Ayilam Ramachandran Rajalakshmy, Titone Rossella, Abdallah Joelle T, Rehman Mahad, Cao Mou, Baniasadi Hamid, Robertson Danielle M
Departments of Ophthalmology, The University of Texas Southwestern Medical Center, Dallas, Texas, USA.
bioAffinity Technologies, San Antonio, Texas, USA.
mSphere. 2025 Feb 25;10(2):e0053724. doi: 10.1128/msphere.00537-24. Epub 2025 Jan 10.
(PA) is an opportunistic gram-negative pathogen that can infect the cornea, leading to permanent vision loss. Autophagy is a cannibalistic process that drives cytoplasmic components to the lysosome for degradation and/or recycling. Autophagy has been shown to play a key role in the removal of intracellular pathogens and, as such, is an important component of the innate immune response. Autophagy is intimately linked to mitochondria, organelles that mediate energy homeostasis, immune signaling, and cell death. Using a combination of biochemical and imaging approaches, we investigated the effects of PA on autophagy and host cell mitochondria in relation to pro-inflammatory cytokine expression. Using a standard invasive test strain of PA, we show that PA infection triggers dephosphorylation of the mechanistic target of rapamycin in corneal epithelial cells, leading to the induction of autophagy through ULK1/2. This was associated with robust mitochondrial depolarization, changes in mitochondrial ultrastructure, and an increase in IL-6 and IL-8 secretion. PA infection was also associated with an increase in purine metabolism by host cells. Treatment with the ULK1/2 inhibitor, MRT68921, which blocks phagophore formation, attenuated levels of intracellular PA in corneal epithelial cells. Unexpectedly, treatment of cells with MRT68921 blocked PA-induced mitochondrial depolarization and downregulated purine and pyrimidine metabolism. While MRT68921 attenuated the PA-induced increase in IL-6, it further increased IL-8 and neutrophil chemotaxis. This was associated with the nuclear internalization of NFB. Taken together, these findings highlight a novel mechanism whereby the inhibition of ULK1/2 activity confers mitoprotection during PA infection in corneal epithelial cells.IMPORTANCE (PA) is a common pathogen that can cause severe disease in man. In the eye, PA infection can lead to blindness. In this study, we show that PA induces autophagy, a mechanism whereby cells recycle damaged proteins and organelles. PA infection further depolarizes mitochondria, leading to the release of pro-inflammatory mediators. Unexpectedly, the inhibition of ULK1/2, an enzyme involved in the early stages of autophagy, not only inhibits autophagy but enhances mitochondrial polarization. This leads to a reduction in intracellular levels of PA and changes in the inflammatory milieu. Together, these data suggest that the inhibition of ULK1/2 may be mitoprotective in corneal epithelial cells during PA infection.
绿脓杆菌（PA）是一种机会性革兰氏阴性病原体，可感染角膜，导致永久性视力丧失。自噬是一个自噬过程，可将细胞质成分运送至溶酶体进行降解和/或再循环。自噬已被证明在清除细胞内病原体中起关键作用，因此是固有免疫反应的重要组成部分。自噬与线粒体密切相关，线粒体是介导能量稳态、免疫信号传导和细胞死亡的细胞器。我们结合生化和成像方法，研究了PA对自噬和宿主细胞线粒体的影响以及与促炎细胞因子表达的关系。使用PA的标准侵袭测试菌株，我们发现PA感染会触发角膜上皮细胞中雷帕霉素作用靶点的去磷酸化，通过ULK1/2诱导自噬。这与强烈的线粒体去极化、线粒体超微结构变化以及IL-6和IL-8分泌增加有关。PA感染还与宿主细胞嘌呤代谢增加有关。用ULK1/2抑制剂MRT68921处理可阻断吞噬体形成，降低角膜上皮细胞内PA水平。出乎意料的是，用MRT68921处理细胞可阻断PA诱导的线粒体去极化，并下调嘌呤和嘧啶代谢。虽然MRT68921减弱了PA诱导的IL-6增加，但它进一步增加了IL-8和中性粒细胞趋化性。这与NF-κB的核内化有关。综上所述，这些发现突出了一种新机制，即抑制ULK1/2活性在角膜上皮细胞PA感染期间赋予线粒体保护作用。重要性：绿脓杆菌（PA）是一种常见病原体，可导致人类严重疾病。在眼部，PA感染可导致失明。在本研究中，我们发现PA诱导自噬，这是一种细胞回收受损蛋白质和细胞器的机制。PA感染进一步使线粒体去极化，导致促炎介质释放。出乎意料的是，抑制参与自噬早期阶段的酶ULK1/2不仅抑制自噬，还增强线粒体极化。这导致细胞内PA水平降低和炎症环境改变。总之，这些数据表明，在PA感染期间，抑制ULK1/2可能对角膜上皮细胞具有线粒体保护作用。
