The impact of CodeChi on the physicochemical properties and 3D organization of the peritrophic matrix from the model pest beetle Tribolium castaneum

大多数昆虫的中肠都有一层半透性的细胞外伪膜，称为周转基质(PM)，它具有改善肠道功能、对肠道病原体和寄生虫产生屏障以及保护其免受机械和化学损伤等重要功能。最近，研究还集中在PM在肠道天然免疫和昆虫-病原体相互作用中的作用。理解所有这些不同的功能需要对PM的组成及其3D组织的构建原则有更深入的了解。PM由不同的成分组成，这些成分赋予相应功能所需的特征属性。除了几丁质，N-乙酰氨基葡萄糖(GlcNAc)的聚合物似乎在某种程度上被脱乙酰化，各种几丁质相关的PM蛋白(PMPs)，其中一些被广泛糖基化，被认为是PM的主要结构元素PM的不同组成部分究竟是如何相互作用的，以及它们以何种方式相互影响，仍然是一个猜测。此外，几丁质被脱乙酰酶和几丁质酶修饰在组织PM的3D结构中的作用在很大程度上是未知的。我们假设，由几丁质脱乙酰酶和几丁质酶编写并被PMPs识别的脱乙酰度和聚合度以及脱乙酰方式(我们称之为Codechi)对PM在不同中肠区域的物理化学性质和3D组织有重要影响，从而影响屏障功能。在本项目中，我们将分析鞘翅目害虫赤霉菌中不同的PM成分。我们将重点研究几丁质纤维和PM蛋白(PMP)的结构与功能关系，这两种蛋白具有不同类型和数量的几丁质结合结构域(CBD)。我们的目标是了解PMPs如何帮助组织PM的纤维网络，从而产生一种通透性屏障，随着中肠前部和后部排除尺寸的减小，正如我们在卡氏锥虫幼虫身上所展示的那样。我们将确定在PM的不同中肠区域存在哪种类型的几丁质异构体，破译中肠特异的几丁质脱乙酰酶和几丁质酶的功能，并检测不同PMP的结合模式及其在三维组织几丁质纤维中的功能。最后，我们将阐明在不同数量的PMP中发现的不同类型的CBD之间在结合亲和力和选择性方面的可能差异。在此过程中，我们旨在解释PMPS如何组织PM中的3D网络，并控制其沿中肠的不同属性。这将为以PM为靶标的害虫防治活性化合物的开发提供基础知识。

The midgut of most insects is lined by a semipermeable, extracellular pseudo-membrane called peritrophic matrix (PM), which has several important functions such as improving the intestinal performance, generating a barrier against intestinal pathogens and parasites, and protecting from mechanical and chemical damage. More recently, research has also focused on the PM’s role in gut innate immunity and insect-pathogen interactions. Understanding all these different functions requires a deeper insight into PM composition and into the construction principles of its 3D organization. The PM is composed of varying constituents that confer characteristic properties needed for the respective function. Next to chitin, a polymer of N-acetylglucosamine (GlcNAc), which appears to be deacetylated to some extent, various chitin-associated PM proteins (PMPs), some of them extensively glycosylated, are considered major structural elements of the PM. It still remains speculative how the different components of the PM exactly interact and in which way they influence each other. In addition, the roles of chitin modification by deacetylases and chitinases in organizing the 3D structure of the PM are largely unknown. We hypothesize that the degree of deacetylation and polymerization as well as patterns of deacetylation (which we term CodeChi), written by chitin deacetylases and chitinases, and recognized by PMPs, have a major impact on physicochemical properties and 3D organization of the PM in different midgut regions and thus affect the barrier function. In this project, we will analyze different PM constituents in the coleopteran pest model Tribolium castaneum. We will focus on the structure-function relationships of chitin fibrils and PM proteins (PMPs), which have different types and numbers of chitin binding domains (CBDs). We aim to understand how PMPs help to organize the PM’s fibrillary network, so that a permeability barrier with decreasing exclusion sizes from anterior to posterior midgut regions is generated, as we have demonstrated it in larvae of T. castaneum. We will determine which type of chitin isomorph is present in the PM at different midgut regions, decipher the function of midgut-specific chitin deacetylases and chitinases, and examine the binding mode of different PMPs and their function in 3D organizing chitin fibrils. Finally, we will elucidate probable differences in binding affinities and selectivities between the various types of CBDs that are found in PMPs in different numbers. In doing so, we aim to explain how PMPs organize the 3D network in the PM and control its different properties along the midgut. This will finally provide fundamental knowledge for the development of active compounds targeting the PM for pest control.