The ESX-1 virulence factors down-regulate miR-147-3p in Mycobacterium marinum-infected macrophages.

As important virulence factors of Mycobacterium tuberculosis (Mtb), EsxA and EsxB not only play a role in phagosome rupture and Mtb cytosolic translocation, but also function as modulators of host immune responses through modulating numerous microRNAs (miRNAs). Recently, we have found that mycobacterial infection down-regulated miR-148a-3p (now termed as miR-148) in macrophages in an ESX-1-dependent manner. Up-regulation of miR-148 reduced mycobacterial intracellular survival. Here, we have investigated miR-147-3p (now termed as miR-147), a negative regulator of inflammatory cytokines (e.g. IL-6 and IL-10), in mycobacterial infection. We infected murine macrophages RAW264.7 with Mycobacterium marinum (Mm), a surrogate model organism of Mtb, and found that the esxBA-knockout strain (MmDeltaesxBA) up-regulated miR-147 to a level that is significantly higher than that induced by Mm wild type (MmWT) or by the MmDeltaesxBA complementary strain MmDeltaesxBA/pesxBA, suggesting that the ESX-1 system (potentially EsxBA and/or other co-dependently secreted factors) are the negative regulators of miR-147. MiR-147 was also down-regulated by directly incubating the macrophages with the purified recombinant proteins EsxA, EsxB or EsxBA heterodimer, which further confirms the role of EsxBA proteins in down-regulation of miR-147. Up-regulation of miR-147 inhibited the production of IL-6 and IL-10 and significantly reduced Mm intracellular survival. Interestingly, the inhibitors of either miR-147 or miR-148 reciprocally compromised the effects of the mimics of their counterparts on Mm intracellular survival. This suggests that miR-147 and miR-148 share converged downstream pathways in response to mycobacterial infection, which was supported by the data that miR-147 up-regulation inhibited the TLR4/NF-kappaB pathway.