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ABSTRACT
Leishmania is an obligate intracellular parasite that replicates inside phagolysosomes or parasitophorous vacuoles (PV) of the monocyte/macrophage lineage. It reprograms macrophages and produces a metabolic state conducive to successful infection and multiplication. MicroRNAs (miRNAs), a class of small 22 to 24 nucleotide noncoding regulatory RNAs alter the gene expression and consequently proteome output by targeting mRNAs, may play a regulatory role in modulating host cell functions. In the present study, we demonstrate the novel regulatory role of host microRNA, MIR30A-3p in modulation of host cell macroautophagy/autophagy after infection with L. donovani. Our in vitro studies showed that MIR30A-3p expression was significantly enhanced after L. donovani infection in a time-dependent manner. Transient transfection with a MIR30A-3p inhibitor followed by L. donovani infection promoted the autophagic response and decreased the intracellular parasite burden in both THP-1 cells and human monocyte-derived macrophages (HsMDM). BECN1/Beclin 1, the mammalian ortholog of yeast Vps30/Atg6, is a key autophagy-promoting protein that plays a key role in the regulation of cell death and survival. We report BECN1-dependent modulation of host cell autophagy in response to L. donovani infection. Pretreatment of L. donovani-infected macrophages with the MIR30A-3p mimic decreased and with antagomir increased the expression of BECN1 protein. We demonstrate that BECN1 is a potential target of MIR30A-3p and this miRNA negatively regulates BECN1 expression. Our present study reveals for the first time a novel role of MIR30A-3p in regulating autophagy-mediated L. donovani elimination by targeting BECN1. The present study has significant impact for the treatment of visceral leishmaniasis.
Abbreviations
| HsMDMs | = | human monocyte-derived macrophages |
| LD | = | Leishmania donovani |
| MAP1LC3B/LC3B | = | microtubule-associated protein 1 light chain 3 β |
| miRNAs | = | microRNAs |
| MOI | = | multiplicity of infection |
| PBS | = | phosphate-buffered saline |
| PFA | = | paraformaldehyde |
| PV | = | parasitophorous vacuole |
| qRT-PCR | = | quantitative reverse transcriptase-polymerase chain reaction |
| Rapa | = | rapamycin |
| SDS-PAGE | = | sodium dodecyl sulfate-polyacrylamide gel electrophoresis |
| VL | = | visceral leishmaniasis |
Disclosure of potential conflicts of interest
No potential conflicts of interest were disclosed.
Acknowledgments
We acknowledge the technical support provided by Mr. Madavan Vasudevan, Bionivid Technology Private Limited, Bangalore, India during microarray data analyses.
Funding
Rentala Madhubala is a JC Bose National Fellow. The funding was from the Department of Science and Technology, Ministry of Science and Technology (DST), India (D.O. No. SB/SO/HS/009/2013) to RMB. Alok Singh is supported by a fellowship from Council of Scientific and Industrial Research, India. DST-PURSE grant supported the publication charges.
