Shrimp Immunity: Genetic and Functional Studies of Diversity in an Antimicrobial Peptide.

先天的非适应性免疫反应是所有动物抵抗病原微生物入侵的第一道防线。 即使在脊椎动物中，其复杂的T和B淋巴细胞为基础的适应性免疫反应，它总是先天免疫系统，首先响应免疫挑战。 然而，在无脊椎动物中，先天非适应性免疫不仅是它们的第一道防线，而且是它们唯一的免疫防线。 先天免疫系统利用一系列细胞和体液防御机制。 在体液免疫效应物中有抗微生物肽（AMP），其基本上已在整个动物界和植物中被鉴定。 AMP不仅分布广泛，而且结构多样。 在对无脊椎动物的免疫研究方面，昆虫（特别是果蝇）受到了大多数关注，并且对该门中的抗细菌和抗真菌反应（包括AMP的参与）有很多了解。然而，甲壳类，尽管它们在环境中的重要性，作为水产养殖和野生捕捞的食物来源，得到了相对较少的关注。最近，在太平洋白色对虾（Litopenaeus vannamei）中发现了一个新的抗菌分子家族--Penaeidins。 Penaeidin在颗粒血细胞内合成，最初鉴定出3种不同的Penaeidin，其中一种（Penaeidin 3）中发现了3种微小的结构变体。从我的研究的初步结果表明，有至少4类penaeidin，并有广泛的结构变化，在每一类。该项目的目标是了解penaeidin的多样性的性质和来源，并确定已定义的新（第四）类penaeidin的抗微生物活性。该项目将通过研究Penaeidin多样性的遗传基础来实现第一个目标。 假设是，观察到的变化的penaeidin，作为多个类和每个类内的变体，是遗传编码。 建立重组对虾基因组文库，克隆对虾毒素基因，定位并测序。 比较penaeidin基因的结构与来自血细胞cDNA文库的penaeidin克隆的序列将允许对penaeidin变异性的遗传基础进行全面分析。 将定义penaeidin基因的数量，推导替代RNA加工途径（替代外显子使用）的贡献，以及任何其他变异来源对观察到的penaeidin多样性的贡献。 第二个目标将与蒙彼利埃大学的Evelyn Bachere博士合作实现。 新定义的penaeidin类（penaeidin 4）将在酵母中表达为重组形式，纯化，并表征其抗微生物功能谱。 在抗微生物肽中，Penaeidin显示出非常高水平的变异性，甚至在单个个体内。 该项目将有助于我们了解这种变异性的基础及其对甲壳动物免疫的意义，这些肽的抗微生物活性谱。

Innate, non-adaptive immune reactions are the first line of defense against invading pathogenic microorganisms in all animals. Even in vertebrates, with their complex T and B lymphocyte-based adaptive immune responses, it is always the innate immune system that responds first to immune challenge. However, in invertebrates innate non-adaptive immunity is not only their first line of defense but their only line of immune defense. The innate immune system utilizes a battery of defense mechanisms, both cellular and humoral. Amongst the humoral immune effectors are the anti-microbial peptides (AMP) which have been identified essentially throughout the animal kingdom and in plants as well. Not only are AMP widely distributed, but they also show a large diversity of structures. In terms of immune studies on invertebrates, the insects (especially Drosophila) have received the majority of attention, and much is known about anti-bacterial and anti-fungal responses (including the involvement of AMPs) in this phylum. However, the crustacea, despite their importance in the environment and as both an aquacultured and a wild-caught food source, have received comparatively little attention. Recently, a new family of anti-microbial molecules, the penaeidins, has been identified in the Pacific white shrimp, Litopenaeus vannamei. The penaeidins are synthesized within the granular hemocytes, and initially 3 distinct classes of penaeidin were identified, with 3 minor structural variants being found within one of the classes (penaeidin 3). Preliminary results from my research indicate that there are at least 4 classes of penaeidin, and that there is extensive variation in structure within each class. The objectives of this project are to understand the nature and source of diversity in the penaeidins and to define the anti-microbial activities of the new (fourth) class of penaeidin that has been defined. The project will address the first objective by examining the genetic basis of penaeidin diversity. The hypothesis is that the observed variability of the penaeidins, both as multiple classes and as variants within each class, is genetically encoded. A recombinant shrimp genomic library will be created and the penaeidin genes will be cloned, mapped and sequenced. Comparison of the structure of the penaeidin gene(s) with the sequences of the penaeidin clones from hemocyte cDNA libraries will permit a full analysis of the genetic basis of penaeidin variability. The number of penaeidin genes will be defined, the contribution of alternative RNA processing pathways (alternative exon usage), and the contribution of any other source of variation to the observed diversity of penaeidins, will be deduced. The second objective will be approached in collaboration with Dr. Evelyn Bachere, of the Universite Montpellier. The newly defined class of penaeidins (penaeidin 4) will be expressed as a recombinant form in yeast, purified, and characterized for the spectrum of its anti-microbial functions. Penaeidins show, amongst the anti-microbial peptides, very high levels of variability, even within a single individual. This project will contribute to our understanding of the basis of this variability and its significance to crustacean immunity in terms of the spectrum of anti-microbial activities of these peptides.