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The ESX-1 Virulence Factors Downregulate miR-147-3p in Mycobacterium marinum-Infected Macrophages
Xiaoshu Zuo1,2, Lin Wang1, Yanqing Bao1
1Department of Biological Sciences, Border Biomedical Research Center, University of Texas at El Paso, El Paso, Texas, USA.
Abstract:
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.
Insights
Mycobacterium tuberculosis virulence factors EsxA and EsxB downregulate miR-147, a microRNA that inhibits inflammatory cytokines and reduces bacterial survival. Upregulated miR-147 and miR-148 share pathways to control mycobacterial infection.
Area of Science:
- Microbiology
- Immunology
- Molecular Biology
Background:
- EsxA and EsxB are key virulence factors of Mycobacterium tuberculosis, influencing host immune responses via microRNA (miRNA) modulation.
- Mycobacterial infection was previously shown to downregulate miR-148 in macrophages in an ESX-1-dependent manner, with miR-148 upregulation reducing bacterial survival.
Purpose of the Study:
- To investigate the role of miR-147, a negative regulator of inflammatory cytokines, in mycobacterial infection.
- To determine if the ESX-1 secretion system influences miR-147 levels and to elucidate the functional consequences of miR-147 modulation.
Main Methods:
- Murine RAW264.7 macrophages were infected with Mycobacterium marinum wild-type, esxBA-knockout, and complemented strains.
- Macrophages were incubated with purified recombinant EsxA, EsxB, or EsxBA heterodimer.
- MicroRNA levels, cytokine production (IL-6, IL-10), bacterial intracellular survival, and the Toll-like receptor 4/NF-κB pathway were assessed.
Main Results:
- The esxBA-knockout M. marinum strain significantly upregulated miR-147 compared to wild-type or complemented strains, indicating ESX-1 system's negative regulation of miR-147.
- Recombinant EsxA, EsxB, and EsxBA proteins downregulated miR-147 in macrophages.
- Upregulated miR-147 inhibited IL-6 and IL-10 production and reduced M. marinum intracellular survival.
- Inhibitors of miR-147 or miR-148 reciprocally affected each other's impact on bacterial survival, suggesting shared downstream pathways.
- miR-147 upregulation was shown to inhibit the Toll-like receptor 4/NF-κB pathway.
Conclusions:
- The ESX-1 secretion system, including EsxBA proteins, negatively regulates miR-147 expression during mycobacterial infection.
- miR-147 acts as a host defense mechanism by suppressing inflammatory cytokines and reducing intracellular mycobacterial survival.
- miR-147 and miR-148 converge on common pathways to control mycobacterial infection, potentially through modulation of the TLR4/NF-κB signaling cascade.
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