MPB,a Novel Berberine Derivative,enhances Lysosomal And Bactericidal Properties Via TGF-β-activated Kinase 1-dependent Activating The Transcription Factor EB

Background:As conventional antibiotics are not effective in the treatment of infections caused by bacterial pathogens,novel therapeutics to improve host cell’s intracellular defense systems and combat infections are urgently required.Lysosomes maintain cellular function involving cell secretion,plasma membrane repair,and energy metabolism by acting as a degradative and recycling organelle in the cytosol.For macrophages,the lysosome plays a direct role in the processing and clearance of endocytosed pathogens from the cell.Therefore,small molecules that can boost lysosome function and bactericidal ability to cope with subsequent infection are urgently needed.Natural anti-diarrhea drug Berberine(BBR)has been extensively used in China for decades with a confirmed safety.The potent anti-inflammatory activity of BBR prompted us to construct a library of BRR analogues in order to find new candidate drug for the prevention and treatment of infectious diseases.However,whether BBR or its analogues could enhance lysosomal function and bactericidal activity in host cells remains largely unknown.Purpose:To delineate the molecular mechanism by which MPB enhances bactericidal properties in host cells through lysosomal-dependent pathway,and provide a theoretical basis and novel therapeutic strategies for the new anti-bacterial drug development.Methods:BMDM cells infected with enteroinvasive Escherichia coli(EIEC)-EGFP or SYT09-methicillin-resistant Staphylococcus aureus(MRSA)were used to examine the effect of a series of novel Berberine derivatives on the clearance of infected bacteria.The effects of MPB on the proliferation and apoptosis of macrophages were determined by MTS and flow cytometer assay.Changes in lysosomal-related genes were measured by qRT-PCR.Lysosome biogenesis was examined by Lysotracker Red staining and Lysosome function was investigated by DQ-Red BSA and NAG assay.EGFP-TFEB stable expressing HeLa cells and Nuclear/Cytosol Fractionation Kit were used to detect TFEB nuclear translocation in macrophages.Changes of phosphorylation levels of AMPK and JNK were determined by immunoblotting.Results:We found that a novel Berberine derivative MPB,2,3-Methylenedioxy-9-[(adamantane-1-carbonyl)oxy]-10-methoxyprotoBerberine chloride,could efficiently reduce the intracellular survival of EIEC-EGFP and SYT09-MRSA in BMDM.MPB had no effect on the viability of MRSA or EIEC and little cytotoxic effects on macrophages within 5-40μM.MPB-induced lysosome-based degradation strengthens its bacterial killing.MPB causes nuclear translocation of transcription factor EB(TFEB),which boosted expression of lysosome genes.TFEB silencing repressed the MPB-mediated enhancements in degradation and bacterial eradication.MPB switched on TFEB nuclear translocation by coupling two parallel signaling pathways.MPB-triggered JNK activation led to 14-3-38 releasing from TFEB,which in turn cause TFEB nuclear translocation.In addition,MPB induced AMPK activation and subsequent inhibition of mTOR activity,which also contributed to TFEB nuclear translocation.Importantly,genetically or pharmaceutically inhibition of TAK1 remarkably reduced MPB action.MPB acted through TAK1 at lysine 158 to activate JNK and AMPK thus induced TFEB-dependent bactericidal activity in macrophages.Therefore,our study reveals a novel mechanism by which MPB controls JNK and AMPK phosphorylation cascades to activate lysosomal function and bactericidal activity via TAK1 K15 8-dependent manner,which may offer insight into novel therapeutic strategies to control bacterial infection.Conclusion:1.MPB triggers lysosome-based degradation and clearance of MRSA and EIEC in macrophages via inducing nuclear translocation of TFEB.2.MPB acts through TAK1 at lysine 158 to activate JNK and AMPK thus induced TFEB-dependent bactericidal activity in macrophages.