A human lectin array for characterizing host-pathogen interactions

寄生和感染人类宿主的细菌、病毒、真菌和寄生虫表面的糖类形成了识别这些微生物的重要手段。凝集素是在人类细胞和血液中发现的糖结合蛋白，可以区分人类细胞和许多类型微生物上的不同糖结构。这种形式的识别可以作为区分自我和非我的一种有用的手段，而非我又可以作为先天免疫的基础。例如，血液中的可溶性凝集素与细菌上的糖结合，激活补体结合的特殊途径，直接攻击和杀死细菌。巨噬细胞上的细胞表面凝集素与细菌和病毒上的糖结合，导致它们被内化和破坏。凝集素还可以启动保护性炎症反应，其中免疫细胞被招募到感染部位。为了完成这些功能，人类系统中的凝集素根据其表面糖识别和攻击潜在的病原体。然而，一些细菌，如组成微生物区系的共生体，不会受到攻击。一些微生物还发展出劫持宿主凝集素的能力，以此作为进入和攻击宿主细胞的一种方式。通过生物化学、结构生物学、遗传学和基因组学的结合，人类凝集素的多个家族已经被定义，并且许多被很好地描述。然而，知道受体和它们相互作用的糖的类型并不足以让我们预测哪些受体会与哪些微生物结合，以及后果会是什么。关键的问题是微生物上的糖是非常多样化的，许多还没有完全被表征。我们提出的工作旨在通过开发凝集素阵列来解决这个问题，作为一种工具来筛选人类抗菌素凝集素的谱系。该阵列将由来自人类凝集素主要家族的固定的碳水化合物结合结构域组成，这样凝集素小组就可以用荧光标记的病毒、细菌和真菌进行探测。在一个单独的实验中，就可以看到哪些受体能够识别和结合每个单独的微生物。我们最近创造了第一个哺乳动物凝集素阵列，包含奶牛凝集素，以证明这种方法的可行性。这项拟议的研究将提供我们第一个关于人类宿主如何与致病和非致病微生物上的糖相互作用的总体观点，以便它能够对不同的挑战做出不同的反应。将开发的凝集素阵列是一个发现工具，将有助于识别可能负责对微生物的早期反应的受体。一旦阵列建立起来，一个潜在的应用将是快速筛选新出现的病原体，看看它们可以与哪些受体结合，以便提供关于它们如何进入细胞以及先天免疫反应是否可以有效控制感染的信息。人类阵列与我们的原型动物凝集素阵列的比较也将提供一些常见类型的相互作用的指示，这些相互作用可能有助于微生物在物种之间移动。人类群体中的遗传变异会导致一些糖结合受体的氨基酸序列发生变化，这可能会影响它们的糖结合特性，从而改变个体对特定微生物的反应方式。在人类凝集素阵列的进一步发展中，我们将研究大规模基因组研究中确定的序列变异对人类凝集素小组相互作用的影响。我们还将通过检查目标微生物表面的哪些糖与特定的凝集素相互作用来跟踪阵列筛选的结果，填补我们关于这些糖结合受体如何区分自我和非我的知识的重要空白。

Sugars on the surface of bacteria, viruses, fungi and parasites that inhabit and infect human hosts form an important means of identifying these micro-organisms. Lectins, which are sugar-binding proteins found on human cells and in the blood, can distinguish between the different sugar structures on human cells and those on many types of micro-organisms. This form of recognition can be useful as a means of differentiating self from non-self, which can in turn serve as a basis for innate immunity. For example, soluble lectins in the blood bind sugars on bacteria and activate a special pathway of complement fixation to directly attack and kill them. Cell-surface lectins on macrophages bind sugars on bacteria and viruses, causing them to be internalized and destroyed. Lectins can also initiate protective inflammatory responses in which immune cells are recruited to sites of infection.To fulfil these functions, lectins in the human system are hard-wired to recognize and attack potential pathogens based on their surface sugars. However, some bacteria, such as commensals that constitute the microbiota, are not attacked. Some microbes have also developed the ability to hijack host lectins as a way to enter and attack host cells. By a combination of biochemistry, structural biology, genetics and genomics, multiple families of human lectins have been defined and many are well characterized. However, knowing the receptors and the types of sugars that they interact with is not sufficient to allow us to predict which receptors will bind to what micro-organisms and what the consequences will be. The key problem is that sugars on micro-organisms are very diverse and many are not fully characterized.The work that we propose is designed to address this problem by developing a lectin array as a tool for screening the human repertoire of lectins against micro-organisms. The array will consist of the immobilized carbohydrate-binding domains from the major families of human lectins, so that the panel of lectins can be probed with fluorescently-labelled viruses, bacteria and fungi. In a single experiment, it will then be possible to see which receptors are able to recognize and bind to each individual microbe. We have recently created the first mammalian lectin array, containing cow lectins, to demonstrate the feasibility of this approach. The proposed studies will provide our first overall view of how the human host interacts with sugars on both pathogenic and non-pathogenic micro-organisms so that it can respond differently to different challenges.The lectin array to be developed is a discovery tool that will help to identify receptors that may be responsible for early responses to micro-organisms. Once the array is set up, one potential application will be to screen novel and emergent pathogens quickly to see which receptors they can bind to, in order to provide information about how they may enter cells and whether the innate immune response may effectively control an infection. Comparison of the human array with our prototype animal lectin array will also provide some indication of common types of interactions that might help micro-organisms move between species. Genetic variations in the human population result in changes in the amino acid sequences of some of the sugar-binding receptors, which can affect their sugar-binding properties and thus change the way that individuals respond to particular microbes. In a further development of the human lectin array, we will examine the effects of sequence variations, identified in large-scale genomic studies, on interactions of the panel of human lectins. We will also follow up results of array screening by examining which sugars on the surfaces of the target micro-organisms interact with specific lectins, filling important gaps in our knowledge of how these sugar-binding receptors distinguish between self and non-self.