Abstract
Exosomes are extracellular vesicles of endocytic origin containing molecular signatures implying the cell of origin; thus, they offer a unique opportunity to discover biomarkers of disease. Plasmodium vivax, responsible for more than half of all malaria cases outside Africa, is a major obstacle in the goal of malaria elimination due to the presence of dormant liver stages (hypnozoites), which after the initial infection may reactivate to cause disease. Hypnozoite infection is asymptomatic and there are currently no diagnostic tools to detect their presence. The human liver-chimeric (FRG huHep) mouse is a robust P. vivax infection model for exo-erythrocytic development of liver stages, including hypnozoites. We studied the proteome of plasma-derived exosomes isolated from P. vivax infected FRG huHep mice with the objective of identifying liver-stage expressed parasite proteins indicative of infection. Proteomic analysis of these exosomes showed the presence of 290 and 234 proteins from mouse and human origin, respectively, including canonical exosomal markers. Human proteins include proteins previously detected in liver-derived exosomes, highlighting the potential of this chimeric mouse model to study plasma exosomes derived unequivocally from human hepatocytes. Noticeably, we identified 17 parasite proteins including enzymes, surface proteins, components of the endocytic pathway and translation machinery, as well as uncharacterized proteins. Western blot analysis validated the presence of human arginase-I and an uncharacterized P. vivax protein in plasma-derived exosomes. This study represents a proof-of-principle that plasma-derived exosomes from P. vivax infected FRG-huHep mice contain human hepatocyte and P. vivax proteins with the potential to unveil biological features of liver infection and identify biomarkers of hypnozoite infection.
Highlights
Plasmodium vivax is a eukaryotic parasite that causes vivax malaria, a disease previously considered to be a benign form of malaria but recognized to be associated with severe disease and responsible for considerable morbidity and mortality in endemic regions (Mueller et al, 2009; Kevin Baird, 2013)
Taking into account the liver infection kinetics previously established by Mikolajczak et al (2015), we analyzed plasma samples of P. vivax infected mice after 8, 10, 16, and 21 dpi in order to get insight into possible changes in the protein content of plasma-derived exosomes associated with the infection by specific parasite liver stages (EI2)
size-exclusion chromatography (SEC) fractions were evaluated in a bead-based flow cytometry assay for the detection of CD5L, an exosomal marker identified in mass spectrometry (MS) studies of exosomes isolated from plasma
Summary
Plasmodium vivax is a eukaryotic parasite that causes vivax malaria, a disease previously considered to be a benign form of malaria but recognized to be associated with severe disease and responsible for considerable morbidity and mortality in endemic regions (Mueller et al, 2009; Kevin Baird, 2013). Current diagnostic tools are unable to detect asymptomatic patients harboring hypnozoites in their liver, implying the existence of a large reservoir of parasites This is detrimental for people suffering the symptoms of relapsing malaria, but represents a major obstacle toward malaria elimination. Without a sensible diagnostic tool detecting asymptomatic hypnozoites carriers, mosquitos feeding in these individuals will continue to spread P. vivax
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