Dataset Information

Plasma-derived extracellular vesicles from Plasmodium vivax patients signal spleen fibroblasts via NF-kB facilitating parasite cytoadherence

ABSTRACT: Extracellular vesicles are major components of circulating plasma holding insights into pathological processes. Here, we demonstrate high levels of EVs in plasma from patients with Plasmodium vivax (PvEVs), the most widely distributed human malaria parasite. Moreover, mass spectrometry analysis identified parasite proteins in PvEVs and their in vivo distribution demonstrated major spleen tropism. PvEVs were preferentially taken up by human spleen fibroblasts (hSFs) and this uptake induced specific and dose-dependent upregulation of ICAM-1 associated with the translocation of NF-kB to the nucleus. After activation of hSFs by PvEVs, P. vivax-infected reticulocytes from patients showed specific adhesion properties reversed by inhibiting NF-kB translocation to the nucleus. Together, these data provide novel physiological EV-based insights into the mechanisms of human malaria pathology and support the existence of P. vivax-adherent parasite subpopulations in the microvasculature of the human spleen

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human) Plasmodium Vivax

TISSUE(S): Blood Plasma, Extracellular Vesicle

DISEASE(S): Malaria

SUBMITTER: Eva Borràs

LAB HEAD: Eduard Sabido

PROVIDER: PXD018337 | Pride | 2020-06-08

REPOSITORIES: Pride

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Plasma-derived extracellular vesicles from Plasmodium vivax patients signal spleen fibroblasts via NF-kB facilitating parasite cytoadherence.

Toda Haruka H Diaz-Varela Miriam M Segui-Barber Joan J Roobsoong Wanlapa W Baro Barbara B Garcia-Silva Susana S Galiano Alicia A Gualdrón-López Melisa M Almeida Anne C G ACG Brito Marcelo A M MAM de Melo Gisely Cardoso GC Aparici-Herraiz Iris I Castro-Cavadía Carlos C Monteiro Wuelton Marcelo WM Borràs Eva E Sabidó Eduard E Almeida Igor C IC Chojnacki Jakub J Martinez-Picado Javier J Calvo Maria M Armengol Pilar P Carmona-Fonseca Jaime J Yasnot Maria Fernanda MF Lauzurica Ricardo R Marcilla Antonio A Peinado Hector H Galinski Mary R MR Lacerda Marcus V G MVG Sattabongkot Jetsumon J Fernandez-Becerra Carmen C Del Portillo Hernando A HA

Nature communications 20200602 1

Plasmodium vivax is the most widely distributed human malaria parasite. Previous studies have shown that circulating microparticles during P. vivax acute attacks are indirectly associated with severity. Extracellular vesicles (EVs) are therefore major components of circulating plasma holding insights into pathological processes. Here, we demonstrate that plasma-derived EVs from Plasmodium vivax patients (PvEVs) are preferentially uptaken by human spleen fibroblasts (hSFs) as compared to the upta ...[more]

PMID: 32487994

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Project description:Plasmodium vivax is the most geographically widespread human malaria parasite causing approximately 130-435 million infections annually. It is an economic burden in many parts of the world and poses a public health challenge along with the other Plasmodium sp. The biology of this parasite is very little understood. Emerging evidences of severe complications due to infections by this parasite provides an impetus to focus research on the same. Investigating this parasite directly from the infected patients is the most feasible way to study its biology and any pathogenic mechanisms which may exist. Gene expression studies of this parasite directly obtained from the patients has provided evidence of gene regulation resulting in varying amount of transcript levels in the different blood stages. However, the mechanisms regulating gene expression in malaria parasites are not well understood. Discovery of natural antisense transcripts (NATs) in P. falciparum has suggested that these might play an important role in regulating gene expression. We report here the genome-wide occurrence of NATs in P. vivax parasites from patients with differing clinical symptoms. A total of 1348 NATs against annotated gene loci have been detected using a custom designed strand specific microarray. Majority of NATs identified from this study shows positive correlation with the expression pattern of the sense transcript. Our data also shows condition specific expression patterns of varying S and AS transcript levels. Genes with AS transcripts enrich to various biological processes. This is the first report detailing the presence of NATs from clinical isolates of P. vivax. The data suggests differential regulation of gene expression in diverse clinical conditions and would lead to future detailed investigations of genome regulation. Plasmodium vivax isolates were collected from patients (n = 8) with differing clinical conditions.The patients exhibited symptoms categorized as un-complicated (n =1) or complicated malaria (n = 7). Criteria for determination of complicated disease were based on World Health Organization year 2010 guidelines. Microarray array based transcriptional profiling was carried out to detect prevalence of natural antisense transcripts.

Project description:Malaria is by far the world’s most significant tropical infectious disease and over the last a few decades large-scale malaria epidemics have happened in almost all continents. Plasmodium falciparum and Plasmodium vivax account for over 90% of the total malaria cases worldwide. The estimated number of annual clinical cases of vivax malaria is even higher than that of falciparum malaria, but yet the morbidity associated with this infection and its spectrum of disease is largely neglected. Identification of serum/plasma proteins, which exhibit altered abundance at the onset and during the acute phase of any infection, could be informative to understand the pathobiology of different infectious diseases and host responses against the invading pathogens. To this end, in recent years, quite a few research groups including us have investigated alterations in serum/plasma proteome in severe and non-severe falciparum malaria (and also vivax malaria) to study malaria pathogenesis. In all these studies, serum/plasma proteome of the malaria patients have been analyzed during the febrile stages of the infection, either at the onset of the disease or at the fastigium stage. However, temporal profiling of serum/plasma proteome during acute and remission stages in malaria, which can provide snapshots of the transient and enduring alterations in serum proteome during the febrile, defervescence and convalescent stages has not been reported hitherto. Here, we report, for the first time, serum proteomic alterations in a longitudinal cohort of P. vivax infected patients to elucidate host responses when fever is established (temperature of the body reaches above higher normal level), during the stage when the temperature comes down to normal, and also during the gradual recovery of health after the illness. The three stages discussed in our study have been categorically chosen depending upon the clinical course of uncomplicated vivax malaria. Analysis of the early febrile stage represents host proteome profile immediately after onset of the infection, without any effect of anti-malarial drugs. The second, defervescence stage, reflects any immediate change in blood proteome at early recovery phase, while the convalescent stage indicates a phase after administration of 14 days radical cure treatment with primaquine and a complete recovery, when none of the patients displayed any apparent symptoms of malaria. We have also performed an extensive quantitative proteomics analysis to compare the serum proteome profiles of vivax malaria patients with low and moderately-high parasitemia with healthy community controls. Isobaric tags for relative and absolute quantitation (iTRAQ) and 2-D fluorescence difference gel electrophoresis (2D-DIGE)-based quantitative proteomics approaches were used in the discovery-phase of the study, and some selected differentially abundant serum proteins were validated further by using ELISA. Interestingly, some of the serum proteins like Serum amyloid A, Apolipoprotein A1, C-reactive protein, Titin and Haptoglobin, were found to be sequentially altered with respect to increased parasite counts, while many of the quantified candidates such as Hemopexin, Vitronectin, Clusterin and Apolipoprotein E exhibited nearly equal levels of differential serum abundance in different parasitemic malaria patients. Analysis of a longitudinal cohort of malaria patients indicated reversible alterations in serum levels of some proteins such as Haptoglobin, Apolipoprotein E, Apolipoprotein A1, Carbonic anhydrase 1, and Hemoglobin subunit alpha upon treatment; however, the levels of a few other proteins did not return to the baseline even during the convalescent phase of the infection. Identification of the differentially abundant serum proteins and associated physiological pathways in vivax malaria along with phase-specific protein profiles during the acute and convalescent phases of the infection can effectively enhance our understanding of P. vivax disease biology and host immune responses.

Project description:Plasmodium vivax is a protozoan parasite responsible for the great majority of Malaria cases outside Sub-Saharan Africa. This parasite is characterized by the presence of latent liver stage forms called hypnozoites which cause relapsing blood infections estimated to contribute up to 79% of vivax malaria cases in endemic regions. P. vivax hypnozoites are considered a major bottleneck for the malaria elimination goal not only due to symptomatic recurrent blood infections experienced by individuals carrying this parasite form but also for continue silent parasite transmission among the population. Importantly, diagnose of hypnozoites cannot be achieved with the current diagnostic tools. Extracellular vesicles (EVs) are nanovesicles secreted by all cell types involved in intercellular communication. EVs are characterized by the presence of molecules from the cell of origin being thus considered as a fingerprint of cell physiological status. Due to this property and to the great EVs stability in biofluids, they are emerging as promising biomarkers for non-invasive diagnosis of a large range of diseases. We have previously reported that plasma-derived EVs from P. vivax infected liver chimeric mice (FRG huHep), a model of liver stage infection of this parasite, contain P. vivax proteins. In this project, we have performed a proteomic characterization of EVs derived from liver chimeric FRG huHEP mice. Pharmacological treatment of FRG huHep mice with a schizontycidal experimental drug (MMV48) was performed to generate infections enriched with latent hypnozoites, an unexplored approach to study EVs specific from this liver stage. The identification of parasite proteins associated to EVs in this model represents a significant advance in the road to identify P. vivax hypnozoite biomarkers that can be implemented for the future diagnose of hypnozoites carriers in endemic regions. Biological samples used • P. vivax FRG huHep mice in vivo model: Female FRG KO mice engrafted with human hepatocytes (FRG KO huHep) were infected with P. vivax as previously described[8]. Mice were divided in 5 experimental groups. Group 1 (3 mice) was uninfected, Group 2 (6 mice) was inoculated with P. vivax sporozoites by the bite of 20 mosquitos and euthanized after 8 days post-inoculation; Group 3 (6 mice) was infected by intravenous injection of P. vivax sporozoites (0.8 million) and euthanized after 8 days post-infection; Group 4 (6 mice) was infected by intravenous injection of P. vivax sporozoites (1 million sporozoites) and euthanized after 21 days post-infection; Group 5 (3 mice) was infected by intravenous injection (1 million sporozoites) and treated with Tafenoquine (10 mg/kg) at 14 dpi and euthanized at 21 days post-infection. MMV048 mice treatment (30 mg/kg) was performed as follows: 3 mice of Group 2 and 3 received intravenous injections of the drug at 4 days post-infection. 3 mice of Group 4 received intravenous injections of the drug at 17 days post-infection. 3 DMSO-treated animals were used as controls in groups 2, 3 and 4. After euthanasia, whole blood was extracted by cardiac venipuncture for plasma collection as previously described. Plasma-derived EVs were purified as follows: Plasma were thawed on ice, centrifuged at 2000 x g for 10 min and supernatant collected. EVs were purified by SEC using commercial Sepharose (iZON) following manufacturer instructions. SEC fractions were characterized for the presence of EV markers CD9 and plasma-derived EVs marker CD5L in a BBA and by NTA. Protein concentration was determined by BCA (Thermo Scientific).