The ESX-1 Virulence Factors Downregulate miR-147-3p in Mycobacterium marinum-Infected Macrophages.

As important virulence factors of Mycobacterium tuberculosis, EsxA and EsxB not only play a role in phagosome rupture and M. tuberculosis cytosolic translocation but also function as modulators of host immune responses by modulating numerous microRNAs (miRNAs). Recently, we have found that mycobacterial infection downregulated miR-148a-3p (now termed miR-148) in macrophages in an ESX-1-dependent manner. The upregulation of miR-148 reduced mycobacterial intracellular survival. Here, we investigated miR-147-3p (now termed miR-147), a negative regulator of inflammatory cytokines (e.g., interleukin-6 [IL-6] and IL-10), in mycobacterial infection. We infected murine RAW264.7 macrophages with Mycobacterium marinum, a surrogate model organism for M. tuberculosis, and found that the esxBA-knockout strain (M. marinum ΔesxBA) upregulated miR-147 to a level that was significantly higher than that induced by the M. marinum wild-type (WT) strain or by the M. marinum ΔesxBA complemented strain, M. marinum ΔesxBA/pesxBA, suggesting that the ESX-1 system (potentially EsxBA and/or other codependently secreted factors) is the negative regulator of miR-147. miR-147 was also downregulated by directly incubating the macrophages with the purified recombinant EsxA or EsxB protein or the EsxBA heterodimer, which further confirms the role of the EsxBA proteins in the downregulation of miR-147. The upregulation of miR-147 inhibited the production of IL-6 and IL-10 and significantly reduced M. marinum intracellular survival. Interestingly, inhibitors of either miR-147 or miR-148 reciprocally compromised the effects of the mimics of their counterparts on M. marinum intracellular survival. This suggests that miR-147 and miR-148 share converged downstream pathways in response to mycobacterial infection, which was supported by data indicating that miR-147 upregulation inhibits the Toll-like receptor 4/NF-κB pathway.