Mechanism of EGFR activation by a novel fungal pore-forming toxin

粘膜(口腔、阴道、胃肠道)上皮在保护人类免受多种感染方面具有极其重要的作用，因为它是大多数微生物遇到的初始组织。这种微生物-上皮细胞的接触要么导致无害的“共生”微生物的静止，要么在致病的“致病”微生物的情况下激活免疫和损害保护机制。当这些正常的宿主反应被扰乱时，可能会出现大量的并发症，其影响超出感染范围，因为包括癌症和自身免疫性疾病在内的疾病与微生物与其宿主之间的异常相互作用有关。鉴于这些相互作用的重要性，上皮细胞具有不同的机制，使黏膜组织能够区分共生和致病微生物。白色念珠菌是一种微生物，它可以作为共生微生物在健康人中存在，但成为一种危险的病原体，在不健康的人中会导致严重和潜在的致命疾病。事实上，念珠菌感染现在是第三种最常见的医院获得性血液感染，对医疗保健服务是一个严重的负担。鉴于绝大多数白色念珠菌感染是通过粘膜表面获得的，了解这些表面的上皮组织如何检测并将这种医学上重要的病原体限制在共生状态是至关重要的。确定这些机制不仅对真菌感染，而且对控制粘膜表面的其他微生物都有相当大的价值。我们最近的工作有助于理解上皮细胞识别致病白念珠菌的机制，以及这如何在宿主中导致损伤诱导和免疫激活。我们之前的研究表明，白念珠菌感染会导致上皮细胞损伤，并通过分泌免疫激活剂来诱导局部粘膜免疫，从而保护机体免受白念珠菌感染。最近，我们发现，为了激活这一保护性免疫过程，上皮细胞靶向于白念珠菌“入侵”形式的菌丝上的一种特定蛋白(Ece1p)。我们现在证明，Ece1p蛋白的一个特定区域(称为Ece1-III)在高浓度下作为一种新的成孔毒素(PFT)发挥作用。这是第一次在人类致病真菌中发现PFT。Ece1-III的成孔能力使白色念珠菌能够引起上皮损伤，并被上皮细胞识别以激活免疫。此外，我们发现，这种新发现的真菌PFT激活了一种特定的上皮表面受体(EGFR)，从而诱导上皮免疫和保护反应，以保护免受真菌诱导的损伤。我们已经证明，免疫激活是Ece1-III和EGFR直接相互作用的结果。然而，我们发现Ece1-III也能够通过一系列复杂的事件间接激活EGFR，这些事件发生在上皮细胞表面，作为细胞损伤反应的一部分。这些间接导致EGFR激活的表面事件的性质尚不清楚，尽管孔的形成似乎对它们的激活是必不可少的。鉴于EGFR是介导上皮免疫和损伤保护的关键受体，本研究的目的是准确地确定这种新型真菌PFT的孔道形成是如何间接激活EGFR的，并证明这些激活事件在活体白色念珠菌黏膜感染过程中的重要性。本项目将为了解真菌PFT的作用机制和我们在黏膜免疫过程中对致病真菌的上皮组织反应提供新的见解。此外，它将确定新的宿主-微生物相互作用，不仅可以针对新的、更有效的抗真菌疗法，而且还可以定义创新的方法，以对抗细菌PFTs和一般的粘膜感染的作用。

The mucosal (oral, vaginal, gastrointestinal) epithelium is of immense importance in protecting humans against a multitude of infections as it is the initial tissue encountered by the majority of microbes. This microbial-epithelial encounter results in either quiescence in the case of harmless 'commensal' microbes, or activation of immunity and damage protection mechanisms in the case of disease-causing 'pathogenic' microbes. When these normal host responses are disrupted, a plethora of complications can arise that have implications beyond infection, as conditions including cancer and autoimmune disease are associated with abnormal interactions between a microbe and its host. Given the importance of these interactions, epithelial cells possess distinct mechanisms enabling mucosal tissues to distinguish between commensal and pathogenic microbes.The fungus Candida albicans is an example of a microbe that can exist as a commensal in healthy people but becomes a dangerous pathogen causing severe and potentially fatal disease in unhealthy people. Indeed, Candida infections are now the third most common hospital-acquired bloodstream infection and are a serious burden to healthcare provision. Given that the vast majority of C. albicans infections are acquired through mucosal surfaces, it is of paramount importance to understand how epithelial tissues at these surfaces detect and restrict this medically-important pathogen to the commensal state. Identifying these mechanisms is of considerable value not only for fungal infections but also for controlling other microbes at mucosal surfaces. Our recent work has been instrumental in understanding the mechanisms by which epithelial cells identify disease-causing C. albicans and how this causes damage induction and immune activation in the host. We previously showed that C. albicans infection leads to epithelial damage and the secretion of immune activators to induce local mucosal immunity to protect against C. albicans infection. Recently, we found that to activate this protective immune process, epithelial cells target a specific protein (Ece1p) on the 'invasive' form of C. albicans known as hyphae. We now demonstrate that a specific region of the Ece1p protein (named Ece1-III) acts as a novel pore-forming toxin (PFT) when present at high concentration. This is the first PFT identified in a human pathogenic fungus. The pore forming abilities of Ece1-III enable C. albicans to cause epithelial damage and are recognised by epithelial cells to activate immunity. Furthermore, we show that this newly identified fungal PFT activates a specific epithelial surface receptor (EGFR), which induces epithelial immunity and the protective responses that protect against fungal-induced damage. We have demonstrated that immune activation occurs as a result of direct interaction between Ece1-III and EGFR. However, we found that Ece1-III is also able to activate EGFR indirectly through a complex series of events that take place at the epithelial cell surface as part of a response to cell damage. The nature of these surface events leading indirectly to EGFR activation is unknown, although pore formation seems to be essential for their activation. Given that EGFR is the key receptor mediating both epithelial immunity and damage protection, the aim of this proposal is to determine precisely how pore-formation by this novel fungal PFT indirectly activates EGFR and to demonstrate the importance of these activation events during C. albicans mucosal infection in vivo.This project will provide new insights into the mechanism of action of fungal PFTs and to our understanding of epithelial tissue responses to 'pathogenic' fungi during mucosal immunity. Further, it will identify novel host-microbe interactions that can be targeted not only for new, more effective antifungal therapies, but also for defining innovative approaches to combat the action of bacterial PFTs and mucosal infections in general.