Interaction of malaria parasites with host late endocytic and autophagic pathways is essential for Plasmodium liver stage development

RESUMO: A Malária é causada por parasitas do género Plasmodium, sendo a doença parasitária mais fatal para o ser humano. Apesar de, durante o século passado, o desenvolvimento económico e a implementação de diversas medidas de controlo, tenham permitido erradicar a doença em muitos países, a Malária continua a ser um problema de saúde grave, em particular nos países em desenvolvimento. A Malária é transmitida através da picada de uma fêmea de mosquito do género Anopheles. Durante a picada, os esporozoítos são injetados na pele do hospedeiro, seguindo-se a fase hepática e obrigatória do ciclo de vida. No fígado, os esporozoítos infetam os hepatócitos onde se replicam, dentro de um vacúolo parasitário (VP) e de uma forma imunitária silenciosa, em centenas de merozoitos. Estas novas formas do parasita são as responsáveis por infetar os eritrócitos, iniciando a fase sanguínea da doença, onde se os primeiros sintomas se manifestam, tais como a característica febre cíclica. A fase hepática da doença é a menos estudada e compreendida. Mais ainda, as interações entre o VP e os organelos da células hospedeira estão ainda pouco caracterizados. Assim, neste estudo, as interações entre os organelos endocíticos e autofágicos da célula hospedeira e o VP foram dissecados, observando-se que os anfisomas, que são organelos resultantes da intersecção do dois processos de tráfego intracelular, interagem com o parasita. Descobrimos que a autofagia tem também uma importante função imunitária durante a fase hepática inicial, ao passo, que durante o desenvolvimento do parasita, já numa fase mais tardia, o parasita depende da interação com os endossomas tardios e anfisomas para crescer. Vesiculas de BSA, EGF e LC3, foram, também, observadas dentro do VP, sugerindo que os parasitas são capazes de internalizar material endocítico e autofágico do hospedeiro. Mais ainda, mostramos que esta interação depende da cinase PIKfyve, responsável pela conversão do fosfoinositidio-3-fosfato no fosfoinositidio-3,5-bifosfato, uma vez que inibindo esta cinase o parasita não é capaz de crescer normalmente. Finalmente, mostramos que a proteína TRPML1, uma proteína efetora do fosfoinositidio-3,5-bifosfato, e envolvida no processo de fusão das membranas dos organelos endocíticos e autofágicos, também é necessária para o crescimento do parasita. Desta forma, o nosso estudo sugere que a membrana do VP funde com vesiculas endocíticas e autofágicas tardias, de uma forma dependente do fositidio-3,5-bifosfato e do seu effetor TRPML1, permitindo a troca de material com a célula hospedeira. Concluindo, os nossos resultados evidenciam que o processo autofágico que ocorre na célula hospedeira tem um papel duplo durante a fase hepática da malaria. Enquanto numa fase inicial os hepatócitos usam o processo autofágico como forma de defesa contra o parasita, já durante a fase de replicação o VP funde com vesiculas autofágicas e endocíticas de forma a obter os nutrientes necessários ao seu desenvolvimento.--------- ABSTRACT: Malaria, which is caused by parasites of the genus Plasmodium, is the most deadly parasitic infection in humans. Although economic development and the implementation of control measures during the last century have erradicated the disease from many areas of the world, it remains a serious human health issue, particularly in developing countries. Malaria is transmitted by female mosquitoes of the genus Anopheles. During the mosquito blood meal, Plasmodium spp. sporozoites are injected into the skin dermis of the vertebrate host, followed by an obligatory liver stage. Upon entering the liver, Plasmodium parasites infect hepatocytes and silently replicate inside a host cell-derived parasitophorous vacuole (PV) into thousands of merozoites. These new parasite forms can infect red blood cells initiating the the blood stage of the disease which shows the characteristic febrile malaria episodes. The liver stage is the least characterized step of the malaria infection. Moreover, the interactions between the Plasmodium spp. PV and the host cell trafficking pathways are poorly understood. We dissected the interaction between Plasmodium parasites and the host cell endocytic and autophagic pathways and we found that both pathways intersect and interconnect in the close vicinity of the parasite PV, where amphisomes are formed and accumulate. Interestingly, we observed a clearance function for autophagy in hepatocytes infected with Plasmodium berghei parasites at early infection times, whereas during late liver stage development late endosomes and amphisomes are required for parasite growth. Moreover, we found the presence of internalized BSA, EGF and LC3 inside parasite vacuoles, suggesting that the parasites uptake endocytic and autophagic cargo. Furthermore, we showed that the interaction between the PV and host traffic pathways is dependent on the kinase PIKfyve, which converts the phosphoinositide PI(3)P into PI(3,5)P2, since PIKfyve inhibition caused a reduction in parasite growth. Finally, we showed that the PI(3,5)P2 effector protein TRPML1, which is involved in late endocytic and autophagic membrane fusion, is also required for parasite development. Thus, our studies suggest that the parasite parasitophorous vacuole membrane (PVM) is able to fuse with late endocytic and autophagic vesicles in a PI(3,5)P2- and TRPML1-dependent manner, allowing the exchange of material between the host cell and the parasites, necessary for the rapid development of the latter that is seen during the liver stage of infection. In conclusion, we present evidence supporting a specific and essential dual role of host autophagy during the course of Plasmodium liver infection. Whereas in the initial hours of infection the host cell uses autophagy as a cell survival mechanism to fight the infection, during the replicative phase the PV fuses with host autophagic and endocytic vesicles to obtain nutrients required for parasite growth.

the liver endoplasmic reticulum in malaria liver infection

Malaria, a disease caused by Plasmodium, represents a major health problem with a still disconcertingly high mortality rate (655 000 malaria deaths were estimated by the World Health Organization in 2012), mainly in Africa [1]. After a bite by an infected Anopheles mosquito occurs, Plasmodium sporozoites reach their target organ, the liver, within minutes. After traversing several hepatocytes, the parasite invades a final one and establishes a parasitophorous vacuole, where it replicates exponentially generating thousands of infective merozoites, the red blood cell infectious forms that are released in the blood stream. The liver stage is the first obligatory phase of malaria infection and, although no symptoms are associated with it, it is absolutely crucial to the establishment of a successful infection.(...)

Unraveling maternal and fetal genetic factors protecting from Pregnancy Associated Malaria in the mouse

Malaria is one of the most devastating diseases in the world. In Plasmodium endemic regions, pregnant women are among the most vulnerable groups. Pregnancy Associated Malaria (PAM) threatens both maternal and foetal lives. Despite differences between human and mouse placentas PAM mouse models recapitulate key pathological features of human PAM. Here we describe new PAM models of mid gestation infection in the C57BL/6 mouse.(...)

A Natural Protective Mechanism Against Malaria

Malaria is an infectious disease of humans and other animals including birds, reptiles and most mammals. It is transmitted via the inoculation of Plasmodium sporozoites into the skin through the bite of an infected female Anopheles mosquito. Although every year, around 700.000 lives are perished, mainly children under the age of 3-5 years old, to Plasmodium infection this deadly parasite has a relatively low efficiency of transmission from mosquitoes into humans.(...)

Host genetic factors in mouse malaria liver stage infection

Fundação para a Ciência e a Tecnologia

Maladies infectieuses [Infectious diseases]

In 2008, several publications have highlighted the role of climate change and globalization on the epidemiology of infectious diseases. Studies have shown the extension towards Europe of diseases such as Crimea-Congo fever (Kosovo, Turkey and Bulgaria), leismaniosis (Cyprus) and chikungunya virus infection (Italy). The article also contains comments on Plasmodium knowlesi, a newly identified cause of severe malaria in humans, as well as an update on human transmission of the H5NI avian influenza virus. It also mentions new data on Bell's palsy as well as two vaccines (varicella-zoster and pneumococcus), and provides a list of recent guidelines for the treatment of common infectious diseases.

Effect of single nucleotide polymorphisms in cytochrome P450 isoenzyme and N-acetyltransferase 2 genes on the metabolism of artemisinin-based combination therapies in malaria patients from Cambodia and Tanzania.

The pharmacogenetics of antimalarial agents are poorly known, although the application of pharmacogenetics might be critical in optimizing treatment. This population pharmacokinetic-pharmacogenetic study aimed at assessing the effects of single nucleotide polymorphisms (SNPs) in cytochrome P450 isoenzyme genes (CYP, namely, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, and CYP3A5) and the N-acetyltransferase 2 gene (NAT2) on the pharmacokinetics of artemisinin-based combination therapies in 150 Tanzanian patients treated with artemether-lumefantrine, 64 Cambodian patients treated with artesunate-mefloquine, and 61 Cambodian patients treated with dihydroartemisinin-piperaquine. The frequency of SNPs varied with the enzyme and the population. Higher frequencies of mutant alleles were found in Cambodians than Tanzanians for CYP2C9*3, CYP2D6*10 (100C → T), CYP3A5*3, NAT2*6, and NAT2*7. In contrast, higher frequencies of mutant alleles were found in Tanzanians for CYP2D6*17 (1023C → T and 2850C → T), CYP3A4*1B, NAT2*5, and NAT2*14. For 8 SNPs, no significant differences in frequencies were observed. In the genetic-based population pharmacokinetic analyses, none of the SNPs improved model fit. This suggests that pharmacogenetic data need not be included in appropriate first-line treatments with the current artemisinin derivatives and quinolines for uncomplicated malaria in specific populations. However, it cannot be ruled out that our results represent isolated findings, and therefore more studies in different populations, ideally with the same artemisinin-based combination therapies, are needed to evaluate the influence of pharmacogenetic factors on the clearance of antimalarials.

Epidemiological traits of the malaria-like parasite Polychromophilus murinus in the Daubenton¿s bat Myotis daubentonii.

BackgroundThe great diversity of bat haemosporidians is being uncovered with the help of molecular tools. Yet most of these studies provide only snapshots in time of the parasites discovered. Polychromophilus murinus, a malaria-like blood parasite, specialised on temperate-zone bats is a species that is being `rediscovered¿. This study describes the infection dynamics over time and between host sex and age classes.MethodsFor three years we followed the members of three breeding colonies of Myotis daubentonii in Western Switzerland and screened them for the prevalence and parasitemia of P. murinus using both molecular tools and traditional microscopy. In order to identify more susceptible classes of hosts, we measured, sexed and aged all individuals. During one year, we additionally measured body temperature and haematocrit values.ResultsJuvenile bats demonstrated much higher parasitemia than any other age class sampled, suggesting that first exposure to the parasite is very early in life during which infections are also at their most intense. Moreover, in subadults there was a clear negative correlation between body condition and intensity of infection, whereas a weak positive correlation was observed in adults. Neither body temperature, nor haematocrit, two proxies used for pathology, could be linked to intensities of infection.ConclusionIf both weaker condition and younger age are associated with higher infection intensity, then the highest selection pressure exerted by P. murinus should be at the juvenile stage. Confusion over the identities and nomenclature of malarial-like parasites requires that molecular barcodes are coupled to accurate morphological descriptions.

Malaria vaccine candidate: design of a multivalent subunit α-helical coiled coil poly-epitope.

A new strategy for the rapid identification of new malaria antigens based on protein structural motifs was previously described. We identified and evaluated the malaria vaccine potential of fragments of several malaria antigens containing α-helical coiled coil protein motifs. By taking advantage of the relatively short size of these structural fragments, we constructed different poly-epitopes in which 3 or 4 of these segments were joined together via a non-immunogenic linker. Only peptides that are targets of human antibodies with anti-parasite in vitro biological activities were incorporated. One of the constructs, P181, was well recognized by sera and peripheral blood mononuclear cells (PBMC) of adults living in malaria-endemic areas. Affinity purified antigen-specific human antibodies and sera from P181-immunized mice recognised native proteins on malaria-infected erythrocytes in both immunofluorescence and western blot assays. In addition, specific antibodies inhibited parasite development in an antibody dependent cellular inhibition (ADCI) assay. Naturally induced antigen-specific human antibodies were at high titers and associated with clinical protection from malaria in longitudinal follow-up studies in Senegal.

Residual antimalarials in malaria patients from Tanzania--implications on drug efficacy assessment and spread of parasite resistance.

Repeated antimalarial treatment for febrile episodes and self-treatment are common in malaria-endemic areas. The intake of antimalarials prior to participating in an in vivo study may alter treatment outcome and affect the interpretation of both efficacy and safety outcomes. We report the findings from baseline plasma sampling of malaria patients prior to inclusion into an in vivo study in Tanzania and discuss the implications of residual concentrations of antimalarials in this setting. In an in vivo study conducted in a rural area of Tanzania in 2008, baseline plasma samples from patients reporting no antimalarial intake within the last 28 days were screened for the presence of 14 antimalarials (parent drugs or metabolites) using liquid chromatography-tandem mass spectrometry. Among the 148 patients enrolled, 110 (74.3%) had at least one antimalarial in their plasma: 80 (54.1%) had lumefantrine above the lower limit of calibration (LLC = 4 ng/mL), 7 (4.7%) desbutyl-lumefantrine (4 ng/mL), 77 (52.0%) sulfadoxine (0.5 ng/mL), 15 (10.1%) pyrimethamine (0.5 ng/mL), 16 (10.8%) quinine (2.5 ng/mL) and none chloroquine (2.5 ng/mL). The proportion of patients with detectable antimalarial drug levels prior to enrollment into the study is worrying. Indeed artemether-lumefantrine was supposed to be available only at government health facilities. Although sulfadoxine-pyrimethamine is only recommended for intermittent preventive treatment in pregnancy (IPTp), it was still widely used in public and private health facilities and sold in drug shops. Self-reporting of previous drug intake is unreliable and thus screening for the presence of antimalarial drug levels should be considered in future in vivo studies to allow for accurate assessment of treatment outcome. Furthermore, persisting sub-therapeutic drug levels of antimalarials in a population could promote the spread of drug resistance. The knowledge on drug pressure in a given population is important to monitor standard treatment policy implementation.

Antibody levels against GLURP R2, MSP1 block 2 hybrid and AS202.11 and the risk of malaria in children living in hyperendemic (Burkina Faso) and hypo-endemic (Ghana) areas.

Differences in parasite transmission intensity influence the process of acquisition of host immunity to Plasmodium falciparum malaria and ultimately, the rate of malaria related morbidity and mortality. Potential vaccines being designed to complement current intervention efforts therefore need to be evaluated against different malaria endemicity backgrounds. The associations between antibody responses to the chimeric merozoite surface protein 1 block 2 hybrid (MSP1 hybrid), glutamate-rich protein region 2 (GLURP R2) and the peptide AS202.11, and the risk of malaria were assessed in children living in malaria hyperendemic (Burkina Faso, n = 354) and hypo-endemic (Ghana, n = 209) areas. Using the same reagent lots and standardized protocols for both study sites, immunoglobulin (Ig) M, IgG and IgG sub-class levels to each antigen were measured by ELISA in plasma from the children (aged 6-72 months). Associations between antibody levels and risk of malaria were assessed using Cox regression models adjusting for covariates. There was a significant association between GLURP R2 IgG3 and reduced risk of malaria after adjusting age of children in both the Burkinabe (hazard ratio 0.82; 95 % CI 0.74-0.91, p < 0.0001) and the Ghanaian (HR 0.48; 95 % CI 0.25-0.91, p = 0.02) cohorts. MSP1 hybrid IgM was associated (HR 0.85; 95 % CI 0.73-0.98, p = 0.02) with reduced risk of malaria in Burkina Faso cohort while IgG against AS202.11 in the Ghanaian children was associated with increased risk of malaria (HR 1.29; 95 % CI 1.01-1.65, p = 0.04). These findings support further development of GLURP R2 and MSP1 block 2 hybrid, perhaps as a fusion vaccine antigen targeting malaria blood stage that can be deployed in areas of varying transmission intensity.

Aspects of spatial and habitat ecology of multiple Anopheles species (Diptera: Culicidae): malaria vectors in the highlands and foothills of Ecuador

The resurgence of malaria in highland regions of Africa, Oceania and recently in South America underlines the importance of the study of the ecology of highland mosquito vectors of malaria. Since the incidence of malaria is limited by the distribution of its vectors, the purpose of this PhD thesis was to examine aspects of the ecology of Anopheles mosquitoes in the Andes of Ecuador, South America. A historical literature and archival data review (Chapter 2) indicated that Anopheles pseudopunctipennis transmitted malaria in highland valleys of Ecuador prior to 1950, although it was eliminated through habitat removal and the use of chemical insecticides. Other anopheline species were previously limited to low-altitude regions, except in a few unconfirmed cases. A thorough larval collection effort (n=438 attempted collection sites) in all road-accessible parts of Ecuador except for the lowland Amazon basin was undertaken between 2008 - 2010 (Chapter 3). Larvae were identified morphologically and using molecular techniques (mitochondrial COl gene), and distribution maps indicated that all five species collected (Anopheles albimanus, An. pseudopunctipennis, Anopheles punctimacula, Anopheles oswaldoi s.l. and Anopheles eiseni) were more widespread throughout highland regions than previously recorded during the 1940s, with higher maximum altitudes for all except An. pseudopunctipennis (1541 m, 1930 m, 1906 m, 1233 m and 1873 m, respectively). During larval collections, to characterize species-specific larval habitat, a variety of abiotic and biotic habitat parameters were measured and compared between species-present and species-absent sites using chi-square tests and stepwise binary logistic regression analyses (Chapter 4). An. albimanus was significantly associated with permanent pools with sand substrates and An. pseudopunctipennis with gravel and boulder substrates. Both species were significantly associated with floating cyanobacterial mats and warmer temperatures, which may limit their presence in cooler highland regions. Anopheles punctimacula was collected more often than expected from algae-free, shaded pools with higher-than-average calculated dissolved oxygen. Anopheles oswaldoi s.l., the species occurring on the Amazonian side of the Andes, was associated with permanent, anthropogenic habitats such as roadside ditches and ponds. To address the hypothesis that human land use change is responsible for the emergence of multiple highland Anopheles species by creating larval habitat, common land uses in the western Andes were surveyed for standing water and potential larval habitat suitability (Chapter 5). Rivers and road edges provided large amounts of potentially suitable anopheline habitat in the western Andes, while cattle pasture also created potentially suitable habitat in irrigation canals and watering ponds. Other common land uses surveyed (banana farms, sugarcane plantations, mixed tree plantations, and empty lots) were usually established on steep slopes and had very little standing water present. Using distribution and larval habitat data, a GIS-based larval habitat distribution model for the common western species was constructed in ArcGIS v.l 0 (ESRI 2010) using derived data layers from field measurements and other sources (Chapter 6). The additive model predicted 76.4 - 97.9% of the field-observed collection localities of An. albimanus, An. pseudopunctipennis and An. punctimacula, although it could not accurately distinguish between species-absent and speciespresent sites due to its coarse scale. The model predicted distributional expansion and/or shift of one or more anopheline species into the following highland valleys with climate warming: Mira/Chota, Imbabura province, Tumbaco, Pichincha province, Pallatanga and Sibambe, Chimborazo province, and Yungilla, Azuay province. These valleys may serve as targeted sites of future monitoring to prevent highland epidemics of malaria. The human perceptions of malaria and mosquitoes in relation to land management practices were assessed through an interview-based survey (n=262) in both highlands and lowlands, of male and female land owners and managers of five property types (Chapter 7). Although respondents had a strong understanding of where the disease occurs in their own country and of the basic relationship among standing water, mosquitoes and malaria, about half of respondents in potential risk areas denied the current possibility of malaria infection on their own property. As well, about half of respondents with potential anopheline larval habitat did not report its presence, likely due to a highly specific definition of suitable mosquito habitat. Most respondents who are considered at risk of malaria currently use at least one type of mosquito bite prevention, most commonly bed nets. In conclusion, this interdisciplinary thesis examines the occurrence of Anopheles species in the lowland transition area and highlands in Ecuador, from a historic, geographic, ecological and sociological perspective.

Bionomía del vector de la malaria Anopheles vestitipennis Dyar & Knab (Díptera: Culicidae) en la cuenca del Río Polochic, Alta Verapaz, Guatemala, C.A. [por] Jaime Abraham Juárez Sandoval

Tesis (Maestría en Ciencias con Especialidad en Entomología Médica) U.A.N.L.

Zoning of the commercial poultry industry in Ontario as a method of avian influenza mitigation

L’Organisation mondiale de la santé animale (OIE) est l’institution internationale responsable de la mise en place des mesures sanitaires associées aux échanges commerciaux d’animaux vivants. Le zonage est une méthode de contrôle recommandée par l’OIE pour certaines maladies infectieuses, dont l’influenza aviaire. Les éclosions d’influenza aviaire été extrêmement coûteuses pour l’industrie avicole partout dans le monde. Afin d’évaluer la possibilité d’user de cette approche en Ontario, les données sur les sites de production avicole ont été fournies par les fédérations d’éleveurs de volailles ce cette province. L’information portant sur les industries associées à la production avicole, soit les meuneries, les abattoirs, les couvoirs, et les usines de classification d’œufs, a été obtenue par l’entremise de plusieurs sources, dont des représentants de l’industrie avicole. Des diagrammes de flux a été crée afin de comprendre les interactions entre les sites de production et les industries associées à ceux-ci. Ces industries constituaient les éléments de bas nécessaires au zonage. Cette analyse a permis de créer une base de données portant sur intrants et extrants de production pour chaque site d’élevage avicole, ainsi que pour les sites de production des industries associées à l’aviculture. À l’aide du logiciel ArcGIS, cette information a été fusionnée à des données géospatiales de Statistique Canada de l’Ontario et du Québec. La base de données résultante a permis de réaliser les essais de zonage. Soixante-douze essais ont été réalisés. Quatre ont été retenus car celles minimisaient de façon similaire les pertes de production de l’industrie. Ces essais montrent que la méthode utilisée pour l’étude du zonage peut démontrer les déficits et les surplus de production de l’industrie avicole commerciale en Ontario. Ceux-ci pourront servir de point de départ lors des discussions des intervenants de l’industrie avicole, étant donné que la coopération et la communication sont essentielles au succès du zonage.