Listeria hijacks host mitophagy through a novel mitophagy receptor to evade killing.

Cells use mitophagy to remove damaged or unwanted mitochondria to maintain homeostasis. Here we report that the intracellular bacterial pathogen Listeria monocytogenes exploits host mitophagy to evade killing. We found that L. monocytogenes induced mitophagy in macrophages through the virulence factor listeriolysin O (LLO). We discovered that NLRX1, the only Nod-like receptor (NLR) family member with a mitochondrial targeting sequence, contains an LC3-interacting region (LIR) and directly associated with LC3 through the LIR. NLRX1 and its LIR motif were essential for L. monocytogenes-induced mitophagy. NLRX1 deficiency and use of a mitophagy inhibitor both increased mitochondrial production of reactive oxygen species and thereby suppressed the survival of L. monocytogenes. Mechanistically, L. monocytogenes and LLO induced oligomerization of NLRX1 to promote binding of its LIR motif to LC3 for induction of mitophagy. Our study identifies NLRX1 as a novel mitophagy receptor and discovers a previously unappreciated strategy used by pathogens to hijack a host cell homeostasis system for their survival.

Two functional microRNA-126s repress a novel target gene p21-activated kinase 1 to regulate vascular integrity in zebrafish.

RATIONALE: MicroRNAs (miRNAs) are key regulators of vascular development and diseases. The function and underlying mechanism of endothelial miRNAs have not been fully defined. OBJECTIVE: To investigate the role of endothelial miR-126 in zebrafish vascular development. METHODS AND RESULTS: Two homologs of miR-126, miR-126a (namely miR-126 in previous literature) and miR-126b, with only 1 nucleotide difference in their mature sequences, were identified in zebrafish genome. In vitro analysis showed that both precursors could sufficiently produce mature functional miRNAs. Expression analyses by Northern blot and quantitative RT-PCR showed that both miR-126s accumulated significantly 12 hours after fertilization and were specifically expressed in endothelial cells of zebrafish. Inhibition of miR-126a or miR-126b with specific morpholinos caused cranial hemorrhage, and simultaneous inhibition of both miR-126s resulted in a pronounced hemorrhage in higher percentage of embryos. Bioinformatics prediction showed that the targets of miR-126a/b partially overlapped but essentially differed. p21-activated kinase1 (pak1) was identified as a novel target of miR-126a/b, and pak1 3' untranslated region was differently regulated by these 2 miRNAs. Quantitative RT-PCR, in situ hybridization, and Western blot analyses showed that the level of pak1 was reduced when miR-126a/b were overexpressed. Notably, pak1 expression in endothelial cells was increased when miR-126a/b were knocked down. Furthermore, overexpression of the active form of human pak1 caused cranial hemorrhage, and knockdown pak1 effectively rescued the hemorrhage caused by inhibiting miR-126a/b. CONCLUSIONS: Two functional endothelial cell-specific miRNAs, miR-126a and miR-126b, synergistically regulate zebrafish vascular integrity, and pak1 is a critical target of miR-126a/b in vascular development.

Rapamycin and CHIR99021 Coordinate Robust Cardiomyocyte Differentiation From Human Pluripotent Stem Cells Via Reducing p53-Dependent Apoptosis.

BACKGROUND: Cardiomyocytes differentiated from human pluripotent stem cells can serve as an unexhausted source for a cellular cardiac disease model. Although small molecule-mediated cardiomyocyte differentiation methods have been established, the differentiation efficiency is relatively unsatisfactory in multiple lines due to line-to-line variation. Additionally, hurdles including line-specific low expression of endogenous growth factors and the high apoptotic tendency of human pluripotent stem cells also need to be overcome to establish robust and efficient cardiomyocyte differentiation. METHODS AND RESULTS: We used the H9-human cardiac troponin T-eGFP reporter cell line to screen for small molecules that promote cardiac differentiation in a monolayer-based and growth factor-free differentiation model. We found that collaterally treating human pluripotent stem cells with rapamycin and CHIR99021 during the initial stage was essential for efficient and reliable cardiomyocyte differentiation. Moreover, this method maintained consistency in efficiency across different human embryonic stem cell and human induced pluripotent stem cell lines without specifically optimizing multiple parameters (the efficiency in H7, H9, and UQ1 human induced pluripotent stem cells is 98.3%, 93.3%, and 90.6%, respectively). This combination also increased the yield of cardiomyocytes (1:24) and at the same time reduced medium consumption by about 50% when compared with the previous protocols. Further analysis indicated that inhibition of the mammalian target of rapamycin allows efficient cardiomyocyte differentiation through overcoming p53-dependent apoptosis of human pluripotent stem cells during high-density monolayer culture via blunting p53 translation and mitochondrial reactive oxygen species production. CONCLUSIONS: We have demonstrated that mammalian target of rapamycin exerts a stage-specific and multifaceted regulation over cardiac differentiation and provides an optimized approach for generating large numbers of functional cardiomyocytes for disease modeling and in vitro drug screening.