Significance of Corneal Cell Invasion by Bacteria

摘要： 铜绿假单胞菌是致盲眼病的主要原因。导致疾病发生的原因是侵袭 角膜上皮细胞，正如我们在体外和体内所展示的那样。在这里，我们报告了铜绿假单胞菌可以 在角膜上皮细胞内建立多个小生境，其中一些定植于细胞质和其他空泡--通常 在同一间牢房里。报告表达结果显示这些交替位置的表型状态不同 提示细胞质细菌“急性”感染模式和“慢性”(生物被膜形成)模式 那些在液泡中的。虽然对这两种状态之间的转换的遗传调控已经被广泛研究 在体外，这可能如何在细胞内发挥作用尚不清楚。我们已公布的和初步的数据显示，“急性” 和“慢性”模式合作，以确定角膜上皮细胞的反应，以允许铜绿假单胞菌穿透 多层角膜上皮细胞，以定植角膜，并引起病理。在这里，我们将测试 假设它们独特的细胞内生态位有助于它们在角膜感染期间的合作努力。 这三个目标将探索细胞内多样化对细菌和宿主角膜的意义。 上皮细胞，并对疾病的发病机制。方法将利用利用资金开发的多种新工具 包括细菌结构、成像技术和感染模型。两位顾问 是细菌基因调控方面的领先专家，分别涵盖这两种表型。重要的是，我们的数据 提示液泡型铜绿假单胞菌转变为生物膜，这可能很难杀死，甚至难以培养。一个 液泡将生物膜包裹在两种寄主膜内--液泡和质膜。这种细胞内表型 仅此一项就可以解释为什么铜绿假单胞菌感染是出了名的难以管理，以及为什么疾病可以 尽管溃疡已明显消毒，但仍在继续。

Summary: P. aeruginosa is a leading cause of blinding eye disease. Contributing to disease pathogenesis is invasion of corneal epithelial cells, as we have demonstrated in vitro and in vivo. Here, we report that P. aeruginosa can establish multiple niches inside corneal epithelial cells, some colonizing the cytoplasm and other vacuoles – often in the same cell. Reporter expression results show different phenotypic states in these alternate locations, the pattern suggesting “acute” infection mode for cytoplasmic bacteria and “chronic” (biofilm forming) mode for those in vacuoles. While genetic regulation of the switch between these two states has been extensively studied in vitro, how this might work inside a cell is unknown. Our published and preliminary data show that “acute” and “chronic” modes cooperate to determine corneal epithelial cell responses, to allow P. aeruginosa to penetrate the multilayered corneal epithelium, to colonize the cornea, and to cause pathology. Here, we will test the hypothesis that their unique intracellular niches contribute to their cooperative efforts during corneal infection. The three aims will explore significance of intracellular diversification to the bacteria, to the host corneal epithelial cell, and to disease pathogenesis. Approaches will utilize multiple novel tools developed using funding from this grant, including bacterial constructs, imaging technologies, and infection models. The two consultants are leading experts in bacterial gene regulation, respectively covering both phenotypes. Importantly, our data suggest that vacuolar P. aeruginosa transition into biofilms, which can be difficult to kill or even to culture. A vacuole would enclose a biofilm inside two host membranes - vacuolar and plasma. This intracellular phenotype alone could explain why P. aeruginosa infections are notoriously difficult to manage and why disease can continue despite apparent sterilization of the ulcer.