Dataset Information

Mass Spectrometry Identification of Biomarkers in Extracellular Vesicles From Plasmodium vivax Liver Hypnozoite Infections.

ABSTRACT: Latent liver stages termed hypnozoites cause relapsing Plasmodium vivax malaria infection and represent a major obstacle in the goal of malaria elimination. Hypnozoites are clinically undetectable, and presently, there are no biomarkers of this persistent parasite reservoir in the human liver. Here, we have identified parasite and human proteins associated with extracellular vesicles (EVs) secreted from in vivo infections exclusively containing hypnozoites. We used P. vivax-infected human liver-chimeric (huHEP) FRG KO mice treated with the schizonticidal experimental drug MMV048 as hypnozoite infection model. Immunofluorescence-based quantification of P. vivax liver forms showed that MMV048 removed schizonts from chimeric mice livers. Proteomic analysis of EVs derived from FRG huHEP mice showed that human EV cargo from infected FRG huHEP mice contain inflammation markers associated with active schizont replication and identified 66 P. vivax proteins. To identify hypnozoite-specific proteins associated with EVs, we mined the proteome data from MMV048-treated mice and performed an analysis involving intragroup and intergroup comparisons across all experimental conditions followed by a peptide compatibility analysis with predicted spectra to warrant robust identification. Only one protein fulfilled this stringent top-down selection, a putative filamin domain-containing protein. This study sets the stage to unveil biological features of human liver infections and identify biomarkers of hypnozoite infection associated with EVs.

SUBMITTER: Gualdron-Lopez M

PROVIDER: S-EPMC9520272 | biostudies-literature | 2022 Oct

REPOSITORIES: biostudies-literature

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Mass Spectrometry Identification of Biomarkers in Extracellular Vesicles From Plasmodium vivax Liver Hypnozoite Infections.

Gualdrón-López Melisa M Díaz-Varela Miriam M Zanghi Gigliola G Aparici-Herraiz Iris I Steel Ryan W J RWJ Schäfer Carola C Cuscó Pol P Chuenchob Vorada V Kangwangransan Niwat N Billman Zachary P ZP Olsen Tayla M TM González Juan R JR Roobsoong Wanlapa W Sattabongkot Jetsumon J Murphy Sean C SC Mikolajczak Sebastian A SA Borràs Eva E Sabidó Eduard E Fernandez-Becerra Carmen C Flannery Erika L EL Kappe Stefan H I SHI Del Portillo Hernando A HA

Molecular & cellular proteomics : MCP 20220824 10

Latent liver stages termed hypnozoites cause relapsing Plasmodium vivax malaria infection and represent a major obstacle in the goal of malaria elimination. Hypnozoites are clinically undetectable, and presently, there are no biomarkers of this persistent parasite reservoir in the human liver. Here, we have identified parasite and human proteins associated with extracellular vesicles (EVs) secreted from in vivo infections exclusively containing hypnozoites. We used P. vivax-infected human liver- ...[more]

PMID: 36030044

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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).

Project description:Plasmodium vivax, one species of parasite causing human malaria, forms a dormant liver stage, termed the hypnozoite, which activate weeks, months or years after the primary infection, causing relapse episodes. Relapses significantly contribute to the vivax malaria burden and are only killed with drugs of the 8-aminoquinoline class, which are contraindicated in many vulnerable populations. Development of new therapies targeting hypnozoites is hindered, in part, by the lack of robust methods to continuously culture and characterize this parasite. As a result, the determinants of relapse periodicity and the molecular processes that drive hypnozoite formation, persistence, and activation are largely unknown. While previous reports have described vastly different liver-stage growth metrics attributable to which hepatocyte donor lot is used to initiate culture, a comprehensive assessment of how different P. vivax patient isolates behave in the same lots at the same time is logistically challenging. Using our primary human hepatocyte-based P. vivax liver-stage culture platform, we aimed to simultaneously test the effects of how hepatocyte donor lot and P. vivax patient isolate influence the fate of sporozoites and growth of liver schizonts. We found that, while environmental factors such as hepatocyte donor lot can modulate hypnozoite formation rate, the P. vivax case is also an important determinant of the proportion of hypnozoites observed in culture. In addition, we found schizont growth to be mostly influenced by hepatocyte donor lot. These results suggest that, while host hepatocytes harbor characteristics making them more- or less-supportive of a quiescent versus growing intracellular parasite, sporozoite fating toward hypnozoites is isolate-specific. Future studies involving these host-parasite interactions, including characterization of individual P. vivax strains, should consider the impact of culture conditions on hypnozoite formation, in order to better understand this important part of the parasite's lifecycle.

Project description:Most cancer-related deaths are caused by distant metastases, which are tumour cells that have escaped from a primary tumour and passed into the bloodstream to colonize a new organ. In this context, communication between tumour and stromal cells is essential. Indeed, tumor cells interact with cells in the tumor microenvironment and are able to modify them to their advantage. Both extracellular vesicles (EVs) and exosomes are heterogeneous populations of small vesicles present in the tumor microenvironment and in body fluids that have recently emerged as powerful mediators involved in this communication and their transport in fluids. Tumor cells release large quantities of exosomes containing tumor markers, which can then spread to distant locations. The exosomes are of endosomal origin. They are composed of proteins, lipids, RNA and DNA, and they circulate in the bloodstream. They can be internalized by specific distant cells and thus deliver a functional message. It has recently been shown that tumor exosomes containing pro-metastatic factors form pre-metastatic niches, before the tumor cells actually arrive, while determining the metastatic organotropism of tumors. These properties are now opening up new avenues of research in tumor biomarkers. In recent years, several studies have highlighted different markers contained specifically in exosomes derived from cancer cells. Consequently, exosomes are considered as potential reservoirs of tumor biomarkers that could be clinically useful for the non-invasive diagnosis of cancer, with the advantage of being performed by liquid biopsy. The study of microRNA (miRNA) is of particular interest. Indeed, miRNAs are small non-coding RNAs (between 21 and 25 nucleotides) involved in the regulation of gene expression and which are frequently deregulated in cancer. Several studies underline that the variation of free miRNAs in the blood is correlated with the progression of the disease, particularly in colon cancer. However, the stability of free miRNAs is controversial. Therefore, exosomes represent a very advantageous means of transporting miRNAs in the blood, as they are able to protect miRNAs from degradation by RNAase. The hypothesis of the project is that circulating exosomes derived from tumours contain markers including specific miRNAs that could be used as biomarkers of early prognosis (survival and progression), easily measured in blood samples from patients with colon cancer. But other molecules contained in exosomes could also be of interest.

Dataset Information

Mass Spectrometry Identification of Biomarkers in Extracellular Vesicles From Plasmodium vivax Liver Hypnozoite Infections.

Publications

Mass Spectrometry Identification of Biomarkers in Extracellular Vesicles From Plasmodium vivax Liver Hypnozoite Infections.

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