parasites (Haemosporida): A comparative analysis of different polymerase chain reaction assays in detection of mixed infections.

    PubMed

    Bernotienė, Rasa; Palinauskas, Vaidas; Iezhova, Tatjana; Murauskaitė, Dovilė; Valkiūnas, Gediminas

    2016-04-01

    Mixed infections of different species and genetic lineages of haemosporidian parasites (Haemosporida) predominate in wildlife, and such infections are particularly virulent. However, currently used polymerase chain reaction (PCR)-based detection methods often do not read mixed infections. Sensitivity of different PCR assays in detection of mixed infections has been insufficiently tested, but this knowledge is essential in studies addressing parasite diversity in wildlife. Here, we applied five different PCR assays, which are broadly used in wildlife avian haemosporidian research, and compared their sensitivity in detection of experimentally designed mixed infections of Haemoproteus and Plasmodium parasites. Three of these PCR assays use primer sets that amplify fragments of cytochrome b gene (cyt b), one of cytochrome oxidase subunit I (COI) gene, and one target apicoplast genome. We collected blood from wild-caught birds and, using microscopic and PCR-based methods applied in parallel, identified single infections of ten haemosporidian species with similar parasitemia. Then, we prepared 15 experimental mixes of different haemosporidian parasites, which often are present simultaneously in wild birds. Similar concentration of total DNA was used in each parasite lineage during preparation of mixes. Positive amplifications were sequenced, and the presence of mixed infections was reported by visualising double-base calling in sequence electropherograms. This study shows that the use of each single PCR assay markedly underestimates biodiversity of haemosporidian parasites. The application of at least 3 PCR assays in parallel detected the majority, but still not all lineages present in mixed infections. We determined preferences of different primers in detection of parasites belonging to different genera of haemosporidians during mixed infections. PMID:26821298

  10. Genetic diversity of avian haemosporidians in Malaysia: cytochrome b lineages of the genera Plasmodium and Haemoproteus (Haemosporida) from Selangor.

    PubMed

    Ivanova, Karina; Zehtindjiev, Pavel; Mariaux, Jean; Georgiev, Boyko B

    2015-04-01

    The knowledge of the diversity of haemosporidian parasites is of primary importance as their representatives include agents of bird malaria. We investigated the occurrence of Haemoproteus spp. and Plasmodium spp. in bird populations from a single locality in the State of Selangor, Peninsular Malaysia, and report on the parasite prevalence of the two genera. A combination of methods (molecular and morphological) was used for detecting these parasites. Seventy-nine bird individuals were caught using mist-nets in July and August 2010 at Gombak Field Station of the University of Malaya, Kuala Lumpur. In total, 23 birds were identified as positive for Haemoproteus or Plasmodium infection and one individual was recognized as carrying mixed infection. The total prevalence of haemosporidians in the collected samples was 30.3%. Infections with parasites of the genus Haemoproteus were predominant compared to those of the genus Plasmodium. In total, 10 new cyt b lineages of Haemoproteus spp. and 3 new cyt b lineages of Plasmodium spp. were recorded in this study. From all recorded haemosporidian lineages (16 in total), 3 were known from previous studies - hCOLL2, hYWT2 and pNILSUN1. Two of them are linked with their corresponding morphospecies - Haemoproteus pallidus (COLL2) and Haemoproteus motacillae (YWT2). The morphological analysis in the present study confirmed the results obtained by the PCR method relative to prevalence, with 25.3% total prevalence of Haemoproteus and Plasmodium parasites. The intensities of infection varied between 0.01% and 19%. Most infections were light, with intensities below 0.1%. The present study is the first molecular survey of the protozoan blood parasites of the order Haemosporida recorded in Malaysia. PMID:25577987

  11. Phylogeny of haemosporidian blood parasites revealed by a multi-gene approach.

    PubMed

    Borner, Janus; Pick, Christian; Thiede, Jenny; Kolawole, Olatunji Matthew; Kingsley, Manchang Tanyi; Schulze, Jana; Cottontail, Veronika M; Wellinghausen, Nele; Schmidt-Chanasit, Jonas; Bruchhaus, Iris; Burmester, Thorsten

    2016-01-01

    The apicomplexan order Haemosporida is a clade of unicellular blood parasites that infect a variety of reptilian, avian and mammalian hosts. Among them are the agents of human malaria, parasites of the genus Plasmodium, which pose a major threat to human health. Illuminating the evolutionary history of Haemosporida may help us in understanding their enormous biological diversity, as well as tracing the multiple host switches and associated acquisitions of novel life-history traits. However, the deep-level phylogenetic relationships among major haemosporidian clades have remained enigmatic because the datasets employed in phylogenetic analyses were severely limited in either gene coverage or taxon sampling. Using a PCR-based approach that employs a novel set of primers, we sequenced fragments of 21 nuclear genes from seven haemosporidian parasites of the genera Leucocytozoon, Haemoproteus, Parahaemoproteus, Polychromophilus and Plasmodium. After addition of genomic data from 25 apicomplexan species, the unreduced alignment comprised 20,580 bp from 32 species. Phylogenetic analyses were performed based on nucleotide, codon and amino acid data employing Bayesian inference, maximum likelihood and maximum parsimony. All analyses resulted in highly congruent topologies. We found consistent support for a basal position of Leucocytozoon within Haemosporida. In contrast to all previous studies, we recovered a sister group relationship between the genera Polychromophilus and Plasmodium. Within Plasmodium, the sauropsid and mammal-infecting lineages were recovered as sister clades. Support for these relationships was high in nearly all trees, revealing a novel phylogeny of Haemosporida, which is robust to the choice of the outgroup and the method of tree inference. PMID:26364971

  12. Different meal, same flavor: cospeciation and host switching of haemosporidian parasites in some non-passerine birds

    PubMed Central

    2014-01-01

    Background Previous studies have shown that haemosporidian parasites (Haemoproteus (Parahaemoproteus) and Plasmodium) infecting passerine birds have an evolutionary history of host switching with little cospeciation, in particular at low taxonomic levels (e.g., below the family level), which is suggested as the main speciation mechanism of this group of parasites. Recent studies have characterized diverse clades of haemosporidian parasites (H. (Haemoproteus) and H. (Parahaemoproteus)) infecting non-passerine birds (e.g., Columbiformes, Pelecaniiformes). Here, we explore the cospeciation history of H. (Haemoproteus) and H. (Parahaemoproteus) parasites with their non-passerine hosts. Methods We sequenced the mtDNA cyt b gene of both haemosporidian parasites and their avian non-passerine hosts. We built Bayesian phylogenetic hypotheses and created concensus phylograms that were subsequently used to conduct cospeciation analyses. We used both a global cospeciation test, PACo, and an event-cost algorithm implemented in CoRe-PA. Results The global test suggests that H. (Haemoproteus) and H. (Parahaemoproteus) parasites have a diversification history dominated by cospeciation events particularly at the family level. Host-parasite links from the PACo analysis show that host switching events are common within families (i.e., among genera and among species within genera), and occasionally across different orders (e.g., Columbiformes to Pelecaniiformes). Event-cost analyses show that haemosporidian coevolutionary history is dominated by host switching and some codivergence, but with duplication events also present. Genetic lineages unique to raptor species (e.g., FALC11) commonly switch between Falconiformes and Strigiformes. Conclusions Our results corroborate previous findings that have detected a global cospeciation signal at the family taxonomic level, and they also support a history of frequent switching closer to the tips of the host phylogeny, which seems to be the main diversification mechanism of haemosporidians. Such dynamic host-parasite associations are relevant to the epidemiology of emerging diseases because low parasite host specificity is a prerequisite for the emergence of novel diseases. The evidence on host distributions suggests that haemosporidian parasites have the potential to rapidly develop novel host-associations. This pattern has also been recorded in fish-monogenean interactions, suggesting a general diversification mechanism for parasites when host choice is not restricted by ecological barriers. PMID:24957563

  13. In vitro development of Haemoproteus columbae (Haemosporida: Haemoproteidae), with perspectives for genomic studies of avian haemosporidian parasites.

    PubMed

    Coral, Arelis A; Valkiūnas, Gediminas; González, Angie D; Matta, Nubia E

    2015-10-01

    The evolutionary origin of wildlife and human malaria parasites (Plasmodium spp.) has been discussed for several decades. The lack of genomic data about species of wildlife haemosporidian parasites related to Plasmodium limits the number of taxa available for phylogenetic analysis. Genomic data about avian parasites of the genus Haemoproteus parasites, the sister genus to Plasmodium are still not available, mainly due to difficulties in obtaining pure DNA of parasites inhabiting nucleated avian host cells. Recent studies show that microgametes of Haemoproteus (Parahaemoproteus) spp. develop in vitro and can be isolated by simple centrifugation, allowing the isolation of pure parasite DNA for genomic studies. However, in vitro development of Haemoproteus (Haemoproteus) spp. has not been investigated, and it is unclear if microgametes of these parasites also can be obtained under in vitro conditions. Here, we provide the first data about the in vitro development of Haemoproteus (Haemoproteus) columbae, a widespread avian haemosporidian parasite, which is specific to pigeons and doves (Columbiformes) and is transmitted by hippoboscid flies (Diptera, Hippoboscidae). In vitro gametogenesis and ookinete development of H. columbae were studied using a strain isolated from a feral Rock Pigeon (Columba livia) in Bogotá-Colombia. The morphological events leading to exflagellation, fertilization and ookinete formation, as well as the rate of development of these stages were followed in vitro at 40 °C, 19 °C and 15 °C for 48 h. Macrogametes, microgametes, zygotes and initial stages of ookinete development were observed in all temperatures, but mature ookinetes were seen only at 40 °C. The largest diversity of sporogonic stages of H. columbae were present at 40 °C however, exflagellation, fertilization of macrogametes and development of immature ookinetes were also observed at 15 °C and 19 °C. Morphological and morphometric features of these stages in vitro were described and illustrated. This study demonstrates a requirement of high temperature for the successful development of mature ookinetes of H. columbae, but not gametes. We show that 1) parasites of the H. (Haemoproteus) subgenus exflagellate in vitro at 15-19 °C, as is the case in H. (Parahaemoproteus) spp. and 2) in vitro exflagellation can be used to obtain pure DNA for genomic studies. PMID:26297677

  14. Spatially variable coevolution between a haemosporidian parasite and the MHC of a widely distributed passerine.

    PubMed

    Jones, Matthew R; Cheviron, Zachary A; Carling, Matthew D

    2015-03-01

    The environment shapes host-parasite interactions, but how environmental variation affects the diversity and composition of parasite-defense genes of hosts is unresolved. In vertebrates, the highly variable major histocompatibility complex (MHC) gene family plays an essential role in the adaptive immune system by recognizing pathogen infection and initiating the cellular immune response. Investigating MHC-parasite associations across heterogeneous landscapes may elucidate the role of spatially fluctuating selection in the maintenance of high levels of genetic variation at the MHC. We studied patterns of association between an avian haemosporidian blood parasite and the MHC of rufous-collared sparrows (Zonotrichia capensis) that inhabit environments with widely varying haemosporidian infection prevalence in the Peruvian Andes. MHC diversity peaked in populations with high infection prevalence, although intra-individual MHC diversity was not associated with infection status. MHC nucleotide and protein sequences associated with infection absence tended to be rare, consistent with negative frequency-dependent selection. We found an MHC variant associated with a ?26% decrease in infection probability at middle elevations (1501-3100m) where prevalence was highest. Several other variants were associated with a significant increase in infection probability in low haemosporidian prevalence environments, which can be interpreted as susceptibility or quantitative resistance. Our study highlights important challenges in understanding MHC evolution in natural systems, but may point to a role of negative frequency-dependent selection and fluctuating spatial selection in the evolution of Z.capensisMHC. PMID:25798222

  15. Spatially variable coevolution between a haemosporidian parasite and the MHC of a widely distributed passerine

    PubMed Central

    Jones, Matthew R; Cheviron, Zachary A; Carling, Matthew D

    2015-01-01

    The environment shapes host–parasite interactions, but how environmental variation affects the diversity and composition of parasite-defense genes of hosts is unresolved. In vertebrates, the highly variable major histocompatibility complex (MHC) gene family plays an essential role in the adaptive immune system by recognizing pathogen infection and initiating the cellular immune response. Investigating MHC-parasite associations across heterogeneous landscapes may elucidate the role of spatially fluctuating selection in the maintenance of high levels of genetic variation at the MHC. We studied patterns of association between an avian haemosporidian blood parasite and the MHC of rufous-collared sparrows (Zonotrichia capensis) that inhabit environments with widely varying haemosporidian infection prevalence in the Peruvian Andes. MHC diversity peaked in populations with high infection prevalence, although intra-individual MHC diversity was not associated with infection status. MHC nucleotide and protein sequences associated with infection absence tended to be rare, consistent with negative frequency-dependent selection. We found an MHC variant associated with a ∽26% decrease in infection probability at middle elevations (1501–3100 m) where prevalence was highest. Several other variants were associated with a significant increase in infection probability in low haemosporidian prevalence environments, which can be interpreted as susceptibility or quantitative resistance. Our study highlights important challenges in understanding MHC evolution in natural systems, but may point to a role of negative frequency-dependent selection and fluctuating spatial selection in the evolution of Z. capensisMHC. PMID:25798222

  16. Multiple lineages of Avian malaria parasites (Plasmodium) in the Galapagos Islands and evidence for arrival via migratory birds.

    PubMed

    Levin, I I; Zwiers, P; Deem, S L; Geest, E A; Higashiguchi, J M; Iezhova, T A; Jimnez-Uzctegui, G; Kim, D H; Morton, J P; Perlut, N G; Renfrew, R B; Sari, E H R; Valkiunas, G; Parker, P G

    2013-12-01

    Haemosporidian parasites in the genus Plasmodium were recently detected through molecular screening in the Galapagos Penguin (Spheniscus mendiculus). We summarized results of an archipelago-wide screen of 3726 endemic birds representing 22 species for Plasmodium spp. through a combination of molecular and microscopy techniques. Three additional Plasmodium lineages were present in Galapagos. Lineage A-infected penguins, Yellow Warblers (Setophaga petechia aureola), and one Medium Ground Finch (Geospiza fortis) and was detected at multiple sites in multiple years [corrected]. The other 3 lineages were each detected at one site and at one time; apparently, they were transient infections of parasites not established on the archipelago. No gametocytes were found in blood smears of infected individuals; thus, endemic Galapagos birds may be dead-end hosts for these Plasmodium lineages. Determining when and how parasites and pathogens arrive in Galapagos is key to developing conservation strategies to prevent and mitigate the effects of introduced diseases. To assess the potential for Plasmodium parasites to arrive via migratory birds, we analyzed blood samples from 438 North American breeding Bobolinks (Dolichonyx oryzivorus), the only songbird that regularly migrates through Galapagos. Two of the ephemeral Plasmodium lineages (B and C) found in Galapagos birds matched parasite sequences from Bobolinks. Although this is not confirmation that Bobolinks are responsible for introducing these lineages, evidence points to higher potential arrival rates of avian pathogens than previously thought. Linajes Mltiples de Parsitos de Malaria Aviar (Plasmodium) en las Islas Galpagos y Evidencia de su Arribo por Medio de Aves Migratorias. PMID:24033638

  17. Plasmodium ovale: Parasite and Disease

    PubMed Central

    Collins, William E.; Jeffery, Geoffrey M.

    2005-01-01

    Humans are infected by four recognized species of malaria parasites. The last of these to be recognized and described is Plasmodium ovale. Like the other malaria parasites of primates, this parasite is only transmitted via the bites of infected Anopheles mosquitoes. The prepatent period in the human ranges from 12 to 20 days. Some forms in the liver have delayed development, and relapse may occur after periods of up to 4 years after infection. The developmental cycle in the blood lasts approximately 49 h. An examination of records from induced infections indicated that there were an average of 10.3 fever episodes of ?101F and 4.5 fever episodes of ?104F. Mean maximum parasite levels were 6,944/?l for sporozoite-induced infections and 7,310/?l for trophozoite-induced infections. Exoerythrocytic stages have been demonstrated in the liver of humans, chimpanzees, and Saimiri monkeys following injection of sporozoites. Many different Anopheles species have been shown to be susceptible to infection with P. ovale, including A. gambiae, A. atroparvus, A. dirus, A. freeborni, A. albimanus, A. quadrimaculatus, A. stephensi, A. maculatus, A. subpictus, and A. farauti. An enzyme-linked immunosorbent assay has been developed to detect mosquitoes infected with P. ovale using a monoclonal antibody directed against the circumsporozoite protein. Plasmodium ovale is primarily distributed throughout sub-Saharan Africa. It has also been reported from numerous islands in the western Pacific. In more recent years, there have been reports of its distribution on the Asian mainland. Whether or not it will become a major public health problem there remains to be seen. The diagnosis of P. ovale is based primarily on the characteristics of the blood stages and its differentiation from P. vivax. The sometimes elliptical shape of the infected erythrocyte is often diagnostic when combined with other, subtler differences in morphology. The advent of molecular techniques, primarily PCR, has made diagnostic confirmation possible. The development of techniques for the long-term frozen preservation of malaria parasites has allowed the development diagnostic reference standards for P. ovale. Infections in chimpanzees are used to provide reference and diagnostic material for serologic and molecular studies because this parasite has not been shown to develop in other nonhuman primates, nor has it adapted to in vitro culture. There is no evidence to suggest that P. ovale is closely related phylogenetically to any other of the primate malaria parasites that have been examined. PMID:16020691

  18. Local parasite lineage sharing in temperate grassland birds provides clues about potential origins of Galapagos avian Plasmodium.

    PubMed

    Levin, Iris I; Colborn, Rachel E; Kim, Daniel; Perlut, Noah G; Renfrew, Rosalind B; Parker, Patricia G

    2016-02-01

    Oceanic archipelagos are vulnerable to natural introduction of parasites via migratory birds. Our aim was to characterize the geographic origins of two Plasmodium parasite lineages detected in the Galapagos Islands and in North American breeding bobolinks (Dolichonyx oryzivorus) that regularly stop in Galapagos during migration to their South American overwintering sites. We used samples from a grassland breeding bird assemblage in Nebraska, United States, and parasite DNA sequences from the Galapagos Islands, Ecuador, to compare to global data in a DNA sequence registry. Homologous DNA sequences from parasites detected in bobolinks and more sedentary birds (e.g., brown-headed cowbirds Molothrus ater, and other co-occurring bird species resident on the North American breeding grounds) were compared to those recovered in previous studies from global sites. One parasite lineage that matched between Galapagos birds and the migratory bobolink, Plasmodium lineage B, was the most common lineage detected in the global MalAvi database, matching 49 sequences from unique host/site combinations, 41 of which were of South American origin. We did not detect lineage B in brown-headed cowbirds. The other Galapagos-bobolink match, Plasmodium lineage C, was identical to two other sequences from birds sampled in California. We detected a close variant of lineage C in brown-headed cowbirds. Taken together, this pattern suggests that bobolinks became infected with lineage B on the South American end of their migratory range, and with lineage C on the North American breeding grounds. Overall, we detected more parasite lineages in bobolinks than in cowbirds. Galapagos Plasmodium had similar host breadth compared to the non-Galapagos haemosporidian lineages detected in bobolinks, brown-headed cowbirds, and other grassland species. This study highlights the utility of global haemosporidian data in the context of migratory bird-parasite connectivity. It is possible that migratory bobolinks bring parasites to the Galapagos and that these parasites originate from different biogeographic regions representing both their breeding and overwintering sites. PMID:26865960

  19. Helminth Parasites Alter Protection against Plasmodium Infection

    PubMed Central

    Salazar-Castañon, Víctor H.; Legorreta-Herrera, Martha

    2014-01-01

    More than one-third of the world's population is infected with one or more helminthic parasites. Helminth infections are prevalent throughout tropical and subtropical regions where malaria pathogens are transmitted. Malaria is the most widespread and deadliest parasitic disease. The severity of the disease is strongly related to parasite density and the host's immune responses. Furthermore, coinfections between both parasites occur frequently. However, little is known regarding how concomitant infection with helminths and Plasmodium affects the host's immune response. Helminthic infections are frequently massive, chronic, and strong inductors of a Th2-type response. This implies that infection by such parasites could alter the host's susceptibility to subsequent infections by Plasmodium. There are a number of reports on the interactions between helminths and Plasmodium; in some, the burden of Plasmodium parasites increased, but others reported a reduction in the parasite. This review focuses on explaining many of these discrepancies regarding helminth-Plasmodium coinfections in terms of the effects that helminths have on the immune system. In particular, it focuses on helminth-induced immunosuppression and the effects of cytokines controlling polarization toward the Th1 or Th2 arms of the immune response. PMID:25276830

  20. Structure and organization of an avian haemosporidian assemblage in a Neotropical savanna in Brazil.

    PubMed

    Fecchio, Alan; Lima, Marcos Robalinho; Svensson-Coelho, Maria; Marini, Miguel ngelo; Ricklefs, Robert E

    2013-02-01

    Studies on avian haemosporidia are on the rise, but we still lack a basic understanding of how ecological and evolutionary factors mold the distributions of haemosporidia among species in the same bird community. We studied the structure and organization of a local avian haemosporidian assemblage (genera Plasmodium and Haemoproteus) in the Cerrado biome of Central Brazil for 5 years. We obtained 790 blood samples from 54 bird species of which 166 (21%) were infected with haemosporidians based on molecular diagnostics. Partial sequences of the parasite cytochrome b gene revealed 18 differentiated avian haemosporidian lineages. We also analysed the relationship of life-history traits (i.e., nesting height, migration status, nest type, sociality, body mass, and embryo development period) of the 14 most abundant bird species with the prevalence of avian haemosporidia. It was found that host species that bred socially presented a higher prevalence of Haemoproteus (Parahaemoproteus) than bird species that bred in pairs. Thus, aspects of host behaviour could be responsible for differential exposure to vectors. The assemblage of avian haemosporidia studied here also confirms a pattern that is emerging in recent studies using molecular markers to identify avian haemosporidians, namely that many lineages are host generalists. PMID:22939119

  1. Prevalence and Lineage Diversity of Avian Haemosporidians from Three Distinct Cerrado Habitats in Brazil

    PubMed Central

    Belo, Nayara O.; Pinheiro, Renato T.; Reis, Elivnia S.; Ricklefs, Robert E.; Braga, rika M.

    2011-01-01

    Habitat alteration can disrupt hostparasite interactions and lead to the emergence of new diseases in wild populations. The cerrado habitat of Brazil is being fragmented and degraded rapidly by agriculture and urbanization. We screened 676 wild birds from three habitats (intact cerrado, disturbed cerrado and transition area Amazonian rainforest-cerrado) for the presence of haemosporidian parasites (Plasmodium and Haemoproteus) to determine whether different habitats were associated with differences in the prevalence and diversity of infectious diseases in natural populations. Twenty one mitochondrial lineages, including 11 from Plasmodium and 10 from Haemoproteus were identified. Neither prevalence nor diversity of infections by Plasmodium spp. or Haemoproteus spp. differed significantly among the three habitats. However, 15 of the parasite lineages had not been previously described and might be restricted to these habitats or to the region. Six haemosporidian lineages previously known from other regions, particularly the Caribbean Basin, comprised 5080% of the infections in each of the samples, indicating a regional relationship between parasite distribution and abundance. PMID:21408114

  2. Avian haemosporidian persistence and co-infection in great tits at the individual level

    PubMed Central

    2013-01-01

    Background Many studies have tracked the distribution and persistence of avian haemosporidian communities across space and time at the population level, but few studies have investigated these aspects of infection at the individual level over time. Important aspects of parasite infection at the individual level can be missed if only trends at the population level are studied. This study aimed to determine how persistent Haemosporida are in great tit individuals recaptured over several years, whether parasitaemia differed by parasite lineage (mitochondrial cytochrome b haplotype) and how co-infection (i.e. concurrent infection with multiple genera of parasites) affects parasitaemia and body mass. Methods Parasite prevalence was determined by polymerase chain reaction (PCR), quantitative PCR were used to assess parasitaemia and sequencing was employed to determine the identity of the lineages using the MalAvi database. Results Haemosporidian prevalence was high over sampled years with 98% of 55 recaptured individuals showing infection in at least one year of capture. Eighty-two percent of all positive individuals suffered co-infection, with an overall haemosporidian lineage diversity of seventeen. Plasmodium and Haemoproteus parasites were found to be highly persistent, with lineages from these genera consistently found in individuals across years and with no differences in individual parasitaemia being recorded at subsequent captures. Conversely, Leucocytozoon parasites showed higher turnover with regard to lineage changes or transitions in infection status (infected vs non-infected) across years. Parasitaemia was found to be lineage specific and there was no relationship between Plasmodium parasitaemia or host body condition and the presence of Leucocytozoon parasites. Conclusions The findings of this study suggest that different genera of haemosporidian parasites interact differently with their host and other co-infecting parasites, influencing parasite persistence most likely through inter-parasite competition or host-parasite immune interactions. Even-though co-infections do not seem to result in increased virulence (higher parasitaemia or poorer host body condition), further investigation into infection potential of these parasites, both individually and as co-infections, is necessary. PMID:23360530

  3. Central carbon metabolism of Plasmodium parasites

    PubMed Central

    Olszewski, Kellen L.; Llins, Manuel

    2010-01-01

    The central role of metabolic perturbation to the pathology of malaria, the promise of antimetabolites as antimalarial drugs and a basic scientific interest in understanding this fascinating example of highly divergent microbial metabolism has spurred a major and concerted research effort towards elucidating the metabolic network of the Plasmodium parasites. Central carbon metabolism, broadly comprising the flow of carbon from nutrients into biomass, has been a particular focus due to clear and early indications that it plays an essential role in this network. Decades of painstaking efforts have significantly clarified our understanding of these pathways of carbon flux, and this foundational knowledge, coupled with the advent of advanced analytical technologies, have set the stage for the development of a holistic, network-level model of plasmodial carbon metabolism. In this review we summarize the current state of knowledge regarding central carbon metabolism and suggest future avenues of research. We focus primarily on the blood stages of Plasmodium falciparum, the most lethal of the human malaria parasites, but also integrate results from simian, avian and rodent models of malaria that were a major focus of early investigations into plasmodial metabolism. PMID:20849882

  4. Mitosis in the Human Malaria Parasite Plasmodium falciparum ▿

    PubMed Central

    Gerald, Noel; Mahajan, Babita; Kumar, Sanjai

    2011-01-01

    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

  5. On the study of the transmission networks of blood parasites from SW Spain: diversity of avian haemosporidians in the biting midge Culicoides circumscriptus and wild birds

    PubMed Central

    2013-01-01

    Background Blood-sucking flying insects play a key role in the transmission of pathogens of vector-borne diseases. However, at least for the case of avian malaria parasites, the vast majority of studies focus on the interaction between parasites and vertebrate hosts, but there is a lack of information regarding the interaction between the parasites and the insect vectors. Here, we identified the presence of malaria and malaria-like parasite lineages harbored by the potential vector Culicoides circumscriptus (Kieffer). Also, we identified some nodes of the transmission network connecting parasite lineages, potential insect vectors and avian hosts by comparing Haemoproteus and Plasmodium lineages isolated from insects with those infecting wild birds in this and previous studies. Methods Using a molecular approach, we analysed the presence of blood parasites in a total of 97 biting midges trapped in the Doñana National Park (SW Spain) and surrounding areas. Also, 123 blood samples from 11 bird species were analyzed for the presence of blood parasite infections. Blood parasites Haemoproteus and Plasmodium were identified by amplification of a 478 bp fragment of the mitochondrial cytochrome b gen. Results Thirteen biting midges harboured blood parasites including six Haemoproteus and two Plasmodium lineages, supporting the potential role of these insects on parasite transmission. Moreover, ten (8.1%) birds carried blood parasites. Seven Plasmodium and one Haemoproteus lineages were isolated from birds. Overall, six new Haemoproteus lineages were described in this study. Also, we identified the transmission networks of some blood parasites. Two Haemoproteus lineages, hCIRCUM03 and GAGLA03, were identical to those isolated from Corvus monedula in southern Spain and Garrulus glandarius in Bulgaria, respectively. Furthermore, the new Haemoproteus lineage hCIRCUM05 showed a 99% similarity with a lineage found infecting captive penguins in Japan. Conclusions The comparison of the parasite lineages isolated in this study with those previously found infecting birds allowed us to identify some potential nodes in the transmission network of avian blood parasite lineages. These results highlight the complexity of the transmission networks of blood parasites in the wild that may involve a high diversity of susceptible birds and insect vectors. PMID:23856348

  6. Avian Plasmodium in Culex and Ochlerotatus Mosquitoes from Southern Spain: Effects of Season and Host-Feeding Source on Parasite Dynamics

    PubMed Central

    Ferraguti, Martina; Martínez-de la Puente, Josué; Muñoz, Joaquín; Roiz, David; Ruiz, Santiago; Soriguer, Ramón; Figuerola, Jordi

    2013-01-01

    Haemosporidians, a group of vector-borne parasites that include Plasmodium, infect vertebrates including birds. Although mosquitoes are crucial elements in the transmission of avian malaria parasites, little is known of their ecology as vectors. We examined the presence of Plasmodium and Haemoproteus lineages in five mosquito species belonging to the genera Culex and Ochlerotatus to test for the effect of vector species, season and host-feeding source on the transmission dynamics of these pathogens. We analyzed 166 blood-fed individually and 5,579 unfed mosquitoes (grouped in 197 pools) from a locality in southern Spain. In all, 15 Plasmodium and two Haemoproteus lineages were identified on the basis of a fragment of 478 bp of the mitochondrial cytochrome b gene. Infection prevalence of blood parasites in unfed mosquitoes varied between species (range: 0–3.2%) and seasons. The feeding source was identified in 91 mosquitoes where 78% were identified as bird. We found that i) several Plasmodium lineages are shared among different Culex species and one Plasmodium lineage is shared between Culex and Ochlerotatus genera; ii) mosquitoes harboured Haemoproteus parasites; iii) pools of unfed females of mostly ornithophilic Culex species had a higher Plasmodium prevalence than the only mammophylic Culex species studied. However, the mammophylic Ochlerotatus caspius had in pool samples the greatest Plasmodium prevalence. This relative high prevalence may be determined by inter-specific differences in vector survival, susceptibility to infection but also the possibility that this species feeds on birds more frequently than previously thought. Finally, iv) infection rate of mosquitoes varies between seasons and reaches its maximum prevalence during autumn and minimum prevalence in spring. PMID:23823127

  7. The Plasmodium bottleneck: malaria parasite losses in the mosquito vector

    PubMed Central

    Smith, Ryan C; Vega-Rodrguez, Joel; Jacobs-Lorena, Marcelo

    2014-01-01

    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

  8. The Plasmodium bottleneck: malaria parasite losses in the mosquito vector.

    PubMed

    Smith, Ryan C; Vega-Rodrguez, Joel; Jacobs-Lorena, Marcelo

    2014-04-29

    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:24789556

  9. The Plasmodium bottleneck: malaria parasite losses in the mosquito vector.

    PubMed

    Smith, Ryan C; Vega-Rodrguez, Joel; Jacobs-Lorena, Marcelo

    2014-08-01

    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

  10. Cultivation in vitro of the quartan malaria parasite Plasmodium inui.

    PubMed

    Nguyen-Dinh, P; Campbell, C C; Collins, W E

    1980-09-12

    The simian guartan malaria parasite Plasmodium inui (OS strain) was cultured in a continuous flow system with rhesus monkey erythrocytes and RPMI 1640nmedium supplemented with Hepes buffer and rhesus serum. Over a 10-week period, the growth of the parasite permitted a 61,000-fold cumulative dilution of the original inoculum. After 5 weeks in culture, the parasites were still infective to the monkey Saimiri sciureus and to Anopheles freeborni mosquitoes. PMID:6773146

  11. Comparative Genomics and Systems Biology of Malaria Parasites Plasmodium

    PubMed Central

    Cai, Hong; Zhou, Zhan; Gu, Jianying; Wang, Yufeng

    2013-01-01

    Malaria is a serious infectious disease that causes over one million deaths yearly. It is caused by a group of protozoan parasites in the genus Plasmodium. No effective vaccine is currently available and the elevated levels of resistance to drugs in use underscore the pressing need for novel antimalarial targets. In this review, we survey omics centered developments in Plasmodium biology, which have set the stage for a quantum leap in our understanding of the fundamental processes of the parasite life cycle and mechanisms of drug resistance and immune evasion. PMID:24298232

  12. Molecular epidemiology of the emerging human malaria parasite Plasmodium knowlesi

    PubMed Central

    Hakimi, Hassan; Kawai, Satoru; Kawazu, Shin-ichiro

    2014-01-01

    Malaria is the most important parasitic disease with global concern. Plasmodium knowlesi recently has emerged from its natural simian host as a significant cause of human malaria, particularly in Malaysian Borneo. Therefore, it has been added as the fifth human Plasmodium specie which is widely distributed in Southeast Asia. Recent developments of new molecular tools enhanced our understanding about the key features of this malaria parasite. Here, we review some of the ways in which molecular approaches might be used for epidemiology of P. knowlesi and finally lead to an efficient control of malaria. PMID:24754022

  13. MalAvi: a public database of malaria parasites and related haemosporidians in avian hosts based on mitochondrial cytochrome b lineages.

    PubMed

    Bensch, Staffan; Hellgren, Olof; Prez-Tris, Javier

    2009-09-01

    Research in avian blood parasites has seen a remarkable increase since the introduction of polymerase chain reaction-based methods for parasite identification. New data are revealing complex multihost-multiparasite systems which are difficult to understand without good knowledge of the host range and geographical distribution of the parasite lineages. However, such information is currently difficult to obtain from the literature, or from general repositories such as GenBank, mainly because (i) different research groups use different parasite lineage names, (ii) GenBank entries frequently refer only to the first host and locality at which each parasite was sampled, and (iii) different researchers use different gene fragments to identify parasite lineages. We propose a unified database of avian blood parasites of the genera Plasmodium, Haemoproteus and Leucocytozoon identified by a partial region of their cytochrome b sequences. The database uses a standardized nomenclature to remove synonymy, and concentrates all available information about each parasite in a public reference site, thereby facilitating access to all researchers. Initial data include a list of host species and localities, as well as genetic markers that can be used for phylogenetical analyses. The database is free to download and will be regularly updated by the authors. Prior to publication of new lineages, we encourage researchers to assign names to match the existing database. We anticipate that the value of the database as a source for determining host range and geographical distribution of the parasites will grow with its size and substantially enhance the understanding of this remarkably diverse group of parasites. PMID:21564906

  14. African origin of the malaria parasite Plasmodium vivax

    PubMed Central

    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; Frnert, 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.

    2014-01-01

    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

  15. African origin of the malaria parasite Plasmodium vivax.

    PubMed

    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; Frnert, 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

    2014-01-01

    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

  16. Nycteria parasites of Afrotropical insectivorous bats.

    PubMed

    Schaer, Juliane; Reeder, DeeAnn M; Vodzak, Megan E; Olival, Kevin J; Weber, Natalie; Mayer, Frieder; Matuschewski, Kai; Perkins, Susan L

    2015-05-01

    Parasitic protozoan parasites have evolved many co-evolutionary paths towards stable transmission to their host population. Plasmodium spp., the causative agents of malaria, and related haemosporidian parasites are dipteran-borne eukaryotic pathogens that actively invade and use vertebrate erythrocytes for gametogenesis and asexual development, often resulting in substantial morbidity and mortality of the infected hosts. Here, we present results of a survey of insectivorous bats from tropical Africa, including new isolates of species of the haemosporidian genus Nycteria. A hallmark of these parasites is their capacity to infect bat species of distinct families of the two evolutionary distant chiropteran suborders. We did detect Nycteria parasites in both rhinolophid and nycterid bat hosts in geographically separate areas of Sub-Saharan Africa, however our molecular phylogenetic analyses support the separation of the parasites into two distinct clades corresponding to their host genera, suggestive of ancient co-divergence and low levels of host switching. For one clade of these parasites, cytochrome b genes could not be amplified and cytochrome oxidase I sequences showed unusually high rates of evolution, suggesting that the mitochondrial genome of these parasites may have either been lost or substantially altered. This haemosporidian parasite-mammalian host system also highlights that sequential population expansion in the liver and gametocyte formation is a successful alternative to intermediate erythrocytic replication cycles. PMID:25765623

  17. Fate of haem iron in the malaria parasite Plasmodium falciparum.

    PubMed Central

    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

    2002-01-01

    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

  18. CD8+ T Cell Responses to Plasmodium and Intracellular Parasites

    PubMed Central

    Villarino, Nicolas; Schmidt, Nathan W.

    2013-01-01

    Parasitic protozoa are major threats to human health affecting millions of people around the world. Control of these infections by the host immune system relies on a myriad of immunological mechanisms that includes both humoral and cellular immunity. CD8+ T cells contribute to the control of these parasitic infections in both animals and humans. Here, we will focus on the CD8+ T cell response against a subset of these protozoa: Plasmodium, Toxoplasma gondii, Leishmania and Trypanosoma cruzi, with an emphasis on experimental rodent systems. It is evident a complex interaction occurs between CD8+ T cells and the invading protozoa. A detailed understanding of how CD8+ T cells mediate protection should provide the basis for the development of effective vaccines that prevent and control infections by these parasites. PMID:24741372

  19. CD8(+) T Cell Responses to Plasmodium and Intracellular Parasites.

    PubMed

    Villarino, Nicolas; Schmidt, Nathan W

    2013-08-01

    Parasitic protozoa are major threats to human health affecting millions of people around the world. Control of these infections by the host immune system relies on a myriad of immunological mechanisms that includes both humoral and cellular immunity. CD8(+) T cells contribute to the control of these parasitic infections in both animals and humans. Here, we will focus on the CD8(+) T cell response against a subset of these protozoa: Plasmodium, Toxoplasma gondii, Leishmania and Trypanosoma cruzi, with an emphasis on experimental rodent systems. It is evident a complex interaction occurs between CD8(+) T cells and the invading protozoa. A detailed understanding of how CD8(+) T cells mediate protection should provide the basis for the development of effective vaccines that prevent and control infections by these parasites. PMID:24741372

  20. Calcium regulation in the intraerythrocytic malaria parasite Plasmodium falciparum.

    PubMed

    Alleva, L M; Kirk, K

    2001-10-01

    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. PMID:11606221

  1. Continuous in vitro propagation of the malaria parasite Plasmodium vivax.

    PubMed

    Golenda, C F; Li, J; Rosenberg, R

    1997-06-24

    The difficulty in controlling Plasmodium vivax, the most common cause of human malaria, has been complicated by growing drug resistance. We have established a method to cycle parasite generations in continuous culture using human blood cells. Chesson strain parasites were passaged from owl monkey erythrocytes to human reticulocytes in McCoy's 5A medium modified with L-glutamine with 25 mM Hepes buffer supplemented with 20% AB+ human serum. Reticulocytes were separated by differential centrifugation in homologous plasma from the peripheral blood of a hemochromatosis patient. Parasites were grown during each 48-hr cycle in a static candle jar environment until the beginning of schizogony, at about 36-40 hr, when reticulocytes were added and cultures transferred to a shaker for 10-12 hr. The addition of a concentration of 10% reticulocytes resulted in stabilizing parasite densities between 0.28 and 0.57 after cycle 3 and increasing the total number of parasites at least 2-fold with each generational cycle. Cultured parasites successfully infected an owl monkey. The morphology of cultured parasites was typical of P. vivax, with highly ameboid trophozoites evident; however, infected erythrocytes were enlarged and distorted on thin film preparations. The species identity of cultivated parasites was confirmed by analysis of the A and C 18S rRNA genes from genomic DNA and expression of only the A gene during erythrocytic asexual growth. The ability to culture P. vivax opens new opportunities to develop vaccines, test drugs, and clone parasites for genome sequencing. PMID:9192643

  2. Compensatory evolution in the human malaria parasite Plasmodium ovale.

    PubMed

    McCutchan, Thomas F; Rathore, Dharmendar; Li, Jun

    2004-01-01

    The fixation of neutral compensatory mutations in a population depends on the effective population size of the species, which can fluctuate dramatically within a few generations, the mutation rate, and the selection intensity associated with the individual mutations. We observe compensatory mutations and intermediate states in populations of the malaria parasite Plasmodium ovale. The appearance of compensatory mutations and intermediate states in P. ovale raises interesting questions about population structure that could have considerable impact on the control of the associated disease. PMID:15020451

  3. Blood parasites in northern goshawk (Accipiter gentilis) with an emphasis to Leucocytozoon toddi.

    PubMed

    Hanel, Jan; Dolealov, Jana; Stehlkov, rka; Modr, David; Chudoba, Josef; Synek, Petr; Votpka, Jan

    2016-01-01

    Haemosporidians and trypanosomes of the northern goshawk (Accipiter gentilis) population in the Czech Republic were studied by morphological and molecular methods. Despite the wide distribution of these medium-large birds of prey, virtually nothing is known about their blood parasites. During a 5-year period, altogether 88 nestlings and 15 adults were screened for haemosporidians and trypanosomes by microscopic examination of blood smears and by nested PCR. Both methods revealed consistently higher prevalence of blood protists in adults, Leucocytozoon (80.0% in adults vs. 13.6% in nestlings), Haemoproteus (60.0 vs. 2.3%), Plasmodium (6.7 vs. 0%), and Trypanosoma (60.0 vs. 2.3%). Altogether, five haemosporidian lineages were detected by cytochrome b sequencing. Two broadly distributed and host nonspecific lineages, Plasmodium (TURDUS1) and Leucocytozoon (BT2), were detected only sporadically, while three newly described northern goshawk host-specific Leucocytozoon lineages (ACGE01-03) represent the absolute majority of the haemosporidians identified by molecular methods. Our findings support evidences that in falconiform birds the Leucocytozoon toddi group is formed by several host-specific clusters, with Leucocytozoon buteonis in buzzards and Leucocytozoon mathisi in hawks. Between-year comparisons revealed that the infection status of adults remained predominantly unchanged and individuals stayed uninfected or possessed the same parasite lineages; however, two gains and one loss of blood parasite taxa were also recorded. PMID:26365666

  4. Targeting NAD+ Metabolism in the Human Malaria Parasite Plasmodium falciparum

    PubMed Central

    O'Hara, Jessica K.; Kerwin, Lewis J.; Cobbold, Simon A.; Tai, Jonathan; Bedell, Thomas A.; Reider, Paul J.; Llins, Manuel

    2014-01-01

    Nicotinamide adenine dinucleotide (NAD+) is an essential metabolite utilized as a redox cofactor and enzyme substrate in numerous cellular processes. Elevated NAD+ levels have been observed in red blood cells infected with the malaria parasite Plasmodium falciparum, but little is known regarding how the parasite generates NAD+. Here, we employed a mass spectrometry-based metabolomic approach to confirm that P. falciparum lacks the ability to synthesize NAD+ de novo and is reliant on the uptake of exogenous niacin. We characterized several enzymes in the NAD+ pathway and demonstrate cytoplasmic localization for all except the parasite nicotinamidase, which concentrates in the nucleus. One of these enzymes, the P. falciparum nicotinate mononucleotide adenylyltransferase (PfNMNAT), is essential for NAD+ metabolism and is highly diverged from the human homolog, but genetically similar to bacterial NMNATs. Our results demonstrate the enzymatic activity of PfNMNAT in vitro and demonstrate its ability to genetically complement the closely related Escherichia coli NMNAT. Due to the similarity of PfNMNAT to the bacterial enzyme, we tested a panel of previously identified bacterial NMNAT inhibitors and synthesized and screened twenty new derivatives, which demonstrate a range of potency against live parasite culture. These results highlight the importance of the parasite NAD+ metabolic pathway and provide both novel therapeutic targets and promising lead antimalarial compounds. PMID:24747974

  5. Transient transfection of Plasmodium vivax blood-stage parasites.

    PubMed

    Sanchez, Cecilia P; Pfahler, Judith; Del Portillo, Hernando A; Lanzer, Michael

    2013-01-01

    Here, we describe the methodology for transient transfection of Plasmodium vivax. The ability to genetically manipulate P. vivax has rendered this important human malaria parasite more amenable to molecular investigation. However, a systematic analysis of this parasite and its disease-mediating interactions with the human host still awaits further technological breakthroughs, foremost the establishment of a continuous in vitro culture system. Nevertheless, the first steps towards domesticating P. vivax for research purposes have been made. Transfection will eventually help to better understand the unique pathogenic features displayed by P. vivax, such as the host cell specificity for reticulocytes and the occurrence of relapses. Transfection will also be an invaluable tool for studies related to the emerging drug resistance, and it will help identify and validate novel targets for rational intervention. PMID:22990776

  6. Comparative genomics of the neglected human malaria parasite Plasmodium vivax

    PubMed Central

    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; Kanga, 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.

    2008-01-01

    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

  7. Visualization of Malaria Parasites in the Skin Using the Luciferase Transgenic Parasite, Plasmodium berghei.

    PubMed

    Matsuoka, Hiroyuki; Tomita, Hiroyuki; Hattori, Ryuta; Arai, Meiji; Hirai, Makoto

    2015-03-01

    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. PMID:25859153

  8. Genome sequence of the human malaria parasite Plasmodium falciparum

    PubMed Central

    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

    2013-01-01

    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 hostparasite 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. PMID:12368864

  9. Clinical proteomics of the neglected human malarial parasite Plasmodium vivax.

    PubMed

    Acharya, Pragyan; Pallavi, Rani; Chandran, Syama; Dandavate, Vrushali; Sayeed, Syed Khund; Rochani, Ankit; Acharya, Jyoti; Middha, Sheetal; Kochar, Sanjay; Kochar, Dhanpat; Ghosh, Susanta Kumar; Tatu, Utpal

    2011-01-01

    Recent reports highlight the severity and the morbidity of disease caused by the long neglected malaria parasite Plasmodium vivax. Due to inherent difficulties in the laboratory-propagation of P. vivax, the biology of this parasite has not been adequately explored. While the proteome of P. falciparum, the causative agent of cerebral malaria, has been extensively explored from several sources, there is limited information on the proteome of P. vivax. We have, for the first time, examined the proteome of P. vivax isolated directly from patients without adaptation to laboratory conditions. We have identified 153 proteins from clinical P. vivax, majority of which do not show homology to any previously known gene products. We also report 29 new proteins that were found to be expressed in P. vivax for the first time. In addition, several proteins previously implicated as anti-malarial targets, were also found in our analysis. Most importantly, we found several unique proteins expressed by P. vivax.This study is an important step in providing insight into physiology of the parasite under clinical settings. PMID:22028927

  10. Clinical Proteomics of the Neglected Human Malarial Parasite Plasmodium vivax

    PubMed Central

    Chandran, Syama; Dandavate, Vrushali; Sayeed, Syed Khund; Rochani, Ankit; Acharya, Jyoti; Middha, Sheetal; Kochar, Sanjay; Kochar, Dhanpat; Ghosh, Susanta Kumar; Tatu, Utpal

    2011-01-01

    Recent reports highlight the severity and the morbidity of disease caused by the long neglected malaria parasite Plasmodium vivax. Due to inherent difficulties in the laboratory-propagation of P. vivax, the biology of this parasite has not been adequately explored. While the proteome of P. falciparum, the causative agent of cerebral malaria, has been extensively explored from several sources, there is limited information on the proteome of P. vivax. We have, for the first time, examined the proteome of P. vivax isolated directly from patients without adaptation to laboratory conditions. We have identified 153 proteins from clinical P. vivax, majority of which do not show homology to any previously known gene products. We also report 29 new proteins that were found to be expressed in P. vivax for the first time. In addition, several proteins previously implicated as anti-malarial targets, were also found in our analysis. Most importantly, we found several unique proteins expressed by P. vivax.This study is an important step in providing insight into physiology of the parasite under clinical settings. PMID:22028927

  11. Food vacuole proteome of the malarial parasite Plasmodium falciparum.

    PubMed

    Lamarque, Mauld; Tastet, Christophe; Poncet, Jol; Demettre, Edith; Jouin, Patrick; Vial, Henri; Dubremetz, Jean-Franois

    2008-09-01

    The Plasmodium falciparum food vacuole (FV) is a lysosome-like organelle where erythrocyte hemoglobin digestion occurs. It is a favorite target in the development of antimalarials. We have used a tandem mass spectrometry approach to investigate the proteome of an FV-enriched fraction and identified 116 proteins. The electron microscopy analysis and the Western blot data showed that the major component of the fraction was the FV and, as expected, the majority of previously known FV markers were recovered. Of particular interest, several proteins involved in vesicle-mediated trafficking were identified, which are likely to play a key role in FV biogenesis and/or FV protein trafficking. Recovery of parasite surface proteins lends support to the cytostomal pathway of hemoglobin ingestion as a FV trafficking route. We have identified 32 proteins described as hypothetical in the databases. This insight into FV protein content provides new clues towards understanding the biological function of this organelle in P. falciparum. PMID:21136929

  12. Alternative Protein Secretion in the Malaria Parasite Plasmodium falciparum

    PubMed Central

    Thavayogarajah, Thuvaraka; Gangopadhyay, Preetish; Rahlfs, Stefan; Becker, Katja; Lingelbach, Klaus; Przyborski, Jude M.; Holder, Anthony A.

    2015-01-01

    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

  13. Age of the last common ancestor of extant Plasmodium parasite lineages.

    PubMed

    Hayakawa, Toshiyuki; Tachibana, Shin-Ichiro; Hikosaka, Kenji; Arisue, Nobuko; Matsui, Atsushi; Horii, Toshihiro; Tanabe, Kazuyuki

    2012-07-01

    Parasites of the genus Plasmodium infect all classes of amniotes (mammals, birds and reptiles) and display host specificity in their infections. It is therefore generally believed that Plasmodium parasites co-evolved intimately with their hosts. Here, we report that based on an evolutionary analysis using 22 genes in the nuclear genome, extant lineages of Plasmodium parasites originated roughly in the Oligocene epoch after the emergence of their hosts. This timing on the age of the common ancestor of extant Plasmodium parasites suggest the importance of host switches and lends support to the evolutionary scenario of a "malaria big bang" that was proposed based on the evolutionary analysis using the mitochondrial genome. PMID:22555021

  14. Protease-associated cellular networks in malaria parasite Plasmodium falciparum

    PubMed Central

    2011-01-01

    Background Malaria continues to be one of the most severe global infectious diseases, responsible for 1-2 million deaths yearly. The rapid evolution and spread of drug resistance in parasites has led to an urgent need for the development of novel antimalarial targets. Proteases are a group of enzymes that play essential roles in parasite growth and invasion. The possibility of designing specific inhibitors for proteases makes them promising drug targets. Previously, combining a comparative genomics approach and a machine learning approach, we identified the complement of proteases (degradome) in the malaria parasite Plasmodium falciparum and its sibling species [1-3], providing a catalog of targets for functional characterization and rational inhibitor design. Network analysis represents another route to revealing the role of proteins in the biology of parasites and we use this approach here to expand our understanding of the systems involving the proteases of P. falciparum. Results We investigated the roles of proteases in the parasite life cycle by constructing a network using protein-protein association data from the STRING database [4], and analyzing these data, in conjunction with the data from protein-protein interaction assays using the yeast 2-hybrid (Y2H) system [5], blood stage microarray experiments [6-8], proteomics [9-12], literature text mining, and sequence homology analysis. Seventy-seven (77) out of 124 predicted proteases were associated with at least one other protein, constituting 2,431 protein-protein interactions (PPIs). These proteases appear to play diverse roles in metabolism, cell cycle regulation, invasion and infection. Their degrees of connectivity (i.e., connections to other proteins), range from one to 143. The largest protease-associated sub-network is the ubiquitin-proteasome system which is crucial for protein recycling and stress response. Proteases are also implicated in heat shock response, signal peptide processing, cell cycle progression, transcriptional regulation, and signal transduction networks. Conclusions Our network analysis of proteases from P. falciparum uses a so-called guilt-by-association approach to extract sets of proteins from the proteome that are candidates for further study. Novel protease targets and previously unrecognized members of the protease-associated sub-systems provide new insights into the mechanisms underlying parasitism, pathogenesis and virulence. PMID:22369208

  15. The ecology of host immune responses to chronic avian haemosporidian infection.

    PubMed

    Ellis, Vincenzo A; Kunkel, Melanie R; Ricklefs, Robert E

    2014-11-01

    Host responses to parasitism in the wild are often studied in the context of single host-parasite systems, which provide little insight into the ecological dynamics of host-parasite interactions within a community. Here we characterized immune system responses to mostly low-intensity, chronic infection by haemosporidian parasites in a sample of 424 individuals of 22 avian host species from the same local assemblage in the Missouri Ozarks. Two types of white blood cells (heterophils and lymphocytes) were elevated in infected individuals across species, as was the acute-phase protein haptoglobin, which is associated with inflammatory immune responses. Linear discriminant analysis indicated that individuals infected by haemosporidians occupied a subset of the overall white blood cell multivariate space that was also occupied by uninfected individuals, suggesting that these latter individuals might have harbored other pathogens or that parasites more readily infect individuals with a specific white blood cell profile. DNA sequence-defined lineages of haemosporidian parasites were sparsely distributed across the assemblage of hosts. In one well-sampled host species, the red-eyed vireo (Vireo olivaceus), heterophils were significantly elevated in individuals infected with one but not another of two common parasite lineages. Another well-sampled host, the yellow-breasted chat (Icteria virens), exhibited no differences in immune response to different haemosporidian lineages. Our results indicate that while immune responses to infection may be generalized across host species, parasite-specific immune responses may also occur. PMID:25179282

  16. Structure, Function and Inhibition of the Phosphoethanolamine Methyltransferases of the Human Malaria Parasites Plasmodium vivax and Plasmodium knowlesi

    PubMed Central

    Garg, Aprajita; Lukk, Tiit; Kumar, Vidya; Choi, Jae-Yeon; Augagneur, Yoann; Voelker, Dennis R.; Nair, Satish; Mamoun, Choukri Ben

    2015-01-01

    Phosphoethanolamine methyltransferases (PMTs) catalyze the three-step methylation of phosphoethanolamine to form phosphocholine, a critical step in the synthesis of phosphatidylcholine in a select number of eukaryotes including human malaria parasites, nematodes and plants. Genetic studies in the malaria parasite Plasmodium falciparum have shown that the methyltransferase PfPMT plays a critical function in parasite development and differentiation. The presence of PMT orthologs in other malaria parasites that infect humans and their absence in mammals make them ideal targets for the development of selective antimalarials with broad specificity against different Plasmodium species. Here we describe the X-ray structures and biochemical properties of PMT orthologs from Plasmodium vivax and Plasmodium knowlesi and show that both enzymes are inhibited by amodiaquine and NSC158011, two drugs with potent antimalarial activity. Metabolic studies in a yeast mutant that relies on PkPMT or PvPMT for survival demonstrated that these compounds inhibit phosphatidylcholine biosynthesis from ethanolamine. Our structural and functional data provide insights into the mechanism of catalysis and inhibition of PMT enzymes and set the stage for a better design of more specific and selective antimalarial drugs. PMID:25761669

  17. Structure, Function and Inhibition of the Phosphoethanolamine Methyltransferases of the Human Malaria Parasites Plasmodium vivax and Plasmodium knowlesi

    SciTech Connect

    Garg, Aprajita; Lukk, Tiit; Kumar, Vidya; Choi, Jae-Yeon; Augagneur, Yoann; Voelker, Dennis R.; Nair, Satish; Mamoun, Choukri Ben

    2015-03-12

    Phosphoethanolamine methyltransferases (PMTs) catalyze the three-step methylation of phosphoethanolamine to form phosphocholine, a critical step in the synthesis of phosphatidylcholine in a select number of eukaryotes including human malaria parasites, nematodes and plants. Genetic studies in the malaria parasite Plasmodium falciparum have shown that the methyltransferase PfPMT plays a critical function in parasite development and differentiation. The presence of PMT orthologs in other malaria parasites that infect humans and their absence in mammals make them ideal targets for the development of selective antimalarials with broad specificity against different Plasmodium species. Here we describe the X-ray structures and biochemical properties of PMT orthologs from Plasmodium vivax and Plasmodium knowlesi and show that both enzymes are inhibited by amodiaquine and NSC158011, two drugs with potent antimalarial activity. Metabolic studies in a yeast mutant that relies on PkPMT or PvPMT for survival demonstrated that these compounds inhibit phosphatidylcholine biosynthesis from ethanolamine. Our structural and functional data provide insights into the mechanism of catalysis and inhibition of PMT enzymes and set the stage for a better design of more specific and selective antimalarial drugs.

  18. Structure, Function and Inhibition of the Phosphoethanolamine Methyltransferases of the Human Malaria Parasites Plasmodium vivax and Plasmodium knowlesi

    DOE PAGESBeta

    Garg, Aprajita; Lukk, Tiit; Kumar, Vidya; Choi, Jae-Yeon; Augagneur, Yoann; Voelker, Dennis R.; Nair, Satish; Mamoun, Choukri Ben

    2015-03-12

    Phosphoethanolamine methyltransferases (PMTs) catalyze the three-step methylation of phosphoethanolamine to form phosphocholine, a critical step in the synthesis of phosphatidylcholine in a select number of eukaryotes including human malaria parasites, nematodes and plants. Genetic studies in the malaria parasite Plasmodium falciparum have shown that the methyltransferase PfPMT plays a critical function in parasite development and differentiation. The presence of PMT orthologs in other malaria parasites that infect humans and their absence in mammals make them ideal targets for the development of selective antimalarials with broad specificity against different Plasmodium species. Here we describe the X-ray structures and biochemical properties ofmore » PMT orthologs from Plasmodium vivax and Plasmodium knowlesi and show that both enzymes are inhibited by amodiaquine and NSC158011, two drugs with potent antimalarial activity. Metabolic studies in a yeast mutant that relies on PkPMT or PvPMT for survival demonstrated that these compounds inhibit phosphatidylcholine biosynthesis from ethanolamine. Our structural and functional data provide insights into the mechanism of catalysis and inhibition of PMT enzymes and set the stage for a better design of more specific and selective antimalarial drugs.« less

  19. Malaria parasites (Plasmodium spp.) infecting introduced, native and endemic New Zealand birds.

    PubMed

    Howe, Laryssa; Castro, Isabel C; Schoener, Ellen R; Hunter, Stuart; Barraclough, Rosemary K; Alley, Maurice R

    2012-02-01

    Avian malaria is caused by intracellular mosquito-transmitted protist parasites in the order Haemosporida, genus Plasmodium. Although Plasmodium species have been diagnosed as causing death in several threatened species in New Zealand, little is known about their ecology and epidemiology. In this study, we examined the presence, microscopic characterization and sequence homology of Plasmodium spp. isolates collected from a small number of New Zealand introduced, native and endemic bird species. We identified 14 Plasmodium spp. isolates from 90 blood or tissue samples. The host range included four species of passerines (two endemic, one native, one introduced), one species of endemic pigeon and two species of endemic kiwi. The isolates were associated into at least four distinct clusters including Plasmodium (Huffia) elongatum, a subgroup of Plasmodium elongatum, Plasmodium relictum and Plasmodium (Noyvella) spp. The infected birds presented a low level of peripheral parasitemia consistent with chronic infection (11/15 blood smears examined). In addition, we report death due to overwhelming parasitemia in a blackbird, a great spotted kiwi and a hihi. These deaths were attributed to infections with either Plasmodium spp. lineage LINN1 or P. relictum lineage GRW4. To the authors' knowledge, this is the first published report of Plasmodium spp. infection in great spotted and brown kiwi, kereru and kokako. Currently, we are only able to speculate on the origin of these 14 isolates but consideration must be made as to the impact they may have on threatened endemic species, particularly due to the examples of mortality. PMID:21842389

  20. Enlightening the malaria parasite life cycle: bioluminescent Plasmodium in fundamental and applied research.

    PubMed

    Siciliano, Giulia; Alano, Pietro

    2015-01-01

    The unicellular protozoan parasites of the genus Plasmodium impose on human health worldwide the enormous burden of malaria. The possibility to genetically modify several species of malaria parasites represented a major advance in the possibility to elucidate their biology and is now turning laboratory lines of transgenic Plasmodium into precious weapons to fight malaria. Amongst the various genetically modified plasmodia, transgenic parasite lines expressing bioluminescent reporters have been essential to unveil mechanisms of parasite gene expression and to develop in vivo imaging approaches in mouse malaria models. Mainly the human malaria parasite Plasmodium falciparum and the rodent parasite P. berghei have been engineered to express bioluminescent reporters in almost all the developmental stages of the parasite along its complex life cycle between the insect and the vertebrate hosts. Plasmodium lines expressing conventional and improved luciferase reporters are now gaining a central role to develop cell based assays in the much needed search of new antimalarial drugs and to open innovative approaches for both fundamental and applied research in malaria. PMID:26029172

  1. Enlightening the malaria parasite life cycle: bioluminescent Plasmodium in fundamental and applied research

    PubMed Central

    Siciliano, Giulia; Alano, Pietro

    2015-01-01

    The unicellular protozoan parasites of the genus Plasmodium impose on human health worldwide the enormous burden of malaria. The possibility to genetically modify several species of malaria parasites represented a major advance in the possibility to elucidate their biology and is now turning laboratory lines of transgenic Plasmodium into precious weapons to fight malaria. Amongst the various genetically modified plasmodia, transgenic parasite lines expressing bioluminescent reporters have been essential to unveil mechanisms of parasite gene expression and to develop in vivo imaging approaches in mouse malaria models. Mainly the human malaria parasite Plasmodium falciparum and the rodent parasite P. berghei have been engineered to express bioluminescent reporters in almost all the developmental stages of the parasite along its complex life cycle between the insect and the vertebrate hosts. Plasmodium lines expressing conventional and improved luciferase reporters are now gaining a central role to develop cell based assays in the much needed search of new antimalarial drugs and to open innovative approaches for both fundamental and applied research in malaria. PMID:26029172

  2. Reduced erythrocyte susceptibility and increased host clearance of young parasites slows Plasmodium growth in a murine model of severe malaria

    NASA Astrophysics Data System (ADS)

    Khoury, David S.; Cromer, Deborah; Best, Shannon E.; James, Kylie R.; Sebina, Ismail; Haque, Ashraful; Davenport, Miles P.

    2015-05-01

    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.

  3. Vertebrate host specificity of two avian malaria parasites of the subgenus Novyella: Plasmodium nucleophilum and Plasmodium vaughani.

    PubMed

    Iezhova, Tatjana A; Valkinas, Gediminas; Bairlein, Franz

    2005-04-01

    The susceptibility of wild-caught European passeriform birds to naturally isolated malaria parasites, Plasmodium (Novyella) nucleophilum and Plasmodium (Novyella) vaughani, was studied by means of intramuscular subinoculation of infected citrated blood. Plasmodium nucleophilum of the great tit, Parus major, was transmitted to 3 great tits, but 3 blackcaps (Sylvia atricapilla) were not susceptible. Plasmodium vaughani of the robin, Erithacus rubecula, was transmitted to 1 robin and 1 blackcap, but 1 dunnock, Prunella modularis, was not susceptible. The prepatent period was between 8 and 10 days in all experimental infections. Maximum experimental parasitemia (3.4% of red cells) was detected in great tits infected with P. nucleophilum 23 days postexposure. A light (<0.01%) transient parasitemia of P. vaughani developed in the robin and blackcap. This study is in accord with former experimental observations on host specificity of P. nucleophilum and P. vaughani, which are characterized by a wide, but selective, range of avian hosts. Two new host-parasite associations were recorded. PMID:15986631

  4. Transglutaminase in Plasmodium parasites: activity and putative role in oocysts and blood stages.

    PubMed

    Adini, A; Krugliak, M; Ginsburg, H; Li, L; Lavie, L; Warburg, A

    2001-10-01

    Transglutaminase was identified in malaria parasites by immunofluorescence microscopy using alpha-transglutaminase antiserum. Functional enzyme was demonstrated in vivo and in vitro using labeled polyamines that become incorporated into protein substrates through TGase activity. In Plasmodium falciparum intraerythrocytic parasites, transglutaminase activity was stage-dependent: it was weak in ring-forms but much stronger in trophozoites and schizonts. High levels of activity were detected in P. gallinaceum zygotes and ookinetes and in capsules of oocysts developing on mosquito midguts. Unlike most known transglutaminases, the enzymatic activity in Plasmodium was Ca(2+)-independent. Furthermore, levels of activity were similar at 37 and 26 degrees C. Parasite transglutaminase may be responsible for the modification of erythrocytic cytoskeleton in infected cells and it may facilitate the construction of oocyst capsules by cross-linking mosquito-derived basement membrane components with Plasmodium-derived proteins. PMID:11606226

  5. Parasite-induced ER stress response in hepatocytes facilitates Plasmodium liver stage infection.

    PubMed

    Incio, Patricia; Zuzarte-Lus, Vanessa; Ruivo, Margarida T G; Falkard, Brie; Nagaraj, Nagarjuna; Rooijers, Koos; Mann, Matthias; Mair, Gunnar; Fidock, David A; Mota, Maria M

    2015-08-01

    Upon infection of a mammalian host, Plasmodium parasites first replicate inside hepatocytes, generating thousands of new parasites. Although Plasmodium intra-hepatic development represents a substantial metabolic challenge to the host hepatocyte, how infected cells respond to and integrate this stress remains poorly understood. Here, we present proteomic and transcriptomic analyses, revealing that the endoplasmic reticulum (ER)-resident unfolded protein response (UPR) is activated in host hepatocytes upon Plasmodium berghei infection. The expression of XBP1s--the active form of the UPR mediator XBP1--and the liver-specific UPR mediator CREBH is induced by P. berghei infection in vivo. Furthermore, this UPR induction increases parasite liver burden. Altogether, our data suggest that ER stress is a central feature of P. berghei intra-hepatic development, contributing to the success of infection. PMID:26113366

  6. Low- and High-Tech Approaches to Control Plasmodium Parasite Transmission by Anopheles Mosquitoes

    PubMed Central

    Cirimotich, Chris M.; Clayton, April M.; Dimopoulos, George

    2011-01-01

    Current efforts have proven inadequate to stop the transmission of Plasmodium parasites, and hence the spread of malaria, by Anopheles mosquitoes. Therefore, a novel arsenal of strategies for inhibiting Plasmodium infection of mosquitoes is urgently needed. In this paper, we summarize research on two approaches to malaria control, a low-tech strategy based on parasite inhibition by the mosquito's natural microflora, and a high-tech strategy using genetic modification of mosquitoes that renders them resistant to infection and discuss advantages and disadvantages for both approaches. PMID:21876705

  7. Features of autophagic cell death in Plasmodium liver-stage parasites.

    PubMed

    Eickel, Nina; Kaiser, Gesine; Prado, Monica; Burda, Paul-Christian; Roelli, Matthias; Stanway, Rebecca R; Heussler, Volker T

    2013-04-01

    Analyzing molecular determinants of Plasmodium parasite cell death is a promising approach for exploring new avenues in the fight against malaria. Three major forms of cell death (apoptosis, necrosis and autophagic cell death) have been described in multicellular organisms but which cell death processes exist in protozoa is still a matter of debate. Here we suggest that all three types of cell death occur in Plasmodium liver-stage parasites. Whereas typical molecular markers for apoptosis and necrosis have not been found in the genome of Plasmodium parasites, we identified genes coding for putative autophagy-marker proteins and thus concentrated on autophagic cell death. We characterized the Plasmodium berghei homolog of the prominent autophagy marker protein Atg8/LC3 and found that it localized to the apicoplast. A relocalization of PbAtg8 to autophagosome-like vesicles or vacuoles that appear in dying parasites was not, however, observed. This strongly suggests that the function of this protein in liver-stage parasites is restricted to apicoplast biology. PMID:23388496

  8. Features of autophagic cell death in Plasmodium liver-stage parasites

    PubMed Central

    Eickel, Nina; Kaiser, Gesine; Prado, Monica; Burda, Paul-Christian; Roelli, Matthias; Stanway, Rebecca R.; Heussler, Volker T.

    2013-01-01

    Analyzing molecular determinants of Plasmodium parasite cell death is a promising approach for exploring new avenues in the fight against malaria. Three major forms of cell death (apoptosis, necrosis and autophagic cell death) have been described in multicellular organisms but which cell death processes exist in protozoa is still a matter of debate. Here we suggest that all three types of cell death occur in Plasmodium liver-stage parasites. Whereas typical molecular markers for apoptosis and necrosis have not been found in the genome of Plasmodium parasites, we identified genes coding for putative autophagy-marker proteins and thus concentrated on autophagic cell death. We characterized the Plasmodium berghei homolog of the prominent autophagy marker protein Atg8/LC3 and found that it localized to the apicoplast. A relocalization of PbAtg8 to autophagosome-like vesicles or vacuoles that appear in dying parasites was not, however, observed. This strongly suggests that the function of this protein in liver-stage parasites is restricted to apicoplast biology. PMID:23388496

  9. Quantitative Bioluminescent Imaging of Pre-Erythrocytic Malaria Parasite Infection Using Luciferase-Expressing Plasmodium yoelii

    PubMed Central

    Miller, Jessica L.; Murray, Sara; Vaughan, Ashley M.; Harupa, Anke; Sack, Brandon; Baldwin, Michael; Crispe, Ian N.; Kappe, Stefan H. I.

    2013-01-01

    The liver stages of Plasmodium parasites are important targets for the development of anti-malarial vaccine candidates and chemoprophylaxis approaches that aim to prevent clinical infection. Analyzing the impact of interventions on liver stages in the murine malaria model system Plasmodium yoelii has been cumbersome and requires terminal procedures. In vivo imaging of bioluminescent parasites has previously been shown to be an effective and non-invasive alternative to monitoring liver stage burden in the Plasmodium berghei model. Here we report the generation and characterization of a transgenic P. yoelii parasite expressing the reporter protein luciferase throughout the parasite life cycle. In vivo bioluminescent imaging of these parasites allows for quantitative analysis of P. yoelii liver stage burden and parasite development, which is comparable to quantitative RT-PCR analysis of liver infection. Using this system, we show that both BALB/cJ and C57BL/6 mice show comparable susceptibility to P. yoelii infection with sporozoites and that bioluminescent imaging can be used to monitor protective efficacy of attenuated parasite immunizations. Thus, this rapid, simple and noninvasive method for monitoring P. yoelii infection in the liver provides an efficient system to screen and evaluate the effects of anti-malarial interventions in vivo and in real-time. PMID:23593316

  10. Transport of lactate and pyruvate in the intraerythrocytic malaria parasite, Plasmodium falciparum.

    PubMed Central

    Elliott, J L; Saliba, K J; Kirk, K

    2001-01-01

    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

  11. Analysis of malaria parasite phenotypes using experimental genetic crosses of Plasmodium falciparum

    PubMed Central

    Ranford-Cartwright, Lisa C.; Mwangi, Jonathan M.

    2012-01-01

    We review the principles of linkage analysis of experimental genetic crosses and their application to Plasmodium falciparum. Three experimental genetic crosses have been performed using the human malaria parasite P. falciparum. Linkage analysis of the progeny of these crosses has been used to identify parasite genes important in phenotypes such as drug resistance, parasite growth and virulence, and transmission to mosquitoes. The construction and analysis of genetic maps has been used to characterise recombination rates across the parasite genome and to identify hotspots of recombination. PMID:22475816

  12. Host compatibility rather than vectorhost-encounter rate determines the host range of avian Plasmodium parasites

    PubMed Central

    Medeiros, Matthew C. I.; Hamer, Gabriel L.; Ricklefs, Robert E.

    2013-01-01

    Blood-feeding arthropod vectors are responsible for transmitting many parasites between vertebrate hosts. While arthropod vectors often feed on limited subsets of potential host species, little is known about the extent to which this influences the distribution of vector-borne parasites in some systems. Here, we test the hypothesis that different vector species structure parasitehost relationships by restricting access of certain parasites to a subset of available hosts. Specifically, we investigate how the feeding patterns of Culex mosquito vectors relate to distributions of avian malaria parasites among hosts in suburban Chicago, IL, USA. We show that Plasmodium lineages, defined by cytochrome b haplotypes, are heterogeneously distributed across avian hosts. However, the feeding patterns of the dominant vectors (Culex restuans and Culex pipiens) are similar across these hosts, and do not explain the distributions of Plasmodium parasites. Phylogenetic similarity of avian hosts predicts similarity in their Plasmodium parasites. This effect was driven primarily by the general association of Plasmodium parasites with particular host superfamilies. Our results suggest that a mosquito-imposed encounter rate does not limit the distribution of avian Plasmodium parasites across hosts. This implies that compatibility between parasites and their avian hosts structure Plasmodium host range. PMID:23595266

  13. Expression profiling of Plasmodium berghei HSP70 genes for generation of bright red fluorescent parasites.

    PubMed

    Hliscs, Marion; Nahar, Carolin; Frischknecht, Friedrich; Matuschewski, Kai

    2013-01-01

    Live cell imaging of recombinant malarial parasites encoding fluorescent probes provides critical insights into parasite-host interactions and life cycle progression. In this study, we generated a red fluorescent line of the murine malarial parasite Plasmodium berghei. To allow constitutive and abundant expression of the mCherry protein we profiled expression of all members of the P. berghei heat shock protein 70 (HSP70) family. We identified PbHSP70/1, an invariant ortholog of Plasmodium falciparum HSP70-1, as the protein with the highest expression levels during Plasmodium blood, mosquito, and liver infection. Stable allelic insertion of a mCherry expression cassette into the PbHsp70/1 locus created constitutive red fluorescent P. berghei lines, termed Pbred. We show that these parasites can be used for live imaging of infected host cells and organs, including hepatocytes, erythrocytes, and whole Anopheles mosquitoes. Quantification of the fluorescence intensity of several Pbred parasite stages revealed significantly enhanced signal intensities in comparison to GFP expressed under the control of the constitutive EF1alpha promoter. We propose that systematic transcript profiling permits generation of reporter parasites, such as the Pbred lines described herein. PMID:24013507

  14. Host genotype by parasite genotype interactions underlying the resistance of anopheline mosquitoes to Plasmodium falciparum

    PubMed Central

    Lambrechts, Louis; Halbert, Jean; Durand, Patrick; Gouagna, Louis C; Koella, Jacob C

    2005-01-01

    Background Most studies on the resistance of mosquitoes to their malaria parasites focus on the response of a mosquito line or colony against a single parasite genotype. In natural situations, however, it may be expected that mosquito-malaria relationships are based, as are many other host-parasite systems, on host genotype by parasite genotype interactions. In such systems, certain hosts are resistant to one subset of the parasite's genotypes, while other hosts are resistant to a different subset. Methods To test for genotype by genotype interactions between malaria parasites and their anopheline vectors, different genetic backgrounds (families consisting of the F1 offspring of individual females) of the major African vector Anopheles gambiae were challenged with several isolates of the human malaria parasite Plasmodium falciparum (obtained from naturally infected children in Kenya). Results Averaged across all parasites, the proportion of infected mosquitoes and the number of oocysts found in their midguts were similar in all mosquito families. Both indices of resistance, however, differed considerably among isolates of the parasite. In particular, no mosquito family was most resistant to all parasites, and no parasite isolate was most infectious to all mosquitoes. Conclusions These results suggest that the level of mosquito resistance depends on the interaction between its own and the parasite's genotype. This finding thus emphasizes the need to take into account the range of genetic diversity exhibited by mosquito and malaria field populations in ideas and studies concerning the control of malaria. PMID:15644136

  15. The Host Targeting motif in exported Plasmodium proteins is cleaved in the parasite endoplasmic reticulum

    PubMed Central

    Osborne, Andrew R.; Speicher, Kaye D.; Tamez, Pamela A.; Bhattacharjee, Souvik; Speicher, David W.; Haldar, Kasturi

    2010-01-01

    During the blood stage of its lifecycle, the malaria parasite resides and replicates inside a membrane vacuole within its host cell, the human erythrocyte. The parasite exports many proteins across the vacuole membrane and into the host cell cytoplasm. Most exported proteins are characterized by the presence of a Host Targeting (HT) motif, also referred to as a Plasmodium Export Element (PEXEL), which corresponds to the consensus sequence RxLxE/D/Q. During export the HT motif is cleaved by an unknown protease. Here, we generate parasite lines expressing HT motif containing proteins that are localized to different compartments within the parasite or host cell. We find that the HT motif in a protein that is retained in the parasite endoplasmic reticulum, is cleaved and N-acetylated as efficiently as a protein that is exported. This shows that cleavage of the HT motif occurs early in the secretory pathway, in the parasite endoplasmic reticulum. PMID:20117149

  16. The host targeting motif in exported Plasmodium proteins is cleaved in the parasite endoplasmic reticulum.

    PubMed

    Osborne, Andrew R; Speicher, Kaye D; Tamez, Pamela A; Bhattacharjee, Souvik; Speicher, David W; Haldar, Kasturi

    2010-05-01

    During the blood stage of its lifecycle, the malaria parasite resides and replicates inside a membrane vacuole within its host cell, the human erythrocyte. The parasite exports many proteins across the vacuole membrane and into the host cell cytoplasm. Most exported proteins are characterized by the presence of a host targeting (HT) motif, also referred to as a Plasmodium export element (PEXEL), which corresponds to the consensus sequence RxLxE/D/Q. During export the HT motif is cleaved by an unknown protease. Here, we generate parasite lines expressing HT motif containing proteins that are localized to different compartments within the parasite or host cell. We find that the HT motif in a protein that is retained in the parasite endoplasmic reticulum is cleaved and N-acetylated as efficiently as a protein that is exported. This shows that cleavage of the HT motif occurs early in the secretory pathway, in the parasite endoplasmic reticulum. PMID:20117149

  17. Cultivation in vitro of the vivax-type malaria parasite Plasmodium cynomolgi.

    PubMed

    Nguyen-Dinh, P; Gardner, A L; Campbell, C C; Skinner, J C; Collins, W E

    1981-06-01

    The vivax-type simian malaria parasite Plasmodium cynomologi was cultured in vitro by both the candle jar method and the continuous flow technique, with rhesus monkey erythrocytes and RPMI 1640 medium supplemented with Hepes buffer and human serum. After 6 weeks in culture, the growth of the parasite had permitted a 5 X 10(6) cumulative dilution of the original inoculum. Cultured parasites remained infective to rhesus monkeys and exhibited a reversible decrease in the ameboid behavior of their trophozoites. PMID:7233207

  18. Using expression information to discover new drug and vaccine targets in the malaria parasite Plasmodium falciparum.

    PubMed

    Young, Jason A; Winzeler, Elizabeth A

    2005-01-01

    The recent completion of the malaria parasite Plasmodium falciparum genome has opened the door for applying a variety of genomic-based systems biology approaches that complement existing gene-by-gene methods of investigation. Transcriptomic analyses of P. falciparum using DNA microarrays has allowed for the rapid elucidation of gene function, parasite drug response, and in vivo expression profiles, as well as general mechanisms guiding the parasite life cycle that are vital to disease pathogenesis. The results of these studies have identified promising novel gene targets for the development of new drug and vaccine therapies. PMID:15723602

  19. Sexual development in Plasmodium parasites: knowing when it's time to commit.

    PubMed

    Josling, Gabrielle A; Llinás, Manuel

    2015-09-01

    Malaria is a devastating infectious disease that is caused by blood-borne apicomplexan parasites of the genus Plasmodium. These pathogens have a complex lifecycle, which includes development in the anopheline mosquito vector and in the liver and red blood cells of mammalian hosts, a process which takes days to weeks, depending on the Plasmodium species. Productive transmission between the mammalian host and the mosquito requires transitioning between asexual and sexual forms of the parasite. Blood- stage parasites replicate cyclically and are mostly asexual, although a small fraction of these convert into male and female sexual forms (gametocytes) in each reproductive cycle. Despite many years of investigation, the molecular processes that elicit sexual differentiation have remained largely unknown. In this Review, we highlight several important recent discoveries that have identified epigenetic factors and specific transcriptional regulators of gametocyte commitment and development, providing crucial insights into this obligate cellular differentiation process. PMID:26272409

  20. Organellar proteomics reveals hundreds of novel nuclear proteins in the malaria parasite Plasmodium falciparum

    PubMed Central

    2012-01-01

    Background The post-genomic era of malaria research provided unprecedented insights into the biology of Plasmodium parasites. Due to the large evolutionary distance to model eukaryotes, however, we lack a profound understanding of many processes in Plasmodium biology. One example is the cell nucleus, which controls the parasite genome in a development- and cell cycle-specific manner through mostly unknown mechanisms. To study this important organelle in detail, we conducted an integrative analysis of the P. falciparum nuclear proteome. Results We combined high accuracy mass spectrometry and bioinformatic approaches to present for the first time an experimentally determined core nuclear proteome for P. falciparum. Besides a large number of factors implicated in known nuclear processes, one-third of all detected proteins carry no functional annotation, including many phylum- or genus-specific factors. Importantly, extensive experimental validation using 30 transgenic cell lines confirmed the high specificity of this inventory, and revealed distinct nuclear localization patterns of hitherto uncharacterized proteins. Further, our detailed analysis identified novel protein domains potentially implicated in gene transcription pathways, and sheds important new light on nuclear compartments and processes including regulatory complexes, the nucleolus, nuclear pores, and nuclear import pathways. Conclusion Our study provides comprehensive new insight into the biology of the Plasmodium nucleus and will serve as an important platform for dissecting general and parasite-specific nuclear processes in malaria parasites. Moreover, as the first nuclear proteome characterized in any protist organism, it will provide an important resource for studying evolutionary aspects of nuclear biology. PMID:23181666

  1. DNA Repair Mechanisms and Their Biological Roles in the Malaria Parasite Plasmodium falciparum

    PubMed Central

    Lee, Andrew H.; Symington, Lorraine S.

    2014-01-01

    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

  2. Within-host Competition Does Not Select for Virulence in Malaria Parasites; Studies with Plasmodium yoelii

    PubMed Central

    Abkallo, Hussein M.; Tangena, Julie-Anne; Tang, Jianxia; Kobayashi, Nobuyuki; Inoue, Megumi; Zoungrana, Augustin; Colegrave, Nick; Culleton, Richard

    2015-01-01

    In endemic areas with high transmission intensities, malaria infections are very often composed of multiple genetically distinct strains of malaria parasites. It has been hypothesised that this leads to intra-host competition, in which parasite strains compete for resources such as space and nutrients. This competition may have repercussions for the host, the parasite, and the vector in terms of disease severity, vector fitness, and parasite transmission potential and fitness. It has also been argued that within-host competition could lead to selection for more virulent parasites. Here we use the rodent malaria parasite Plasmodium yoelii to assess the consequences of mixed strain infections on disease severity and parasite fitness. Three isogenic strains with dramatically different growth rates (and hence virulence) were maintained in mice in single infections or in mixed strain infections with a genetically distinct strain. We compared the virulence (defined as harm to the mammalian host) of mixed strain infections with that of single infections, and assessed whether competition impacted on parasite fitness, assessed by transmission potential. We found that mixed infections were associated with a higher degree of disease severity and a prolonged infection time. In the mixed infections, the strain with the slower growth rate was often responsible for the competitive exclusion of the faster growing strain, presumably through host immune-mediated mechanisms. Importantly, and in contrast to previous work conducted with Plasmodium chabaudi, we found no correlation between parasite virulence and transmission potential to mosquitoes, suggesting that within-host competition would not drive the evolution of parasite virulence in P. yoelii. PMID:25658331

  3. CD47 regulates the phagocytic clearance and replication of the Plasmodium yoelii malaria parasite.

    PubMed

    Banerjee, Rajdeep; Khandelwal, Sanjay; Kozakai, Yukiko; Sahu, Bikash; Kumar, Sanjai

    2015-03-10

    Several Plasmodium species exhibit a strong age-based preference for the red blood cells (RBC) they infect, which in turn is a major determinant of disease severity and pathogenesis. The molecular basis underlying this age constraint on the use of RBC and its influence on parasite burden is poorly understood. CD47 is a marker of self on most cells, including RBC, which, in conjunction with signal regulatory protein alpha (expressed on macrophages), prevents the clearance of cells by the immune system. In this report, we have investigated the role of CD47 on the growth and survival of nonlethal Plasmodium yoelii 17XNL (PyNL) malaria in C57BL/6 mice. By using a quantitative biotin-labeling procedure and a GFP-expressing parasite, we demonstrate that PyNL parasites preferentially infect high levels of CD47 (CD47(hi))-expressing young RBC. Importantly, C57BL/6 CD47(-/-) mice were highly resistant to PyNL infection and developed a 9.3-fold lower peak parasitemia than their wild-type (WT) counterparts. The enhanced resistance to malaria observed in CD47(-/-) mice was associated with a higher percentage of splenic F4/80(+) cells, and these cells had a higher percentage of phagocytized parasitized RBC than infected WT mice during the acute phase of infection, when parasitemia was rapidly rising. Furthermore, injection of CD47-neutralizing antibody caused a significant reduction in parasite burden in WT C57BL/6 mice. Together, these results strongly suggest that CD47(hi) young RBC may provide a shield to the malaria parasite from clearance by the phagocytic cells, which may be an immune escape mechanism used by Plasmodium parasites that preferentially infect young RBC. PMID:25713361

  4. CD47 regulates the phagocytic clearance and replication of the Plasmodium yoelii malaria parasite

    PubMed Central

    Banerjee, Rajdeep; Khandelwal, Sanjay; Kozakai, Yukiko; Sahu, Bikash; Kumar, Sanjai

    2015-01-01

    Several Plasmodium species exhibit a strong age-based preference for the red blood cells (RBC) they infect, which in turn is a major determinant of disease severity and pathogenesis. The molecular basis underlying this age constraint on the use of RBC and its influence on parasite burden is poorly understood. CD47 is a marker of self on most cells, including RBC, which, in conjunction with signal regulatory protein alpha (expressed on macrophages), prevents the clearance of cells by the immune system. In this report, we have investigated the role of CD47 on the growth and survival of nonlethal Plasmodium yoelii 17XNL (PyNL) malaria in C57BL/6 mice. By using a quantitative biotin-labeling procedure and a GFP-expressing parasite, we demonstrate that PyNL parasites preferentially infect high levels of CD47 (CD47hi)-expressing young RBC. Importantly, C57BL/6 CD47?/? mice were highly resistant to PyNL infection and developed a 9.3-fold lower peak parasitemia than their wild-type (WT) counterparts. The enhanced resistance to malaria observed in CD47?/? mice was associated with a higher percentage of splenic F4/80+ cells, and these cells had a higher percentage of phagocytized parasitized RBC than infected WT mice during the acute phase of infection, when parasitemia was rapidly rising. Furthermore, injection of CD47-neutralizing antibody caused a significant reduction in parasite burden in WT C57BL/6 mice. Together, these results strongly suggest that CD47hi young RBC may provide a shield to the malaria parasite from clearance by the phagocytic cells, which may be an immune escape mechanism used by Plasmodium parasites that preferentially infect young RBC. PMID:25713361

  5. The remarkable journey of adaptation of the Plasmodium falciparum malaria parasite to New World anopheline mosquitoes

    PubMed Central

    Molina-Cruz, Alvaro; Barillas-Mury, Carolina

    2014-01-01

    Plasmodium falciparum originated in Africa, dispersed around the world as a result of human migration and had to adapt to several different indigenous anopheline mosquitoes. Anophelines from the New World are evolutionary distant form African ones and this probably resulted in a more stringent selection of Plasmodium as it adapted to these vectors. It is thought that Plasmodium has been genetically selected by some anopheline species through unknown mechanisms. The mosquito immune system can greatly limit infection and P. falciparum evolved a strategy to evade these responses, at least in part mediated by Pfs47, a highly polymorphic gene. We propose that adaptation of P. falciparum to new vectors may require evasion of their immune system. Parasites with a Pfs47 haplotype compatible with the indigenous mosquito vector would be able to survive and be transmitted. The mosquito antiplasmodial response could be an important determinant of P. falciparum population structure and could affect malaria transmission in the Americas. PMID:25185006

  6. Artesunate Tolerance in Transgenic Plasmodium falciparum Parasites Overexpressing a Tryptophan-Rich Protein▿†

    PubMed Central

    Deplaine, Guillaume; Lavazec, Catherine; Bischoff, Emmanuel; Natalang, Onguma; Perrot, Sylvie; Guillotte-Blisnick, Micheline; Coppée, Jean-Yves; Pradines, Bruno; Mercereau-Puijalon, Odile; David, Peter H.

    2011-01-01

    Due to their rapid, potent action on young and mature intraerythrocytic stages, artemisinin derivatives are central to drug combination therapies for Plasmodium falciparum malaria. However, the evidence for emerging parasite resistance/tolerance to artemisinins in southeast Asia is of great concern. A better understanding of artemisinin-related drug activity and resistance mechanisms is urgently needed. A recent transcriptome study of parasites exposed to artesunate led us to identify a series of genes with modified levels of expression in the presence of the drug. The gene presenting the largest mRNA level increase, Pf10_0026 (PArt), encoding a hypothetical protein of unknown function, was chosen for further study. Immunodetection with PArt-specific sera showed that artesunate induced a dose-dependent increase of the protein level. Bioinformatic analysis showed that PArt belongs to a Plasmodium-specific gene family characterized by the presence of a tryptophan-rich domain with a novel hidden Markov model (HMM) profile. Gene disruption could not be achieved, suggesting an essential function. Transgenic parasites overexpressing PArt protein were generated and exhibited tolerance to a spike exposure to high doses of artesunate, with increased survival and reduced growth retardation compared to that of wild-type-treated controls. These data indicate the involvement of PArt in parasite defense mechanisms against artesunate. This is the first report of genetically manipulated parasites displaying a stable and reproducible decreased susceptibility to artesunate, providing new possibilities to investigate the parasite response to artemisinins. PMID:21464256

  7. High diversity of West African bat malaria parasites and a tight link with rodent Plasmodium taxa

    PubMed Central

    Schaer, Juliane; Perkins, Susan L.; Decher, Jan; Leendertz, Fabian H.; Fahr, Jakob; Weber, Natalie; Matuschewski, Kai

    2013-01-01

    As the only volant mammals, bats are captivating for their high taxonomic diversity, for their vital roles in ecosystemsparticularly as pollinators and insectivoresand, 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

  8. High diversity of West African bat malaria parasites and a tight link with rodent Plasmodium taxa.

    PubMed

    Schaer, Juliane; Perkins, Susan L; Decher, Jan; Leendertz, Fabian H; Fahr, Jakob; Weber, Natalie; Matuschewski, Kai

    2013-10-22

    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

  9. Human red blood cell-adapted Plasmodium knowlesi parasites: a new model system for malaria research.

    PubMed

    Grüring, Christof; Moon, Robert W; Lim, Caeul; Holder, Anthony A; Blackman, Michael J; Duraisingh, Manoj T

    2014-05-01

    Plasmodium knowlesi is a simian malaria parasite primarily infecting macaque species in Southeast Asia. Although its capacity to infect humans has been recognized since the early part of the last century, it has recently become evident that human infections are widespread and potentially life threatening. Historically, P.  knowlesi has proven to be a powerful tool in early studies of malaria parasites, providing key breakthroughs in understanding many aspects of Plasmodium biology. However, the necessity to grow the parasite either in macaques or in vitro using macaque blood restricted research to laboratories with access to these resources. The recent adaptation of P.  knowlesi to grow and proliferate in vitro in human red blood cells (RBCs) is therefore a substantial step towards revitalizing and expanding research on P.  knowlesi. Furthermore, the development of a highly efficient transfection system to genetically modify the parasite makes P.  knowlesi an ideal model to study parasite biology. In this review, we elaborate on the importance of P.  knowlesi in earlier phases of malaria research and highlight the future potential of the newly available human adapted P.  knowlesi parasite lines. PMID:24506567

  10. The malarial parasite Plasmodium falciparum imports the human protein peroxiredoxin 2 for peroxide detoxification.

    PubMed

    Koncarevic, Sasa; Rohrbach, Petra; Deponte, Marcel; Krohne, Georg; Prieto, Judith Helena; Yates, John; Rahlfs, Stefan; Becker, Katja

    2009-08-11

    Coevolution of the malarial parasite and its human host has resulted in a complex network of interactions contributing to the homeodynamics of the host-parasite unit. As a rapidly growing and multiplying organism, Plasmodium falciparum depends on an adequate antioxidant defense system that is efficient despite the absence of genuine catalase and glutathione peroxidase. Using different experimental approaches, we demonstrate that P. falciparum imports the human redox-active protein peroxiredoxin 2 (hPrx-2, hTPx1) into its cytosol. As shown by confocal microscopy and immunogold electron microscopy, hPrx-2 is also present in the Maurer's clefts, organelles that are described as being involved in parasite protein export. Enzyme kinetic analyses prove that hPrx-2 accepts Plasmodium cytosolic thioredoxin 1 as a reducing substrate. hPrx-2 accounts for roughly 50% of thioredoxin peroxidase activity in parasite extracts, thus indicating a functional role of hPrx-2 as an enzymatic scavenger of peroxides in the parasite. Under chloroquine treatment, a drug promoting oxidative stress, the abundance of hPrx-2 in the parasite increases significantly. P. falciparum has adapted to adopt the hPrx-2, thereby using the host protein for its own purposes. PMID:19666612

  11. Plasmodium yoelii Vitamin B5 Pantothenate Transporter Candidate is Essential for Parasite Transmission to the Mosquito

    PubMed Central

    Hart, Robert J.; Lawres, Lauren; Fritzen, Emma; Mamoun, Choukri Ben; Aly, Ahmed S. I.

    2014-01-01

    In nearly all non-photosynthetic cells, pantothenate (vitamin B5) transport and utilization are prerequisites for the synthesis of the universal essential cofactor Coenzyme A (CoA). Early studies showed that human malaria parasites rely on the uptake of pantothenate across the parasite plasma membrane for survival within erythrocytes. Recently, a P. falciparum candidate pantothenate transporter (PAT) was characterized by functional complementation in yeast. These studies revealed that PfPAT mediated survival of yeast cells in low pantothenate concentrations and restored sensitivity of yeast cells lacking pantothenate uptake to fenpropimorph. In addition, PfPAT was refractory to deletion in P. falciparum in vitro, but nothing is known about the in vivo functions of PAT in Plasmodium life cycle stages. Herein, we used gene-targeting techniques to delete PAT in Plasmodium yoelii. Parasites lacking PAT displayed normal asexual and sexual blood stage development compared to wild-type (WT) and WT-like p230p(-) parasites. However, progression from the ookinete to the oocyst stage and sporozoite formation were completely abolished in pat(-) parasites. These studies provide the first evidence for an essential role of a candidate pantothenate transport in malaria transmission to Anopheles mosquitoes. This will set the stage for the development of PAT inhibitors against multiple parasite life cycle stages. PMID:25012929

  12. Plasmodium yoelii vitamin B5 pantothenate transporter candidate is essential for parasite transmission to the mosquito.

    PubMed

    Hart, Robert J; Lawres, Lauren; Fritzen, Emma; Ben Mamoun, Choukri; Aly, Ahmed S I

    2014-01-01

    In nearly all non-photosynthetic cells, pantothenate (vitamin B5) transport and utilization are prerequisites for the synthesis of the universal essential cofactor Coenzyme A (CoA). Early studies showed that human malaria parasites rely on the uptake of pantothenate across the parasite plasma membrane for survival within erythrocytes. Recently, a P. falciparum candidate pantothenate transporter (PAT) was characterized by functional complementation in yeast. These studies revealed that PfPAT mediated survival of yeast cells in low pantothenate concentrations and restored sensitivity of yeast cells lacking pantothenate uptake to fenpropimorph. In addition, PfPAT was refractory to deletion in P. falciparum in vitro, but nothing is known about the in vivo functions of PAT in Plasmodium life cycle stages. Herein, we used gene-targeting techniques to delete PAT in Plasmodium yoelii. Parasites lacking PAT displayed normal asexual and sexual blood stage development compared to wild-type (WT) and WT-like p230p(-) parasites. However, progression from the ookinete to the oocyst stage and sporozoite formation were completely abolished in pat(-) parasites. These studies provide the first evidence for an essential role of a candidate pantothenate transport in malaria transmission to Anopheles mosquitoes. This will set the stage for the development of PAT inhibitors against multiple parasite life cycle stages. PMID:25012929

  13. Antimitotic herbicides bind to an unidentified site on malarial parasite tubulin and block development of liver-stage Plasmodium parasites.

    PubMed

    Dempsey, Enda; Prudncio, Miguel; Fennell, Brian J; Gomes-Santos, Carina S; Barlow, James W; Bell, Angus

    2013-04-01

    Malarial parasites are exquisitely susceptible to a number of microtubule inhibitors but most of these compounds also affect human microtubules. Herbicides of the dinitroaniline and phosphorothioamidate classes however affect some plant and protozoal cells but not mammalian ones. We have previously shown that these herbicides block schizogony in erythrocytic parasites of the most lethal human malaria, Plasmodium falciparum, disrupt their mitotic spindles, and bind selectively to parasite tubulin. Here we show for the first time that the antimitotic herbicides also block the development of malarial parasites in the liver stage. Structure-based design of novel antimalarial agents binding to tubulin at the herbicide site, which presumably exists on (some) parasite and plant tubulins but not mammalian ones, can therefore constitute an important transmission blocking approach. The nature of this binding site is controversial, with three overlapping but non-identical locations on ?-tubulin proposed in the literature. We tested the validity of the three sites by (i) using site-directed mutagenesis to introduce six amino acid changes designed to occlude them, (ii) producing the resulting tubulins recombinantly in Escherichia coli and (iii) measuring the affinity of the herbicides amiprophosmethyl and oryzalin for these proteins in comparison with wild-type tubulins by fluorescence quenching. The changes had little or no effect, with dissociation constants (Kd) no more than 1.3-fold (amiprophosmethyl) or 1.6-fold (oryzalin) higher than wild-type. We conclude that the herbicides impair Plasmodium liver stage as well as blood stage development but that the location of their binding site on malarial parasite tubulin remains to be proven. PMID:23523992

  14. Investigations into Outbreaks of Black Fly Attacks and Subsequent Avian Haemosporidians in Backyard-Type Poultry and Other Exposed Avian Species.

    PubMed

    Jones, Kelli; Johnson, Nora; Yang, Sharon; Stokes, John; Smith, Whitney; Wills, Robert; Goddard, Jerome; Varela-Stokes, Andrea

    2015-03-01

    In late spring of 2009 and 2010, there were reports of severe black fly (Simulium spp., shown in Fig. 1) outbreaks in various counties in Mississippi, especially those in and around the Mississippi River Delta. Complaints were of black flies attacking multiple species of backyard poultry and causing high morbidity and mortality in affected flocks. At several affected locations, black flies were readily observed swarming around and feeding on birds. A large number of these parasites were easily trapped on fly strips (Fig. 2). Multifocal to coalescing cutaneous hemorrhagic lesions, consistent with fly bites, were seen on the birds. Upon necropsy examination, a large number of black flies were also observed in the digestive tract (Fig. 3). Although black flies may cause disease directly, such as cardiopulmonary collapse and anaphylactoid reactions, detection of Leucocytozoon in blood smears (Fig. 4) of affected birds prompted further investigations of this protozoan as a cause of disease. Leucocytozoon spp. are known to be transmitted by black flies and may be associated with morbidity and mortality in birds such as poultry. From June 2009 through July 2012, the investigation included a total collection of 1068 individual blood samples, representing 371 individual premises in 89 counties/parishes across Mississippi (59), Alabama (10), Louisiana (4), and Tennessee (16). Of the 371 premises where blood samples were collected, 96 (26%) were either positive or highly suspected to be positive for Leucocytozoon spp. by blood smear analysis, and 5 (1.2%) were positive for Haemoproteus spp. by blood smear analysis. Attempts to diagnose Leucocytozoon spp. by PCR analysis and sequencing were complicated by coinfections with two closely related haemosporidians (Haemoproteus spp. and Plasmodium spp.). A novel technique involving flow cytometry was also explored. This study discusses the black fly field outbreak, the involvement of haemosporidians, molecular methods for detection of both the black flies and blood parasites, and initial attempts at flow cytometry. PMID:26292530

  15. Implications of Plasmodium parasite infected mosquitoes on an insular avifauna: the case of Socorro Island, Mxico.

    PubMed

    Carlson, Jenny S; Martnez-Gmez, Juan E; Cornel, Anthony; Loiseau, Claire; Sehgal, Ravinder N M

    2011-06-01

    Avian malaria (Plasmodium spp.) has been implicated in the decline of avian populations in the Hawaiian Islands and it is generally agreed that geographically isolated and immunologically nave bird populations are particularly vulnerable to the pathogenic effects of invasive malaria parasites. In order to assess the potential disease risk of malaria to the avifauna of Socorro Island, Mxico, we surveyed for Plasmodium isolates from 1,300 resident field-caught mosquitoes. Most of them were identified as Aedes (Ochlerotatus) taeniorhynchus (Wiedemann, 1821), which were abundant in the salt marshes. We also collected Culex quinquefasciatus Say, 1823 close to human dwellings. Mitochondrial ND5 and COII gene sequences of Ae. taeniorhynchus were analyzed and compared to corresponding sequences of mosquitoes of the Galpagos Islands, Latin America, and the North American mainland. Aedes lineages from Socorro Island clustered most closely with a lineage from the continental U.S. Plasmodium spp. DNA was isolated from both species of mosquitoes. From 38 positive pools, we isolated 11 distinct mitochondrial Cytb lineages of Plasmodium spp. Seven of the Plasmodium lineages represent previously documented avian infective strains while four were new lineages. Our results confirm a potential risk for the spread of avian malaria and underscore the need to monitor both the mosquito and avian populations as a necessary conservation measure to protect endangered bird species on Socorro Island. PMID:21635660

  16. The Calcium Signaling Toolkit of the Apicomplexan Parasites Toxoplasma gondii and Plasmodium spp

    PubMed Central

    Lourido, Sebastian; Moreno, Silvia N.J.

    2015-01-01

    Apicomplexan parasites have complex life cycles, frequently split between different hosts and reliant on rapid responses as the parasites react to changing environmental conditions. Calcium ion (Ca2+) signaling is consequently essential for the cellular and developmental changes that support apicomplexan parasitism. Apicomplexan genomes reveal a rich repertoire of genes involved in calcium signaling, although many of the genes responsible for observed physiological changes remain unknown. There is evidence, for example, for the presence of a nifedipine-sensitive calcium entry mechanism in Toxoplasma, but the molecular components involved in Ca2+ entry in both Toxoplasma and Plasmodium, have not been identified. The major calcium stores are the endoplasmic reticulum (ER), the acidocalcisomes, and the plant-like vacuole in Toxoplasma, or the food vacuole in Plasmodium spp. Pharmacological evidence suggests that Ca2+ release from intracellular stores may be mediated by inositol 1,4,5-trisphosphate (IP3) or cyclic ADP ribose (cADPR) although there is no molecular evidence for the presence of receptors for these second messengers in the parasites. Several Ca2+-ATPases are present in apicomplexans and a putative mitochondrial Ca2+/H+ exchanger has been identified. Apicomplexan genomes contain numerous genes encoding Ca2+-binding proteins, with the notable expansion of calcium-dependent protein kinases (CDPKs), whose study has revealed novel roles in gliding motility, microneme secretion, host cell invasion and egress, and parasite differentiation. Microneme secretion has also been shown to depend on the C2 domain containing protein DOC2 in both Plasmodium spp. and Toxoplasma, providing further evidence for the complex transduction of Ca2+ signals in these organisms. The characterization of these pathways could lead to the discovery of novel drug targets and to a better understanding of the role of Ca2+ in these parasites. PMID:25605521

  17. Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

    PubMed Central

    Claessens, Antoine; Rowe, J. Alexandra

    2012-01-01

    Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the disease, is characterised by an accumulation of Plasmodium falciparum infected red blood cells (iRBC) at pigmented trophozoite stage in the microvasculature of the brain2-4. This microvessel obstruction (sequestration) leads to acidosis, hypoxia and harmful inflammatory cytokines (reviewed in 5). Sequestration is also found in most microvascular tissues of the human body2, 3. The mechanism by which iRBC attach to the blood vessel walls is still poorly understood. The immortalized Human Brain microvascular Endothelial Cell line (HBEC-5i) has been used as an in vitro model of the blood-brain barrier6. However, Plasmodium falciparum iRBC attach only poorly to HBEC-5i in vitro, unlike the dense sequestration that occurs in cerebral malaria cases. We therefore developed a panning assay to select (enrich) various P. falciparum strains for adhesion to HBEC-5i in order to obtain populations of high-binding parasites, more representative of what occurs in vivo. A sample of a parasite culture (mixture of iRBC and uninfected RBC) at the pigmented trophozoite stage is washed and incubated on a layer of HBEC-5i grown on a Petri dish. After incubation, the dish is gently washed free from uRBC and unbound iRBC. Fresh uRBC are added to the few iRBC attached to HBEC-5i and incubated overnight. As schizont stage parasites burst, merozoites reinvade RBC and these ring stage parasites are harvested the following day. Parasites are cultured until enough material is obtained (typically 2 to 4 weeks) and a new round of selection can be performed. Depending on the P. falciparum strain, 4 to 7 rounds of selection are needed in order to get a population where most parasites bind to HBEC-5i. The binding phenotype is progressively lost after a few weeks, indicating a switch in variant surface antigen gene expression, thus regular selection on HBEC-5i is required to maintain the phenotype. In summary, we developed a selection assay rendering P. falciparum parasites a more "cerebral malaria adhesive" phenotype. We were able to select 3 out of 4 P. falciparum strains on HBEC-5i. This assay has also successfully been used to select parasites for binding to human dermal and pulmonary endothelial cells. Importantly, this method can be used to select tissue-specific parasite populations in order to identify candidate parasite ligands for binding to brain endothelium. Moreover, this assay can be used to screen for putative anti-sequestration drugs7. PMID:22230803

  18. An Ancient Protein Phosphatase, SHLP1, Is Critical to Microneme Development in Plasmodium Ookinetes and Parasite Transmission

    PubMed Central

    Patzewitz, Eva-Maria; Guttery, DavidS.; Poulin, Benoit; Ramakrishnan, Chandra; Ferguson, DavidJ.P.; Wall, RichardJ.; Brady, Declan; Holder, AnthonyA.; Sz?r, Balzs; Tewari, Rita

    2013-01-01

    Summary Signaling pathways controlled by reversible protein phosphorylation (catalyzed by kinases and phosphatases) in the malaria parasite Plasmodium are of great interest, for both increased understanding of parasite biology and identification of novel drug targets. Here, we report a functional analysis in Plasmodium of an ancient bacterial Shewanella-like protein phosphatase (SHLP1) found only in bacteria, fungi, protists, and plants. SHLP1 is abundant in asexual blood stages and expressed at all stages of the parasite life cycle. shlp1 deletion results in a reduction in ookinete (zygote) development, microneme formation, and complete ablation of oocyst formation, thereby blocking parasite transmission. This defect is carried by the female gamete and can be rescued by direct injection of mutant ookinetes into the mosquito hemocoel, where oocysts develop. This study emphasizes the varied functions of SHLP1 in Plasmodium ookinete biology and suggests that it could be a novel drug target for blocking parasite transmission. PMID:23434509

  19. Cultivation of Plasmodium falciparum parasites in a serum-free medium.

    PubMed

    Ofulla, A V; Okoye, V C; Khan, B; Githure, J I; Roberts, C R; Johnson, A J; Martin, S K

    1993-09-01

    The elimination of serum from Plasmodium falciparum culture media could decrease costs, enhance procurement, and improve the feasibility of large-scale production of parasite material. We provide a semi-defined, serum-free formulation, of commercially available constituents that supports P. falciparum parasite growth at rates comparable with those obtained with serum-supplemented media. The medium is composed of RPMI 1640 to which HEPES, extra glucose, bicarbonate, and hypoxanthine have been added. Bovine albumin and serum-derived, lipids-cholesterol-rich mixture are then used in place of serum. PMID:8396859

  20. Towards genome-wide experimental genetics in the in vivo malaria model parasite Plasmodium berghei.

    PubMed

    Matz, Joachim M; Kooij, Taco W A

    2015-03-01

    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. PMID:25789828

  1. Towards genome-wide experimental genetics in the in vivo malaria model parasite Plasmodium berghei

    PubMed Central

    Matz, Joachim M.; Kooij, Taco W. A.

    2015-01-01

    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. PMID:25789828

  2. Prevalence, transmission, and genetic diversity of blood parasites infecting tundra-nesting geese in Alaska

    USGS Publications Warehouse

    Ramey, Andy M.; Reed, John A.; Schmutz, Joel A.; Fondell, Tom F.; Meixell, Brandt W.; Hupp, Jerry W.; Ward, David H.; Terenzi, John; Ely, Craig R.

    2014-01-01

    A total of 842 blood samples collected from five species of tundra-nesting geese in Alaska was screened for haemosporidian parasites using molecular techniques. Parasites of the generaLeucocytozoonDanilewsky, 1890,HaemoproteusKruse, 1890, andPlasmodiumMarchiafava and Celli, 1885 were detected in 169 (20%), 3 (<1%), and 0 (0%) samples, respectively. Occupancy modeling was used to estimate prevalence ofLeucocytozoonparasites and assess variation relative to species, age, sex, geographic area, year, and decade. Species, age, and decade were identified as important in explaining differences in prevalence ofLeucocytozoonparasites.Leucocytozoonparasites were detected in goslings sampled along the Arctic Coastal Plain using both historic and contemporary samples, which provided support for transmission in the North American Arctic. In contrast, lack of detection ofHaemoproteusandPlasmodiumparasites in goslings (n= 238) provided evidence to suggest that the transmission of parasites of these genera may not occur among waterfowl using tundra habitats in Alaska, or alternatively, may only occur at low levels. Five haemosporidian genetic lineages shared among different species of geese sampled from two geographic areas were indicative of interspecies parasite transmission and supported broad parasite or vector distributions. However, identicalLeucocytozoonandHaemoproteuslineages on public databases were limited to waterfowl hosts suggesting constraints in the range of parasite hosts.

  3. Quantitative Time-course Profiling of Parasite and Host Cell Proteins in the Human Malaria Parasite Plasmodium falciparum*

    PubMed Central

    Foth, Bernardo Javier; Zhang, Neng; Chaal, Balbir Kaur; Sze, Siu Kwan; Preiser, Peter Rainer; Bozdech, Zbynek

    2011-01-01

    Studies of the Plasmodium falciparum transcriptome have shown that the tightly controlled progression of the parasite through the intra-erythrocytic developmental cycle (IDC) is accompanied by a continuous gene expression cascade in which most expressed genes exhibit a single transcriptional peak. Because the biochemical and cellular functions of most genes are mediated by the encoded proteins, understanding the relationship between mRNA and protein levels is crucial for inferring biological activity from transcriptional gene expression data. Although studies on other organisms show that <50% of protein abundance variation may be attributable to corresponding mRNA levels, the situation in Plasmodium is further complicated by the dynamic nature of the cyclic gene expression cascade. In this study, we simultaneously determined mRNA and protein abundance profiles for P. falciparum parasites during the IDC at 2-hour resolution based on oligonucleotide microarrays and two-dimensional differential gel electrophoresis protein gels. We find that most proteins are represented by more than one isoform, presumably because of post-translational modifications. Like transcripts, most proteins exhibit cyclic abundance profiles with one peak during the IDC, whereas the presence of functionally related proteins is highly correlated. In contrast, the abundance of most parasite proteins peaks significantly later (median 11 h) than the corresponding transcripts and often decreases slowly in the second half of the IDC. Computational modeling indicates that the considerable and varied incongruence between transcript and protein abundance may largely be caused by the dynamics of translation and protein degradation. Furthermore, we present cyclic abundance profiles also for parasite-associated human proteins and confirm the presence of five human proteins with a potential role in antioxidant defense within the parasites. Together, our data provide fundamental insights into transcript-protein relationships in P. falciparum that are important for the correct interpretation of transcriptional data and that may facilitate the improvement and development of malaria diagnostics and drug therapy. PMID:21558492

  4. Plasticity in transmission strategies of the malaria parasite, Plasmodium chabaudi: environmental and genetic effects

    PubMed Central

    Cameron, Angus; Reece, Sarah E; Drew, Damien R; Haydon, Daniel T; Yates, Andrew J

    2013-01-01

    Parasites may alter their behaviour to cope with changes in the within-host environment. In particular, investment in transmission may alter in response to the availability of parasite resources or host immune responses. However, experimental and theoretical studies have drawn conflicting conclusions regarding parasites' optimal (adaptive) responses to deterioration in habitat quality. We analyse data from acute infections with six genotypes of the rodent malaria species Plasmodium chabaudi to quantify how investment in transmission (gametocytes) is influenced by the within-host environment. Using a minimum of modelling assumptions, we find that proportional investment in gametocytogenesis increases sharply with host anaemia and also increases at low parasite densities. Further, stronger dependence of investment on parasite density is associated with greater virulence of the parasite genotype. Our study provides a robust quantitative framework for studying parasites' responses to the host environment and whether these responses are adaptive, which is crucial for predicting the short-term and evolutionary impact of transmission-blocking treatments for parasitic diseases. PMID:23467678

  5. Disruption of Parasite hmgb2 Gene Attenuates Plasmodium berghei ANKA Pathogenicity.

    PubMed

    Briquet, Sylvie; Lawson-Hogban, Nadou; Boisson, Bertrand; Soares, Miguel P; Pronet, Roger; Smith, Leanna; Mnard, Robert; Huerre, Michel; Mcheri, Salah; Vaquero, Catherine

    2015-07-01

    Eukaryotic high-mobility-group-box (HMGB) proteins are nuclear factors involved in chromatin remodeling and transcription regulation. When released into the extracellular milieu, HMGB1 acts as a proinflammatory cytokine that plays a central role in the pathogenesis of several immune-mediated inflammatory diseases. We found that the Plasmodium genome encodes two genuine HMGB factors, Plasmodium HMGB1 and HMGB2, that encompass, like their human counterparts, a proinflammatory domain. Given that these proteins are released from parasitized red blood cells, we then hypothesized that Plasmodium HMGB might contribute to the pathogenesis of experimental cerebral malaria (ECM), a lethal neuroinflammatory syndrome that develops in C57BL/6 (susceptible) mice infected with Plasmodium berghei ANKA and that in many aspects resembles human cerebral malaria elicited by P. falciparum infection. The pathogenesis of experimental cerebral malaria was suppressed in C57BL/6 mice infected with P. berghei ANKA lacking the hmgb2 gene (?hmgb2 ANKA), an effect associated with a reduction of histological brain lesions and with lower expression levels of several proinflammatory genes. The incidence of ECM in pbhmgb2-deficient mice was restored by the administration of recombinant PbHMGB2. Protection from experimental cerebral malaria in ?hmgb2 ANKA-infected mice was associated with reduced sequestration in the brain of CD4(+) and CD8(+) T cells, including CD8(+) granzyme B(+) and CD8(+) IFN-?(+) cells, and, to some extent, neutrophils. This was consistent with a reduced parasite sequestration in the brain, lungs, and spleen, though to a lesser extent than in wild-type P. berghei ANKA-infected mice. In summary, Plasmodium HMGB2 acts as an alarmin that contributes to the pathogenesis of cerebral malaria. PMID:25916985

  6. Disruption of Parasite hmgb2 Gene Attenuates Plasmodium berghei ANKA Pathogenicity

    PubMed Central

    Lawson-Hogban, Nadou; Boisson, Bertrand; Soares, Miguel P.; Pronet, Roger; Smith, Leanna; Mnard, Robert; Huerre, Michel; Mcheri, Salah

    2015-01-01

    Eukaryotic high-mobility-group-box (HMGB) proteins are nuclear factors involved in chromatin remodeling and transcription regulation. When released into the extracellular milieu, HMGB1 acts as a proinflammatory cytokine that plays a central role in the pathogenesis of several immune-mediated inflammatory diseases. We found that the Plasmodium genome encodes two genuine HMGB factors, Plasmodium HMGB1 and HMGB2, that encompass, like their human counterparts, a proinflammatory domain. Given that these proteins are released from parasitized red blood cells, we then hypothesized that Plasmodium HMGB might contribute to the pathogenesis of experimental cerebral malaria (ECM), a lethal neuroinflammatory syndrome that develops in C57BL/6 (susceptible) mice infected with Plasmodium berghei ANKA and that in many aspects resembles human cerebral malaria elicited by P. falciparum infection. The pathogenesis of experimental cerebral malaria was suppressed in C57BL/6 mice infected with P. berghei ANKA lacking the hmgb2 gene (?hmgb2 ANKA), an effect associated with a reduction of histological brain lesions and with lower expression levels of several proinflammatory genes. The incidence of ECM in pbhmgb2-deficient mice was restored by the administration of recombinant PbHMGB2. Protection from experimental cerebral malaria in ?hmgb2 ANKA-infected mice was associated with reduced sequestration in the brain of CD4+ and CD8+ T cells, including CD8+ granzyme B+ and CD8+ IFN-?+ cells, and, to some extent, neutrophils. This was consistent with a reduced parasite sequestration in the brain, lungs, and spleen, though to a lesser extent than in wild-type P. berghei ANKA-infected mice. In summary, Plasmodium HMGB2 acts as an alarmin that contributes to the pathogenesis of cerebral malaria. PMID:25916985

  7. Population genetic analysis of Plasmodium falciparum parasites using a customized Illumina GoldenGate genotyping assay.

    PubMed

    Campino, Susana; Auburn, Sarah; Kivinen, Katja; Zongo, Issaka; Ouedraogo, Jean-Bosco; Mangano, Valentina; Djimde, Abdoulaye; Doumbo, Ogobara K; Kiara, Steven M; Nzila, Alexis; Borrmann, Steffen; Marsh, Kevin; Michon, Pascal; Mueller, Ivo; Siba, Peter; Jiang, Hongying; Su, Xin-Zhuan; Amaratunga, Chanaki; Socheat, Duong; Fairhurst, Rick M; Imwong, Mallika; Anderson, Timothy; Nosten, Franois; White, Nicholas J; Gwilliam, Rhian; Deloukas, Panos; MacInnis, Bronwyn; Newbold, Christopher I; Rockett, Kirk; Clark, Taane G; Kwiatkowski, Dominic P

    2011-01-01

    The diversity in the Plasmodium falciparum genome can be used to explore parasite population dynamics, with practical applications to malaria control. The ability to identify the geographic origin and trace the migratory patterns of parasites with clinically important phenotypes such as drug resistance is particularly relevant. With increasing single-nucleotide polymorphism (SNP) discovery from ongoing Plasmodium genome sequencing projects, a demand for high SNP and sample throughput genotyping platforms for large-scale population genetic studies is required. Low parasitaemias and multiple clone infections present a number of challenges to genotyping P. falciparum. We addressed some of these issues using a custom 384-SNP Illumina GoldenGate assay on P. falciparum DNA from laboratory clones (long-term cultured adapted parasite clones), short-term cultured parasite isolates and clinical (non-cultured isolates) samples from East and West Africa, Southeast Asia and Oceania. Eighty percent of the SNPs (n?=?306) produced reliable genotype calls on samples containing as little as 2 ng of total genomic DNA and on whole genome amplified DNA. Analysis of artificial mixtures of laboratory clones demonstrated high genotype calling specificity and moderate sensitivity to call minor frequency alleles. Clear resolution of geographically distinct populations was demonstrated using Principal Components Analysis (PCA), and global patterns of population genetic diversity were consistent with previous reports. These results validate the utility of the platform in performing population genetic studies of P. falciparum. PMID:21673999

  8. Plasmodium Apicoplast Gln-tRNAGln Biosynthesis Utilizes a Unique GatAB Amidotransferase Essential for Erythrocytic Stage Parasites.

    PubMed

    Mailu, Boniface M; Li, Ling; Arthur, Jen; Nelson, Todd M; Ramasamy, Gowthaman; Fritz-Wolf, Karin; Becker, Katja; Gardner, Malcolm J

    2015-12-01

    The malaria parasite Plasmodium falciparum apicoplast indirect aminoacylation pathway utilizes a non-discriminating glutamyl-tRNA synthetase to synthesize Glu-tRNA(Gln) and a glutaminyl-tRNA amidotransferase to convert Glu-tRNA(Gln) to Gln-tRNA(Gln). Here, we show that Plasmodium falciparum and other apicomplexans possess a unique heterodimeric glutamyl-tRNA amidotransferase consisting of GatA and GatB subunits (GatAB). We localized the P. falciparum GatA and GatB subunits to the apicoplast in blood stage parasites and demonstrated that recombinant GatAB converts Glu-tRNA(Gln) to Gln-tRNA(Gln) in vitro. We demonstrate that the apicoplast GatAB-catalyzed reaction is essential to the parasite blood stages because we could not delete the Plasmodium berghei gene encoding GatA in blood stage parasites in vivo. A phylogenetic analysis placed the split between Plasmodium GatB, archaeal GatE, and bacterial GatB prior to the phylogenetic divide between bacteria and archaea. Moreover, Plasmodium GatA also appears to have emerged prior to the bacterial-archaeal phylogenetic divide. Thus, although GatAB is found in Plasmodium, it emerged prior to the phylogenetic separation of archaea and bacteria. PMID:26318454

  9. Centrins, cell cycle regulation proteins in human malaria parasite Plasmodium falciparum.

    PubMed

    Mahajan, Babita; Selvapandiyan, Angamuthu; Gerald, Noel J; Majam, Victoria; Zheng, Hong; Wickramarachchi, Thilan; Tiwari, Jawahar; Fujioka, Hisashi; Moch, J Kathleen; Kumar, Nirbhay; Aravind, L; Nakhasi, Hira L; Kumar, Sanjai

    2008-11-14

    Molecules and cellular mechanisms that regulate the process of cell division in malaria parasites remain poorly understood. In this study we isolate and characterize the four Plasmodium falciparum centrins (PfCENs) and, by growth complementation studies, provide evidence for their involvement in cell division. Centrins are cytoskeleton proteins with key roles in cell division, including centrosome duplication, and possess four Ca(2+)-binding EF hand domains. By means of phylogenetic analysis, we were able to decipher the evolutionary history of centrins in eukaryotes with particular emphasis on the situation in apicomplexans and other alveolates. Plasmodium possesses orthologs of four distinct centrin paralogs traceable to the ancestral alveolate, including two that are unique to alveolates. By real time PCR and/or immunofluorescence, we determined the expression of PfCEN mRNA or protein in sporozoites, asexual blood forms, gametocytes, and in the oocysts developing inside mosquito mid-gut. Immunoelectron microscopy studies showed that centrin is expressed in close proximity with the nucleus of sporozoites and asexual schizonts. Furthermore, confocal and widefield microscopy using the double staining with alpha-tubulin and centrin antibodies strongly suggested that centrin is associated with the parasite centrosome. Following the episomal expression of the four PfCENs in a centrin knock-out Leishmania donovani parasite line that exhibited a severe growth defect, one of the PfCENs was able to partially restore Leishmania growth rate and overcome the defect in cytokinesis in such mutant cell line. To our knowledge, this study is the first characterization of a Plasmodium molecule that is involved in the process of cell division. These results provide the opportunity to further explore the role of centrins in cell division in malaria parasites and suggest novel targets to construct genetically modified, live attenuated malaria vaccines. PMID:18693242

  10. The multifunctional autophagy pathway in the human malaria parasite, Plasmodium falciparum.

    PubMed

    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

    2014-01-01

    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. PMID:24275162

  11. Direct Tests of Enzymatic Heme Degradation by the Malaria Parasite Plasmodium falciparum*

    PubMed Central

    Sigala, Paul A.; Crowley, Jan R.; Hsieh, Samantha; Henderson, Jeffrey P.; Goldberg, Daniel E.

    2012-01-01

    Malaria parasites generate vast quantities of heme during blood stage infection via hemoglobin digestion and limited de novo biosynthesis, but it remains unclear if parasites metabolize heme for utilization or disposal. Recent in vitro experiments with a heme oxygenase (HO)-like protein from Plasmodium falciparum suggested that parasites may enzymatically degrade some heme to the canonical HO product, biliverdin (BV), or its downstream metabolite, bilirubin (BR). To directly test for BV and BR production by P. falciparum parasites, we DMSO-extracted equal numbers of infected and uninfected erythrocytes and developed a sensitive LC-MS/MS assay to quantify these tetrapyrroles. We found comparable low levels of BV and BR in both samples, suggesting the absence of HO activity in parasites. We further tested live parasites by targeted expression of a fluorescent BV-binding protein within the parasite cytosol, mitochondrion, and plant-like plastid. This probe could detect exogenously added BV but gave no signal indicative of endogenous BV production within parasites. Finally, we recombinantly expressed and tested the proposed heme degrading activity of the HO-like protein, PfHO. Although PfHO bound heme and protoporphyrin IX with modest affinity, it did not catalyze heme degradation in vivo within bacteria or in vitro in UV absorbance and HPLC assays. These observations are consistent with PfHO's lack of a heme-coordinating His residue and suggest an alternative function within parasites. We conclude that P. falciparum parasites lack a canonical HO pathway for heme degradation and thus rely fully on alternative mechanisms for heme detoxification and iron acquisition during blood stage infection. PMID:22992734

  12. The multifunctional autophagy pathway in the human malaria parasite, Plasmodium falciparum

    PubMed Central

    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

    2014-01-01

    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 A1 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. PMID:24275162

  13. Large-scale growth of the Plasmodium falciparum malaria parasite in a wave bioreactor.

    PubMed

    Dalton, John P; Demanga, Corine G; Reiling, Sarah J; Wunderlich, Juliane; Eng, Jenny W L; Rohrbach, Petra

    2012-01-01

    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. PMID:22326740

  14. The mechanism of erythrocyte invasion by the malarial parasite, Plasmodium falciparum.

    PubMed

    Farrow, Rachel E; Green, Judith; Katsimitsoulia, Zoe; Taylor, William R; Holder, Anthony A; Molloy, Justin E

    2011-12-01

    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. PMID:22001249

  15. Wild Anopheles funestus Mosquito Genotypes Are Permissive for Infection with the Rodent Malaria Parasite, Plasmodium berghei

    PubMed Central

    Coulibaly, Boubacar; Sacko, Madjou; Dao, Adama; Niar, Oumou; Riehle, Michelle M.; Traor, Sekou F.; Vernick, Kenneth D.

    2013-01-01

    Background Malaria parasites undergo complex developmental transitions within the mosquito vector. A commonly used laboratory model for studies of mosquito-malaria interaction is the rodent parasite, P. berghei. Anopheles funestus is a major malaria vector in sub-Saharan Africa but has received less attention than the sympatric species, Anopheles gambiae. The imminent completion of the A. funestus genome sequence will provide currently lacking molecular tools to describe malaria parasite interactions in this mosquito, but previous reports suggested that A. funestus is not permissive for P. berghei development. Methods An A. funestus population was generated in the laboratory by capturing female wild mosquitoes in Mali, allowing them to oviposit, and rearing the eggs to adults. These F1 progeny of wild mosquitoes were allowed to feed on mice infected with a fluorescent P. berghei strain. Fluorescence microscopy was used to track parasite development inside the mosquito, salivary gland sporozoites were tested for infectivity to mice, and parasite development in A. funestus was compared to A. gambiae. Results P. berghei oocysts were detectable on A. funestus midguts by 7 days post-infection. By 1820 days post-infection, sporozoites had invaded the median and distal lateral lobes of the salivary glands, and hemocoel sporozoites were observed in the hemolymph. Mosquitoes were capable of infecting mice via bite, demonstrating that A. funestus supports the complete life cycle of P. berghei. In a random sample of wild mosquito genotypes, A. funestus prevalence of infection and the characteristics of parasite development were similar to that observed in A. gambiae-P. berghei infections. Conclusions The data presented in this study establish an experimental laboratory model for Plasmodium infection of A. funestus, an important vector of human malaria. Studying A. funestus-Plasmodium interactions is now feasible in a laboratory setting. This information lays the groundwork for exploitation of the awaited genome sequence of A. funestus. PMID:23593423

  16. In situ hybridization and sequence analysis reveal an association of Plasmodium spp. with mortalities in wild passerine birds in Austria.

    PubMed

    Dinhopl, Nora; Nedorost, Nora; Mostegl, Meike M; Weissenbacher-Lang, Christiane; Weissenböck, Herbert

    2015-04-01

    Native European passerine birds are frequently clinically inapparent carriers of haemosporidian parasites of the genus Plasmodium. Clinical disease and death are only exceptionally reported. In the present study, tissue samples of 233 wild passerine birds found dead in Eastern Austria were examined by in situ hybridization (ISH) and partial cytochrome B gene sequence analysis for the presence, abundance and taxonomic assignment of Plasmodium spp. In 34 cases (14.6%), ISH yielded a positive result with large numbers of developmental stages in different cell types of the spleen, liver, brain and lung. The abundance of the tissue stages, which was comparable to fatal cases of avian malaria in penguins, suggested a major contribution to the cause of death. Genetic analysis revealed infections with representatives of three different valid species of Plasmodium, Plasmodium elongatum, Plasmodium lutzi and Plasmodium vaughani. Genetically identical parasite lineages had been found in a previous study in penguins kept in the Vienna zoo, providing evidence for the role of wild birds as reservoir hosts. Further, this study provides evidence that several species of Plasmodium are able to abundantly proliferate in endemic wild birds ultimately resulting in mortalities. PMID:25636246

  17. Genome-wide mapping of DNA methylation in the human malaria parasite Plasmodium falciparum.

    PubMed

    Ponts, Nadia; Fu, Lijuan; Harris, Elena Y; Zhang, Jing; Chung, Duk-Won D; Cervantes, Michael C; Prudhomme, Jacques; Atanasova-Penichon, Vessela; Zehraoui, Enric; Bunnik, Evelien M; Rodrigues, Elisandra M; Lonardi, Stefano; Hicks, Glenn R; Wang, Yinsheng; Le Roch, Karine G

    2013-12-11

    Cytosine DNA methylation is an epigenetic mark in most eukaryotic cells that regulates numerous processes, including gene expression and stress responses. We performed a genome-wide analysis of DNA methylation in the human malaria parasite Plasmodium falciparum. We mapped the positions of methylated cytosines and identified a single functional DNA methyltransferase (Plasmodium falciparum DNA methyltransferase; PfDNMT) that may mediate these genomic modifications. These analyses revealed that the malaria genome is asymmetrically methylated and shares common features with undifferentiated plant and mammalian cells. Notably, core promoters are hypomethylated, and transcript levels correlate with intraexonic methylation. Additionally, there are sharp methylation transitions at nucleosome and exon-intron boundaries. These data suggest that DNA methylation could regulate virulence gene expression and transcription elongation. Furthermore, the broad range of action of DNA methylation and the uniqueness of PfDNMT suggest that the methylation pathway is a potential target for antimalarial strategies. PMID:24331467

  18. Malaria parasite colonisation of the mosquito midgut--placing the Plasmodium ookinete centre stage.

    PubMed

    Angrisano, Fiona; Tan, Yan-Hong; Sturm, Angelika; McFadden, Geoffrey I; Baum, Jake

    2012-05-15

    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. PMID:22406332

  19. Proteomic Profiling of Plasmodium Sporozoite Maturation Identifies New Proteins Essential for Parasite Development and Infectivity

    PubMed Central

    Mair, Gunnar R.; Vermunt, Adriaan M. W.; Douradinha, Bruno G.; van Noort, Vera; Huynen, Martijn A.; Luty, Adrian J. F.; Kroeze, Hans; Khan, Shahid M.; Sauerwein, Robert W.; Waters, Andrew P.; Mann, Matthias; Stunnenberg, Hendrik G.

    2008-01-01

    Plasmodium falciparum sporozoites that develop and mature inside an Anopheles mosquito initiate a malaria infection in humans. Here we report the first proteomic comparison of different parasite stages from the mosquitoearly and late oocysts containing midgut sporozoites, and the mature, infectious salivary gland sporozoites. Despite the morphological similarity between midgut and salivary gland sporozoites, their proteomes are markedly different, in agreement with their increase in hepatocyte infectivity. The different sporozoite proteomes contain a large number of stage specific proteins whose annotation suggest an involvement in sporozoite maturation, motility, infection of the human host and associated metabolic adjustments. Analyses of proteins identified in the P. falciparum sporozoite proteomes by orthologous gene disruption in the rodent malaria parasite, P. berghei, revealed three previously uncharacterized Plasmodium proteins that appear to be essential for sporozoite development at distinct points of maturation in the mosquito. This study sheds light on the development and maturation of the malaria parasite in an Anopheles mosquito and also identifies proteins that may be essential for sporozoite infectivity to humans. PMID:18974882

  20. Conditional Degradation of Plasmodium Calcineurin Reveals Functions in Parasite Colonization of both Host and Vector

    PubMed Central

    Philip, Nisha; Waters, AndrewP.

    2015-01-01

    Summary Functional analysis of essential genes in the malarial parasite, Plasmodium, is hindered by lack of efficient strategies for conditional protein regulation. We report the development of a rapid, specific, and inducible chemical-genetic tool in the rodent malaria parasite, P.berghei, in which endogenous proteins engineered to contain the auxin-inducible degron (AID) are selectively degraded upon adding auxin. Application of AID to the calcium-regulated protein phosphatase, calcineurin, revealed functions in host and vector stages of parasite development. Whereas depletion of calcineurin in late-stage schizonts demonstrated its critical role in erythrocyte attachment and invasion invivo, stage-specific depletion uncovered roles in gamete development, fertilization, and ookinete-to-oocyst and sporozoite-to-liver stage transitions. Furthermore, AID technology facilitated concurrent generation and phenotyping of transgenic lines, allowing multiple lines to be assessed simultaneously with significant reductions in animal use. This study highlights the broad applicability of AID for functional analysis of proteins across the Plasmodium life cycle. PMID:26118994

  1. Characterization of a conserved extrachromosomal element isolated from the avian malarial parasite Plasmodium gallinaceum.

    PubMed Central

    Joseph, J T; Aldritt, S M; Unnasch, T; Puijalon, O; Wirth, D F

    1989-01-01

    We have identified a conserved, repeated, and highly transcribed DNA element from the avian malarial parasite Plasmodium gallinaceum. The element produced multiple transcripts in both zygotes and asexual blood stages of this parasite. It was found to be highly conserved in all of five malarial species tested and hybridized at reduced stringency to other members of the phylum Apicomplexa, including the genera Babesia, Eimeria, Toxoplasma, and Theileria. The copy number of the element was about 15, and it had a circularly permuted restriction map with a repeat unit length of about 6.2 kilobases. It could be separated from the main genomic DNA by using sucrose gradients and agarose gels, and it migrated separately from the recognized Plasmodium chromosomes on pulse-field gels. In the accompanying paper (S. M. Aldritt, J. T. Joseph, and D. F. Wirth, Mol. Cell. Biol. 9:3614-3620, 1989), evidence is presented that element contains the mitochondrial genes for the protein cytochrome b and a fragment of the large rRNA. We postulate that this element is an episome in the mitochondria of the obligate parasites belonging to the phylum Apicomplexa. Images PMID:2779561

  2. Plasmodium falciparum heat shock protein 110 stabilizes the asparagine repeatrich parasite proteome during malarial fevers

    PubMed Central

    Muralidharan, Vasant; Oksman, Anna; Pal, Priya; Lindquist, Susan; E. Goldberg, Daniel

    2013-01-01

    One-fourth of Plasmodium falciparum proteins have asparagine repeats that increase the propensity for aggregation, especially at elevated temperatures that occur routinely in malaria-infected patients. We report that a Plasmodium Asn repeat-containing protein (PFI1155w) formed aggregates in mammalian cells at febrile temperatures, as did a yeast Asn/Gln-rich protein (Sup35). Co-expression of the cytoplasmic P. falciparum heat shock protein 110 (PfHsp110c) prevented aggregation. Human or yeast orthologs were much less effective. All-Asn and all-Gln versions of Sup35 were protected from aggregation by PfHsp110c, suggesting that this chaperone is not limited to handling runs of Asn. PfHsp110c gene knockout parasites were not viable and conditional knockdown parasites died slowly in the absence of protein-stabilizing ligand. When exposed to brief heat shock, these knockdowns were unable to prevent aggregation of PFI1155w or Sup35 and died rapidly. We conclude that PfHsp110c protects the parasite from harmful effects of its asparagine repeat-rich proteome during febrile episodes. PMID:23250440

  3. Conditional Degradation of Plasmodium Calcineurin Reveals Functions in Parasite Colonization of both Host and Vector.

    PubMed

    Philip, Nisha; Waters, Andrew P

    2015-07-01

    Functional analysis of essential genes in the malarial parasite, Plasmodium, is hindered by lack of efficient strategies for conditional protein regulation. We report the development of a rapid, specific, and inducible chemical-genetic tool in the rodent malaria parasite, P. berghei, in which endogenous proteins engineered to contain the auxin-inducible degron (AID) are selectively degraded upon adding auxin. Application of AID to the calcium-regulated protein phosphatase, calcineurin, revealed functions in host and vector stages of parasite development. Whereas depletion of calcineurin in late-stage schizonts demonstrated its critical role in erythrocyte attachment and invasion in vivo, stage-specific depletion uncovered roles in gamete development, fertilization, and ookinete-to-oocyst and sporozoite-to-liver stage transitions. Furthermore, AID technology facilitated concurrent generation and phenotyping of transgenic lines, allowing multiple lines to be assessed simultaneously with significant reductions in animal use. This study highlights the broad applicability of AID for functional analysis of proteins across the Plasmodium life cycle. PMID:26118994

  4. Diversity, loss, and gain of malaria parasites in a globally invasive bird.

    PubMed

    Marzal, Alfonso; Ricklefs, Robert E; Valki?nas, Gediminas; Albayrak, Tamer; Arriero, Elena; Bonneaud, Camille; Czirjk, Gbor A; Ewen, John; Hellgren, Olof; Ho?kov, Dita; Iezhova, Tatjana A; Jensen, Henrik; Krianauskien?, Asta; Lima, Marcos R; de Lope, Florentino; Magnussen, Eyfinn; Martin, Lynn B; Mller, Anders P; Palinauskas, Vaidas; Pap, Pter L; Prez-Tris, Javier; Sehgal, Ravinder N M; Soler, Manuel; Szllosi, Eszter; Westerdahl, Helena; Zetindjiev, Pavel; Bensch, Staffan

    2011-01-01

    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

  5. Evidence for intercontinental parasite exchange through molecular detection and characterization of haematozoa in northern pintails (Anas acuta) sampled throughout the North Pacific Basin

    USGS Publications Warehouse

    Ramey, Andy M.; Schmutz, Joel A.; Reed, John A.; Fujita, Go; Scotton, Bradley D.; Casler, Bruce; Fleskes, Joseph P.; Konishi, Kan; Uchida, Kiyoshi; Yabsley, Michael J.

    2015-01-01

    Empirical evidence supports wild birds as playing a role in the interhemispheric exchange of bacteria and viruses; however, data supporting the redistribution of parasites among continents are limited. In this study, the hypothesis that migratory birds contribute to the redistribution of parasites between continents was tested by sampling northern pintails (Anas acuta) at locations throughout the North Pacific Basin in North America and East Asia for haemosporidian infections and assessing the genetic evidence for parasite exchange. Of 878 samples collected from birds in Alaska (USA), California (USA), and Hokkaido (Japan) during August 2011 - May 2012 and screened for parasitic infections using molecular techniques, Leucocytozoon, Haemoproteus, and Plasmodium parasites were detected in 555 (63%), 44 (5%), and 52 (6%) samples, respectively. Using an occupancy modeling approach, the probability of detecting parasites via replicate genetic tests was estimated to be high (p ≥ 0.95). Multi-model inference supported variation of Leucocytozoon parasite prevalence by northern pintail age class and geographic location of sampling in contrast to Haemoproteus and Plasmodium parasites for which there was only support for variation in parasite prevalence by sampling location. Thirty-one unique mitochondrial DNA haplotypes were detected among haematozoa infecting northern pintails including seven lineages shared between samples from North America and Japan. The finding of identical parasite haplotypes at widely distributed geographic locations and general lack of genetic structuring by continent in phylogenies for Leucocytozoon and Plasmodium provides evidence for intercontinental genetic exchange of haemosporidian parasites. Results suggest that migratory birds, including waterfowl, could therefore facilitate the introduction of avian malaria and other haemosporidia to novel hosts and spatially distant regions.

  6. Evidence for intercontinental parasite exchange through molecular detection and characterization of haematozoa in northern pintails (Anas acuta) sampled throughout the North Pacific Basin

    PubMed Central

    Ramey, Andrew M.; Schmutz, Joel A.; Reed, John A.; Fujita, Go; Scotton, Bradley D.; Casler, Bruce; Fleskes, Joseph P.; Konishi, Kan; Uchida, Kiyoshi; Yabsley, Michael J.

    2014-01-01

    Empirical evidence supports wild birds as playing a role in the interhemispheric exchange of bacteria and viruses; however, data supporting the redistribution of parasites among continents are limited. In this study, the hypothesis that migratory birds contribute to the redistribution of parasites between continents was tested by sampling northern pintails (Anas acuta) at locations throughout the North Pacific Basin in North America and East Asia for haemosporidian infections and assessing the genetic evidence for parasite exchange. Of 878 samples collected from birds in Alaska (USA), California (USA), and Hokkaido (Japan) during August 2011May 2012 and screened for parasitic infections using molecular techniques, Leucocytozoon, Haemoproteus, and Plasmodium parasites were detected in 555 (63%), 44 (5%), and 52 (6%) samples, respectively. Using an occupancy modeling approach, the probability of detecting parasites via replicate genetic tests was estimated to be high (??>?0.95). Multi-model inference supported variation of Leucocytozoon parasite prevalence by northern pintail age class and geographic location of sampling in contrast to Haemoproteus and Plasmodium parasites for which there was only support for variation in parasite prevalence by sampling location. Thirty-one unique mitochondrial DNA haplotypes were detected among haematozoa infecting northern pintails including seven lineages shared between samples from North America and Japan. The finding of identical parasite haplotypes at widely distributed geographic locations and general lack of genetic structuring by continent in phylogenies for Leucocytozoon and Plasmodium provides evidence for intercontinental genetic exchange of haemosporidian parasites. Results suggest that migratory birds, including waterfowl, could therefore facilitate the introduction of avian malaria and other haemosporidia to novel hosts and spatially distant regions. PMID:25830100

  7. Bloodmeal Analysis Reveals Avian Plasmodium Infections and Broad Host Preferences of Culicoides (Diptera: Ceratopogonidae) Vectors

    PubMed Central

    Santiago-Alarcon, Diego; Havelka, Peter; Schaefer, Hinrich Martin; Segelbacher, Gernot

    2012-01-01

    Changing environmental conditions and human encroachment on natural habitats bring human populations closer to novel sources of parasites, which might then develop into new emerging diseases. Diseases transmitted by host generalist vectors are of special interest due to their capacity to move pathogens into novel hosts. We hypothesize that humans using forests for recreation are exposed to a broad range of parasites from wild animals and their vectors. A corollary of this is that new vector-host, parasite-host, and vector-parasite associations could eventually develop. Thus, we expect to observe atypical vector-host associations. Using molecular bloodmeal analysis via amplification of the mtDNA COI gene we identified the vertebrate hosts of Culicoides (Diptera: Ceratopogonidae) species in a sub-urban forest of Southwestern Germany. Bloodmeals were also checked for haemosporidian infections by amplifying a fragment of the mtDNA cyt b gene. We identified a total of 20 Culicoides species, thirteen of which fed on humans. From 105 screened bloodmeals we obtained high quality sequences for 77 samples, 73 (94.8%) originated from humans, two from livestock (Bos taurus and Equus caballus), and two from wild birds (Sylvia atricapilla and Turdus merula). We found that four Culicoides species previously assumed to feed exclusively on either birds (C. kibunensis) or domestic mammals (C. chiopterus, C. deltus, C. scoticus) fed also on humans. A total of six Culicoides abdomens were infected with avian haemosporidian parasites (Plasmodium or Haemoproteus), four of those abdomens contained blood derived from humans. Our results suggest that parasites of wild animals may be transferred to humans through infectious bites of Culicoides vectors. Further, we show that Culicoides vectors believed to be a specialist on specific vertebrate groups can have plastic feeding preferences, and that Culicoides are susceptible to infection by Plasmodium parasites, though vector viability must still be experimentally demonstrated. PMID:22363557

  8. Population genomic structure and adaptation in the zoonotic malaria parasite Plasmodium knowlesi.

    PubMed

    Assefa, Samuel; Lim, Caeul; Preston, Mark D; Duffy, Craig W; Nair, Mridul B; Adroub, Sabir A; Kadir, Khamisah A; Goldberg, Jonathan M; Neafsey, Daniel E; Divis, Paul; Clark, Taane G; Duraisingh, Manoj T; Conway, David J; Pain, Arnab; Singh, Balbir

    2015-10-20

    Malaria cases caused by the zoonotic parasite Plasmodium knowlesi are being increasingly reported throughout Southeast Asia and in travelers returning from the region. To test for evidence of signatures of selection or unusual population structure in this parasite, we surveyed genome sequence diversity in 48 clinical isolates recently sampled from Malaysian Borneo and in five lines maintained in laboratory rhesus macaques after isolation in the 1960s from Peninsular Malaysia and the Philippines. Overall genomewide nucleotide diversity (? = 6.03 10(-3)) was much higher than has been seen in worldwide samples of either of the major endemic malaria parasite species Plasmodium falciparum and Plasmodium vivax. A remarkable substructure is revealed within P. knowlesi, consisting of two major sympatric clusters of the clinical isolates and a third cluster comprising the laboratory isolates. There was deep differentiation between the two clusters of clinical isolates [mean genomewide fixation index (FST) = 0.21, with 9,293 SNPs having fixed differences of FST = 1.0]. This differentiation showed marked heterogeneity across the genome, with mean FST values of different chromosomes ranging from 0.08 to 0.34 and with further significant variation across regions within several chromosomes. Analysis of the largest cluster (cluster 1, 38 isolates) indicated long-term population growth, with negatively skewed allele frequency distributions (genomewide average Tajima's D = -1.35). Against this background there was evidence of balancing selection on particular genes, including the circumsporozoite protein (csp) gene, which had the top Tajima's D value (1.57), and scans of haplotype homozygosity implicate several genomic regions as being under recent positive selection. PMID:26438871

  9. Population genomic structure and adaptation in the zoonotic malaria parasite Plasmodium knowlesi

    PubMed Central

    Assefa, Samuel; Lim, Caeul; Preston, Mark D.; Duffy, Craig W.; Nair, Mridul B.; Adroub, Sabir A.; Kadir, Khamisah A.; Goldberg, Jonathan M.; Neafsey, Daniel E.; Divis, Paul; Clark, Taane G.; Duraisingh, Manoj T.; Conway, David J.; Pain, Arnab; Singh, Balbir

    2015-01-01

    Malaria cases caused by the zoonotic parasite Plasmodium knowlesi are being increasingly reported throughout Southeast Asia and in travelers returning from the region. To test for evidence of signatures of selection or unusual population structure in this parasite, we surveyed genome sequence diversity in 48 clinical isolates recently sampled from Malaysian Borneo and in five lines maintained in laboratory rhesus macaques after isolation in the 1960s from Peninsular Malaysia and the Philippines. Overall genomewide nucleotide diversity (π = 6.03 × 10−3) was much higher than has been seen in worldwide samples of either of the major endemic malaria parasite species Plasmodium falciparum and Plasmodium vivax. A remarkable substructure is revealed within P. knowlesi, consisting of two major sympatric clusters of the clinical isolates and a third cluster comprising the laboratory isolates. There was deep differentiation between the two clusters of clinical isolates [mean genomewide fixation index (FST) = 0.21, with 9,293 SNPs having fixed differences of FST = 1.0]. This differentiation showed marked heterogeneity across the genome, with mean FST values of different chromosomes ranging from 0.08 to 0.34 and with further significant variation across regions within several chromosomes. Analysis of the largest cluster (cluster 1, 38 isolates) indicated long-term population growth, with negatively skewed allele frequency distributions (genomewide average Tajima’s D = −1.35). Against this background there was evidence of balancing selection on particular genes, including the circumsporozoite protein (csp) gene, which had the top Tajima’s D value (1.57), and scans of haplotype homozygosity implicate several genomic regions as being under recent positive selection. PMID:26438871

  10. Diversity in mitochondrial metabolic pathways in parasitic protists Plasmodium and Cryptosporidium.

    PubMed

    Mogi, Tatsushi; Kita, Kiyoshi

    2010-09-01

    Apicomplexans are obligate intracellular parasites and occupy diverse niches. They have remodeled mitochondrial carbon and energy metabolism through reductive evolution. Plasmodium lacks mitochondrial pyruvate dehydrogenase and H(+)-translocating NADH dehydrogenase (Complex I, NDH1). The mitochondorion contains a minimal mtDNA ( approximately 6kb) and carries out oxidative phosphorylation in the insect vector stages, by using 2-oxoglutarate as an alternative means of entry into the TCA cycle and a single-subunit flavoprotein as an alternative NADH dehydrogenase (NDH2). In the blood stages of mammalian hosts, mitochondrial enzymes are down-regulated and parasite energy metabolism relies mainly on glycolysis. Mitosomes of Cryptosporidium parvum and Cryptosporidium hominis (human intestine parasites) lack mtDNA, pyruvate dehydrogenase, TCA cycle enzymes except malate-quinone oxidoreductase (MQO), and ATP synthase subunits except alpha and beta. In contrast, mitosomes of Cryptosporidium muris (a rodent gastric parasite) retain all TCA cycle enzymes and functional ATP synthase and carry out oxidative phosphorylation with pyruvate-NADP(+) oxidoreductase (PNO) and a simple and unique respiratory chain consisting of NDH2 and alternative oxidase (AOX). Cryptosporidium and Perkinsus are early branching groups of chromoalveolates (apicomplexa and dinoflagellates, respectively), and both Cryptosporidium mitosome and Perkinsus mitochondrion use PNO, MQO, and AOX. All apicomplexan parasites and dinoflagellates share MQO, which has been acquired from epsilon-proteobacteria via lateral gene transfer. By genome data mining on Plasmodium, Cryptosporidium and Perkinsus, here we summarized their mitochondrial metabolic pathways, which are varied largely from those of mammalian hosts. We hope that our findings will help in understanding the apicomplexan metabolism and development of new chemotherapeutics with novel targets. PMID:20433942

  11. Conserved mosquito/parasite interactions affect development of Plasmodium falciparum in Africa.

    PubMed

    Mendes, Antonio M; Schlegelmilch, Timm; Cohuet, Anna; Awono-Ambene, Parfait; De Iorio, Maria; Fontenille, Didier; Morlais, Isabelle; Christophides, George K; Kafatos, Fotis C; Vlachou, Dina

    2008-05-01

    In much of sub-Saharan Africa, the mosquito Anopheles gambiae is the main vector of the major human malaria parasite, Plasmodium falciparum. Convenient laboratory studies have identified mosquito genes that affect positively or negatively the developmental cycle of the model rodent parasite, P. berghei. Here, we use transcription profiling and reverse genetics to explore whether five disparate mosquito gene regulators of P. berghei development are also pertinent to A. gambiae/P. falciparum interactions in semi-natural conditions, using field isolates of this parasite and geographically related mosquitoes. We detected broadly similar albeit not identical transcriptional responses of these genes to the two parasite species. Gene silencing established that two genes affect similarly both parasites: infections are hindered by the intracellular local activator of actin cytoskeleton dynamics, WASP, but promoted by the hemolymph lipid transporter, ApoII/I. Since P. berghei is not a natural parasite of A. gambiae, these data suggest that the effects of these genes have not been drastically altered by constant interaction and co-evolution of A. gambiae and P. falciparum; this conclusion allowed us to investigate further the mode of action of these two genes in the laboratory model system using a suite of genetic tools and infection assays. We showed that both genes act at the level of midgut invasion during the parasite's developmental transition from ookinete to oocyst. ApoII/I also affects the early stages of oocyst development. These are the first mosquito genes whose significant effects on P. falciparum field isolates have been established by direct experimentation. Importantly, they validate for semi-field human malaria transmission the concept of parasite antagonists and agonists. PMID:18483558

  12. Systems analysis of chaperone networks in the malarial parasite Plasmodium falciparum.

    PubMed

    Pavithra, Soundara Raghavan; Kumar, Ranjit; Tatu, Utpal

    2007-09-01

    Molecular chaperones participate in the maintenance of cellular protein homeostasis, cell growth and differentiation, signal transduction, and development. Although a vast body of information is available regarding individual chaperones, few studies have attempted a systems level analysis of chaperone function. In this paper, we have constructed a chaperone interaction network for the malarial parasite, Plasmodium falciparum. P. falciparum is responsible for several million deaths every year, and understanding the biology of the parasite is a top priority. The parasite regularly experiences heat shock as part of its life cycle, and chaperones have often been implicated in parasite survival and growth. To better understand the participation of chaperones in cellular processes, we created a parasite chaperone network by combining experimental interactome data with in silico analysis. We used interolog mapping to predict protein-protein interactions for parasite chaperones based on the interactions of corresponding human chaperones. This data was then combined with information derived from existing high-throughput yeast two-hybrid assays. Analysis of the network reveals the broad range of functions regulated by chaperones. The network predicts involvement of chaperones in chromatin remodeling, protein trafficking, and cytoadherence. Importantly, it allows us to make predictions regarding the functions of hypothetical proteins based on their interactions. It allows us to make specific predictions about Hsp70-Hsp40 interactions in the parasite and assign functions to members of the Hsp90 and Hsp100 families. Analysis of the network provides a rational basis for the anti-malarial activity of geldanamycin, a well-known Hsp90 inhibitor. Finally, analysis of the network provides a theoretical basis for further experiments designed toward understanding the involvement of this important class of molecules in parasite biology. PMID:17941702

  13. A Plasmodium falciparum host-targeting motif functions in export during blood stage infection of the rodent malarial parasite Plasmodium berghei.

    PubMed

    MacKenzie, Julia J; Gmez, No D; Bhattacharjee, Souvik; Mann, Shaina; Haldar, Kasturi

    2008-01-01

    Plasmodium falciparum (P. falciparum) secretes hundreds of proteins--including major virulence proteins--into the host erythrocyte. In order to reach the host cytoplasm, most P. falciparum proteins contain an N terminal host-targeting (HT) motif composed of 11 amino acids. In silico analyses have suggested that the HT motif is conserved throughout the Plasmodium species but experimental evidence only exists for P. falciparum. Here, we show that in the rodent malaria parasite Plasmodium berghei (P. berghei) a reporter-like green fluorescent protein expressed by the parasite can be exported to the erythrocyte cytoplasm in a HT-specific manner. This provides the first experimental proof that the HT motif can function as a signal for protein delivery to the erythrocyte across Plasmodium species. Further, it suggests that P. berghei may serve as a model for validation of P. falciparum secretome proteins. We also show that tubovesicular membranes extend from the vacuolar parasite into the erythrocyte cytoplasm and speculate that these structures may facilitate protein export to the erythrocyte. PMID:18545649

  14. In vivo transmission blocking activities of artesunate on the avian malaria parasite Plasmodium gallinaceum.

    PubMed

    Kumnuan, Rapeeporn; Pattaradilokrat, Sittiporn; Chumpolbanchorn, Kamlang; Pimnon, Suntorn; Narkpinit, Somphong; Harnyuttanakorn, Pongchai; Saiwichai, Tawee

    2013-11-01

    Infection and transmission of the avian malaria parasite Plasmodium gallinaceum in domestic chickens is associated with high economic burden and presents a major challenge to poultry industry in South East Asia. Development of drugs targeting both asexual blood stage parasites and sexual stages of the avian malarias will be beneficial for malaria treatment and eradication. However, current drugs recommended for treatment of the avian malaria parasites target specifically the asexual blood stage parasites, but have little or no impact to the gametocytes, the major target for development of transmission-blocking strategies. In the present work, we established a simple procedure to evaluate gametocytocidal and transmission blocking activities in a P. gallinaceum-avian model. The assays involved administration of seven consecutive daily doses of test compounds into P. gallinaceum-infected chickens with 10% parasitaemia and 1% gametocytaemia. Our studies indicated that intramuscular injection with seven daily low doses (the minimum effective dose of 10mg/kg) of artesunate blocked the gametocyte production and transmission to the mosquito vector Aedes aegypti. This assay can be further applicable for testing new compounds against P. gallinaceum and for other parasitic protozoa infecting birds. PMID:23937960

  15. Multiple dimensions of epigenetic gene regulation in the malaria parasite Plasmodium falciparum

    PubMed Central

    Ay, Ferhat; Bunnik, Evelien M.; Varoquaux, Nelle; Vert, Jean-Philippe; Noble, William Stafford; Le Roch, Karine G.

    2015-01-01

    Plasmodium falciparum is the most deadly human malarial parasite, responsible for an estimated 207 million cases of disease and 627,000 deaths in 2012. Recent studies reveal that the parasite actively regulates a large fraction of its genes throughout its replicative cycle inside human red blood cells and that epigenetics plays an important role in this precise gene regulation. Here we discuss recent advances in our understanding of three aspects of epigenetic regulation in P. falciparum: changes in histone modifications, nucleosome occupancy and the three-dimensional genome structure. We compare these three aspects of the P. falciparum epigenome to those of other eukaryotes, and show that large-scale compartmentalization is particularly important in determining histone decomposition and gene regulation in P. falciparum. We conclude by presenting a gene regulation model for P. falciparum that combines the described epigenetic factors, and by discussing the implications of this model for the future of malaria research. PMID:25394267

  16. Plasmodium falciparum kelch 13: a potential molecular marker for tackling artemisinin-resistant malaria parasites.

    PubMed

    Mita, Toshihiro; Tachibana, Shin-Ichiro; Hashimoto, Muneaki; Hirai, Makoto

    2016-01-01

    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. PMID:26535806

  17. Radioimmunoassay for detecting antibodies against murine malarial parasite antigens: monoclonal antibodies recognizing Plasmodium yoelii antigens

    SciTech Connect

    Kim, K.J.; Taylor, D.W.; Evans, C.B.; Asofsky, R.

    1980-12-01

    A solid-phase radioimmunoassay (SPRIA) in microtiter wells was established for detecting antibodies against Plasmodium yoelii Ag. The SPRIA was found (1) to require as little as 5 ..mu..g of crude parasite Ag per well, (2) to be able to detect 0.5 ng of monoclonal Ab, and (3) to be 10/sup 4/ times more sensitive than the indirect fluorescent Ab staining technique. In a modification of the above assay using intact RBC as an Ag, hyperimmune serum showed significant binding to the surface of erythrocytes of mice infected with P. yoelii parasites but not to RBC of normal mice. Hybridomas were prepared by fusing infected mouse spleen cells with myeloma cells. Using the SPRIA, hybrids secreting Ab against P. yoelii 17XL Ag were detected.

  18. Gibberellin Biosynthetic Inhibitors Make Human Malaria Parasite Plasmodium falciparum Cells Swell and Rupture to Death

    PubMed Central

    Toyama, Tomoko; Tahara, Michiru; Nagamune, Kisaburo; Arimitsu, Kenji; Hamashima, Yoshio; Palacpac, Nirianne M. Q.; Kawaide, Hiroshi; Horii, Toshihiro; Tanabe, Kazuyuki

    2012-01-01

    Malaria remains as one of the most devastating infectious disease, and continues to exact an enormous toll in medical cost and days of labor lost especially in the tropics. Effective malaria control and eventual eradication remain a huge challenge, with efficacious antimalarials as important intervention/management tool. Clearly new alternative drugs that are more affordable and with fewer side effects are desirable. After preliminary in vitro assays with plant growth regulators and inhibitors, here, we focus on biosynthetic inhibitors of gibberellin, a plant hormone with many important roles in plant growth, and show their inhibitory effect on the growth of both apicomplexa, Plasmodium falciparum and Toxoplasma gondii. Treatment of P. falciparum cultures with the gibberellin biosynthetic inhibitors resulted in marked morphological changes that can be reversed to a certain degree under hyperosmotic environment. These unique observations suggest that changes in the parasite membrane permeability may explain the pleiotropic effects observed within the intracellular parasites. PMID:22412858

  19. Malarial parasite diversity in chimpanzees: the value of comparative approaches to ascertain the evolution of Plasmodium falciparum antigens

    PubMed Central

    2013-01-01

    Background Plasmodium falciparum shares its most recent common ancestor with parasites found in African apes; these species constitute the so-called Laverania clade. In this investigation, the evolutionary history of Plasmodium lineages found in chimpanzees (Pan troglodytes) was explored. Methods Here, the remainders of 74 blood samples collected as part of the chimpanzees routine health examinations were studied. For all positive samples with parasite lineages belonging to the Laverania clade, the complete mitochondrial genome (mtDNA), the gene encoding dihydrofolate reductase-thymidylate synthase (dhfr-ts), the chloroquine resistance transporter (Pfcrt), the circumsporozoite protein (csp), merozoite surface protein 2 (msp2), and the DBL-1 domain from var2CSA were amplified, cloned, and sequenced. Other Plasmodium species were included in the mtDNA, dhfr-ts, and csp analyses. Phylogenetic and evolutionary genetic analyses were performed, including molecular clock analyses on the mtDNA. Results/Conclusions Nine chimpanzees were malaria positive (12.2%); four of those infections were identified as P. falciparum, two as a Plasmodium reichenowi-like parasite or Plasmodium sp., one as Plasmodium gaboni, and two as Plasmodium malariae. All P. falciparum isolates were resistant to chloroquine indicating that the chimpanzees acquired such infections from humans in recent times. Such findings, however, are not sufficient for implicating chimpanzees as an animal reservoir for P. falciparum. Timing estimates support that the Laverania clade has co-existed with hominids for a long-period of time. The proposed species P. gaboni, Plasmodium billbrayi, and Plasmodium billcollinsi are monophyletic groups supporting that they are indeed different species. An expanded CSP phylogeny is presented, including all the Laverania species and other malarial parasites. Contrasting with other Plasmodium, the Laverania csp exhibits great conservation at the central tandem repeat region. Msp2 and var2CSA, however, show extended recent polymorphism in P. falciparum that likely originated after the P. reichenowi-P. falciparum split. The accumulation of such diversity may indicate adaptation to the human host. These examples support the notion that comparative approaches among P. falciparum and its related species will be of great value in understanding the evolution of proteins that are important in parasite invasion of the human red blood cell, as well as those involved in malaria pathogenesis. PMID:24044371

  20. Protein Export Marks the Early Phase of Gametocytogenesis of the Human Malaria Parasite Plasmodium falciparum*

    PubMed Central

    Silvestrini, Francesco; Lasonder, Edwin; Olivieri, Anna; Camarda, Grazia; van Schaijk, Ben; Sanchez, Massimo; Younis Younis, Sumera; Sauerwein, Robert; Alano, Pietro

    2010-01-01

    Despite over a century of study of malaria parasites, parts of the Plasmodium falciparum life cycle remain virtually unknown. One of these is the early gametocyte stage, a round shaped cell morphologically similar to an asexual trophozoite in which major cellular transformations ensure subsequent development of the elongated gametocyte. We developed a protocol to obtain for the first time highly purified preparations of early gametocytes using a transgenic line expressing a green fluorescent protein from the onset of gametocytogenesis. We determined the cellular proteome (1427 proteins) of this parasite stage by high accuracy tandem mass spectrometry and newly determined the proteomes of asexual trophozoites and mature gametocytes, identifying altogether 1090 previously undetected parasite proteins. Quantitative label-free comparative proteomics analysis determined enriched protein clusters for the three parasite developmental stages. Gene set enrichment analysis on the 251 proteins enriched in the early gametocyte proteome revealed that proteins putatively exported and involved in erythrocyte remodeling are the most overrepresented protein set in these stages. One-tenth of the early gametocyte-enriched proteome is constituted of putatively exported proteins, here named PfGEXPs (P. falciparum gametocyte-exported proteins). N-terminal processing and N-acetylation at a conserved leucine residue within the Plasmodium export element pentamotif were detected by mass spectrometry for three such proteins in the early but not in the mature gametocyte sample, further supporting a specific role in protein export in early gametocytogenesis. Previous reports and results of our experiments confirm that the three proteins are indeed exported in the erythrocyte cytoplasm. This work indicates that protein export profoundly marks early sexual differentiation in P. falciparum, probably contributing to host cell remodeling in this phase of the life cycle, and that gametocyte-enriched molecules are recruited to modulate this process in gametocytogenesis. PMID:20332084

  1. Structural and functional characterization of Falcipain-2, a hemoglobinase from the malarial parasite Plasmodium falciparum.

    PubMed

    Hogg, Tanis; Nagarajan, Krishna; Herzberg, Saskia; Chen, Lili; Shen, Xu; Jiang, Hualiang; Wecke, Maria; Blohmke, Christoph; Hilgenfeld, Rolf; Schmidt, Christian L

    2006-09-01

    Malaria is caused by protozoan erythrocytic parasites of the Plasmodium genus, with Plasmodium falciparum being the most dangerous and widespread disease-causing species. Falcipain-2 (FP-2) of P. falciparum is a papain-family (C1A) cysteine protease that plays an important role in the parasite life cycle by degrading erythrocyte proteins, most notably hemoglobin. Inhibition of FP-2 and its paralogues prevents parasite maturation, suggesting these proteins may be valuable targets for the design of novel antimalarial drugs, but lack of structural knowledge has impeded progress toward the rational discovery of potent, selective, and efficacious inhibitors. As a first step toward this goal, we present here the crystal structure of mature FP-2 at 3.1 A resolution, revealing novel structural features of the FP-2 subfamily proteases including a dynamic beta-hairpin hemoglobin binding motif, a flexible N-terminal alpha-helical extension, and a unique active-site cleft. We also demonstrate by biochemical methods that mature FP-2 can proteolytically process its own precursor in trans at neutral to weakly alkaline pH, that the binding of hemoglobin to FP-2 is strictly pH-dependent, and that FP-2 preferentially binds methemoglobin over hemoglobin. Because the specificity and proteolytic activity of FP-2 toward its multiple targets appears to be pH-dependent, we suggest that environmental pH may play an important role in orchestrating FP-2 function over the different life stages of the parasite. Moreover, it appears that selectivity of FP-2 for methemoglobin may represent an evolutionary adaptation to oxidative stress conditions within the host cell. PMID:16777845

  2. Droplet microfluidics platform for highly sensitive and quantitative detection of malaria-causing Plasmodium parasites based on enzyme activity measurement.

    PubMed

    Juul, Sissel; Nielsen, Christine J F; Labouriau, Rodrigo; Roy, Amit; Tesauro, Cinzia; Jensen, Pia W; Harmsen, Charlotte; Kristoffersen, Emil L; Chiu, Ya-Ling; Frhlich, Rikke; Fiorani, Paola; Cox-Singh, Janet; Tordrup, David; Koch, Jrn; Bienvenu, Anne-Lise; Desideri, Alessandro; Picot, Stephane; Petersen, Eskild; Leong, Kam W; Ho, Yi-Ping; Stougaard, Magnus; Knudsen, Birgitta R

    2012-12-21

    We present an attractive new system for the specific and sensitive detection of the malaria-causing Plasmodium parasites. The system relies on isothermal conversion of single DNA cleavage-ligation events catalyzed specifically by the Plasmodium enzyme topoisomerase I to micrometer-sized products detectable at the single-molecule level. Combined with a droplet microfluidics lab-on-a-chip platform, this design allowed for sensitive, specific, and quantitative detection of all human-malaria-causing Plasmodium species in single drops of unprocessed blood with a detection limit of less than one parasite/?L. Moreover, the setup allowed for detection of Plasmodium parasites in noninvasive saliva samples from infected patients. During recent years malaria transmission has declined worldwide, and with this the number of patients with low-parasite density has increased. Consequently, the need for accurate detection of even a few parasites is becoming increasingly important for the continued combat against the disease. We believe that the presented droplet microfluidics platform, which has a high potential for adaptation to point-of-care setups suitable for low-resource settings, may contribute significantly to meet this demand. Moreover, potential future adaptation of the presented setup for the detection of other microorganisms may form the basis for the development of a more generic platform for diagnosis, fresh water or food quality control, or other purposes within applied or basic science. PMID:23121492

  3. Diagnosis of Plasmodium falciparum infection in man: detection of parasite antigens by ELISA*

    PubMed Central

    Mackey, L. J.; McGregor, I. A.; Paounova, N.; Lambert, P. H.

    1982-01-01

    An ELISA method has been developed for the diagnosis of Plasmodium falciparum infection in man. Parasites from in vitro cultures of P. falciparum were used as source of antigen for the solid phase and the source of specific antibody was immune Gambian sera; binding of antibody in antigen-coated wells was registered by means of alkaline phosphatase-conjugated anti-human IgG. Parasites were detected on the basis of inhibition of antibody-binding. The test was applied to the detection of parasites in human red blood cells (RBC) from in vitro cultures of P. falciparum and in RBC from infected Gambians; RBC from 100 Geneva blood donors served as normal, uninfected controls. In titration experiments, the degree of antibody-binding inhibition correlated with the number of parasites in the test RBC. Parasites were detected at a level of 8 parasites/106 RBC. Samples of RBC were tested from 126 Gambians with microscopically proven infection; significant antibody-binding inhibition was found in 86% of these cases, where parasitaemia ranged from 10 to 125 000/?l of blood. The presence of high-titre antibody in the test preparations was found to reduce the sensitivity of parasite detection in infected RBC from in vitro cultures mixed with equal volumes of different antibody-containing sera. The sensitivity was restored in most cases by recovering the RBC by centrifugation before testing. In a preliminary experiment, there was no significant difference in antibody-binding inhibition using fresh infected RBC and RBC dried on filter-paper and recovered by elution, although there was greater variation in the latter samples. PMID:7044589

  4. Protein export in Plasmodium parasites: from the endoplasmic reticulum to the vacuolar export machine.

    PubMed

    Crabb, Brendan S; de Koning-Ward, Tania F; Gilson, Paul R

    2010-04-01

    It is somewhat paradoxical that the malaria parasite's survival strategy involves spending almost all of its blood-stage existence residing behind a two-membrane barrier in a host red blood cell, yet giving considerable attention to exporting parasite-encoded proteins back across these membranes. These exported proteins are thought to play diverse roles and are crucial in pathogenic processes, such as re-modelling of the erythrocyte cytoskeleton and mediating the export of a major virulence protein known as Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), and in metabolic processes such as nutrient uptake and solute exchange. Despite these varied roles most exported proteins have at least one common link; they share a trafficking pathway that begins with entry into the endoplasmic reticulum and concludes with passage across the vacuole membrane via a proteinaceous translocon known as the Plasmodium translocon of exported proteins (PTEX). In this commentary we review recent advances in our understanding of this export pathway and suggest several models by which different aspects of the process may be interconnected. PMID:20170656

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

    PubMed

    Mikolajczak, Sebastian A; Lakshmanan, Viswanathan; Fishbaugher, Matthew; Camargo, Nelly; Harupa, Anke; Kaushansky, Alexis; Douglass, Alyse N; Baldwin, Michael; Healer, Julie; O'Neill, Matthew; Phuong, Thuan; Cowman, Alan; Kappe, Stefan H I

    2014-09-01

    Immunization with live-attenuated Plasmodium sporozoites completely protects against malaria infection. Genetic engineering offers a versatile platform to create live-attenuated sporozoite vaccine candidates. We previously generated a genetically attenuated parasite (GAP) by deleting the P52 and P36 genes in the NF54 wild-type (WT) strain of Plasmodium falciparum (Pf p52(-)/p36(-) GAP). Preclinical assessment of p52(-)/p36(-) GAP in a humanized mouse model indicated an early and severe liver stage growth defect. However, human exposure to >200 Pf p52(-)/p36(-) GAP-infected mosquito bites in a safety trial resulted in peripheral parasitemia in one of six volunteers, revealing that this GAP was incompletely attenuated. We have now created a triple gene deleted GAP by additionally removing the SAP1 gene (Pf p52(-)/p36(-)/sap1(-) GAP) and employed flippase (FLP)/flippase recognition target (FRT) recombination for drug selectable marker cassette removal. This next-generation GAP was indistinguishable from WT parasites in blood stage and mosquito stage development. Using an improved humanized mouse model transplanted with human hepatocytes and human red blood cells, we show that despite a high-dose sporozoite challenge, Pf p52(-)/p36(-)/sap1(-) GAP did not transition to blood stage infection and appeared to be completely attenuated. Thus, clinical testing of Pf p52(-)/p36(-)/sap1(-) GAP assessing safety, immunogenicity, and efficacy against sporozoite challenge is warranted. PMID:24827907

  6. Hostile Takeover by Plasmodium: Reorganization of Parasite and Host Cell Membranes during Liver Stage Egress

    PubMed Central

    Graewe, Stefanie; Rankin, Kathleen E.; Lehmann, Christine; Deschermeier, Christina; Hecht, Leonie; Froehlke, Ulrike; Stanway, Rebecca R.; Heussler, Volker

    2011-01-01

    The protozoan parasite Plasmodium is transmitted by female Anopheles mosquitoes and undergoes obligatory development within a parasitophorous vacuole in hepatocytes before it is released into the bloodstream. The transition to the blood stage was previously shown to involve the packaging of exoerythrocytic merozoites into membrane-surrounded vesicles, called merosomes, which are delivered directly into liver sinusoids. However, it was unclear whether the membrane of these merosomes was derived from the parasite membrane, the parasitophorous vacuole membrane or the host cell membrane. This knowledge is required to determine how phagocytes will be directed against merosomes. Here, we fluorescently label the candidate membranes and use live cell imaging to show that the merosome membrane derives from the host cell membrane. We also demonstrate that proteins in the host cell membrane are lost during merozoite liberation from the parasitophorous vacuole. Immediately after the breakdown of the parasitophorous vacuole membrane, the host cell mitochondria begin to degenerate and protein biosynthesis arrests. The intact host cell plasma membrane surrounding merosomes allows Plasmodium to mask itself from the host immune system and bypass the numerous Kupffer cells on its way into the bloodstream. This represents an effective strategy for evading host defenses before establishing a blood stage infection. PMID:21909271

  7. Autophagy-Related Atg8 Localizes to the Apicoplast of the Human Malaria Parasite Plasmodium falciparum

    PubMed Central

    Kitamura, Kei; Kishi-Itakura, Chieko; Tsuboi, Takafumi; Sato, Shigeharu; Kita, Kiyoshi; Ohta, Nobuo; Mizushima, Noboru

    2012-01-01

    Autophagy is a membrane-mediated degradation process, which is governed by sequential functions of Atg proteins. Although Atg proteins are highly conserved in eukaryotes, protozoa possess only a partial set of Atg proteins. Nonetheless, almost all protozoa have the complete factors belonging to the Atg8 conjugation system, namely, Atg3, Atg4, Atg7, and Atg8. Here, we report the biochemical properties and subcellular localization of the Atg8 protein of the human malaria parasite Plasmodium falciparum (PfAtg8). PfAtg8 is expressed during intra-erythrocytic development and associates with membranes likely as a lipid-conjugated form. Fluorescence microscopy and immunoelectron microscopy show that PfAtg8 localizes to the apicoplast, a four membrane-bound non-photosynthetic plastid. Autophagosome-like structures are not observed in the erythrocytic stages. These data suggest that, although Plasmodium parasites have lost most Atg proteins during evolution, they use the Atg8 conjugation system for the unique organelle, the apicoplast. PMID:22900071

  8. Does haemosporidian infection affect hematological and biochemical profiles of the endangered Black-fronted piping-guan (Aburria jacutinga)?

    PubMed Central

    Ferreira Junior, Francisco Carlos; Andery, Danielle de Assis; Horta, Rodrigo Santos; Peixoto, Renata Barbosa; Lacorte, Gustavo Augusto; Moreira, Patrcia de Abreu; Paes Leme, Fabola de Oliveira; Melo, Marlia Martins; Martins, Nelson Rodrigo da Silva

    2013-01-01

    Infectious diseases can cause deleterious effects on bird species, leading to population decline and extinction. Haemosporidia can be recognized by their negative effects on host fitness, including reproductive success and immune responses. In captivity, outbreaks of haemosporidian infection have been observed in birds in zoos and aviaries. The endemic Brazilian Atlantic rainforest species Aburria jacutinga is one of the most endangered species in the Cracidae family, and wild populations of this species are currently found mainly in conservation areas in only two Brazilian states. In this study, we aimed to evaluate the effects of avian haemosporidia on hematological and biochemical parameters in two captive populations of A. jacutinga. Forty-two animals were assessed, and the haemosporidian prevalence was similar for males and females. The occurrence of haemosporidian infection in captive A. jacutinga observed in this study was similar to results found in other captive and wild birds in Brazil. We found three different lineages of haemosporidia. Two lineages were identified as Plasmodium sp., one of which was previously detected in Europe and Asia, and the other is a new lineage closely related to P. gallinaceum. A new third lineage was identified as Haemoproteus sp. We found no significant differences in hematological and biochemical values between infected and non-infected birds, and the haemosporidian lineage did not seem to have an impact on the clinical and physiological parameters of A. jacutinga. This is the first report on an evaluation of natural haemosporidian infections diagnosed by microscopic and molecular methods in A. jacutinga by hematology, blood biochemistry, and serum protein values. Determining physiological parameters, occurrence and an estimation of the impact of haemosporidia in endangered avian species may contribute to the management of species rehabilitation and conservation. PMID:23638382

  9. The antimalarial action of desferal involves a direct access route to erythrocytic (Plasmodium falciparum) parasites.

    PubMed Central

    Loyevsky, M; Lytton, S D; Mester, B; Libman, J; Shanzer, A; Cabantchik, Z I

    1993-01-01

    We designed the N-methylanthranilic-desferrioxamine (MA-DFO) as a fluorescent iron (III) chelator with improved membrane permeation properties. Upon binding of iron (III), MA-DFO fluorescence is quenched, thus allowing traceability of drug-iron (III) interactions. MA-DFO is well tolerated by mammalian cells in culture. Its antimalarial activity is pronounced: IC50 values on in vitro (24-h) growth of Plasmodium falciparum were 3 +/- 1 microM for MA-DFO compared with 30 +/- 8 for DFO. The onset of growth inhibition of rings or trophozoites occurs 2-4 h after exposure to 13 microM MA-DFO. This effect is commensurate with MA-DFO permeation into infected cells. In a 24-h exposure to MA-DFO or DFO, trophozoites take up either compound to approximately 10% of the external concentration, rings to 5%, and noninfected cells to < 1%. Red cells encapsulated with millimolar concentrations of DFO or MA-DFO fully support parasite invasion and growth. We conclude that extracellular MA-DFO and DFO gain selective access into parasites by bypassing the host. The rate-limiting step is permeation through the parasite membrane, which MA-DFO accomplishes faster than DFO, in accordance with its higher hydrophobicity. These views are consistent with the proposed duct, which apparently provides parasitized cells with a window to the external medium. PMID:8423220

  10. The apicoplast: a plastid in Plasmodium falciparum and other Apicomplexan parasites.

    PubMed

    Foth, Bernardo J; McFadden, Geoffrey I

    2003-01-01

    Apicomplexan parasites cause severe diseases such as malaria, toxoplasmosis, and coccidiosis (caused by Plasmodium spp., Toxoplasma, and Eimeria, respectively). These parasites contain a relict plastid-termed "apicoplast"--that originated from the engulfment of an organism of the red algal lineage. The apicoplast is indispensable but its exact role in parasites is unknown. The apicoplast has its own genome and expresses a small number of genes, but the vast majority of the apicoplast proteome is encoded in the nuclear genome. The products of these nuclear genes are posttranslationally targeted to the organelle via the secretory pathway courtesy of a bipartite N-terminal leader sequence. Apicoplasts are nonphotosynthetic but retain other typical plastid functions such as fatty acid, isoprenoid and heme synthesis, and products of these pathways might be exported from the apicoplast for use by the parasite. Apicoplast pathways are essentially prokaryotic and therefore excellent drug targets. Some antibiotics inhibiting these molecular processes are already in chemotherapeutic use, whereas many new drugs will hopefully spring from our growing understanding of this intriguing organelle. PMID:12722949

  11. Haemosporidian infections in the Tengmalm's Owl (Aegolius funereus) and potential insect vectors of their transmission.

    PubMed

    Synek, Petr; Popelková, Alena; Koubínová, Darina; Šťastný, Karel; Langrová, Iva; Votýpka, Jan; Munclinger, Pavel

    2016-01-01

    Sedentary bird species are suitable model hosts for identifying potential vectors of avian blood parasites. We studied haemosporidian infections in the Tengmalm's Owl (Aegolius funereus) in the Ore Mountains of the Czech Republic using molecular detection methods. Sex of owl nestlings was scored using molecular sexing based on fragment analysis of PCR-amplified CHD1 introns. Observed infection prevalences in nestlings and adult owls were 51 and 86 %, respectively. Five parasite lineages were detected. Most of the infections comprised the Leucocytozoon AEFUN02 and STOCC06 lineages that probably refer to distinct Leucocytozoon species. Other lineages were detected only sporadically. Mixed infections were found in 49 % of samples. The main factor affecting the probability of infection was host age. No effect of individual sex on infection probability was evidenced. The youngest infected nestling was 12 days old. High parasite prevalence in the Tengmalm's Owl nestlings suggests that insect vectors must enter nest boxes to transmit parasites before fledging. Hence, we placed sticky insect traps into modified nest boxes, collected potential insect vectors, and examined them for the presence of haemosporidian parasites using molecular detection. We trapped 201 insects which were determined as biting midges from the Culicoides genus and two black fly species, Simulium (Nevermannia) vernum and Simulium (Eusimulium) angustipes. Six haemosporidian lineages were detected in the potential insect vectors, among which the Leucocytozoon lineage BT2 was common to the Tengmalm's Owl and the trapped insects. However, we have not detected the most frequently encountered Tengmalm's Owl Leucocytozoon lineages AEFUN02 and STOCC06 in insects. PMID:26365667

  12. Long-term live imaging reveals cytosolic immune responses of host hepatocytes against Plasmodium infection and parasite escape mechanisms

    PubMed Central

    Prado, Monica; Eickel, Nina; De Niz, Mariana; Heitmann, Anna; Agop-Nersesian, Carolina; Wacker, Rahel; Schmuckli-Maurer, Jacqueline; Caldelari, Reto; Janse, Chris J; Khan, Shahid M; May, Jürgen; Meyer, Christian G; Heussler, Volker T

    2015-01-01

    Plasmodium parasites are transmitted by Anopheles mosquitoes to the mammalian host and actively infect hepatocytes after passive transport in the bloodstream to the liver. In their target host hepatocyte, parasites reside within a parasitophorous vacuole (PV). In the present study it was shown that the parasitophorous vacuole membrane (PVM) can be targeted by autophagy marker proteins LC3, ubiquitin, and SQSTM1/p62 as well as by lysosomes in a process resembling selective autophagy. The dynamics of autophagy marker proteins in individual Plasmodium berghei-infected hepatocytes were followed by live imaging throughout the entire development of the parasite in the liver. Although the host cell very efficiently recognized the invading parasite in its vacuole, the majority of parasites survived this initial attack. Successful parasite development correlated with the gradual loss of all analyzed autophagy marker proteins and associated lysosomes from the PVM. However, other autophagic events like nonselective canonical autophagy in the host cell continued. This was indicated as LC3, although not labeling the PVM anymore, still localized to autophagosomes in the infected host cell. It appears that growing parasites even benefit from this form of nonselective host cell autophagy as an additional source of nutrients, as in host cells deficient for autophagy, parasite growth was retarded and could partly be rescued by the supply of additional amino acid in the medium. Importantly, mouse infections with P. berghei sporozoites confirmed LC3 dynamics, the positive effect of autophagy activation on parasite growth, and negative effects upon autophagy inhibition. PMID:26208778

  13. Genome-wide functional analysis of Plasmodium protein phosphatases reveals key regulators of parasite development and differentiation.

    PubMed

    Guttery, David S; Poulin, Benoit; Ramaprasad, Abhinay; Wall, Richard J; Ferguson, David J P; Brady, Declan; Patzewitz, Eva-Maria; Whipple, Sarah; Straschil, Ursula; Wright, Megan H; Mohamed, Alyaa M A H; Radhakrishnan, Anand; Arold, Stefan T; Tate, Edward W; Holder, Anthony A; Wickstead, Bill; Pain, Arnab; Tewari, Rita

    2014-07-01

    Reversible protein phosphorylation regulated by kinases and phosphatases controls many cellular processes. Although essential functions for the malaria parasite kinome have been reported, the roles of most protein phosphatases (PPs) during Plasmodium development are unknown. We report a functional analysis of the Plasmodium berghei protein phosphatome, which exhibits high conservation with the P. falciparum phosphatome and comprises 30 predicted PPs with differential and distinct expression patterns during various stages of the life cycle. Gene disruption analysis of P. berghei PPs reveals that half of the genes are likely essential for asexual blood stage development, whereas six are required for sexual development/sporogony in mosquitoes. Phenotypic screening coupled with transcriptome sequencing unveiled morphological changes and altered gene expression in deletion mutants of two N-myristoylated PPs. These findings provide systematic functional analyses of PPs in Plasmodium, identify how phosphatases regulate parasite development and differentiation, and can inform the identification of drug targets for malaria. PMID:25011111

  14. Genome-wide Functional Analysis of Plasmodium Protein Phosphatases Reveals Key Regulators of Parasite Development and Differentiation

    PubMed Central

    Guttery, DavidS.; Poulin, Benoit; Ramaprasad, Abhinay; Wall, RichardJ.; Ferguson, DavidJ.P.; Brady, Declan; Patzewitz, Eva-Maria; Whipple, Sarah; Straschil, Ursula; Wright, MeganH.; Mohamed, AlyaaM.A.H.; Radhakrishnan, Anand; Arold, StefanT.; Tate, EdwardW.; Holder, AnthonyA.; Wickstead, Bill; Pain, Arnab; Tewari, Rita

    2014-01-01

    Summary Reversible protein phosphorylation regulated by kinases and phosphatases controls many cellular processes. Although essential functions for the malaria parasite kinome have been reported, the roles of most protein phosphatases (PPs) during Plasmodium development are unknown. We report a functional analysis of the Plasmodium berghei protein phosphatome, which exhibits high conservation with the P.falciparum phosphatome and comprises 30 predicted PPs with differential and distinct expression patterns during various stages of the life cycle. Gene disruption analysis of P.berghei PPs reveals that half of the genes are likely essential for asexual bloodstage development, whereas six are required for sexual development/sporogony in mosquitoes. Phenotypic screening coupled with transcriptome sequencing unveiled morphological changes and altered gene expression in deletion mutants of two N-myristoylated PPs. These findings provide systematic functional analyses of PPs in Plasmodium, identify how phosphatases regulate parasite development and differentiation, and can inform the identification of drug targets for malaria. PMID:25011111

  15. Arteether resistance reversal by ketoconazole/fluconazole in rodent malaria parasite Plasmodium vinckei.

    PubMed

    Chandra, Ramesh; Puri, S K

    2015-03-01

    Artemisinin and its derivative arteether (ART) are fast acting antimalarial drugs against chloroquine-resistant. There are several partner drugs that are identified as a potential drug for artemisinin combination therapy (ACT) to develop as the antimalarial drug. Limited studies have been carried out in ART drug combination that may have more promising as ACT for resistant Plasmodium parasite. Here, we are the first to show the ART drug resistance reversal in Plasmodium vinckei by using antifungal azole compounds ketoconazole (KTZ) and fluconazole (FCZ). Our previous study has shown that higher antioxidant enzyme, glutathione, and less hemozoin may be correlated with ART resistance in P. vinckei (PvAR). We further hypothesized that glutathione and heme catabolism may be interfered by KTZ and FCZ, resulting in an increased efficacy of ART in PvAR parasite. The results of present study demonstrate synergetic effect of KTZ and FCZ against PvAR parasite, since none of the mice developed infection up to day 10 after combination with ART. These results further showed that ED90 of ART was reduced from 17.23 to 2.19 and 2.56 mg/kg when used in combination with KTZ and FCZ, respectively. Resultant, activity enhancement index (AEI) of ART is significantly increased to 8.60 and 6.73 with partner agents. These studies propose the possibility of ART drug combination that may be helpful in prolonging the life of drug and a promising lead to reduce the chance of resistance development of artemisinin and its derivative. PMID:25616344

  16. Pivotal and distinct role for Plasmodium actin capping protein alpha during blood infection of the malaria parasite

    PubMed Central

    Ganter, Markus; Rizopoulos, Zaira; Schler, Herwig; Matuschewski, Kai

    2015-01-01

    Accurate regulation of microfilament dynamics is central to cell growth, motility and response to environmental stimuli. Stabilizing and depolymerizing proteins control the steady-state levels of filamentous (F-) actin. Capping protein (CP) binds to free barbed ends, thereby arresting microfilament growth and restraining elongation to remaining free barbed ends. In all CPs characterized to date, alpha and beta subunits form the active heterodimer. Here, we show in a eukaryotic parasitic cell that the two CP subunits can be functionally separated. Unlike the beta subunit, the CP alpha subunit of the apicomplexan parasite Plasmodium is refractory to targeted gene deletion during blood infection in the mammalian host. Combinatorial complementation of Plasmodium berghei CP genes with the orthologs from Plasmodium falciparum verified distinct activities of CP alpha and CP alpha/beta during parasite life cycle progression. Recombinant Plasmodium?CP alpha could be produced in Escherichia coli in the absence of the beta subunit and the protein displayed F-actin capping activity. Thus, the functional separation of two CP subunits in a parasitic eukaryotic cell and the F-actin capping activity of CP alpha expand the repertoire of microfilament regulatory mechanisms assigned to CPs. PMID:25565321

  17. Blood Parasites in Owls with Conservation Implications for the Spotted Owl (Strix occidentalis)

    PubMed Central

    Ishak, Heather D.; Dumbacher, John P.; Anderson, Nancy L.; Keane, John J.; Valkiūnas, Gediminas; Haig, Susan M.; Tell, Lisa A.; Sehgal, Ravinder N. M.

    2008-01-01

    The three subspecies of Spotted Owl (Northern, Strix occidentalis caurina; California, S. o. occidentalis; and Mexican, S. o. lucida) are all threatened by habitat loss and range expansion of the Barred Owl (S. varia). An unaddressed threat is whether Barred Owls could be a source of novel strains of disease such as avian malaria (Plasmodium spp.) or other blood parasites potentially harmful for Spotted Owls. Although Barred Owls commonly harbor Plasmodium infections, these parasites have not been documented in the Spotted Owl. We screened 111 Spotted Owls, 44 Barred Owls, and 387 owls of nine other species for haemosporidian parasites (Leucocytozoon, Plasmodium, and Haemoproteus spp.). California Spotted Owls had the greatest number of simultaneous multi-species infections (44%). Additionally, sequencing results revealed that the Northern and California Spotted Owl subspecies together had the highest number of Leucocytozoon parasite lineages (n = 17) and unique lineages (n = 12). This high level of sequence diversity is significant because only one Leucocytozoon species (L. danilewskyi) has been accepted as valid among all owls, suggesting that L. danilewskyi is a cryptic species. Furthermore, a Plasmodium parasite was documented in a Northern Spotted Owl for the first time. West Coast Barred Owls had a lower prevalence of infection (15%) when compared to sympatric Spotted Owls (S. o. caurina 52%, S. o. occidentalis 79%) and Barred Owls from the historic range (61%). Consequently, Barred Owls on the West Coast may have a competitive advantage over the potentially immune compromised Spotted Owls. PMID:18509541

  18. Blood parasites in Owls with conservation implications for the Spotted Owl (Strix occidentalis)

    USGS Publications Warehouse

    Ishak, H.D.; Dumbacher, J.P.; Anderson, N.L.; Keane, J.J.; Valkiunas, G.; Haig, S.M.; Tell, L.A.; Sehgal, R.N.M.

    2008-01-01

    The three subspecies of Spotted Owl (Northern, Strix occidentalis courina; California, S. o. occidentalis; and Mexican, S. o. lucida) are all threatened by habitat loss and range expansion of the Barred Owl (S. varia). An unaddressed threat is whether Barred Owls could be a source of novel strains of disease such as avian malaria (Plasmodium spp.) or other blood parasites potentially harmful for Spotted Owls. Although Barred Owls commonly harbor Plasmodium infections, these parasites have not been documented in the Spotted Owl. We screened 111 Spotted Owls, 44 Barred Owls, and 387 owls of nine other species for haemosporidian parasites (Leucocytozoon, Plasmodium, and Haemoproteus spp.). California Spotted Owls had the greatest number of simultaneous multi-species infections (44%). Additionally, sequencing results revealed that the Northern and California Spotted Owl subspecies together had the highest number of Leucocytozoon parasite lineages (n=17) and unique lineages (n=12). This high level of sequence diversity is significant because only one leucocytozoon species (L. danilewskyi) has been accepted as valid among all owls, suggesting that L. danilewskyi is a cryptic species. Furthermore, a Plasmodium parasite was documented in a Northern Spotted Owl for the first time. West Coast Barred Owls had a lower prevalence of infection (15%) when compared to sympatric Spotted Owls (S. o. caurina 52%, S. o. occidentalis 79%) and Barred Owls from the historic range (61%). Consequently, Barred Owls on the West Coast may have a competitive advantage over the potentially immune compromised Spotted Owls. ?? 2008 Ishak et al.

  19. Preferential binding of 4-hydroxynonenal to lysine residues in specific parasite proteins in plakortin-treated Plasmodium falciparum-parasitized red blood cells

    PubMed Central

    Schwarzer, Evelin; Gallo, Valentina; Valente, Elena; Ulliers, Daniela; Taglialatela-Scafati, Orazio; Arese, Paolo; Skorokhod, Oleksii A.

    2015-01-01

    The data show the frequencies by which the amino acid residues lysine, histidine and cysteine of six proteins of the malaria parasite Plasmodium falciparum are post-translationally modified by the lipoperoxydation endproduct 4-hydroxynonenal after challenging the parasitized red blood cell with plakortin. Plakortin is an antimalarial endoperoxide whose molecular anti-parasitic effect is described in Skorokhod et al. (2015) [1]. Plakortin did not elicit hemoglobin leakage from host red blood cells and did not oxidize reduced glutathione. PMID:26702418

  20. Prevalence and diversity of Plasmodium and Haemoproteus parasites in the globally-threatened Aquatic Warbler Acrocephalus paludicola.

    PubMed

    Neto, Jlio Manuel; Prez-Rodrguez, Antn; Haase, Martin; Flade, Martin; Bensch, Staffan

    2015-08-01

    The diversity and prevalence of malaria parasites of the genera Plasmodium and Haemoproteus were determined in the globally-threatened Aquatic Warbler Acrocephalus paludicola. Birds were sampled during migration in Portugal and at the wintering quarters in Senegal and parasites were detected using molecular methods. Only three generalist parasite lineages (Plasmodium) were found. There were no significant differences in the prevalence of parasites between sexes in Europe, but adults had higher prevalence than first-year birds, and birds in Europe had higher prevalence than those captured in Africa. When comparing with other Acrocephalus species and taking sample size into account, Aquatic Warblers had the lowest prevalence and, together with another threatened species, the Seychelles Warbler Acrocephalus sechellensis, the lowest diversity of malaria parasites. We hypothesize that the low diversity of parasites and absence of specialist lineages of Aquatic Warblers are caused by its small population size and fragmented distribution. Furthermore, Aquatic Warblers' extreme habitat specialization may decrease their exposure to malaria parasites, but other explanations such as high mortality (which would constraint the sampling of infected birds) or, in contrast, very efficient immunological system in clearing the infections cannot be ruled out. This study contributes to explain variation in prevalence and diversity of malaria parasites among hosts. PMID:25924680

  1. Persistence of Plasmodium falciparum parasites in infected pregnant Mozambican women after delivery.

    PubMed

    Serra-Casas, Elisa; Menndez, Clara; Dobao, Carlota; Bardaj, Azucena; Quint, Lloren; Quint, Llorenc; Ordi, Jaume; Sigauque, Betuel; Cister, Pau; Mandomando, Inacio; Alonso, Pedro L; Mayor, Alfredo

    2011-01-01

    Pregnant women are susceptible to Plasmodium falciparum parasites that sequester in the placenta. The massive accumulation of infected erythrocytes in the placenta has been suggested to trigger the deleterious effects of malaria in pregnant women and their offspring. The risk of malaria is also high during the postpartum period, although mechanisms underlying this susceptibility are not known. Here, we aimed to identify host factors contributing to the risk of postpartum infections and to determine the origin of postpartum parasites by comparing their genotypes with those present at the time of delivery. To address this, blood samples were collected at delivery (n = 402) and postpartum (n = 354) from Mozambican women enrolled in a trial of intermittent preventive treatment in pregnancy (IPTp). P. falciparum was detected by real-time quantitative PCR (qPCR), and the parasite merozoite surface protein 1 (msp-1) and msp-2 genes were genotyped. Fifty-seven out of 354 (16%) women were infected postpartum as assessed by qPCR, whereas prevalence by optical microscopy was only 4%. Risk of postpartum infection was lower in older women (odds ratio [OR] = 0.34, 95% confidence interval [CI] = 0.15 to 0.81) and higher in women with a placental infection at delivery (OR = 4.20, 95% CI = 2.19 to 8.08). Among 24 women with matched infections, 12 (50%) were infected postpartum with at least one parasite strain that was also present in their placentas. These results suggest that parasites infecting pregnant women persist after delivery and increase the risk of malaria during the postpartum period. Interventions that reduce malaria during pregnancy may translate into a lower risk of postpartum infection. PMID:21041485

  2. Host cell deformability is linked to transmission in the human malaria parasite Plasmodium falciparum

    PubMed Central

    Aingaran, Mythili; Zhang, Rou; Law, Sue KaYee; Peng, Zhangli; Undisz, Andreas; Meyer, Evan; Diez-Silva, Monica; Burke, Thomas A.; Spielmann, Tobias; Lim, Chwee Teck; Suresh, Subra; Dao, Ming; Marti, Matthias

    2012-01-01

    SUMMARY Gametocyte maturation in Plasmodium falciparum is a critical step in the transmission of malaria. While the majority of parasites proliferate asexually in red blood cells, a small fraction of parasites undergo sexual conversion and mature over two weeks to become competent for transmission to a mosquito vector. Immature gametocytes sequester in deep tissues while mature stages must be able to circulate, pass the spleen and present themselves to the mosquito vector in order to complete transmission. Sequestration of asexual red blood cell stage parasites has been investigated in great detail. These studies have demonstrated that induction of cytoadherence properties through specific receptor-ligand interactions coincides with a significant increase in host cell stiffness. In contrast, the adherence and biophysical properties of gametocyte-infected red blood cells have not been studied systematically. Utilizing a transgenic line for 3D live imaging, in vitro capillary assays and 3D finite element whole cell modeling, we studied the role of cellular deformability in determining the circulatory characteristics of gametocytes. Our analysis shows that the red blood cell deformability of immature gametocytes displays an overall decrease followed by rapid restoration in mature gametocytes. Intriguingly, simulations suggest that along with deformability variations, the morphological changes of the parasite may play an important role in tissue distribution in vivo. Taken together we present a model, which suggests that mature but not immature gametocytes circulate in the peripheral blood for uptake in the mosquito blood meal and transmission to another human host thus ensuring long term survival of the parasite. PMID:22417683

  3. Enzymatic Characterization of Recombinant Food Vacuole Plasmepsin 4 from the Rodent Malaria Parasite Plasmodium berghei

    PubMed Central

    Liu, Peng; Robbins, Arthur H.; Marzahn, Melissa R.; McClung, Scott H.; Yowell, Charles A.; Stevens, Stanley M.; Dame, John B.; Dunn, Ben M.

    2015-01-01

    The rodent malaria parasite Plasmodium berghei is a practical model organism for experimental studies of human malaria. Plasmepsins are a class of aspartic proteinase isoforms that exert multiple pathological effects in malaria parasites. Plasmepsins residing in the food vacuole (FV) of the parasite hydrolyze hemoglobin in red blood cells. In this study, we cloned PbPM4, the FV plasmepsin gene of P. berghei that encoded an N-terminally truncated pro-segment and the mature enzyme from genomic DNA. We over-expressed this PbPM4 zymogen as inclusion bodies (IB) in Escherichia coli, and purified the protein following in vitro IB refolding. Auto-maturation of the PbPM4 zymogen to mature enzyme was carried out at pH 4.5, 5.0, and 5.5. Interestingly, we found that the PbPM4 zymogen exhibited catalytic activity regardless of the presence of the pro-segment. We determined the optimal catalytic conditions for PbPM4 and studied enzyme kinetics on substrates and inhibitors of aspartic proteinases. Using combinatorial chemistry-based peptide libraries, we studied the active site preferences of PbPM4 at subsites S1, S2, S3, S1’, S2’ and S3’. Based on these results, we designed and synthesized a selective peptidomimetic compound and tested its inhibition of PbPM4, seven FV plasmepsins from human malaria parasites, and human cathepsin D (hcatD). We showed that this compound exhibited a >10-fold selectivity to PbPM4 and human malaria parasite plasmepsin 4 orthologs versus hcatD. Data from this study furthesr our understanding of enzymatic characteristics of the plasmepsin family and provides leads for anti-malarial drug design. PMID:26510189

  4. Plasmodium falciparum: modifications of the in vitro culture conditions improving parasitic yields.

    PubMed

    Zolg, J W; MacLeod, A J; Dickson, I H; Scaife, J G

    1982-12-01

    We attempted to optimize some of the variables involved in the in vitro culturing of Plasmodium falciparum. Irrespective of the isolates used, suspension cultures in glucose-enriched RPMI-1640 medium buffered with TES yielded about twice the amount of parasites than could be obtained from static, thin-layer cultures with HEPES-buffered RPMI-1640 without additional glucose. In suspension cultures, methylcellulose (1 mg/ml) was added to protect the erythrocytes. In addition the erythrocytes were found to be more suitable for culturing P. falciparum when stored as a concentrate in saline-adenine-glucose than as whole blood in citrate-phosphate-dextrose. With a cloned isolate of P. falciparum (Tak9/clone 96) a further stimulation of the final parasitemia could be achieved by supplementing the medium with hypoxanthine (50 micrograms/ml) and reduced glutathione (600 micrograms/ml). Moreover, we identified hypoxanthine and glutathione as two of the factors critical for the ability of human serum to support the growth of the parasites. These modifications give a two- to four-fold increase in the final parasitemia over the original Trager-Jensen culture method, thus allowing more parasites to be isolated for biochemical studies. PMID:6757399

  5. The Clp Chaperones and Proteases of the Human Malaria Parasite Plasmodium falciparum

    SciTech Connect

    Bakkouri, Majida El; Pow, Andre; Mulichak, Anne; Cheung, Kevin L.Y.; Artz, Jennifer D.; Amani, Mehrnaz; Fell, Stuart; de Koning-Ward, Tania F.; Goodman, C. Dean; McFadden, Geoffrey I.; Ortega, Joaquin; Hui, Raymond; Houry, Walid A.

    2015-02-09

    The Clp chaperones and proteases play an important role in protein homeostasis in the cell. They are highly conserved across prokaryotes and found also in the mitochondria of eukaryotes and the chloroplasts of plants. They function mainly in the disaggregation, unfolding and degradation of native as well as misfolded proteins. Here, we provide a comprehensive analysis of the Clp chaperones and proteases in the human malaria parasite Plasmodium falciparum. The parasite contains four Clp ATPases, which we term PfClpB1, PfClpB2, PfClpC and PfClpM. One PfClpP, the proteolytic subunit, and one PfClpR, which is an inactive version of the protease, were also identified. Expression of all Clp chaperones and proteases was confirmed in blood-stage parasites. The proteins were localized to the apicoplast, a non-photosynthetic organelle that accommodates several important metabolic pathways in P. falciparum, with the exception of PfClpB2 (also known as Hsp101), which was found in the parasitophorous vacuole. Both PfClpP and PfClpR form mostly homoheptameric rings as observed by size-exclusion chromatography, analytical ultracentrifugation and electron microscopy. The X-ray structure of PfClpP showed the protein as a compacted tetradecamer similar to that observed for Streptococcus pneumoniae and Mycobacterium tuberculosis ClpPs. Our data suggest the presence of a ClpCRP complex in the apicoplast of P. falciparum.

  6. The MYST family histone acetyltransferase regulates gene expression and cell cycle in malaria parasite Plasmodium falciparum.

    PubMed

    Miao, Jun; Fan, Qi; Cui, Long; Li, Xiaolian; Wang, Haiyan; Ning, Gang; Reese, Joseph C; Cui, Liwang

    2010-11-01

    Histone lysine acetylation, normally associated with euchromatin and active genes, is regulated by different families of histone acetyltransferases (HATs). A single Plasmodium falciparum MYST (PfMYST) HAT was expressed as a long and a short version in intraerythrocytic stages. Whereas the recombinant PfMYST expressed in prokaryotes and insect cells did not show HAT activity, recombinant PfMYST purified from the parasites exhibited a predilection to acetylate histone H4 in vitro at K5, K8, K12 and K16. Tagging PfMYST with the green fluorescent protein at the C-terminus showed that PfMYST protein was localized in both the nucleus and cytoplasm. Consistent with the importance of H4 acetylation in var gene expression, PfMYST was recruited to the active var promoter. Attempts to disrupt PfMYST were not successful, suggesting that PfMYST is essential for asexual intraerythrocytic growth. However, overexpression of the long, active or a truncated, non-active version of PfMYST by stable integration of the expression cassette in the parasite genome resulted in changes of H4 acetylation and cell cycle progression. Furthermore, parasites with PfMYST overexpression showed changes in sensitivity to DNA-damaging agents. Collectively, this study showed that PfMYST plays important roles in cellular processes such as gene activation, cell cycle control and DNA repair. PMID:20807207

  7. Artemisinin-resistant Plasmodium falciparum parasites exhibit altered patterns of development in infected erythrocytes.

    PubMed

    Hott, Amanda; Casandra, Debora; Sparks, Kansas N; Morton, Lindsay C; Castanares, Geocel-Grace; Rutter, Amanda; Kyle, Dennis E

    2015-01-01

    Artemisinin derivatives are used in combination with other antimalarial drugs for treatment of multidrug-resistant malaria worldwide. Clinical resistance to artemisinin recently emerged in southeast Asia, yet in vitro phenotypes for discerning mechanism(s) of resistance remain elusive. Here, we describe novel phenotypic resistance traits expressed by artemisinin-resistant Plasmodium falciparum. The resistant parasites exhibit altered patterns of development that result in reduced exposure to drug at the most susceptible stage of development in erythrocytes (trophozoites) and increased exposure in the most resistant stage (rings). In addition, a novel in vitro delayed clearance assay (DCA) that assesses drug effects on asexual stages was found to correlate with parasite clearance half-life in vivo as well as with mutations in the Kelch domain gene associated with resistance (Pf3D7_1343700). Importantly, all of the resistance phenotypes were stable in cloned parasites for more than 2 years without drug pressure. The results demonstrate artemisinin-resistant P. falciparum has evolved a novel mechanism of phenotypic resistance to artemisinin drugs linked to abnormal cell cycle regulation. These results offer insights into a novel mechanism of drug resistance in P. falciparum and new tools for monitoring the spread of artemisinin resistance. PMID:25779582

  8. Artemisinin-Resistant Plasmodium falciparum Parasites Exhibit Altered Patterns of Development in Infected Erythrocytes

    PubMed Central

    Hott, Amanda; Casandra, Debora; Sparks, Kansas N.; Morton, Lindsay C.; Castanares, Geocel-Grace; Rutter, Amanda

    2015-01-01

    Artemisinin derivatives are used in combination with other antimalarial drugs for treatment of multidrug-resistant malaria worldwide. Clinical resistance to artemisinin recently emerged in southeast Asia, yet in vitro phenotypes for discerning mechanism(s) of resistance remain elusive. Here, we describe novel phenotypic resistance traits expressed by artemisinin-resistant Plasmodium falciparum. The resistant parasites exhibit altered patterns of development that result in reduced exposure to drug at the most susceptible stage of development in erythrocytes (trophozoites) and increased exposure in the most resistant stage (rings). In addition, a novel in vitro delayed clearance assay (DCA) that assesses drug effects on asexual stages was found to correlate with parasite clearance half-life in vivo as well as with mutations in the Kelch domain gene associated with resistance (Pf3D7_1343700). Importantly, all of the resistance phenotypes were stable in cloned parasites for more than 2 years without drug pressure. The results demonstrate artemisinin-resistant P. falciparum has evolved a novel mechanism of phenotypic resistance to artemisinin drugs linked to abnormal cell cycle regulation. These results offer insights into a novel mechanism of drug resistance in P. falciparum and new tools for monitoring the spread of artemisinin resistance. PMID:25779582

  9. Immunogenicity of whole-parasite vaccines against Plasmodium falciparum involves malarial hemozoin and host TLR9.

    PubMed

    Coban, Cevayir; Igari, Yoshikatsu; Yagi, Masanori; Reimer, Thornik; Koyama, Shohei; Aoshi, Taiki; Ohata, Keiichi; Tsukui, Toshihiro; Takeshita, Fumihiko; Sakurai, Kazuo; Ikegami, Takahisa; Nakagawa, Atsushi; Horii, Toshihiro; Nuez, Gabriel; Ishii, Ken J; Akira, Shizuo

    2010-01-21

    Although whole-parasite vaccine strategies for malaria infection have regained attention, their immunological mechanisms of action remain unclear. We find that immunization of mice with a crude blood stage extract of the malaria parasite Plasmodium falciparum elicits parasite antigen-specific immune responses via Toll-like receptor (TLR) 9 and that the malarial heme-detoxification byproduct, hemozoin (HZ), but not malarial DNA, produces a potent adjuvant effect. Malarial and synthetic (s)HZ bound TLR9 directly to induce conformational changes in the receptor. The adjuvant effect of sHZ depended on its method of synthesis and particle size. Although natural HZ acts as a TLR9 ligand, the adjuvant effects of synthetic HZ are independent of TLR9 or the NLRP3-inflammasome but are dependent on MyD88. The adjuvant function of sHZ was further validated in a canine antiallergen vaccine model. Thus, HZ can influence adaptive immune responses to malaria infection and may have therapeutic value in vaccine adjuvant development. PMID:20114028

  10. The human malaria parasite Plasmodium falciparum possesses two distinct dihydrolipoamide dehydrogenases.

    PubMed

    McMillan, Paul J; Stimmler, Luciana M; Foth, Bernardo J; McFadden, Geoffrey I; Müller, Sylke

    2005-01-01

    The Plasmodium falciparum genome contains genes encoding three alpha-ketoacid dehydrogenase multienzyme complexes (KADHs) that have central metabolic functions. The parasites possess two distinct genes encoding dihydrolipoamide dehydrogenases (LipDH), which are indispensable subunits of KADHs. This situation is reminiscent of that in plants, where two distinct LipDHs are found in mitochondria and chloroplasts, respectively, that are part of the organelle-specific KADHs. In this study, we show by reverse transcription polymerase chain reaction (RT-PCR) that the genes encoding subunits of all three KADHs, including both LipDHs, are transcribed during the erythrocytic development of P. falciparum. Protein expression of mitochondrial LipDH and mitochondrial branched chain alpha-ketoacid dihydrolipoamide transacylase in these parasite stages was confirmed by Western blotting. The localization of the two LipDHs to the parasite's apicoplast and mitochondrion, respectively, was shown by expressing the LipDH N-terminal presequences fused to green fluorescent protein in erythrocytic stages of P. falciparum and by immunofluorescent colocalization with organelle-specific markers. Biochemical characterization of recombinantly expressed mitochondrial LipDH revealed that the protein has kinetic and physicochemical characteristics typical of these flavo disulphide oxidoreductases. We propose that the mitochondrial LipDH is part of the mitochondrial alpha-ketoglutarate dehydrogenase and branched chain alpha-ketoacid dehydrogenase complexes and that the apicoplast LipDH is an integral part of the pyruvate dehydrogenase complex which occurs only in the apicoplast in P. falciparum. PMID:15612914

  11. A flow cytometric assay to quantify invasion of red blood cells by rodent Plasmodium parasites in vivo

    PubMed Central

    2014-01-01

    Background 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. Methods 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. Results 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. Conclusions 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 alternative method of estimating blood parasitaemia. PMID:24628989

  12. NMR and crystallographic structures of the FK506 binding domain of human malarial parasite Plasmodium vivax FKBP35.

    PubMed

    Alag, Reema; Qureshi, Insaf A; Bharatham, Nagakumar; Shin, Joon; Lescar, Julien; Yoon, Ho Sup

    2010-08-01

    The emergence of drug-resistant malaria parasites is the major threat to effective malaria control, prompting a search for novel compounds with mechanisms of action that are different from the traditionally used drugs. The immunosuppressive drug FK506 shows an antimalarial activity. The mechanism of the drug action involves the molecular interaction with the parasite target proteins PfFKBP35 and PvFKBP35, which are novel FK506 binding protein family (FKBP) members from Plasmodium falciparum and Plasmodium vivax, respectively. Currently, molecular mechanisms of the FKBP family proteins in the parasites still remain elusive. To understand their functions, here we have determined the structures of the FK506 binding domain of Plasmodium vivax (PvFKBD) in unliganded form by NMR spectroscopy and in complex with FK506 by X-ray crystallography. We found out that PvFKBP35 exhibits a canonical FKBD fold and shares kinetic profiles similar to those of PfFKBP35, the homologous protein in P. falciparum, indicating that the parasite FKBP family members play similar biological roles in their life cycles. Despite the similarity, differences were observed in the ligand binding modes between PvFKBD and HsFKBP12, a human FKBP homolog, which could provide insightful information into designing selective antimalarial drug against the parasites. PMID:20572013

  13. Comparative genomics of transcriptional control in the human malaria parasite Plasmodium falciparum.

    PubMed

    Coulson, Richard M R; Hall, Neil; Ouzounis, Christos A

    2004-08-01

    The life cycle of the parasite Plasmodium falciparum, responsible for the most deadly form of human malaria, requires specialized protein expression for survival in the mammalian host and insect vector. To identify components of processes controlling gene expression during its life cycle, the malarial genome--along with seven crown eukaryote group genomes--was queried with a reference set of transcription-associated proteins (TAPs). Following clustering on the basis of sequence similarity of the TAPs with their homologs, and together with hidden Markov model profile searches, 156 P. falciparum TAPs were identified. This represents about a third of the number of TAPs usually found in the genome of a free-living eukaryote. Furthermore, the P. falciparum genome appears to contain a low number of sequences, which are highly conserved and abundant within the kingdoms of free-living eukaryotes, that contribute to gene-specific transcriptional regulation. However, in comparison with these other eukaryotic genomes, the CCCH-type zinc finger (common in proteins modulating mRNA decay and translation rates) was found to be the most abundant in the P. falciparum genome. This observation, together with the paucity of malarial transcriptional regulators identified, suggests Plasmodium protein levels are primarily determined by posttranscriptional mechanisms. PMID:15256513

  14. Human Monoclonal Antibodies to Pf 155, a Major Antigen of Malaria Parasite Plasmodium falciparum

    NASA Astrophysics Data System (ADS)

    Udomsangpetch, Rachanee; Lundgren, Katarina; Berzins, Klavs; Wahlin, Birgitta; Perlmann, Hedvig; Troye-Blomberg, Marita; Carlsson, Jan; Wahlgren, Mats; Perlmann, Peter; Bjorkman, Anders

    1986-01-01

    Pf 155, a protein of the human malaria parasite Plasmodium falciparum, is strongly immunogenic in humans and is believed to be a prime candidate for the preparation of a vaccine. Human monoclonal antibodies to Pf 155 were obtained by cloning B cells that had been prepared from an immune donor and transformed with Epstein-Barr virus. When examined by indirect immunofluorescence, these antibodies stained the surface of infected erythrocytes, free merozoites, segmented schizonts, and gametocytes. They bound to a major polypeptide with a relative molecular weight of 155K and to two minor ones (135K and 120K), all having high affinity for human glycophorin. The antibodies strongly inhibited merozoite reinvasion in vitro, suggesting that they might be appropriate reagents for therapeutic administration in vivo.

  15. Defining the protein interaction network of human malaria parasite Plasmodium falciparum.

    PubMed

    Ramaprasad, Abhinay; Pain, Arnab; Ravasi, Timothy

    2012-02-01

    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. PMID:22178265

  16. Genome-wide mapping of DNA methylation in the human malaria parasite Plasmodium falciparum

    PubMed Central

    Ponts, Nadia; Fu, Lijuan; Harris, Elena Y.; Zhang, Jing; Chung, Duk-Won D.; Cervantes, Michael C.; Prudhomme, Jacques; Atanasova-Penichon, Vessela; Zehraoui, Enric; Bunnik, Evelien; Rodrigues, Elisandra M.; Lonardi, Stefano; Hicks, Glenn R.; Wang, Yinsheng; Le Roch, Karine G.

    2014-01-01

    SUMMARY Cytosine DNA methylation is an epigenetic mark in most eukaryotic cells that regulates numerous processes, including gene expression and stress responses. We performed a genome-wide analysis of DNA methylation in the human malaria parasite Plasmodium falciparum. We mapped the positions of methylated cytosines and identified a single functional DNA methyltransferase, PfDNMT, that may mediate these genomic modifications. These analyses revealed that the malaria genome is asymmetrically methylated, in which only one DNA strand is methylated, and shares common features with undifferentiated plant and mammalian cells. Notably, core promoters are hypomethylated and transcript levels correlate with intra-exonic methylation. Additionally, there are sharp methylation transitions at nucleosome and exon-intron boundaries. These data suggest that DNA methylation could regulate virulence gene expression and transcription elongation. Furthermore, the broad range of action of DNA methylation and uniqueness of PfDNMT suggest that the methylation pathway is a potential target for anti-malarial strategies. PMID:24331467

  17. K13-propeller polymorphisms in Plasmodium falciparum parasites from sub-Saharan Africa.

    PubMed

    Kamau, Edwin; Campino, Susana; Amenga-Etego, Lucas; Drury, Eleanor; Ishengoma, Deus; Johnson, Kimberly; Mumba, Dieudonne; Kekre, Mihir; Yavo, William; Mead, Daniel; Bouyou-Akotet, Marielle; Apinjoh, Tobias; Golassa, Lemu; Randrianarivelojosia, Milijaona; Andagalu, Ben; Maiga-Ascofare, Oumou; Amambua-Ngwa, Alfred; Tindana, Paulina; Ghansah, Anita; MacInnis, Bronwyn; Kwiatkowski, Dominic; Djimde, Abdoulaye A

    2015-04-15

    Mutations in the Plasmodium falciparum K13-propeller domain have recently been shown to be important determinants of artemisinin resistance in Southeast Asia. This study investigated the prevalence of K13-propeller polymorphisms across sub-Saharan Africa. A total of 1212 P. falciparum samples collected from 12 countries were sequenced. None of the K13-propeller mutations previously reported in Southeast Asia were found, but 22 unique mutations were detected, of which 7 were nonsynonymous. Allele frequencies ranged between 1% and 3%. Three mutations were observed in >1 country, and the A578S was present in parasites from 5 countries. This study provides the baseline prevalence of K13-propeller mutations in sub-Saharan Africa. PMID:25367300

  18. Dihydroorotase of human malarial parasite Plasmodium falciparum differs from host enzyme.

    PubMed

    Krungkrai, Sudaratana R; Wutipraditkul, Nuchanat; Krungkrai, Jerapan

    2008-02-15

    Plasmodium falciparum, the causative agent of human malaria, is totally dependent on de novo pyrimidine biosynthetic pathway. A gene encoding P. falciparum dihydroorotase (pfDHOase) was cloned and expressed in Escherichia coli as monofunctional enzyme. PfDHOase revealed a molecular mass of 42kDa. In gel filtration chromatography, the major enzyme activity eluted at 40kDa, indicating that it functions in a monomeric form. This was similarly observed using the native enzyme purified from P. falciparum. Interestingly, kinetic parameters of the enzyme and inhibitory effect by orotate and its 5-substituted derivatives parallel that found in mammalian type I DHOase. Thus, the malarial enzyme shares characteristics of both type I and type II DHOases. This study provides the monofunctional property of the parasite DHOase lending further insights into its differences from the human enzyme which forms part of a multifunctional protein. PMID:18082626

  19. Optimized Pan-species and Speciation Duplex Real-time PCR Assays for Plasmodium Parasites Detection in Malaria Vectors

    PubMed Central

    Sandeu, Maurice Marcel; Moussiliou, Azizath; Moiroux, Nicolas; Padonou, Gilles G.; Massougbodji, Achille; Corbel, Vincent; Tuikue Ndam, Nicaise

    2012-01-01

    Background An accurate method for detecting malaria parasites in the mosquitos vector remains an essential component in the vector control. The Enzyme linked immunosorbent assay specific for circumsporozoite protein (ELISA-CSP) is the gold standard method for the detection of malaria parasites in the vector even if it presents some limitations. Here, we optimized multiplex real-time PCR assays to accurately detect minor populations in mixed infection with multiple Plasmodium species in the African malaria vectors Anopheles gambiae and Anopheles funestus. Methods Complementary TaqMan-based real-time PCR assays that detect Plasmodium species using specific primers and probes were first evaluated on artificial mixtures of different targets inserted in plasmid constructs. The assays were further validated in comparison with the ELISA-CSP on 200 field caught Anopheles gambiae and Anopheles funestus mosquitoes collected in two localities in southern Benin. Results The validation of the duplex real-time PCR assays on the plasmid mixtures demonstrated robust specificity and sensitivity for detecting distinct targets. Using a panel of mosquito specimen, the real-time PCR showed a relatively high sensitivity (88.6%) and specificity (98%), compared to ELISA-CSP as the referent standard. The agreement between both methods was excellent (??=?0.8, P<0.05). The relative quantification of Plasmodium DNA between the two Anopheles species analyzed showed no significant difference (P?=?0, 2). All infected mosquito samples contained Plasmodium falciparum DNA and mixed infections with P. malariae and/or P. ovale were observed in 18.6% and 13.6% of An. gambiae and An. funestus respectively. Plasmodium vivax was found in none of the mosquito samples analyzed. Conclusion This study presents an optimized method for detecting the four Plasmodium species in the African malaria vectors. The study highlights substantial discordance with traditional ELISA-CSP pointing out the utility of employing an accurate molecular diagnostic tool for detecting malaria parasites in field mosquito populations. PMID:23285168

  20. Cloning, expression and functional characterization of heme detoxification protein (HDP) from the rodent malaria parasite Plasmodium vinckei.

    PubMed

    Soni, Awakash; Goyal, Manish; Prakash, Kirtika; Bhardwaj, Jyoti; Siddiqui, Arif Jamal; Puri, Sunil K

    2015-07-15

    Malaria parasite resides within the host red blood cells, where it degrades vast amount of haemoglobin. During haemoglobin degradation, toxic free heme is liberated which subsequently gets converted into hemozoin. This process is facilitated by action of various proteins viz. heme detoxification protein (HDP), and histidine rich proteins II and III (HRP II & III). Out of these, HDP is the most potent in hemozoin formation and plays indispensible role for parasite survival. Despite this, the detailed study of HDP from rodent and simian parasite has not been performed till date. Here, we have cloned and sequenced hdp gene from different malaria parasites Plasmodium vinckei, Plasmodium yoelii, Plasmodium knowlesi, and Plasmodium cynomolgi. Furthermore, HDP from P. vinckei (PvHDP) was over-expressed and purified for detailed characterization. The PvHDP is cytosolic, expressed throughout the intra erythrocytic stages and its expression is higher in late trophozoite and schizont stages of parasite. The PvHDP interacts with free heme (KD=89 nM) and efficiently converts heme into hemozoin in a time and concentration dependent manner. Moreover, PvHDP showed activity in acidic pH and over a broad range of temperature. Histidine modification of PvHDP using DEPC showed reduction in heme binding and hemozoin formation, thus emphasizing the importance of histidine residues in heme binding and subsequent hemozoin production. Furthermore, applicability of PvHDP to screen anti-plasmodial agents (targeting heme to hemozoin conversion) was also determined using chloroquine, and mefloquine as reference antimalarials. Results showed that these drugs inhibit heme polymerization effectively in a concentration dependent manner. In conclusion, our study identified and biochemically characterized HDP from rodent malaria parasite P. vinckei and this will help to develop a high throughput assay to evaluate new antimalarials targeting hemozoin pathway. PMID:25891072

  1. Extensive lysine acetylation occurs in evolutionarily conserved metabolic pathways and parasite-specific functions during Plasmodium falciparum intraerythrocytic development

    PubMed Central

    Miao, Jun; Lawrence, Matthew; Jeffers, Victoria; Zhao, Fangqing; Parker, Daniel; Ge, Ying; Sullivan, William J.; Cui, Liwang

    2013-01-01

    Summary Lysine acetylation has emerged as a major posttranslational modification involved in diverse cellular functions. Using a combination of immunoisolation and liquid chromatography coupled to accurate mass spectrometry, we determined the first acetylome of the human malaria parasite Plasmodium falciparum during its active proliferation in erythrocytes with 421 acetylation sites identified in 230 proteins. Lysine-acetylated proteins are distributed in the nucleus, cytoplasm, mitochondrion, and apicoplast. Whereas occurrence of lysine acetylation in a similarly wide range of cellular functions suggests conservation of lysine acetylation through evolution, the Plasmodium acetylome also revealed significant divergence from those of other eukaryotes and even the closely-related parasite Toxoplasma. This divergence is reflected in the acetylation of a large number of Plasmodium-specific proteins and different acetylation sites in evolutionarily conserved acetylated proteins. A prominent example is the abundant acetylation of proteins in the glycolysis pathway but relatively deficient acetylation of enzymes in the citrate cycle. Using specific transgenic lines and inhibitors, we determined that the acetyltransferase PfMYST and lysine deacetylases play important roles in regulating the dynamics of cytoplasmic protein acetylation. The Plasmodium acetylome provides an exciting start point for further exploration of functions of acetylation in the biology of malaria parasites. PMID:23796209

  2. A Next-generation Genetically Attenuated Plasmodium falciparum Parasite Created by Triple Gene Deletion

    PubMed Central

    Mikolajczak, Sebastian A; Lakshmanan, Viswanathan; Fishbaugher, Matthew; Camargo, Nelly; Harupa, Anke; Kaushansky, Alexis; Douglass, Alyse N; Baldwin, Michael; Healer, Julie; O'Neill, Matthew; Phuong, Thuan; Cowman, Alan; Kappe, Stefan H I

    2014-01-01

    Immunization with live-attenuated Plasmodium sporozoites completely protects against malaria infection. Genetic engineering offers a versatile platform to create live-attenuated sporozoite vaccine candidates. We previously generated a genetically attenuated parasite (GAP) by deleting the P52 and P36 genes in the NF54 wild-type (WT) strain of Plasmodium falciparum (Pf p52−/p36− GAP). Preclinical assessment of p52−/p36− GAP in a humanized mouse model indicated an early and severe liver stage growth defect. However, human exposure to >200 Pf p52−/p36− GAP-infected mosquito bites in a safety trial resulted in peripheral parasitemia in one of six volunteers, revealing that this GAP was incompletely attenuated. We have now created a triple gene deleted GAP by additionally removing the SAP1 gene (Pf p52−/p36−/sap1− GAP) and employed flippase (FLP)/flippase recognition target (FRT) recombination for drug selectable marker cassette removal. This next-generation GAP was indistinguishable from WT parasites in blood stage and mosquito stage development. Using an improved humanized mouse model transplanted with human hepatocytes and human red blood cells, we show that despite a high-dose sporozoite challenge, Pf p52−/p36−/sap1− GAP did not transition to blood stage infection and appeared to be completely attenuated. Thus, clinical testing of Pf p52−/p36−/sap1− GAP assessing safety, immunogenicity, and efficacy against sporozoite challenge is warranted. PMID:24827907

  3. Real-Time Imaging of the Intracellular Glutathione Redox Potential in the Malaria Parasite Plasmodium falciparum

    PubMed Central

    Kasozi, Denis; Mohring, Franziska; Rahlfs, Stefan; Meyer, Andreas J.; Becker, Katja

    2013-01-01

    In the malaria parasite Plasmodium falciparum, the cellular redox potential influences signaling events, antioxidant defense, and mechanisms of drug action and resistance. Until now, the real-time determination of the redox potential in malaria parasites has been limited because conventional approaches disrupt sub-cellular integrity. Using a glutathione biosensor comprising human glutaredoxin-1 linked to a redox-sensitive green fluorescent protein (hGrx1-roGFP2), we systematically characterized basal values and drug-induced changes in the cytosolic glutathione-dependent redox potential (EGSH) of drug-sensitive (3D7) and resistant (Dd2) P. falciparum parasites. Via confocal microscopy, we demonstrated that hGrx1-roGFP2 rapidly detects EGSH changes induced by oxidative and nitrosative stress. The cytosolic basal EGSH of 3D7 and Dd2 were estimated to be −314.2±3.1 mV and −313.9±3.4 mV, respectively, which is indicative of a highly reducing compartment. We furthermore monitored short-, medium-, and long-term changes in EGSH after incubation with various redox-active compounds and antimalarial drugs. Interestingly, the redox cyclers methylene blue and pyocyanin rapidly changed the fluorescence ratio of hGrx1-roGFP2 in the cytosol of P. falciparum, which can, however, partially be explained by a direct interaction with the probe. In contrast, quinoline and artemisinin-based antimalarial drugs showed strong effects on the parasites' EGSH after longer incubation times (24 h). As tested for various conditions, these effects were accompanied by a drop in total glutathione concentrations determined in parallel with alternative methods. Notably, the effects were generally more pronounced in the chloroquine-sensitive 3D7 strain than in the resistant Dd2 strain. Based on these results hGrx1-roGFP2 can be recommended as a reliable and specific biosensor for real-time spatiotemporal monitoring of the intracellular EGSH in P. falciparum. Applying this technique in further studies will enhance our understanding of redox regulation and mechanisms of drug action and resistance in Plasmodium and might also stimulate redox research in other pathogens. PMID:24348249

  4. Natural infection of Plasmodium brasilianum in humans: Man and monkey share quartan malaria parasites in the Venezuelan Amazon

    PubMed Central

    Lalremruata, Albert; Magris, Magda; Vivas-Martínez, Sarai; Koehler, Maike; Esen, Meral; Kempaiah, Prakasha; Jeyaraj, Sankarganesh; Perkins, Douglas Jay; Mordmüller, Benjamin; Metzger, Wolfram G.

    2015-01-01

    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

  5. Use of Malachite Green-Loop Mediated Isothermal Amplification for Detection of Plasmodium spp. Parasites

    PubMed Central

    Lucchi, Naomi W.; Ljolje, Dragan; Silva-Flannery, Luciana; Udhayakumar, Venkatachalam

    2016-01-01

    Malaria elimination efforts are hampered by the lack of sensitive tools to detect infections with low-level parasitemia, usually below the threshold of standard diagnostic methods, microscopy and rapid diagnostic tests. 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 run the test. However, the use of specialized equipment, as described by many groups, reduces the versatility of the LAMP technique as a simple tool for use in endemic countries. In this study, the use of the malachite green (MG) dye, as a visual endpoint readout, together with a simple mini heat block was evaluated for the detection of malaria parasites. The assay was performed for 1 hour at 63°C and the results scored by 3 independent human readers. The limit of detection of the assay was determined using well-quantified Plasmodium spp. infected reference samples and its utility in testing clinical samples was determined using 190 pre-treatment specimens submitted for reference diagnosis of imported malaria in the United States. Use of a simplified boil and spin methods of DNA extraction from whole blood and filter paper was also investigated. We demonstrate the accurate and sensitive detection of malaria parasites using this assay with a detection limit ranging between 1–8 parasites/μL, supporting its applicability for the detection of infections with low parasite burden. This assay is compatible with the use of a simple boil and spin sample preparation method from both whole blood and filter papers without a loss of sensitivity. The MG-LAMP assay described here has great potential to extend the reach of molecular tools to settings where they are needed. PMID:26967908

  6. Use of Malachite Green-Loop Mediated Isothermal Amplification for Detection of Plasmodium spp. Parasites.

    PubMed

    Lucchi, Naomi W; Ljolje, Dragan; Silva-Flannery, Luciana; Udhayakumar, Venkatachalam

    2016-01-01

    Malaria elimination efforts are hampered by the lack of sensitive tools to detect infections with low-level parasitemia, usually below the threshold of standard diagnostic methods, microscopy and rapid diagnostic tests. 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 run the test. However, the use of specialized equipment, as described by many groups, reduces the versatility of the LAMP technique as a simple tool for use in endemic countries. In this study, the use of the malachite green (MG) dye, as a visual endpoint readout, together with a simple mini heat block was evaluated for the detection of malaria parasites. The assay was performed for 1 hour at 63°C and the results scored by 3 independent human readers. The limit of detection of the assay was determined using well-quantified Plasmodium spp. infected reference samples and its utility in testing clinical samples was determined using 190 pre-treatment specimens submitted for reference diagnosis of imported malaria in the United States. Use of a simplified boil and spin methods of DNA extraction from whole blood and filter paper was also investigated. We demonstrate the accurate and sensitive detection of malaria parasites using this assay with a detection limit ranging between 1-8 parasites/μL, supporting its applicability for the detection of infections with low parasite burden. This assay is compatible with the use of a simple boil and spin sample preparation method from both whole blood and filter papers without a loss of sensitivity. The MG-LAMP assay described here has great potential to extend the reach of molecular tools to settings where they are needed. PMID:26967908

  7. Development of the piggyBac transposable system for Plasmodium berghei and its application for random mutagenesis in malaria parasites

    PubMed Central

    2011-01-01

    Background The genome of a number of species of malaria parasites (Plasmodium spp.) has been sequenced in the hope of identifying new drug and vaccine targets. However, almost one-half of predicted Plasmodium genes are annotated as hypothetical and are difficult to analyse in bulk due to the inefficiency of current reverse genetic methodologies for Plasmodium. Recently, it has been shown that the transposase piggyBac integrates at random into the genome of the human malaria parasite P. falciparum offering the possibility to develop forward genetic screens to analyse Plasmodium gene function. This study reports the development and application of the piggyBac transposition system for the rodent malaria parasite P. berghei and the evaluation of its potential as a tool in forward genetic studies. P. berghei is the most frequently used malaria parasite model in gene function analysis since phenotype screens throughout the complete Plasmodium life cycle are possible both in vitro and in vivo. Results We demonstrate that piggyBac based gene inactivation and promoter-trapping is both easier and more efficient in P. berghei than in the human malaria parasite, P. falciparum. Random piggyBac-mediated insertion into genes was achieved after parasites were transfected with the piggyBac donor plasmid either when transposase was expressed either from a helper plasmid or a stably integrated gene in the genome. Characterization of more than 120 insertion sites demonstrated that more than 70 most likely affect gene expression classifying their protein products as non-essential for asexual blood stage development. The non-essential nature of two of these genes was confirmed by targeted gene deletion one of which encodes P41, an ortholog of a human malaria vaccine candidate. Importantly for future development of whole genome phenotypic screens the remobilization of the piggyBac element in parasites that stably express transposase was demonstrated. Conclusion These data demonstrate that piggyBac behaved as an efficient and random transposon in P. berghei. Remobilization of piggyBac element shows that with further development the piggyBac system can be an effective tool to generate random genome-wide mutation parasite libraries, for use in large-scale phenotype screens in vitro and in vivo. PMID:21418605

  8. Plasmodium falciparum parasites lacking histidine-rich protein 2 and 3: a review and recommendations for accurate reporting

    PubMed Central

    2014-01-01

    Malaria rapid diagnostic tests (RDTs) play a critical role in malaria case management, surveillance and case investigations. Test performance is largely determined by design and quality characteristics, such as detection sensitivity, specificity, and thermal stability. However, parasite characteristics such as variable or absent expression of antigens targeted by RDTs can also affect RDT performance. Plasmodium falciparum parasites lacking the PfHRP2 protein, the most common target antigen for detection of P. falciparum, have been reported in some regions. Therefore, accurately mapping the presence and prevalence of P. falciparum parasites lacking pfhrp2 would be an important step so that RDTs targeting alternative antigens, or microscopy, can be preferentially selected for use in such regions. Herein the available evidence and molecular basis for identifying malaria parasites lacking PfHRP2 is reviewed, and a set of recommended procedures to apply for future investigations for parasites lacking PfHRP2, is proposed. PMID:25052298

  9. Processing of Plasmodium falciparum Merozoite Surface Protein MSP1 Activates a Spectrin-Binding Function Enabling Parasite Egress from RBCs

    PubMed Central

    Das, Sujaan; Hertrich, Nadine; Perrin, Abigail J.; Withers-Martinez, Chrislaine; Collins, Christine R.; Jones, Matthew L.; Watermeyer, Jean M.; Fobes, Elmar T.; Martin, Stephen R.; Saibil, Helen R.; Wright, Gavin J.; Treeck, Moritz; Epp, Christian; Blackman, Michael J.

    2015-01-01

    Summary The malaria parasite Plasmodium falciparum replicates within erythrocytes, producing progeny merozoites that are released from infected cells via a poorly understood process called egress. The most abundant merozoite surface protein, MSP1, is synthesized as a large precursor that undergoes proteolytic maturation by the parasite protease SUB1 just prior to egress. The function of MSP1 and its processing are unknown. Here we show that SUB1-mediated processing of MSP1 is important for parasite viability. Processing modifies the secondary structure of MSP1 and activates its capacity to bind spectrin, a molecular scaffold protein that is the major component of the host erythrocyte cytoskeleton. Parasites expressing an inefficiently processed MSP1 mutant show delayed egress, and merozoites lacking surface-bound MSP1 display a severe egress defect. Our results indicate that interactions between SUB1-processed merozoite surface MSP1 and the spectrin network of the erythrocyte cytoskeleton facilitate host erythrocyte rupture to enable parasite egress. PMID:26468747

  10. Within-host competition and drug resistance in the human malaria parasite Plasmodium falciparum.

    PubMed

    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

    2016-03-16

    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. PMID:26984625

  11. Clonal reproduction shapes evolution in the lizard malaria parasite Plasmodium floridense.

    PubMed

    Falk, Bryan G; Glor, Richard E; Perkins, Susan L

    2015-06-01

    The preponderant clonal evolution hypothesis (PCE) predicts that frequent clonal reproduction (sex between two clones) in many pathogens capable of sexual recombination results in strong linkage disequilibrium and the presence of discrete genetic subdivisions characterized by occasional gene flow. We expand on the PCE and predict that higher rates of clonal reproduction will result in: (1) morphologically cryptic species that exhibit (2) low within-species variation and (3) recent between-species divergence. We tested these predictions in the Caribbean lizard malaria parasite Plasmodium floridense using 63 single-infection samples in lizards collected from across the parasite's range, and sequenced them at two mitochondrial, one apicoplast, and five nuclear genes. We identified 11 provisionally cryptic species within P. floridense, each of which exhibits low intraspecific variation and recent divergence times between species (some diverged approximately 110,000 years ago). Our results are consistent with the hypothesis that clonal reproduction can profoundly affect diversification of species capable of sexual recombination, and suggest that clonal reproduction may have led to a large number of unrecognized pathogen species. The factors that may influence the rates of clonal reproduction among pathogens are unclear, and we discuss how prevalence and virulence may relate to clonal reproduction. PMID:25959003

  12. Plasmodium falciparum: gp195 tripeptide repeat-specific monoclonal antibody inhibits parasite growth in vitro.

    PubMed

    Locher, C P; Tam, L Q; Chang, S P; McBride, J S; Siddiqui, W A

    1996-10-01

    Seven monoclonal antibodies (mAbs) were produced to the precursor of the merozoite surface antigens (MSA-1 or gp195) using the Plasmodium falciparum Uganda-Palo Alto isolate. Three mAbs (CE2, DB8, and EB2) reacted with epitopes on the 83-kDa N-terminal processing fragment by immunoprecipitation of radiolabeled proteins and in immunoblots of native and recombinant proteins. Three other mAbs (BC9, AG5, and AD9) reacted with epitopes on the 42- and 19-kDa C-terminal processing fragments while one remaining mAb (24A1.7) reacted with only 150- and 110-kDa intermediate processing fragments. Epitopes were mapped to either conserved or dimorphic regions of the expressed protein when parasite isolates with known MSA-1 alleles were examined by indirect immunofluorescence. Moreover, one mAb (CE2), specific for the variable tripeptide repeat region SAQ(SGT)5, was growth inhibitory for P. falciparum in vitro. Growth inhibition by the mAb was concentration dependent and its parasite-neutralizing properties were not enhanced when used in combination with other gp195-specific mAbs. These results may be useful in the elucidation of biological variation of field isolates and in the definition of immunologically relevant epitopes in a gp195-based malaria vaccine. PMID:8888734

  13. Crystal structure of the aquaglyceroporin PfAQP from the malarial parasite Plasmodium falciparum.

    PubMed

    Newby, Zachary E R; O'Connell, Joseph; Robles-Colmenares, Yaneth; Khademi, Shahram; Miercke, Larry J; Stroud, Robert M

    2008-06-01

    The 2.05-A resolution structure of the aquaglyceroporin from the malarial parasite Plasmodium falciparum (PfAQP), a protein important in the parasite's life cycle, has been solved. The structure provides key evidence for the basis of water versus glycerol selectivity in aquaporin family members. Unlike its closest homolog of known structure, GlpF, the channel conducts both glycerol and water at high rates, framing the question of what determines high water conductance in aquaporin channels. The universally conserved arginine in the selectivity filter is constrained by only two hydrogen bonds in GlpF, whereas there are three in all water-selective aquaporins and in PfAQP. The decreased cost of dehydrating the triply-satisfied arginine cation may provide the basis for high water conductance. The two Asn-Pro-Ala (NPA) regions of PfAQP, which bear rare substitutions to Asn-Leu-Ala (NLA) and Asn-Pro-Ser (NPS), participate in preserving the orientation of the selectivity filter asparagines in the center of the channel. PMID:18500352

  14. Select pyrimidinones inhibit the propagation of the malarial parasite, Plasmodium falciparum.

    PubMed

    Chiang, Annette N; Valderramos, Juan-Carlos; Balachandran, Raghavan; Chovatiya, Raj J; Mead, Brian P; Schneider, Corinne; Bell, Samantha L; Klein, Michael G; Huryn, Donna M; Chen, Xiaojiang S; Day, Billy W; Fidock, David A; Wipf, Peter; Brodsky, Jeffrey L

    2009-02-15

    Plasmodium falciparum, the Apicomplexan parasite that is responsible for the most lethal forms of human malaria, is exposed to radically different environments and stress factors during its complex lifecycle. In any organism, Hsp70 chaperones are typically associated with tolerance to stress. We therefore reasoned that inhibition of P. falciparum Hsp70 chaperones would adversely affect parasite homeostasis. To test this hypothesis, we measured whether pyrimidinone-amides, a new class of Hsp70 modulators, could inhibit the replication of the pathogenic P. falciparum stages in human red blood cells. Nine compounds with IC(50) values from 30 nM to 1.6 micrOM were identified. Each compound also altered the ATPase activity of purified P. falciparum Hsp70 in single-turnover assays, although higher concentrations of agents were required than was necessary to inhibit P. falciparum replication. Varying effects of these compounds on Hsp70s from other organisms were also observed. Together, our data indicate that pyrimidinone-amides constitute a novel class of anti-malarial agents. PMID:19195901

  15. Mitochondrial Membrane Potential in a Small Subset of Artemisinin-Induced Dormant Plasmodium falciparum Parasites In Vitro.

    PubMed

    Peatey, Christopher L; Chavchich, Marina; Chen, Nanhua; Gresty, Karryn J; Gray, Karen-Ann; Gatton, Michelle L; Waters, Norman C; Cheng, Qin

    2015-08-01

    Artemisinin-induced dormancy is a proposed mechanism for failures of monotherapy and is linked with artemisinin resistance in Plasmodium falciparum. The biological characterization and dynamics of dormant parasites are not well understood. Here we report that after dihydroartemisinin treatment in vitro, a small subset of morphologically dormant parasites was stained with rhodamine 123 (RH), a mitochondrial membrane potential marker, and persisted to recovery. RH-positive parasites sorted with fluorescence-activated cell sorting resumed growth at 10,000/well whereas RH-negative parasites failed to recover at 5 million/well. Furthermore, transcriptional activity for mitochondrial enzymes was detected only in RH-positive dormant parasites. Importantly, after treatment of dormant parasites with different concentrations of atovaquone, a mitochondrial inhibitor, the recovery of dormant parasites was delayed or stopped. This demonstrates that mitochondrial activity is critical for survival and regrowth of dormant parasites and that RH staining provides a means of identifying these parasites. These findings provide novel paths for studying and eradicating this dormant stage. PMID:25635122

  16. Humoral immune responses to a recombinant Plasmodium vivax tryptophan-rich antigen among Plasmodium vivax-infected patients and its localization in the parasite.

    PubMed

    Siddiqui, Asim A; Khan, Fozia; Sharma, Yagya D

    2015-02-01

    Our recent studies have focused on the identification and characterization of the tryptophan-rich proteins of the Plasmodium vivax parasite where their role in the elicitation of humoral and cellular responses and erythrocyte-binding activity was investigated. Here, we report the humoral responses of a 32.4-kDa P. vivax tryptophan-rich antigen (PvTRAg32.4) among the sera of P. vivax-infected patients. PvTRAg32.4 also contains an unusually high percentage of tryptophan residues (10.7 %) that are positionally conserved with its orthologues in Plasmodium yoelii (PypAg1 and PypAg2) and Plasmodium falciparum (PfTryThrA and PfMATRA). Thirty-four of the 40 (85.0 %) P. vivax isolates showed seropositivity to recombinant PvTRAg32.4 by ELISA. The mean??SD values of optical density (OD) for P. vivax subjects and nave individuals were 1.02??0.36 and 0.26??0.11, respectively. In the Western blot analysis, majority of the subjects studied (n?=?44) showed reactivity to the recombinant, purified PvTRAg32.4. This antigen does not show binding to the erythrocytes, but the immunofluorescence data reveals that it is expressed in the erythrocytic stages of the parasite. Sequence analysis of the clinical isolates from various parts of the country shows that PvTRAg32.4 is highly conserved. Functional in-depth characterization of more such type of novel proteins in the parasite is warranted for the development of successful malaria intervention methods. PMID:25467946

  17. In silico multiple-targets identification for heme detoxification in the human malaria parasite Plasmodium falciparum.

    PubMed

    Phaiphinit, Suthat; Pattaradilokrat, Sittiporn; Lursinsap, Chidchanok; Plaimas, Kitiporn

    2016-01-01

    Detoxification of hemoglobin byproducts or free heme is an essential step and considered potential targets for anti-malaria drug development. However, most of anti-malaria drugs are no longer effective due to the emergence and spread of the drug resistant malaria parasites. Therefore, it is an urgent need to identify potential new targets and even for target combinations for effective malaria drug design. In this work, we reconstructed the metabolic networks of Plasmodium falciparum and human red blood cells for the simulation of steady mass and flux flows of the parasite's metabolites under the blood environment by flux balance analysis (FBA). The integrated model, namely iPF-RBC-713, was then adjusted into two stage-specific metabolic models, which first was for the pathological stage metabolic model of the parasite when invaded the red blood cell without any treatment and second was for the treatment stage of the parasite when a drug acted by inhibiting the hemozoin formation and caused high production rate of heme toxicity. The process of identifying target combinations consisted of two main steps. Firstly, the optimal fluxes of reactions in both the pathological and treatment stages were computed and compared to determine the change of fluxes. Corresponding enzymes of the reactions with zero fluxes in the treatment stage but non-zero fluxes in the pathological stage were predicted as a preliminary list of potential targets in inhibiting heme detoxification. Secondly, the combinations of all possible targets listed in the first step were examined to search for the best promising target combinations resulting in more effective inhibition of the detoxification to kill the malaria parasites. Finally, twenty-three enzymes were identified as a preliminary list of candidate targets which mostly were in pyruvate metabolism and citrate cycle. The optimal set of multiple targets for blocking the detoxification was a set of heme ligase, adenosine transporter, myo-inositol 1-phosphate synthase, ferrodoxim reductase-like protein and guanine transporter. In conclusion, the method has shown an effective and efficient way to identify target combinations which are obviously useful in the development of novel antimalarial drug combinations. PMID:26626103

  18. Immune characterization of Plasmodium falciparum parasites with a shared genetic signature in a region of decreasing transmission.

    PubMed

    Bei, Amy K; Diouf, Ababacar; Miura, Kazutoyo; Larremore, Daniel B; Ribacke, Ulf; Tullo, Gregory; Moss, Eli L; Neafsey, Daniel E; Daniels, Rachel F; Zeituni, Amir E; Nosamiefan, Iguosadolo; Volkman, Sarah K; Ahouidi, Ambroise D; Ndiaye, Daouda; Dieye, Tandakha; Mboup, Souleymane; Buckee, Caroline O; Long, Carole A; Wirth, Dyann F

    2015-01-01

    As the intensity of malaria transmission has declined, Plasmodium falciparum parasite populations have displayed decreased clonal diversity resulting from the emergence of many parasites with common genetic signatures (CGS). We have monitored such CGS parasite clusters from 2006 to 2013 in This, Senegal, using the molecular barcode. The first, and one of the largest observed clusters of CGS parasites, was present in 24% of clinical isolates in 2008, declined to 3.4% of clinical isolates in 2009, and then disappeared. To begin to explore the relationship between the immune responses of the population and the emergence and decline of specific parasite genotypes, we have determined whether antibodies to CGS parasites correlate with their prevalence. We measured (i) antibodies capable of inhibiting parasite growth in culture and (ii) antibodies recognizing the surfaces of infected erythrocytes (RBCs). IgG obtained from volunteers in 2009 showed increased reactivity to the surfaces of CGS-parasitized erythrocytes over IgG from 2008. Since P. falciparum EMP-1 (PfEMP-1) is a major variant surface antigen, we used var Ups quantitative reverse transcription-PCR (qRT-PCR) and sequencing with degenerate DBL1? domain primers to characterize the var genes expressed by CGS parasites after short-term in vitro culture. CGS parasites show upregulation of UpsA var genes and 2-cysteine-containing PfEMP-1 molecules and express the same dominant var transcript. Our work indicates that the CGS parasites in this cluster express similar var genes, more than would be expected by chance in the population, and that there is year-to-year variation in immune recognition of surface antigens on CGS parasite-infected erythrocytes. This study lays the groundwork for detailed investigations of the mechanisms driving the expansion or contraction of specific parasite clones in the population. PMID:25368109

  19. Monoclonal antibodies to stage-specific, species-specific, and cross-reactive antigens of the rodent malarial parasite, Plasmodium yoelii.

    PubMed Central

    Taylor, D W; Kim, K J; Munoz, P A; Evans, C B; Asofsky, R

    1981-01-01

    Eighteen hybridoma cell lines were used to study species-specific, stage-specific, and serological cross-reactive antigens of the rodent malarial parasite, Plasmodium yoelii. Specificity and location of plasmodial antigens were determined by indirect fluorescent-antibody analysis. Results showed that a minimum of 12 distinct plasmodial antigens could be distinguished by the 18 hybridomas. Antigens were found on the surface or within the cytoplasm of the parasite, but not on the surface of erythrocytes from infected animals. The majority (11 of 12) of antigens were present in all erythrocytic stages of the parasite, but one was stage-specific for merozoites. Additional studies showed that 6 of 18 of the monoclonal antibodies identified species-specific antigens, 2 of 18 recognized antigens confined to related rodent malarial parasites (Plasmodium berghei, Plasmodium vinckei, and Plasmodium chabaudi), whereas 8 of 18 detected cross-reactive antigens common to rodent, primate (Plasmodium knowlesi, Plasmodium falciparum), and avian (Plasmodium gallinaceum) malarias. Images PMID:6166558

  20. Novel S-adenosyl-L-methionine decarboxylase inhibitors as potent antiproliferative agents against intraerythrocytic Plasmodium falciparum parasites?

    PubMed Central

    le Roux, Dina; Burger, Pieter B.; Niemand, Jandeli; Grobler, Anne; Urbn, Patricia; Fernndez-Busquets, Xavier; Barker, Robert H.; Serrano, Adelfa E.; I. Louw, Abraham; Birkholtz, Lyn-Marie

    2013-01-01

    S-adenosyl-l-methionine decarboxylase (AdoMetDC) in the polyamine biosynthesis pathway has been identified as a suitable drug target in Plasmodium falciparum parasites, which causes the most lethal form of malaria. Derivatives of an irreversible inhibitor of this enzyme, 5?-{[(Z)-4-amino-2-butenyl]methylamino}-5?-deoxyadenosine (MDL73811), have been developed with improved pharmacokinetic profiles and activity against related parasites, Trypanosoma brucei. Here, these derivatives were assayed for inhibition of AdoMetDC from P. falciparum parasites and the methylated derivative, 8-methyl-5?-{[(Z)-4-aminobut-2-enyl]methylamino}-5?-deoxyadenosine (Genz-644131) was shown to be the most active. The in vitro efficacy of Genz-644131 was markedly increased by nanoencapsulation in immunoliposomes, which specifically targeted intraerythrocytic P. falciparum parasites. PMID:24596666

  1. In Vitro Plasmodium falciparum Drug Sensitivity Assay: Inhibition of Parasite Growth by Incorporation of Stomatocytogenic Amphiphiles into the Erythrocyte Membrane

    PubMed Central

    Ziegler, Hanne L.; Stærk, Dan; Christensen, Jette; Hviid, Lars; Hägerstrand, Henry; Jaroszewski, Jerzy W.

    2002-01-01

    Lupeol, which shows in vitro inhibitory activity against Plasmodium falciparum 3D7 strain with a 50% inhibitory concentration (IC50) of 27.7 ± 0.5 μM, was shown to cause a transformation of the human erythrocyte shape toward that of stomatocytes. Good correlation between the IC50 value and the membrane curvature changes caused by lupeol was observed. Preincubation of erythrocytes with lupeol, followed by extensive washing, made the cells unsuitable for parasite growth, suggesting that the compound incorporates into erythrocyte membrane irreversibly. On the other hand, lupeol-treated parasite culture continued to grow well in untreated erythrocytes. Thus, the antiplasmodial activity of lupeol appears to be indirect, being due to stomatocytic transformation of the host cell membrane and not to toxic effects via action on a drug target within the parasite. A number of amphiphiles that cause stomatocyte formation, but not those causing echinocyte formation, were shown to inhibit growth of the parasites, apparently via a mechanism similar to that of lupeol. Since antiplasmodial agents that inhibit parasite growth through erythrocyte membrane modifications must be regarded as unsuitable as leads for development of new antimalarial drugs, care must be exercised in the interpretation of results of screening of plant extracts and natural product libraries by an in vitro Plasmodium toxicity assay. PMID:11959580

  2. Genetic variability and population structure of Plasmodium falciparum parasite populations from different malaria ecological regions of Kenya.

    PubMed

    Ingasia, Luicer A; Cheruiyot, Jelagat; Okoth, Sheila Akinyi; Andagalu, Ben; Kamau, Edwin

    2016-04-01

    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 introduction of the artemether-lumefantrine is important in refining the spread of drug resistant strains and malaria transmission for more effective control and eventual elimination of malaria in Kenya. PMID:26472129

  3. The avian malaria parasite Plasmodium gallinaceum causes marked structural changes on the surface of its host erythrocyte

    PubMed Central

    Nagao, Eriko; Arie, Takayuki; Dorward, David W.; Fairhurst, Rick M.; Dvorak, James A.

    2008-01-01

    Using a combination of atomic force, scanning and transmission electron microscopy, we found that avian erythrocytes infected with the avian malaria parasite Plasmodium gallinaceum develop ~60 nm wide and ~430 nm long furrow-like structures on the surface. Furrows begin to appear during the early trophozoite stage of the parasites development. They remain constant in size and density during the course of parasite maturation and are uniformly distributed in random orientations over the erythrocyte surface. In addition, the density of furrows is directly proportional to the number of parasites contained within the erythrocyte. These findings suggest that parasite-induced intraerythrocytic processes are involved in modifying the surface of host erythrocytes. These processes may be analogous to those of the human malaria parasite P. falciparum, which induces knob-like protrusions that mediate the pathogenic adherence of parasitized erythrocytes to microvessels. Although P. gallinaceum-infected erythrocytes do not seem to adhere to microvessels in the host chicken, the furrows might be involved in the pathogenesis of P. gallinaceum infections by some other mechanism involving host-pathogen interactions. PMID:18442920

  4. A more appropriate white blood cell count for estimating malaria parasite density in Plasmodium vivax patients in northeastern Myanmar.

    PubMed

    Liu, Huaie; Feng, Guohua; Zeng, Weilin; Li, Xiaomei; Bai, Yao; Deng, Shuang; Ruan, Yonghua; Morris, James; Li, Siman; Yang, Zhaoqing; Cui, Liwang

    2016-04-01

    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. PMID:26802490

  5. Novel antimalarial aminoquinolines: heme binding and effects on normal or Plasmodium falciparum-parasitized human erythrocytes.

    PubMed

    Omodeo-Sal, Fausta; Cortelezzi, Lucia; Basilico, Nicoletta; Casagrande, Manolo; Sparatore, Anna; Taramelli, Donatella

    2009-10-01

    Two new quinolizidinyl-alkyl derivatives of 7-chloro-4-aminoquinoline, named AM-1 and AP4b, which are highly effective in vitro against both the D10 (chloroquine [CQ] susceptible) and W2 (CQ resistant) strains of Plasmodium falciparum and in vivo in the rodent malaria model, have been studied for their ability to bind to and be internalized by normal or parasitized human red blood cells (RBC) and for their effects on RBC membrane stability. In addition, an analysis of the heme binding properties of these compounds and of their ability to inhibit beta-hematin formation in vitro has been performed. Binding of AM1 or AP4b to RBC is rapid, dose dependent, and linearly related to RBC density. Their accumulation in parasitized RBC (pRBC) is increased twofold compared to levels in normal RBC. Binding of AM1 or AP4b to both normal and pRBC is higher than that of CQ, in agreement with the lower pKa and higher lipophilicity of the compounds. AM1 or AP4b is not hemolytic per se and is less hemolytic than CQ when hemolysis is accelerated (induced) by hematin. Moreover, AM-1 and AP4b bind heme with a stoichiometry of interaction similar to that of CQ (about 1:1.7) but with a lower affinity. They both inhibit dose dependently the formation of beta-hematin in vitro with a 50% inhibitory concentration comparable to that of CQ. Taken together, these results suggest that the antimalarial activity of AM1 or AP4b is likely due to inhibition of hemozoin formation and that the efficacy of these compounds against the CQ-resistant strains can be ascribed to their hydrophobicity and capacity to accumulate in the vacuolar lipid (elevated lipid accumulation ratios). PMID:19651905

  6. Antigenic Diversity of the Plasmodium vivax Circumsporozoite Protein in Parasite Isolates of Western Colombia

    PubMed Central

    Hernndez-Martnez, Miguel ngel; Escalante, Ananas A.; Arvalo-Herrera, Myriam; Herrera, Scrates

    2011-01-01

    Circumsporozoite (CS) protein is a malaria antigen involved in sporozoite invasion of hepatocytes, and thus considered to have good vaccine potential. We evaluated the polymorphism of the Plasmodium vivax CS gene in 24 parasite isolates collected from malaria-endemic areas of Colombia. We sequenced 27 alleles, most of which (25/27) corresponded to the VK247 genotype and the remainder to the VK210 type. All VK247 alleles presented a mutation (Gly ? Asn) at position 28 in the N-terminal region, whereas the C-terminal presented three insertions: the ANKKAGDAG, which is common in all VK247 isolates; 12 alleles presented the insertion GAGGQAAGGNAANKKAGDAG; and 5 alleles presented the insertion GGNAGGNA. Both repeat regions were polymorphic in gene sequence and size. Sequences coding for B-, T-CD4+, and T-CD8+ cell epitopes were found to be conserved. This study confirms the high polymorphism of the repeat domain and the highly conserved nature of the flanking regions. PMID:21292878

  7. Molecular basis of fosmidomycin's action on the human malaria parasite Plasmodium falciparum

    PubMed Central

    Umeda, Tomonobu; Tanaka, Nobutada; Kusakabe, Yoshio; Nakanishi, Masayuki; Kitade, Yukio; Nakamura, Kazuo T.

    2011-01-01

    The human malaria parasite Plasmodium falciparum is responsible for the deaths of more than a million people each year. Fosmidomycin has been proven to be efficient in the treatment of P. falciparum malaria by inhibiting 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), an enzyme of the non-mevalonate pathway, which is absent in humans. However, the structural details of DXR inhibition by fosmidomycin in P. falciparum are unknown. Here, we report the crystal structures of fosmidomycin-bound complete quaternary complexes of PfDXR. Our study revealed that (i) an intrinsic flexibility of the PfDXR molecule accounts for an induced-fit movement to accommodate the bound inhibitor in the active site and (ii) a cis arrangement of the oxygen atoms of the hydroxamate group of the bound inhibitor is essential for tight binding of the inhibitor to the active site metal. We expect the present structures to be useful guides for the design of more effective antimalarial compounds. PMID:22355528

  8. Functional Analysis of Protein Kinase CK2 of the Human Malaria Parasite Plasmodium falciparum?

    PubMed Central

    Holland, Zo; Prudent, Renaud; Reiser, Jean-Baptiste; Cochet, Claude; Doerig, Christian

    2009-01-01

    Protein kinase CK2 (casein kinase 2) is a eukaryotic serine/threonine protein kinase with multiple substrates and roles in diverse cellular processes, including differentiation, proliferation, and translation. The mammalian holoenzyme consists of two catalytic alpha or alpha? subunits and two regulatory beta subunits. We report the identification and characterization of a Plasmodium falciparum CK2? orthologue, PfCK2?, and two PfCK2? orthologues, PfCK2?1 and PfCK2?2. Recombinant PfCK2? possesses protein kinase activity, exhibits similar substrate and cosubstrate preferences to those of CK2? subunits from other organisms, and interacts with both of the PfCK2? subunits in vitro. Gene disruption experiments show that the presence of PfCK2? is crucial to asexual blood stage parasites and thereby validate the enzyme as a possible drug target. PfCK2? is amenable to inhibitor screening, and we report differential susceptibility between the human and P. falciparum CK2? enzymes to a small molecule inhibitor. Taken together, our data identify PfCK2? as a potential target for antimalarial chemotherapeutic intervention. PMID:19114502

  9. Genomic sequencing of Plasmodium falciparum malaria parasites from Senegal reveals the demographic history of the population.

    PubMed

    Chang, Hsiao-Han; Park, Daniel J; Galinsky, Kevin J; Schaffner, Stephen F; Ndiaye, Daouda; Ndir, Omar; Mboup, Souleymane; Wiegand, Roger C; Volkman, Sarah K; Sabeti, Pardis C; Wirth, Dyann F; Neafsey, Daniel E; Hartl, Daniel L

    2012-11-01

    Malaria is a deadly disease that causes nearly one million deaths each year. To develop methods to control and eradicate malaria, it is important to understand the genetic basis of Plasmodium falciparum adaptations to antimalarial treatments and the human immune system while taking into account its demographic history. To study the demographic history and identify genes under selection more efficiently, we sequenced the complete genomes of 25 culture-adapted P. falciparum isolates from three sites in Senegal. We show that there is no significant population structure among these Senegal sampling sites. By fitting demographic models to the synonymous allele-frequency spectrum, we also estimated a major 60-fold population expansion of this parasite population ?20,000-40,000 years ago. Using inferred demographic history as a null model for coalescent simulation, we identified candidate genes under selection, including genes identified before, such as pfcrt and PfAMA1, as well as new candidate genes. Interestingly, we also found selection against G/C to A/T changes that offsets the large mutational bias toward A/T, and two unusual patterns: similar synonymous and nonsynonymous allele-frequency spectra, and 18% of genes having a nonsynonymous-to-synonymous polymorphism ratio >1. PMID:22734050

  10. Polysome profiling reveals translational control of gene expression in the human malaria parasite Plasmodium falciparum

    PubMed Central

    2013-01-01

    Background In eukaryotic organisms, gene expression is regulated at multiple levels during the processes of transcription and translation. The absence of a tight regulatory network for transcription in the human malaria parasite suggests that gene expression may largely be controlled at post-transcriptional and translational levels. Results In this study, we compare steady-state mRNA and polysome-associated mRNA levels of Plasmodium falciparum at different time points during its asexual cell cycle. For more than 30% of its genes, we observe a delay in peak transcript abundance in the polysomal fraction as compared to the steady-state mRNA fraction, suggestive of strong translational control. Our data show that key regulatory mechanisms could include inhibitory activity of upstream open reading frames and translational repression of the major virulence gene family by intronic transcripts. In addition, we observe polysomal mRNA-specific alternative splicing events and widespread transcription of non-coding transcripts. Conclusions These different layers of translational regulation are likely to contribute to a complex network that controls gene expression in this eukaryotic pathogen. Disrupting the mechanisms involved in such translational control could provide novel anti-malarial strategies. PMID:24267660

  11. Serologic Markers in Relation to Parasite Exposure History Help to Estimate Transmission Dynamics of Plasmodium vivax

    PubMed Central

    Yildiz Zeyrek, Fadile; Palacpac, Nirianne; Yuksel, Fehmi; Yagi, Masanori; Honjo, Kaori; Fujita, Yukiko; Arisue, Nobuko; Takeo, Satoru; Tanabe, Kazuyuki; Horii, Toshihiro; Tsuboi, Takafumi; Ishii, Ken J.; Coban, Cevayir

    2011-01-01

    Plasmodium vivax infection has been gaining attention because of its re-emergence in several parts of the world. Southeastern Turkey is one of the places in which persistent focal malaria caused exclusively by P. vivax parasites occurs. Although control and elimination studies have been underway for many years, no detailed study has been conducted to understand the mechanisms underlying the ineffective control of malaria in this region. Here, for the first time, using serologic markers we try to extract as much information as possible in this region to get a glimpse of P. vivax transmission. We conducted a sero-immunological study, evaluating antibody responses of individuals living in Sanliurfa to four different P. vivax antigens; three blood-stage antigens (PvMSP119, PvAMA1-ecto, and PvSERA4) and one pre-erythrocytic stage antigen (PvCSP). The results suggest that a prior history of malaria infection and age can be determining factors for the levels and sustainability of naturally acquired antibodies. Significantly higher antibody responses to all the studied antigens were observed in blood smear-negative individuals with a prior history of malaria infection. Moreover, these individuals were significantly older than blood smear-negative individuals with no prior history of infection. These data from an area of sole P. vivax-endemic region may have important implications for the global malaria control/elimination programs and vaccine design. PMID:22140521

  12. Antisense long noncoding RNAs regulate var gene activation in the malaria parasite Plasmodium falciparum

    PubMed Central

    Amit-Avraham, Inbar; Pozner, Guy; Eshar, Shiri; Fastman, Yair; Kolevzon, Netanel; Yavin, Eylon; Dzikowski, Ron

    2015-01-01

    The virulence of Plasmodium falciparum, the causative agent of the deadliest form of human malaria, is attributed to its ability to evade human immunity through antigenic variation. These parasites alternate between expression of variable antigens, encoded by members of a multicopy gene family named var. Immune evasion through antigenic variation depends on tight regulation of var gene expression, ensuring that only a single var gene is expressed at a time while the rest of the family is maintained transcriptionally silent. Understanding how a single gene is chosen for activation is critical for understanding mutually exclusive expression but remains a mystery. Here, we show that antisense long noncoding RNAs (lncRNAs) initiating from var introns are associated with the single active var gene at the time in the cell cycle when the single var upstream promoter is active. We demonstrate that these antisense transcripts are incorporated into chromatin, and that expression of these antisense lncRNAs in trans triggers activation of a silent var gene in a sequence- and dose-dependent manner. On the other hand, interference with these lncRNAs using complement peptide nucleic acid molecules down-regulated the active var gene, erased the epigenetic memory, and induced expression switching. Altogether, our data provide evidence that these antisense lncRNAs play a key role in regulating var gene activation and mutually exclusive expression. PMID:25691743

  13. Deletion of Plasmodium berghei-Specific CD4+ T Cells Adoptively Transferred into Recipient Mice after Challenge with Homologous Parasite

    NASA Astrophysics Data System (ADS)

    Hirunpetcharat, Chakrit; Good, Michael F.

    1998-02-01

    The immune response to malaria parasites includes T cell responses that reduce parasites by effector T cell responses and by providing help for antibody responses. Some parasites are more sensitive to antibody and others are more sensitive to cell-mediated immunity. We demonstrate that cultured CD4+ T cells that produce interferon CD4+ and interleukin 2, but not interleukin 4, in response to stimulation with the rodent parasite Plasmodium berghei can reduce but not eliminate parasites in vivo after adoptive transfer. Although cells can persist in vivo for up to 9 months in uninfected mice, infection results in elimination of up to 99% of specific T cells in different tissues, as judged by tracking T cells labeled with the fluorescent dye 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester. T cells specific for ovalbumin are unaffected. In vivo activation and division of transferred T cells per se are not responsible for deletion because T cells positive for 5-(and -6)-carboxyfluorescein diacetate succinimidyl ester divide up to six times within 7 days in uninfected mice and are not deleted. Understanding the factors responsible for parasite-mediated specific deletion of T cells would enhance our knowledge of parasite immunity.

  14. Plasmodium falciparum: stage-specific ribosomal RNA as a potential target for monitoring parasite development in Anopheles stephensi.

    PubMed

    Li, J; Wirtz, R A; Schneider, I; Muratova, O V; McCutchan, T F; Appiah, A; Hollingdale, M R

    1993-02-01

    The transcriptional switch of Plasmodium falciparum ribosomal RNA A gene to the C gene was demonstrated during the developmental transition from the vertebrate blood stage to the invertebrate sporozoite stage. Expression of the sporozoite specific C gene in infected mosquitoes was not detected until Day 10 postinfectious blood meal, the time of mature oocyst formation on the midgut. As a potential target for monitoring malaria parasite development in mosquitoes, oligonucleotide probes based on sequences of small subunit ribosomal RNA were evaluated for specificity and sensitivity by filter blot hybridization against different species and stages of malaria parasites. Probes PfC02 and PfA02 were selected as the most sensitive for sporozoite and blood stage parasites, respectively. Filter blot hybridization using probe PfC02 resulted in sensitivity comparable with microscopic dissection in single mosquitoes, detecting mosquitoes with an average of 1.2 oocysts per gut or as few as 800 salivary gland sporozoites. PMID:8467898

  15. Plasmodium falciparum Bloom homologue, a nucleocytoplasmic protein, translocates in 3' to 5' direction and is essential for parasite growth.

    PubMed

    Rahman, Farhana; Tarique, Mohammed; Tuteja, Renu

    2016-05-01

    Malaria caused by Plasmodium, particularly Plasmodium falciparum, is the most serious and widespread parasitic disease of humans. RecQ helicase family members are essential in homologous recombination-based error-free DNA repair processes in all domains of life. RecQ helicases present in each organism differ and several homologues have been identified in various multicellular organisms. These proteins are involved in various pathways of DNA metabolism by providing duplex unwinding function. Five members of RecQ family are present in Homo sapiens but P. falciparum contains only two members of this family. Here we report the detailed biochemical and functional characterization of the Bloom (Blm) homologue (PfBlm) from P. falciparum 3D7 strain. Purified PfBlm exhibits ATPase and 3' to 5' direction specific DNA helicase activity. The calculated average reaction rate of ATPase was ~13 pmol of ATP hydrolyzed/min/pmol of enzyme. The immunofluorescence assay results show that PfBlm is expressed in all the stages of intraerythrocytic development of the P. falciparum 3D7 strain. In some stages of development in addition to nucleus PfBlm also localizes in the cytoplasm. The gene disruption studies of PfBlm by dsRNA showed that it is required for the ex-vivo intraerythrocytic development of the parasite P. falciparum 3D7 strain. The dsRNA mediated inhibition of parasite growth suggests that a variety of pathways are affected resulting in curtailing of the parasite growth. This study will be helpful in unravelling the basic mechanism of DNA transaction in the malaria parasite and additionally it may provide leads to understand the parasite specific characteristics of this protein. PMID:26917473

  16. Dominant negative mutant of Plasmodium?Rad51 causes reduced parasite burden in host by abrogating DNA double-strand break repair.

    PubMed

    Roy, Nabamita; Bhattacharyya, Sunanda; Chakrabarty, Swati; Laskar, Shyamasree; Babu, Somepalli Mastan; Bhattacharyya, Mrinal Kanti

    2014-10-01

    Malaria parasites survive through repairing a plethora of DNA double-stranded breaks (DSBs) experienced during their asexual growth. In Plasmodium?Rad51 mediated homologous recombination (HR) mechanism and homology-independent alternative end-joining mechanism have been identified. Here we address whether loss of HR activity can be compensated by other DSB repair mechanisms. Creating a transgenic Plasmodium line defective in HR function, we demonstrate that HR is the most important DSB repair pathway in malarial parasite. Using mouse malaria model we have characterized the dominant negative effect of PfRad51(K143R) mutant on Plasmodium?DSB repair and host-parasite interaction. Our work illustrates that Plasmodium berghei harbouring the mutant protein (PfRad51(K143R)) failed to repair DSBs as evidenced by hypersensitivity to DNA-damaging agent. Mice infected with mutant parasites lived significantly longer with markedly reduced parasite burden. To better understand the effect of mutant PfRad51(K143R) on HR, we used yeast as a surrogate model and established that the presence of PfRad51(K143R) completely inhibited DNA repair, gene conversion and gene targeting. Biochemical experiment confirmed that very low level of mutant protein was sufficient for complete disruption of wild-type PfRad51 activity. Hence our work provides evidence that HR pathway of Plasmodium could be efficiently targeted to curb malaria. PMID:25145341

  17. Biochemical characterization of FIKK8 – A unique protein kinase from the malaria parasite Plasmodium falciparum and other apicomplexans

    PubMed Central

    Osman, Khan T.; Lou, Hua Jane; Qiu, Wei; Brand, Verena; Edwards, Aled M.; Turk, Benjamin E.; Hui, Raymond

    2015-01-01

    FIKKs are protein kinases with distinctive sequence motifs found exclusively in Apicomplexa. Here, we report on the biochemical characterization of Plasmodium falciparum FIKK8 (PfFIKK8) and its Cryptosporidium parvum orthologue (CpFIKK) – the only member of the family predicted to be cytosolic and conserved amongst non-Plasmodium parasites. Recombinant protein samples of both were catalytically active. We characterized their phosphorylation ability using an enzymatic assay and substrate specificities using an arrayed positional scanning peptide library. Our results show that FIKK8 targets serine, preferably with arginine in the +3 and −3 positions. Furthermore, the soluble and active FIKK constructs in our experiments contained an N-terminal extension (NTE) conserved in FIKK8 orthologues from other apicomplexan species. Based on our results, we propose that this NTE is an integral feature of the FIKK subfamily. PMID:26112892

  18. Plasmodium falciparum: food vacuole localization of nitric oxide-derived species in intraerythrocytic stages of the malaria parasite

    PubMed Central

    Ostera, Graciela; Tokumasu, Fuyuki; Oliveira, Fabiano; Sa, Juliana; Furuya, Tetsuya; Teixeira, Clarissa; Dvorak, James

    2008-01-01

    Nitric oxide (NO) has diverse biological functions. Numerous studies have documented NOs biosynthetic pathway in a wide variety of organisms. Little is known, however, about NO production in intraerythrocytic Plasmodium falciparum. Using diaminorhodamine-4-methyl acetoxymethylester (DAR-4M AM), a fluorescent indicator, we obtained direct evidence of NO and NO-derived reactive nitrogen species (RNS) production in intraerythrocytic P. falciparum parasites, as well as in isolated food vacuoles from trophozoite stage parasites. We preliminarily identified two gene sequences that might be implicated in NO synthesis in intraerythrocytic P. falciparum. We showed localization of the protein product of one of these two genes, a molecule that is structurally similar to a plant nitrate reductase, in trophozoite food vacuole membranes. We confirmed previous reports on the antiproliferative effect of NOS (nitric oxide synthase) inhibitors in P.falciparum cultures; however, we did not obtain evidence that NOS inhibitors had the ability to inhibit RNS production or that there is an active NOS in mature forms of the parasite. We concluded that a nitrate reductase activity produce NO and NO-derived RNS in or around the food vacuole in P. falciparum parasites. The food vacuole is a critical parasitic compartment involved in hemoglobin degradation, heme detoxification and a target for antimalarial drug action. Characterization of this relatively unexplored synthetic activity could provide important clues into poorly understood metabolic processes of the malaria parasite, PMID:18504040

  19. Yeast-based high-throughput screen identifies Plasmodium falciparum equilibrative nucleoside transporter 1 inhibitors that kill malaria parasites.

    PubMed

    Frame, I J; Deniskin, Roman; Rinderspacher, Alison; Katz, Francine; Deng, Shi-Xian; Moir, Robyn D; Adjalley, Sophie H; Coburn-Flynn, Olivia; Fidock, David A; Willis, Ian M; Landry, Donald W; Akabas, Myles H

    2015-03-20

    Equilibrative transporters are potential drug targets; however, most functional assays involve radioactive substrate uptake that is unsuitable for high-throughput screens (HTS). We developed a robust yeast-based growth assay that is potentially applicable to many equilibrative transporters. As proof of principle, we applied our approach to Equilibrative Nucleoside Transporter 1 of the malarial parasite Plasmodium falciparum (PfENT1). PfENT1 inhibitors might serve as novel antimalarial drugs since PfENT1-mediated purine import is essential for parasite proliferation. To identify PfENT1 inhibitors, we screened 64?560 compounds and identified 171 by their ability to rescue the growth of PfENT1-expressing fui1? yeast in the presence of a cytotoxic PfENT1 substrate, 5-fluorouridine (5-FUrd). In secondary assays, nine of the highest activity compounds inhibited PfENT1-dependent growth of a purine auxotrophic yeast strain with adenosine as the sole purine source (IC50 0.2-2 ?M). These nine compounds completely blocked [(3)H]adenosine uptake into PfENT1-expressing yeast and erythrocyte-free trophozoite-stage parasites (IC50 5-50 nM), and inhibited chloroquine-sensitive and -resistant parasite proliferation (IC50 5-50 ?M). Wild-type (WT) parasite IC50 values were up to 4-fold lower compared to PfENT1-knockout (pfent1?) parasites. pfent1? parasite killing showed a delayed-death phenotype not observed with WT. We infer that, in parasites, the compounds inhibit both PfENT1 and a secondary target with similar efficacy. The secondary target identity is unknown, but its existence may reduce the likelihood of parasites developing resistance to PfENT1 inhibitors. Our data support the hypothesis that blocking purine transport through PfENT1 may be a novel and compelling approach for antimalarial drug development. PMID:25602169

  20. Transport of nucleosides across the Plasmodium falciparum parasite plasma membrane has characteristics of PfENT1.

    PubMed

    Downie, Megan J; Saliba, Kevin J; Howitt, Susan M; Bröer, Stefan; Kirk, Kiaran

    2006-05-01

    Like all parasitic protozoa, the human malaria parasite Plasmodium falciparum lacks the enzymes required for de novo synthesis of purines and it is therefore reliant upon the salvage of these compounds from the external environment. P. falciparum equilibrative nucleoside transporter 1 (PfENT1) is a nucleoside transporter that has been localized to the plasma membrane of the intraerythrocytic form of the parasite. In this study we have characterized the transport of purine and pyrimidine nucleosides across the plasma membrane of 'isolated' trophozoite-stage P. falciparum parasites and compared the transport characteristics of the parasite with those of PfENT1 expressed in Xenopus oocytes. The transport of nucleosides into the parasite: (i) was, in the case of adenosine, inosine and thymidine, very fast, equilibrating within a few seconds; (ii) was of low affinity [K(m) (adenosine) = 1.45 +/- 0.25 mM; K(m) (thymidine) = 1.11 +/- 0.09 mM]; and (iii) showed 'cross-competition' for adenosine, inosine and thymidine, but not cytidine. The kinetic characteristics of nucleoside transport in intact parasites matched very closely those of PfENT1 expressed in Xenopus oocytes [K(m) (adenosine) = 1.86 +/- 0.28 mM; K(m) (thymidine) = 1.33 +/- 0.17 mM]. Furthermore, PfENT1 transported adenosine, inosine and thymidine, with a cross-competition profile the same as that seen for isolated parasites. The data are consistent with PfENT1 serving as a major route for the uptake of nucleosides across the parasite plasma membrane. PMID:16629674

  1. Variation in susceptibility of African Plasmodium falciparum malaria parasites to TEP1 mediated killing in Anopheles gambiae mosquitoes.

    PubMed

    Eldering, Maarten; Morlais, Isabelle; van Gemert, Geert-Jan; van de Vegte-Bolmer, Marga; Graumans, Wouter; Siebelink-Stoter, Rianne; Vos, Martijn; Abate, Luc; Roeffen, Will; Bousema, Teun; Levashina, Elena A; Sauerwein, Robert W

    2016-01-01

    Anopheles gambiae s.s. mosquitoes are efficient vectors for Plasmodium falciparum, although variation exists in their susceptibility to infection. This variation depends partly on the thioester-containing protein 1 (TEP1) and TEP depletion results in significantly elevated numbers of oocysts in susceptible and resistant mosquitoes. Polymorphism in the Plasmodium gene coding for the surface protein Pfs47 modulates resistance of some parasite laboratory strains to TEP1-mediated killing. Here, we examined resistance of P. falciparum isolates of African origin (NF54, NF165 and NF166) to TEP1-mediated killing in a susceptible Ngousso and a refractory L3-5 strain of A. gambiae. All parasite clones successfully developed in susceptible mosquitoes with limited evidence for an impact of TEP1 on transmission efficiency. In contrast, NF166 and NF165 oocyst densities were strongly reduced in refractory mosquitoes and TEP1 silencing significantly increased oocyst densities. Our results reveal differences between African P. falciparum strains in their capacity to evade TEP1-mediated killing in resistant mosquitoes. There was no significant correlation between Pfs47 genotype and resistance of a given P. falciparum isolate for TEP1 killing. These data suggest that polymorphisms in this locus are not the sole mediators of immune evasion of African malaria parasites. PMID:26861587

  2. Identification of New Human Malaria Parasite Plasmodium falciparum Dihydroorotate Dehydrogenase Inhibitors by Pharmacophore and Structure-Based Virtual Screening.

    PubMed

    Pavadai, Elumalai; El Mazouni, Farah; Wittlin, Sergio; de Kock, Carmen; Phillips, Margaret A; Chibale, Kelly

    2016-03-28

    Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH), a key enzyme in the de novo pyrimidine biosynthesis pathway, which the Plasmodium falciparum relies on exclusively for survival, has emerged as a promising target for antimalarial drugs. In an effort to discover new and potent PfDHODH inhibitors, 3D-QSAR pharmacophore models were developed based on the structures of known PfDHODH inhibitors and the validated Hypo1 model was used as a 3D search query for virtual screening of the National Cancer Institute database. The virtual hit compounds were further filtered based on molecular docking and Molecular Mechanics/Generalized Born Surface Area binding energy calculations. The combination of the pharmacophore and structure-based virtual screening resulted in the identification of nine new compounds that showed >25% inhibition of PfDHODH at a concentration of 10 μM, three of which exhibited IC50 values in the range of 0.38-20 μM. The most active compound, NSC336047, displayed species-selectivity for PfDHODH over human DHODH and inhibited parasite growth with an IC50 of 26 μM. In addition to this, 13 compounds inhibited parasite growth with IC50 values of ≤50 μM, 4 of which showed IC50 values in the range of 5-12 μM. These compounds could be further explored in the identification and development of more potent PfDHODH and parasite growth inhibitors. PMID:26915022

  3. Variation in susceptibility of African Plasmodium falciparum malaria parasites to TEP1 mediated killing in Anopheles gambiae mosquitoes

    PubMed Central

    Eldering, Maarten; Morlais, Isabelle; van Gemert, Geert-Jan; van de Vegte-Bolmer, Marga; Graumans, Wouter; Siebelink-Stoter, Rianne; Vos, Martijn; Abate, Luc; Roeffen, Will; Bousema, Teun; Levashina, Elena A.; Sauerwein, Robert W.

    2016-01-01

    Anopheles gambiae s.s. mosquitoes are efficient vectors for Plasmodium falciparum, although variation exists in their susceptibility to infection. This variation depends partly on the thioester-containing protein 1 (TEP1) and TEP depletion results in significantly elevated numbers of oocysts in susceptible and resistant mosquitoes. Polymorphism in the Plasmodium gene coding for the surface protein Pfs47 modulates resistance of some parasite laboratory strains to TEP1-mediated killing. Here, we examined resistance of P. falciparum isolates of African origin (NF54, NF165 and NF166) to TEP1-mediated killing in a susceptible Ngousso and a refractory L35 strain of A. gambiae. All parasite clones successfully developed in susceptible mosquitoes with limited evidence for an impact of TEP1 on transmission efficiency. In contrast, NF166 and NF165 oocyst densities were strongly reduced in refractory mosquitoes and TEP1 silencing significantly increased oocyst densities. Our results reveal differences between African P. falciparum strains in their capacity to evade TEP1-mediated killing in resistant mosquitoes. There was no significant correlation between Pfs47 genotype and resistance of a given P. falciparum isolate for TEP1 killing. These data suggest that polymorphisms in this locus are not the sole mediators of immune evasion of African malaria parasites. PMID:26861587

  4. Plasmodium falciparum heat shock protein 110 stabilizes the asparagine repeat-rich parasite proteome during malarial fevers.

    PubMed

    Muralidharan, Vasant; Oksman, Anna; Pal, Priya; Lindquist, Susan; Goldberg, Daniel E

    2012-01-01

    One-fourth of Plasmodium falciparum proteins have asparagine repeats that increase the propensity for aggregation, especially at elevated temperatures that occur routinely in malaria-infected patients. Here we report that a Plasmodium Asn repeat-containing protein (PFI1155w) formed aggregates in mammalian cells at febrile temperatures, as did a yeast Asn/Gln-rich protein (Sup35). Co-expression of the cytoplasmic P. falciparum heat shock protein 110 (PfHsp110c) prevented aggregation. Human or yeast orthologs were much less effective. All-Asn and all-Gln versions of Sup35 were protected from aggregation by PfHsp110c, suggesting that this chaperone is not limited to handling runs of asparagine. PfHsp110c gene-knockout parasites were not viable and conditional knockdown parasites died slowly in the absence of protein-stabilizing ligand. When exposed to brief heat shock, these knockdowns were unable to prevent aggregation of PFI1155w or Sup35 and died rapidly. We conclude that PfHsp110c protects the parasite from harmful effects of its asparagine repeat-rich proteome during febrile episodes. PMID:23250440

  5. Genetic polymorphism and natural selection in the malaria parasite Plasmodium falciparum.

    PubMed Central

    Escalante, A A; Lal, A A; Ayala, F J

    1998-01-01

    We have studied the genetic polymorphism at 10 Plasmodium falciparum loci that are considered potential targets for specific antimalarial vaccines. The polymorphism is unevenly distributed among the loci; loci encoding proteins expressed on the surface of the sporozoite or the merozoite (AMA-1, CSP, LSA-1, MSP-1, MSP-2, and MSP-3) are more polymorphic than those expressed during the sexual stages or inside the parasite (EBA-175, Pfs25, PF48/45, and RAP-1). Comparison of synonymous and nonsynonymous substitutions indicates that natural selection may account for the polymorphism observed at seven of the 10 loci studied. This inference depends on the assumption that synonymous substitutions are neutral, which we test by analyzing codon bias and G+C content in a set of 92 gene loci. We find evidence for an overall trend towards increasing A+T richness, but no evidence for mutation bias. Although the neutrality of synonymous substitutions is not definitely established, this trend towards an A+T rich genome cannot explain the accumulation of substitutions at least in the case of four genes (AMA-1, CSP, LSA-1, and PF48/45) because the Gleft and right arrow C transversions are more frequent than expected. Moreover, the Tajima test manifests positive natural selection for the MSP-1 and, less strongly, MSP-3 polymorphisms; the McDonald-Kreitman test manifests natural selection at LSA-1 and PF48/45. We conclude that there is definite evidence for positive natural selection in the genes encoding AMA-1, CSP, LSA-1, MSP-1, and Pfs48/45. For four other loci, EBA-175, MSP-2, MSP-3, and RAP-1, the evidence is limited. No evidence for natural selection is found for Pfs25. PMID:9584096

  6. Glutathione S-transferase of the malarial parasite Plasmodium falciparum: characterization of a potential drug target.

    PubMed

    Harwaldt, Petra; Rahlfs, Stefan; Becker, Katja

    2002-05-01

    Glutathione S-transferases (GSTs), which occur abundantly in most organisms, are essentially involved in the intracellular detoxification of numerous substances including chemotherapeutic agents, and thus play a major role in the development of drug resistance. A gene encoding a protein with sequence identity of up to 37% with known GSTs was identified on chromosome 14 of the malarial parasite Plasmodium falciparum. It was amplified using gametocyte cDNA and expressed in Escherichia coli as a hexahistidyl-tagged protein of 26 kDa subunit size. The homodimeric enzyme (PfGST) was found to catalyse the glutathione (GSH)-dependent modification of 1-chloro-2,4-dinitrobenzene and other typical GST substrates such as o-nitrophenyl acetate, ethacrynic acid, and cumene hydroperoxide. The Km value for GSH was 164+/-20 microM. PfGST was inhibited by cibacron blue (Ki=0.5 microM), S-hexylglutathione (Ki=35 microM), and protoporphyrin IX (Ki=10 microM). Hemin, a most toxic compound for parasitised erythrocytes, was found to be an uncompetitive ligand of PfGST with a Ki of 6.5 microM. Based on the activity of PfGST in extracts of P. falciparum, the enzyme represents 1 to 10% of cellular protein and might therefore serve as an efficient in vivo buffer for parasitotoxic hemin. Destabilising ligands of GST are thus expected to be synergistic with the antimalarial drug chloroquine, which itself was found to be a very weak inhibitor of PfGST (IC50>200 microM). X-ray quality crystals of PfGST (250x200x50 microm) will serve as starting point for structure-based drug design. PMID:12108547

  7. Long term persistence of clonal malaria parasite Plasmodium falciparum lineages in the Colombian Pacific region

    PubMed Central

    2013-01-01

    Background Resistance to chloroquine and antifolate drugs has evolved independently in South America, suggesting that genotype - phenotype studies aimed at understanding the genetic basis of resistance to these and other drugs should be conducted in this continent. This research was conducted to better understand the population structure of Colombian Plasmodium falciparum in preparation for such studies. Results A set of 384 SNPs were genotyped in blood spot DNA samples from 447 P. falciparum infected subjects collected over a ten year period from four provinces of the Colombian Pacific coast to evaluate clonality, population structure and linkage disequilibrium (LD). Most infections (81%) contained a single predominant clone. These clustered into 136 multilocus genotypes (MLGs), with 32% of MLGs recovered from multiple (2 28) independent subjects. We observed extremely low genotypic richness (R?=?0.42) and long persistence of MLGs through time (median?=?537 days, range?=?1 2,997 days). There was a high probability (>5%) of sampling parasites from the same MLG in different subjects within 28 days, suggesting caution is needed when using genotyping methods to assess treatment success in clinical drug trials. Panmixia was rejected as four well differentiated subpopulations (FST?=?0.084 - 0.279) were identified. These occurred sympatrically but varied in frequency within the four provinces. Linkage disequilibrium (LD) decayed more rapidly (r2?=?0.17 for markers <10 kb apart) than observed previously in South American samples. Conclusions We conclude that Colombian populations have several advantages for association studies, because multiple clone infections are uncommon and LD decays over the scale of one or a few genes. However, the extensive population structure and low genotype richness will need to be accounted for when designing and analyzing association studies. PMID:23294725

  8. Calorimetric studies of ligands binding to glutathione S-transferase from the malarial parasite Plasmodium falciparum.

    PubMed

    Quesada-Soriano, Indalecio; Barn, Carmen; Garca-Maroto, Federico; Aguilera, Ana M; Garca-Fuentes, Lus

    2013-03-19

    Glutathione S-transferase, from the malarial parasite Plasmodium falciparum (PfGST), exerts a protective role in the organism and is thus considered an interesting target for antimalarial drug development. In contrast to other GSTs, it is present in solution as a tetramer and a dimer in equilibrium, which is induced by glutathione (GSH). These properties prevent a calorimetric titration from being conducted upon binding of ligands to this protein's G-site. Thermodynamic characterization can be an optimal strategy for antimalarial drug development, and isothermal titration calorimetry (ITC) is the only technique that allows the separation of the binding energy into both enthalpic and entropic contributions. This information facilitates an understanding of the changes in the drugs' substituents, improving their affinity and specificity. In this study, we have applied a nontypical ITC procedure, based on the dissociation of the ligand-protein complex, to calorimetrically study the binding of the GSH substrate, and the glutathione sulfonate competitive inhibitor, to dimeric PfGST over a temperature range of 15-37 C. The optimal experimental conditions for applying this procedure have been optimized by studying the dimer to tetramer conversion using size exclusion chromatography. The binding of these ligands to dimeric PfGST is noncooperative, the affinity of glutathione sulfonate being approximately 2 orders of magnitude higher than that of its natural substrate GSH. The binding of both ligands is enthalpically favorable and entropically unfavorable at all the studied temperatures. These results demonstrate that, although PfGST presents differences when compared to other known GSTs, these ligands bind to its dimeric form with a similar affinity and energetic balance. However, in contrast to that of other GSTs, the binding of GSH to protein, in the absence of the ligand, is slow. PMID:23439010

  9. Proteomic Analysis of Detergent-resistant Membrane Microdomains in Trophozoite Blood Stage of the Human Malaria Parasite Plasmodium falciparum*

    PubMed Central

    Yam, Xue Yan; Birago, Cecilia; Fratini, Federica; Di Girolamo, Francesco; Raggi, Carla; Sargiacomo, Massimo; Bachi, Angela; Berry, Laurence; Fall, Gamou; Curr, Chiara; Pizzi, Elisabetta; Breton, Catherine Braun; Ponzi, Marta

    2013-01-01

    Intracellular pathogens contribute to a significant proportion of infectious diseases worldwide. The successful strategy of evading the immune system by hiding inside host cells is common to all the microorganism classes, which exploit membrane microdomains, enriched in cholesterol and sphingolipids, to invade and colonize the host cell. These assemblies, with distinct biochemical properties, can be isolated by means of flotation in sucrose density gradient centrifugation because they are insoluble in nonionic detergents at low temperature. We analyzed the protein and lipid contents of detergent-resistant membranes from erythrocytes infected by Plasmodium falciparum, the most deadly human malaria parasite. Proteins associated with membrane microdomains of trophic parasite blood stages (trophozoites) include an abundance of chaperones, molecules involved in vesicular trafficking, and enzymes implicated in host hemoglobin degradation. About 60% of the identified proteins contain a predicted localization signal suggesting a role of membrane microdomains in protein sorting/trafficking. To validate our proteomic data, we raised antibodies against six Plasmodium proteins not characterized previously. All the selected candidates were recovered in floating low-density fractions after density gradient centrifugation. The analyzed proteins localized either to internal organelles, such as the mitochondrion and the endoplasmic reticulum, or to exported membrane structures, the parasitophorous vacuole membrane and Maurer's clefts, implicated in targeting parasite proteins to the host erythrocyte cytosol or surface. The relative abundance of cholesterol and phospholipid species varies in gradient fractions containing detergent-resistant membranes, suggesting heterogeneity in the lipid composition of the isolated microdomain population. This study is the first report showing the presence of cholesterol-rich microdomains with distinct properties and subcellular localization in trophic stages of Plasmodium falciparum. PMID:24045696

  10. Proteomic analysis of detergent-resistant membrane microdomains in trophozoite blood stage of the human malaria parasite Plasmodium falciparum.

    PubMed

    Yam, Xue Yan; Birago, Cecilia; Fratini, Federica; Di Girolamo, Francesco; Raggi, Carla; Sargiacomo, Massimo; Bachi, Angela; Berry, Laurence; Fall, Gamou; Curr, Chiara; Pizzi, Elisabetta; Breton, Catherine Braun; Ponzi, Marta

    2013-12-01

    Intracellular pathogens contribute to a significant proportion of infectious diseases worldwide. The successful strategy of evading the immune system by hiding inside host cells is common to all the microorganism classes, which exploit membrane microdomains, enriched in cholesterol and sphingolipids, to invade and colonize the host cell. These assemblies, with distinct biochemical properties, can be isolated by means of flotation in sucrose density gradient centrifugation because they are insoluble in nonionic detergents at low temperature. We analyzed the protein and lipid contents of detergent-resistant membranes from erythrocytes infected by Plasmodium falciparum, the most deadly human malaria parasite. Proteins associated with membrane microdomains of trophic parasite blood stages (trophozoites) include an abundance of chaperones, molecules involved in vesicular trafficking, and enzymes implicated in host hemoglobin degradation. About 60% of the identified proteins contain a predicted localization signal suggesting a role of membrane microdomains in protein sorting/trafficking. To validate our proteomic data, we raised antibodies against six Plasmodium proteins not characterized previously. All the selected candidates were recovered in floating low-density fractions after density gradient centrifugation. The analyzed proteins localized either to internal organelles, such as the mitochondrion and the endoplasmic reticulum, or to exported membrane structures, the parasitophorous vacuole membrane and Maurer's clefts, implicated in targeting parasite proteins to the host erythrocyte cytosol or surface. The relative abundance of cholesterol and phospholipid species varies in gradient fractions containing detergent-resistant membranes, suggesting heterogeneity in the lipid composition of the isolated microdomain population. This study is the first report showing the presence of cholesterol-rich microdomains with distinct properties and subcellular localization in trophic stages of Plasmodium falciparum. PMID:24045696

  11. Plasmodium vivax: ookinete destruction and oocyst development arrest are responsible for Anopheles albimanus resistance to circumsporozoite phenotype VK247 parasites.

    PubMed

    Gonzalez-Ceron, L; Rodriguez, M H; Santillan, F; Chavez, B; Nettel, J A; Hernandez-Avila, J E; Kain, K C

    2001-07-01

    Anopheles albimanus and An. pseudopunctipennis differ in their susceptibilities to Plasmodium vivax circumsporozoite phenotypes. An. pseudopunctipennis is susceptible to phenotype VK247 but almost refractory to VK210. In contrast, An. albimanus is almost refractory to VK247 but susceptible to VK210. To investigate the site in the mosquito and the parasite stage at which resistance mechanisms affect VK247 development in An. albimanus, parasite development was followed in a series of experiments in which both mosquitoes species were simultaneously infected with blood from patients. Parasite phenotype was determined in mature oocysts and salivary gland sporozoites by use of immunofluorescence and Western blot assays and/or gene identification. Ookinete maturation and their densities within the bloodmeal bolus were similar in both mosquito species. Ookinete densities on the internal midgut surface of An. albimanus were 4.7 times higher than those in An. pseudopunctipennis; however, the densities of developing oocysts on the external midgut surface were 6.12 times higher in the latter species. Electron microscopy observation of ookinetes in An. albimanus midgut epithelium indicated severe parasite damage. These results indicate that P. vivax VK247 parasites are destroyed at different parasite stages during migration in An. albimanus midguts. A portion, accumulated on the internal midgut surface, is probably destroyed by the mosquito's digestive enzymes and another portion is most likely destroyed by mosquito defense molecules within the midgut epithelium. A third group, reaching the external midgut surface, initiates oocyst development, but over 90% of them interrupt their development and die. The identification of mechanisms that participate in parasite destruction could provide new elements to construct transgenic mosquitoes resistant to malaria parasites. PMID:11527438

  12. Recognition of Human Erythrocyte Receptors by the Tryptophan-Rich Antigens of Monkey Malaria Parasite Plasmodium knowlesi

    PubMed Central

    Tyagi, Kriti; Gupta, Deepali; Saini, Ekta; Choudhary, Shilpa; Jamwal, Abhishek; Alam, Mohd. Shoeb; Zeeshan, Mohammad; Tyagi, Rupesh K.; Sharma, Yagya D.

    2015-01-01

    Background The monkey malaria parasite Plasmodium knowlesi also infect humans. There is a lack of information on the molecular mechanisms that take place between this simian parasite and its heterologous human host erythrocytes leading to this zoonotic disease. Therefore, we investigated here the binding ability of P. knowlesi tryptophan-rich antigens (PkTRAgs) to the human erythrocytes and sharing of the erythrocyte receptors between them as well as with other commonly occurring human malaria parasites. Methods Six PkTRAgs were cloned and expressed in E.coli as well as in mammalian CHO-K1 cell to determine their human erythrocyte binding activity by cell-ELISA, and in-vitro rosetting assay, respectively. Results Three of six PkTRAgs (PkTRAg38.3, PkTRAg40.1, and PkTRAg67.1) showed binding to human erythrocytes. Two of them (PkTRAg40.1 and PkTRAg38.3) showed cross-competition with each other as well as with the previously described P.vivax tryptophan-rich antigens (PvTRAgs) for human erythrocyte receptors. However, the third protein (PkTRAg67.1) utilized the additional but different human erythrocyte receptor(s) as it did not cross-compete for erythrocyte binding with either of these two PkTRAgs as well as with any of the PvTRAgs. These three PkTRAgs also inhibited the P.falciparum parasite growth in in-vitro culture, further indicating the sharing of human erythrocyte receptors by these parasite species and the biological significance of this receptor-ligand interaction between heterologous host and simian parasite. Conclusions Recognition and sharing of human erythrocyte receptor(s) by PkTRAgs with human parasite ligands could be part of the strategy adopted by the monkey malaria parasite to establish inside the heterologous human host. PMID:26393350

  13. The Plasmodium vivax Merozoite Surface Protein 3? Sequence Reveals Contrasting Parasite Populations in Southern and Northwestern Thailand

    PubMed Central

    Kuamsab, Napaporn; Sattabongkot, Jetsumon; Sirichaisinthop, Jeeraphat; Jongwutiwes, Somchai; Cui, Liwang

    2014-01-01

    Background Malaria control efforts have a significant impact on the epidemiology and parasite population dynamics. In countries aiming for malaria elimination, malaria transmission may be restricted to limited transmission hot spots, where parasite populations may be isolated from each other and experience different selection forces. Here we aim to examine the Plasmodium vivax population divergence in geographically isolated transmission zones in Thailand. Methodology We employed the P. vivax merozoite surface protein 3? (PvMSP3?) as a molecular marker for characterizing P. vivax populations based on the extensive diversity of this gene in Southeast Asian parasite populations. To examine two parasite populations with different transmission levels in Thailand, we obtained 45 P. vivax isolates from Tak Province, northwestern Thailand, where the annual parasite incidence (API) was more than 2%, and 28 isolates from Yala and Narathiwat Provinces, southern Thailand, where the API was less than 0.02%. We sequenced the PvMSP3? gene and examined its genetic diversity and molecular evolution between the parasite populations. Principal Findings Of 58 isolates containing single PvMSP3? alleles, 31 sequence types were identified. The overall haplotype diversity was 0.770.06 and nucleotide diversity 0.08770.0054. The northwestern vivax malaria population exhibited extensive haplotype diversity (HD) of PvMSP3? (HD?=?1.0). In contrast, the southern parasite population displayed a single PvMSP3? allele (HD?=?0), suggesting a clonal population expansion. This result revealed that the extent of allelic diversity in P. vivax populations in Thailand varies among endemic areas. Conclusion Malaria parasite populations in a given region may vary significantly in genetic diversity, which may be the result of control and influenced by the magnitude of malaria transmission intensity. This is an issue that should be taken into account for the implementation of P. vivax control measures such as drug policy and vaccine development. PMID:25412166

  14. Plasmodium simium, a Plasmodium vivax-Related Malaria Parasite: Genetic Variability of Duffy Binding Protein II and the Duffy Antigen/Receptor for Chemokines

    PubMed Central

    Camargos Costa, Daniela; Pereira de Assis, Gabriela Mara; de Souza Silva, Flvia Alessandra; Arajo, Flvia Carolina; de Souza Junior, Jlio Csar; Braga Hirano, Zelinda Maria; Satiko Kano, Flora; Nbrega de Sousa, Tas; Carvalho, Luzia Helena; Ferreira Alves de Brito, Cristiana

    2015-01-01

    Plasmodium simium is a parasite from New World monkeys that is most closely related to the human malaria parasite Plasmodium vivax; it also naturally infects humans. The blood-stage infection of P. vivax depends on Duffy binding protein II (PvDBPII) and its cognate receptor on erythrocytes, the Duffy antigen receptor for chemokines (hDARC), but there is no information on the P. simium erythrocytic invasion pathway. The genes encoding P. simium DBP (PsDBPII) and simian DARC (sDARC) were sequenced from Southern brown howler monkeys (Alouatta guariba clamitans) naturally infected with P. simium because P. simium may also depend on the DBPII/DARC interaction. The sequences of DBP binding domains from P. vivax and P. simium were highly similar. However, the genetic variability of PsDBPII was lower than that of PvDBPII. Phylogenetic analyses demonstrated that these genes were strictly related and clustered in the same clade of the evolutionary tree. DARC from A. clamitans was also sequenced and contained three new non-synonymous substitutions. None of these substitutions were located in the N-terminal domain of DARC, which interacts directly with DBPII. The interaction between sDARC and PvDBPII was evaluated using a cytoadherence assay of COS7 cells expressing PvDBPII on their surfaces. Inhibitory binding assays in vitro demonstrated that antibodies from monkey sera blocked the interaction between COS-7 cells expressing PvDBPII and hDARC-positive erythrocytes. Taken together, phylogenetic analyses reinforced the hypothesis that the host switch from humans to monkeys may have occurred very recently in evolution, which sheds light on the evolutionary history of new world plasmodia. Further invasion studies would confirm whether P. simium depends on DBP/DARC to trigger internalization into red blood cells. PMID:26107662

  15. Solution NMR structure of a sheddase inhibitor prodomain from the malarial parasite Plasmodium falciparum.

    PubMed

    He, Yanan; Chen, Yihong; Oganesyan, Natalia; Ruan, Biao; O'Brochta, David; Bryan, Philip N; Orban, John

    2012-12-01

    Plasmodium subtilisin 2 (Sub2) is a multidomain protein that plays an important role in malaria infection. Here, we describe the solution NMR structure of a conserved region of the inhibitory prodomain of Sub2 from Plasmodium falciparum, termed prosub2. Despite the absence of any detectable sequence homology, the protozoan prosub2 has structural similarity to bacterial and mammalian subtilisin-like prodomains. Comparison with the three-dimensional structures of these other prodomains suggests a likely binding interface with the catalytic domain of Sub2 and provides insights into the locations of primary and secondary processing sites in Plasmodium prodomains. PMID:23011838

  16. Parasitological and new molecular-phylogenetic characterization of the malaria parasite Plasmodium tejerai in South American penguins.

    PubMed

    Silveira, Patricia; Belo, Nayara O; Lacorte, Gustavo A; Kolesnikovas, Cristiane K M; Vanstreels, Ralph E T; Steindel, Mrio; Cato-Dias, Jos Luiz; Valki?nas, Gediminas; Braga, Erika M

    2013-04-01

    This study is the first report on mortality of Spheniscus magellanicus, penguin of South America, caused by Plasmodium tejerai, which was identified using morphological and molecular analyses. Blood stages (trophozoites, meronts and gametocytes) were reported and illustrated. The necropsy revealed marked splenomegaly and pulmonary edema, as well as moderate hepatomegaly and hydropericardium. The histopathology revealed the presence of tissue meronts in the macrophages and endothelial cells of multiple organs. The molecular analyses showed 5.6% of genetic divergence in cytochrome b gene between P. tejerai and Plasmodium relictum. Morphology of blood and tissue stages of P. tejerai is similar to P. relictum; the distinction between these two species requires experience in the identification of avian Plasmodium species. Molecular studies associated with reliably identified morphological species are useful for barcoding and comparisons with previous studies of wildlife malaria infections as well as for posterior phylogenetic and phylogeographic studies. S. magellanicus is a new host record of P. tejerai, which is the virulent parasite and worth more attention in avian conservation and veterinary medicine projects in South America. PMID:23269202

  17. SNP Genotyping Identifies New Signatures of Selection in a Deep Sample of West African Plasmodium falciparum Malaria Parasites

    PubMed Central

    Amambua-Ngwa, Alfred; Park, Daniel J.; Volkman, Sarah K.; Barnes, Kayla G.; Bei, Amy K.; Lukens, Amanda K.; Sene, Papa; Van Tyne, Daria; Ndiaye, Daouda; Wirth, Dyann F.; Conway, David J.; Neafsey, Daniel E.; Schaffner, Stephen F.

    2012-01-01

    We used a high-density single-nucleotide polymorphism array to genotype 75 Plasmodium falciparum isolates recently collected from Senegal and The Gambia to search for signals of selection in this malaria endemic region. We found little geographic or temporal stratification of the genetic diversity among the sampled parasites. Through application of the iHS and REHH haplotype-based tests for positive selection, we found evidence of recent selective sweeps at a known drug resistance locus, at several known antigenic loci, and at several genomic regions not previously identified as sites of recent selection. We discuss the value of deep population-specific genomic analyses for identifying selection signals within sampled endemic populations of parasites, which may correspond to local selection pressures such as distinctive therapeutic regimes or mosquito vectors. PMID:22688945

  18. Matrix Metalloproteinase-9 and Haemozoin: Wedding Rings for Human Host and Plasmodium falciparum Parasite in Complicated Malaria

    PubMed Central

    Prato, Mauro; Giribaldi, Giuliana

    2011-01-01

    It is generally accepted that the combination of both Plasmodium falciparum parasite and human host factors is involved in the pathogenesis of complicated severe malaria, including cerebral malaria (CM). Among parasite products, the malarial pigment haemozoin (HZ) has been shown to impair the functions of mononuclear and endothelial cells. Different CM models were associated with enhanced levels of matrix metalloproteinases (MMPs), a family of proteolytic enzymes able to disrupt subendothelial basement membrane and tight junctions and shed, activate, or inactivate cytokines, chemokines, and other MMPs through cleavage from their precursors. Among MMPs, a good candidate for targeted therapy might be MMP-9, whose mRNA and protein expression enhancement as well as direct proenzyme activation by HZ have been recently investigated in a series of studies by our group and others. In the present paper the role of HZ and MMP-9 in complicated malaria, as well as their interactions, will be discussed. PMID:21760809

  19. Profiling the anti-protozoal activity of anti-cancer HDAC inhibitors against Plasmodium and Trypanosoma parasites.

    PubMed

    Engel, Jessica A; Jones, Amy J; Avery, Vicky M; Sumanadasa, Subathdrage D M; Ng, Susanna S; Fairlie, David P; Adams, Tina S; Andrews, Katherine T

    2015-12-01

    Histone deacetylase (HDAC) enzymes work together with histone acetyltransferases (HATs) to reversibly acetylate both histone and non-histone proteins. As a result, these enzymes are involved in regulating chromatin structure and gene expression as well as other important cellular processes. HDACs are validated drug targets for some types of cancer, with four HDAC inhibitors clinically approved. However, they are also showing promise as novel drug targets for other indications, including malaria and other parasitic diseases. In this study the invitro activity of four anti-cancer HDAC inhibitors was examined against parasites that cause malaria and trypanosomiasis. Three of these inhibitors, suberoylanilide hydroxamic acid (SAHA; vorinostat()), romidepsin (Istodax()) and belinostat (Beleodaq()), are clinically approved for the treatment of T-cell lymphoma, while the fourth, panobinostat, has recently been approved for combination therapy use in certain patients with multiple myeloma. All HDAC inhibitors were found to inhibit the growth of asexual-stage Plasmodium falciparum malaria parasites in the nanomolar range (IC50 10-200nM), while only romidepsin was active at sub-?M concentrations against bloodstream form Trypanosoma brucei brucei parasites (IC50 35nM). The compounds were found to have some selectivity for malaria parasites compared with mammalian cells, but were not selective for trypanosome parasites versus mammalian cells. All compounds caused hyperacetylation of histone and non-histone proteins in P.falciparum asexual stage parasites and inhibited deacetylase activity in P.falciparum nuclear extracts in addition to recombinant PfHDAC1 activity. P.falciparum histone hyperacetylation data indicate that HDAC inhibitors may differentially affect the acetylation profiles of histone H3 and H4. PMID:26199860

  20. Profiling the anti-protozoal activity of anti-cancer HDAC inhibitors against Plasmodium and Trypanosoma parasites

    PubMed Central

    Engel, Jessica A.; Jones, Amy J.; Avery, Vicky M.; Sumanadasa, Subathdrage D.M.; Ng, Susanna S.; Fairlie, David P.; Adams, Tina S.; Andrews, Katherine T.

    2015-01-01

    Histone deacetylase (HDAC) enzymes work together with histone acetyltransferases (HATs) to reversibly acetylate both histone and non-histone proteins. As a result, these enzymes are involved in regulating chromatin structure and gene expression as well as other important cellular processes. HDACs are validated drug targets for some types of cancer, with four HDAC inhibitors clinically approved. However, they are also showing promise as novel drug targets for other indications, including malaria and other parasitic diseases. In this study the in vitro activity of four anti-cancer HDAC inhibitors was examined against parasites that cause malaria and trypanosomiasis. Three of these inhibitors, suberoylanilide hydroxamic acid (SAHA; vorinostat®), romidepsin (Istodax®) and belinostat (Beleodaq®), are clinically approved for the treatment of T-cell lymphoma, while the fourth, panobinostat, has recently been approved for combination therapy use in certain patients with multiple myeloma. All HDAC inhibitors were found to inhibit the growth of asexual-stage Plasmodium falciparum malaria parasites in the nanomolar range (IC50 10–200 nM), while only romidepsin was active at sub-μM concentrations against bloodstream form Trypanosoma brucei brucei parasites (IC50 35 nM). The compounds were found to have some selectivity for malaria parasites compared with mammalian cells, but were not selective for trypanosome parasites versus mammalian cells. All compounds caused hyperacetylation of histone and non-histone proteins in P. falciparum asexual stage parasites and inhibited deacetylase activity in P. falciparum nuclear extracts in addition to recombinant PfHDAC1 activity. P. falciparum histone hyperacetylation data indicate that HDAC inhibitors may differentially affect the acetylation profiles of histone H3 and H4. PMID:26199860

  1. Manifold habitat effects on the prevalence and diversity of avian blood parasites

    PubMed Central

    Sehgal, Ravinder N.M.

    2015-01-01

    Habitats are rapidly changing across the planet and the consequences will have major and long-lasting effects on wildlife and their parasites. Birds harbor many types of blood parasites, but because of their relatively high prevalence and ease of diagnosis, it is the haemosporidiansPlasmodium, Haemoproteus, and Leucocytozoon – that are the best studied in terms of ecology and evolution. For parasite transmission to occur, environmental conditions must be permissive, and given the many constraints on the competency of parasites, vectors and hosts, it is rather remarkable that these parasites are so prevalent and successful. Over the last decade, a rapidly growing body of literature has begun to clarify how environmental factors affect birds and the insects that vector their hematozoan parasites. Moreover, several studies have modeled how anthropogenic effects such as global climate change, deforestation and urbanization will impact the dynamics of parasite transmission. This review highlights recent research that impacts our understanding of how habitat and environmental changes can affect the distribution, diversity, prevalence and parasitemia of these avian blood parasites. Given the importance of environmental factors on transmission, it remains essential that researchers studying avian hematozoa document abiotic factors such as temperature, moisture and landscape elements. Ultimately, this continued research has the potential to inform conservation policies and help avert the loss of bird species and threatened habitats. PMID:26835250

  2. Manifold habitat effects on the prevalence and diversity of avian blood parasites.

    PubMed

    Sehgal, Ravinder N M

    2015-12-01

    Habitats are rapidly changing across the planet and the consequences will have major and long-lasting effects on wildlife and their parasites. Birds harbor many types of blood parasites, but because of their relatively high prevalence and ease of diagnosis, it is the haemosporidians - Plasmodium, Haemoproteus, and Leucocytozoon - that are the best studied in terms of ecology and evolution. For parasite transmission to occur, environmental conditions must be permissive, and given the many constraints on the competency of parasites, vectors and hosts, it is rather remarkable that these parasites are so prevalent and successful. Over the last decade, a rapidly growing body of literature has begun to clarify how environmental factors affect birds and the insects that vector their hematozoan parasites. Moreover, several studies have modeled how anthropogenic effects such as global climate change, deforestation and urbanization will impact the dynamics of parasite transmission. This review highlights recent research that impacts our understanding of how habitat and environmental changes can affect the distribution, diversity, prevalence and parasitemia of these avian blood parasites. Given the importance of environmental factors on transmission, it remains essential that researchers studying avian hematozoa document abiotic factors such as temperature, moisture and landscape elements. Ultimately, this continued research has the potential to inform conservation policies and help avert the loss of bird species and threatened habitats. PMID:26835250

  3. Maintenance of phenotypic diversity within a set of virulence encoding genes of the malaria parasite Plasmodium falciparum.

    PubMed

    Holding, Thomas; Recker, Mario

    2015-12-01

    Infection by the human malaria parasite Plasmodium falciparum results in a broad spectrum of clinical outcomes, ranging from severe and potentially life-threatening malaria to asymptomatic carriage. In a process of naturally acquired immunity, individuals living in malaria-endemic regions build up a level of clinical protection, which attenuates infection severity in an exposure-dependent manner. Underlying this shift in the immunoepidemiology as well as the observed range in malaria pathogenesis is the var multigene family and the phenotypic diversity embedded within. The var gene-encoded surface proteins Plasmodium falciparum erythrocyte membrane protein 1 mediate variant-specific binding of infected red blood cells to a diverse set of host receptors that has been linked to specific disease manifestations, including cerebral and pregnancy-associated malaria. Here, we show that cross-reactive immune responses, which minimize the within-host benefit of each additionally expressed gene during infection, can cause selection for maximum phenotypic diversity at the genome level. We further show that differential functional constraints on protein diversification stably maintain uneven ratios between phenotypic groups, in line with empirical observation. Our results thus suggest that the maintenance of phenotypic diversity within P. falciparum is driven by an evolutionary trade-off that optimizes between within-host parasite fitness and between-host selection pressure. PMID:26674193

  4. Baculovirus-Vectored Multistage Plasmodium vivax Vaccine Induces Both Protective and Transmission-Blocking Immunities against Transgenic Rodent Malaria Parasites

    PubMed Central

    Mizutani, Masanori; Iyori, Mitsuhiro; Blagborough, Andrew M.; Fukumoto, Shinya; Funatsu, Tomohiro; Sinden, Robert E.

    2014-01-01

    A multistage malaria vaccine targeting the pre-erythrocytic and sexual stages of Plasmodium could effectively protect individuals against infection from mosquito bites and provide transmission-blocking (TB) activity against the sexual stages of the parasite, respectively. This strategy could help prevent malaria infections in individuals and, on a larger scale, prevent malaria transmission in communities of endemicity. Here, we describe the development of a multistage Plasmodium vivax vaccine which simultaneously expresses P. vivax circumsporozoite protein (PvCSP) and P25 (Pvs25) protein of this species as a fusion protein, thereby acting as a pre-erythrocytic vaccine and a TB vaccine, respectively. A new-concept vaccine platform based on the baculovirus dual-expression system (BDES) was evaluated. The BDES-Pvs25-PvCSP vaccine displayed correct folding of the Pvs25-PvCSP fusion protein on the viral envelope and was highly expressed upon transduction of mammalian cells in vitro. This vaccine induced high levels of antibodies to Pvs25 and PvCSP and elicited protective (43%) and TB (82%) efficacies against transgenic P. berghei parasites expressing the corresponding P. vivax antigens in mice. Our data indicate that our BDES, which functions as both a subunit and DNA vaccine, can offer a promising multistage vaccine capable of delivering a potent antimalarial pre-erythrocytic and TB response via a single immunization regimen. PMID:25092912

  5. Maintenance of phenotypic diversity within a set of virulence encoding genes of the malaria parasite Plasmodium falciparum

    PubMed Central

    Holding, Thomas; Recker, Mario

    2015-01-01

    Infection by the human malaria parasite Plasmodium falciparum results in a broad spectrum of clinical outcomes, ranging from severe and potentially life-threatening malaria to asymptomatic carriage. In a process of naturally acquired immunity, individuals living in malaria-endemic regions build up a level of clinical protection, which attenuates infection severity in an exposure-dependent manner. Underlying this shift in the immunoepidemiology as well as the observed range in malaria pathogenesis is the var multigene family and the phenotypic diversity embedded within. The var gene-encoded surface proteins Plasmodium falciparum erythrocyte membrane protein 1 mediate variant-specific binding of infected red blood cells to a diverse set of host receptors that has been linked to specific disease manifestations, including cerebral and pregnancy-associated malaria. Here, we show that cross-reactive immune responses, which minimize the within-host benefit of each additionally expressed gene during infection, can cause selection for maximum phenotypic diversity at the genome level. We further show that differential functional constraints on protein diversification stably maintain uneven ratios between phenotypic groups, in line with empirical observation. Our results thus suggest that the maintenance of phenotypic diversity within P. falciparum is driven by an evolutionary trade-off that optimizes between within-host parasite fitness and between-host selection pressure. PMID:26674193

  6. Expression of PD-1/LAG-3 and cytokine production by CD4(+) T cells during infection with Plasmodium parasites.

    PubMed

    Doe, Henrietta T; Kimura, Daisuke; Miyakoda, Mana; Kimura, Kazumi; Akbari, Masoud; Yui, Katsuyuki

    2016-02-01

    CD4(+) T cells play critical roles in protection against the blood stage of malarial infection; however, their uncontrolled activation can be harmful to the host. In this study, in which rodent models of Plasmodium parasites were used, the expression of inhibitory receptors on activated CD4(+) T cells and their cytokine production was compared with their expression in a bacterial and another protozoan infection. CD4(+) T cells from mice infected with P. yoelii 17XL, P yoelii 17XNL, P. chabaudi, P. vinckei and P. berghei expressed the inhibitory receptors, PD-1 and LAG-3, as early as 6 days after infection, whereas those from either Listeria monocytogenes- or Leishmania major-infected mice did not. In response to T-cell receptor stimulation, CD4(+) T cells from mice infected with all the pathogens under study produced high concentrations of IFN-?. IL-2 production was reduced in mice infected with Plasmodium species, but not in those infected with Listeria or Leishmania. In vitro blockade of the interaction between PD-1 and its ligands resulted in increased IFN-? production in response to Plasmodium antigens, implying that PD-1 expressed on activated CD4(+) T cells actively inhibits T cell immune responses. Studies using Myd88(-/-) , Trif(-/-) and Irf3(-/-) mice showed that induction of these CD4(+) T cells and their ability to produce cytokines is largely independent of TLR signaling. These studies suggest that expression of the inhibitory receptors PD-1 and LAG-3 on CD4(+) T cells and their reduced IL-2 production are common characteristic features of Plasmodium infection. PMID:26696540

  7. Detailed characterization of a cyclophilin from the human malaria parasite Plasmodium falciparum.

    PubMed Central

    Berriman, M; Fairlamb, A H

    1998-01-01

    Cyclosporin (Cs) A has pronounced antimalarial activity in vitro and in vivo. In other organisms, the drug is thought to exert its effects either by inhibiting the peptidylprolyl cis/trans isomerase activity of cyclophilin (CyP) or by forming a CyP-CsA complex that inhibits the phosphatase activity of calcineurin. We have cloned and overexpressed in Escherichia coli a gene encoding a CyP from Plasmodium falciparum (PfCyP19) that is located on chromosome 3. The sequence of PfCyP19 shows remarkable sequence identity with human CyPA and, unlike the two previously identified CyPs from P. falciparum, PfCyP19 has no signal peptide or N-terminal sequence extension, suggesting a cytosolic localization. All the residues implicated in the recognition of the synthetic substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide are conserved, resulting in characteristically high Michaelis-Menten and specificity constants (Km>>120 microM, kcat/Km=1.2x10(7) M-1.s-1 respectively). As the first line in the functional characterization of this enzyme, we have assessed its binding affinity for CsA. In accordance with its tryptophan-containing CsA-binding domain, PfCyP19 binds CsA with high affinity (Kd=13 nM, Ki=6.9 nM). Twelve CsA analogues were also found to possess Ki values similar to CsA, with the notable exceptions of Val2-Cs (Ki=218 nM) and Thr2-Cs (Ki=690 nM). The immunosuppressants rapamycin and FK506 were inactive as inhibitors, consistent with other members of the CyP family of rotamases. The CsA analogues were also assessed as inhibitors of P. falciparum growth in vitro. Although their antimalarial activity did not correlate with inhibition of enzyme activity, residues 3 and 4 of CsA appeared to be important for inhibition of parasite growth and residues 1 and 2 for PfCyP19 inhibition. We propose that a malarial CyP-CsA complex presents residues 3 and 4 as part of an 'effector surface' for recognition by a downstream target, similar to the proposed mechanism for T-cell immunosuppression. PMID:9716503

  8. Genome-Wide Collation of the Plasmodium falciparum WDR Protein Superfamily Reveals Malarial Parasite-Specific Features.

    PubMed

    Chahar, Priyanka; Kaushik, Manjeri; Gill, Sarvajeet Singh; Gakhar, Surendra Kumar; Gopalan, Natrajan; Datt, Manish; Sharma, Amit; Gill, Ritu

    2015-01-01

    Despite a significant drop in malaria deaths during the past decade, malaria continues to be one of the biggest health problems around the globe. WD40 repeats (WDRs) containing proteins comprise one of the largest and functionally diverse protein superfamily in eukaryotes, acting as scaffolds for assembling large protein complexes. In the present study, we report an extensive in silico analysis of the WDR gene family in human malaria parasite Plasmodium falciparum. Our genome-wide identification has revealed 80 putative WDR genes in P. falciparum (PfWDRs). Five distinct domain compositions were discovered in Plasmodium as compared to the human host. Notably, 31 PfWDRs were annotated/re-annotated on the basis of their orthologs in other species. Interestingly, most PfWDRs were larger as compared to their human homologs highlighting the presence of parasite-specific insertions. Fifteen PfWDRs appeared specific to the Plasmodium with no assigned orthologs. Expression profiling of PfWDRs revealed a mixture of linear and nonlinear relationships between transcriptome and proteome, and only nine PfWDRs were found to be stage-specific. Homology modeling identified conservation of major binding sites in PfCAF-1 and PfRACK. Protein-protein interaction network analyses suggested that PfWDRs are highly connected proteins with ~1928 potential interactions, supporting their role as hubs in cellular networks. The present study highlights the roles and relevance of the WDR family in P. falciparum, and identifies unique features that lay a foundation for further experimental dissection of PfWDRs. PMID:26043001

  9. Genome-Wide Collation of the Plasmodium falciparum WDR Protein Superfamily Reveals Malarial Parasite-Specific Features

    PubMed Central

    Chahar, Priyanka; Kaushik, Manjeri; Gill, Sarvajeet Singh; Gakhar, Surendra Kumar; Gopalan, Natrajan; Datt, Manish; Sharma, Amit; Gill, Ritu

    2015-01-01

    Despite a significant drop in malaria deaths during the past decade, malaria continues to be one of the biggest health problems around the globe. WD40 repeats (WDRs) containing proteins comprise one of the largest and functionally diverse protein superfamily in eukaryotes, acting as scaffolds for assembling large protein complexes. In the present study, we report an extensive in silico analysis of the WDR gene family in human malaria parasite Plasmodium falciparum. Our genome-wide identification has revealed 80 putative WDR genes in P. falciparum (PfWDRs). Five distinct domain compositions were discovered in Plasmodium as compared to the human host. Notably, 31 PfWDRs were annotated/re-annotated on the basis of their orthologs in other species. Interestingly, most PfWDRs were larger as compared to their human homologs highlighting the presence of parasite-specific insertions. Fifteen PfWDRs appeared specific to the Plasmodium with no assigned orthologs. Expression profiling of PfWDRs revealed a mixture of linear and nonlinear relationships between transcriptome and proteome, and only nine PfWDRs were found to be stage-specific. Homology modeling identified conservation of major binding sites in PfCAF-1 and PfRACK. Protein-protein interaction network analyses suggested that PfWDRs are highly connected proteins with ~1928 potential interactions, supporting their role as hubs in cellular networks. The present study highlights the roles and relevance of the WDR family in P. falciparum, and identifies unique features that lay a foundation for further experimental dissection of PfWDRs. PMID:26043001

  10. Identification and Functional Analysis of the Primary Pantothenate Transporter, PfPAT, of the Human Malaria Parasite Plasmodium falciparum*

    PubMed Central

    Augagneur, Yoann; Jaubert, Lise; Schiavoni, Matthieu; Pachikara, Niseema; Garg, Aprajita; Usmani-Brown, Sahar; Wesolowski, Donna; Zeller, Skye; Ghosal, Abhisek; Cornillot, Emmanuel; Said, Hamid M.; Kumar, Priti; Altman, Sidney; Ben Mamoun, Choukri

    2013-01-01

    The human malaria parasite Plasmodium falciparum is absolutely dependent on the acquisition of host pantothenate for its development within human erythrocytes. Although the biochemical properties of this transport have been characterized, the molecular identity of the parasite-encoded pantothenate transporter remains unknown. Here we report the identification and functional characterization of the first protozoan pantothenate transporter, PfPAT, from P. falciparum. We show using cell biological, biochemical, and genetic analyses that this transporter is localized to the parasite plasma membrane and plays an essential role in parasite intraerythrocytic development. We have targeted PfPAT to the yeast plasma membrane and showed that the transporter complements the growth defect of the yeast fen2Δ pantothenate transporter-deficient mutant and mediates the entry of the fungicide drug, fenpropimorph. Our studies in P. falciparum revealed that fenpropimorph inhibits the intraerythrocytic development of both chloroquine- and pyrimethamine-resistant P. falciparum strains with potency equal or better than that of currently available pantothenate analogs. The essential function of PfPAT and its ability to deliver both pantothenate and fenpropimorph makes it an attractive target for the development and delivery of new classes of antimalarial drugs. PMID:23729665

  11. Drug-induced permeabilization of parasite's digestive vacuole is a key trigger of programmed cell death in Plasmodium falciparum

    PubMed Central

    Ch'Ng, J-H; Liew, K; Goh, A S-P; Sidhartha, E; Tan, K S-W

    2011-01-01

    Having previously characterized chloroquine (CQ)-induced programmed cell death (PCD) hallmarks in the malaria parasite Plasmodium falciparum and delineating a pathway linking these features, the roles of non-classical mediators were investigated in this paper. It was shown that the later stages of this pathway are Ca2+-dependent and transcriptionally regulated. Moreover, it was demonstrated for the first time that micromolar concentrations of CQ partially permeabilized the parasite's digestive vacuole (DV) membrane and that this important upstream event appears to precede mitochondrial dysfunction. This permeabilization of the DV occurred without rupture of the DV membrane and was reminiscent of lysosome-mediated cell death in mammalian cells. As such micromolar concentrations of CQ are found in the patient's plasma after initial CQ loading, this alludes to a clinically relevant antimalarial mechanism of the drug which has yet to be recognized. Furthermore, other non-antimalarial' lysosomotropic compounds were also shown to cause DV permeabilization, triggering PCD in both CQ-sensitive and -resistant parasites. These findings present new avenues for antimalarial developments, which induce DV destabilization to kill parasites. PMID:21993392

  12. Mutations in the Plasmodium falciparum chloroquine resistance transporter, PfCRT, enlarge the parasite's food vacuole and alter drug sensitivities.

    PubMed

    Pulcini, Serena; Staines, Henry M; Lee, Andrew H; Shafik, Sarah H; Bouyer, Guillaume; Moore, Catherine M; Daley, Daniel A; Hoke, Matthew J; Altenhofen, Lindsey M; Painter, Heather J; Mu, Jianbing; Ferguson, David J P; Llins, Manuel; Martin, Rowena E; Fidock, David A; Cooper, Roland A; Krishna, Sanjeev

    2015-01-01

    Mutations in the Plasmodium falciparum chloroquine resistance transporter, PfCRT, are the major determinant of chloroquine resistance in this lethal human malaria parasite. Here, we describe P. falciparum lines subjected to selection by amantadine or blasticidin that carry PfCRT mutations (C101F or L272F), causing the development of enlarged food vacuoles. These parasites also have increased sensitivity to chloroquine and some other quinoline antimalarials, but exhibit no or minimal change in sensitivity to artemisinins, when compared with parental strains. A transgenic parasite line expressing the L272F variant of PfCRT confirmed this increased chloroquine sensitivity and enlarged food vacuole phenotype. Furthermore, the introduction of the C101F or L272F mutation into a chloroquine-resistant variant of PfCRT reduced the ability of this protein to transport chloroquine by approximately 93 and 82%, respectively, when expressed in Xenopus oocytes. These data provide, at least in part, a mechanistic explanation for the increased sensitivity of the mutant parasite lines to chloroquine. Taken together, these findings provide new insights into PfCRT function and PfCRT-mediated drug resistance, as well as the food vacuole, which is an important target of many antimalarial drugs. PMID:26420308

  13. Inhibition of malaria parasite Plasmodium falciparum development by crotamine, a cell penetrating peptide from the snake venom.

    PubMed

    El Chamy Maluf, S; Dal Mas, C; Oliveira, E B; Melo, P M; Carmona, A K; Gazarini, M L; Hayashi, M A F

    2016-04-01

    We show here that crotamine, a polypeptide from the South American rattlesnake venom with cell penetrating and selective anti-fungal and anti-tumoral properties, presents a potent anti-plasmodial activity in culture. Crotamine inhibits the development of the Plasmodium falciparum parasites in a dose-dependent manner [IC50 value of 1.87μM], and confocal microscopy analysis showed a selective internalization of fluorescent-labeled crotamine into P. falciparum infected erythrocytes, with no detectable fluorescence in uninfected healthy erythrocytes. In addition, similarly to the crotamine cytotoxic effects, the mechanism underlying the anti-plasmodial activity may involve the disruption of parasite acidic compartments H(+) homeostasis. In fact, crotamine promoted a reduction of parasites organelle fluorescence loaded with the lysosomotropic fluorochrome acridine orange, in the same way as previously observed mammalian tumoral cells. Taken together, we show for the first time crotamine not only compromised the metabolism of the P. falciparum, but this toxin also inhibited the parasite growth. Therefore, we suggest this snake polypeptide as a promising lead molecule for the development of potential new molecules, namely peptidomimetics, with selectivity for infected erythrocytes and ability to inhibit the malaria infection by its natural affinity for acid vesicles. PMID:26806200

  14. Specialist enemies, generalist weapons and the potential spread of exotic pathogens: malaria parasites in a highly invasive bird.

    PubMed

    Clark, Nicholas J; Olsson-Pons, Sophie; Ishtiaq, Farah; Clegg, Sonya M

    2015-12-01

    Pathogens can influence the success of invaders. The Enemy Release Hypothesis predicts invaders encounter reduced pathogen abundance and diversity, while the Novel Weapons Hypothesis predicts invaders carry novel pathogens that spill over to competitors. We tested these hypotheses using avian malaria (haemosporidian) infections in the invasive myna (Acridotheres tristis), which was introduced to southeastern Australia from India and was secondarily expanded to the eastern Australian coast. Mynas and native Australian birds were screened in the secondary introduction range for haemosporidians (Plasmodium and Haemoproteus spp.) and results were combined with published data from the myna's primary introduction and native ranges. We compared malaria prevalence and diversity across myna populations to test for Enemy Release and used phylogeographic analyses to test for exotic strains acting as Novel Weapons. Introduced mynas carried significantly lower parasite diversity than native mynas and significantly lower Haemoproteus prevalence than native Australian birds. Despite commonly infecting native species that directly co-occur with mynas, Haemoproteus spp. were only recorded in introduced mynas in the primary introduction range and were apparently lost during secondary expansion. In contrast, Plasmodium infections were common in all ranges and prevalence was significantly higher in both introduced and native mynas than in native Australian birds. Introduced mynas carried several exotic Plasmodium lineages that were shared with native mynas, some of which also infected native Australian birds and two of which are highly invasive in other bioregions. Our results suggest that introduced mynas may benefit through escape from Haemoproteus spp. while acting as important reservoirs for Plasmodium spp., some of which are known exotic lineages. PMID:26433143

  15. The relationship between the Plasmodium falciparum parasite ratio in childhood and climate estimates of malaria transmission in Kenya

    PubMed Central

    Omumbo, Judith A; Hay, Simon I; Guerra, Carlos A; Snow, Robert W

    2004-01-01

    Background Plasmodium falciparum morbid and fatal risks are considerably higher in areas supporting parasite prevalence ≥25%, when compared with low transmission areas supporting parasite prevalence below 25%. Recent descriptions of the health impacts of malaria in Africa are based upon categorical descriptions of a climate-driven fuzzy model of suitability (FCS) for stable transmission developed by the Mapping Malaria Risk in Africa collaboration (MARA). Methods An electronic and national search was undertaken to identify community-based parasite prevalence surveys in Kenya. Data from these surveys were matched using ArcView 3.2 to extract spatially congruent estimates of the FCS values generated by the MARA model. Levels of agreement between three classes used during recent continental burden estimations of parasite prevalence (0%, >0 – <25% and ≥25%) and three classes of FCS (0, >0 – <0.75 and ≥0.75) were tested using the kappa (k) statistic and examined as continuous variables to define better levels of agreement. Results Two hundred and seventeen independent parasite prevalence surveys undertaken since 1980 were identified during the search. Overall agreement between the three classes of parasite prevalence and FCS was weak although significant (k = 0.367, p < 0.0001). The overall correlation between the FCS and the parasite ratio when considered as continuous variables was also positive (0.364, p < 0.001). The margins of error were in the stable, endemic (parasite ratio ≥25%) class with 42% of surveys represented by an FCS <0.75. Reducing the FCS value criterion to ≥0.6 improved the classification of stable, endemic parasite ratio surveys. Zero values of FCS were not adequate discriminators of zero parasite prevalence. Conclusion Using the MARA model to categorically distinguish populations at differing intensities of malaria transmission in Kenya may under-represent those who are exposed to stable, endemic transmission and over-represent those at no risk. The MARA approach to defining FCS values of suitability for stable transmission represents our only contemporary continental level map of malaria in Africa but there is a need to redefine Africa's population at risk in accordance with both climatic and non-climatic determinants of P. falciparum transmission intensity to provide a more informed approach to estimating the morbid and fatal consequences of infection across the continent. PMID:15202945

  16. Probable macrophage origin of the lipopolysaccharide-induced cytostatic effect on intra-erythrocytic malarial parasites (Plasmodium vinckei)

    PubMed Central

    Rzepczyk, Christine M.

    1982-01-01

    This study showed that intra-erythrocytic Plasmodium vinckei parasites taken from either normal, irradiated, nude or splenectomized mice 78 hr after the injection of a small dose of bacterial lipopolysaccharide (LPS) incorporate hypoxanthine more slowly in an in vitro assay than parasites from saline-treated controls. The incorporation by parasites of isoleucine, which was also measured in some experiments, was similarly affected. However, this cytostatic effect on parasite metabolism was found to be markedly reduced in experiments with mice which had received an intravenous injection of silica dust 2830 hr before being injected with LPS. These findings indicate that macrophages, being radioresistant and silica-sensitive, are the source of the cytostatic effect. The present results also imply that T cells are not required in the response, and they show that the host cells mediating this response are not restricted to the spleen. It was also shown that an intravenous injection of a small dose of LPS into mice infected with P. vinckei 24 hr previously, could temporarily arrest the rise in parasitaemia in these animals, thereby prolonging their survival. This protection afforded by LPS was also found to be radioresistant and T-independent. It is suggested that the effect on parasitaemia seen in vivo and the cytostatic effect in vitro are both due to the release of a soluble factor from macrophages which is ultimately capable of causing intra-erythrocytic parasite death. P. vinckei-infected mice exhibited symptoms of endotoxaemia following the injection of LPS. However, no clear relationship was noted between the severity of the illness in the host and the cytostatic effect on the parasites. PMID:6282737

  17. Target evaluation of deoxyhypusine synthase from Theileria parva the neglected animal parasite and its relationship to Plasmodium.

    PubMed

    Njuguna, James T; von Koschitzky, Imke; Gerhardt, Heike; Lmmerhofer, Michael; Choucry, Ali; Pink, Mario; Schmitz-Spahnke, Simone; Bakheit, Mohammed A; Strube, Christina; Kaiser, Annette

    2014-08-01

    East Coast fever (ECF) is a tick-borne disease caused by the parasite Theileria parva which infects cattle. In Sub-Saharan Africa it leads to enormous economic costs. After a bite of a tick, sporozoites invade the host lymphocytes and develop into schizonts. At this stage the parasite transforms host lymphocytes resulting in the clonal expansion of infected lymphocytes. Animals develop a lymphoma like disorder after infection which is rapidly fatal. Hitherto, a few drugs of the quinone type can cure the disease. However, therapy can only be successful after early diagnosis. The genera Theileria and Plasmodium, which includes the causative agent of human malaria, are closely related apicomplexan parasites. Enzymes of the hypusine pathway, a posttranslational modification in eukaryotic initiation factor EIF-5A, have shown to be druggable targets in Plasmodium. We identified the first enzyme of the hypusine pathway from T. parva, the deoxyhypusine synthase (DHS), which is located on chromosome 2 of the Muguga strain. Transcription is significantly increased in schizonts. The expressed T. parva DHS reveals an open reading frame (ORF) of 370 amino acids after expression in Escherichia coli Rosetta cells with a molecular size of 41.26 kDa and a theoretical pI of 5.26. Screening of the Malaria Box which consists of 400 active compounds resulted in a novel heterocyclic compound with a guanyl spacer which reduced the activity of T. parva DHS to 45%. In sum, the guanyl residue seems to be an important lead structure for inhibition of Theileria DHS. Currently, more different guanyl analogues from the Malaria Box are tested in inhibitor experiments to determine their efficacy. PMID:24909679

  18. Studies on the effects of sida acuta and vetiveria zizanioides against the malarial vector, anopheles stephensi and malarial parasite, plasmodium berghei

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Methanolic extracts of Sida acuta and Vetiveria zizanioides leaves and root were studied for toxicity to Anopheles stephensi mosquitoes and to the malaria parasite Plasmodium berghei in mice. The extracts reduced parasitemia levels in mice by 17-69%, depending on extract concentration. Median le...

  19. Global warming will reshuffle the areas of high prevalence and richness of three genera of avian blood parasites.

    PubMed

    Prez-Rodrguez, Antn; de la Hera, Ivn; Fernndez-Gonzlez, Sofa; Prez-Tris, Javier

    2014-08-01

    The importance of parasitism for host populations depends on local parasite richness and prevalence: usually host individuals face higher infection risk in areas where parasites are most diverse, and host dispersal to or from these areas may have fitness consequences. Knowing how parasites are and will be distributed in space and time (in a context of global change) is thus crucial from both an ecological and a biological conservation perspective. Nevertheless, most research articles focus just on elaborating models of parasite distribution instead of parasite diversity. We produced distribution models of the areas where haemosporidian parasites are currently highly diverse (both at community and at within-host levels) and prevalent among Iberian populations of a model passerine host: the blackcap Sylvia atricapilla; and how these areas are expected to vary according to three scenarios of climate change. On the basis of these models, we analysed whether variation among populations in parasite richness or prevalence are expected to remain the same or change in the future, thereby reshuffling the geographic mosaic of host-parasite interactions as we observe it today. Our models predict a rearrangement of areas of high prevalence and richness of parasites in the future, with Haemoproteus and Leucocytozoon parasites (today the most diverse genera in blackcaps) losing areas of high diversity and Plasmodium parasites (the most virulent ones) gaining them. Likewise, the prevalence of multiple infections and parasite infracommunity richness would be reduced. Importantly, differences among populations in the prevalence and richness of parasites are expected to decrease in the future, creating a more homogeneous parasitic landscape. This predicts an altered geographic mosaic of host-parasite relationships, which will modify the interaction arena in which parasite virulence evolves. PMID:24488566

  20. In Vitro Activity of wALADin Benzimidazoles against Different Life Cycle Stages of Plasmodium Parasites

    PubMed Central

    Lentz, Christian S.; Sattler, Julia M.; Fendler, Martina; Gottwalt, Simon; Halls, Victoria S.; Strassel, Silke; Arriens, Sandra; Hannam, Jeffrey S.; Specht, Sabine; Famulok, Michael; Mueller, Ann-Kristin; Hoerauf, Achim

    2014-01-01

    wALADin1 benzimidazoles are specific inhibitors of ?-aminolevulinic acid dehydratase from Wolbachia endobacteria of filarial nematodes. We report that wALADin1 and two derivatives killed blood stage Plasmodium falciparum in vitro (50% inhibitory concentrations, 39, 7.7, and 12.8 ?M, respectively). One of these derivatives inhibited gliding motility of Plasmodium berghei ANKA infectious sporozoites with nanomolar affinity and blocked invasion into hepatocytes but did not affect intrahepatocytic replication. Hence, wALADin1 benzimidazoles are tools to study gliding motility and potential antiplasmodial drug candidates. PMID:25313210

  1. Earthworm-mediated synthesis of silver nanoparticles: A potent tool against hepatocellular carcinoma, Plasmodium falciparum parasites and malaria mosquitoes.

    PubMed

    Jaganathan, Anitha; Murugan, Kadarkarai; Panneerselvam, Chellasamy; Madhiyazhagan, Pari; Dinesh, Devakumar; Vadivalagan, Chithravel; Aziz, Al Thabiani; Chandramohan, Balamurugan; Suresh, Udaiyan; Rajaganesh, Rajapandian; Subramaniam, Jayapal; Nicoletti, Marcello; Higuchi, Akon; Alarfaj, Abdullah A; Munusamy, Murugan A; Kumar, Suresh; Benelli, Giovanni

    2016-06-01

    The development of parasites and pathogens resistant to synthetic drugs highlighted the needing of novel, eco-friendly and effective control approaches. Recently, metal nanoparticles have been proposed as highly effective tools towards cancer cells and Plasmodium parasites. In this study, we synthesized silver nanoparticles (EW-AgNP) using Eudrilus eugeniae earthworms as reducing and stabilizing agents. EW-AgNP showed plasmon resonance reduction in UV-vis spectrophotometry, the functional groups involved in the reduction were studied by FTIR spectroscopy, while particle size and shape was analyzed by FESEM. The effect of EW-AgNP on in vitro HepG2 cell proliferation was measured using MTT assays. Apoptosis assessed by flow cytometry showed diminished endurance of HepG2 cells and cytotoxicity in a dose-dependent manner. EW-AgNP were toxic to Anopheles stephensi larvae and pupae, LC50 were 4.8ppm (I), 5.8ppm (II), 6.9ppm (III), 8.5ppm (IV), and 15.5ppm (pupae). The antiplasmodial activity of EW-AgNP was evaluated against CQ-resistant (CQ-r) and CQ-sensitive (CQ-s) strains of Plasmodium falciparum. EW-AgNP IC50 were 49.3μg/ml (CQ-s) and 55.5μg/ml (CQ-r), while chloroquine IC50 were 81.5μg/ml (CQ-s) and 86.5μg/ml (CQ-r). EW-AgNP showed a valuable antibiotic potential against important pathogenic bacteria and fungi. Concerning non-target effects of EW-AgNP against mosquito natural enemies, the predation efficiency of the mosquitofish Gambusia affinis towards the II and II instar larvae of A. stephensi was 68.50% (II) and 47.00% (III), respectively. In EW-AgNP-contaminated environments, predation was boosted to 89.25% (II) and 70.75% (III), respectively. Overall, this research highlighted the EW-AgNP potential against hepatocellular carcinoma, Plasmodium parasites and mosquito vectors, with little detrimental effects on mosquito natural enemies. PMID:26873539

  2. Potential of lichen secondary metabolites against Plasmodium liver stage parasites with FAS-II as the potential target.

    PubMed

    Lauinger, Ina L; Vivas, Livia; Perozzo, Remo; Stairiker, Christopher; Tarun, Alice; Zloh, Mire; Zhang, Xujie; Xu, Hua; Tonge, Peter J; Franzblau, Scott G; Pham, Duc-Hung; Esguerra, Camila V; Crawford, Alexander D; Maes, Louis; Tasdemir, Deniz

    2013-06-28

    Chemicals targeting the liver stage (LS) of the malaria parasite are useful for causal prophylaxis of malaria. In this study, four lichen metabolites, evernic acid (1), vulpic acid (2), psoromic acid (3), and (+)-usnic acid (4), were evaluated against LS parasites of Plasmodium berghei. Inhibition of P. falciparum blood stage (BS) parasites was also assessed to determine stage specificity. Compound 4 displayed the highest LS activity and stage specificity (LS IC50 value 2.3 μM, BS IC50 value 47.3 μM). The compounds 1-3 inhibited one or more enzymes (PfFabI, PfFabG, and PfFabZ) from the plasmodial fatty acid biosynthesis (FAS-II) pathway, a potential drug target for LS activity. To determine species specificity and to clarify the mechanism of reported antibacterial effects, 1-4 were also evaluated against FabI homologues and whole cells of various pathogens (S. aureus, E. coli, M. tuberculosis). Molecular modeling studies suggest that lichen acids act indirectly via binding to allosteric sites on the protein surface of the FAS-II enzymes. Potential toxicity of compounds was assessed in human hepatocyte and cancer cells (in vitro) as well as in a zebrafish model (in vivo). This study indicates the therapeutic and prophylactic potential of lichen metabolites as antibacterial and antiplasmodial agents. PMID:23806111

  3. Expression of senescent antigen on erythrocytes infected with a knobby variant of the human malaria parasite Plasmodium falciparum

    SciTech Connect

    Winograd, E.; Greenan, J.R.T.; Sherman, I.W.

    1987-04-01

    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.

  4. Multicolor bioluminescence boosts malaria research: quantitative dual-color assay and single-cell imaging in Plasmodium falciparum parasites.

    PubMed

    Cevenini, Luca; Camarda, Grazia; Michelini, Elisa; Siciliano, Giulia; Calabretta, Maria Maddalena; Bona, Roberta; Kumar, T R Santha; Cara, Andrea; Branchini, Bruce R; Fidock, David A; Roda, Aldo; Alano, Pietro

    2014-09-01

    New reliable and cost-effective antimalarial drug screening assays are urgently needed to identify drugs acting on different stages of the parasite Plasmodium falciparum, and particularly those responsible for human-to-mosquito transmission, that is, the P. falciparum gametocytes. Low Z' factors, narrow dynamic ranges, and/or extended assay times are commonly reported in current gametocyte assays measuring gametocyte-expressed fluorescent or luciferase reporters, endogenous ATP levels, activity of gametocyte enzymes, or redox-dependent dye fluorescence. We hereby report on a dual-luciferase gametocyte assay with immature and mature P. falciparum gametocyte stages expressing red and green-emitting luciferases from Pyrophorus plagiophthalamus under the control of the parasite sexual stage-specific pfs16 gene promoter. The assay was validated with reference antimalarial drugs and allowed to quantitatively and simultaneously measure stage-specific drug effects on parasites at different developmental stages. The optimized assay, requiring only 48 h incubation with drugs and using a cost-effective luminogenic substrate, significantly reduces assay cost and time in comparison to state-of-the-art analogous assays. The assay had a Z' factor of 0.71 ± 0.03, and it is suitable for implementation in 96- and 384-well microplate formats. Moreover, the use of a nonlysing D-luciferin substrate significantly improved the reliability of the assay and allowed one to perform, for the first time, P. falciparum bioluminescence imaging at single-cell level. PMID:25102353

  5. KAI407, a potent non-8-aminoquinoline compound that kills Plasmodium cynomolgi early dormant liver stage parasites in vitro.

    PubMed

    Zeeman, Anne-Marie; van Amsterdam, Sandra M; McNamara, Case W; Voorberg-van der Wel, Annemarie; Klooster, Els J; van den Berg, Alexander; Remarque, Edmond J; Plouffe, David M; van Gemert, Geert-Jan; Luty, Adrian; Sauerwein, Robert; Gagaring, Kerstin; Borboa, Rachel; Chen, Zhong; Kuhen, Kelli; Glynne, Richard J; Chatterjee, Arnab K; Nagle, Advait; Roland, Jason; Winzeler, Elizabeth A; Leroy, Didier; Campo, Brice; Diagana, Thierry T; Yeung, Bryan K S; Thomas, Alan W; Kocken, Clemens H M

    2014-01-01

    Preventing relapses of Plasmodium vivax malaria through a radical cure depends on use of the 8-aminoquinoline primaquine, which is associated with safety and compliance issues. For future malaria eradication strategies, new, safer radical curative compounds that efficiently kill dormant liver stages (hypnozoites) will be essential. A new compound with potential radical cure activity was identified using a low-throughput assay of in vitro-cultured hypnozoite forms of Plasmodium cynomolgi (an excellent and accessible model for Plasmodium vivax). In this assay, primary rhesus hepatocytes are infected with P. cynomolgi sporozoites, and exoerythrocytic development is monitored in the presence of compounds. Liver stage cultures are fixed after 6 days and stained with anti-Hsp70 antibodies, and the relative proportions of small (hypnozoite) and large (schizont) forms relative to the untreated controls are determined. This assay was used to screen a series of 18 known antimalarials and 14 new non-8-aminoquinolines (preselected for blood and/or liver stage activity) in three-point 10-fold dilutions (0.1, 1, and 10 ?M final concentrations). A novel compound, designated KAI407 showed an activity profile similar to that of primaquine (PQ), efficiently killing the earliest stages of the parasites that become either primary hepatic schizonts or hypnozoites (50% inhibitory concentration [IC50] for hypnozoites, KAI407, 0.69 ?M, and PQ, 0.84 ?M; for developing liver stages, KAI407, 0.64 ?M, and PQ, 0.37 ?M). When given as causal prophylaxis, a single oral dose of 100 mg/kg of body weight prevented blood stage parasitemia in mice. From these results, we conclude that KAI407 may represent a new compound class for P. vivax malaria prophylaxis and potentially a radical cure. PMID:24366744

  6. KAI407, a Potent Non-8-Aminoquinoline Compound That Kills Plasmodium cynomolgi Early Dormant Liver Stage Parasites In Vitro

    PubMed Central

    Zeeman, Anne-Marie; van Amsterdam, Sandra M.; McNamara, Case W.; Voorberg-van der Wel, Annemarie; Klooster, Els J.; van den Berg, Alexander; Remarque, Edmond J.; Plouffe, David M.; van Gemert, Geert-Jan; Luty, Adrian; Sauerwein, Robert; Gagaring, Kerstin; Borboa, Rachel; Chen, Zhong; Kuhen, Kelli; Glynne, Richard J.; Chatterjee, Arnab K.; Nagle, Advait; Roland, Jason; Winzeler, Elizabeth A.; Leroy, Didier; Campo, Brice; Diagana, Thierry T.; Yeung, Bryan K. S.; Thomas, Alan W.

    2014-01-01

    Preventing relapses of Plasmodium vivax malaria through a radical cure depends on use of the 8-aminoquinoline primaquine, which is associated with safety and compliance issues. For future malaria eradication strategies, new, safer radical curative compounds that efficiently kill dormant liver stages (hypnozoites) will be essential. A new compound with potential radical cure activity was identified using a low-throughput assay of in vitro-cultured hypnozoite forms of Plasmodium cynomolgi (an excellent and accessible model for Plasmodium vivax). In this assay, primary rhesus hepatocytes are infected with P. cynomolgi sporozoites, and exoerythrocytic development is monitored in the presence of compounds. Liver stage cultures are fixed after 6 days and stained with anti-Hsp70 antibodies, and the relative proportions of small (hypnozoite) and large (schizont) forms relative to the untreated controls are determined. This assay was used to screen a series of 18 known antimalarials and 14 new non-8-aminoquinolines (preselected for blood and/or liver stage activity) in three-point 10-fold dilutions (0.1, 1, and 10 μM final concentrations). A novel compound, designated KAI407 showed an activity profile similar to that of primaquine (PQ), efficiently killing the earliest stages of the parasites that become either primary hepatic schizonts or hypnozoites (50% inhibitory concentration [IC50] for hypnozoites, KAI407, 0.69 μM, and PQ, 0.84 μM; for developing liver stages, KAI407, 0.64 μM, and PQ, 0.37 μM). When given as causal prophylaxis, a single oral dose of 100 mg/kg of body weight prevented blood stage parasitemia in mice. From these results, we conclude that KAI407 may represent a new compound class for P. vivax malaria prophylaxis and potentially a radical cure. PMID:24366744

  7. Human malarial parasite, Plasmodium falciparum, displays capacitative calcium entry: 2-aminoethyl diphenylborinate blocks the signal transduction pathway of melatonin action on the P. falciparum cell cycle.

    PubMed

    Beraldo, Flvio H; Mikoshiba, Katsuhiko; Garcia, Clia R S

    2007-11-01

    The malarial parasite senses the environment to modulate its own cycle. Knowledge of the mechanisms for regulation signaling processes at the invasion, maturation, as well as division of Plasmodium falciparum before reinvasion would represent a major breakthrough and, therefore, might open new avenues for therapy. We have previously reported that melatonin modulates the circadian rhythm of malarial parasites through the activation of phospholipase C (PLC), production of InsP3, and induction of calcium release from intracellular stores. To further investigate the molecular mechanism of melatonin's action, we have used the InsP3 modulator 2-aminoethyl diphenylborinate (2-APB) given in a culture of P. falciparum parasites. Here we show that the melatonin acts on Plasmodium cell cycle through InsP3 signaling as 2-APB blocks melatonin's effect on calcium release. The function of the InsP3 signaling can be regarded as an important event for parasite invasion and maturation process, since addition of the PLC inhibitor, U73122 into Plasmodium-infected red blood cells impairs parasite invasion in vitro. By using 8BrcAMP, we also report here that Plasmodia displays a 'capacitative calcium entry' mechanism for amplification of calcium signals throughout the cytoplasm. PMID:17910604

  8. Ubiquitous Hepatocystis infections, but no evidence of Plasmodium falciparum-like malarial parasites in wild greater spot-nosed monkeys (Cercopithecus nictitans)?

    PubMed Central

    Ayouba, Ahidjo; Mouacha, Fatima; Learn, Gerald H.; Mpoudi-Ngole, Eitel; Rayner, Julian C.; Sharp, Paul M.; Hahn, Beatrice H.; Delaporte, Eric; Peeters, Martine

    2013-01-01

    Western gorillas (Gorilla gorilla) have been identified as the natural reservoir of the parasites that were the immediate precursor of Plasmodium falciparum infecting humans. Recently, a P. falciparum-like sequence was reported in a sample from a captive greater spot-nosed monkey (Cercopithecus nictitans), and was taken to indicate that this species may also be a natural reservoir for P. falciparum-related parasites. To test this hypothesis we screened blood samples from 292 wild C. nictitans monkeys that had been hunted for bushmeat in Cameroon. We detected Hepatocystis spp. in 49% of the samples, as well as one sequence from a clade of Plasmodium spp. previously found in birds, lizards and bats. However, none of the 292 wild C. nictitans harbored P. falciparum-like parasites. PMID:22691606

  9. Effect of thioredoxin peroxidase-1 gene disruption on the liver stages of the rodent malaria parasite Plasmodium berghei.

    PubMed

    Usui, Miho; Masuda-Suganuma, Hirono; Fukumoto, Shinya; Angeles, Jose Ma M; Hakimi, Hassan; Inoue, Noboru; Kawazu, Shin-Ichiro

    2015-06-01

    Phenotypic observation of thioredoxin peroxidase-1 (TPx-1) gene-disrupted Plasmodium berghei (TPx-1 KO) in the liver-stage was performed with an in vitro infection system in order to investigate defective liver-stage development in a mouse infection model. Indirect immunofluorescence microscopy assay with anti-circumsporozoite protein antibody revealed that in the liver schizont stage, TPx-1 KO parasite cells were significantly smaller than cells of the wild-type parent strain (WT). Indirect immunofluorescence microscopy assay with anti-merozoite surface protein-1 antibody, which was used to evaluate late schizont-stage development, indicated that TPx-1 KO schizont development was similar to WT strain development towards the merozoite-forming stage (mature schizont). However, fewer merozoites were produced in the mature TPx-1 KO schizont than in the mature WT schizont. Taken together, the results suggest that TPx-1 may be involved in merozoite formation during liver schizont development. PMID:25284813

  10. Predicted conformations for the immunodominant region of the circumsporozoite protein of the human malaria parasite Plasmodium falciparum.

    PubMed Central

    Gibson, K D; Scheraga, H A

    1986-01-01

    The circumsporozoite protein of the human malaria parasite Plasmodium falciparum contains multiple tandem repeats of the amino acid sequence Asn-Ala-Asn-Pro. The repeated sequence encompasses the immunodominant region of the protein, and antibodies raised against it are potent inhibitors of invasion and development of sporozoites in cultured hepatocytes. Using a modified build-up procedure, we have explored a large number of possible helical and near-helical conformations of a terminally blocked tetraicosapeptide, consisting of six repeats of the sequence Asn-Ala-Asn-Pro, and conclude that two helical conformations are energetically favored to the exclusion of all others. One of these conformations is longer, thinner, and left-handed and is likely to be adopted in nonpolar environments, while the other is shorter, broader, and right-handed and should be favored in aqueous solutions. We propose that the immunodominant region of the circumsporozoite protein of P. falciparum adopts one of these conformations in vivo. PMID:2426702

  11. Immunogenicity and protective efficacy of a recombinant yellow fever vaccine against the murine malarial parasite Plasmodium yoelii.

    PubMed

    Stoyanov, Cristina T; Boscardin, Silvia B; Deroubaix, Stephanie; Barba-Spaeth, Giovanna; Franco, David; Nussenzweig, Ruth S; Nussenzweig, Michel; Rice, Charles M

    2010-06-23

    The live-attenuated yellow fever vaccine (YF17D) is one of the safest and most effective vaccines available today. Here, YF17D was genetically altered to express the circumsporozoite protein (CSP) from the murine malarial parasite Plasmodium yoelii. Reconstituted recombinant virus was viable and exhibited robust CSP expression. Immunization of nave mice resulted in extensive proliferation of adoptively transferred CSP-specific transgenic CD8(+) T-cells. A single immunization of nave mice with recombinant YF17D resulted in robust production of IFN-gamma by CD8(+) T-cells and IFN-gamma and IL-2 by CD4(+) T-cells. A prime-boost regimen consisting of recombinant virus followed by a low-dose of irradiated sporozoites conferred protection against challenge with P. yoelii. Taken together, these results show that recombinant YF17D can efficiently express CSP in culture, and prime a protective immune response in vivo. PMID:20451637

  12. A Unique Virulence Gene Occupies a Principal Position in Immune Evasion by the Malaria Parasite Plasmodium falciparum

    PubMed Central

    Heinberg, Adina R.; Wele, Mamadou; Chen, Qijun; Deitsch, Kirk W.

    2015-01-01

    Mutually exclusive gene expression, whereby only one member of a multi-gene family is selected for activation, is used by the malaria parasite Plasmodium falciparum to escape the human immune system and perpetuate long-term, chronic infections. A family of genes called var encodes the chief antigenic and virulence determinant of P. falciparum malaria. var genes are transcribed in a mutually exclusive manner, with switching between active genes resulting in antigenic variation. While recent work has shed considerable light on the epigenetic basis for var gene activation and silencing, how switching is controlled remains a mystery. In particular, switching seems not to be random, but instead appears to be coordinated to result in timely activation of individual genes leading to sequential waves of antigenically distinct parasite populations. The molecular basis for this apparent coordination is unknown. Here we show that var2csa, an unusual and highly conserved var gene, occupies a unique position within the var gene switching hierarchy. Induction of switching through the destabilization of var specific chromatin using both genetic and chemical methods repeatedly led to the rapid and exclusive activation of var2csa. Additional experiments demonstrated that these represent “true” switching events and not simply de-silencing of the var2csa promoter, and that activation is limited to the unique locus on chromosome 12. Combined with translational repression of var2csa transcripts, frequent “default” switching to this locus and detection of var2csa untranslated transcripts in non-pregnant individuals, these data suggest that var2csa could play a central role in coordinating switching, fulfilling a prediction made by mathematical models derived from population switching patterns. These studies provide the first insights into the mechanisms by which var gene switching is coordinated as well as an example of how a pharmacological agent can disrupt antigenic variation in Plasmodium falciparum. PMID:25993442

  13. Kinetics of B cell responses to Plasmodium falciparum erythrocyte membrane protein 1 in Ghanaian women naturally exposed to malaria parasites.

    PubMed

    Ampomah, Paulina; Stevenson, Liz; Ofori, Michael F; Barfod, Lea; Hviid, Lars

    2014-06-01

    Naturally acquired protective immunity to Plasmodium falciparum malaria takes years to develop. It relies mainly on Abs, particularly IgG specific for Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) proteins on the infected erythrocyte surface. It is only partially understood why acquisition of clinical protection takes years to develop, but it probably involves a range of immune-evasive parasite features, not least of which are PfEMP1 polymorphism and clonal variation. Parasite-induced subversion of immunological memory and expansion of "atypical" memory B cells may also contribute. In this first, to our knowledge, longitudinal study of its kind, we measured B cell subset composition, as well as PfEMP1-specific Ab levels and memory B cell frequencies, in Ghanaian women followed from early pregnancy up to 1 y after delivery. Cell phenotypes and Ag-specific B cell function were assessed three times during and after pregnancy. Levels of IgG specific for pregnancy-restricted, VAR2CSA-type PfEMP1 increased markedly during pregnancy and declined after delivery, whereas IgG levels specific for two PfEMP1 proteins not restricted to pregnancy did not. Changes in VAR2CSA-specific memory B cell frequencies showed typical primary memory induction among primigravidae and recall expansion among multigravidae, followed by contraction postpartum in all. No systematic changes in the frequencies of memory B cells specific for the two other PfEMP1 proteins were identified. The B cell subset analysis confirmed earlier reports of high atypical memory B cell frequencies among residents of P. falciparum-endemic areas, and indicated an additional effect of pregnancy. Our study provides new knowledge regarding immunity to P. falciparum malaria and underpins efforts to develop PfEMP1-based vaccines against this disease. PMID:24760153

  14. A Unique Virulence Gene Occupies a Principal Position in Immune Evasion by the Malaria Parasite Plasmodium falciparum.

    PubMed

    Ukaegbu, Uchechi E; Zhang, Xu; Heinberg, Adina R; Wele, Mamadou; Chen, Qijun; Deitsch, Kirk W

    2015-05-01

    Mutually exclusive gene expression, whereby only one member of a multi-gene family is selected for activation, is used by the malaria parasite Plasmodium falciparum to escape the human immune system and perpetuate long-term, chronic infections. A family of genes called var encodes the chief antigenic and virulence determinant of P. falciparum malaria. var genes are transcribed in a mutually exclusive manner, with switching between active genes resulting in antigenic variation. While recent work has shed considerable light on the epigenetic basis for var gene activation and silencing, how switching is controlled remains a mystery. In particular, switching seems not to be random, but instead appears to be coordinated to result in timely activation of individual genes leading to sequential waves of antigenically distinct parasite populations. The molecular basis for this apparent coordination is unknown. Here we show that var2csa, an unusual and highly conserved var gene, occupies a unique position within the var gene switching hierarchy. Induction of switching through the destabilization of var specific chromatin using both genetic and chemical methods repeatedly led to the rapid and exclusive activation of var2csa. Additional experiments demonstrated that these represent "true" switching events and not simply de-silencing of the var2csa promoter, and that activation is limited to the unique locus on chromosome 12. Combined with translational repression of var2csa transcripts, frequent "default" switching to this locus and detection of var2csa untranslated transcripts in non-pregnant individuals, these data suggest that var2csa could play a central role in coordinating switching, fulfilling a prediction made by mathematical models derived from population switching patterns. These studies provide the first insights into the mechanisms by which var gene switching is coordinated as well as an example of how a pharmacological agent can disrupt antigenic variation in Plasmodium falciparum. PMID:25993442

  15. Identification and initial characterization of three novel cyclin-related proteins of the human malaria parasite Plasmodium falciparum.

    PubMed

    Merckx, Anaïs; Le Roch, Karine; Nivez, Marie-Paule; Dorin, Dominique; Alano, Pietro; Gutierrez, Gustavo J; Nebreda, Angel R; Goldring, Dean; Whittle, Christina; Patterson, Shelley; Chakrabarti, Debopam; Doerig, Christian

    2003-10-10

    The molecular mechanisms regulating cell proliferation and development during the life cycle of malaria parasites remain to be elucidated. The peculiarities of the cell cycle organization during Plasmodium falciparum schizogony suggest that the modalities of cell cycle control in this organism may differ from those in other eukaryotes. Indeed, existing data concerning Plasmodium cell cycle regulators such as cyclin-dependent kinases reveal structural and functional properties that are divergent from those of their homologues in other systems. The work presented here lies in the context of the exploitation of the recently available P. falciparum genome sequence toward the characterization of putative cell cycle regulators. We describe the in silico identification of three open reading frames encoding proteins with maximal homology to various members of the cyclin family and demonstrate that the corresponding polypeptides are expressed in the erythrocytic stages of the infection. We present evidence that these proteins possess cyclin activity by demonstrating either their association with histone H1 kinase activity in parasite extracts or their ability to activate PfPK5, a P. falciparum cyclin-dependent kinase homologue, in vitro. Furthermore, we show that RINGO, a protein with no sequence homology to cyclins but that is nevertheless a strong activator of mammalian CDK1/2, is also a strong activator of PfPK5 in vitro. This raises the possibility that "cryptic" cell cycle regulators may be found among the 50% of the open reading frames in the P. falciparum genome that display no homology to any known proteins. PMID:12869562

  16. Delayed parasite clearance after treatment with dihydroartemisinin-piperaquine in Plasmodium falciparum malaria patients in central Vietnam.

    PubMed

    Thriemer, Kamala; Hong, Nguyen Van; Rosanas-Urgell, Anna; Phuc, Bui Quang; Ha, Do Manh; Pockele, Evi; Guetens, Pieter; Van, Nguyen Van; Duong, Tran Thanh; Amambua-Ngwa, Alfred; D'Alessandro, Umberto; Erhart, Annette

    2014-12-01

    Reduced susceptibility of Plasmodium falciparum toward artemisinin derivatives has been reported from the Thai-Cambodian and Thai-Myanmar borders. Following increasing reports from central Vietnam of delayed parasite clearance after treatment with dihydroartemisinin-piperaquine (DHA-PPQ), the current first-line treatment, we carried out a study on the efficacy of this treatment. Between September 2012 and February 2013, we conducted a 42-day in vivo and in vitro efficacy study in Quang Nam Province. Treatment was directly observed, and blood samples were collected twice daily until parasite clearance. In addition, genotyping, quantitative PCR (qPCR), and in vitro sensitivity testing of isolates was performed. The primary endpoints were parasite clearance rate and time. The secondary endpoints included PCR-corrected and uncorrected cure rates, qPCR clearance profiles, in vitro sensitivity results (for chloroquine, dihydroartemisinin, and piperaquine), and genotyping for mutations in the Kelch 13 propeller domain. Out of 672 screened patients, 95 were recruited and 89 available for primary endpoint analyses. The median parasite clearance time (PCT) was 61.7 h (interquartile range [IQR], 47.6 to 83.2 h), and the median parasite clearance rate had a slope half-life of 6.2 h (IQR, 4.4 to 7.5 h). The PCR-corrected efficacy rates were estimated at 100% at day 28 and 97.7% (95% confidence interval, 91.2% to 99.4%) at day 42. At day 3, the P. falciparum prevalence by qPCR was 2.5 times higher than that by microscopy. The 50% inhibitory concentrations (IC50s) of isolates with delayed clearance times (≥ 72 h) were significantly higher than those with normal clearance times for all three drugs. Delayed parasite clearance (PCT, ≥ 72 h) was significantly higher among day 0 samples carrying the 543 mutant allele (47.8%) than those carrying the wild-type allele (1.8%; P = 0.048). In central Vietnam, the efficacy of DHA-PPQ is still satisfactory, but the parasite clearance time and rate are indicative of emerging artemisinin resistance. (This study has been registered at ClinicalTrials.gov under registration no. NCT01775592.). PMID:25224002

  17. Multiple stiffening effects of nanoscale knobs on human red blood cells infected with Plasmodium falciparum malaria parasite.

    PubMed

    Zhang, Yao; Huang, Changjin; Kim, Sangtae; Golkaram, Mahdi; Dixon, Matthew W A; Tilley, Leann; Li, Ju; Zhang, Sulin; Suresh, Subra

    2015-05-12

    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. PMID:25918423

  18. Molecular Evidence of Plasmodium vivax Mono and Mixed Malaria Parasite Infections in Duffy-Negative Native Cameroonians

    PubMed Central

    Ngassa Mbenda, Huguette Gaelle; Das, Aparup

    2014-01-01

    The malaria parasite Plasmodium vivax is known to be majorly endemic to Asian and Latin American countries with no or very few reports of Africans infected with this parasite. Since the human Duffy antigens act as receptors for P. vivax to invade human RBCs and Africans are generally Duffy-negative, non-endemicity of P. vivax in Africa has been attributed to this fact. However, recent reports describing P. vivax infections in Duffy-negative Africans from West and Central parts of Africa have been surfaced including a recent report on P. vivax infection in native Cameroonians. In order to know if Cameroonians living in the southern regions are also susceptible to P. vivax infection, we collected finger-prick blood samples from 485 malarial symptomatic patients in five locations and followed PCR diagnostic assays with DNA sequencing of the 18S ribosomal RNA gene. Out of the 201 malaria positive cases detected, 193 were pure P. falciparum, six pure P. vivax and two mixed parasite infections (P. falciparum + P. vivax). The eight P. vivax infected samples (six single + two mixed) were further subjected to DNA sequencing of the P. vivax multidrug resistance 1 (pvmdr1) and the P.vivax circumsporozoite (pvcsp) genes. Alignment of the eight Cameroonian pvmdr1 sequences with the reference sequence showed high sequence similarities, reconfirming P. vivax infection in all the eight patients. DNA sequencing of the pvcsp gene indicated all the eight P. vivax to be of VK247 type. Interestingly, DNA sequencing of a part of the human Duffy gene covering the promoter region in the eight P. vivax-infected Cameroonians to identify the T-33C mutation revealed all these patients as Duffy-negative. The results provide evidence of single P. vivax as well as mixed malaria parasite infection in native Cameroonians and add knowledge to the growing evidences of P. vivax infection in Duffy-negative Africans. PMID:25084090

  19. Multiple stiffening effects of nanoscale knobs on human red blood cells infected with Plasmodium falciparum malaria parasite

    PubMed Central

    Zhang, Yao; Kim, Sangtae; Golkaram, Mahdi; Dixon, Matthew W. A.; Tilley, Leann; Li, Ju; Zhang, Sulin; Suresh, Subra

    2015-01-01

    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 2448 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. PMID:25918423

  20. Molecular evidence of Plasmodium vivax mono and mixed malaria parasite infections in Duffy-negative native Cameroonians.

    PubMed

    Ngassa Mbenda, Huguette Gaelle; Das, Aparup

    2014-01-01

    The malaria parasite Plasmodium vivax is known to be majorly endemic to Asian and Latin American countries with no or very few reports of Africans infected with this parasite. Since the human Duffy antigens act as receptors for P. vivax to invade human RBCs and Africans are generally Duffy-negative, non-endemicity of P. vivax in Africa has been attributed to this fact. However, recent reports describing P. vivax infections in Duffy-negative Africans from West and Central parts of Africa have been surfaced including a recent report on P. vivax infection in native Cameroonians. In order to know if Cameroonians living in the southern regions are also susceptible to P. vivax infection, we collected finger-prick blood samples from 485 malarial symptomatic patients in five locations and followed PCR diagnostic assays with DNA sequencing of the 18S ribosomal RNA gene. Out of the 201 malaria positive cases detected, 193 were pure P. falciparum, six pure P. vivax and two mixed parasite infections (P. falciparum + P. vivax). The eight P. vivax infected samples (six single + two mixed) were further subjected to DNA sequencing of the P. vivax multidrug resistance 1 (pvmdr1) and the P.vivax circumsporozoite (pvcsp) genes. Alignment of the eight Cameroonian pvmdr1 sequences with the reference sequence showed high sequence similarities, reconfirming P. vivax infection in all the eight patients. DNA sequencing of the pvcsp gene indicated all the eight P. vivax to be of VK247 type. Interestingly, DNA sequencing of a part of the human Duffy gene covering the promoter region in the eight P. vivax-infected Cameroonians to identify the T-33C mutation revealed all these patients as Duffy-negative. The results provide evidence of single P. vivax as well as mixed malaria parasite infection in native Cameroonians and add knowledge to the growing evidences of P. vivax infection in Duffy-negative Africans. PMID:25084090

  1. PfPDE1, a novel cGMP-specific phosphodiesterase from the human malaria parasite Plasmodium falciparum

    PubMed Central

    2005-01-01

    This is the first report of molecular characterization of a novel cyclic nucleotide PDE (phosphodiesterase), isolated from the human malaria parasite Plasmodium falciparum and designated PfPDE1. PfPDE1 cDNA encodes an 884-amino-acid protein, including six putative transmembrane domains in the N-terminus followed by a catalytic domain. The PfPDE1 gene is a single-copy gene consisting of two exons and a 170 bp intron. PfPDE1 transcripts were abundant in the ring form of the asexual blood stages of the parasite. The C-terminal catalytic domain of PfPDE1, produced in Escherichia coli, specifically hydrolysed cGMP with a Km value of 0.65 μM. Among the PDE inhibitors tested, a PDE5 inhibitor, zaprinast, was the most effective, having an IC50 value of 3.8 μM. The non-specific PDE inhibitors IBMX (3-isobutyl-1-methylxanthine), theophylline and the antimalarial chloroquine had IC50 values of over 100 μM. Membrane fractions prepared from P. falciparum at mixed asexual blood stages showed potent cGMP hydrolytic activity compared with cytosolic fractions. This hydrolytic activity was sensitive to zaprinast with an IC50 value of 4.1 μM, but insensitive to IBMX and theophylline. Furthermore, an in vitro antimalarial activity assay demonstrated that zaprinast inhibited the growth of the asexual blood parasites, with an ED50 value of 35 μM. The impact of cyclic nucleotide signalling on the cellular development of this parasite has previously been discussed. Thus this enzyme is suggested to be a novel potential target for the treatment of the disease malaria. PMID:16038615

  2. Parasite maturation and host serum iron influence the labile iron pool of erythrocyte stage Plasmodium falciparum

    PubMed Central

    Clark, Martha; Fisher, Nancy C.; Kasthuri, Raj; Hand, Carla Cerami

    2014-01-01

    Summary Iron is a critical and tightly regulated nutrient for both the malaria parasite and its human host. The importance of the relationship between host iron and the parasite has been underscored recently by studies showing that host iron supplementation may increase the risk of falciparum malaria. It is unclear what host iron sources the parasite is able to access. We developed a flow cytometry-based method for measuring the labile iron pool (LIP) of parasitized erythrocytes using the nucleic acid dye STYO 61 and the iron sensitive dye, calcein acetoxymethyl ester (CA-AM). This new approach enabled us to measure the LIP of P. falciparum through the course of its erythrocytic life cycle and in response to the addition of host serum iron sources. We found that the LIP increases as the malaria parasite develops from early ring to late schizont stage, and that the addition of either transferrin or ferric citrate to culture media increases the LIP of trophozoites. Our method for detecting the LIP within malaria parasitized RBCs provides evidence that the parasite is able to access serum iron sources as part of the host vs. parasite arms race for iron. PMID:23398516

  3. Molecular cloning, characterization and expression profile of a glutathione peroxidase-like thioredoxin peroxidase (TPxGl) of the rodent malaria parasite Plasmodium berghei.

    PubMed

    Haselton, Kyle J; David, Robin; Fell, Katherine; Schulte, Emily; Dybas, Matthew; Olsen, Kenneth W; Kanzok, Stefan M

    2015-06-01

    Glutathione peroxidases (GPx) comprise an important group of redox active proteins with diverse functions, including antioxidant defense and signaling. Although the genome of the malaria parasite Plasmodium does not contain a genuine GPx gene a glutathione peroxidase-like thioredoxin peroxidase (TPx(Gl)) has recently been identified and biochemically characterized in the human malaria parasite P. falciparum. To gain more insight into the potential biological function of this enzyme we have cloned and expressed TPx(Gl) of the rodent model system P. berghei (PbTPx(Gl)). Biochemical characterization confirmed that the protein is redox active with the P. berghei thioredoxin system. We compared PbTPx(Gl) to recently characterized thioredoxin-dependent GPx-type proteins of other organisms, and generated the first hypothetical 3D model of a Plasmodium TPx(Gl), which shows the conservation of the thioredoxin-fold as well as the spatial orientation of a classic GPx catalytic tetrad. In vivo studies indicate that PbTPx(Gl) is continuously expressed in all P. berghei asexual blood stages, gametocytes and in early mosquito-stage parasites. Confocal microscopy suggest a cytoplasmic localization of PbTPx(Gl) in all investigated parasite life stages, specifically in mature ookinetes. Our data provides new insights into the structure and ubiquitous expression of Plasmodium TPx(Gl) and warrants further investigation into this potentially important redox enzyme. PMID:24637102

  4. Spleen-Dependent Regulation of Antigenic Variation in Malaria Parasites: Plasmodium knowlesi SICAvar Expression Profiles in Splenic and Asplenic Hosts

    PubMed Central

    Lapp, Stacey A.; Korir-Morrison, Cindy; Jiang, Jianlin; Bai, Yaohui; Corredor, Vladimir; Galinski, Mary R.

    2013-01-01

    Background Antigenic variation by malaria parasites was first described in Plasmodium knowlesi, which infects humans and macaque monkeys, and subsequently in P. falciparum, the most virulent human parasite. The schizont-infected cell agglutination (SICA) variant proteins encoded by the SICAvar multigene family in P. knowlesi, and Erythrocyte Membrane Protein-1 (EMP-1) antigens encoded by the var multigene family in P. falciparum, are expressed at the surface of infected erythrocytes, are associated with virulence, and serve as determinants of naturally acquired immunity. A parental P. knowlesi clone, Pk1(A+), and a related progeny clone, Pk1(B+)1+, derived by an in vivo induced variant antigen switch, were defined by the expression of distinct SICA variant protein doublets of 210/190 and 205/200 kDa, respectively. Passage of SICA[+] infected erythrocytes through splenectomized rhesus monkeys results in the SICA[-] phenotype, defined by the lack of surface expression and agglutination with variant specific antisera. Principal Findings We have investigated SICAvar RNA and protein expression in Pk1(A+), Pk1(B+)1+, and SICA[-] parasites. The Pk1(A+) and Pk1(B+)1+ parasites express different distinct SICAvar transcript and protein repertoires. By comparison, SICA[-] parasites are characterized by a vast reduction in SICAvar RNA expression, the lack of full-length SICAvar transcript signals on northern blots, and correspondingly, the absence of any SICA protein detected by mass spectrometry. Significance SICA protein expression may be under transcriptional as well as post-transcriptional control, and we show for the first time that the spleen, an organ central to blood-stage immunity in malaria, exerts an influence on these processes. Furthermore, proteomics has enabled the first in-depth characterization of SICA[+] protein phenotypes and we show that the in vivo switch from Pk1(A+) to Pk1(B+)1+ parasites resulted in a complete change in SICA profiles. These results emphasize the importance of studying antigenic variation in the context of the host environment. PMID:24205067

  5. The Evolutionary History of Plasmodium vivax as Inferred from Mitochondrial Genomes: Parasite Genetic Diversity in the Americas

    PubMed Central

    Taylor, Jesse E.; Pacheco, M. Andrena; Bacon, David J.; Beg, Mohammad A.; Machado, Ricardo Luiz; Fairhurst, Rick M.; Herrera, Socrates; Kim, Jung-Yeon; Menard, Didier; Pvoa, Marinete Marins; Villegas, Leopoldo; Mulyanto; Snounou, Georges; Cui, Liwang; Zeyrek, Fadile Yildiz; Escalante, Ananias A.

    2013-01-01

    Plasmodium vivax is the most prevalent human malaria parasite in the Americas. Previous studies have contrasted the genetic diversity of parasite populations in the Americas with those in Asia and Oceania, concluding that New World populations exhibit low genetic diversity consistent with a recent introduction. Here we used an expanded sample of complete mitochondrial genome sequences to investigate the diversity of P. vivax in the Americas as well as in other continental populations. We show that the diversity of P. vivax in the Americas is comparable to that in Asia and Oceania, and we identify several divergent clades circulating in South America that may have resulted from independent introductions. In particular, we show that several haplotypes sampled in Venezuela and northeastern Brazil belong to a clade that diverged from the other P. vivax lineages at least 30,000 years ago, albeit not necessarily in the Americas. We propose that, unlike in Asia where human migration increases local genetic diversity, the combined effects of the geographical structure and the low incidence of vivax malaria in the Americas has resulted in patterns of low local but high regional genetic diversity. This could explain previous views that P. vivax in the Americas has low genetic diversity because these were based on studies carried out in limited areas. Further elucidation of the complex geographical pattern of P. vivax variation will be important both for diversity assessments of genes encoding candidate vaccine antigens and in the formulation of control and surveillance measures aimed at malaria elimination. PMID:23733143

  6. An Adjustable Gas-Mixing Device to Increase Feasibility of In Vitro Culture of Plasmodium falciparum Parasites in the Field

    PubMed Central

    Volkman, Sarah K.; Ahouidi, Ambroise D.; Ndiaye, Daouda; Mboup, Souleymane; Wirth, Dyann F.

    2014-01-01

    A challenge to conducting high-impact and reproducible studies of the mechanisms of P. falciparum drug resistance, invasion, virulence, and immunity is the lack of robust and sustainable in vitro culture in the field. While the technology exists and is routinely utilized in developed countries, various factorsfrom cost, to supply, to qualitymake it hard to implement in malaria endemic countries. Here, we design and rigorously evaluate an adjustable gas-mixing device for the in vitro culture of P. falciparum parasites in the field to circumvent this challenge. The device accurately replicates the gas concentrations needed to culture laboratory isolates, short-term adapted field isolates, cryopreserved previously non-adapted isolates, as well as to adapt ex vivo isolates to in vitro culture in the field. We also show an advantage over existing alternatives both in cost and in supply. Furthermore, the adjustable nature of the device makes it an ideal tool for many applications in which varied gas concentrations could be critical to culture success. This adjustable gas-mixing device will dramatically improve the feasibility of in vitro culture of Plasmodium falciparum parasites in malaria endemic countries given its numerous advantages. PMID:24603696

  7. Purification of a recombinant histidine-tagged lactate dehydrogenase from the malaria parasite, Plasmodium vivax, and characterization of its properties.

    PubMed

    Sundaram, Balamurugan; Varadarajan, Nandan Mysore; Subramani, Pradeep Annamalai; Ghosh, Susanta Kumar; Nagaraj, Viswanathan Arun

    2014-12-01

    Lactate dehydrogenase (LDH) of the malaria parasite, Plasmodium vivax (Pv), serves as a drug target and immunodiagnostic marker. The LDH cDNA generated from total RNA of a clinical isolate of the parasite was cloned into pRSETA plasmid. Recombinant his-tagged PvLDH was over-expressed in E. coli Rosetta2DE3pLysS and purified using Ni(2+)-NTA resin giving a yield of 25-30mg/litre bacterial culture. The recombinant protein was enzymatically active and its catalytic efficiency for pyruvate was 5.4נ10(8)min(-1)M(-1), 14.5 fold higher than a low yield preparation reported earlier to obtain PvLDH crystal structure. The enzyme activity was inhibited by gossypol and sodium oxamate. The recombinant PvLDH was reactive in lateral flow immunochromatographic assays detecting pan- and vivax-specific LDH. The soluble recombinant PvLDH purified using heterologous expression system can facilitate the generation of vivax LDH-specific monoclonals and the screening of chemical compound libraries for PvLDH inhibitors. PMID:25048245

  8. Crystallization and preliminary crystallographic analysis of orotidine 5′-monophosphate decarboxylase from the human malaria parasite Plasmodium falciparum

    SciTech Connect

    Krungkrai, Sudaratana R.; Tokuoka, Keiji; Kusakari, Yukiko; Inoue, Tsuyoshi; Adachi, Hiroaki; Matsumura, Hiroyoshi; Takano, Kazufumi; Murakami, Satoshi; Mori, Yusuke; Kai, Yasushi; Krungkrai, Jerapan; Horii, Toshihiro

    2006-06-01

    Orotidine 5′-monophosphate decarboxylase of human malaria parasite P. falciparum was crystallized by the seeding method in a hanging drop using PEG 3000 as a precipitant. A complete set of diffraction data from a native crystal was collected to 2.7 Å resolution at 100 K using synchrotron radiation. Orotidine 5′-monophosphate (OMP) decarboxylase (OMPDC; EC 4.1.1.23) catalyzes the final step in the de novo synthesis of uridine 5′-monophosphate (UMP) and defects in the enzyme are lethal in the malaria parasite Plasmodium falciparum. Active recombinant P. falciparum OMPDC (PfOMPDC) was crystallized by the seeding method in a hanging drop using PEG 3000 as a precipitant. A complete set of diffraction data from a native crystal was collected to 2.7 Å resolution at 100 K using synchrotron radiation at the Swiss Light Source. The crystal exhibits trigonal symmetry (space group R3), with hexagonal unit-cell parameters a = b = 201.81, c = 44.03 Å. With a dimer in the asymmetric unit, the solvent content is 46% (V{sub M} = 2.3 Å{sup 3} Da{sup −1})

  9. Evaluation of a range of antimicrobial agents against the parasitic protozoa, Plasmodium falciparum, Babesia rodhaini and Theileria parva in vitro.

    PubMed

    McColm, A A; McHardy, N

    1984-08-01

    Eighteen antimicrobials commonly used in tissue culture were screened in three different protozoan test systems in order to establish their suitability for routine inclusion in protozoal cultivation systems. The human malaria parasite, Plasmodium falciparum, was inhibited by more than half the antibiotics tested at concentrations recommended for normal tissue culture use. Eight compounds were well tolerated and thus could be used prophylactically to prevent microbial contamination. These antimicrobials were the bactericidal aminoglycoside antibiotics, streptomycin, gentamicin and kanamycin, the bacteriostatic protein synthesis inhibitors, chloramphenicol and chlortetracycline and the antifungals, 5-fluorocytosine, nystatin and amphotericin B. Babesia rodhaini and Theileria parva were less sensitive than P. falciparum and tolerated all 18 compounds at concentrations well above 100 micrograms ml-1. Extension of the study to examine direct antiprotozoal action of these and other antimicrobials not normally used in culture confirmed that P. falciparum was significantly more sensitive than the other parasites. Tylosin, rifamycin, gramicidin D and valinomycin were all strongly antimalarial with IC50 values of 0.245, 1.20, 1.3 X 10(-3) and 1.9 X 10(-3) micrograms ml-1 respectively. This compares with a value of 1.35 X 10(-2) micrograms ml-1 for the standard antimalarial, chloroquine. Only valinomycin and, more particularly, gramicidin D were significantly active against B. rodhaini and T. parva. Gramicidin D was more effective, but more toxic, than the standard antiprotozoal agents tested at curing in vivo malarial and babesial infections in mice. PMID:6206808

  10. Recombinant plasmepsin 1 from the human malaria parasite Plasmodium falciparum: Enzymatic characterization, active site inhibitor design, and structural analysis

    PubMed Central

    Liu, Peng; Marzahn, Melissa R.; Robbins, Arthur H.; Gutirrez-de-Tern, Hugo; Rodrguez, David; McClung, Scott; Stevens, Stanley M.; Yowell, Charles A.; Dame, John B.; McKenna, Robert; Dunn, Ben M.

    2009-01-01

    A mutated form of truncated proplasmepsin 1 (proPfPM1) from the human malaria parasite Plasmodium falciparum, proPfPM1 K110pN, was generated and overexpressed in E. coli. The auto-maturation process was carried out at pH 4.0 and 4.5, and the optimal catalytic pH of the resulting mature PfPM1 was determined to be pH 5.5. This mature PfPM1 showed comparable binding affinity to peptide substrates and inhibitors with the naturally-occurring form isolated from parasites. The S3-S3 subsite preferences of the recombinant mature PfPM1 were explored using combinatorial chemistry based peptide libraries. Based on the results, a peptidomimetic inhibitor (compound 1) was designed and yielded 5-fold selectivity for binding to PfPM1 versus the homologous human cathepsin D (hcatD). The 2.8 structure of the PfPMP2-compound 1 complex is reported. Modeling studies were conducted using a series of peptidomimetic inhibitors (compounds 16, Table 3) and three plasmepsins: the crystal structure of PfPM2, and homology derived models of PfPM1 and PfPM4. PMID:19271776

  11. Transmission stages of Plasmodium: does the parasite use the one same signal, provided both by the host and the vector, for gametocytogenesis and sporozoite maturation?

    PubMed

    Milon, G; David, P H

    1999-09-01

    Among the microorganisms that strictly depend upon other organisms (hosts or vectors) for achieving their life cycle, protozoan and metazoan parasites have been often primarily distinguished through the major pathogenic processes they could induce. A variety of different mechanisms linked to parasitism can indeed systemically (e.g. Plasmodium falciparum) or locally (e.g. Toxoplasma gondii) induce important alterations of tissue homeostasis. But more than obvious pathogenicity, it is the capacity to be transmitted that is essential for parasite survival and there is increasing evidence that certain parasites can achieve their life cycle to the point of transmission in the absence of clinically detectable processes. For this, constitutive microenvironments of the host or vector can be exploited. Moreover, parasites are sometimes able to highjack effectors of the host's immune response towards conditioning the microenvironments which are permissive to differentiation of transmissible developmental stages. Based on a few examples taken from studies on the transmission stages of Leishmania, Toxoplasma and Plasmodium, we have here attempted to formulate a few hypothesis on the biology of the transmission stages of P. falciparum, i.e. on gametocytogenesis and sporozoite maturation. As discussants, we may have been somewhat dwarfed by issues evoked by the organizers of this meeting in the title of the session, i.e. 'Vector-parasite-man interactions'!... In reaction, we may have taken refuge in somewhat over-selective comments, biased by the objects of our personal research.... PMID:10697849

  12. Resistance of early midgut stages of natural Plasmodium falciparum parasites to high temperatures in experimentally infected Anopheles gambiae (Diptera: Culicidae).

    PubMed

    Okech, Bernard A; Gouagna, Louis C; Kabiru, Ephantus W; Walczak, Elizabeth; Beier, John C; Yan, Guiyun; Githure, John I

    2004-08-01

    We studied the effects of high temperature, 30 and 32 versus 27 C on early Plasmodium falciparum development in Anopheles gambiae experimentally infected with gametocytes from 30 volunteers with mean density of 264.1 gametocytes/microl blood (range: 16-1,536/microl). From several batches of mosquitoes, fed by membrane feeding, midguts of individual mosquitoes were dissected at 24 hr for ookinete enumeration and at 7 days to quantify oocysts. There were temperature-related differences in mean ookinete intensity per mosquito midgut, with 9.71 +/- 1.6 at 27 C, 9.85 +/- 2.32 at 30 C, and 3.89 +/- 0.81 at 32 C. The prevalence of oocyst infection decreased with an increase in temperatures from 15.9 to 8.5 to 6.4% at 27, 30, and 32 C, respectively. The average oocyst intensities for the infected mosquitoes increased with temperatures from 2.9 at 27 C to 3.5 at 30 C, and to 3.3 at 32 C. However, the success of infections was reduced at 30 and 32 C, and resulted in greater losses during consecutive inter-stage parasite development. The most significant impact of high temperatures occurred at the transition between macrogametocytes and ookinetes, whereas the transition between ookinetes and oocysts apparently was not affected. In contrast to other reports, exposure of mosquitoes infected with natural parasites to high temperatures did not eliminate preoocyst stages, as has been observed from laboratory studies using the NF-54 strain of P. falciparum. This observation of parasite resistance to high temperatures is consistent with the natural situation in tropical environments where perennial malaria transmission occurs during hot dry seasons. PMID:15357066

  13. Plasmodium falciparum synthetic LbL microparticle vaccine elicits protective neutralizing antibody and parasite-specific cellular immune responses

    PubMed Central

    Powell, Thomas J.; Tang, Jie; DeRome, Mary E.; Mitchell, Robert A.; Jacobs, Andrea; Deng, Yanhong; Palath, Naveen; Cardenas, Edwin; Boyd, James G.; Nardin, Elizabeth

    2013-01-01

    Epitopes of the circumsporozoite (CS) protein of Plasmodium falciparum, the most pathogenic species of the malaria parasite, have been shown to elicit protective immunity in experimental animals and human volunteers. The mechanisms of immunity include parasite-neutralizing antibodies that can inhibit parasite motility in the skin at the site of infection and in the bloodstream during transit to the hepatocyte host cell and also block interaction with host cell receptors on hepatocytes. In addition, specific CD4+ and CD8+ cellular mechanisms target the intracellular hepatic forms, thus preventing release of erythrocytic stage parasites from the infected hepatocyte and the ensuing blood stage cycle responsible for clinical disease. An innovative method for producing particle vaccines, layer-by-layer (LbL) fabrication of polypeptide films on solid CaCO3 cores, was used to produce synthetic malaria vaccines containing a tri-epitope CS peptide T1BT* comprising the antibody epitope of the CS repeat region (B) and two T-cell epitopes, the highly conserved T1 epitope and the universal epitope T*. Mice immunized with microparticles loaded with T1BT* peptide developed parasite-neutralizing antibodies and malaria-specific T-cell responses including cytotoxic effector T-cells. Protection from liver stage infection following challenge with live sporozoites from infected mosquitoes correlated with neutralizing antibody levels. Although some immunized mice with low or undetectable neutralizing antibodies were also protected, depletion of T-cells prior to challenge resulted in the majority of mice remaining resistant to challenge. In addition, mice immunized with microparticles bearing only T-cell epitopes were not protected, demonstrating that cellular immunity alone was not sufficient for protective immunity. Although the microparticles without adjuvant were immunogenic and protective, a simple modification with the lipopeptide TLR2 agonist Pam3Cys increased the potency and efficacy of the LbL vaccine candidate. This study demonstrates the potential of LbL particles as promising malaria vaccine candidates using the T1BT* epitopes from the P. falciparum CS protein. PMID:23481177

  14. Melatonin and IP3-induced Ca2+ release from intracellular stores in the malaria parasite Plasmodium falciparum within infected red blood cells.

    PubMed

    Alves, Eduardo; Bartlett, Paula J; Garcia, Celia R S; Thomas, Andrew P

    2011-02-18

    IP(3)-dependent Ca(2+) signaling controls a myriad of cellular processes in higher eukaryotes and similar signaling pathways are evolutionarily conserved in Plasmodium, the intracellular parasite that causes malaria. We have reported that isolated, permeabilized Plasmodium chabaudi, releases Ca(2+) upon addition of exogenous IP(3). In the present study, we investigated whether the IP(3) signaling pathway operates in intact Plasmodium falciparum, the major disease-causing human malaria parasite. P. falciparum-infected red blood cells (RBCs) in the trophozoite stage were simultaneously loaded with the Ca(2+) indicator Fluo-4/AM and caged-IP(3). Photolytic release of IP(3) elicited a transient Ca(2+) increase in the cytosol of the intact parasite within the RBC. The intracellular Ca(2+) pools of the parasite were selectively discharged, using thapsigargin to deplete endoplasmic reticulum (ER) Ca(2+) and the antimalarial chloroquine to deplete Ca(2+) from acidocalcisomes. These data show that the ER is the major IP(3)-sensitive Ca(2+) store. Previous work has shown that the human host hormone melatonin regulates P. falciparum cell cycle via a Ca(2+)-dependent pathway. In the present study, we demonstrate that melatonin increases inositol-polyphosphate production in intact intraerythrocytic parasite. Moreover, the Ca(2+) responses to melatonin and uncaging of IP(3) were mutually exclusive in infected RBCs. Taken together these data provide evidence that melatonin activates PLC to generate IP(3) and open ER-localized IP(3)-sensitive Ca(2+) channels in P. falciparum. This receptor signaling pathway is likely to be involved in the regulation and synchronization of parasite cell cycle progression. PMID:21149448

  15. Polymorphism in dhfr/dhps genes, parasite density and ex vivo response to pyrimethamine in Plasmodium falciparum malaria parasites in Thies, Senegal.

    PubMed

    Ndiaye, Daouda; Dieye, Baba; Ndiaye, Yaye D; Van Tyne, Daria; Daniels, Rachel; Bei, Amy K; Mbaye, Aminata; Valim, Clarissa; Lukens, Amanda; Mboup, Souleymane; Ndir, Omar; Wirth, Dyann F; Volkman, Sarah

    2013-12-01

    Resistance to sulfadoxine-pyrimethamine (SP) in Plasmodium falciparum malaria parasites is associated with mutations in the dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes, and these mutations have spread resistance worldwide. SP, used for several years in Senegal, has been recommended for intermittent preventive treatment for malaria in pregnancy (IPTp) and has been widely implemented since 2003 in this country. There is currently limited data on SP resistance from molecular marker genotyping, and no data on pyrimethamine ex vivo sensitivity in Senegal. Molecular markers of SP resistance and pyrimethamine ex vivo sensitivity were investigated in 416 parasite samples collected from the general population, from the Thies region between 2003 and 2011. The prevalence of the N51I/C59R/S108N triple mutation in dhfr increased from 40% in 2003 to 93% in 2011. Furthermore, the prevalence of the dhfr N51I/C59R/S108N and dhps A437G quadruple mutation increased, from 20% to 66% over the same time frame, then down to 44% by 2011. There was a significant increase in the prevalence of the dhfr triple mutation, as well as an association between dhfr genotypes and pyrimethamine response. Conversely, dhps mutations in codons 436 and 437 did not show consistent variation between 2003 and 2011. These findings suggest that regular screening for molecular markers of antifolate resistance and ex vivo drug response monitoring should be incorporated with ongoing in vivo efficacy monitoring in areas where IPTp-SP is implemented and where pyrimethamine and sulfa drugs are still widely administered in the general population. PMID:24533303

  16. Perturbations of Plasmodium Puf2 Expression and RNA-seq of Puf2-Deficient Sporozoites Reveal a Critical Role in Maintaining RNA Homeostasis and Parasite Transmissibility

    PubMed Central

    Lindner, Scott E.; Mikolajczak, Sebastian A.; Vaughan, Ashley M.; Moon, Wonjong; Joyce, Brad R.; Sullivan, William J.; Kappe, Stefan H. I.

    2013-01-01

    Summary Malaria's cycle of infection requires parasite transmission between a mosquito vector and a mammalian host. We here demonstrate that the Plasmodium yoelii Pumilio-FBF family member Puf2 allows the sporozoite to remain infectious in the mosquito salivary glands while awaiting transmission. Puf2 mediates this solely through its RNA-Binding Domain (RBD) likely by stabilizing or hastening the degradation of specific mRNAs. Puf2 traffics to sporozoite cytosolic granules, which are negative for several markers of stress granules and P-bodies, and disappear rapidly after infection of hepatocytes. In contrast to previously described Plasmodium berghei pbpuf2? parasites, pypuf2? sporozoites have no apparent defect in host infection when tested early in salivary gland residence, but become progressively noninfectious and prematurely transform into EEFs during prolonged salivary gland residence. The premature overexpression of Puf2 in oocysts causes striking deregulation of sporozoite maturation and infectivity while extension of Puf2 expression in liver stages causes no defect, suggesting that the presence of Puf2 alone is not sufficient for its functions. Finally, by conducting the first comparative RNA-seq analysis of Plasmodium sporozoites, we find that Puf2 may play a role in directly or indirectly maintaining the homeostasis of specific transcripts. These findings uncover requirements for maintaining a window of opportunity for the malaria parasite to accommodate the unpredictable moment of transmission from mosquito to mammalian host. PMID:23356439

  17. Artemisinin activity-based probes identify multiple molecular targets within the asexual stage of the malaria parasites Plasmodium falciparum 3D7

    PubMed Central

    Ismail, Hanafy M.; Barton, Victoria; Phanchana, Matthew; Charoensutthivarakul, Sitthivut; Wong, Michael H. L.; Hemingway, Janet; Biagini, Giancarlo A.; O’Neill, Paul M.; Ward, Stephen A.

    2016-01-01

    The artemisinin (ART)-based antimalarials have contributed significantly to reducing global malaria deaths over the past decade, but we still do not know how they kill parasites. To gain greater insight into the potential mechanisms of ART drug action, we developed a suite of ART activity-based protein profiling probes to identify parasite protein drug targets in situ. Probes were designed to retain biological activity and alkylate the molecular target(s) of Plasmodium falciparum 3D7 parasites in situ. Proteins tagged with the ART probe can then be isolated using click chemistry before identification by liquid chromatography–MS/MS. Using these probes, we define an ART proteome that shows alkylated targets in the glycolytic, hemoglobin degradation, antioxidant defense, and protein synthesis pathways, processes essential for parasite survival. This work reveals the pleiotropic nature of the biological functions targeted by this important class of antimalarial drugs. PMID:26858419

  18. Translational Control of UIS4 Protein of the Host-Parasite Interface Is Mediated by the RNA Binding Protein Puf2 in Plasmodium berghei Sporozoites

    PubMed Central

    Silva, Patrícia A. G. C.; Guerreiro, Ana; Santos, Jorge M.; Braks, Joanna A. M.; Janse, Chris J.; Mair, Gunnar R.

    2016-01-01

    UIS4 is a key protein component of the host-parasite interface in the liver stage of the rodent malaria parasite Plasmodium berghei and required for parasite survival after invasion. In the infectious sporozoite, UIS4 protein has variably been shown to be translated but also been reported to be translationally repressed. Here we show that uis4 mRNA translation is regulated by the P. berghei RNA binding protein Pumilio-2 (PbPuf2 or Puf2 from here on forward) in infectious salivary gland sporozoites in the mosquito vector. Using RNA immunoprecipitation we show that uis4 mRNA is bound by Puf2 in salivary gland sporozoites. In the absence of Puf2, uis4 mRNA translation is de-regulated and UIS4 protein expression upregulated in salivary gland sporozoites. Here, using RNA immunoprecipitation, we reveal the first Puf2-regulated mRNA in this parasite. PMID:26808677

  19. Artemisinin activity-based probes identify multiple molecular targets within the asexual stage of the malaria parasites Plasmodium falciparum 3D7.

    PubMed

    Ismail, Hanafy M; Barton, Victoria; Phanchana, Matthew; Charoensutthivarakul, Sitthivut; Wong, Michael H L; Hemingway, Janet; Biagini, Giancarlo A; O'Neill, Paul M; Ward, Stephen A

    2016-02-23

    The artemisinin (ART)-based antimalarials have contributed significantly to reducing global malaria deaths over the past decade, but we still do not know how they kill parasites. To gain greater insight into the potential mechanisms of ART drug action, we developed a suite of ART activity-based protein profiling probes to identify parasite protein drug targets in situ. Probes were designed to retain biological activity and alkylate the molecular target(s) of Plasmodium falciparum 3D7 parasites in situ. Proteins tagged with the ART probe can then be isolated using click chemistry before identification by liquid chromatography-MS/MS. Using these probes, we define an ART proteome that shows alkylated targets in the glycolytic, hemoglobin degradation, antioxidant defense, and protein synthesis pathways, processes essential for parasite survival. This work reveals the pleiotropic nature of the biological functions targeted by this important class of antimalarial drugs. PMID:26858419

  20. Biliverdin targets enolase and eukaryotic initiation factor 2 (eIF2α) to reduce the growth of intraerythrocytic development of the malaria parasite Plasmodium falciparum

    PubMed Central

    Alves, Eduardo; Maluf, Fernando V.; Bueno, Vânia B.; Guido, Rafael V. C.; Oliva, Glaucius; Singh, Maneesh; Scarpelli, Pedro; Costa, Fahyme; Sartorello, Robson; Catalani, Luiz H.; Brady, Declan; Tewari, Rita; Garcia, Celia R. S.

    2016-01-01

    In mammals, haem degradation to biliverdin (BV) through the action of haem oxygenase (HO) is a critical step in haem metabolism. The malaria parasite converts haem into the chemically inert haemozoin to avoid toxicity. We discovered that the knock-out of HO in P. berghei is lethal; therefore, we investigated the function of biliverdin (BV) and haem in the parasite. Addition of external BV and haem to P. falciparum-infected red blood cell (RBC) cultures delays the progression of parasite development. The search for a BV molecular target within the parasites identified P. falciparum enolase (Pf enolase) as the strongest candidate. Isothermal titration calorimetry using recombinant full-length Plasmodium enolase suggested one binding site for BV. Kinetic assays revealed that BV is a non-competitive inhibitor. We employed molecular modelling studies to predict the new binding site as well as the binding mode of BV to P. falciparum enolase. Furthermore, addition of BV and haem targets the phosphorylation of Plasmodium falciparum eIF2α factor, an eukaryotic initiation factor phosphorylated by eIF2α kinases under stress conditions. We propose that BV targets enolase to reduce parasite glycolysis rates and changes the eIF2α phosphorylation pattern as a molecular mechanism for its action. PMID:26915471

  1. Biliverdin targets enolase and eukaryotic initiation factor 2 (eIF2?) to reduce the growth of intraerythrocytic development of the malaria parasite Plasmodium falciparum.

    PubMed

    Alves, Eduardo; Maluf, Fernando V; Bueno, Vnia B; Guido, Rafael V C; Oliva, Glaucius; Singh, Maneesh; Scarpelli, Pedro; Costa, Fahyme; Sartorello, Robson; Catalani, Luiz H; Brady, Declan; Tewari, Rita; Garcia, Celia R S

    2016-01-01

    In mammals, haem degradation to biliverdin (BV) through the action of haem oxygenase (HO) is a critical step in haem metabolism. The malaria parasite converts haem into the chemically inert haemozoin to avoid toxicity. We discovered that the knock-out of HO in P. berghei is lethal; therefore, we investigated the function of biliverdin (BV) and haem in the parasite. Addition of external BV and haem to P. falciparum-infected red blood cell (RBC) cultures delays the progression of parasite development. The search for a BV molecular target within the parasites identified P. falciparum enolase (Pf enolase) as the strongest candidate. Isothermal titration calorimetry using recombinant full-length Plasmodium enolase suggested one binding site for BV. Kinetic assays revealed that BV is a non-competitive inhibitor. We employed molecular modelling studies to predict the new binding site as well as the binding mode of BV to P. falciparum enolase. Furthermore, addition of BV and haem targets the phosphorylation of Plasmodium falciparum eIF2? factor, an eukaryotic initiation factor phosphorylated by eIF2? kinases under stress conditions. We propose that BV targets enolase to reduce parasite glycolysis rates and changes the eIF2? phosphorylation pattern as a molecular mechanism for its action. PMID:26915471

  2. Efficacy of a Plasmodium vivax malaria vaccine using ChAd63 and modified vaccinia Ankara expressing thrombospondin-related anonymous protein as assessed with transgenic Plasmodium berghei parasites.

    PubMed

    Bauza, Karolis; Malinauskas, Tomas; Pfander, Claudia; Anar, Burcu; Jones, E Yvonne; Billker, Oliver; Hill, Adrian V S; Reyes-Sandoval, Arturo

    2014-03-01

    Plasmodium vivax is the world's most widely distributed malaria parasite and a potential cause of morbidity and mortality for approximately 2.85 billion people living mainly in Southeast Asia and Latin America. Despite this dramatic burden, very few vaccines have been assessed in humans. The clinically relevant vectors modified vaccinia virus Ankara (MVA) and the chimpanzee adenovirus ChAd63 are promising delivery systems for malaria vaccines due to their safety profiles and proven ability to induce protective immune responses against Plasmodium falciparum thrombospondin-related anonymous protein (TRAP) in clinical trials. Here, we describe the development of new recombinant ChAd63 and MVA vectors expressing P. vivax TRAP (PvTRAP) and show their ability to induce high antibody titers and T cell responses in mice. In addition, we report a novel way of assessing the efficacy of new candidate vaccines against P. vivax using a fully infectious transgenic Plasmodium berghei parasite expressing P. vivax TRAP to allow studies of vaccine efficacy and protective mechanisms in rodents. Using this model, we found that both CD8+ T cells and antibodies mediated protection against malaria using virus-vectored vaccines. Our data indicate that ChAd63 and MVA expressing PvTRAP are good preerythrocytic-stage vaccine candidates with potential for future clinical application. PMID:24379295

  3. Supraorbital Postmortem Brain Sampling for Definitive Quantitative Confirmation of Cerebral Sequestration of Plasmodium falciparum Parasites

    PubMed Central

    Milner, Danny A.; Valim, Clarissa; Luo, Robert; Playforth, Krupa B.; Kamiza, Steve; Molyneux, Malcolm E.; Seydel, Karl B.; Taylor, Terrie E.

    2012-01-01

    Background The conventional clinical case definition of cerebral malaria (CM) is imprecise but specificity is improved by a definitive clinical feature such as retinopathy or confirming sequestration of parasites in a post-mortem examination of the brain. A full autopsy is often not possible, since it is costly and may encounter resistance of the deceased's family. Methods We have assessed the use of a cytological smear of brain tissue, obtained post-mortem by supraorbital sampling, for the purpose of quantifying cerebral sequestration in children with fatal malaria in Blantyre, Malawi. We have compared this method to histological quantification of parasites at autopsy. Results The number of parasites present on cytological smears correlated with the proportion of vessels parasitized as assessed by histology of fixed and stained brain tissue. Use of cytological results in addition to the standard clinical case definition increases the specificity of the clinical case definition alone from 48.3% to 100% with a minimal change in sensitivity. Conclusions Post-mortem supraorbital sampling of brain tissue improves the specificity of the diagnosis of fatal cerebral malaria and provides accurate quantitative estimates of cerebral sequestration. This tool can be of great value in clinical, pathogenetic, and epidemiological research studies on cerebral malaria. PMID:22291197

  4. Impact of Mosquito Bites on Asexual Parasite Density and Gametocyte Prevalence in Asymptomatic Chronic Plasmodium falciparum Infections and Correlation with IgE and IgG Titers

    PubMed Central

    Lawaly, Ramatoulaye; Konate, Lassana; Marrama, Laurence; Dia, Ibrahima; Diallo, Diawo; Dine Sarr, Fatoumata; Schneider, Bradley S.; Casademont, Isabelle; Diallo, Mawlouth; Brey, Paul T.; Sakuntabhai, Anavaj; Mecheri, Salah

    2012-01-01

    An immunomodulatory role of arthropod saliva has been well documented, but evidence for an effect on Plasmodium sp. infectiousness remains controversial. Mosquito saliva may orient the immune response toward a Th2 profile, thereby priming a Th2 response against subsequent antigens, including Plasmodium. Orientation toward a Th1 versus a Th2 profile promotes IgG and IgE proliferation, respectively, where the former is crucial for the development of an efficient antiparasite immune response. Here we assessed the direct effect of mosquito bites on the density of Plasmodium falciparum asexual parasites and the prevalence of gametocytes in chronic, asymptomatic infections in a longitudinal cohort study of seasonal transmission. We additionally correlated these parasitological measures with IgE and IgG antiparasite and anti-salivary gland extract titers. The mosquito biting density was positively correlated with the asexual parasite density but not asexual parasite prevalence and was negatively correlated with gametocyte prevalence. Individual anti-salivary gland IgE titers were also negatively correlated with gametocyte carriage and were strongly positively correlated with antiparasite IgE titers, consistent with the hypothesis that mosquito bites predispose individuals to develop an IgE antiparasite response. We provide evidence that mosquito bites have an impact on asymptomatic infections and differentially so for the production of asexual and sexual parasites. An increased research focus on the immunological impact of mosquito bites during asymptomatic infections is warranted, to establish whether strategies targeting the immune response to saliva can reduce the duration of infection and the onward transmission of the parasite. PMID:22451520

  5. SYBR Green Real-Time PCR-RFLP Assay Targeting the Plasmodium Cytochrome B Gene A Highly Sensitive Molecular Tool for Malaria Parasite Detection and Species Determination

    PubMed Central

    Xu, Weiping; Morris, Ulrika; Aydin-Schmidt, Berit; Msellem, Mwinyi I.; Shakely, Delr; Petzold, Max; Bjrkman, Anders; Mrtensson, Andreas

    2015-01-01

    A prerequisite for reliable detection of low-density Plasmodium infections in malaria pre-elimination settings is the availability of ultra-sensitive and high-throughput molecular tools. We developed a SYBR Green real-time PCR restriction fragment length polymorphism assay (cytb-qPCR) targeting the cytochrome b gene of the four major human Plasmodium species (P. falciparum, P. vivax, P. malariae, and P. ovale) for parasite detection and species determination with DNA extracted from dried blood spots collected on filter paper. The performance of cytb-qPCR was first compared against four reference PCR methods using serially diluted Plasmodium samples. The detection limit of the cytb-qPCR was 1 parasite/?l (p/?l) for P. falciparum and P. ovale, and 2 p/?l for P. vivax and P. malariae, while the reference PCRs had detection limits of 0.510 p/?l. The ability of the PCR methods to detect low-density Plasmodium infections was then assessed using 2977 filter paper samples collected during a cross-sectional survey in Zanzibar, a malaria pre-elimination setting in sub-Saharan Africa. Field samples were defined as final positive if positive in at least two of the five PCR methods. Cytb-qPCR preformed equal to or better than the reference PCRs with a sensitivity of 100% (65/65; 95%CI 94.5100%) and a specificity of 99.9% (2910/2912; 95%CI 99.7100%) when compared against final positive samples. The results indicate that the cytb-qPCR may represent an opportunity for improved molecular surveillance of low-density Plasmodium infections in malaria pre-elimination settings. PMID:25774805

  6. Novel Antimalarial Aminoquinolines: Heme Binding and Effects on Normal or Plasmodium falciparum-Parasitized Human Erythrocytes ▿

    PubMed Central

    Omodeo-Salè, Fausta; Cortelezzi, Lucia; Basilico, Nicoletta; Casagrande, Manolo; Sparatore, Anna; Taramelli, Donatella

    2009-01-01

    Two new quinolizidinyl-alkyl derivatives of 7-chloro-4-aminoquinoline, named AM-1 and AP4b, which are highly effective in vitro against both the D10 (chloroquine [CQ] susceptible) and W2 (CQ resistant) strains of Plasmodium falciparum and in vivo in the rodent malaria model, have been studied for their ability to bind to and be internalized by normal or parasitized human red blood cells (RBC) and for their effects on RBC membrane stability. In addition, an analysis of the heme binding properties of these compounds and of their ability to inhibit beta-hematin formation in vitro has been performed. Binding of AM1 or AP4b to RBC is rapid, dose dependent, and linearly related to RBC density. Their accumulation in parasitized RBC (pRBC) is increased twofold compared to levels in normal RBC. Binding of AM1 or AP4b to both normal and pRBC is higher than that of CQ, in agreement with the lower pKa and higher lipophilicity of the compounds. AM1 or AP4b is not hemolytic per se and is less hemolytic than CQ when hemolysis is accelerated (induced) by hematin. Moreover, AM-1 and AP4b bind heme with a stoichiometry of interaction similar to that of CQ (about 1:1.7) but with a lower affinity. They both inhibit dose dependently the formation of beta-hematin in vitro with a 50% inhibitory concentration comparable to that of CQ. Taken together, these results suggest that the antimalarial activity of AM1 or AP4b is likely due to inhibition of hemozoin formation and that the efficacy of these compounds against the CQ-resistant strains can be ascribed to their hydrophobicity and capacity to accumulate in the vacuolar lipid (elevated lipid accumulation ratios). PMID:19651905

  7. Expression of Plasmodium vivax crt-o Is Related to Parasite Stage but Not Ex Vivo Chloroquine Susceptibility

    PubMed Central

    Pava, Zuleima; Handayuni, Irene; Wirjanata, Grennady; To, Sheren; Trianty, Leily; Noviyanti, Rintis; Poespoprodjo, Jeanne Rini; Auburn, Sarah; Price, Ric N.

    2015-01-01

    Chloroquine (CQ)-resistant Plasmodium vivax is present in most countries where P. vivax infection is endemic, but the underlying molecular mechanisms responsible remain unknown. Increased expression of P. vivax crt-o (pvcrt-o) has been correlated with in vivo CQ resistance in an area with low-grade resistance. We assessed pvcrt-o expression in isolates from Papua (Indonesia), where P. vivax is highly CQ resistant. Ex vivo drug susceptibilities to CQ, amodiaquine, piperaquine, mefloquine, and artesunate were determined using a modified schizont maturation assay. Expression levels of pvcrt-o were measured using a novel real-time quantitative reverse transcription-PCR method. Large variations in pvcrt-o expression were observed across the 51 isolates evaluated, with the fold change in expression level ranging from 0.01 to 59 relative to that seen with the P. vivax β-tubulin gene and from 0.01 to 24 relative to that seen with the P. vivax aldolase gene. Expression was significantly higher in isolates with the majority of parasites at the ring stage of development (median fold change, 1.7) compared to those at the trophozoite stage (median fold change, 0.5; P < 0.001). Twenty-nine isolates fulfilled the criteria for ex vivo drug susceptibility testing and showed high variability in CQ responses (median, 107.9 [range, 6.5 to 345.7] nM). After controlling for the parasite stage, we found that pvcrt-o expression levels did not correlate with the ex vivo response to CQ or with that to any of the other antimalarials tested. Our results highlight the importance of development-stage composition for measuring pvcrt-o expression and suggest that pvcrt-o transcription is not a primary determinant of ex vivo drug susceptibility. A comprehensive transcriptomic approach is warranted for an in-depth investigation of the role of gene expression levels and P. vivax drug resistance. PMID:26525783

  8. Analysis of the spatial and temporal arrangement of transcripts over intergenic regions in the human malarial parasite Plasmodium falciparum

    PubMed Central

    2013-01-01

    Background The ability of the human malarial parasite Plasmodium falciparum to invade, colonise and multiply within diverse host environments, as well as to manifest its virulence within the human host, are activities tightly linked to the temporal and spatial control of gene expression. Yet, despite the wealth of high throughput transcriptomic data available for this organism there is very little information regarding the location of key transcriptional landmarks or their associated cis-acting regulatory elements. Here we provide a systematic exploration of the size and organisation of transcripts within intergenic regions to yield surrogate information regarding transcriptional landmarks, and to also explore the spatial and temporal organisation of transcripts over these poorly characterised genomic regions. Results Utilising the transcript data for a cohort of 105 genes we demonstrate that the untranscribed regions of mRNA are large and apportioned predominantly to the 5? end of the open reading frame. Given the relatively compact size of the P. falciparum genome, we suggest that whilst transcriptional units are likely to spatially overlap, temporal co-transcription of adjacent transcriptional units is actually limited. Critically, the size of intergenic regions is directly dependent on the orientation of the two transcriptional units arrayed over them, an observation we extend to an analysis of the complete sequences of twelve additional organisms that share moderately compact genomes. Conclusions Our study provides a theoretical framework that extends our current understanding of the transcriptional landscape across the P. falciparum genome. Demonstration of a consensus gene-spacing rule that is shared between P. falciparum and ten other moderately compact genomes of apicomplexan parasites reveals the potential for our findings to have a wider impact across a phylum that contains many organisms important to human and veterinary health. PMID:23601558

  9. Expression of Plasmodium vivax crt-o Is Related to Parasite Stage but Not Ex Vivo Chloroquine Susceptibility.

    PubMed

    Pava, Zuleima; Handayuni, Irene; Wirjanata, Grennady; To, Sheren; Trianty, Leily; Noviyanti, Rintis; Poespoprodjo, Jeanne Rini; Auburn, Sarah; Price, Ric N; Marfurt, Jutta

    2015-01-01

    Chloroquine (CQ)-resistant Plasmodium vivax is present in most countries where P. vivax infection is endemic, but the underlying molecular mechanisms responsible remain unknown. Increased expression of P. vivax crt-o (pvcrt-o) has been correlated with in vivo CQ resistance in an area with low-grade resistance. We assessed pvcrt-o expression in isolates from Papua (Indonesia), where P. vivax is highly CQ resistant. Ex vivo drug susceptibilities to CQ, amodiaquine, piperaquine, mefloquine, and artesunate were determined using a modified schizont maturation assay. Expression levels of pvcrt-o were measured using a novel real-time quantitative reverse transcription-PCR method. Large variations in pvcrt-o expression were observed across the 51 isolates evaluated, with the fold change in expression level ranging from 0.01 to 59 relative to that seen with the P. vivax ?-tubulin gene and from 0.01 to 24 relative to that seen with the P. vivax aldolase gene. Expression was significantly higher in isolates with the majority of parasites at the ring stage of development (median fold change, 1.7) compared to those at the trophozoite stage (median fold change, 0.5; P < 0.001). Twenty-nine isolates fulfilled the criteria for ex vivo drug susceptibility testing and showed high variability in CQ responses (median, 107.9 [range, 6.5 to 345.7] nM). After controlling for the parasite stage, we found that pvcrt-o expression levels did not correlate with the ex vivo response to CQ or with that to any of the other antimalarials tested. Our results highlight the importance of development-stage composition for measuring pvcrt-o expression and suggest that pvcrt-o transcription is not a primary determinant of ex vivo drug susceptibility. A comprehensive transcriptomic approach is warranted for an in-depth investigation of the role of gene expression levels and P. vivax drug resistance. PMID:26525783

  10. Characterisation of a sexual stage-specific gene encoding ORC1 homologue in the human malaria parasite Plasmodium falciparum.

    PubMed

    Li, Ji-Liang; Cox, Lynne S

    2003-03-01

    The origin recognition complex (ORC) is a multisubunit protein composed of six polypeptides that binds to replication origins and is essential for the initiation of chromosomal DNA replication. Using the Vectorette technique, we have isolated a novel gene encoding an ORC1-like protein from the human malaria parasite Plasmodium falciparum. The gene has no introns and encodes a protein (PfORC1) of 1189 amino acid residues with a predicted molecular mass of 139 kDa. PfORC1 contains all conserved sequences in the ORC1/Cdc6/Cdc18 family and displays the highest homology to the Schizosaccharomyces pombe ORC1. However, PfORC1 possesses an extensive N-terminal segment with several interesting features including multiple potential phosphorylation sites, a large proportion of charged amino acids, four copies of a heptamer repeat, two nuclear localisation signals, and a leucine zipper motif. Southern blot analyses show that the Pforc1 gene is present as a single copy per haploid genome and is located on chromosome 12. A 5600 nucleotide transcript of this gene is expressed predominantly in the sexual erythrocytic stage, indicating that PfORC1 may be involved in gametogenesis during which DNA is quickly replicated. PMID:12543146

  11. Evidence that the Malaria Parasite Plasmodium falciparum Putative Rhoptry Protein 2 Localizes to the Golgi Apparatus throughout the Erythrocytic Cycle

    PubMed Central

    Halle, Stphanie; Richard, Dave

    2015-01-01

    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

  12. Synthetic indole and melatonin derivatives exhibit antimalarial activity on the cell cycle of the human malaria parasite Plasmodium falciparum.

    PubMed

    Schuck, Desire C; Jordo, Alessandro K; Nakabashi, Myna; Cunha, Anna C; Ferreira, Vitor F; Garcia, Clia R S

    2014-05-01

    Discovering the mechanisms by which cell signaling controls the cell cycle of the human malaria parasite Plasmodium falciparum is fundamental to designing more effective antimalarials. To better understand the impacts of melatonin structure and function on the cell cycle of P.falciparum, we have synthesized two families of structurally-related melatonin compounds (7-11 and 12-16). All synthesized melatonin analogs were assayed in P.falciparum culture and their antimalarial activities were measured by flow cytometry. We have found that the chemical modification of the carboxamide group attached at C-3 position of the indole ring of melatonin (6) was crucial for the action of the indole-related compounds on the P.falciparum cell cycle. Among the melatonin derivatives, only the compounds 12, 13 and 14 were capable of inhibiting the P.falciparum growth in low micromolar IC50. These results open good perspectives for the development of new drugs with novel mechanisms of action. PMID:24699367

  13. Distribution of drug resistance genotypes in Plasmodium falciparum in an area of limited parasite diversity in Saudi Arabia.

    PubMed

    Bin Dajem, Saad M; Al-Farsi, Hissa M; Al-Hashami, Zainab S; Al-Sheikh, Adel Ali H; Al-Qahtani, Ahmed; Babiker, Hamza A

    2012-05-01

    Two hundred and three Plasmodium falciparum isolates from Jazan area, southwest Saudi Arabia, were typed for Pfcrt, Pfmdr1, dhps, and dhfr mutations associated with resistance to chloroquine, mefloquine, halofantrine, artemisinin, sulfadoxine-pyrimethamine, and the neutral polymorphic gene Pfg377. A large proportion (33%) of isolates harbored double mutant dhfr genotype (51I,59C,108N). However, only one isolate contained mutation dhps-437G. For Pfcrt, almost all examined isolates (163; 99%) harbored the mutant genotype (72C,73V,74I,75E,76T), whereas only 49 (31%) contained the mutant Pfmdr1 genotype (86Y,184F,1034S,1042N), 109 (66%) harbored the single mutant genotype (86N,184F,1034S,1042N), and no mutations were seen in codons 1034, 1042, and 1246. Nonetheless, three new single-nucleotide polymorphisms were detected at codons 182, 192, and 102. No differences were seen in distribution of drug resistance genes among Saudis and expatriates. There was a limited multiplicity (5%), mean number of clones (1.05), and two dominant multilocus genotypes among infected individuals in Jazan. A pattern consistent with limited cross-mating and recombination among local parasite was apparent. PMID:22556074

  14. Landscape and Dynamics of Transcription Initiation in the Malaria Parasite Plasmodium falciparum.

    PubMed

    Adjalley, Sophie H; Chabbert, Christophe D; Klaus, Bernd; Pelechano, Vicent; Steinmetz, Lars M

    2016-03-15

    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. PMID:26947071

  15. The Mu subunit of Plasmodium falciparum clathrin-associated adaptor protein 2 modulates in vitro parasite response to artemisinin and quinine.

    PubMed

    Henriques, Gisela; van Schalkwyk, Donelly A; Burrow, Rebekah; Warhurst, David C; Thompson, Eloise; Baker, David A; Fidock, David A; Hallett, Rachel; Flueck, Christian; Sutherland, Colin J

    2015-05-01

    The emergence of drug-resistant parasites is a serious threat faced by malaria control programs. Understanding the genetic basis of resistance is critical to the success of treatment and intervention strategies. A novel locus associated with antimalarial resistance, ap2-mu (encoding the mu chain of the adaptor protein 2 [AP2] complex), was recently identified in studies on the rodent malaria parasite Plasmodium chabaudi (pcap2-mu). Furthermore, analysis in Kenyan malaria patients of polymorphisms in the Plasmodium falciparum ap2-mu homologue, pfap2-mu, found evidence that differences in the amino acid encoded by codon 160 are associated with enhanced parasite survival in vivo following combination treatments which included artemisinin derivatives. Here, we characterize the role of pfap2-mu in mediating the in vitro antimalarial drug response of P. falciparum by generating transgenic parasites constitutively expressing codon 160 encoding either the wild-type Ser (Ser160) or the Asn mutant (160Asn) form of pfap2-mu. Transgenic parasites carrying the pfap2-mu 160Asn allele were significantly less sensitive to dihydroartemisinin using a standard 48-h in vitro test, providing direct evidence of an altered parasite response to artemisinin. Our data also provide evidence that pfap2-mu variants can modulate parasite sensitivity to quinine. No evidence was found that pfap2-mu variants contribute to the slow-clearance phenotype exhibited by P. falciparum in Cambodian patients treated with artesunate monotherapy. These findings provide compelling evidence that pfap2-mu can modulate P. falciparum responses to multiple drugs. We propose that this gene should be evaluated further as a potential molecular marker of antimalarial resistance. PMID:25691625

  16. The Mu Subunit of Plasmodium falciparum Clathrin-Associated Adaptor Protein 2 Modulates In Vitro Parasite Response to Artemisinin and Quinine

    PubMed Central

    Henriques, Gisela; van Schalkwyk, Donelly A.; Burrow, Rebekah; Warhurst, David C.; Thompson, Eloise; Baker, David A.; Fidock, David A.; Hallett, Rachel; Flueck, Christian

    2015-01-01

    The emergence of drug-resistant parasites is a serious threat faced by malaria control programs. Understanding the genetic basis of resistance is critical to the success of treatment and intervention strategies. A novel locus associated with antimalarial resistance, ap2-mu (encoding the mu chain of the adaptor protein 2 [AP2] complex), was recently identified in studies on the rodent malaria parasite Plasmodium chabaudi (pcap2-mu). Furthermore, analysis in Kenyan malaria patients of polymorphisms in the Plasmodium falciparum ap2-mu homologue, pfap2-mu, found evidence that differences in the amino acid encoded by codon 160 are associated with enhanced parasite survival in vivo following combination treatments which included artemisinin derivatives. Here, we characterize the role of pfap2-mu in mediating the in vitro antimalarial drug response of P. falciparum by generating transgenic parasites constitutively expressing codon 160 encoding either the wild-type Ser (Ser160) or the Asn mutant (160Asn) form of pfap2-mu. Transgenic parasites carrying the pfap2-mu 160Asn allele were significantly less sensitive to dihydroartemisinin using a standard 48-h in vitro test, providing direct evidence of an altered parasite response to artemisinin. Our data also provide evidence that pfap2-mu variants can modulate parasite sensitivity to quinine. No evidence was found that pfap2-mu variants contribute to the slow-clearance phenotype exhibited by P. falciparum in Cambodian patients treated with artesunate monotherapy. These findings provide compelling evidence that pfap2-mu can modulate P. falciparum responses to multiple drugs. We propose that this gene should be evaluated further as a potential molecular marker of antimalarial resistance. PMID:25691625

  17. Highly Sensitive Quantitative Real-Time PCR for the Detection of Plasmodium Liver-Stage Parasite Burden following Low-Dose Sporozoite Challenge

    PubMed Central

    Schussek, Sophie; Groves, Penny L.; Apte, Simon H.; Doolan, Denise L.

    2013-01-01

    The pre-erythrocytic stages of Plasmodium spp. are increasingly recognised as ideal targets for prophylactic vaccines and drug treatments. Intense research efforts in the last decade have been focused on in vitro culture and in vivo detection and quantification of liver stage parasites to assess the effects of candidate vaccines or drugs. Typically, the onset of blood stage parasitaemia is used as a surrogate endpoint to estimate the efficacy of vaccines and drugs targeting pre-erythrocytic parasite stages in animal models. However, this provides no information on the parasite burden in the liver after vaccination or treatment and therefore does not detect partial efficacy of any vaccine or drug candidates. Herein, we describe a quantitative RT-PCR method adapted to detect and quantitate Plasmodium yoelii liver stages in mice with increased sensitivity even after challenge with as few as 50 cryopreserved sporozoites (corresponding to approximately 5-10 freshly isolated sporozoites). We have validated our quantitative RT-PCR assay according to the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines and established high reproducibility and accuracy. Our assay provides a rapid and reproducible assessment of liver stage parasite burden in rodent malaria models, thereby facilitating the evaluation of the efficacy of anti-malarial drugs or prophylactic vaccines with high precision and efficacy. PMID:24098596

  18. Generation of an antibody that recognizes Plasmodium chabaudi cysteine protease (chabaupain-1) in both sexual and asexual parasite life cycle and evaluation of chabaupain-1 vaccine potential.

    PubMed

    Armada, Ana; Gazarini, Marcos L; Gonalves, Ldia M; Antunes, Sandra; Custdio, Ana; Rodrigues, Armanda; Almeida, Antnio J; Silveira, Henrique; Rosrio, Virglio do; Santos-Gomes, Gabriela; Domingos, Ana

    2013-09-01

    Malaria cysteine proteases have been shown to be immunogenic and are being exploited as serodiagnostic markers, drug and vaccine targets. Several Plasmodium spp. cysteine proteases have been described and the best characterized of these are the falcipains, a family of papain-family enzymes. Falcipain-2 and falcipain-3 act in concert with other proteases to hydrolyze host erythrocyte hemoglobin in the parasite food vacuole. Falcipain-1 has less similarity to the other falcipains and its physiological role in parasite asexual blood stage still remains uncertain. Immunolocalization studies using an antibody developed against the Plasmodium chabaudi recombinant chabaupain-1, the falcipain-1 ortholog, were performed confirming its cellular localization in both erythrocyte and mosquito ookinete stage. Immunostaining of chabaupain-1 preferentially in apical portion of parasite ookinete suggests that this protease may be related with parasite egression from mosquito midgut. Immune responses to chabaupain-1 were evaluated using two different adjuvants, chitosan nanoparticles and hydroxide aluminum. Mice immunized with the recombinant protein alone or in association with nanoparticles were challenged with P. chabaudi showing that immunization with the recombinant protein confers partial protection to blood stage infection in BALB/c animal model. PMID:23830988

  19. Interrogating alkyl and arylalkylpolyamino (bis)urea and (bis)thiourea isosteres as potent antimalarial chemotypes against multiple lifecycle forms of Plasmodium falciparum parasites

    PubMed Central

    Verlinden, Bianca K.; de Beer, Marna; Pachaiyappan, Boobalan; Besaans, Ethan; Andayi, Warren A.; Reader, Janette; Niemand, Jandeli; van Biljon, Riette; Guy, Kiplin; Egan, Timothy; Woster, Patrick M.; Birkholtz, Lyn-Marie

    2015-01-01

    A new series of potent potent aryl/alkylated (bis)urea- and (bis)thiourea polyamine analogues were synthesized and evaluated in vitro for their antiplasmodial activity. Altering the carbon backbone and terminal substituents increased the potency of analogues in the compound library 3-fold, with the most active compounds, 15 and 16, showing half-maximal inhibitory concentrations (IC50 values) of 28 and 30 nM, respectively, against various Plasmodium falciparum parasite strains without any cross-resistance. In vitro evaluation of the cytotoxicity of these analogues revealed marked selectivity towards targeting malaria parasites compared to mammalian HepG2 cells (>5000-fold lower IC50 against the parasite). Preliminary biological evaluation of the polyamine analogue antiplasmodial phenotype revealed that (bis)urea compounds target parasite asexual proliferation, whereas (bis)thiourea compounds of the same series have the unique ability to block transmissible gametocyte forms of the parasite, indicating pluripharmacology against proliferative and non-proliferative forms of the parasite. In this manuscript, we describe these results and postulate a refined structure-activity relationship (SAR) model for antiplasmodial polyamine analogues. The terminally aryl/alkylated (bis)urea- and (bis)thiourea-polyamine analogues featuring a 3-5-3 or 3-6-3 carbon backbone represent a structurally novel and distinct class of potential antiplasmodials with activities in the low nanomolar range, and high selectivity against various lifecycle forms of P. falciparum parasites. PMID:25684422

  20. Functional dissection of proliferating-cell nuclear antigens (1 and 2) in human malarial parasite Plasmodium falciparum: possible involvement in DNA replication and DNA damage response.

    PubMed

    Mitra, Pallabi; Banu, Khadija; Deshmukh, Abhijit S; Subbarao, Naidu; Dhar, Suman Kumar

    2015-08-15

    Eukaryotic PCNAs (proliferating-cell nuclear antigens) play diverse roles in nucleic acid metabolism in addition to DNA replication. Plasmodium falciparum, which causes human malaria, harbours two PCNA homologues: PfPCNA1 and PfPCNA2. The functional role of two distinct PCNAs in the parasite still eludes us. In the present study, we show that, whereas both PfPCNAs share structural and biochemical properties, only PfPCNA1 functionally complements the ScPCNA mutant and forms distinct replication foci in the parasite, which PfPCNA2 fails to do. Although PfPCNA1 appears to be the primary replicative PCNA, both PfPCNA1 and PfPCNA2 participate in an active DDR (DNA-damage-response) pathway with significant accumulation in the parasite upon DNA damage induction. Interestingly, PfPCNA genes were found to be regulated not at the transcription level, but presumably at the protein stability level upon DNA damage. Such regulation of PCNA has not been shown in eukaryotes before. Moreover, overexpression of PfPCNA1 and PfPCNA2in the parasite confers a survival edge on the parasite in a genotoxic environment. This is the first evidence of a PfPCNA-mediated DDR in the parasite and gives new insights and rationale for the presence of two PCNAs as a parasite survival strategy and its probable success. PMID:26251451

  1. Wolbachia infections are virulent and inhibit the human malaria parasite Plasmodium falciparum in Anopheles gambiae.

    PubMed

    Hughes, Grant L; Koga, Ryuichi; Xue, Ping; Fukatsu, Takema; Rasgon, Jason L

    2011-05-01

    Endosymbiotic Wolbachia bacteria are potent modulators of pathogen infection and transmission in multiple naturally and artificially infected insect species, including important vectors of human pathogens. Anopheles mosquitoes are naturally uninfected with Wolbachia, and stable artificial infections have not yet succeeded in this genus. Recent techniques have enabled establishment of somatic Wolbachia infections in Anopheles. Here, we characterize somatic infections of two diverse Wolbachia strains (wMelPop and wAlbB) in Anopheles gambiae, the major vector of human malaria. After infection, wMelPop disseminates widely in the mosquito, infecting the fat body, head, sensory organs and other tissues but is notably absent from the midgut and ovaries. Wolbachia initially induces the mosquito immune system, coincident with initial clearing of the infection, but then suppresses expression of immune genes, coincident with Wolbachia replication in the mosquito. Both wMelPop and wAlbB significantly inhibit Plasmodium falciparum oocyst levels in the mosquito midgut. Although not virulent in non-bloodfed mosquitoes, wMelPop exhibits a novel phenotype and is extremely virulent for approximately 12-24 hours post-bloodmeal, after which surviving mosquitoes exhibit similar mortality trajectories to control mosquitoes. The data suggest that if stable transinfections act in a similar manner to somatic infections, Wolbachia could potentially be used as part of a strategy to control the Anopheles mosquitoes that transmit malaria. PMID:21625582

  2. High-resolution three-dimensional imaging of red blood cells parasitized by Plasmodium falciparum and in situ hemozoin crystals using optical diffraction tomography

    PubMed Central

    Kim, Kyoohyun; Yoon, HyeOk; Diez-Silva, Monica; Dao, Ming; Dasari, Ramachandra R.; Park, YongKeun

    2013-01-01

    Abstract. We present high-resolution optical tomographic images of human red blood cells (RBC) parasitized by malaria-inducing Plasmodium falciparum (Pf)-RBCs. Three-dimensional (3-D) refractive index (RI) tomograms are reconstructed by recourse to a diffraction algorithm from multiple two-dimensional holograms with various angles of illumination. These 3-D RI tomograms of Pf-RBCs show cellular and subcellular structures of host RBCs and invaded parasites in fine detail. Full asexual intraerythrocytic stages of parasite maturation (ring to trophozoite to schizont stages) are then systematically investigated using optical diffraction tomography algorithms. These analyses provide quantitative information on the structural and chemical characteristics of individual host Pf-RBCs, parasitophorous vacuole, and cytoplasm. The in situ structural evolution and chemical characteristics of subcellular hemozoin crystals are also elucidated. PMID:23797986

  3. High-resolution three-dimensional imaging of red blood cells parasitized by Plasmodium falciparum and in situ hemozoin crystals using optical diffraction tomography

    NASA Astrophysics Data System (ADS)

    Kim, Kyoohyun; Yoon, HyeOk; Diez-Silva, Monica; Dao, Ming; Dasari, Ramachandra R.; Park, YongKeun

    2014-01-01

    We present high-resolution optical tomographic images of human red blood cells (RBC) parasitized by malaria-inducing Plasmodium falciparum (Pf)-RBCs. Three-dimensional (3-D) refractive index (RI) tomograms are reconstructed by recourse to a diffraction algorithm from multiple two-dimensional holograms with various angles of illumination. These 3-D RI tomograms of Pf-RBCs show cellular and subcellular structures of host RBCs and invaded parasites in fine detail. Full asexual intraerythrocytic stages of parasite maturation (ring to trophozoite to schizont stages) are then systematically investigated using optical diffraction tomography algorithms. These analyses provide quantitative information on the structural and chemical characteristics of individual host Pf-RBCs, parasitophorous vacuole, and cytoplasm. The in situ structural evolution and chemical characteristics of subcellular hemozoin crystals are also elucidated.

  4. The Clp Chaperones and Proteases of the Human Malaria Parasite Plasmodium falciparum

    SciTech Connect

    M El Bakkouri; A Pow; A Mulichak; K Cheung; J Artz; M Amani; S Fell; T de Koning-Ward; C Goodman; et al.

    2011-12-31

    The Clpchaperones and proteases play an important role in protein homeostasis in the cell. They are highly conserved across prokaryotes and found also in the mitochondria of eukaryotes and the chloroplasts of plants. They function mainly in the disaggregation, unfolding and degradation of native as well as misfolded proteins. Here, we provide a comprehensive analysis of the Clpchaperones and proteases in the humanmalariaparasitePlasmodiumfalciparum. The parasite contains four Clp ATPases, which we term PfClpB1, PfClpB2, PfClpC and PfClpM. One PfClpP, the proteolytic subunit, and one PfClpR, which is an inactive version of the protease, were also identified. Expression of all Clpchaperones and proteases was confirmed in blood-stage parasites. The proteins were localized to the apicoplast, a non-photosynthetic organelle that accommodates several important metabolic pathways in P. falciparum, with the exception of PfClpB2 (also known as Hsp101), which was found in the parasitophorous vacuole. Both PfClpP and PfClpR form mostly homoheptameric rings as observed by size-exclusion chromatography, analytical ultracentrifugation and electron microscopy. The X-ray structure of PfClpP showed the protein as a compacted tetradecamer similar to that observed for Streptococcus pneumoniae and Mycobacterium tuberculosis ClpPs. Our data suggest the presence of a ClpCRP complex in the apicoplast of P. falciparum.

  5. A whole parasite vaccine to control the blood stages of Plasmodium: the case for lateral thinking.

    PubMed

    Good, Michael F

    2011-08-01

    Now, 27 years following the cloning of malaria antigens with the promise of the rapid development of a malaria vaccine, we face significant obstacles that are belatedly being addressed. Poor immunogenicity of subunit vaccine antigens and significant antigenic diversity of target epitopes represent major hurdles for which there are no clear strategies for a way forward within the current paradigm. Thus, a different paradigm - a vaccine that uses the whole organism - is now being examined. Although most advances in this approach relate to a vaccine for the pre-erythrocytic stages (sporozoites, liver stages), this opinion paper will outline the possibilities of developing a whole parasite vaccine for the blood stage and address some of the challenges for this strategy, which are entirely different to the challenges for a subunit vaccine. It is the view of the author that both vaccine paradigms should be pursued, but that success will come more quickly using the paranormal approach of exposing individuals to ultra-low doses of whole attenuated or killed parasites. PMID:21514227

  6. Imputation-based population genetics analysis of Plasmodium falciparum malaria parasites.

    PubMed

    Samad, Hanif; Coll, Francesc; Preston, Mark D; Ocholla, Harold; Fairhurst, Rick M; Clark, Taane G

    2015-04-01

    Whole-genome sequencing technologies are being increasingly applied to Plasmodium falciparum clinical isolates to identify genetic determinants of malaria pathogenesis. However, genome-wide discovery methods, such as haplotype scans for signatures of natural selection, are hindered by missing genotypes in sequence data. Poor correlation between single nucleotide polymorphisms (SNPs) in the P. falciparum genome complicates efforts to apply established missing-genotype imputation methods that leverage off patterns of linkage disequilibrium (LD). The accuracy of state-of-the-art, LD-based imputation methods (IMPUTE, Beagle) was assessed by measuring allelic r2 for 459 P. falciparum samples from malaria patients in 4 countries: Thailand, Cambodia, Gambia, and Malawi. In restricting our analysis to 86 k high-quality SNPs across the populations, we found that the complete-case analysis was restricted to 21k SNPs (24.5%), despite no single SNP having more than 10% missing genotypes. The accuracy of Beagle in filling in missing genotypes was consistently high across all populations (allelic r2, 0.87-0.96), but the performance of IMPUTE was mixed (allelic r2, 0.34-0.99) depending on reference haplotypes and population. Positive selection analysis using Beagle-imputed haplotypes identified loci involved in resistance to chloroquine (crt) in Thailand, Cambodia, and Gambia, sulfadoxine-pyrimethamine (dhfr, dhps) in Cambodia, and artemisinin (kelch13) in Cambodia. Tajima's D-based analysis identified genes under balancing selection that encode well-characterized vaccine candidates: apical merozoite antigen 1 (ama1) and merozoite surface protein 1 (msp1). In contrast, the complete-case analysis failed to identify any well-validated drug resistance or candidate vaccine loci, except kelch13. In a setting of low LD and modest levels of missing genotypes, using Beagle to impute P. falciparum genotypes is a viable strategy for conducting accurate large-scale population genetics and association analyses, and supporting global surveillance for drug resistance markers and candidate vaccine antigens. PMID:25928499

  7. A Bacterial Phosphatase-Like Enzyme of the Malaria Parasite Plasmodium falciparum Possesses Tyrosine Phosphatase Activity and Is Implicated in the Regulation of Band 3 Dynamics during Parasite Invasion

    PubMed Central

    Fernandez-Pol, Sebastian; Slouka, Zdenek; Bhattacharjee, Souvik; Fedotova, Yana; Freed, Stefan; An, Xiuli; Holder, Anthony A.; Campanella, Estela; Low, Philip S.

    2013-01-01

    Eukaryotic parasites of the genus Plasmodium cause malaria by invading and developing within host erythrocytes. Here, we demonstrate that PfShelph2, a gene product of Plasmodium falciparum that belongs to the Shewanella-like phosphatase (Shelph) subfamily, selectively hydrolyzes phosphotyrosine, as shown for other previously studied Shelph family members. In the extracellular merozoite stage, PfShelph2 localizes to vesicles that appear to be distinct from those of rhoptry, dense granule, or microneme organelles. During invasion, PfShelph2 is released from these vesicles and exported to the host erythrocyte. In vitro, PfShelph2 shows tyrosine phosphatase activity against the host erythrocyte protein Band 3, which is the most abundant tyrosine-phosphorylated species of the erythrocyte. During P. falciparum invasion, Band 3 undergoes dynamic and rapid clearance from the invasion junction within 1 to 2 s of parasite attachment to the erythrocyte. Release of Pfshelph2 occurs after clearance of Band 3 from the parasite-host cell interface and when the parasite is nearly or completely enclosed in the nascent vacuole. We propose a model in which the phosphatase modifies Band 3 in time to restore its interaction with the cytoskeleton and thus reestablishes the erythrocyte cytoskeletal network at the end of the invasion process. PMID:23825180

  8. Lineage-specific positive selection at the merozoite surface protein 1 (msp1) locus of Plasmodium vivax and related simian malaria parasites

    PubMed Central

    2010-01-01

    Background The 200 kDa merozoite surface protein 1 (MSP-1) of malaria parasites, a strong vaccine candidate, plays a key role during erythrocyte invasion and is a target of host protective immune response. Plasmodium vivax, the most widespread human malaria parasite, is closely related to parasites that infect Asian Old World monkeys, and has been considered to have become a parasite of man by host switch from a macaque malaria parasite. Several Asian monkey parasites have a range of natural hosts. The same parasite species shows different disease manifestations among host species. This suggests that host immune responses to P. vivax-related malaria parasites greatly differ among host species (albeit other factors). It is thus tempting to invoke that a major immune target parasite protein such as MSP-1 underwent unique evolution, depending on parasite species that exhibit difference in host range and host specificity. Results We performed comparative phylogenetic and population genetic analyses of the gene encoding MSP-1 (msp1) from P. vivax and nine P. vivax-related simian malaria parasites. The inferred phylogenetic tree of msp1 significantly differed from that of the mitochondrial genome, with a striking displacement of P. vivax from a position close to P. cynomolgi in the mitochondrial genome tree to an outlier of Asian monkey parasites. Importantly, positive selection was inferred for two ancestral branches, one leading to P. inui and P. hylobati and the other leading to P. vivax, P. fieldi and P. cynomolgi. This ancestral positive selection was estimated to have occurred three to six million years ago, coinciding with the period of radiation of Asian macaques. Comparisons of msp1 polymorphisms between P. vivax, P. inui and P. cynomolgi revealed that while some positively selected amino acid sites or regions are shared by these parasites, amino acid changes greatly differ, suggesting that diversifying selection is acting species-specifically on msp1. Conclusions The present results indicate that the msp1 locus of P. vivax and related parasite species has lineage-specific unique evolutionary history with positive selection. P. vivax and related simian malaria parasites offer an interesting system toward understanding host species-dependent adaptive evolution of immune-target surface antigen genes such as msp1. PMID:20167126

  9. Habitat fragmentation and ecological traits influence the prevalence of avian blood parasites in a tropical rainforest landscape.

    PubMed

    Laurance, Susan G W; Jones, Dean; Westcott, David; McKeown, Adam; Harrington, Graham; Hilbert, David W

    2013-01-01

    In the tropical rainforests of northern Australia, we investigated the effects of habitat fragmentation and ecological parameters on the prevalence of blood-borne parasites (Plasmodium and Haemoproteus) in bird communities. Using mist-nets on forest edges and interiors, we sampled bird communities across six study sites: 3 large fragments (20-85 ha) and 3 continuous-forest sites. From 335 mist-net captures, we recorded 28 bird species and screened 299 bird samples with PCR to amplify and detect target DNA. Of the 28 bird species sampled, 19 were infected with Plasmodium and/or Haemoproteus and 9 species were without infection. Over one third of screened birds (99 individuals) were positive for Haemoproteus and/or Plasmodium. In forest fragments, bird capture rates were significantly higher than in continuous forests, but bird species richness did not differ. Unexpectedly, we found that the prevalence of the dominant haemosporidian infection, Haemoproteus, was significantly higher in continuous forest than in habitat fragments. Further, we found that ecological traits such as diet, foraging height, habitat specialisation and distributional ranges were significantly associated with blood-borne infections. PMID:24124541

  10. Habitat Fragmentation and Ecological Traits Influence the Prevalence of Avian Blood Parasites in a Tropical Rainforest Landscape

    PubMed Central

    Laurance, Susan G. W.; Jones, Dean; Westcott, David; Mckeown, Adam; Harrington, Graham; Hilbert, David W.

    2013-01-01

    In the tropical rainforests of northern Australia, we investigated the effects of habitat fragmentation and ecological parameters on the prevalence of blood-borne parasites (Plasmodium and Haemoproteus) in bird communities. Using mist-nets on forest edges and interiors, we sampled bird communities across six study sites: 3 large fragments (2085 ha) and 3 continuous-forest sites. From 335 mist-net captures, we recorded 28 bird species and screened 299 bird samples with PCR to amplify and detect target DNA. Of the 28 bird species sampled, 19 were infected with Plasmodium and/or Haemoproteus and 9 species were without infection. Over one third of screened birds (99 individuals) were positive for Haemoproteus and/or Plasmodium. In forest fragments, bird capture rates were significantly higher than in continuous forests, but bird species richness did not differ. Unexpectedly, we found that the prevalence of the dominant haemosporidian infection, Haemoproteus, was significantly higher in continuous forest than in habitat fragments. Further, we found that ecological traits such as diet, foraging height, habitat specialisation and distributional ranges were significantly associated with blood-borne infections. PMID:24124541

  11. Effects of Age, Hemoglobin Type and Parasite Strain on IgG Recognition of Plasmodium falciparumInfected Erythrocytes in Malian Children

    PubMed Central

    Zeituni, Amir E.; Miura, Kazutoyo; Diakite, Mahamadou; Doumbia, Saibou; Moretz, Samuel E.; Diouf, Ababacar; Tullo, Gregory; Lopera-Mesa, Tatiana M.; Bess, Cameron D.; Mita-Mendoza, Neida K.; Anderson, Jennifer M.; Fairhurst, Rick M.; Long, Carole A.

    2013-01-01

    Background Naturally-acquired antibody responses to antigens on the surface of Plasmodium falciparum-infected red blood cells (iRBCs) have been implicated in antimalarial immunity. To profile the development of this immunity, we have been studying a cohort of Malian children living in an area with intense seasonal malaria transmission. Methodology/Principal Findings We collected plasma from a sub-cohort of 176 Malian children aged 3-11 years, before (May) and after (December) the 2009 transmission season. To measure the effect of hemoglobin (Hb) type on antibody responses, we enrolled age-matched HbAA, HbAS and HbAC children. To quantify antibody recognition of iRBCs, we designed a high-throughput flow cytometry assay to rapidly test numerous plasma samples against multiple parasite strains. We evaluated antibody reactivity of each plasma sample to 3 laboratory-adapted parasite lines (FCR3, D10, PC26) and 4 short-term-cultured parasite isolates (2 Malian and 2 Cambodian). 97% of children recognized ?1 parasite strain and the proportion of IgG responders increased significantly during the transmission season for most parasite strains. Both strain-specific and strain-transcending IgG responses were detected, and varied by age, Hb type and parasite strain. In addition, the breadth of IgG responses to parasite strains increased with age in HbAA, but not in HbAS or HbAC, children. Conclusions/Significance Our assay detects both strain-specific and strain-transcending IgG responses to iRBCs. The magnitude and breadth of these responses varied not only by age, but also by Hb type and parasite strain used. These findings indicate that studies of acquired humoral immunity should account for Hb type and test large numbers of diverse parasite strains. PMID:24124591

  12. The Plasmodium Class XIV Myosin, MyoB, Has a Distinct Subcellular Location in Invasive and Motile Stages of the Malaria Parasite and an Unusual Light Chain*

    PubMed Central

    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.

    2015-01-01

    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

  13. In Silico Screening on the Three-dimensional Model of the Plasmodium vivax SUB1 Protease Leads to the Validation of a Novel Anti-parasite Compound*

    PubMed Central

    Bouillon, Anthony; Giganti, David; Benedet, Christophe; Gorgette, Olivier; Pêtres, Stéphane; Crublet, Elodie; Girard-Blanc, Christine; Witkowski, Benoit; Ménard, Didier; Nilges, Michael; Mercereau-Puijalon, Odile; Stoven, Véronique; Barale, Jean-Christophe

    2013-01-01

    Widespread drug resistance calls for the urgent development of new antimalarials that target novel steps in the life cycle of Plasmodium falciparum and Plasmodium vivax. The essential subtilisin-like serine protease SUB1 of Plasmodium merozoites plays a dual role in egress from and invasion into host erythrocytes. It belongs to a new generation of attractive drug targets against which specific potent inhibitors are actively searched. We characterize here the P. vivax SUB1 enzyme and show that it displays a typical auto-processing pattern and apical localization in P. vivax merozoites. To search for small PvSUB1 inhibitors, we took advantage of the similarity of SUB1 with bacterial subtilisins and generated P. vivax SUB1 three-dimensional models. The structure-based virtual screening of a large commercial chemical compounds library identified 306 virtual best hits, of which 37 were experimentally confirmed inhibitors and 5 had Ki values of <50 μm for PvSUB1. Interestingly, they belong to different chemical families. The most promising competitive inhibitor of PvSUB1 (compound 2) was equally active on PfSUB1 and displayed anti-P. falciparum and Plasmodium berghei activity in vitro and in vivo, respectively. Compound 2 inhibited the endogenous PfSUB1 as illustrated by the inhibited maturation of its natural substrate PfSERA5 and inhibited parasite egress and subsequent erythrocyte invasion. These data indicate that the strategy of in silico screening of three-dimensional models to select for virtual inhibitors combined with stringent biological validation successfully identified several inhibitors of the PvSUB1 enzyme. The most promising hit proved to be a potent cross-inhibitor of PlasmodiumSUB1, laying the groundwork for the development of a globally active small compound antimalarial. PMID:23653352

  14. Transgenic Parasites Stably Expressing Full-Length Plasmodium falciparum Circumsporozoite Protein as a Model for Vaccine Down-Selection in Mice Using Sterile Protection as an Endpoint

    PubMed Central

    Porter, Michael D.; Nicki, Jennifer; Pool, Christopher D.; DeBot, Margot; Illam, Ratish M.; Brando, Clara; Bozick, Brooke; De La Vega, Patricia; Angra, Divya; Spaccapelo, Roberta; Crisanti, Andrea; Murphy, Jittawadee R.; Bennett, Jason W.; Schwenk, Robert J.; Ockenhouse, Christian F.

    2013-01-01

    Circumsporozoite protein (CSP) of Plasmodium falciparum is a protective human malaria vaccine candidate. There is an urgent need for models that can rapidly down-select novel CSP-based vaccine candidates. In the present study, the mouse-mosquito transmission cycle of a transgenic Plasmodium berghei malaria parasite stably expressing a functional full-length P. falciparum CSP was optimized to consistently produce infective sporozoites for protection studies. A minimal sporozoite challenge dose was established, and protection was defined as the absence of blood-stage parasites 14 days after intravenous challenge. The specificity of protection was confirmed by vaccinating mice with multiple CSP constructs of differing lengths and compositions. Constructs that induced high NANP repeat-specific antibody titers in enzyme-linked immunosorbent assays were protective, and the degree of protection was dependent on the antigen dose. There was a positive correlation between antibody avidity and protection. The antibodies in the protected mice recognized the native CSP on the parasites and showed sporozoite invasion inhibitory activity. Passive transfer of anti-CSP antibodies into naive mice also induced protection. Thus, we have demonstrated the utility of a mouse efficacy model to down-select human CSP-based vaccine formulations. PMID:23536694

  15. Protein phosphatase beta, a putative type-2A protein phosphatase from the human malaria parasite Plasmodium falciparum.

    PubMed

    Li, J L; Baker, D A

    1997-10-01

    Protein phosphatases play a critical role in the regulation of the eukaryotic cell cycle and signal transduction. A putative protein serine/threonine phosphatase gene has been isolated from the human malaria parasite Plasmodium falciparum. The gene has an unusual intron that contains four repeats of 32 nucleotides and displays a high degree of size polymorphism among different strains of P. falciparum. The open reading frame reconstituted by removal of the intron encodes a protein of 466 amino acids with a predicted molecular mass of approximately 53.7 kDa. The encoded protein, termed protein phosphatase beta (PP-beta), is composed of two distinct domains. The C-terminal domain comprises 315 amino acids and exhibits a striking similarity to the catalytic subunits of the type-2A protein phosphatases. Database searches revealed that the catalytic domain has the highest similarity to Schizosaccharomyces pombe Ppa1 (58% identity and 73% similarity). However, it contains a hydrophilic insert consisting of five amino acids. The N-terminal domain comprises 151 amino acid residues and exhibits several striking features, including high levels of charged amino acids and asparagine, and multiple consensus phosphorylation sites for a number of protein kinases. An overall structural comparison of PP-beta with other members of the protein phosphatase 2A group revealed that PP-beta is more closely related to Saccharomyces cerevisiae PPH22. Southern blots of genomic DNA digests and chromosomal separations showed that PP-beta is a single-copy gene and is located on chromosome 9. A 2800-nucleotide transcript of this gene is expressed specifically in the sexual erythrocytic stage (gametocytes). The results indicate that PP-beta may be involved in sexual stage development. PMID:9363759

  16. Genome-Wide Analysis of Selection on the Malaria Parasite Plasmodium falciparum in West African Populations of Differing Infection Endemicity

    PubMed Central

    Mobegi, Victor A.; Duffy, Craig W.; Amambua-Ngwa, Alfred; Loua, Kovana M.; Laman, Eugene; Nwakanma, Davis C.; MacInnis, Bronwyn; Aspeling-Jones, Harvey; Murray, Lee; Clark, Taane G.; Kwiatkowski, Dominic P.; Conway, David J.

    2014-01-01

    Locally varying selection on pathogens may be due to differences in drug pressure, host immunity, transmission opportunities between hosts, or the intensity of between-genotype competition within hosts. Highly recombining populations of the human malaria parasite Plasmodium falciparum throughout West Africa are closely related, as gene flow is relatively unrestricted in this endemic region, but markedly varying ecology and transmission intensity should cause distinct local selective pressures. Genome-wide analysis of sequence variation was undertaken on a sample of 100 P. falciparum clinical isolates from a highly endemic region of the Republic of Guinea where transmission occurs for most of each year and compared with data from 52 clinical isolates from a previously sampled population from The Gambia, where there is relatively limited seasonal malaria transmission. Paired-end short-read sequences were mapped against the 3D7 P. falciparum reference genome sequence, and data on 136,144 single nucleotide polymorphisms (SNPs) were obtained. Within-population analyses identifying loci showing evidence of recent positive directional selection and balancing selection confirm that antimalarial drugs and host immunity have been major selective agents. Many of the signatures of recent directional selection reflected by standardized integrated haplotype scores were population specific, including differences at drug resistance loci due to historically different antimalarial use between the countries. In contrast, both populations showed a similar set of loci likely to be under balancing selection as indicated by very high Tajimas D values, including a significant overrepresentation of genes expressed at the merozoite stage that invades erythrocytes and several previously validated targets of acquired immunity. Between-population FST analysis identified exceptional differentiation of allele frequencies at a small number of loci, most markedly for five SNPs covering a 15-kb region within and flanking the gdv1 gene that regulates the early stages of gametocyte development, which is likely related to the extreme differences in mosquito vector abundance and seasonality that determine the transmission opportunities for the sexual stage of the parasite. PMID:24644299

  17. [Specific antibodies against recombinant MSP1 of Plasmodium falciparum strongly inhibit the parasite growth in vitro].

    PubMed

    Zhang, D M; Pan, W Q; Lu, D R

    2002-05-01

    In order to produce large amounts of protein for vaccine trials, a synthetic msp1-42 gene was inserted into Pichia pastoris expression vector and the plasmid was introduced into Pichia pastoris SMD1168 by electroporation. The expressed MSP1-42 was secreted into the protein-free medium. To measure the conformational properties of MSP1-42,16 monoclonal antibodies (11 recognizing conformational epitopes) were allowed to interact with the Pichia-derived MSP1-42, and all antibodies specific for conserved and K1 protype interacted with the protein. Interestingly, three monoclonal antibodies (e.g. 9.8, 13.1 and 7.3), that were shown not to interact with CHO-derived MSP1, could interact with the Pichia-derived MSP1-42. Rabbits were immunized with recombinant MSP1-42 formulated with CFA adjuvant four times. The rabbits were bled on the day 3 after last immunization, and total IgG isolated by protein A column from the immunized rabbits was shown to strongly inhibit the parasite growth in vitro dose-dependently, whereas IgG from rabbit with adjuvant had no inhibition. PMID:12019444

  18. Plasmodium falciparum PfSET7: enzymatic characterization and cellular localization of a novel protein methyltransferase in sporozoite, liver and erythrocytic stage parasites.

    PubMed

    Chen, Patty B; Ding, Shuai; Zangh, Gigliola; Soulard, Valrie; DiMaggio, Peter A; Fuchter, Matthew J; Mecheri, Salah; Mazier, Dominique; Scherf, Artur; Malmquist, Nicholas A

    2016-01-01

    Epigenetic control via reversible histone methylation regulates transcriptional activation throughout the malaria parasite genome, controls the repression of multi-copy virulence gene families and determines sexual stage commitment. Plasmodium falciparum encodes ten predicted SET domain-containing protein methyltransferases, six of which have been shown to be refractory to knock-out in blood stage parasites. We have expressed and purified the first recombinant malaria methyltransferase in sufficient quantities to perform a full enzymatic characterization and reveal the ill-defined PfSET7 is an AdoMet-dependent histone H3 lysine methyltransferase with highest activity towards lysines 4 and 9. Steady-state kinetics of the PfSET7 enzyme are similar to previously characterized histone methyltransferase enzymes from other organisms, however, PfSET7 displays specific protein substrate preference towards nucleosomes with pre-existing histone H3 lysine 14 acetylation. Interestingly, PfSET7 localizes to distinct cytoplasmic foci adjacent to the nucleus in erythrocytic and liver stage parasites, and throughout the cytoplasm in salivary gland sporozoites. Characterized recombinant PfSET7 now allows for target based inhibitor discovery. Specific PfSET7 inhibitors can aid in further investigating the biological role of this specific methyltransferase in transmission, hepatic and blood stage parasites, and may ultimately lead to the development of suitable antimalarial drug candidates against this novel class of essential parasite enzymes. PMID:26902486

  19. Plasmodium falciparum PfSET7: enzymatic characterization and cellular localization of a novel protein methyltransferase in sporozoite, liver and erythrocytic stage parasites

    PubMed Central

    Chen, Patty B.; Ding, Shuai; Zanghì, Gigliola; Soulard, Valérie; DiMaggio, Peter A.; Fuchter, Matthew J.; Mecheri, Salah; Mazier, Dominique; Scherf, Artur; Malmquist, Nicholas A.

    2016-01-01

    Epigenetic control via reversible histone methylation regulates transcriptional activation throughout the malaria parasite genome, controls the repression of multi-copy virulence gene families and determines sexual stage commitment. Plasmodium falciparum encodes ten predicted SET domain-containing protein methyltransferases, six of which have been shown to be refractory to knock-out in blood stage parasites. We have expressed and purified the first recombinant malaria methyltransferase in sufficient quantities to perform a full enzymatic characterization and reveal the ill-defined PfSET7 is an AdoMet-dependent histone H3 lysine methyltransferase with highest activity towards lysines 4 and 9. Steady-state kinetics of the PfSET7 enzyme are similar to previously characterized histone methyltransferase enzymes from other organisms, however, PfSET7 displays specific protein substrate preference towards nucleosomes with pre-existing histone H3 lysine 14 acetylation. Interestingly, PfSET7 localizes to distinct cytoplasmic foci adjacent to the nucleus in erythrocytic and liver stage parasites, and throughout the cytoplasm in salivary gland sporozoites. Characterized recombinant PfSET7 now allows for target based inhibitor discovery. Specific PfSET7 inhibitors can aid in further investigating the biological role of this specific methyltransferase in transmission, hepatic and blood stage parasites, and may ultimately lead to the development of suitable antimalarial drug candidates against this novel class of essential parasite enzymes. PMID:26902486

  20. Hemolytic and antimalarial effects of tight-binding glyoxalase 1 inhibitors on the host-parasite unit of erythrocytes infected with Plasmodium falciparum

    PubMed Central

    Wezena, Cletus A.; Urscher, Miriam; Vince, Robert; More, Swati S.; Deponte, Marcel

    2016-01-01

    Glyoxalases prevent the formation of advanced glycation end products by converting glycolysis-derived methylglyoxal to d-lactate with the help of glutathione. Vander Jagt and colleagues previously showed that erythrocytes release about thirty times more d-lactate after infection with the human malaria parasite Plasmodium falciparum. Functional glyoxalases in the host-parasite unit might therefore be crucial for parasite survival. Here, we determined the antimalarial and hemolytic activity of two tight-binding glyoxalase inhibitors using infected and uninfected erythrocytes. In addition, we synthesized and analyzed a set of diester derivates of both tight-binding inhibitors resulting in up to threefold lower IC50 values and an altered methemoglobin formation and hemolytic activity depending on the type of ester. Inhibitor treatments of uninfected erythrocytes revealed an extremely slow inactivation of the host cell glyoxalase, irrespective of inhibitor modifications, and a potential dispensability of the host cell enzyme for parasite survival. Our study highlights the benefits and drawbacks of different esterifications of glutathione-derived inhibitors and demonstrates the suitability of glyoxalase inhibitors as a tool for deciphering the relevance and mode of action of different glyoxalase systems in a host-parasite unit. PMID:26972115

  1. Exploring the diversity and distribution of neotropical avian malaria parasites--a molecular survey from Southeast Brazil.

    PubMed

    Lacorte, Gustavo A; Flix, Gabriel M F; Pinheiro, Rafael R B; Chaves, Anderson V; Almeida-Neto, Gilberto; Neves, Frederico S; Leite, Lemuel O; Santos, Fabrcio R; Braga, Erika M

    2013-01-01

    Southeast Brazil is a neotropical region composed of a mosaic of different tropical habitats and mountain chains, which allowed for the formation of bird-rich communities with distinct ecological niches. Although this region has the potential to harbor a remarkable variety of avian parasites, there is a lack of information about the diversity of malarial parasites. We used molecular approaches to characterize the lineage diversity of Plasmodium and Haemoproteus in bird communities from three different habitats in southeast Brazil based on the prevalence, richness and composition of lineages. We observed an overall prevalence of 35.3%, with a local prevalence ranging from 17.2% to 54.8%. Moreover, no significant association between prevalence and habitat type could be verified (p>0.05). We identified 89 Plasmodium and 22 Haemoproteus lineages, with 86% of them described for the first time here, including an unusual infection of a non-columbiform host by a Haemoproteus (Haemoproteus) parasite. The composition analyses of the parasite communities showed that the lineage composition from Brazilian savannah and tropical dry forest was similar, but it was different from the lineage composition of Atlantic rainforest, reflecting the greater likeness of the former habitats with respect to seasonality and forest density. No significant effects of habitat type on lineage richness were observed based on GLM analyses. We also found that sites whose samples had a greater diversity of bird species showed a greater diversity of parasite lineages, providing evidence that areas with high bird richness also have high parasite richness. Our findings point to the importance of the neotropical region (southeast Brazil) as a major reservoir of new haemosporidian lineages. PMID:23469235

  2. Plasmodium Immunomics

    PubMed Central

    Doolan, Denise L.

    2010-01-01

    The Plasmodium parasite, the causative agent of malaria, is an excellent model for immunomic-based approaches to vaccine development. The Plasmodium parasite has a complex life cycle with multiple stages and stage-specific expression of ~ 5,300 putative proteins. No malaria vaccine has yet been licensed. Many believe that an effective vaccine will need to target several antigens and multiple stages, and will require the generation of both antibody and cellular immune responses. Vaccine efforts to date have been stage-specific and based on only a very limited number of proteins representing < 0.5% of the genome. The recent availability of comprehensive genomic, proteomic and transcriptomic datasets from human and selected non-human primate and rodent malarias provide a foundation to exploit for vaccine development. This information can be mined to identify promising vaccine candidate antigens, by proteome-wide screening of antibody and T cell reactivity using specimens from individuals exposed to malaria and technology platforms such as protein arrays, high throughput protein production and epitope prediction algorithms. Such antigens could be incorporated into a rational vaccine development process that targets specific stages of the Plasmodium parasite life cycle with immune responses implicated in parasite elimination and control. Immunomic approaches which enable the selection of the best possible targets by prioritizing antigens according to clinically relevant criteria may overcome the problem of poorly immunogenic, poorly protective vaccines that has plagued malaria vaccine developers for the past 25 years. Herein, current progress and perspectives regarding Plasmodium immunomics are reviewed. PMID:20816843

  3. Naturally Acquired Antibodies Specific for Plasmodium falciparum Reticulocyte-Binding Protein Homologue 5 Inhibit Parasite Growth and Predict Protection From Malaria

    PubMed Central

    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.

    2014-01-01

    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 premalaria 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

  4. Changes in the transcriptome of the malaria parasite Plasmodium falciparum during the initial phase of transmission from the human to the mosquito

    PubMed Central

    2013-01-01

    Background The transmission of the malaria parasite Plasmodium falciparum from the human to the mosquito is mediated by dormant sexual precursor cells, the gametocytes, which become activated in the mosquito midgut. Because gametocytes are the only parasite stages able to establish an infection in the mosquito, they play a crucial role in spreading the tropical disease. The human-to-mosquito transmission triggers important molecular changes in the gametocytes, which initiate gametogenesis and prepare the parasite for life-cycle progression in the insect vector. Results To better understand gene regulations during the initial phase of malaria parasite transmission, we focused on the transcriptome changes that occur within the first half hour of parasite development in the mosquito. Comparison of mRNA levels of P. falciparum gametocytes before and 30 min following activation using suppression subtractive hybridization (SSH) identified 126 genes, which changed in expression during gametogenesis. Among these, 17.5% had putative functions in signaling, 14.3% were assigned to cell cycle and gene expression, 8.7% were linked to the cytoskeleton or inner membrane complex, 7.9% were involved in proteostasis and 6.4% in metabolism, 12.7% were cell surface-associated proteins, 11.9% were assigned to other functions, and 20.6% represented genes of unknown function. For 40% of the identified genes there has as yet not been any protein evidence. For a subset of 27 genes, transcript changes during gametogenesis were studied in detail by real-time RT-PCR. Of these, 22 genes were expressed in gametocytes, and for 15 genes transcript expression in gametocytes was increased compared to asexual blood stage parasites. Transcript levels of seven genes were particularly high in activated gametocytes, pointing at functions downstream of gametocyte transmission to the mosquito. For selected genes, a regulated expression during gametogenesis was confirmed on the protein level, using quantitative confocal microscopy. Conclusions The obtained transcriptome data demonstrate the regulations of gene expression immediately following malaria parasite transmission to the mosquito. Our findings support the identification of proteins important for sexual reproduction and further development of the mosquito midgut stages and provide insights into the genetic basis of the rapid adaption of Plasmodium to the insect vector. PMID:23586929

  5. Parasites

    MedlinePLUS

    ... U V W X Y Z Parasites Topics Malaria An ancient disease that affects millions of people ... source: Global Health - Division of Parasitic Diseases and Malaria Notice: Linking to a non-federal site does ...

  6. Prospects and Pitfalls of Pregnancy-Associated Malaria Vaccination Based on the Natural Immune Response to Plasmodium falciparum VAR2CSA-Expressing Parasites

    PubMed Central

    Kane, Elizabeth G.; Taylor-Robinson, Andrew W.

    2011-01-01

    Pregnancy-associated malaria, a manifestation of severe malaria, is the cause of up to 200,000 infant deaths a year, through the effects of placental insufficiency leading to growth restriction and preterm delivery. Development of a vaccine is one strategy for control. Plasmodium falciparum-infected red blood cells accumulate in the placenta through specific binding of pregnancy-associated parasite variants that express the VAR2CSA antigen to chondroitin sulphate A on the surface of syncytiotrophoblast cells. Parasite accumulation, accompanied by an inflammatory infiltrate, disrupts the cytokine balance of pregnancy with the potential to cause placental damage and compromise foetal growth. Multigravid women develop immunity towards VAR2CSA-expressing parasites in a gravidity-dependent manner which prevents unfavourable pregnancy outcomes. Although current vaccine design, targeting VAR2CSA antigens, has succeeded in inducing antibodies artificially, this candidate may not provide protection during the first trimester and may only protect those women living in areas endemic for malaria. It is concluded that while insufficient information about placental-parasite interactions is presently available to produce an effective vaccine, incremental progress is being made towards achieving this goal. PMID:22363896

  7. Malaria Parasite Invasion of the Mosquito Salivary Gland Requires Interaction between the Plasmodium TRAP and the Anopheles Saglin Proteins

    PubMed Central

    Ghosh, Anil K.; Devenport, Martin; Jethwaney, Deepa; Kalume, Dario E.; Pandey, Akhilesh; Anderson, Vernon E.; Sultan, Ali A.; Kumar, Nirbhay; Jacobs-Lorena, Marcelo

    2009-01-01

    SM1 is a twelve-amino-acid peptide that binds tightly to the Anopheles salivary gland and inhibits its invasion by Plasmodium sporozoites. By use of UV-crosslinking experiments between the peptide and its salivary gland target protein, we have identified the Anopheles salivary protein, saglin, as the receptor for SM1. Furthermore, by use of an anti-SM1 antibody, we have determined that the peptide is a mimotope of the Plasmodium sporozoite Thrombospondin Related Anonymous Protein (TRAP). TRAP binds to saglin with high specificity. Point mutations in TRAP's binding domain A abrogate binding, and binding is competed for by the SM1 peptide. Importantly, in vivo down-regulation of saglin expression results in strong inhibition of salivary gland invasion. Together, the results suggest that saglin/TRAP interaction is crucial for salivary gland invasion by Plasmodium sporozoites. PMID:19148273

  8. The Puf-family RNA-binding protein PfPuf2 regulates sexual development and sex differentiation in the malaria parasite Plasmodium falciparum.

    PubMed

    Miao, Jun; Li, Jinfang; Fan, Qi; Li, Xiaolian; Li, Xinyi; Cui, Liwang

    2010-04-01

    Translation regulation plays an important role during gametocytogenesis in the malaria parasite, a process that is obligatory for the transmission of the parasite through mosquito vectors. In this study we determined the function of PfPuf2, a member of the Puf family of translational repressors, in gametocytogenesis of Plasmodium falciparum. Tagging of the endogenous PfPuf2 protein with green fluorescent protein showed that PfPuf2 was expressed in both male and female gametocytes, and the protein was localized in the cytoplasm of the parasite. Targeted disruption of the PfPuf2 gene did not affect asexual growth of the parasite, but promoted the formation of gametocytes and differentiation of male gametocytes. Complementation studies were performed to confirm that the resultant phenotypic changes were due to disruption of the PfPuf2 gene. Episomal expression of PfPuf2 under its cognate promoter almost restored the gametocytogenesis rate in a PfPuf2 disruptant to the level of the wild-type parasite. It also partially restored the effect of PfPuf2 disruption on male-female sex ratio. In addition, episomal overexpression of PfPuf2 under its cognate promoter but with a higher concentration of the selection drug or under the constitutive hsp86 promoter in both the PfPuf2-disruptant and wild-type 3D7 lines, further dramatically reduced gametocytogenesis rates and sex ratios. These findings suggest that in this early branch of eukaryotes the function of PfPuf2 is consistent with the ancestral function of suppressing differentiation proposed for Puf-family proteins. PMID:20197405

  9. Immunization of mice with live-attenuated late liver stage-arresting Plasmodium yoelii parasites generates protective antibody responses to preerythrocytic stages of malaria.

    PubMed

    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

    2014-12-01

    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. PMID:25267837

  10. Polymorphisms in the K13-propeller gene in artemisinin-susceptible Plasmodium falciparum parasites from Bougoula-Hameau and Bandiagara, Mali.

    PubMed

    Ouattara, Amed; Kone, Aminatou; Adams, Matthew; Fofana, Bakary; Maiga, Amelia Walling; Hampton, Shay; Coulibaly, Drissa; Thera, Mahamadou A; Diallo, Nouhoum; Dara, Antoine; Sagara, Issaka; Gil, Jose Pedro; Bjorkman, Anders; Takala-Harrison, Shannon; Doumbo, Ogobara K; Plowe, Christopher V; Djimde, Abdoulaye A

    2015-06-01

    Artemisinin-resistant Plasmodium falciparum malaria has been documented in southeast Asia and may already be spreading in that region. Molecular markers are important tools for monitoring the spread of antimalarial drug resistance. Recently, single-nucleotide polymorphisms (SNPs) in the PF3D7_1343700 kelch propeller (K13-propeller) domain were shown to be associated with artemisinin resistance in vivo and in vitro. The prevalence and role of K13-propeller mutations are poorly known in sub-Saharan Africa. K13-propeller mutations were genotyped by direct sequencing of nested polymerase chain reaction (PCR) amplicons from dried blood spots of pre-treatment falciparum malaria infections collected before and after the use of artemisinin-based combination therapy (ACT) as first-line therapy in Mali. Although K13-propeller mutations previously associated with delayed parasite clearance in Cambodia were not identified, 26 K13-propeller mutations were identified in both recent samples and pre-ACT infections. Parasite clearance time was comparable between infections with non-synonymous K13-propeller mutations and infections with the reference allele. These findings suggest that K13-propeller mutations are present in artemisinin-sensitive parasites and that they preceded the wide use of ACTs in Mali. PMID:25918205

  11. Antibodies to the Plasmodium falciparum Proteins MSPDBL1 and MSPDBL2 Opsonize Merozoites, Inhibit Parasite Growth, and Predict Protection From Clinical Malaria.

    PubMed

    Chiu, Chris Y H; Hodder, Anthony N; Lin, Clara S; Hill, Danika L; Li Wai Suen, Connie S N; Schofield, Louis; Siba, Peter M; Mueller, Ivo; Cowman, Alan F; Hansen, Diana S

    2015-08-01

    Increasing evidence suggests that antibodies against merozoite surface proteins (MSPs) play an important role in clinical immunity to malaria. Two unusual members of the MSP-3 family, merozoite surface protein duffy binding-like (MSPDBL)1 and MSPDBL2, have been shown to be extrinsically associated to MSP-1 on the parasite surface. In addition to a secreted polymorphic antigen associated with merozoite (SPAM) domain characteristic of MSP-3 family members, they also contain Duffy binding-like (DBL) domain and were found to bind to erythrocytes, suggesting that they play a role in parasite invasion. Antibody responses to these proteins were investigated in a treatment-reinfection study conducted in an endemic area of Papua New Guinea to determine their contribution to naturally acquired immunity. Antibodies to the SPAM domains of MSPDBL1 and MSPDBL2 as well as the DBL domain of MSPDBL1 were found to be associated with protection from Plasmodium falciparum clinical episodes. Moreover, affinity-purified anti-MSPDBL1 and MSPDBL2 were found to inhibit in vitro parasite growth and had strong merozoite opsonizing capacity, suggesting that protection targeting these antigens results from ≥2 distinct effector mechanisms. Together these results indicate that MSPDBL1 and MSPDBL2 are important targets of naturally acquired immunity and might constitute potential vaccine candidates. PMID:25646353

  12. Plasmodium falciparum var Gene Expression Homogeneity as a Marker of the Host-Parasite Relationship under Different Levels of Naturally Acquired Immunity to Malaria

    PubMed Central

    Warimwe, George M.; Recker, Mario; Kiragu, Esther W.; Buckee, Caroline O.; Wambua, Juliana; Musyoki, Jennifer N.; Marsh, Kevin; Bull, Peter C.

    2013-01-01

    Acquired immunity to Plasmodium falciparum infection causes a change from frequent, sometimes life-threatening, malaria in young children to asymptomatic, chronic infections in older children and adults. Little is known about how this transition occurs but antibodies to the extremely diverse PfEMP1 parasite antigens are thought to play a role. PfEMP1 is encoded by a family of 60 var genes that undergo clonal antigenic variation, potentially creating an antigenically heterogeneous infecting population of parasites within the host. Previous theoretical work suggests that antibodies to PfEMP1 may play a role in orchestrating their expression within infections leading to sequential, homogeneous expression of var genes, and prolonged infection chronicity. Here, using a cloning and sequencing approach we compare the var expression homogeneity (VEH) between isolates from children with asymptomatic and clinical infections. We show that asymptomatic infections have higher VEH than clinical infections and a broader host antibody response. We discuss this in relation to the potential role of host antibodies in promoting chronicity of infection and parasite survival through the low transmission season. PMID:23922996

  13. Deletion of Plasmodium falciparum Histidine-Rich Protein 2 (pfhrp2) and Histidine-Rich Protein 3 (pfhrp3) Genes in Colombian Parasites

    PubMed Central

    Murillo Solano, Claribel; Akinyi Okoth, Sheila; Abdallah, Joseph F.; Pava, Zuleima; Dorado, Erika; Incardona, Sandra; Huber, Curtis S.; Macedo de Oliveira, Alexandre; Bell, David; Udhayakumar, Venkatachalam; Barnwell, John W.

    2015-01-01

    A number of studies have analyzed the performance of malaria rapid diagnostic tests (RDTs) in Colombia with discrepancies in performance being attributed to a combination of factors such as parasite levels, interpretation of RDT results and/or the handling and storage of RDT kits. However, some of the inconsistencies observed with results from Plasmodium falciparum histidine-rich protein 2 (PfHRP2)-based RDTs could also be explained by the deletion of the gene that encodes the protein, pfhrp2, and its structural homolog, pfhrp3, in some parasite isolates. Given that pfhrp2- and pfhrp3-negative P. falciparum isolates have been detected in the neighboring Peruvian and Brazilian Amazon regions, we hypothesized that parasites with deletions of pfhrp2 and pfhrp3 may also be present in Colombia. In this study we tested 100 historical samples collected between 1999 and 2009 from six Departments in Colombia for the presence of pfhrp2, pfhrp3 and their flanking genes. Seven neutral microsatellites were also used to determine the genetic background of these parasites. In total 18 of 100 parasite isolates were found to have deleted pfhrp2, a majority of which (14 of 18) were collected from Amazonas Department, which borders Peru and Brazil. pfhrp3 deletions were found in 52 of the100 samples collected from all regions of the country. pfhrp2 flanking genes PF3D7_0831900 and PF3D7_0831700 were deleted in 22 of 100 and in 1 of 100 samples, respectively. pfhrp3 flanking genes PF3D7_1372100 and PF3D7_1372400 were missing in 55 of 100 and in 57 of 100 samples. Structure analysis of microsatellite data indicated that Colombian samples tested in this study belonged to four clusters and they segregated mostly based on their geographic region. Most of the pfhrp2-deleted parasites were assigned to a single cluster and originated from Amazonas Department although a few pfhrp2-negative parasites originated from the other three clusters. The presence of a high proportion of pfhrp2-negative isolates in the Colombian Amazon may have implications for the use of PfHRP2-based RDTs in the region and may explain inconsistencies observed when PfHRP2-based tests and assays are performed. PMID:26151448

  14. Deletion of Plasmodium falciparum Histidine-Rich Protein 2 (pfhrp2) and Histidine-Rich Protein 3 (pfhrp3) Genes in Colombian Parasites.

    PubMed

    Murillo Solano, Claribel; Akinyi Okoth, Sheila; Abdallah, Joseph F; Pava, Zuleima; Dorado, Erika; Incardona, Sandra; Huber, Curtis S; Macedo de Oliveira, Alexandre; Bell, David; Udhayakumar, Venkatachalam; Barnwell, John W

    2015-01-01

    A number of studies have analyzed the performance of malaria rapid diagnostic tests (RDTs) in Colombia with discrepancies in performance being attributed to a combination of factors such as parasite levels, interpretation of RDT results and/or the handling and storage of RDT kits. However, some of the inconsistencies observed with results from Plasmodium falciparum histidine-rich protein 2 (PfHRP2)-based RDTs could also be explained by the deletion of the gene that encodes the protein, pfhrp2, and its structural homolog, pfhrp3, in some parasite isolates. Given that pfhrp2- and pfhrp3-negative P. falciparum isolates have been detected in the neighboring Peruvian and Brazilian Amazon regions, we hypothesized that parasites with deletions of pfhrp2 and pfhrp3 may also be present in Colombia. In this study we tested 100 historical samples collected between 1999 and 2009 from six Departments in Colombia for the presence of pfhrp2, pfhrp3 and their flanking genes. Seven neutral microsatellites were also used to determine the genetic background of these parasites. In total 18 of 100 parasite isolates were found to have deleted pfhrp2, a majority of which (14 of 18) were collected from Amazonas Department, which borders Peru and Brazil. pfhrp3 deletions were found in 52 of the 100 samples collected from all regions of the country. pfhrp2 flanking genes PF3D7_0831900 and PF3D7_0831700 were deleted in 22 of 100 and in 1 of 100 samples, respectively. pfhrp3 flanking genes PF3D7_1372100 and PF3D7_1372400 were missing in 55 of 100 and in 57 of 100 samples. Structure analysis of microsatellite data indicated that Colombian samples tested in this study belonged to four clusters and they segregated mostly based on their geographic region. Most of the pfhrp2-deleted parasites were assigned to a single cluster and originated from Amazonas Department although a few pfhrp2-negative parasites originated from the other three clusters. The presence of a high proportion of pfhrp2-negative isolates in the Colombian Amazon may have implications for the use of PfHRP2-based RDTs in the region and may explain inconsistencies observed when PfHRP2-based tests and assays are performed. PMID:26151448

  15. Ycf93 (Orf105), a Small Apicoplast-Encoded Membrane Protein in the Relict Plastid of the Malaria Parasite Plasmodium falciparum That Is Conserved in Apicomplexa

    PubMed Central

    Goodman, Christopher D.; McFadden, Geoffrey I.

    2014-01-01

    Malaria parasites retain a relict plastid (apicoplast) from a photosynthetic ancestor shared with dinoflagellate algae. The apicoplast is a useful drug target; blocking housekeeping pathways such as genome replication and translation in the organelle kills parasites and protects against malaria. The apicoplast of Plasmodium falciparum encodes 30 proteins and a suite of rRNAs and tRNAs that facilitate their expression. orf105 is a hypothetical apicoplast gene that would encode a small protein (PfOrf105) with a predicted C-terminal transmembrane domain. We produced antisera to a predicted peptide within PfOrf105. Western blot analysis confirmed expression of orf105 and immunofluorescence localised the gene product to the apicoplast. Pforf105 encodes a membrane protein that has an apparent mass of 17.5 kDa and undergoes substantial turnover during the 48-hour asexual life cycle of the parasite in blood stages. The effect of actinonin, an antimalarial with a putative impact on post-translational modification of apicoplast proteins like PfOrf105, was examined. Unlike other drugs perturbing apicoplast housekeeping that induce delayed death, actinonin kills parasites immediately and has an identical drug exposure phenotype to the isopentenyl diphosphate synthesis blocker fosmidomycin. Open reading frames of similar size to PfOrf105, which also have predicted C-terminal trans membrane domains, occur in syntenic positions in all sequenced apicoplast genomes from Phylum Apicomplexa. We therefore propose to name these genes ycf93 (hypothetical chloroplast reading frame 93) according to plastid gene nomenclature convention for conserved proteins of unknown function. PMID:24705170

  16. A programmed cell death pathway in the malaria parasite Plasmodium falciparum has general features of mammalian apoptosis but is mediated by clan CA cysteine proteases

    PubMed Central

    Ch'ng, J-H; Kotturi, S R; Chong, A G-L; Lear, M J; Tan, K S-W

    2010-01-01

    Several recent discoveries of the hallmark features of programmed cell death (PCD) in Plasmodium falciparum have presented the possibility of revealing novel targets for antimalarial therapy. Using a combination of cell-based assays, flow cytometry and fluorescence microscopy, we detected features including mitochondrial dysregulation, activation of cysteine proteases and in situ DNA fragmentation in parasites induced with chloroquine (CQ) and staurosporine (ST). The use of the pan-caspase inhibitor, z-Val-Ala-Asp-fmk (zVAD), and the mitochondria outer membrane permeabilization (MOMP) inhibitor, 4-hydroxy-tamoxifen, enabled the characterization of a novel CQ-induced pathway linking cysteine protease activation to downstream mitochondrial dysregulation, amplified protease activity and DNA fragmentation. The PCD features were observed only at high (?M) concentrations of CQ. The use of a new synthetic coumarin-labeled chloroquine (CM-CQ) showed that these features may be associated with concentration-dependent differences in drug localization. By further using cysteine protease inhibitors z-Asp-Glu-Val-Asp-fmk (zDEVD), z-Phe-Ala-fmk (zFA), z-Phe-Phe-fmk (zFF), z-Leu-Leu-Leu-fmk (zLLL), E64d and CA-074, we were able to implicate clan CA cysteine proteases in CQ-mediated PCD. Finally, CQ induction of two CQ-resistant parasite strains, 7G8 and K1, reveals the existence of PCD features in these parasites, the extent of which was less than 3D7. The use of the chemoreversal agent verapamil implicates the parasite digestive vacuole in mediating CQ-induced PCD. PMID:21364634

  17. Combining Parasite Lactate Dehydrogenase-Based and Histidine-Rich Protein 2-Based Rapid Tests To Improve Specificity for Diagnosis of Malaria Due to Plasmodium knowlesi and Other Plasmodium Species in Sabah, Malaysia

    PubMed Central

    William, Timothy; Barber, Bridget E.; Parameswaran, Uma; Bird, Elspeth; Piera, Kim; Aziz, Ammar; Dhanaraj, Prabakaran; Yeo, Tsin W.; Anstey, Nicholas M.

    2014-01-01

    Plasmodium knowlesi causes severe and fatal malaria in Malaysia. Microscopic misdiagnosis is common and may delay appropriate treatment. P. knowlesi can cross-react with species-specific parasite lactate dehydrogenase (pLDH) monoclonal antibodies used in rapid diagnostic tests (RDTs) to detect P. falciparum and P. vivax. At one tertiary-care hospital and two district hospitals in Sabah, we prospectively evaluated two combination RDTs for malaria diagnosis by using both a pan-Plasmodium-pLDH (pan-pLDH)/P. falciparum-specific-pLDH (Pf-pLDH) RDT (OptiMAL-IT) and a non-P. falciparum VOM-pLDH/Pf-HRP2 RDT (CareStart). Differential cross-reactivity among these combinations was hypothesized to differentiate P. knowlesi from other Plasmodium monoinfections. Among 323 patients with PCR-confirmed P. knowlesi (n = 193), P. falciparum (n = 93), and P. vivax (n = 37) monoinfections, the VOM-pLDH individual component had the highest sensitivity for nonsevere (35%; 95% confidence interval [CI], 27 to 43%) and severe (92%; CI, 81 to 100%) P. knowlesi malaria. CareStart demonstrated a P. knowlesi sensitivity of 42% (CI, 34 to 49%) and specificity of 74% (CI, 65 to 82%), a P. vivax sensitivity of 83% (CI, 66 to 93%) and specificity of 71% (CI, 65 to 76%), and a P. falciparum sensitivity of 97% (CI, 90 to 99%) and specificity of 99% (CI, 97 to 100%). OptiMAL-IT demonstrated a P. knowlesi sensitivity of 32% (CI, 25 to 39%) and specificity of 21% (CI, 15 to 29%), a P. vivax sensitivity of 60% (CI, 42 to 75%) and specificity of 97% (CI, 94 to 99%), and a P. falciparum sensitivity of 82% (CI, 72 to 89%) and specificity of 39% (CI, 33 to 46%). The combination of CareStart plus OptiMAL-IT for P. knowlesi using predefined criteria gave a sensitivity of 25% (CI, 19 to 32%) and specificity of 97% (CI, 92 to 99%). Combining two RDT combinations was highly specific for P. knowlesi malaria diagnosis; however, sensitivity was poor. The specificity of pLDH RDTs was decreased for P. vivax and P. falciparum because of P. knowlesi cross-reactivity and cautions against their use alone in areas where P. knowlesi malaria is endemic. Sensitive P. knowlesi-specific RDTs and/or alternative molecular diagnostic tools are needed in areas where P. knowlesi malaria is endemic. PMID:24696029

  18. Single-stranded DNA binding protein from human malarial parasite Plasmodium falciparum is encoded in the nucleus and targeted to the apicoplast.

    PubMed

    Prusty, Dhaneswar; Dar, Ashraf; Priya, Rashmi; Sharma, Atul; Dana, Srikanta; Choudhury, Nirupam Roy; Rao, N Subba; Dhar, Suman Kumar

    2010-11-01

    Apicoplast, an essential organelle of human malaria parasite Plasmodium falciparum contains a ?35?kb circular genome and is a possible target for therapy. Proteins required for the replication and maintenance of the apicoplast DNA are not clearly known. Here we report the presence of single-stranded DNA binding protein (SSB) in P falciparum. PfSSB is targeted to the apicoplast and it binds to apicoplast DNA. A strong ssDNA binding activity specific to SSB was also detected in P. falciparum lysate. Both the recombinant and endogenous proteins form tetramers and the homology modelling shows the presence of an oligosaccharide/oligonucleotide-binding fold responsible for ssDNA binding. Additionally, we used SSB as a tool to track the mechanism of delayed death phenomena shown by apicoplast targeted drugs ciprofloxacin and tetracycline. We find that the transport of PfSSB is severely affected during the second life cycle following drug treatment. Moreover, the translation of PfSSB protein and not the transcription of PfSSB seem to be down-regulated specifically during second life cycle although there is no considerable change in protein expression profile between drug-treated and untreated parasites. These results suggest dual control of translocation and translation of apicoplast targeted proteins behind the delayed death phenomena. PMID:20571080

  19. Structural Insights into Substrate Binding by PvFKBP35, a Peptidylprolyl cis-trans Isomerase from the Human Malarial Parasite Plasmodium vivax

    PubMed Central

    Alag, Reema; Balakrishna, Asha Manikkoth; Rajan, Sreekanth; Qureshi, Insaf A.; Shin, Joon; Lescar, Julien; Grber, Gerhard

    2013-01-01

    The immunosuppressive drug FK506 binding proteins (FKBPs), an immunophilin family with the immunosuppressive drug FK506 binding property, exhibit peptidylprolyl cis-trans isomerase (PPIase) activity. While the cyclophilin-catalyzed peptidylprolyl isomerization of X-Pro peptide bonds has been extensively studied, the mechanism of the FKBP-mediated peptidylprolyl isomerization remains uncharacterized. Thus, to investigate the binding of FKBP with its substrate and the underlying catalytic mechanism of the FKBP-mediated proline isomerization, here we employed the FK506 binding domain (FKBD) of the human malarial parasite Plasmodium vivax FK506 binding protein 35 (PvFKBP35) and examined the details of the molecular interaction between the isomerase and a peptide substrate. The crystallographic structures of apo PvFKBD35 and its complex with the tetrapeptide substrate succinyl-Ala-Leu-Pro-Phe-p-nitroanilide (sALPFp) determined at 1.4 ? and 1.65 ? resolutions, respectively, showed that the substrate binds to PvFKBD35 in a cis conformation. Nuclear magnetic resonance (NMR) studies demonstrated the chemical shift perturbations of D55, H67, V73, and I74 residues upon the substrate binding. In addition, the X-ray crystal structure, along with the mutational studies, shows that Y100 is a key residue for the catalytic activity. Taken together, our results provide insights into the catalytic mechanism of PvFKBP35-mediated cis-trans isomerization of substrate and ultimately might aid designing substrate mimetic inhibitors targeting the malarial parasite FKBPs. PMID:23435727

  20. Overexpression, purification and assessment of cyclosporin binding of a family of cyclophilins and cyclophilin-like proteins of the human malarial parasite Plasmodium falciparum.

    PubMed

    Marn-Menndez, Alejandro; Bell, Angus

    2011-08-01

    Malaria represents a global health, economic and social burden of enormous magnitude. Chemotherapy is at the moment a largely effective weapon against the disease, but the appearance of drug-resistant parasites is reducing the effectiveness of most drugs. Finding new drug-target candidates is one approach to the development of new drugs. The family of cyclophilins may represent a group of potential targets. They are involved in protein folding and regulation due to their peptidyl-prolyl cis-trans isomerase and/or chaperone activities. They also mediate the action of the immunosuppressive drug cyclosporin A, which additionally has strong antimalarial activity. In the genome database of the most lethal human malarial parasite Plasmodium falciparum, 11 genes apparently encoding cyclophilin or cyclophilin-like proteins were found, but most of these have not yet been characterized. Previously a pET vector conferring a C-terminal His? tag was used for recombinant expression and purification of one member of the P. falciparum cyclophilin family in Escherichia coli. The approach here was to use an identical method to produce all of the other members of this family and thereby allow the most consistent functional comparisons. We were successful in generating all but three of the family, plus a single amino-acid mutant, in the same recombinant form as either full-length proteins or isolated cyclophilin-like domains. The recombinant proteins were assessed by thermal melt assay for correct folding and cyclosporin A binding. PMID:21549842

  1. Structural insights into substrate binding by PvFKBP35, a peptidylprolyl cis-trans isomerase from the human malarial parasite Plasmodium vivax.

    PubMed

    Alag, Reema; Balakrishna, Asha Manikkoth; Rajan, Sreekanth; Qureshi, Insaf A; Shin, Joon; Lescar, Julien; Grber, Gerhard; Yoon, Ho Sup

    2013-04-01

    The immunosuppressive drug FK506 binding proteins (FKBPs), an immunophilin family with the immunosuppressive drug FK506 binding property, exhibit peptidylprolyl cis-trans isomerase (PPIase) activity. While the cyclophilin-catalyzed peptidylprolyl isomerization of X-Pro peptide bonds has been extensively studied, the mechanism of the FKBP-mediated peptidylprolyl isomerization remains uncharacterized. Thus, to investigate the binding of FKBP with its substrate and the underlying catalytic mechanism of the FKBP-mediated proline isomerization, here we employed the FK506 binding domain (FKBD) of the human malarial parasite Plasmodium vivax FK506 binding protein 35 (PvFKBP35) and examined the details of the molecular interaction between the isomerase and a peptide substrate. The crystallographic structures of apo PvFKBD35 and its complex with the tetrapeptide substrate succinyl-Ala-Leu-Pro-Phe-p-nitroanilide (sALPFp) determined at 1.4 ? and 1.65 ? resolutions, respectively, showed that the substrate binds to PvFKBD35 in a cis conformation. Nuclear magnetic resonance (NMR) studies demonstrated the chemical shift perturbations of D55, H67, V73, and I74 residues upon the substrate binding. In addition, the X-ray crystal structure, along with the mutational studies, shows that Y100 is a key residue for the catalytic activity. Taken together, our results provide insights into the catalytic mechanism of PvFKBP35-mediated cis-trans isomerization of substrate and ultimately might aid designing substrate mimetic inhibitors targeting the malarial parasite FKBPs. PMID:23435727

  2. Crystallization and preliminary X-ray analysis of the aspartic protease plasmepsin 4 from the malarial parasite Plasmodium malariae

    SciTech Connect

    Madabushi, Amrita; Chakraborty, Sibani; Fisher, S. Zo; Clemente, Jos C.; Yowell, Charles; Agbandje-McKenna, Mavis; Dame, John B.; Dunn, Ben M.; McKenna, Robert

    2005-02-01

    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 resolution is reported.

  3. Genetically attenuated parasite vaccines induce contact-dependent CD8+ T cell killing of Plasmodium yoelii liver stage-infected hepatocytes.

    PubMed

    Trimnell, Adama; Takagi, Akihide; Gupta, Megha; Richie, Thomas L; Kappe, Stefan H; Wang, Ruobing

    2009-11-01

    The production of IFN-gamma by CD8(+) T cells is an important hallmark of protective immunity induced by irradiation-attenuated sporozoites against malaria. Here, we demonstrate that protracted sterile protection conferred by a Plasmodium yoelii genetically attenuated parasite (PyGAP) vaccine was completely dependent on CD8(+) T lymphocytes but only partially dependent on IFN-gamma. We used live cell imaging to document that CD8(+) CTL from PyGAP-immunized mice directly killed hepatocyte infected with a liver stage parasite. Immunization studies with perforin and IFN-gamma knockout mice also indicated that the protection was largely dependent on perforin-mediated effector mechanisms rather than on IFN-gamma. This was further supported by our observation that both liver and spleen CD8(+) T cells from PyGAP-immunized mice induced massive apoptosis of liver stage-infected hepatocytes in vitro without the release of detectable IFN-gamma and TNF-alpha. Conversely, CD8(+) T cells isolated from naive mice that had survived wild-type P. yoelii sporozoite infection targeted mainly sporozoite-traversed and uninfected hepatocytes, revealing an immune evasion strategy that might be used by wild-type parasites to subvert host immune responses during natural infection. However, CTLs from wild-type sporozoite-challenged mice could recognize and kill infected hepatocytes that were pulsed with circumsporozoite protein. Additionally, protection in PyGAP-immunized mice directly correlated with the magnitude of effector memory CD8(+) T cells. Our findings implicate CTLs as key immune effectors in a highly protective PyGAP vaccine for malaria and emphasize the critical need to define surrogate markers for correlates of protection, apart from IFN-gamma. PMID:19812194

  4. Molecular evolution and intragenic recombination of the merozoite surface protein MSP-3alpha from the malaria parasite Plasmodium vivax in Thailand.

    PubMed

    Mascorro, C N; Zhao, K; Khuntirat, B; Sattabongkot, J; Yan, G; Escalante, A A; Cui, L

    2005-07-01

    The merozoite surface antigens of malaria parasites are prime anti-morbidity/mortality vaccine candidates. However, their highly polymorphic nature requires extensive surveys of parasite populations to validate vaccine designs. Previous studies have found 3 molecular types (A, B and C) of the Plasmodium vivax merozoite surface protein 3a (PvMSP-3alpha) among parasite field populations. Here we analysed complete PvMSP-3alpha sequences from 17 clinical P. vivax isolates from Thailand and found that the nucleotide diversity was as high as that from samples widely separated by time and space. The polymorphic sites were not randomly distributed but concentrated in the N-terminal Ala-rich domain (block 2A), which is partially deleted in type B and C sequences. The size variations among type A sequences were due to small indels occurring in block 2A, whereas type B and C sequences were uniform in length with each type having a different large deletion. Analysis of synonymous and non-synonymous substitutions suggested that different selection forces were operating on different regions of the molecule. The numerous recombination sites detected within the Ala-rich domain suggested that intragenic recombination was at least partially responsible for the observed genetic diversity of the PvMSP-3alpha gene. Phylogenetic analysis failed to link any alleles to a specific geographical origin, even when different domains of PvMSP-3alpha were used for analysis. The highly polymorphic nature and lack of geographical clustering of isolates suggest that more systematic investigations of the PvMSP-3alpha gene are needed to explore its evolution and vaccine potential. PMID:16038393

  5. Effect of anti-mosquito antibodies on the infectivity of the rodent malaria parasite Plasmodium berghei to Anopheles farauti.

    PubMed

    Ramasamy, M S; Ramasamy, R

    1990-04-01

    The effect of mouse anti-mosquito antibodies, present in the bloodmeal, on the infectivity of Plasmodium berghei Vincke to Anopheles farauti Laveran was investigated. Significantly fewer oocysts developed in mosquitoes feeding on mice immunized with sugar-fed mosquito midgut antigens than in mosquitoes feeding on control mice. Mosquitoes feeding on mice immunized with the midgut antigens derived from sugar-fed mosquitoes also showed reduced mortality and had lower infection rates than those fed on unimmunized mice. Blood-fed midgut antigen was less effective in producing these effects than sugar-fed midgut antigen. PMID:2132980

  6. Fucoxanthin, tetraprenylated toluquinone and toluhydroquinone metabolites from Sargassum heterophyllum inhibit the in vitro growth of the malaria parasite Plasmodium falciparum.

    PubMed

    Afolayan, Anthonia F; Bolton, John J; Lategan, Carmen A; Smith, Peter J; Beukes, Denzil R

    2008-01-01

    In the course of our search for antimalarial leads from marine algae, four metabolites, sargaquinoic acid, sargahydroquinoic acid, sargaquinal and fucoxanthin, were isolated from the South African alga Sargassum heterophyllum. Fucoxanthin and sargaquinal showed good antiplasmodial activity toward a chloroquine-sensitive strain (D10) of Plasmodium falciparum (IC50 1.5 and 2.0 microM, respectively), while sargaquinoic acid and sargahydroquinoic acid were only moderately active (IC50 12.0 and 15.2 microM, respectively). PMID:19227833

  7. Counter-regulatory anti-parasite cytokine responses during concurrent Plasmodium yoelii and intestinal helminth infections in mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Malaria and helminth infections are two of the most prevalent parasitic diseases in tropical areas. While concomitant infection is common, mechanisms contributing to altered disease outcomes during co-infection remain poorly defined. We have previously reported exacerbation of normally non-lethal ...

  8. Antiplasmodial activity of iron(II) and ruthenium(II) organometallic complexes against Plasmodium falciparum blood parasites.

    PubMed

    Souza, Nicolli Bellotti de; Aguiar, Anna Caroline Campos; Oliveira, Alane Cabral de; Top, Siden; Pigeon, Pascal; Jaouen, Gérard; Goulart, Marilia Oliveira Fonseca; Krettli, Antoniana Ursine

    2015-12-01

    This work reports the in vitro activity against Plasmodium falciparumblood forms (W2 clone, chloroquine-resistant) of tamoxifen-based compounds and their ferrocenyl (ferrocifens) and ruthenocenyl (ruthenocifens) derivatives, as well as their cytotoxicity against HepG2 human hepatoma cells. Surprisingly with these series, results indicate that the biological activity of ruthenocifens is better than that of ferrocifens and other tamoxifen-like compounds. The synthesis of a new metal-based compound is also described. It was shown, for the first time, that ruthenocifens are good antiplasmodial prototypes. Further studies will be conducted aiming at a better understanding of their mechanism of action and at obtaining new compounds with better therapeutic profile. PMID:26602875

  9. Antiplasmodial activity of iron(II) and ruthenium(II) organometallic complexes against Plasmodium falciparum blood parasites

    PubMed Central

    de Souza, Nicolli Bellotti; Aguiar, Anna Caroline Campos; de Oliveira, Alane Cabral; Top, Siden; Pigeon, Pascal; Jaouen, Gérard; Goulart, Marilia Oliveira Fonseca; Krettli, Antoniana Ursine

    2015-01-01

    This work reports the in vitro activity against Plasmodium falciparumblood forms (W2 clone, chloroquine-resistant) of tamoxifen-based compounds and their ferrocenyl (ferrocifens) and ruthenocenyl (ruthenocifens) derivatives, as well as their cytotoxicity against HepG2 human hepatoma cells. Surprisingly with these series, results indicate that the biological activity of ruthenocifens is better than that of ferrocifens and other tamoxifen-like compounds. The synthesis of a new metal-based compound is also described. It was shown, for the first time, that ruthenocifens are good antiplasmodial prototypes. Further studies will be conducted aiming at a better understanding of their mechanism of action and at obtaining new compounds with better therapeutic profile. PMID:26602875

  10. ?2-Macroglobulin Can Crosslink Multiple Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) Molecules and May Facilitate Adhesion of Parasitized Erythrocytes

    PubMed Central

    Stevenson, Liz; Laursen, Erik; Cowan, Graeme J.; Bandoh, Betty; Barfod, Lea; Cavanagh, David R.; Andersen, Gregers R.; Hviid, Lars

    2015-01-01

    Rosetting, the adhesion of Plasmodium falciparum-infected erythrocytes to uninfected erythrocytes, involves clonal variants of the parasite protein P. falciparum erythrocyte membrane protein 1 (PfEMP1) and soluble serum factors. While rosetting is a well-known phenotypic marker of parasites associated with severe malaria, the reason for this association remains unclear, as do the molecular details of the interaction between the infected erythrocyte (IE) and the adhering erythrocytes. Here, we identify for the first time a single serum factor, the abundant serum protease inhibitor ?2-macroglobulin (?2M), which is both required and sufficient for rosetting mediated by the PfEMP1 protein HB3VAR06 and some other rosette-mediating PfEMP1 proteins. We map the ?2M binding site to the C terminal end of HB3VAR06, and demonstrate that ?2M can bind at least four HB3VAR06 proteins, plausibly augmenting their combined avidity for host receptors. IgM has previously been identified as a rosette-facilitating soluble factor that acts in a similar way, but it cannot induce rosetting on its own. This is in contrast to ?2M and probably due to the more limited cross-linking potential of IgM. Nevertheless, we show that IgM works synergistically with ?2M and markedly lowers the concentration of ?2M required for rosetting. Finally, HB3VAR06+ IEs share the capacity to bind ?2M with subsets of genotypically distinct P. falciparum isolates forming rosettes in vitro and of patient parasite isolates ex vivo. Together, our results are evidence that P. falciparum parasites exploit ?2M (and IgM) to expand the repertoire of host receptors available for PfEMP1-mediated IE adhesion, such as the erythrocyte carbohydrate moieties that lead to formation of rosettes. It is likely that this mechanism also affects IE adhesion to receptors on vascular endothelium. The study opens opportunities for broad-ranging immunological interventions targeting the ?2M(and IgM-) binding domains of PfEMP1, which would be independent of the host receptor specificity of clinically important PfEMP1 antigens. PMID:26134405

  11. Blood parasites from California ducks and geese

    USGS Publications Warehouse

    Herman, C.M.

    1951-01-01

    Blood smears were procured from 1,011 geese and ducks of 19 species from various locations in California. Parasites were found in 28 individuals. The parasites observed included Haemoproteus hermani, Leucocytozoon simondi, microfilaria, Plasmodium relictum (=P. biziurae), and Plasmodium sp. with elongate gametocytes. This is the first report of a natural infection with a Plasmodium in North American wild ducks.

  12. Malaria Parasite-Infected Erythrocytes Secrete PfCK1, the Plasmodium Homologue of the Pleiotropic Protein Kinase Casein Kinase 1

    PubMed Central

    Dorin-Semblat, Dominique; Demarta-Gatsi, Claudia; Hamelin, Romain; Armand, Florence; Carvalho, Teresa Gil; Moniatte, Marc; Doerig, Christian

    2015-01-01

    Casein kinase 1 (CK1) is a pleiotropic protein kinase implicated in several fundamental processes of eukaryotic cell biology. Plasmodium falciparum encodes a single CK1 isoform, PfCK1, that is expressed at all stages of the parasite’s life cycle. We have previously shown that the pfck1 gene cannot be disrupted, but that the locus can be modified if no loss-of-function is incurred, suggesting an important role for this kinase in intra-erythrocytic asexual proliferation. Here, we report on the use of parasite lines expressing GFP- or His-tagged PfCK1 from the endogenous locus to investigate (i) the dynamics of PfCK1 localisation during the asexual cycle in red blood cells, and (ii) potential interactors of PfCK1, so as to gain insight into the involvement of the enzyme in specific cellular processes. Immunofluorescence analysis reveals a dynamic localisation of PfCK1, with evidence for a pool of the enzyme being directed to the membrane of the host erythrocyte in the early stages of infection, followed by a predominantly intra-parasite localisation in trophozoites and schizonts and association with micronemes in merozoites. Furthermore, we present strong evidence that a pool of enzymatically active PfCK1 is secreted into the culture supernatant, demonstrating that PfCK1 is an ectokinase. Our interactome experiments and ensuing kinase assays using recombinant PfCK1 to phosphorylate putative interactors in vitro suggest an involvement of PfCK1 in many cellular processes such as mRNA splicing, protein trafficking, ribosomal, and host cell invasion. PMID:26629826

  13. In Vitro and Molecular Surveillance for Antimalarial Drug Resistance in Plasmodium falciparum Parasites in Western Kenya Reveals Sustained Artemisinin Sensitivity and Increased Chloroquine Sensitivity.

    PubMed

    Lucchi, Naomi W; Komino, Franklin; Okoth, Sheila Akinyi; Goldman, Ira; Onyona, Philip; Wiegand, Ryan E; Juma, Elizabeth; Shi, Ya Ping; Barnwell, John W; Udhayakumar, Venkatachalam; Kariuki, Simon

    2015-12-01

    Malaria control is hindered by the evolution and spread of resistance to antimalarials, necessitating multiple changes to drug policies over time. A comprehensive antimalarial drug resistance surveillance program is vital for detecting the potential emergence of resistance to antimalarials, including current artemisinin-based combination therapies. An antimalarial drug resistance surveillance study involving 203 Plasmodium falciparum malaria-positive children was conducted in western Kenya between 2010 and 2013. Specimens from enrolled children were analyzed in vitro for sensitivity to chloroquine (CQ), amodiaquine (AQ), mefloquine (MQ), lumefantrine, and artemisinin derivatives (artesunate and dihydroartemisinin) and for drug resistance allele polymorphisms in P. falciparum crt (Pfcrt), Pfmdr-1, and the K13 propeller domain (K13). We observed a significant increase in the proportion of samples with the Pfcrt wild-type (CVMNK) genotype, from 61.2% in 2010 to 93.0% in 2013 (P < 0.0001), and higher proportions of parasites with elevated sensitivity to CQ in vitro. The majority of isolates harbored the wild-type N allele in Pfmdr-1 codon 86 (93.5%), with only 7 (3.50%) samples with the N86Y mutant allele (the mutant nucleotide is underlined). Likewise, most isolates harbored the wild-type Pfmdr-1 D1246 allele (79.8%), with only 12 (6.38%) specimens with the D1246Y mutant allele and 26 (13.8%) with mixed alleles. All the samples had a single copy of the Pfmdr-1 gene (mean of 0.907 0.141 copies). None of the sequenced parasites had mutations in K13. Our results suggest that artemisinin is likely to remain highly efficacious and that CQ sensitivity appears to be on the rise in western Kenya. PMID:26392510

  14. Partial suppression of malaria parasites, and of the transmission of malaria, in Aedes aegypti (L.) doubly-infected with Semliki Forest virus and Plasmodium gallinaceum Brumpt*

    PubMed Central

    Bertram, D. S.; Varma, M. G. R.; Baker, J. R.

    1964-01-01

    Laboratory experiments were undertaken with Aedes aegypti infected with both Semliki Forest virus (SFV) from infant mice and Plasmodium gallinaceum from fowls to determine if such double infection of mosquitos suppressed their ability to transmit the malaria parasite, a possibility suggested to explain reduction in malaria transmission in Uganda in 1960 when Anopheles funestus and Anopheles gambiae were transmitting both malaria and o'nyongnyong virus to the African population. In general, transmission of fowl malaria was not prevented by SFV infection in Aedes, although some malariometric indices, and consistently the mean oocyst count, were slightly lower in doubly-infected mosquitos than in controls. In one experiment, however, 60% of the Aedes infected 8 days previously with SFV died within 48 hours of ingesting a malarious meal. Mortality was selectively in favour of the survival of SFV-infected mosquitos negative for, or least heavily infected with, malaria; depression in the presence of the virus of the intensity of malaria infection in the individual Aedes also occurred. Some physiological factora stress in adult life or, possibly more important, suboptimal larval nutritionappears to have been crucial to eliciting the adverse effect on the mosquitos themselves and on their malaria infections. Suppression of the development of a malaria parasite in a mosquito, and of malaria transmission, by concurrent infection of the vector with an arbovirus can happen, but is by no means inevitable. It is shown that a doubly-infected Aedes can transmit both Semliki Forest virus and P. gallinaceum simultaneously. PMID:14278005

  15. Genetic diversity of the Plasmodium falciparum apical membrane antigen I gene in parasite population from the China-Myanmar border area.

    PubMed

    Zhu, Xiaotong; Zhao, Zhenjun; Feng, Yonghui; Li, Peipei; Liu, Fei; Liu, Jun; Yang, Zhaoqing; Yan, Guiyun; Fan, Qi; Cao, Yaming; Cui, Liwang

    2016-04-01

    To investigate the genetic diversity of the Plasmodium falciparum apical membrane antigen 1 (PfAMA1) gene in Southeast Asia, we determined PfAMA1 sequences from 135 field isolates collected from the China-Myanmar border area and compared them with 956 publically available PfAMA1 sequences from seven global P. falciparum populations. This analysis revealed high genetic diversity of PfAMA1 in global P. falciparum populations with a total of 229 haplotypes identified. The genetic diversity of PfAMA1 gene from the China-Myanmar border is not evenly distributed in the different domains of this gene. Sequence diversity in PfAMA1 from the China-Myanmar border is lower than that observed in Thai, African and Oceanian populations, but higher than that in the South American population. This appeared to correlate well with the levels of endemicity of different malaria-endemic regions, where hyperendemic regions favor genetic cross of the parasite isolates and generation of higher genetic diversity. Neutrality tests show significant departure from neutrality in the entire ectodomain and Domain I of PfAMA1 in the China-Myanmar border parasite population. We found evidence supporting a substantial continent-wise genetic structure among P. falciparum populations, with the highest genetic differentiation detected between the China-Myanmar border and the South American populations. Whereas no alleles were unique to a specific region, there were considerable geographical differences in major alleles and their frequencies, highlighting further necessity to include more PfAMA1 alleles in vaccine designs. PMID:26825252

  16. The structurally related auxin and melatonin tryptophan-derivatives and their roles in Arabidopsis thaliana and in the human malaria parasite Plasmodium falciparum.

    PubMed

    Koyama, Fernanda C; Carvalho, Thais L G; Alves, Eduardo; da Silva, Henrique B; de Azevedo, Mauro F; Hemerly, Adriana S; Garcia, Clia R S

    2013-01-01

    Indole compounds are involved in a range of functions in many organisms. In the human malaria parasite Plasmodium falciparum, melatonin and other tryptophan derivatives are able to modulate its intraerythrocytic cycle, increasing the schizont population as well as parasitemia, likely through ubiquitin-proteasome system (UPS) gene regulation. In plants, melatonin regulates root development, in a similar way to that described for indoleacetic acid, suggesting that melatonin and indoleacetic acid could co-participate in some physiological processes due to structural similarities. In the present work, we evaluate whether the chemical structure similarity found in indoleacetic acid and melatonin can lead to similar effects in Arabidopsis thaliana lateral root formation and P. falciparum cell cycle modulation, as well as in the UPS of gene regulation, by qRT-PCR. Our data show that P. falciparum is not able to respond to indoleacetic acid either in the modulation of the intraerythrocytic cycle or in the gene regulation mediated by the UPS as observed for melatonin. The similarities of these indole compounds are not sufficient to confer synergistic functions in P. falciparum cell cycle modulation, but could interplay in A. thaliana lateral root formation. PMID:24102716

  17. Molecular inference of sources and spreading patterns of Plasmodium falciparum malaria parasites in internally displaced persons settlements in Myanmar-China border area.

    PubMed

    Lo, Eugenia; Zhou, Guofa; Oo, Winny; Lee, Ming-Chieh; Baum, Elisabeth; Felgner, Philip L; Yang, Zhaoqing; Cui, Liwang; Yan, Guiyun

    2015-07-01

    In Myanmar, civil unrest and establishment of internally displaced persons (IDP) settlement along the Myanmar-China border have impacted malaria transmission. The growing IDP populations raise deep concerns about health impact on local communities. Microsatellite markers were used to examine the source and spreading patterns of Plasmodium falciparum between IDP settlement and surrounding villages in Myanmar along the China border. Genotypic structure of P. falciparum was compared over the past three years from the same area and the demographic history was inferred to determine the source of recent infections. In addition, we examined if border migration is a factor of P. falciparum infections in China by determining gene flow patterns across borders. Compared to local community, the IDP samples showed a reduced and consistently lower genetic diversity over the past three years. A strong signature of genetic bottleneck was detected in the IDP samples. P. falciparum infections from the border regions in China were genetically similar to Myanmar and parasite gene flow was not constrained by geographical distance. Reduced genetic diversity of P. falciparum suggested intense malaria control within the IDP settlement. Human movement was a key factor to the spread of malaria both locally in Myanmar and across the international border. PMID:25952567

  18. Interaction of Silver Nanoparticles with Triosephosphate Isomerase from Human and Malarial Parasite (Plasmodium falciparum): A Comparative Study.

    PubMed

    De Moor, W; van Marwijk, J; Wilhelmi, B S; Whiteley, C G

    2015-06-01

    Recombinant triosephosphate isomerase from Plasmodium falciparum (PfTIM) and humans (hTIM) were expressed, purified and characterised. High specific activity (1207 U x mg(-1)) with a fold purification of -1.8 and a yield of 48% were obtained for hTIM after gel filtration while, in contrast PfTIM afforded a specific activity of 1387 U x mg(-1) with a fold purification of -6.8 and a yield of 57% after gel filtration and prior to dialysis. PfTIM had an optimal pH and temperature, K(m) and V(max) of 5.25, 25 degrees C, 12.8 mM and 1.13 ?mol x mL(-1) min(-1) respectively while for hTIM the pH and temperature optima, K(m) and V(max) were 6.75, 30 degrees C; 8.2 mM and 1.35 ?mol x ml(-1) min(-1). Polyvinylpyrrolidone stabilised silver nanoparticles (60 nM; 2-6 nm diameter) selectively inhibited PfTIM with a 7-fold decrease in enzyme catalytic efficiency (K(cat)/K(m)) over hTIM. Respective K(i) values were 283 nM [hTIM] and 85.7 nM [PfTIM]. Key structural differences between the two enzyme variants, especially with Cys13 at the dimer interface of PfTIM, were significant enough to suggest unique characteristics allowing for selective targeting of PfTIM by AgNPs. PMID:26353595

  19. Isolation and Characterization of the MSP1 Genes from Plasmodium malariae and Plasmodium ovale

    PubMed Central

    Birkenmeyer, Larry; Muerhoff, A. Scott; Dawson, George J.; Desai, Suresh M.

    2010-01-01

    The merozoite surface protein 1 (MSP1) is the principal surface antigen of the blood stage form of the Plasmodium parasite. Antibodies recognizing MSP1 are frequently detected following Plasmodium infection, making this protein a significant component of malaria vaccines and diagnostic tests. Although the MSP1 gene sequence has been reported for Plasmodium falciparum and Plasmodium vivax, this gene has not been identified for the other two major human-infectious species, Plasmodium malariae and Plasmodium ovale. MSP1 genes from these two species were isolated from Cameroon blood donor samples. The genes are similar in size to known MSP1 genes and encode proteins with interspecies conserved domains homologous to those identified in other Plasmodium species. Sequence and phylogenetic analysis of all available Plasmodium MSP1 amino acid sequences clearly shows that the Po and Pm MSP1 sequences are truly unique within the Plasmodium genus and not simply Pf or Pv variants. PMID:20519591

  20. Ungulate malaria parasites

    PubMed Central

    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

    2016-01-01

    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

  1. Ungulate malaria parasites.

    PubMed

    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

    2016-01-01

    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

  2. Malaria and blood transfusion: major issues of blood safety in malaria-endemic countries and strategies for mitigating the risk of Plasmodium parasites.

    PubMed

    Abdullah, Saleh; Karunamoorthi, Kaliyaperumal

    2016-01-01

    Malaria inflicts humankind over centuries, and it remains as a major threat to both clinical medicine and public health worldwide. Though hemotherapy is a life-sustaining modality, it continues to be a possible source of disease transmission. Hence, hemovigilance is a matter of grave concern in the malaria-prone third-world countries. In order to pursue an effective research on hemovigilance, a comprehensive search has been conducted by using the premier academic-scientific databases, WHO documents, and English-language search engines. One hundred two appropriate articles were chosen for data extraction, with a particular reference to emerging pathogens transmitted through blood transfusion, specifically malaria. Blood donation screening is done through microscopic examination and immunological assays to improve the safety of blood products by detection major blood-borne pathogens, viz., HIV, HBV, HCV, syphilis, and malarial parasites. Transfusion therapy significantly dwindles the preventable morbidity and mortality attributed to various illnesses and diseases, particularly AIDS, tuberculosis, and malaria. Examination of thick and thin blood smears are performed to detect positivity and to identify the Plasmodium species, respectively. However, all of these existing diagnostic tools have their own limitations in terms of sensitivity, specificity, cost-effectiveness, and lack of resources and skilled personnel. Globally, despite the mandate need of screening blood and its components according to the blood-establishment protocols, it is seldom practiced in the low-income/poverty-stricken settings. In addition, each and every single phase of transfusion chain carries sizable inherent risks from donors to recipients. Interestingly, opportunities also lie ahead to enhance the safety of blood-supply chain and patients. It can be achieved through sustainable blood-management strategies like (1) appropriate usage of precise diagnostic tools/techniques, (2) promoting hemovigilance system, and (3) adopting novel processes of inactivation technology. Furthermore, selection of the zero-risk donors could pave the way to build a transmissible malaria-free world in the near future. PMID:26531301

  3. Plasmodium falciparum multidrug resistance protein 1 (pfmrp1) gene and its association with in vitro drug susceptibility of parasite isolates from north-east Myanmar

    PubMed Central

    Gupta, Bhavna; Xu, Shuhui; Wang, Zenglei; Sun, Ling; Miao, Jun; Cui, Liwang; Yang, Zhaoqing

    2014-01-01

    Objectives Plasmodium falciparum multidrug resistance protein 1 (pfmrp1) has recently emerged as an important determinant of drug resistance and mutations in the gene have been associated with several drugs. The aim of this study was to understand the level of genetic diversity in pfmrp1 and to determine the association of different mutations with altered drug susceptibilities of P. falciparum. Methods We analysed 193 sequences of pfmrp1 from South-East Asia, west Asia, Africa, Oceania and South America. We measured the level of genetic diversity and determined signatures of selection on the gene. In vitro susceptibilities of 28 P. falciparum isolates from north-east Myanmar to a panel of seven commonly used antimalarials were determined. Statistical analysis was performed to determine the association of different mutations with in vitro drug susceptibilities. Results A total of 28 single nucleotide polymorphisms were identified in 193 sequences, of which 22 were non-synonymous. Whereas mutations in the pfmrp1 gene were conserved among different countries within a continent, they were different between continents. Seven non-synonymous mutations were identified in the north-east Myanmar isolates; all were relatively frequent in this region as well as in other neighbouring countries. Molecular evolutionary analysis detected signatures of positive selection on the gene. Moreover, some mutations in this gene were found to be associated with reduced susceptibilities to chloroquine, mefloquine, pyronaridine and lumefantrine. Conclusions Evidence of the positive selection of pmfrp1 and its association with the susceptibilities of parasites to multiple drugs signifies its potential as an important candidate for monitoring drug resistance. PMID:24855124

  4. A large proportion of asymptomatic Plasmodium infections with low and sub-microscopic parasite densities in the low transmission setting of Temotu Province, Solomon Islands: challenges for malaria diagnostics in an elimination setting

    PubMed Central

    2010-01-01

    Background Many countries are scaling up malaria interventions towards elimination. This transition changes demands on malaria diagnostics from diagnosing ill patients to detecting parasites in all carriers including asymptomatic infections and infections with low parasite densities. Detection methods suitable to local malaria epidemiology must be selected prior to transitioning a malaria control programme to elimination. A baseline malaria survey conducted in Temotu Province, Solomon Islands in late 2008, as the first step in a provincial malaria elimination programme, provided malaria epidemiology data and an opportunity to assess how well different diagnostic methods performed in this setting. Methods During the survey, 9,491 blood samples were collected and examined by microscopy for Plasmodium species and density, with a subset also examined by polymerase chain reaction (PCR) and rapid diagnostic tests (RDTs). The performances of these diagnostic methods were compared. Results A total of 256 samples were positive by microscopy, giving a point prevalence of 2.7%. The species distribution was 17.5% Plasmodium falciparum and 82.4% Plasmodium vivax. In this low transmission setting, only 17.8% of the P. falciparum and 2.9% of P. vivax infected subjects were febrile (?38C) at the time of the survey. A significant proportion of infections detected by microscopy, 40% and 65.6% for P. falciparum and P. vivax respectively, had parasite density below 100/?L. There was an age correlation for the proportion of parasite density below 100/?L for P. vivax infections, but not for P. falciparum infections. PCR detected substantially more infections than microscopy (point prevalence of 8.71%), indicating a large number of subjects had sub-microscopic parasitemia. The concordance between PCR and microscopy in detecting single species was greater for P. vivax (135/162) compared to P. falciparum (36/118). The malaria RDT detected the 12 microscopy and PCR positive P. falciparum, but failed to detect 12/13 microscopy and PCR positive P. vivax infections. Conclusion Asymptomatic malaria infections and infections with low and sub-microscopic parasite densities are highly prevalent in Temotu province where malaria transmission is low. This presents a challenge for elimination since the large proportion of the parasite reservoir will not be detected by standard active and passive case detection. Therefore effective mass screening and treatment campaigns will most likely need more sensitive assays such as a field deployable molecular based assay. PMID:20822506

  5. A Case Report of Plasmodium Vivax, Plasmodium Falciparum and Dengue Co-Infection in a 6 Months Pregnancy

    PubMed Central

    Pande, A; Guharoy, D

    2013-01-01

    India being a tropical country, parasitic infections especially with Plasmodium species are very common in this region. The present case report is that of Plasmodium vivax, Plasmodium falciparum and dengue co-infection in a 6 months pregnant lady who was timely diagnosed and appropriately treated followed by a complete recovery along with feto-maternal well-being. PMID:24349838

  6. Age-Stratified Profiles of Serum IL-6, IL-10, and TNF-? Cytokines Among Kenyan Children with Schistosoma haematobium, Plasmodium falciparum, and Other Chronic Parasitic Co-Infections.

    PubMed

    Bustinduy, Amaya L; Sutherland, Laura J; Chang-Cojulun, Alicia; Malhotra, Indu; DuVall, Adam S; Fairley, Jessica K; Mungai, Peter L; Muchiri, Eric M; Mutuku, Francis M; Kitron, Uriel; King, Charles H

    2015-05-01

    In a study of children having polyparasitic infections in a Schistosoma haematobium-endemic area, we examined the hypothesis that S. haematobium-positive children, compared with S. haematobium-negative children (anti-soluble worm antigen preparation [SWAP] negative and egg negative) have increased systemic production of pro-inflammatory cytokines (interleukin [IL]-6, tumor necrosis factor [TNF]-?) and decreased down-regulatory IL-10. A total of 804 children, 2-19 years of age, were surveyed between July and December 2009 and tested for S. haematobium, Plasmodium falciparum, filariasis, and soil-transmitted helminth infections. Plasma levels of IL-6, TNF-?, and IL-10 were compared for S. haematobium-positive and S. haematobium-negative children, adjusting for malaria, filaria, and hookworm co-infections, and for nutritional status, age group, sex, and geographic location. IL-10 was significantly elevated among children infected with S. haematobium, showing bimodal peaks in 7-8 and 13-14 years age groups. IL-10 was also higher among children who were acutely malnourished, whereas IL-10 levels were lower in the presence of S. haematobium-filaria co-infection. After adjustment for co-factors, IL-6 was significantly elevated among children of 5-6 years and among those with P. falciparum infection. Lower levels of IL-6 were found in malaria-hookworm co-infection. High levels of TNF-? were found in children aged 11-12 years regardless of infection status. In addition, village of residence was a strong predictor of IL-6 and IL-10 plasma levels. In adolescent children infected with S. haematobium, there is an associated elevation in circulating IL-10 that may reduce the risk of later morbidity. Although we did not find a direct link between S. haematobium infection and circulating pro-inflammatory IL-6 and TNF-? levels, future T-cell stimulation studies may provide more conclusive linkages between infection and cytokine responses in settings that are endemic for multiple parasites and multiple co-infections. PMID:25758654

  7. Plasmodium vivax Tryptophan Rich Antigen PvTRAg36.6 Interacts with PvETRAMP and PvTRAg56.6 Interacts with PvMSP7 during Erythrocytic Stages of the Parasite

    PubMed Central

    Tyagi, Kriti; Hossain, Mohammad Enayet; Thakur, Vandana; Aggarwal, Praveen; Malhotra, Pawan; Mohmmed, Asif; Sharma, Yagya Dutta

    2016-01-01

    Plasmodium vivax is most wide spread and a neglected malaria parasite. There is a lack of information on parasite biology of this species. Genome of this parasite encodes for the largest number of tryptophan-rich proteins belonging to ‘Pv-fam-a’ family and some of them are potential drug/vaccine targets but their functional role(s) largely remains unexplored. Using bacterial and yeast two hybrid systems, we have identified the interacting partners for two of the P. vivax tryptophan-rich antigens called PvTRAg36.6 and PvTRAg56.2. The PvTRAg36.6 interacts with early transcribed membrane protein (ETRAMP) of P.vivax. It is apically localized in merozoites but in early stages it is seen in parasite periphery suggesting its likely involvement in parasitophorous vacuole membrane (PVM) development or maintenance. On the other hand, PvTRAg56.2 interacts with P.vivax merozoite surface protein7 (PvMSP7) and is localized on merozoite surface. Co-localization of PvTRAg56.2 with PvMSP1 and its molecular interaction with PvMSP7 probably suggest that, PvTRAg56.2 is part of MSP-complex, and might assist or stabilize the protein complex at the merozoite surface. In conclusion, the PvTRAg proteins have different sub cellular localizations and specific associated functions during intra-erythrocytic developmental cycle. PMID:26954579

  8. An interplay between 2 signaling pathways: Melatonin-cAMP and IP{sub 3}–Ca{sup 2+} signaling pathways control intraerythrocytic development of the malaria parasite Plasmodium falciparum

    SciTech Connect

    Furuyama, Wakako; Enomoto, Masahiro; Mossaad, Ehab; Kawai, Satoru; Mikoshiba, Katsuhiko; Kawazu, Shin-ichiro

    2014-03-28

    Highlights: • A melatonin receptor antagonist blocked Ca{sup 2+} oscillation in P. falciparum and inhibited parasite growth. • P. falciparum development is controlled by Ca{sup 2+}- and cAMP-signaling pathways. • The cAMP-signaling pathway at ring form and late trophozoite stages governs parasite growth of P. falciparum. - Abstract: Plasmodium falciparum spends most of its asexual life cycle within human erythrocytes, where proliferation and maturation occur. Development into the mature forms of P. falciparum causes severe symptoms due to its distinctive sequestration capability. However, the physiological roles and the molecular mechanisms of signaling pathways that govern development are poorly understood. Our previous study showed that P. falciparum exhibits stage-specific spontaneous Calcium (Ca{sup 2+}) oscillations in ring and early trophozoites, and the latter was essential for parasite development. In this study, we show that luzindole (LZ), a selective melatonin receptor antagonist, inhibits parasite growth. Analyses of development and morphology of LZ-treated P. falciparum revealed that LZ severely disrupted intraerythrocytic maturation, resulting in parasite death. When LZ was added at ring stage, the parasite could not undergo further development, whereas LZ added at the trophozoite stage inhibited development from early into late schizonts. Live-cell Ca{sup 2+} imaging showed that LZ treatment completely abolished Ca{sup 2+} oscillation in the ring forms while having little effect on early trophozoites. Further, the melatonin-induced cAMP increase observed at ring and late trophozoite stage was attenuated by LZ treatment. These suggest that a complex interplay between IP{sub 3}–Ca{sup 2+} and cAMP signaling pathways is involved in intraerythrocytic development of P. falciparum.

  9. Merozoite surface protein-9 of Plasmodium vivax and related simian malaria parasites is orthologous to p101/ABRA of P. falciparum.

    PubMed

    Vargas-Serrato, Esmeralda; Barnwell, John W; Ingravallo, Paul; Perler, Francine B; Galinski, Mary R

    2002-03-01

    Plasmodium vivax merozoite surface protein-9 (Pvmsp-9) is characterized here along with orthologues from the related simian malarias Plasmodium cynomolgi and Plasmodium knowlesi. We show that although the corresponding MSP-9 proteins do not have acidic-basic repeated amino acid (aa) motifs, they are related to the Plasmodium falciparum acidic-basic repeat antigen (ABRA) also known as p101. Recognition of this new interspecies Plasmodium MSP family stems from the prior identification of related MSP termed PvMSP-185, PcyMSP-150, and PkMSP-110 on the surface of P. vivax, P. cynomolgi and P. knowlesi merozoites. A clone containing the nearly complete P. knowlesi gene encoding PkMSP-110/MSP-9 provided a hybridization probe and initial sequence information for the design of primers to obtain the P. vivax and P. cynomolgi orthologues using polymerase chain reaction (PCR) amplification strategies. The P. vivax, P. cynomolgi and P. knowlesi msp-9 genes encode proteins that range in calculated molecular mass from 80 to 107 kDa, have typical eukaryotic signal peptides and diverse repeated motifs present immediately upstream of their termination codon. Another feature conserved among these proteins, including the P. falciparum ABRA protein, is the positions of four cysteine residues near the N-terminus, suggesting this conservation maintains structural and perhaps functional characteristics in the MSP-9 family. Rabbit polyclonal antisera raised against recombinantly expressed N-termini of P. knowlesi and P. vivax MSP-9 cross-react with the counterpart proteins in immunofluorescence and immunoblot assays. Comparative interspecies investigations of the potential role(s) of Plasmodium MSP-9 in merozoite invasion of erythrocytes and as a malaria vaccine candidate can now be pursued. PMID:11849704

  10. Growth inhibition of Plasmodium falciparum involving carbon centered iron-chelate radical (L., X-)-Fe(III) based on pyridoxal-betaine. A novel type of antimalarials active against chloroquine-resistant parasites.

    PubMed

    Iheanacho, E N; Sarel, S; Samuni, A; Avramovici-Grisaru, S; Spira, D T

    1991-01-01

    Malaria parasites have been shown to be more susceptible to oxidative stress than their host erythrocytes. In the present work, a chloroquine resistant malaria parasite, Plasmodium falciparum (FCR-3) was found to be susceptible in vitro to a pyridoxal based iron chelator--(1-[N-ethoxycarbonylmethylpyridoxlidenium]-2-[2'-pyridyl ] hydrazine bromide--(code named L2-9). 2h exposure to 20 microM L2-9 was sufficient to irreversibly inhibit parasite growth. Desferrioxamine blocked the drug effect, indicating the requirement for iron. Oxygen however, was not essential. Spectrophotometric analysis showed that under anoxic conditions, L2-9-Fe(II) chelate undergoes an intramolecular redox reaction which presumably involves a one electron transfer and is expected to result in the formation of free radical. Spin trapping coupled to electron spin resonance (ESR) studies of L2-9-iron chelate showed that L2-9-Fe(II) produced free radicals both in the presence and absence of cells, while L2-9-Fe(III) produced free radicals only in the presence of actively metabolising cells. PMID:1663064

  11. A host-parasite list of the haematozoa of domestic poultry in sub-Saharan Africa and the isolation of Plasmodium durae Herman from turkeys and francolins in South Africa.

    PubMed

    Huchzermeyer, F W

    1993-03-01

    An annotated host-parasite list of the blood parasites of domestic poultry in sub-Saharan Africa is presented. This list contains the haematozoa found in domestic waterfowl (ducks, geese and muscovies) and phasianids (turkey, fowl and peafowl). In South Africa Plasmodium durae was isolated from 4 out of 8 backyard turkeys, from 3 out of 26 Swainson's francolins and from 1 redwing francolin, but not from 20 helmeted guineafowls and 9 greywing francolins. This points at Swainson's and redwing francolins as being the main natural hosts of P. durae in South Africa. The increase in the period of prepatency after intramuscular subinoculation as compared with the intravenous route was found to correspond to that of a 1,000 fold dilution of an intravenous inoculum of parasitized blood. This delay was not due to an intervening cycle of exoerythrocytic schizogony, but to large numbers of the injected erythrocytes apparently not finding their way into the circulation of the new host. PMID:8332314

  12. Bis(benzyl)polyamine analogs inhibit the growth of chloroquine-resistant human malaria parasites (Plasmodium falciparum) in vitro and in combination with alpha-difluoromethylornithine cure murine malaria.

    PubMed Central

    Bitonti, A J; Dumont, J A; Bush, T L; Edwards, M L; Stemerick, D M; McCann, P P; Sjoerdsma, A

    1989-01-01

    A number of bis(benzyl)polyamine analogs were found to be potent inhibitors of both chloroquine-resistant and chloroquine-sensitive strains of the human malaria parasite Plasmodium falciparum in vitro (IC50 values = 0.2-14 microM). Administration of one of the compounds, MDL 27695, which is N,N'-bis(3-[(phenylmethyl)amino]propyl)-1,7-diaminoheptane (C6H5CH2NH(CH2)3NH(CH2)7NH(CH2)3NHCH2C6H5), at 10-15 mg/kg i.p. three times per day for 3 days in combination with 2% alpha-difluoromethylornithine (DFMO; eflornithine) in drinking water effected cures of 47/54 mice infected with Plasmodium berghei. Cured mice were found to be immune upon rechallenge with the same P. berghei strain 4 months after the initial infection and drug-induced cure. MDL 27695 rapidly inhibited the incorporation of [3H]hypoxanthine into P. falciparum RNA and DNA, whereas the incorporation of [3H]isoleucine was not affected until much later. We conclude, therefore, that the major cytotoxic event may be direct binding of MDL 27695 to DNA with subsequent disruption of macromolecular biosynthesis and cell death. These compounds offer a lead in the search for new agents for chemotherapy of malaria. PMID:2463635

  13. Assessment of the induction of dormant ring stages in Plasmodium falciparum parasites by artemisone and artemisone entrapped in Pheroid vesicles in vitro.

    PubMed

    Grobler, Lizette; Chavchich, Marina; Haynes, Richard K; Edstein, Michael D; Grobler, Anne F

    2014-12-01

    The in vitro antimalarial activities of artemisone and artemisone entrapped in Pheroid vesicles were compared, as was their ability to induce dormancy in Plasmodium falciparum. There was no increase in the activity of artemisone entrapped in Pheroid vesicles against multidrug-resistant P. falciparum lines. Artemisone induced the formation of dormant ring stages similar to dihydroartemisinin. Thus, the Pheroid delivery system neither improved the activity of artemisone nor prevented the induction of dormant rings. PMID:25288088

  14. EWGWS insert in Plasmodium falciparum ookinete surface enolase is involved in binding of PWWP containing peptides: Implications to mosquito midgut invasion by the parasite.

    PubMed

    Mukherjee, Debanjan; Mishra, Pushpa; Joshi, Mamata; Thakur, Prasoon Kumar; Hosur, R V; Jarori, Gotam K

    2016-01-01

    There are multiple stages in the life cycle of Plasmodium that invade host cells. Molecular machinery involved is such host-pathogen interactions constitute excellent drug targets and/or vaccine candidates. A screen using a phage display library has previously demonstrated presence of enolase on the surface of the Plasmodium ookinete. Phage-displayed peptides that bound to the ookinete contained a conserved motif (PWWP) in their sequence. Here, direct binding of these peptides with recombinant Plasmodium falciparum enolase (rPfeno) was investigated. These peptides showed specific binding to rPfeno, but failed to bind to other enolases. Plasmodium spp enolases are distinct in having an insert of five amino acids ((104)EWGWS(108)) that is not found in host enolases. The possibility of this insert being the recognition motif for the PWWP containing peptides was examined, (i) by comparing the binding of the peptides with rPfeno and a deletion variant Δ-rPfeno lacking (104)EWGWS(108), (ii) by measuring the changes in proton chemical shifts of PWWP peptides on binding to different enolases and (iii) by inter-molecular docking experiment to locate the peptide binding site. Results from these studies showed that the pentapeptide insert of Pfeno indeed constitutes the binding site for the PWWP domain containing peptide ligands. Search for sequences homologous to phage displayed peptides among peritrophic matrix proteins resulted in identification of perlecan, laminin, peritrophin and spacran. The possibility of these PWWP domain-containing proteins in the peritrophic matrix of insect gut to interact with ookinete cell surface enolase and facilitate the invasion of mosquito midgut epithelium is discussed. PMID:26592350

  15. Highly Dynamic Host Actin Reorganization around Developing Plasmodium Inside Hepatocytes

    PubMed Central

    Gomes-Santos, Carina S. S.; Itoe, Maurice A.; Afonso, Cristina; Henriques, Ricardo; Gardner, Rui; Seplveda, Nuno; Simes, Pedro D.; Raquel, Helena; Almeida, Antnio Paulo; Moita, Luis F.; Frischknecht, Friedrich; Mota, Maria M.

    2012-01-01

    Plasmodium sporozoites are transmitted by Anopheles mosquitoes and infect hepatocytes, where a single sporozoite replicates into thousands of merozoites inside a parasitophorous vacuole. The nature of the Plasmodium-host cell interface, as well as the interactions occurring between these two organisms, remains largely unknown. Here we show that highly dynamic hepatocyte actin reorganization events occur around developing Plasmodium berghei parasites inside human hepatoma cells. Actin reorganization is most prominent between 10 to 16 hours post infection and depends on the actin severing and capping protein, gelsolin. Live cell imaging studies also suggest that the hepatocyte cytoskeleton may contribute to parasite elimination during Plasmodium development in the liver. PMID:22238609

  16. No evidence of blood parasites in Little Auks (Alle alle) breeding on Svalbard.

    PubMed

    Wojczulanis-Jakubas, Katarzyna; Svoboda, Ales; Kruszewicz, Andrzej; Johnsen, Arild

    2010-04-01

    Absence of blood parasites in birds occurring in polar regions is believed to be a common phenomenon but only few surveys have been conducted on truly polar bird species. We examined blood parasites in a seabird with an exclusively high-Arctic breeding distribution, the Little Auk (Alle alle). Polymerase chain reaction was used to detect haemosporidians and traditional optical microscopy was used to detect a wider range of haematozoans in birds breeding on Svalbard (Norway). No blood parasites were found among 100 birds examined. This finding confirms scarcity of haematozoa in polar regions and provides baseline data for the Little Auk. PMID:20688653

  17. The Robust and Modulated Biomarker Network Elicited by the Plasmodium vivax Infection Is Mainly Mediated by the IL-6/IL-10 Axis and Is Associated with the Parasite Load

    PubMed Central

    Guimares da Costa, Allyson; do Valle Antonelli, Lis Ribeiro; Augusto Carvalho Costa, Pedro; Paulo Diniz Pimentel, Joo; Garcia, Nadja Pinto; Monteiro Tarrag, Andra; Socorro Lopes dos Santos, Maria do Perptuo; Nogueira, Paulo Afonso; Hekcmann, Maria Izabel Ovellar; Sadahiro, Aya; Teixeira-Carvalho, Andra; Martins-Filho, Olindo Assis; Malheiro, Adriana

    2014-01-01

    Background. Recent studies have shown that the inflammatory process, including the biomarker production, and the intense activation of innate immune responses are greater in the malaria caused by Plasmodium vivax than other species. Here, we examined the levels of serum biomarkers and their interaction during acute malaria. Material and Methods. Blood samples were collected from P. vivax-infected patients at admission and from healthy donors. Levels of serum biomarkers were measured by Cytometric Bead Assay or ELISA. Results. P. vivax infection triggered the production of both inflammatory and regulatory biomarkers. Levels of IL-6, CXCL-8, IFN-?, IL-5, and IL-10 were higher in P. vivax-infected patients than in healthy donors. On the other hand, malaria patients produced lower levels of TNF-?, IL-12p70, and IL-2 than healthy individuals. While the levels of IL-10 and IL-6 were found independent on the number of malaria episodes, higher levels of these cytokines were seen in patients with higher parasite load. Conclusion. A mixed pattern of proinflammatory and regulatory biomarkers is produced in P. vivax malaria. Analysis of biomarker network suggests that IL-10 and IL-6 are a robust axis in malaria patients and that this interaction seems to be associated with the parasite load. PMID:24741587

  18. Adenine metabolism in Plasmodium falciparum.

    PubMed

    Mehrotra, Sonali; Bopanna, Monnanda P; Bulusu, Vinay; Balaram, Hemalatha

    2010-06-01

    Plasmodium falciparum lacks the de novo purine biosynthesis pathway and relies entirely on the salvage pathway to meet its purine nucleotide requirements. The entire flux for purine nucleotide biosynthesis in the parasite is believed to be through hypoxanthine guanine phosphoribosyltransferase (HGPRT), with the enzymes, adenosine kinase and adenine phosphoribosyltransferase (APRT) being unannotated in the Plasmodium genome database. This manuscript reports on the studies carried out to explore bypass mechanisms, if any, for AMP synthesis in the intraerythrocyitc stages of the parasite life cycle. Uptake and subsequent incorporation of radiolabel adenine in the nucleotide pool of saponin released erythrocyte free parasites implicated the role of parasite encoded enzymes in adenine metabolism. To explore the route for AMP synthesis in the parasite, we have monitored adenine mediated supplementation of metabolic viability in saponin released hadacidin (N-formyl-N-hydroxyglycine) treated parasites. Our results implicate the role of an APRT like activity that enables parasite survival when the flux through the HGPRT pathway is blocked. PMID:20093117

  19. Vector sequence contamination of the Plasmodium vivax sequence database in PlasmoDB and In silico correction of 26 parasite sequences.

    PubMed

    Tao, Zhi-Yong; Sui, Xu; Jun, Cao; Culleton, Richard; Fang, Qiang; Xia, Hui; Gao, Qi

    2015-01-01

    We found a 47 aa protein sequence that occurs 17 times in the Plasmodium vivax nucleotide database published on PlasmoDB. Coding sequence analysis showed multiple restriction enzyme sites within the 141bp nucleotide sequence, and a His6 tag attached to the 3' end, suggesting cloning vector origins. Sequences with vector contamination were submitted to NCBI, and BLASTN was used to cross-examine whole-genome shotgun contigs (WGS) from four recently deposited P. vivax whole genome sequencing projects. There are at least 26 genes listed in the PlasmoDB database that incorporate this cloning vector sequence into their predicted provisional protein products. PMID:26062606

  20. Temporal trends in prevalence of Plasmodium falciparum molecular markers selected for by artemetherlumefantrine treatment in pre-ACT and post-ACT parasites in western Kenya

    PubMed Central

    Achieng, Angela O.; Muiruri, Peninah; Ingasia, Luicer A.; Opot, Benjamin H.; Juma, Dennis W.; Yeda, Redemptah; Ngalah, Bidii S.; Ogutu, Bernhards R.; Andagalu, Ben; Akala, Hoseah M.; Kamau, Edwin

    2015-01-01

    Artemetherlumefantrine (AL) became the first-line treatment for uncomplicated malaria in Kenya in 2006. Studies have shown AL selects for SNPs in pfcrt and pfmdr1 genes in recurring parasites compared to the baseline infections. The genotypes associated with AL selection are K76 in pfcrt and N86, 184F and D1246 in pfmdr1. To assess the temporal change of these genotypes in western Kenya, 47 parasite isolates collected before (pre-ACT; 19952003) and 745 after (post-ACT; 20082014) introduction of AL were analyzed. In addition, the associations of parasite haplotype against the IC50 of artemether and lumefantrine, and clearance rates were determined. Parasite genomic DNA collected between 1995 and 2014 was analyzed by sequencing or PCR-based single-base extension on Sequenom MassARRAY. IC50s were determined for a subset of the samples. One hundred eighteen samples from 2013 to 2014 were from an efficacy trial of which 68 had clearance half-lives. Data revealed there were significant differences between pre-ACT and post-ACT genotypes at the four codons (chi-square analysis; p<0.0001). The prevalence of pfcrt K76 and N86 increased from 6.4% in 19951996 to 93.2% in 2014 and 0.0% in 20022003 to 92.4% in 2014 respectively. Analysis of parasites carrying pure alleles of K+NFD or T+YYY haplotypes revealed that 100.0% of the pre-ACT parasites carried T+YYY and 99.3% of post-ACT parasites carried K+NFD. There was significant correlation (p=0.04) between lumefantrine IC50 and polymorphism at pfmdr1 codon 184. There was no difference in parasite clearance half-lives based on genetic haplotype profiles. This study shows there is a significant change in parasite genotype, with key molecular determinants of AL selection almost reaching saturation. The implications of these findings are not clear since AL remains highly efficacious. However, there is need to closely monitor parasite genotypic, phenotypic and clinical dynamics in response to continued use of AL in western Kenya. PMID:26236581

  1. Temporal trends in prevalence of Plasmodium falciparum molecular markers selected for by artemether-lumefantrine treatment in pre-ACT and post-ACT parasites in western Kenya.

    PubMed

    Achieng, Angela O; Muiruri, Peninah; Ingasia, Luicer A; Opot, Benjamin H; Juma, Dennis W; Yeda, Redemptah; Ngalah, Bidii S; Ogutu, Bernhards R; Andagalu, Ben; Akala, Hoseah M; Kamau, Edwin

    2015-12-01

    Artemether-lumefantrine (AL) became the first-line treatment for uncomplicated malaria in Kenya in 2006. Studies have shown AL selects for SNPs in pfcrt and pfmdr1 genes in recurring parasites compared to the baseline infections. The genotypes associated with AL selection are K76 in pfcrt and N86, 184F and D1246 in pfmdr1. To assess the temporal change of these genotypes in western Kenya, 47 parasite isolates collected before (pre-ACT; 1995-2003) and 745 after (post-ACT; 2008-2014) introduction of AL were analyzed. In addition, the associations of parasite haplotype against the IC50 of artemether and lumefantrine, and clearance rates were determined. Parasite genomic DNA collected between 1995 and 2014 was analyzed by sequencing or PCR-based single-base extension on Sequenom MassARRAY. IC50s were determined for a subset of the samples. One hundred eighteen samples from 2013 to 2014 were from an efficacy trial of which 68 had clearance half-lives. Data revealed there were significant differences between pre-ACT and post-ACT genotypes at the four codons (chi-square analysis; p<0.0001). The prevalence of pfcrt K76 and N86 increased from 6.4% in 1995-1996 to 93.2% in 2014 and 0.0% in 2002-2003 to 92.4% in 2014 respectively. Analysis of parasites carrying pure alleles of K+NFD or T+YYY haplotypes revealed that 100.0% of the pre-ACT parasites carried T+YYY and 99.3% of post-ACT parasites carried K+NFD. There was significant correlation (p=0.04) between lumefantrine IC50 and polymorphism at pfmdr1 codon 184. There was no difference in parasite clearance half-lives based on genetic haplotype profiles. This study shows there is a significant change in parasite genotype, with key molecular determinants of AL selection almost reaching saturation. The implications of these findings are not clear since AL remains highly efficacious. However, there is need to closely monitor parasite genotypic, phenotypic and clinical dynamics in response to continued use of AL in western Kenya. PMID:26236581

  2. Evaluation of parasite subpopulations and genetic diversity of the msp1, msp2 and glurp genes during and following artesunate monotherapy treatment of Plasmodium falciparum malaria in Western Cambodia

    PubMed Central

    2013-01-01

    Background Despite widespread coverage of the emergence of artemisinin resistance, relatively little is known about the parasite populations responsible. The use of PCR genotyping around the highly polymorphic Plasmodium falciparum msp1, msp2 and glurp genes has become well established both to describe variability in alleles within a population of parasites, as well as classify treatment outcome in cases of recurrent disease. The primary objective was to assess the emergence of minority parasite clones during seven days of artesunate (AS) treatment in a location with established artemisinin resistance. An additional objective was to investigate whether the classification of clinical outcomes remained valid when additional genotyping was performed. Methods Blood for parasite genotyping was collected from 143 adult patients presenting with uncomplicated falciparum malaria during a clinical trial of AS monotherapy in Western Cambodia. Nested allelic type-specific amplification of the genes encoding the merozoite surface proteins 1 and 2 (msp1 and msp2) and the glutamate-rich protein (glurp) was performed at baseline, daily during seven days of treatment, and again at failure. Allelic variants were analysed with respect to the size of polymorphisms using Quantity One software to enable identification of polyclonal infections. Results Considerable variation of msp2 alleles but well-conserved msp1 and glurp were identified. At baseline, 31% of infections were polyclonal for one or more genes. Patients with recurrent malaria were significantly more likely to have polyclonal infections than patients without recurrence (seven of nine versus 36 of 127, p?=?0.004). Emergence of minority alleles during treatment was detected in only one of twenty-three cases defined as being artemisinin resistant. Moreover, daily genotyping did not alter the final outcome classification in any recurrent cases. Conclusions The parasites responsible for artemisinin-resistant malaria in a clinical trial in Western Cambodia comprise the dominant clones of acute malaria infections rather than minority clones emerging during treatment. Additional genotyping during therapy was not beneficial. Disproportionately high rates of polyclonal infections in cases of recurrence suggest complex infections lead to poor treatment outcomes. Current research objectives should be broadened to include identification and follow-up of recurrent polyclonal infections so as to define their role as potential agents of emerging resistance. PMID:24206588

  3. Polymorphisms in Plasmodium vivax Circumsporozoite Protein (CSP) Influence Parasite Burden and Cytokine Balance in a Pre-Amazon Endemic Area from Brazil

    PubMed Central

    Ribeiro, Bruno de Paulo; Cassiano, Gustavo Capatti; de Souza, Rodrigo Medeiros; Cysne, Dalila Nunes; Grisotto, Marcos Augusto Grigolin; de Azevedo dos Santos, Ana Paula Silva; Marinho, Cláudio Romero Farias; Machado, Ricardo Luiz Dantas; Nascimento, Flávia Raquel Fernandes

    2016-01-01

    Mechanisms involved in severe P. vivax malaria remain unclear. Parasite polymorphisms, parasite load and host cytokine profile may influence the course of infection. In this study, we investigated the influence of circumsporozoite protein (CSP) polymorphisms on parasite load and cytokine profile in patients with vivax malaria. A cross-sectional study was carried out in three cities: São Luís, Cedral and Buriticupu, Maranhão state, Brazil, areas of high prevalence of P. vivax. Interleukin (IL)-2, IL-4, IL-10, IL-6, IL-17, tumor necrosis factor alpha (TNF-α, interferon gamma (IFN-γ and transforming growth factor beta (TGF-β were quantified in blood plasma of patients and in supernatants from peripheral blood mononuclear cell (PBMC) cultures. Furthermore, the levels of cytokines and parasite load were correlated with VK210, VK247 and P. vivax-like CSP variants. Patients infected with P. vivax showed increased IL-10 and IL-6 levels, which correlated with the parasite load, however, in multiple comparisons, only IL-10 kept this association. A regulatory cytokine profile prevailed in plasma, while an inflammatory profile prevailed in PBMC culture supernatants and these patterns were related to CSP polymorphisms. VK247 infected patients showed higher parasitaemia and IL-6 concentrations, which were not associated to IL-10 anti-inflammatory effect. By contrast, in VK210 patients, these two cytokines showed a strong positive correlation and the parasite load was lower. Patients with the VK210 variant showed a regulatory cytokine profile in plasma, while those infected with the VK247 variant have a predominantly inflammatory cytokine profile and higher parasite loads, which altogether may result in more complications in infection. In conclusion, we propose that CSP polymorphisms is associated to the increase of non-regulated inflammatory immune responses, which in turn may be associated with the outcome of infection. PMID:26943639

  4. Polymorphisms in Plasmodium vivax Circumsporozoite Protein (CSP) Influence Parasite Burden and Cytokine Balance in a Pre-Amazon Endemic Area from Brazil.

    PubMed

    Ribeiro, Bruno de Paulo; Cassiano, Gustavo Capatti; de Souza, Rodrigo Medeiros; Cysne, Dalila Nunes; Grisotto, Marcos Augusto Grigolin; de Azevedo Dos Santos, Ana Paula Silva; Marinho, Cláudio Romero Farias; Machado, Ricardo Luiz Dantas; Nascimento, Flávia Raquel Fernandes

    2016-03-01

    Mechanisms involved in severe P. vivax malaria remain unclear. Parasite polymorphisms, parasite load and host cytokine profile may influence the course of infection. In this study, we investigated the influence of circumsporozoite protein (CSP) polymorphisms on parasite load and cytokine profile in patients with vivax malaria. A cross-sectional study was carried out in three cities: São Luís, Cedral and Buriticupu, Maranhão state, Brazil, areas of high prevalence of P. vivax. Interleukin (IL)-2, IL-4, IL-10, IL-6, IL-17, tumor necrosis factor alpha (TNF-α, interferon gamma (IFN-γ and transforming growth factor beta (TGF-β were quantified in blood plasma of patients and in supernatants from peripheral blood mononuclear cell (PBMC) cultures. Furthermore, the levels of cytokines and parasite load were correlated with VK210, VK247 and P. vivax-like CSP variants. Patients infected with P. vivax showed increased IL-10 and IL-6 levels, which correlated with the parasite load, however, in multiple comparisons, only IL-10 kept this association. A regulatory cytokine profile prevailed in plasma, while an inflammatory profile prevailed in PBMC culture supernatants and these patterns were related to CSP polymorphisms. VK247 infected patients showed higher parasitaemia and IL-6 concentrations, which were not associated to IL-10 anti-inflammatory effect. By contrast, in VK210 patients, these two cytokines showed a strong positive correlation and the parasite load was lower. Patients with the VK210 variant showed a regulatory cytokine profile in plasma, while those infected with the VK247 variant have a predominantly inflammatory cytokine profile and higher parasite loads, which altogether may result in more complications in infection. In conclusion, we propose that CSP polymorphisms is associated to the increase of non-regulated inflammatory immune responses, which in turn may be associated with the outcome of infection. PMID:26943639

  5. Treatment of Plasmodium chabaudi Parasites with Curcumin in Combination with Antimalarial Drugs: Drug Interactions and Implications on the Ubiquitin/Proteasome System

    PubMed Central

    Neto, Zoraima; Machado, Marta; Lindeza, Ana; do Rosrio, Virglio; Gazarini, Marcos L.; Lopes, Dinora

    2013-01-01

    Antimalarial drug resistance remains a major obstacle in malaria control. Evidence from Southeast Asia shows that resistance to artemisinin combination therapy (ACT) is inevitable. Ethnopharmacological studies have confirmed the efficacy of curcumin against Plasmodium spp. Drug interaction assays between curcumin/piperine/chloroquine and curcumin/piperine/artemisinin combinations and the potential of drug treatment to interfere with the ubiquitin proteasome system (UPS) were analyzed. In vivo efficacy of curcumin was studied in BALB/c mice infected with Plasmodium chabaudi clones resistant to chloroquine and artemisinin, and drug interactions were analyzed by isobolograms. Subtherapeutic doses of curcumin, chloroquine, and artemisinin were administered to mice, and mRNA was collected following treatment for RT-PCR analysis of genes encoding deubiquitylating enzymes (DUBs). Curcumin was found be nontoxic in BALB/c mice. The combination of curcumin/chloroquine/piperine reduced parasitemia to 37% seven days after treatment versus the control group's 65%, and an additive interaction was revealed. Curcumin/piperine/artemisinin combination did not show a favorable drug interaction in this murine model of malaria. Treatment of mice with subtherapeutic doses of the drugs resulted in a transient increase in genes encoding DUBs indicating UPS interference. If curcumin is to join the arsenal of available antimalarial drugs, future studies exploring suitable drug partners would be of interest. PMID:23691276

  6. Treatment of Plasmodium chabaudi Parasites with Curcumin in Combination with Antimalarial Drugs: Drug Interactions and Implications on the Ubiquitin/Proteasome System.

    PubMed

    Neto, Zoraima; Machado, Marta; Lindeza, Ana; do Rosrio, Virglio; Gazarini, Marcos L; Lopes, Dinora

    2013-01-01

    Antimalarial drug resistance remains a major obstacle in malaria control. Evidence from Southeast Asia shows that resistance to artemisinin combination therapy (ACT) is inevitable. Ethnopharmacological studies have confirmed the efficacy of curcumin against Plasmodium spp. Drug interaction assays between curcumin/piperine/chloroquine and curcumin/piperine/artemisinin combinations and the potential of drug treatment to interfere with the ubiquitin proteasome system (UPS) were analyzed. In vivo efficacy of curcumin was studied in BALB/c mice infected with Plasmodium chabaudi clones resistant to chloroquine and artemisinin, and drug interactions were analyzed by isobolograms. Subtherapeutic doses of curcumin, chloroquine, and artemisinin were administered to mice, and mRNA was collected following treatment for RT-PCR analysis of genes encoding deubiquitylating enzymes (DUBs). Curcumin was found be nontoxic in BALB/c mice. The combination of curcumin/chloroquine/piperine reduced parasitemia to 37% seven days after treatment versus the control group's 65%, and an additive interaction was revealed. Curcumin/piperine/artemisinin combination did not show a favorable drug interaction in this murine model of malaria. Treatment of mice with subtherapeutic doses of the drugs resulted in a transient increase in genes encoding DUBs indicating UPS interference. If curcumin is to join the arsenal of available antimalarial drugs, future studies exploring suitable drug partners would be of interest. PMID:23691276

  7. Sugar activation and glycosylation in Plasmodium.

    PubMed

    Cova, Marta; Rodrigues, Joo A; Smith, Terry K; Izquierdo, Luis

    2015-01-01

    Glycoconjugates are important mediators of host-pathogen interactions and are usually very abundant in the surface of many protozoan parasites. However, in the particular case of Plasmodium species, previous works show that glycosylphosphatidylinositol anchor modifications, and to an unknown extent, a severely truncated N-glycosylation are the only glycosylation processes taking place in the parasite. Nevertheless, a detailed analysis of the parasite genome and the recent identification of the sugar nucleotide precursors biosynthesized by Plasmodium falciparum support a picture in which several overlooked, albeit not very prominent glycosylations may be occurring during the parasite life cycle. In this work, the authors review recent developments in the characterization of the biosynthesis of glycosylation precursors in the parasite, focusing on the outline of the possible fates of these precursors. PMID:26520586

  8. Plasmodium vivax malaria vaccines

    PubMed Central

    Reyes-Sandoval, Arturo; Bachmann, Martin F

    2013-01-01

    Malaria is one of the few diseases in which morbidity is still measured in hundreds of millions of cases every year. Plasmodium vivax and Plasmodium falciparum are responsible for nearly all the malaria cases in the world and despite difficulties in obtaining an exact number, estimates indicate an astonishing 349552 million clinical cases of malaria due to P. falciparum in 2007 and between 132391 million clinical episodes due to P. vivax in 2009. It is becoming evident that eradication of malaria will be an arduous task and P. vivax will be one of the most difficult species to eliminate and perhaps become the last standing malaria parasite. Indeed, in countries that succeed in decreasing the disease burden, nearly all the remaining malaria cases are caused by P. vivax. Such resilience is mainly due to the sophisticated mechanism that the parasite has evolved to remain dormant for months or years forming hypnozoites, a small structure in the liver that will be a major hurdle in the efforts toward malaria eradication. Furthermore, while clinical trials of vaccines against P. falciparum are making fast progress, a very different picture is seen with P. vivax, where only few candidates are currently active in clinical trials. PMID:23978931

  9. Evidence of tRNA cleavage in apicomplexan parasites: half-tRNAs as new potential regulatory molecules of Toxoplasma gondii and Plasmodium berghei

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Several lines of evidence demonstrated that organisms ranging from bacteria to higher animals possess a regulated endonucleolytic cleavage pathway producing half-tRNA fragments. In the present study, we investigated the occurrence of this phenomenon in two distantly related apicomplexan parasites, T...

  10. Residual Plasmodium falciparum Parasitemia in Kenyan Children After Artemisinin-Combination Therapy Is Associated With Increased Transmission to Mosquitoes and Parasite Recurrence

    PubMed Central

    Beshir, Khalid B.; Sutherland, Colin J.; Sawa, Patrick; Drakeley, Chris J.; Okell, Lucy; Mweresa, Collins K.; Omar, Sabah A.; Shekalaghe, Seif A.; Kaur, Harparkash; Ndaro, Arnold; Chilongola, Jaffu; Schallig, Henk D. F. H.; Sauerwein, Robert W.; Hallett, Rachel L.; Bousema, Teun

    2013-01-01

    Background.?Parasite clearance time after artemisinin-based combination therapy (ACT) may be increasing in Asian and African settings. The association between parasite clearance following ACT and transmissibility is currently unknown. Methods.?We determined parasite clearance dynamics by duplex quantitative polymerase chain reaction (qPCR) in samples collected in the first 3 days after treatment of uncomplicated malaria with ACT. Gametocyte carriage was determined by Pfs25 quantitative nucleic acid sequencebased amplification assays; infectiousness to mosquitoes by membrane-feeding assays on day 7 after treatment. Results.?Residual parasitemia was detected by qPCR in 31.8% (95% confidence interval [CI], 24.639.8) of the children on day 3 after initiation of treatment. Residual parasitemia was associated with a 2-fold longer duration of gametocyte carriage (P = .0007), a higher likelihood of infecting mosquitoes (relative risk, 1.95; 95% CI, 1.173.24; P = .015), and a higher parasite burden in mosquitoes (incidence rate ratio, 2.92; 95% CI, 1.615.31; P < .001). Children with residual parasitemia were also significantly more likely to experience microscopically detectable parasitemia during follow-up (relative risk, 11.25; 95% CI, 4.0831.01; P < .001). Conclusions.?Residual submicroscopic parasitemia is common after ACT and is associated with a higher transmission potential. Residual parasitemia may also have consequences for individual patients because of its higher risk of recurrent parasitemia. PMID:23945376

  11. Effect of anti-mosquito hemolymph antibodies on fecundity and on the infectivity of malarial parasite Plasmodium vivax to Anopheles stephensi (Diptera:Insecta).

    PubMed

    Gulia, Monika; Suneja, Amita; Gakhar, Surendra K

    2002-06-01

    Rabbit antibodies to hemolymph antigens (102.5, 101, 100, 96, 88, 80, 64, 55, 43, 29, and 23 kDa) of Anopheles stephensi reduced fecundity as well as viability in An. stephensi. However, ingestion of these antibodies was not associated with a marked effect on the engorgement of mosquitoes but egg laying was significantly delayed. Antisera raised against hemolymph proteins were also used to identify cross reactive antigens/epitopes present in other tissues by Western blotting, as well as by in vivo ELISA. In addition, a significant reduction in oocyst development was also observed in An. stephensi mosquitoes that ingested anti-hemolymph antibodies along with Plasmodium vivax. The results confirmed the feasibility of targeting mosquito antigens as a novel anti-mosquito strategy, as well as confirmed the usefulness of such antigens for the development of a transmission-blocking vaccine. PMID:12195047

  12. Chloroquine efficacy in Plasmodium berghei NK65-infected ICR mice, with reference to the influence of initial parasite load and starting day of drug administration on the outcome of treatment.

    PubMed

    Ishih, Akira; Suzuki, Tohru; Muregi, Francis W; Matsui, Kae; Terada, Mamoru

    2006-01-01

    We examined whether the initial number of parasites inoculated and the starting day of medication post-infection influenced the antimalarial efficacy of chloroquine (CQ) against Plasmodium berghei NK65 infection in ICR mice. Male ICR mice were inoculated intraperitoneally with 1 x 10(5), 1x10(6), 1 x 10(7), 1 x 10(8) P. berghei NK65-parasitized erythrocytes (pRBC). In the treated group, all mice received an oral dose of 20 mg/kg of CQ base for 4 days starting on day 0 after infection. From day 3, Giemsa-stained thin blood smears from tail vein blood were used to assess parasitemia. Mice in the untreated control in each group showed a progressive increase in parasitemia leading to death. Treatment of mice, inoculated with 1 x 10(5), 1 x 10(6) and 1 x 10(7) pRBC, with CQ showed a marked effect. All the mice survived during the experiment. During the observation period, malaria parasites could not be detected on microscopic examination. Conversely, mice inoculated with 1 x 10(8) pRBC showed little response to CQ treatment, and all mice showed a progressive increase in parasitemia and ultimately died. In another experiment, mice infected with 1 x 10(3) and 1x 10(5) pRBC were treated with an oral four-day dosage of 20 mg/kg of CQ base from days 2, 3 or 4 post-infection. Treatment of mice, inoculated with 1 x 10(3) pRBC, with CQ from days 2 and 3 showed a marked effect. All mice survived during the experiment. However, treatment from day 4 showed a limited derease in parasitemia and all the mice ultimately died. On the other hand, treatment from day 2 showed a marked effect against 1 x 10(5) P. berghei NK65-infected mice, but treatment from days 3 or 4 was only slightly effective and all the mice died with an increasing parasitemia. The present results indicate that in in vivo antimalarial drug-assay systems, several factors, sush as initial parasite load and starting time of treatment may influence the drug response in the host. PMID:16771206

  13. Polymorphisms in Plasmodium falciparum chloroquine resistance transporter and multidrug resistance 1 genes: parasite risk factors that affect treatment outcomes for P. falciparum malaria after artemether-lumefantrine and artesunate-amodiaquine.

    PubMed

    Venkatesan, Meera; Gadalla, Nahla B; Stepniewska, Kasia; Dahal, Prabin; Nsanzabana, Christian; Moriera, Clarissa; Price, Ric N; Mårtensson, Andreas; Rosenthal, Philip J; Dorsey, Grant; Sutherland, Colin J; Guérin, Philippe; Davis, Timothy M E; Ménard, Didier; Adam, Ishag; Ademowo, George; Arze, Cesar; Baliraine, Frederick N; Berens-Riha, Nicole; Björkman, Anders; Borrmann, Steffen; Checchi, Francesco; Desai, Meghna; Dhorda, Mehul; Djimdé, Abdoulaye A; El-Sayed, Badria B; Eshetu, Teferi; Eyase, Frederick; Falade, Catherine; Faucher, Jean-François; Fröberg, Gabrielle; Grivoyannis, Anastasia; Hamour, Sally; Houzé, Sandrine; Johnson, Jacob; Kamugisha, Erasmus; Kariuki, Simon; Kiechel, Jean-René; Kironde, Fred; Kofoed, Poul-Erik; LeBras, Jacques; Malmberg, Maja; Mwai, Leah; Ngasala, Billy; Nosten, Francois; Nsobya, Samuel L; Nzila, Alexis; Oguike, Mary; Otienoburu, Sabina Dahlström; Ogutu, Bernhards; Ouédraogo, Jean-Bosco; Piola, Patrice; Rombo, Lars; Schramm, Birgit; Somé, A Fabrice; Thwing, Julie; Ursing, Johan; Wong, Rina P M; Zeynudin, Ahmed; Zongo, Issaka; Plowe, Christopher V; Sibley, Carol Hopkins

    2014-10-01

    Adequate clinical and parasitologic cure by artemisinin combination therapies relies on the artemisinin component and the partner drug. Polymorphisms in the Plasmodium falciparum chloroquine resistance transporter (pfcrt) and P. falciparum multidrug resistance 1 (pfmdr1) genes are associated with decreased sensitivity to amodiaquine and lumefantrine, but effects of these polymorphisms on therapeutic responses to artesunate-amodiaquine (ASAQ) and artemether-lumefantrine (AL) have not been clearly defined. Individual patient data from 31 clinical trials were harmonized and pooled by using standardized methods from the WorldWide Antimalarial Resistance Network. Data for more than 7,000 patients were analyzed to assess relationships between parasite polymorphisms in pfcrt and pfmdr1 and clinically relevant outcomes after treatment with AL or ASAQ. Presence of the pfmdr1 gene N86 (adjusted hazards ratio = 4.74, 95% confidence interval = 2.29 - 9.78, P < 0.001) and increased pfmdr1 copy number (adjusted hazards ratio = 6.52, 95% confidence interval = 2.36-17.97, P < 0.001 : were significant independent risk factors for recrudescence in patients treated with AL. AL and ASAQ exerted opposing selective effects on single-nucleotide polymorphisms in pfcrt and pfmdr1. Monitoring selection and responding to emerging signs of drug resistance are critical tools for preserving efficacy of artemisinin combination therapies; determination of the prevalence of at least pfcrt K76T and pfmdr1 N86Y should now be routine. PMID:25048375

  14. Polymorphisms in Plasmodium falciparum Chloroquine Resistance Transporter and Multidrug Resistance 1 Genes: Parasite Risk Factors that Affect Treatment Outcomes for P. falciparum Malaria after Artemether-Lumefantrine and Artesunate-Amodiaquine

    PubMed Central

    Venkatesan, Meera; Gadalla, Nahla B.; Stepniewska, Kasia; Dahal, Prabin; Nsanzabana, Christian; Moriera, Clarissa; Price, Ric N.; Mrtensson, Andreas; Rosenthal, Philip J.; Dorsey, Grant; Sutherland, Colin J.; Gurin, Philippe; Davis, Timothy M. E.; Mnard, Didier; Adam, Ishag; Ademowo, George; Arze, Cesar; Baliraine, Frederick N.; Berens-Riha, Nicole; Bjrkman, Anders; Borrmann, Steffen; Checchi, Francesco; Desai, Meghna; Dhorda, Mehul; Djimd, Abdoulaye A.; El-Sayed, Badria B.; Eshetu, Teferi; Eyase, Frederick; Falade, Catherine; Faucher, Jean-Franois; Frberg, Gabrielle; Grivoyannis, Anastasia; Hamour, Sally; Houz, Sandrine; Johnson, Jacob; Kamugisha, Erasmus; Kariuki, Simon; Kiechel, Jean-Ren; Kironde, Fred; Kofoed, Poul-Erik; LeBras, Jacques; Malmberg, Maja; Mwai, Leah; Ngasala, Billy; Nosten, Francois; Nsobya, Samuel L.; Nzila, Alexis; Oguike, Mary; Otienoburu, Sabina Dahlstrm; Ogutu, Bernhards; Oudraogo, Jean-Bosco; Piola, Patrice; Rombo, Lars; Schramm, Birgit; Som, A. Fabrice; Thwing, Julie; Ursing, Johan; Wong, Rina P. M.; Zeynudin, Ahmed; Zongo, Issaka; Plowe, Christopher V.; Sibley, Carol Hopkins

    2014-01-01

    Adequate clinical and parasitologic cure by artemisinin combination therapies relies on the artemisinin component and the partner drug. Polymorphisms in the Plasmodium falciparum chloroquine resistance transporter (pfcrt) and P. falciparum multidrug resistance 1 (pfmdr1) genes are associated with decreased sensitivity to amodiaquine and lumefantrine, but effects of these polymorphisms on therapeutic responses to artesunate-amodiaquine (ASAQ) and artemether-lumefantrine (AL) have not been clearly defined. Individual patient data from 31 clinical trials were harmonized and pooled by using standardized methods from the WorldWide Antimalarial Resistance Network. Data for more than 7,000 patients were analyzed to assess relationships between parasite polymorphisms in pfcrt and pfmdr1 and clinically relevant outcomes after treatment with AL or ASAQ. Presence of the pfmdr1 gene N86 (adjusted hazards ratio = 4.74, 95% confidence interval = 2.29 9.78, P < 0.001) and increased pfmdr1 copy number (adjusted hazards ratio = 6.52, 95% confidence interval = 2.3617.97, P < 0.001) were significant independent risk factors for recrudescence in patients treated with AL. AL and ASAQ exerted opposing selective effects on single-nucleotide polymorphisms in pfcrt and pfmdr1. Monitoring selection and responding to emerging signs of drug resistance are critical tools for preserving efficacy of artemisinin combination therapies; determination of the prevalence of at least pfcrt K76T and pfmdr1 N86Y should now be routine. PMID:25048375

  15. Identifying merozoite surface protein 4 and merozoite surface protein 7 Plasmodium falciparum protein family members specifically binding to human erythrocytes suggests a new malarial parasite-redundant survival mechanism.

    PubMed

    Garcia, Yesid; Puentes, Alvaro; Curtidor, Hernando; Cifuentes, Gladys; Reyes, Claudia; Barreto, Jose; Moreno, Armando; Patarroyo, Manuel E

    2007-11-15

    Plasmodium falciparum merozoite surface proteins (MSP-1 to -11) have been involved in merozoite interaction with the red blood cell (RBC) surface. Peptides covering complete MSP-4 and MSP-7 amino acid sequences were synthesized and tested in RBC binding assays. One MSP-4 high activity binding peptide (HABP) and five MSP-7 HABPs were found having specific binding to RBC surface. MSP-4 and MSP-7 HABP binding was sensitive to enzymatic treatment; they recognized a 52 kDa erythrocyte membrane protein. MSP-4 HABP had low invasion inhibition, suggesting it might bind to RBCs and also be involved in physiological mechanisms, while MSP-7 HABPs displayed different invasion inhibition activity (83-24%) in in vitro tests, suggesting different roles for both proteins during invasion. Structural characteristics found when comparing the MSP-4 HABP with MSP-HABPs displaying epidermal growth factor-like sequences suggested that these redundant MSP-family proteins could be a new parasite strategy for evading host genetic variability and immune pressure. PMID:17944453

  16. Mathematical model of susceptibility, resistance, and resilience in the within-host dynamics between a Plasmodium parasite and the immune system.

    PubMed

    Yan, Yi; Adam, Brian; Galinski, Mary; C Kissinger, Jessica; Moreno, Alberto; Gutierrez, Juan B

    2015-12-01

    We developed a coupled age-structured partial differential equation model to capture the disease dynamics during blood-stage malaria. The addition of age structure for the parasite population, with respect to previous models, allows us to better characterize the interaction between the malaria parasite and red blood cells during infection. Here we prove that the system we propose is well-posed and there exist at least two global states. We further demonstrate that the numerical simulation of the system coincides with clinically observed outcomes of primary and secondary malaria infection. The well-posedness of this system guarantees that the behavior of the model remains smooth, bounded, and continuously dependent on initial conditions; calibration with clinical data will constrain domains of parameters and variables to physiological ranges. PMID:26505135

  17. Effect of anti-mosquito midgut antibodies on development of malaria parasite, Plasmodium vivax and fecundity in vector mosquito Anopheles culicifacies (Diptera: culicidae).

    PubMed

    Chugh, Manoj; Adak, T; Sehrawat, Neelam; Gakhar, S K

    2011-04-01

    The effect of anti-mosquito-midgut antibodies on the development of the malaria parasite, P. vivax was studied by feeding the vector mosquito, An. culicifacies with infected blood supplemented with serum from immunized rabbits. In order to get antisera, rabbits were immunized with midgut proteins of three siblings species of Anopheles culicifacies, reported to exhibit differential vectorial capacity. The mosquitoes that ingested anti-midgut antibodies along with infectious parasites had significantly fewer oocysts compared to the control group of mosquitoes. The immunized rabbits generated high titer of antibodies. Their cross reactivity amongst various tissues of the same species and with other sibling species was also determined. Immunogenic polypeptides expressed in the midgut of glucose or blood fed An. culicifacies sibling species were identified by Western blotting. One immunogenic polypeptide of 62 kDa was exclusively present in the midgut of species A. Similarly, three polypeptides of 97, 94 and 58 kDa and one polypeptide of 23 kDa were present exclusively in species B and C respectively. Immunoelectron microscopy revealed the localization of these antigens on baso-lateral membrane and microvilli. The effects of anti-mosquito midgut antibodies on fecundity, longevity, mortality and engorgement of mosquitoes were studied. Fecundity was also reduced significantly. These observations open an avenue for research toward the development of a vector-based malaria parasite transmission-blocking vaccine. PMID:21614887

  18. A Transmission Model for the Ecology of an Avian Blood Parasite in a Temperate Ecosystem

    PubMed Central

    Murdock, Courtney C.; Foufopoulos, Johannes; Simon, Carl P.

    2013-01-01

    Most of our knowledge about avian haemosporidian parasites comes from the Hawaiian archipelago, where recently introduced Plasmodiumrelictum has contributed to the extinction of many endemic avian species. While the ecology of invasive malaria is reasonably understood, the ecology of endemic haemosporidian infection in mainland systems is poorly understood, even though it is the rule rather than the exception. We develop a mathematical model to explore and identify the ecological factors that most influence transmission of the common avian parasite, Leucocytozoonfringillinarum (Apicomplexa). The model was parameterized from White-crowned Sparrow (Zonotrichialeucophrys) and S. silvestre / craigi black fly populations breeding in an alpine ecosystem. We identify and examine the importance of altricial nestlings, the seasonal relapse of infected birds for parasite persistence across breeding seasons, and potential impacts of seasonal changes in black fly emergence on parasite prevalence in a high elevation temperate system. We also use the model to identify and estimate the parameters most influencing transmission dynamics. Our analysis found that relapse of adult birds and young of the year birds were crucial for parasite persistence across multiple seasons. However, distinguishing between nude nestlings and feathered young of the year was unnecessary. Finally, due to model sensitivity to many black fly parameters, parasite prevalence and sparrow recruitment may be most affected by seasonal changes in environmental temperature driving shifts in black fly emergence and gonotrophic cycles. PMID:24073288

  19. A transmission model for the ecology of an avian blood parasite in a temperate ecosystem.

    PubMed

    Murdock, Courtney C; Foufopoulos, Johannes; Simon, Carl P

    2013-01-01

    Most of our knowledge about avian haemosporidian parasites comes from the Hawaiian archipelago, where recently introduced Plasmodiumrelictum has contributed to the extinction of many endemic avian species. While the ecology of invasive malaria is reasonably understood, the ecology of endemic haemosporidian infection in mainland systems is poorly understood, even though it is the rule rather than the exception. We develop a mathematical model to explore and identify the ecological factors that most influence transmission of the common avian parasite, Leucocytozoonfringillinarum (Apicomplexa). The model was parameterized from White-crowned Sparrow (Zonotrichialeucophrys) and S. silvestre / craigi black fly populations breeding in an alpine ecosystem. We identify and examine the importance of altricial nestlings, the seasonal relapse of infected birds for parasite persistence across breeding seasons, and potential impacts of seasonal changes in black fly emergence on parasite prevalence in a high elevation temperate system. We also use the model to identify and estimate the parameters most influencing transmission dynamics. Our analysis found that relapse of adult birds and young of the year birds were crucial for parasite persistence across multiple seasons. However, distinguishing between nude nestlings and feathered young of the year was unnecessary. Finally, due to model sensitivity to many black fly parameters, parasite prevalence and sparrow recruitment may be most affected by seasonal changes in environmental temperature driving shifts in black fly emergence and gonotrophic cycles. PMID:24073288

  20. Anopheles Midgut FREP1 Mediates Plasmodium Invasion.

    PubMed

    Zhang, Genwei; Niu, Guodong; Franca, Caio M; Dong, Yuemei; Wang, Xiaohong; Butler, Noah S; Dimopoulos, George; Li, Jun

    2015-07-01

    Malaria transmission depends on sexual stage Plasmodium parasites successfully invading Anopheline mosquito midguts following a blood meal. However, the molecular mechanisms of Plasmodium invasion of mosquito midguts have not been fully elucidated. Previously, we showed that genetic polymorphisms in the fibrinogen-related protein 1 (FREP1) gene are significantly associated with Plasmodium falciparum infection in Anopheles gambiae, and FREP1 is important for Plasmodium berghei infection of mosquitoes. Here we identify that the FREP1 protein is secreted from the mosquito midgut epithelium and integrated as tetramers into the peritrophic matrix, a chitinous matrix formed inside the midgut lumen after a blood meal feeding. Moreover, we show that the FREP1 can directly bind Plasmodia sexual stage gametocytes and ookinetes. Notably, ablating FREP1 expression or targeting FREP1 with antibodies significantly decreases P. falciparum infection in mosquito midguts. Our data support that the mosquito-expressed FREP1 mediates mosquito midgut invasion by multiple species of Plasmodium parasites via anchoring ookinetes to the peritrophic matrix and enabling parasites to penetrate the peritrophic matrix and the epithelium. Thus, targeting FREP1 can limit malaria transmission. PMID:25991725

  1. High Levels of Asymptomatic and Subpatent Plasmodium falciparum Parasite Carriage at Health Facilities in an Area of Heterogeneous Malaria Transmission Intensity in the Kenyan Highlands

    PubMed Central

    Stresman, Gillian H.; Stevenson, Jennifer C.; Ngwu, Nnenna; Marube, Elizabeth; Owaga, Chrispin; Drakeley, Chris; Bousema, Teun; Cox, Jonathan

    2014-01-01

    In endemic settings, health facility surveys provide a convenient approach to estimating malaria transmission intensity. Typically, testing for malaria at facilities is performed on symptomatic attendees, but asymptomatic infections comprise a considerable proportion of the parasite reservoir. We sampled individuals attending five health facilities in the western Kenyan highlands. Malaria prevalence by rapid diagnostic test (RDT) was 8.632.9% in the health facilities. Of all polymerase chain reaction-positive participants, 46.4% (95% confidence interval [95% CI] = 42.650.2%) of participants had infections that were RDT-negative and asymptomatic, and 55.9% of those infections consisted of multiple parasite clones as assessed by merozoite surface protein-2 genotyping. Subpatent infections were more common in individuals reporting the use of non-artemisininbased antimalarials in the 2 weeks preceding the survey (odds ratio = 2.49, 95% CI = 1.045.92) compared with individuals not reporting previous use of antimalarials. We observed a large and genetically complex pool of subpatent parasitemia in the Kenya highlands that must be considered in malaria interventions. PMID:25331807

  2. Analysis of gene mutations involved in chloroquine resistance in Plasmodium falciparum parasites isolated from patients in the southwest of Saudi Arabia

    PubMed Central

    Bin Dajem, Saad M.; Al-Qahtani, Ahmed

    2010-01-01

    BACKGROUND AND OBJECTIVES: Chloroquine has been the drug of choice for the treatment of malaria for many decades. We aimed to examine the molecular basis of chloroquine resistance among Plasmodium falciparum isolates from the southwestern region of Saudi Arabia by analyzing the K76T and N86Y mutations in the PfCRT and PfMDR1 genes, respectively. PATIENTS AND METHODS: P falciparum-infected blood spot samples (n=121) were collected on filter papers. DNA was extracted and fragments from the above genes were amplified using nested PCR. The amplicons were digested by ApoI enzyme and sequenced. RESULTS: Of the 121 samples, 95 and 112 samples could be amplified for PfCRT K76T and PfMDR1 N86Y mutations, respectively. All of the samples amplified for the PfCRT K76T mutation were undigestible by ApoI, suggesting the presence of the K76T mutation. For the PfMDR1 N86Y mutation, 65/109 samples (59.6%) were digestible when treated with ApoI in a pattern, suggestive of the presence of the investigated wild allele (N86). However, 44/109 samples (40.4%) were digestible by ApoI, suggesting the presence of the mutated allele (Y) at position 86. DNA sequencing confirmed these results. CONCLUSION: Surprisingly, all isolates exhibited the mutated allele at codon 76 (K76T) of PfCRT. However, the mutated mutant allele at codon 86 (N86Y) of PfMDR1 was found in 40.4% of the samples studied. To our knowledge, this is the first study that has investigated the existence of the mutation in the PfMDR1 gene in the country. This study will contribute to the development of new strategies for therapeutic intervention against malaria in Saudi Arabia. PMID:20427933

  3. Plasmodium falciparum Malaria, Southern Algeria, 2007

    PubMed Central

    Gassen, Ibrahim; Khechache, Yacine; Lamali, Karima; Tchicha, Boualem; Brengues, Ccile; Menegon, Michela; Severini, Carlo; Fontenille, Didier; Harrat, Zoubir

    2010-01-01

    An outbreak of Plasmodium falciparum malaria occurred in Tinzaouatine in southern Algeria in 2007. The likely vector, Anopheles gambiae mosquitoes, had not been detected in Algeria. Genes for resistance to chloroquine were detected in the parasite. The outbreak shows the potential for an increase in malaria vectors in Algeria. PMID:20113565

  4. A case of Plasmodium ovale malaria imported from West Africa.

    PubMed

    Kang, Yunjung; Yang, Jinyoung

    2013-04-01

    Malaria is a parasitic infection caused by Plasmodium species. Most of the imported malaria in Korea are due to Plasmodium vivax and Plasmodium falciparum, and Plasmodium ovale infections are very rare. Here, we report a case of a 24-year-old American woman who acquired P. ovale while staying in Ghana, West Africa for 5 months in 2010. The patient was diagnosed with P. ovale malaria based on a Wright-Giemsa stained peripheral blood smear, Plasmodium genus-specific real-time PCR, Plasmodium species-specific nested PCR, and sequencing targeting 18S rRNA gene. The strain identified had a very long incubation period of 19-24 months. Blood donors who have malaria with a very long incubation period could be a potential danger for propagating malaria. Therefore, we should identify imported P. ovale infections not only by morphological findings but also by molecular methods for preventing propagation and appropriate treatment. PMID:23710090

  5. Mixed-species Plasmodium falciparum and Plasmodium ovale malaria in a paediatric returned traveller

    PubMed Central

    2014-01-01

    Malaria is a common and potentially fatal cause of febrile illness in returned travellers. Endemic areas for different malaria parasites overlap, but mixed species infections are rare. An adolescent male returned from a trip to Ghana in late summer 2013. He subsequently presented with blood smears positive for two species of malaria parasite, Plasmodium falciparum and Plasmodium ovale, on two isolated hospital visits within a six-week period. The epidemiology of mixed infections, likely pathophysiology of his presentation, and the implications for malaria testing and treatment in returned travellers are discussed. PMID:24593188

  6. The evolution and diversity of a low complexity vaccine candidate, merozoite surface protein 9 (MSP-9), in Plasmodium vivax and closely related species.

    PubMed

    Chenet, Stella M; Pacheco, M Andrena; Bacon, David J; Collins, William E; Barnwell, John W; Escalante, Ananias A

    2013-12-01

    The merozoite surface protein-9 (MSP-9) has been considered a target for an anti-malarial vaccine since it is one of many proteins involved in the erythrocyte invasion, a critical step in the parasite life cycle. Orthologs encoding this antigen have been found in all known species of Plasmodium parasitic to primates. In order to characterize and investigate the extent and maintenance of MSP-9 genetic diversity, we analyzed DNA sequences of the following malaria parasite species: Plasmodium falciparum, Plasmodium reichenowi, Plasmodium chabaudi, Plasmodium yoelii, Plasmodium berghei, Plasmodium coatneyi, Plasmodium gonderi, Plasmodium knowlesi, Plasmodium inui, Plasmodium simiovale, Plasmodium fieldi, Plasmodium cynomolgi and Plasmodium vivax and evaluated the signature of natural selection in all MSP-9 orthologs. Our findings suggest that the gene encoding MSP-9 is under purifying selection in P. vivax and closely related species. We further explored how selection affected different regions of MSP-9 by comparing the polymorphisms in P. vivax and P. falciparum, and found contrasting patterns between these two species that suggest differences in functional constraints. This observation implies that the MSP-9 orthologs in human parasites may interact differently with the host immune response. Thus, studies carried out in one species cannot be directly translated into the other. PMID:24044894

  7. Physicochemical Aspects of the Plasmodium chabaudi-Infected Erythrocyte

    PubMed Central

    Hayakawa, Eri H.; Kobayashi, Seiki; Matsuoka, Hiroyuki

    2015-01-01

    Membrane electrochemical potential is a feature of the molecular profile of the cell membrane and the two-dimensional arrangement of its charge-bearing molecules. Plasmodium species, the causative agents of malaria, are intracellular parasites that remodel host erythrocytes by expressing their own proteins on erythrocyte membranes. Although various aspects of the modifications made to the host erythrocyte membrane have been extensively studied in some human Plasmodium species (such as Plasmodium falciparum), details of the structural and molecular biological modifications made to host erythrocytes by nonhuman Plasmodium parasites have not been studied. We employed zeta potential analysis of erythrocytes parasitized by P. chabaudi, a nonhuman Plasmodium parasite. From these measurements, we found that the surface potential shift was more negative for P. chabaudi-infected erythrocytes than for P. falciparum-infected erythrocytes. However, electron microscopic analysis of the surface of P. chabaudi-infected erythrocytes did not reveal any modifications as compared with nonparasitized erythrocytes. These results suggest that differences in the membrane modifications found herein represent unique attributes related to the pathogenesis profiles of the two different malaria parasite species in different host animals and that these features have been acquired through parasite adaptations acquired over long evolutionary time periods. PMID:26557685

  8. Natural microbe-mediated refractoriness to Plasmodium infection in Anopheles gambiae

    PubMed Central

    Cirimotich, Chris M.; Dong, Yuemei; Clayton, April M.; Sandiford, Simone L.; Souza-Neto, Jayme A.; Mulenga, Musapa; Dimopoulos, George

    2014-01-01

    Malaria parasite transmission depends on the successful transition of Plasmodium through discrete developmental stages in the lumen of the mosquito midgut. Like the human intestinal tract, the mosquito midgut contains a diverse microbial flora, which may compromise the ability of Plasmodium to establish infection. We have identified an Enterobacter bacterium isolated from wild mosquito populations in Zambia that renders the mosquito 99% resistant to infection with the human malaria parasite Plasmodium falciparum by interfering with parasite development prior to invasion of the midgut epithelium. Phenotypic analyses showed that the anti-Plasmodium mechanism requires small populations of replicating bacteria and is mediated through a mosquito-independent interaction with the malaria parasite. We show that this anti-Plasmodium effect is largely caused by bacterial generation of reactive oxygen species. PMID:21566196

  9. Optimal strategy for controlling the spread of Plasmodium Knowlesi malaria: Treatment and culling

    NASA Astrophysics Data System (ADS)

    Abdullahi, Mohammed Baba; Hasan, Yahya Abu; Abdullah, Farah Aini

    2015-05-01

    Plasmodium Knowlesi malaria is a parasitic mosquito-borne disease caused by a eukaryotic protist of genus Plasmodium Knowlesi transmitted by mosquito, Anopheles leucosphyrus to human and macaques. We developed and analyzed a deterministic Mathematical model for the transmission of Plasmodium Knowlesi malaria in human and macaques. The optimal control theory is applied to investigate optimal strategies for controlling the spread of Plasmodium Knowlesi malaria using treatment and culling as control strategies. The conditions for optimal control of the Plasmodium Knowlesi malaria are derived using Pontryagin's Maximum Principle. Finally, numerical simulations suggested that the combination of the control strategies is the best way to control the disease in any community.

  10. Plasmodium inui is not closely related to other quartan Plasmodium species.

    PubMed

    Kissinger, J C; Collins, W E; Li, J; McCutchan, T F

    1998-04-01

    Plasmodium inui (Halberstaedter and von Prowazek, 1907), a malarial parasite of Old World monkeys that occurs in isolated pockets throughout the Celebes, Indonesia, Malaysia, and the Philippines, has traditionally been considered to be related more closely to Plasmodium malariae of humans (and its primate counterpart Plasmodium brasilianum), than to other primate Plasmodium species. This inference was made in part because of the similarities in the periodicities or duration of the asexual cycle in the blood, the extended sporogonic cycle, and the longer period of time for development of the pre-erythrocytic stages in the liver. Both P. inui and P. malariae have quartan (72 hr) periodicities associated with their asexual cycle, whereas other primate malarias, such as Plasmodium fragile and Plasmodium cynomolgi, are associated with tertian periodicities (48 hr), and Plasmodiumn knowlesi, with a quotidian (24 hr) periodicity. Phylogenetic analyses of portions of orthologous small subunit ribosomal genes reveal that P. inui is actually more closely related to the Plasmodium species of the "vivax-type" lineage than to P. malariae. Ribosomal sequence analysis of many different, geographically isolated, antigenically distinct P. inui isolates reveals that the isolates are nearly identical in sequence and thus members of the same species. PMID:9576499

  11. Plasmodium falciparum genetic crosses in a humanized mouse model

    PubMed Central

    Vaughan, Ashley M.; Pinapati, Richard S.; Cheeseman, Ian H.; Camargo, Nelly; Fishbaugher, Matthew; Checkley, Lisa A.; Nair, Shalini; Hutyra, Carolyn A.; Nosten, François H.; Anderson, Timothy J. C.; Ferdig, Michael T.; Kappe, Stefan H. I.

    2015-01-01

    Genetic crosses of phenotypically distinct strains of the human malaria parasite Plasmodium falciparum are a powerful tool for identifying genes controlling drug resistance and other key phenotypes. Previous studies relied on the isolation of recombinant parasites from splenectomized chimpanzees, a research avenue that is no longer available. Here, we demonstrate that human-liver chimeric mice support recovery of recombinant progeny for the identification of genetic determinants of parasite traits and adaptations. PMID:26030447

  12. Plasmodium falciparum genetic crosses in a humanized mouse model.

    PubMed

    Vaughan, Ashley M; Pinapati, Richard S; Cheeseman, Ian H; Camargo, Nelly; Fishbaugher, Matthew; Checkley, Lisa A; Nair, Shalini; Hutyra, Carolyn A; Nosten, François H; Anderson, Timothy J C; Ferdig, Michael T; Kappe, Stefan H I

    2015-07-01

    Genetic crosses of phenotypically distinct strains of the human malaria parasite Plasmodium falciparum are a powerful tool for identifying genes controlling drug resistance and other key phenotypes. Previous studies relied on the isolation of recombinant parasites from splenectomized chimpanzees, a research avenue that is no longer available. Here we demonstrate that human-liver chimeric mice support recovery of recombinant progeny for the identification of genetic determinants of parasite traits and adaptations. PMID:26030447

  13. Cytoskeleton of Apicomplexan Parasites

    PubMed Central

    Morrissette, Naomi S.; Sibley, L. David

    2002-01-01

    The Apicomplexa are a phylum of diverse obligate intracellular parasites including Plasmodium spp., the cause of malaria; Toxoplasma gondii and Cryptosporidium parvum, opportunistic pathogens of immunocompromised individuals; and Eimeria spp. and Theileria spp., parasites of considerable agricultural importance. These protozoan parasites share distinctive morphological features, cytoskeletal organization, and modes of replication, motility, and invasion. This review summarizes our current understanding of the cytoskeletal elements, the properties of cytoskeletal proteins, and the role of the cytoskeleton in polarity, motility, invasion, and replication. We discuss the unusual properties of actin and myosin in the Apicomplexa, the highly stereotyped microtubule populations in apicomplexans, and a network of recently discovered novel intermediate filament-like elem