Listeria Monocytogenes Infection of the Brain

描述(由申请人提供)： 单核细胞增多性李斯特菌是一种革兰氏阳性的胞内致病菌，能够侵袭多种宿主细胞。LM感染可导致人类患上严重疾病，最常见的是影响免疫功能低下的个人、孕妇和老年人。特别令人担忧的是，LM有能力入侵中枢神经系统(CNS)，导致危及生命的脑膜炎和脑炎。促进LM脑部感染的必要因素的身份仍不清楚。我们已经发现，一种名为InlF的LM表面蛋白是成功在小鼠体内定植大脑所必需的。亲和层析和质谱分析结果表明，InlF与脑血管内皮细胞表面蛋白、Vimentin、剪接因子SFPQ和核蛋白AHNAK相互作用。这项建议的重点是阐明InlF是如何介导脑部LM感染的。我们推测，InlF与Vimentin相互作用，伴随着SFPQ和AHNAK，介导LM进入脑细胞，在没有Vimentin的情况下，大脑的LM感染将是 由于InlF-Vimentin相互作用对成功的脑定植的重要性，在体内严重受损。我们进一步假设，InlF需要通过血脑屏障才能在大脑中建立LM感染。在Aim I中，将使用庆大霉素保护试验来确定Vimentin对InlF介导的培养细胞系侵袭的需求。使用表达绿色荧光蛋白(GFP)的LM菌株和InlF包裹的绿色荧光珠子的荧光显微镜将被用来更直接地检测InlF与宿主细胞相互作用的能力。此外，还将采用生化方法演示直接蛋白质结合，并确定InlF-Vimentin蛋白质-蛋白质相互作用所涉及的区域。 在AIM II中，InlF-Vimentin相互作用对体内LM感染的贡献将通过Vimentin或AHNAK基因敲除小鼠的感染研究来确定。体外感染分析和荧光显微镜将对培养自正常和基因敲除小鼠的原代脑细胞进行，以确定InlF是否介导不同原代脑细胞类型的感染，以及Vimentin和AHNAK对感染的重要性。在AIM III中，我们将确定InlF是通过直接侵袭还是细胞间扩散来介导BBB通路。我们将对原代小鼠脑微血管内皮细胞和脑内皮细胞系进行庆大霉素保护试验，以确定构成血脑屏障的细胞是否需要InlF直接侵袭。异源扩散试验将被用于评估InlF在细胞间扩散到脑组织中的重要性。最后，感染细胞间传播缺陷的Acta或Acta/inlF LM菌株的小鼠将确定InlF在体内通过直接侵袭与细胞间传播对大脑定植的贡献。TE提议的研究将为促进LM侵入大脑的蛋白质-蛋白质相互作用提供洞察力，并可能确定防止LM和其他细胞内病原体感染大脑的新靶点。

DESCRIPTION (provided by applicant): Listeria monocytogenes (Lm) is a Gram-positive intracellular bacterial pathogen capable of invading numerous host cell types. Lm infections can lead to severe disease in humans and most often affects immunocompromised individuals, pregnant women, and the elderly. Of particular concern is the ability of Lm to invade the central nervous system (CNS), leading to life-threatening meningitis and encephalitis. The identity of factors necessary to facilitate Lm brain infection has remained unclear. We have discovered that an Lm surface protein, InlF, is required for successful colonization of the brain in mice. Moreover, the results of affinity chromatography and mass spectrometry analysis indicate that InlF interacts with the brain endothelial cell surface protein, Vimentin, splicing factor Sfpq and nucleoprotein AHNAK. The focus of this proposal is to elucidate how InlF mediates Lm infection of the brain. We hypothesize that InlF interacts with Vimentin, accompanied by Sfpq and AHNAK, to mediate entry of Lm into brain cells and that in the absence of Vimentin, Lm infection of the brain will be severely compromised in vivo due to the importance of InlF-Vimentin interactions for successful brain colonization. We further hypothesize that InlF is required for passage across the BBB to establish an Lm infection in the brain. In Aim I, gentamicin protection assays will be used to determine the requirement of Vimentin for InlF-mediated invasion of cultured cell lines. Fluorescence microscopy with Lm strains expressing green fluorescent protein (GFP) and InlF-coated green-fluorescent beads will be used to more directly examine the ability of InlF to interact with host cells. In addition, a biochemical approach will be taken to demonstrate direct protein binding and identify the regions involved in the InlF-Vimentin protein-protein interaction. In Aim II, the contribution of InlF-Vimentin interactions to Lm infection in vivo will be determine by infection studies in Vimentin or AHNAK knockout mice. In vitro infection assays and fluorescence microscopy will be performed with primary brain cells cultured from normal and knockout mice to determine if InlF mediates infection of distinct primary brain cell types and the importance of Vimentin and AHNAK for infection. In Aim III, we will determine if InlF mediates BBB passage by direct invasion or cell-to-cell spread. Gentamicin protection assays with primary mouse brain microvascular endothelial cells and brain endothelial cell lines will be performed to determine if InlF is required for direct invasion of cells that constitute the BBB. Heterologous spread assays with Lm infected monocytes and brain endothelial cells will be used to assess the importance of InlF in cell-to-cell spread into the brain. Finally, infection of mice with cell-to-cell spread defective ¿actA or ¿actA/¿inlF Lm strains will determine the in vivo contribution of InlF for colonization of the brain by direct invasion versus cell-to-cell spread. Te proposed studies will provide insights into the protein-protein interactions facilitating Lm invasin of the brain and may identify novel targets for preventing infections of the brain by Lm and other intracellular pathogens.