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.

M. G.-L. was a postdoctoral fellow supported by the Plan Estratégico de Investigación e Innovación en Salud (PERIS, SLT002/16/00179) of the Generalitat de Catalunya, Spain. M. D.-V. was a predoctoral fellow supported by Secretaria d’Universitats i Recerca del Departament d’Economia i Creixement, Generalitat de Catalunya (2017 FI_B2_00029). I. A.-H. is a predoctoral fellow supported by the Ministerio de Economia y Competitividad (FPI BES-2017081657). C. S. was funded by the German Research Foundation (DFG; fellowship SCHA2047/1-1). We acknowledge support from the National Institute of Health R21 program (1R21AI135680-01). The CRG/UPF Proteomics Unit is part of the Spanish Infrastructure for Omics Technologies (ICTS OmicsTech), and it is supported by “Secretaria d’Universitats i Recerca del Departament d’Economia i Coneixement de la Generalitat de Catalunya” (2017SGR595) and acknowledges support of the Spanish Ministry of Science and Innovation to the EMBL partnership. We also acknowledge support from the Spanish Ministry of Science and Innovation through the Centro de Excelencia Severo Ochoa 2019–2023” Program (CEX2018-000806-S) and support from the Generalitat de Catalunya through the CERCA Program. This research is part of the ISGlobal’s Program on the Molecular Mechanisms of Malaria which is partially supported by the Fundación Ramón Areces. Work in the laboratory of Carmen Fernandez-Becerra and Hernando A. del Portillo is funded by the Ministerio Español de Economía y Competitividad (SAF2016 80655-R) and by and by the Ministerio de Ciencia e Innovación (PID2019-111795RB-I00). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.