MicroRNA expression profiling ofLeishmania donovani-infected host cells uncovers the regulatory role of MIR30A-3p in host autophagy

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 monocytederived 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.

Countervailing, time-dependent effects on host autophagy promote intracellular survival of Leishmania

“…To best of our knowledge, the ability of L. donovani to actively inhibit classical mTOR regulated autophagy, at early stages of infection is a novel strategy that promotes pathogen survival (Figs 2 & 3, Supplementary Fig 3). Nevertheless, recent reports and the present findings make it clear that leishmania do activate autophagic machinery in host cells, albeit with delayed kinetics (Fig 1) in the case of L. donovani (13,18). In respect to the latter, it was of interest to establish whether delayed autophagy involves mTOR-dependent cell signaling or an alternate signaling pathway which is mTOR-independent.…”

“…Several species of leishmania have been observed to induce host autophagy, but the molecular mechanism(s) involved in the autophagy response have only recently begun to emerge (12)(13)(14)(15)(16)(17)(18)(19). For example, two recent studies reported a link between leishmania induced autophagy and a differentially expressed set of host micro RNAs (miRNAs), including MIR-30A-3p in infected cells (17,18).…”

“…However, this does not preclude the engagement of other autophagy regulatory pathways. For example, recent findings have shown the role of miRNAs in autophagy regulation, particularly MIR-30A-3p, in leishmania infected macrophages (18). As a corollary to this question, it was of interest to investigate whether early induction of host autophagy would be deleterious to leishmania survival.…”

“…For example, two recent studies reported a link between leishmania induced autophagy and a differentially expressed set of host micro RNAs (miRNAs), including MIR-30A-3p in infected cells (17,18).…”

“…The molecular mechanism(s) involved in leishmania mediated regulation of host autophagy have begun to emerge. For example, recent evidence suggests that select host microRNAs (miRNAs) may participate in regulation of host autophagy in response to leishmania infection (17,18). It has also been shown that autophagy induction through endosomal Toll-like receptors play a role in macrophages conferring resistance against Leishmania major infections.…”