High-Throughput Isolation and Sequencing of Cellular and Exosomal microRNAs from Erythrocytic Stages of Plasmodium Falciparum 3D7 Infection Using Novaseq 6000 Illumina
Authors: Joshi Urja , Shah Sumedha , Nanavaty Vishal , Gupta Sharad , Highland Hyacinth , George Linz-buoy 
Published: December 2024

Falciparum malaria is the main cause of severe malaria, its related complications, and malaria related mortality, our work is to investigate the role of red blood cells' (RBCs') microRNAs (miRNAs) in the host-parasitic interactions in falciparum malaria. microRNAs (miRNAs) have been proven as key mediators in the pathogenesis caused by parasitic protozoan Plasmodium falciparum. However, the role of cellular and exosomal miRNAs and the underlying molecular mechanisms related to cross-kingdom miRNA-mRNA interactions in malaria remain unclear. Thus, this current research focuses on identifying putative novel or known miRNAs and their potential in diagnostic or therapeutic applications. In this study, in-vitro RBC stages of Plasmodium falciparum 3D7 (Pf3D7) established and miRNA profiling carried out. Here, we used differential centrifugation to isolate and purify extracellular vesicles during an in-vitro Pf3D7 culture from the spent culture media samples. Furthermore, the cells and cells derived exosomes were characterized for their abundance, size, morphology, and cell surface markers. Total RNA was isolated from the samples include 1) Control O+ve RBCs 2) Pf3D7 infected RBCs 3) Control O+ve RBCs derived exosomes - CEM-Exo 4) f3D7-iRBCs derived exosomes- IEM- Exo. Followed by, performed high-throughput sequencing of the miRNA transcriptome using the PE 50 illumina (NOVASEQ 6000) platform to identify novel miRNAs as well as potential differences in the expression of defined miRNAs in cellular and exosomal samples between control and infected conditions.

A total of &gt;4GB million PE small RNA-sequnce reads were generated in all samples, with an average length of 22 bp and with a p-value &lt;0.05 and log 2-Fold Change ‘2‘. The RNA types (rRNA and tRNA content also reported &lt;1% in all the samples, as well as unknown RNA content cover 49.7% of total fraction % reported among all the samples is unexpected. Interestingly, Plasmodium falciparum-infected red blood cells (Pf-iRBCs) and control RBCs reported the highest QC qualified read counts and miRNA content (&gt;85% and &gt;75%), respectively. A total of 666 miRNAs were identified in Pf-iRBCs, of which 466 were known miRNAs identified using the miRDeep2 tool, while 172 novel miRNAs were discovered in this study. Here, it is observed that miR-451a, miR-144-3p, miR-15a, miR-16-5p, miR-22-3p, miR-185-5p significantly expressed in Pf-iRBCs with added contribution from miRs 33a-5p, 548 family, 148b-5p, miR-628-5p, miR-1537-3p reported 4 folds higher during the plasmodial infection as compared to control RBCs. At this point, miR-486-5p were significantly dysregulated with some other miRNAs 6765-3p, 6880-5p, 1260b, 7977, 1260a, 4459, 3620-5p, 4298, 7847-3p, 498, 4508, 193b-3p, 501-3p in Pf-iRBCs as compared to control. In this study, the control exosomes, derived from the control RBCs (CEM-Exo) reported 321 known and 50 novel miRNAs as well, but infected exosomes, derived from the Pf3D7-infected RBCs (IEM-Exo) reported a modified miRNA profile. However, miR-451a, 15a-5p, 22-3p, 16-5p, 185-5p, 16-5p, 486-5p were significantly dysregulated during the infection. With this, miRNAs 125a-5p, 143-3p, 191-5p, 100-5p, 10a-5p, 10b-5p, 1-3p, 1260a, 1260b, 7977 found notably upregulated in IEM-Exo versus CEM-Exo. Eight miRNAs 412-5p, 299-5p, 324-3p, 4646-5p, 193b-3p, 4485-5p, 1269b, 3607-5p were shown to be exclusively significantly expressed in IEM-Exo alone. Top 10 miRNAs which upregulated in CEM-Exo are miR-335-3p, miR-429, miR-203a-3p, miR-34a-5p, miR-885-5p, miR-320d, miR-135b-5p, miR-203b-5p, miR-30c-2-3p, miR-4458. These up regulated miRs are involved in the expression and translocation of Olfactory receptors, signal transduction, metabolism of proteins, gene expression particularly transcription, transport of small molecules, disease, DNA repair, cellular response to stimuli, developmental biology, metabolism of proteins and immune system so on. This comparative study between cellular and exosomal samples during control and infectious conditions elucidates the role of miRNAs in understanding the pathophysiology of falciparum malaria. This study investigated the role of differentially expressed miRNAs and determine their role by targeting them in the P. falciparum transcriptome. Further, the identified falciparum genes were studied through pathway prediction and Reactome analysis using the software and database mentioned.