Parasite's Cycle Research Articles

Oriented nucleation of hemozoin at the digestive vacuole membrane in Plasmodium falciparum

Heme detoxification is a critical step in the life cycle of malaria-causing parasites, achieved by crystallization into physiologically insoluble hemozoin. The mode of nucleation has profound implications for understanding the mechanism of action of antimalarial drugs that inhibit hemozoin growth. Several lines of evidence point to involvement of acylglycerol lipids in the nucleation process. Hemozoin crystals have been reported to form within lipid nanospheres; alternatively, it has been found in vitro that they are nucleated at an acylglycerol lipid-water interface. We have applied cryogenic soft X-ray tomography and three-dimensional electron microscopy to address the location and orientation of hemozoin crystals within the digestive vacuole (DV), as a signature of their nucleation and growth processes. Cryogenic soft X-ray tomography in the "water window" is particularly advantageous because contrast generation is based inherently on atomic absorption. We find that hemozoin nucleation occurs at the DV inner membrane, with crystallization occurring in the aqueous rather than lipid phase. The crystal morphology indicates a common {100} orientation facing the membrane as expected of templated nucleation. This is consistent with conclusions reached by X-ray fluorescence and diffraction in a companion work. Uniform dark spheres observed in the parasite were identified as hemoglobin transport vesicles. Their analysis supports a model of hemozoin nucleation primarily in the DV. Modeling of the contrast at the DV membrane indicates a 4-nm thickness with patches about three times thicker, possibly implicated in the nucleation.

Spliced leader RNA silencing (SLS) - a programmed cell death pathway in Trypanosoma brucei that is induced upon ER stress

Trypanosoma brucei is the causative agent of African sleeping sickness. The parasite cycles between its insect (procyclic form) and mammalian hosts (bloodstream form). Trypanosomes lack conventional transcription regulation, and their genes are transcribed in polycistronic units that are processed by trans-splicing and polyadenylation. In trans-splicing, which is essential for processing of each mRNA, an exon, the spliced leader (SL) is added to all mRNAs from a small RNA, the SL RNA. Trypanosomes lack the machinery for the unfolded protein response (UPR), which in other eukaryotes is induced under endoplasmic reticulum (ER) stress. Trypanosomes respond to such stress by changing the stability of mRNAs, which are essential for coping with the stress. However, under severe ER stress that is induced by blocking translocation of proteins to the ER, treatment of cells with chemicals that induce misfolding in the ER, or extreme pH, trypanosomes elicit the spliced leader silencing (SLS) pathway. In SLS, the transcription of the SL RNA gene is extinguished, and tSNAP42, a specific SL RNA transcription factor, fails to bind to its cognate promoter. SLS leads to complete shut-off of trans-splicing. In this review, I discuss the UPR in mammals and compare it to the ER stress response in T. brucei leading to SLS. I summarize the evidence supporting the notion that SLS is a programmed cell death (PCD) pathway that is utilized by the parasites to substitute for the apoptosis observed in higher eukaryotes under prolonged ER stress. I present the hypothesis that SLS evolved to expedite the death process, and rapidly remove from the population unfit parasites that, by elimination via SLS, cause minimal damage to the parasite population.

The Arabidopsis thaliana-Alternaria brassicicola pathosystem: A model interaction for investigating seed transmission of necrotrophic fungi

BackgroundSeed transmission constitutes a major component of the parasitic cycle for several fungal pathogens. However, very little is known concerning fungal or plant genetic factors that impact seed transmission and mechanisms underlying this key biological trait have yet to be clarified. Such lack of available data could be probably explained by the absence of suitable model pathosystem to study plant-fungus interactions during the plant reproductive phase.ResultsHere we report on setting up a new pathosystem that could facilitate the study of fungal seed transmission. Reproductive organs of Arabidopsis thaliana were inoculated with Alternaria brassicicola conidia. Parameters (floral vs fruit route, seed collection date, plant and silique developmental stages) that could influence the seed transmission efficiency were tested to define optimal seed infection conditions. Microscopic observations revealed that the fungus penetrates siliques through cellular junctions, replum and stomata, and into seed coats either directly or through cracks. The ability of the osmosensitive fungal mutant nik1Δ3 to transmit to A. thaliana seeds was analyzed. A significant decrease in seed transmission rate was observed compared to the wild-type parental strain, confirming that a functional osmoregulation pathway is required for efficient seed transmission of the fungus. Similarly, to test the role of flavonoids in seed coat protection against pathogens, a transparent testa Arabidopsis mutant (tt4-1) not producing any flavonoid was used as host plant. Unexpectedly, tt4-1 seeds were infected to a significantly lower extent than wild-type seeds, possibly due to over-accumulation of other antimicrobial metabolites.ConclusionsThe Arabidopsis thaliana-Alternaria brassicicola pathosystem, that have been widely used to study plant-pathogen interactions during the vegetative phase, also proved to constitute a suitable model pathosystem for detailed analysis of plant-pathogen interactions during the reproductive phase. We demonstrated that it provides an excellent system for investigating the impact of different fungal or plant mutations on the seed transmission process and therefore paves the way towards future high-throughput screening of both Arabidopsis and fungal mutant.

Parasitism (Trematoda, Digenea) in medusae from the southwestern Atlantic Ocean: medusa hosts, parasite prevalences, and ecological implications

Digenean are important endoparasites of fish with complex life cycles; some genera include medusae as secondary hosts. Their transmission to fish occurs when fish prey on these jelly hosts. Fish predation on jellyfish is a widespread phenomenon, even though predation by fish on jellyfish has not been determined through parasitism yet. We hypothesized that medusae with high prevalences of digeneans could be important for their transmission to fish. A total of 48,900 specimens of 50 medusa species were analyzed; 2,181 harbored digeneans. Opechona sp. and Monascus filiformis were the most frequent and abundant parasites with the widest range of hosts. Hemiuridae gen. sp. and Bacciger sp. were found in few specimens of some medusa species. Prevalences were unevenly distributed in the region. Three groups with high prevalence values were identified mainly related to frontal areas: Rio de la Plata, Bahia Blanca, and North Patagonian tidal front. Eucheilota ventricularis, Clytia hemisphaerica, Proboscidactyla mutabilis, Liriope tetraphylla, and Aequoerea spp. were the medusae that contributed the most as secondary hosts to M. filiformis and Opechona sp. The high prevalences found in these medusae suggest that may be a fundamental part of the life cycles of both parasites in these areas.

Glycosomal localization of dihydroxyacetone phosphate acyltransferase LmDAT is important for lipophosphoglycan biosynthesis

Protozoan parasites of the genus Leishmania are the causative agents of several human and animal diseases collectively called leishmaniases. The parasite cycles between an insect vector the sand fly, and mammalian host's macrophages. Cell‐surface components protect the parasite from the host immune system and constitute virulence factors. Ether glycerolipids are a significant part of the parasite's membranes and ether glycerolipid based macromolecules are also important virulence factors. Thus, enzymes involved in synthesis of ether lipids in Leshmania may represent a drug target for control of the disease. In L. major, a glycosomal dihydroxyacetonephosphate acyltransferase, LmDAT, initiates the ether lipid biosynthetic pathway. We demonstrated that an active LmDAT is critical for normal growth and survival of the parasite during stationary phase. Deletion of the N‐terminal amino acid residues of the enzyme reveals that this domain may be important for proper folding of the enzyme and hence for its activity. Abrogation of its C‐terminal glycosomal targeting sequence leads to its extraglycosomal localization. However, this mislocalization does not affect its enzymatic activity but alters the synthesis of the ether‐lipid based virulence factor, lipophosphopglycan, demonstrating that glycosomal subcellular localization is critical for the production of ether‐lipids.This work was funded by American Heart Association.

Cryptostatin, a chagasin-family cysteine protease inhibitor ofCryptosporidium parvum

Cysteine proteases of pathogenic protozoan parasites play pivotal roles in the life cycle of parasites, but strict regulation of their activities is also essential for maintenance of parasite physiology and interaction with hosts. In this study, we identified and characterized cryptostatin, a novel inhibitor of cysteine protease (ICP) of Cryptosporidium parvum. Cryptostatin showed low sequence identity to other chagasin-family ICPs, but 3 motifs (NPTTG, GXGG, and RPW/F motifs), which are evolutionarily conserved in chagasin-family ICPs, were found in the sequence. The overall structure of cryptostatin consisted of 8 β-strands that progressed in parallel and closely resembled the immunoglobulin fold. Recombinant cryptostatin inhibited various cysteine proteases, including papain, human cathepsin B, human cathepsin L, and cryptopain-1, with K i's in the picomolar range. Cryptostatin was active over a wide pH range and was highly stable under physiological conditions. The protein was thermostable and retained its inhibitory activity even after incubation at 95°C. Cryptostatin formed tight complexes with cysteine proteases, so the complexes remained intact in the presence of sodium dodecyl sulfate and β-mercaptoethanol, but they were disassembled by boiling. An immunogold electron microscopy analysis demonstrated diffused localization of cryptostatin within oocystes and meronts, but not within trophozoites, which suggests a possible role for cryptostatin in host cell invasion by C. parvum.

The interaction of the novel 30C02 cyst nematode effector protein with a plant β-1,3-endoglucanase may suppress host defence to promote parasitism

Phytoparasitic nematodes secrete an array of effector proteins to modify selected recipient plant cells into elaborate and essential feeding sites. The biological function of the novel 30C02 effector protein of the soybean cyst nematode, Heterodera glycines, was studied using Arabidopsis thaliana as host and the beet cyst nematode, Heterodera schachtii, which contains a homologue of the 30C02 gene. Expression of Hg30C02 in Arabidopsis did not affect plant growth and development but increased plant susceptibility to infection by H. schachtii. The 30C02 protein interacted with a specific (AT4G16260) host plant β-1,3-endoglucanase in both yeast and plant cells, possibly to interfere with its role as a plant pathogenesis-related protein. Interestingly, the peak expression of 30C02 in the nematode and peak expression of At4g16260 in plant roots coincided at around 3–5 d after root infection by the nematode, after which the relative expression of At4g16260 declined significantly. An Arabidopsis At4g16260 T-DNA mutant showed increased susceptibility to cyst nematode infection, and plants that overexpressed At4g16260 were reduced in nematode susceptibility, suggesting a potential role of host β-1,3-endoglucanase in the defence response against H. schachtii infection. Arabidopsis plants that expressed dsRNA and its processed small interfering RNA complementary to the Hg30C02 sequence were not phenotypically different from non-transformed plants, but they exhibited a strong RNA interference-mediated resistance to infection by H. schachtii. The collective results suggest that, as with other pathogens, active suppression of host defence is a critical component for successful parasitism by nematodes and a vulnerable target to disrupt the parasitic cycle.

Avian malaria infections in western European mosquitoes

In the complex life cycle of avian malaria parasites (Plasmodium sp.), we still have a poor understanding on the vector-parasite relationships. This study described the community of potential avian malaria vectors in four Portuguese reedbeds. We tested if their geographical distribution differed, and investigated on their Plasmodium infections. The mosquitoes' feeding preferences were evaluated using CO(2), mice, and birds as baits. The most abundant species were Culex pipiens, Culex theileri, and Ochlerotatus caspius (and, in one site, Coquillettidia richiardii). Plasmodium lineages SGS1 and SYAT05 were found in unengorged Cx. pipiens and Cx. theileri, respectively, suggesting that these mosquitoes were competent vectors of those lineages. The species' abundance was significantly different among sites, which may help to explain the observed differences in the prevalence of SGS1. At the study sites, SGS1 was detected in the most abundant mosquito species and reached a high prevalence in the most abundant passerine species. Probably, this parasite needs abundant hosts in all phases of its cycle to keep a good reservoir of infection in all its stages. Cq. richiardii showed an opportunistic feeding behavior, while Cx. pipiens appeared to be more mammophilic than previously described, perhaps because the used avian bait was not its preferential target. In one of the study sites, mosquitoes seem to be attracted to the Spotless Starling Sturnus unicolor, an abundant bird species that may be an important local reservoir of avian malaria infections. To our knowledge, this is the first report of detection of avian Plasmodium DNA from European mosquitoes.

Biological activities of nitidine, a potential anti-malarial lead compound

BackgroundNitidine is thought to be the main active ingredient in several traditional anti-malarial remedies used in different parts of the world. The widespread use of these therapies stresses the importance of studying this molecule in the context of malaria control. However, little is known about its potential as an anti-plasmodial drug, as well as its mechanism of action.MethodsIn this study, the anti-malarial potential of nitidine was evaluated in vitro on CQ-sensitive and -resistant strains. The nitidine's selectivity index compared with cancerous and non-cancerous cell lines was then determined. In vivo assays were then performed, using the four-day Peter's test methodology. To gain information about nitidine's possible mode of action, its moment of action on the parasite cell cycle was studied, and its localization inside the parasite was determined using confocal microscopy. The in vitro abilities of nitidine to bind haem and to inhibit β-haematin formation were also demonstrated.ResultsNitidine showed similar in vitro activity in CQ-sensitive and resistant strains, and also a satisfying selectivity index (> 10) when compared with a non-cancerous cells line. Its in vivo activity was moderate; however, no sign of acute toxicity was observed during treatment. Nitidine's moment of action on the parasite cycle showed that it could not interfere with DNA replication; this was consistent with the observation that nitidine did not localize in the nucleus, but rather in the cytoplasm of the parasite. Nitidine was able to form a 1-1 complex with haem in vitro and also inhibited β-haematin formation with the same potency as chloroquine.ConclusionNitidine can be considered a potential anti-malarial lead compound. Its ability to complex haem and inhibit β-haematin formation suggests a mechanism of action similar to that of chloroquine. The anti-malarial activity of nitidine could therefore be improved by structural modification of this molecule to increase its penetration of the digestive vacuole in the parasite, where haemoglobin metabolization takes place.

New findings on Simalikalactone D, an antimalarial compound from Quassia amara L. (Simaroubaceae)

Quassia amara L. (Simaroubaceae) is a species widely used as tonic and is claimed to be an efficient antimalarial all over the Northern part of the Amazon basin. Quassinoid compound Simalikalactone D (SkD) has been shown to be one of the molecules responsible for the antiplasmodial activity of a watery preparation made out of juvenile fresh leaves of this plant. Because of its strong antimalarial activity, we decided to have a further insight of SkD pharmacological properties, alone or in association with classical antimalarials. At concentrations of up to 200μM, we showed herein that SkD did not exert any apoptotic or necrotic activities in vitro on lymphoblastic cells. However, an antiproliferative effect was evident at concentrations higher than 45nM. SkD was inefficient at inhibiting heme biomineralization and the new permeability pathways induced by the parasite in the host erythrocyte membrane. With respect to Plasmodium falciparum erythrocytic stages, SkD was almost inactive on earlier and later parasite stages, but potently active at the 30th h of parasite cycle when DNA replicates in mature trophozoites. In vitro combination studies with conventional antimalarial drugs showed that SkD synergizes with atovaquone (ATO). The activity of ATO on the Plasmodium mitochondrial membrane potential was enhanced by SkD, which on its own had a poor effect on this cellular parameter.

A DOC2 Protein Identified by Mutational Profiling Is Essential for Apicomplexan Parasite Exocytosis

Exocytosis is essential to the lytic cycle of apicomplexan parasites and required for the pathogenesis of toxoplasmosis and malaria. DOC2 proteins recruit the membrane fusion machinery required for exocytosis in a Ca(2+)-dependent fashion. Here, the phenotype of a Toxoplasma gondii conditional mutant impaired in host cell invasion and egress was pinpointed to a defect in secretion of the micronemes, an apicomplexan-specific organelle that contains adhesion proteins. Whole-genome sequencing identified the etiological point mutation in TgDOC2.1. A conditional allele of the orthologous gene engineered into Plasmodium falciparum was also defective in microneme secretion. However, the major effect was on invasion, suggesting that microneme secretion is dispensable for Plasmodium egress.

Rhomboid Proteolysis: Why inside the Membrane?

Rhomboid proteins are membrane-immersed enzymes with the remarkable ability to hydrolyze protein segments within the membrane. This form of intramembrane proteolysis plays key roles in cell communication including EGF signalling during animal development, and quorum sensing during bacterial growth. We have been investigating rhomboid protease mechanism using a pure enzyme reconstitution system that we developed, coupled with high-resolution crystal structures of a model bacterial rhomboid protease (1, 2). Intramembrane proteolysis is catalyzed within a hydrated cavity that opens to the outside of the cell and allows water to enter, but is protected laterally from membrane lipids by a ring of protein segments. Using a structure-function approach, we subsequently identified one transmembrane helix and overlying loop that provide a rate-limiting movement to gate substrate entry laterally from the membrane (3). By integrating reconstitution into defined bilayer environments with spectroscopic interrogation, we recently discovered that rhomboid proteases rely on the natural biophysical constraints of the membrane to achieve site-specific proteolysis. Since rhomboid proteins play critical roles in the infective cycles of several protozoan parasites (4,5,6), understanding the protease mechanism may provide therapeutic opportunities (7).1. Urban, S. and M.S. Wolfe. (2005). Proc. Natl. Acad. Sci. USA, 102(6):1883-1888.2. Wu Z., Yan N., Feng L., Oberstein A., Yan H., Baker R. P., Gu L., Jeffrey P.D., Urban S., and Y. Shi. (2006). Nature Structural and Molecular Biology 13: 1084-1091.3. Baker, R.P., Young K., Feng L., Shi Y. and S. Urban. (2007). Proc. Natl. Acad. Sci. USA, 104: 8257-62.4. Baker, R.P., Wijetilaka R, and S. Urban. (2006). PLoS Pathogens 10(2): e113; 922-32.5. Baxt, L., Baker, R.P., Singh, U. and S. Urban. (2008). Genes & Development 22(12): 1636-46.6. Urban S. (2009). Nature Reviews Microbiology 7: 411-23.7. Urban S. (2010). The Biochemical Journal 425(3): 501-512.

O Complexo Teníase Humana-Cisticercose: ainda um sério problema de saúde pública

O presente trabalho tem como objetivo revisar o complexo teníase-cisticercose. Trata-se de um sério problema de saúde pública, representado por entidades zoonóticas das mais importantes na atualidade. O complexo compreende duas doenças distintas, com sintomatologia e epidemiologia totalmente diferentes: a teníase que corresponde à fase final do ciclo do parasita e ocorre apenas no ser humano e a cisticercose que corresponde ao estágio larval da Taenia saginata, a qual parasita bovinos, ou da Taenia solium,que pode acometer suínos e também seres humanos. A cisticercose e a teníase são encontradas com maior frequência em países cujas populações apresentam hábitos de higiene precários e com saneamento básico deficiente. Sua etiologia inclui a Taenia solium e a Taenia saginata que pertencem à classe Cestoidea, ordemCyclophillidea, família Taenidae e gênero Taenia e as respectivas formas larvares, Cysticercus cellulosae e Cysticercus bovis. Na teníase a sintomatologia clínica é bastante variável, de acordo coma idade e o grau de higidez orgânica do hospedeiro. Em condições naturais, os bovinos acometidos por cisticercose não manifestam sinais clínicos, já na cisticercose humana, variam dependendo da localização dos cisticercos. Quando localizados no sistema nervoso central (neurocisticercose) os sinais clínicos podem variar de acordo com o número de cisticercos, seu estado de desenvolvimento, a variedade morfológica, com sua localização e com as reações que provocam no paciente. O controle da teníase-cisticercose depende das condições econômicas, sociais e culturais de cada região e país, tendo a educação sanitária como ferramenta fundamental.

PF4-Related Kinocidins Mediate Plasmodicidal Activity of Platelets

Abstract 1140 Malaria is a thrombocytopenic syndrome. Platelet activation occurs after binding to the knobs of Plasmodium-infected erythrocytes (Srivastava et al 2008). Severely thrombocytopenic thrombopoetin receptor knock-out mice infected with murine malaria have significantly higher mortality as compared to wild-type mice (McMorran et al 2008). Platelets have significant antibacterial properties when added to the bacterial cultures (Yeaman et al 2002). These are mediated by platelet microbicidal proteins, or kinocidins, alpha granule proteins related to the chemokine Platelet Factor 4 (PF4). PF4 possesses antibacterial properties itself. Additionally, plasma PF4 levels are significantly elevated in malaria. We hypothesized that the antimalarial properties of platelets are mediated by PF4-related thrombocidins, and studied growth-inhibition of platelet and PF4 on Plasmodium falciparum, in vitro. Plasmodium falciparum (ITG-2F2 strain) was cultivated in O negative human erythrocytes in a media comprised of RPMI 1640 plus glutamine, hypoxanthine and human serum, at low O2 (1%) and 37°C (Trager and Jensen 1972). Parasite growth was monitored by estimating percent parasitemia of stained smears by light microscopy. The parasite growth cycle was synchronized using gelatin floatation prior to the experiments. To ensure similar baseline parasitemia same stock culture was split between experimental and control cultures. Exp. 1 Washed human platelets were added to the culture of mature stage P. falciparum at a ratio of 1:1 of platelets to erythrocytes, and growth compared to control cultures at 12 and 24 hours. Platelet activation was studied by measuring supernatant PF4 levels (human PF4-specific ELISA [Hyphen Biomed]), and compared to platelet controls, where washed platelets were added to culture media containing only uninfected erythrocytes. Exp. 2 Recombinant human PF4 was added to P. falciparum cultures at a concentration of 2500 IU/ml, and growth compared to control cultures at 12 and 24 hours. Chloroquine-treated cultures represented positive controls in all experiments. The results of the experiments are reported as proportional change in baseline parasitemia, averaged for repeat experiments. These data are based on three sets of experiments. The addition of platelets to parasite cultures synchronized to mature stages resulted in significant growth inhibition at 24 hours as compared to the controls (Decrease to 0.44 of baseline versus 1.5 fold rise in controls; Figure 1). The PF4 levels (IU/ml) were significantly higher in experimental cultures than the platelet controls (mean 440, SD 80 vs. mean 265, SD 35; p=0.032), indicating platelet activation in the presence of Plasmodia. Finally, addition of PF4 to parasite cultures synchronized to mature stages resulted in significant growth inhibition at 24 hours as compared to the controls (Decrease to 0.5 of baseline versus 2.1 fold rise in control; Figure 1). An additional experiment was performed to study the effect of platelets and PF4 on immature stages of parasites. No differences in growth of parasites were observed between experimental and control cultures at 24 hours (1.4, 1.3 and 1.3 fold growth in control, platelet-treated, and PF4-treated cultures).Figure 1.Average change in parasitemia with timeFigure 1. Average change in parasitemia with time The results of this study indicate that the platelets are inhibitory to the growth of Plasmodium falciparum, in vitro. This is accompanied by platelet activation as evidenced by significantly higher PF4 levels in the presence of infected erythrocytes. Furthermore, we demonstrate that PF4 is growth-inhibitory to P. falciparum, likely mediating the plasmodicidal action of the platelets. This effect of platelets is dependent on the growth stage of parasites and appears to affect primarily mature stages. Currently available data do not exclude an intra-erythrocytic effect of PF4, but our data support the more likely hypothesis that the cytotoxic effect of PF4 occurs following the release of merozoites from erythrocytes. Although, supra-physiologic platelet and PF4 concentrations were used in this study, platelets are known to rosette Plasmodium-infected erythrocytes in vivo, particularly in microcirculation, indicating that the ‘local’ concentrations of PF4 are likely higher. Therefore, our data provide additional experimental evidence in support of the role of platelets in malaria innate immunity. Disclosures: No relevant conflicts of interest to declare.

A historical view of alveolar echinococcosis, 160 years after the discovery of the first case in humans: part 1. What have we learnt on the distribution of the disease and on its parasitic agent?

Since the first 2 cases observed in southern Germany and the correct identification of a parasite at the origin of the disease by the famous scientist Rudolf Virchow in 1855, the borders of the endemic area of alveolar echinococcosis (AE) have never stopped to expand. The parasite was successively recognized in Switzerland, then in Russia, Austria and France which were long considered as the only endemic areas for the disease. Cases were disclosed in Turkey in 1939; then much attention was paid to Alaska and to Hokkaido, in Japan. The situation totally changed in 1991 after the recognition of the Chinese endemic areas by the international community of scientists. The world map was completed in the beginning of the 21st century by the identification of AE in most of the countries of central/eastern Europe and Baltic States, and by the recognition of cases in central Asia. Up to now, the disease has however never been reported in the South hemisphere and in the United Kingdom. In the mid-1950s, demonstration by Rausch and Schiller in Alaska, and by Vogel in Germany, of the distinction between 2 parasite species responsible respectively for cystic echinococcosis (“hydatid disease”) and AE put an end to the long-lasting debate between the "dualists", who believed in that theory which eventually proved to be true, and the "unicists", who believed in a single species responsible for both diseases. At the end of the 20th century, molecular biology fully confirmed the "dualist" theory while adding several new species to the initially described E. granulosus; within the past decade, it also confirmed that little variation existed within Echinococcus (E.) multilocularis species, and that AE-looking infection in some intermediate animal hosts on the Tibetan plateau was indeed due to a new species, distinct from E. multilocularis, named E. shiquicus. Since the 1970s, the unique ecological interactions between the landscape, the hosts, and E. multilocularis have progressively been delineated. The important role of the rodent/lagomorph reservoir size for the maintenance of the parasite cycle has been recognized within the last 2 decades of the 20th century. And the discovery of a close relationship between high densities of small mammals and particularities in land use by agriculture/forestry has stressed the responsibility of political/economic decisions on the contamination pressure. Urbanization of foxes in Europe and Japan and the major role of dogs in China represent the new deals at the beginning of the 21st century regarding definitive hosts and prevention measures.

O-GlcNAc modification of the anti-malarial vaccine candidate PfAMA1: in silico-defined structural changes and potential to generate a better vaccine

The complex life cycle of plasmodial parasites makes the selection of a single subunit protein a less than optimal strategy to generate an efficient vaccinal protection against malaria. Moreover, the full protection afforded by malarial proteins carried by intact parasites implies that immune responses against different antigens expressed in different phases of the cycle are required, but also suggests that native malarial antigens are presented to the host immune system in a manner that recombinant proteins do not achieve. The malarial apical membrane antigen 1 (AMA1) represents a suitable vaccine candidate because AMA1 is expressed on sporozoites and merozoites and allows them to invade hepatocytes and erythrocytes, respectively. Anti-AMA1 antibodies and cytotoxic T-cells are therefore expected to interfere both with the primary invasion of hepatocytes by sporozoites and with the later propagation of merozoites in erythrocytes, and thus efficiently counteract parasite development in its human host. AMA1 bears potential glycosylation sites and the human erythrocytic O-linked N-acetylglucosamine transferase (OGT) could glycosylate AMA1 through combinatorial metabolism. This hypothesis was tested in silico by developing binding models of AMA1 with human OGT complexed with UDP-GlcNc, and followed by the binding of O-GlcNAc with the hydroxyl group of AMA1 serine and threonine residues. Our results suggests that AMA1 shows potential for glycosylation at Thr517 and Ser498 and that O-GlcNAc AMA1 may constitute a conformationally more appropriate antigen for developing a protective anti-malarial immune response.

The molecular epidemiology of parasite infections: Tools and applications

Molecular epidemiology, broadly defined, is the application of molecular genetic techniques to the dynamics of disease in a population. In this review, we briefly describe molecular and analytical tools available for molecular epidemiological studies and then provide an overview of how they can be applied to better understand parasitic disease. A range of new molecular tools have been developed in recent years, allowing for the direct examination of parasites from clinical or environmental samples, and providing access to relatively cheap, rapid, high throughput molecular assays. At the same time, new analytical approaches, in particular those derived from coalescent theory, have been developed to provide more robust estimates of evolutionary processes and demographic parameters from multilocus, genotypic data. To date, the primary application of molecular epidemiology has been to provide specific and sensitive identification of parasites and to resolve taxonomic issues, particularly at the species level and below. Population genetic studies have also been used to determine the extent of genetic diversity among populations of parasites and the degree to which this diversity is associated with different host cycles or epidemiologically important phenotypes. Many of these studies have also shed new light on transmission cycles of parasites, particularly the extent to which zoonotic transmission occurs, and on the prevalence and importance of mixed infections with different parasite species or intraspecific variants (polyparasitism). A major challenge, and one which is now being addressed by an increasing number of studies, is to find and utilize genetic markers for complex traits of epidemiological significance, such as drug resistance, zoonotic potential and virulence.

Global kinomic and phospho-proteomic analyses of the human malaria parasite Plasmodium falciparum

The role of protein phosphorylation in the life cycle of malaria parasites is slowly emerging. Here we combine global phospho-proteomic analysis with kinome-wide reverse genetics to assess the importance of protein phosphorylation in Plasmodium falciparum asexual proliferation. We identify 1177 phosphorylation sites on 650 parasite proteins that are involved in a wide range of general cellular activities such as DNA synthesis, transcription and metabolism as well as key parasite processes such as invasion and cyto-adherence. Several parasite protein kinases are themselves phosphorylated on putative regulatory residues, including tyrosines in the activation loop of PfGSK3 and PfCLK3; we show that phosphorylation of PfCLK3 Y526 is essential for full kinase activity. A kinome-wide reverse genetics strategy identified 36 parasite kinases as likely essential for erythrocytic schizogony. These studies not only reveal processes that are regulated by protein phosphorylation, but also define potential anti-malarial drug targets within the parasite kinome.

Proteolytic activity in Hysterothylacium aduncum (Nematoda: Anisakidae), a fish gastrointestinal parasite of worldwide distribution

Proteases have a significant role in the life cycle of parasites and the pathogen–host relationship, being regarded as important virulence factors. In the parasitic nematode Hysterothylacium aduncum proteolytic activity was measured during in vitro development from third larval stage (L3) to mature adult, using DQ red casein as a fluorogenic substrate. Proteolytic activity was detected in all the developmental stages studied and at all pH values within the range employed (2.0–7.5). The assay with specific inhibitors permitted the determination of metalloprotease activity, and, to a lesser extent, that of aspartate- and cysteine-protease. Serine-protease activity was the lowest of those studied. In L3 recently collected from the host fish (L3-0h), the greatest activity was found at an optimum pH of 4.0 and was mainly inhibited by 1,10-phenathroline (metalloprotease inhibitor). This metalloprotease activity in L3-0h (infective stage) may be related to the invasion of the host tissues by this larva. In the other developmental stages, the greatest protease activity was found at pH 5.5, although at pH 4.0 a lower activity peak was detected. On the other hand, our data show that the proteolytic activity of the nematode varies according to the presence of pepsin (an aspartic-protease) in the culture medium. Thus, at pH 4.0, activity was greater in the absence of pepsin, with increasing aspartic-protease activity. Together with the detection of aspartic-, cysteine- and metallo-protease (enzymes involved in digestion in invertebrates) in all the developmental stages of the parasite taking place in the digestive tract of the host fish, this allows us to suggest that the pepsin in the culture medium mimics the predigestion conditions in the habitat of the worm within the host and that the activity detected may have, amongst others, a digestive function.

Effects of the antimalarial drugs ferroquine and artesunate on Plasmodium yoelii yoelii gametocytegenesis and vectorial transmission

Chemistry still has a role in the management of malaria, alongside the mosquito netting soaked in insecticide that is used increasingly, as we continue to await the long anticipated vaccine. During its cycle, the hematozoon parasite develops through three major periods. The first, malarial infection, corresponds to the intrahepatic development of infective forms from the mosquito vector; this period is not sensitive to treatment and is often asymptomatic. The period of erythrocytic schizogony is the most urgent, and treatment activity is primordial. Finally, the phase of sexual reproduction, when gametocytes develop within the erythrocytes ensures the perpetuation of the species when these reach the blood-feeding female anopheles mosquitoes. The aim of our work was to study the effect on gametocytes of drugs known to be effective on the asexual blood forms of the protozoan and thus the potential repercussions on malaria transmission. This experimental study was conducted with an animal model whose parasite cycle and modes of transmission are close to those of human malaria: Plasmodium yoelii, maintained on Swiss mice, with the Anopheles stephensi vector (maintained in an animal facility at the National Museum of Natural History in Paris). Two drugs were tested: ferroquine (a chloroquine derivative with a ferrocene molecule at the lateral carbon chain that restores its efficacy against chloroquine-resistant strains) and artesunate (a derivative of artemisinin, from ginghao, a Chinese plant also known as artemisia annua, or sweet wormwood), a treatment of choice in the combined therapies recommended by WHO. The efficacy of these drugs, prescribed at doses subcurative for the asexual forms, were tested against gametocyte production, quantitatively by counting them in the blood and qualitatively by counting the quantity of oocysts developed on the mosquito's midgut, which are indicators of gametocyte activity. The mice that were parasite-infected and then treated served as their own controls: lots of 30 mosquitoes fed on each mouse before treatment and then 90 minutes and 5 hours after treatment. Quantitatively, the comparison of the blood parasite level and the gametocyte index shows that treated mice had a higher level of circulating gametocytes than untreated parasite infested mice, regardless of drug or dose (5 or 10 mg/kg). For artesunate at 5 mg/kg, we noted that the blood gametocyte level was almost double that of the controls. On the other hand, qualitatively, the first results obtained with optical and electronic microscopy showed morphologic alterations of the circulating gametocytes (pigment clumping and lateralisation within red blood cells) and reduced infectivity of the gametocytes for the mosquitoes that fed at 1 and 5 hours after treatment. We were able to demonstrate statistically that the infectivity of gametocytes, measured by the quantity of oocysts counted in the mosquito midgut, was reduced by 70% for those treated with ferroquine and by 85% for those from mice treated by artesunate. Complementary studies will seek to specify the populations (age) of gametocytes damaged by treatment and the importance and nature of their morphologic alterations.