Among the different clinical manifestations caused by P. vivax, anaemia and splenomegaly are highly prevalent. Noticeably, the bone marrow and the spleen have recently emerged as cryptic niches of natural infections and where a large parasite biomass is hidden. Our group previously demonstrated the presence of P. vivax parasites in the bone marrow of patients during acute attacks (1). Of interest, infection induced bone marrow dyserythropoiesis and altered RNA profiles related to erythropoiesis (2). Moreover, we also demonstrated that P. vivax encodes spleen-dependent genes associated with cytoadhesion and clinical protection (3). The aim of this study is to functionally characterize selected genes from the parasite upregulated in the bone marrow and from spleen-dependent genes through the implementation of the CRISPR-Cas9 technology.
Initially, we performed global transcriptional analysis of bone marrow (BM) samples of patients from Manaus (Brazil) infected with P. vivax. Computational approaches revealed a list of genes preferentially expressed in BM as compared with peripheral blood. We also used a computational approach to select a list of spleen-dependent genes. Due to the lack of a robust in vitro culture system for P. vivax, we have generated a plasmid-construct to express those genes in P. falciparum using the CRISPR-Cas9 system. Transgenic lines are being generated and confirmed through genomic integration, transcript identification and protein expression by western blot analysis. Moreover, confocal immunofluorescence microscopy is being used to determine vivax protein localization. Last, functional binding assays will be performed to unveil the function of the selected genes using spleen fibroblasts and stromal bone marrow cells.
The functional characterization of P. vivax bone marrow and spleen ligands will enable us to gain better understanding about the physiopathology of the parasite and the parasite’s tropism towards these reticulocyte-rich tissues.