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御入侵病原微生物的第一道防线。即使在脊椎动物中，由于其复杂的基于T和B淋巴细胞的适应性免疫反应，先天免疫系统总是首先对免疫挑战做出反应。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防御机制，包括细胞和体液。体液免疫效应物中有抗微生物多肽(AMP)，它们在整个动物界和植物中都得到了基本的鉴定。AMP不仅分布广泛，而且结构也呈现出很大的多样性。在无脊椎动物的免疫研究方面，昆虫(特别是果蝇)受到了大多数人的关注，对这一门的抗细菌和抗真菌反应(包括AMP的参与)也有很多了解。然而，尽管甲壳类在环境中很重要，而且作为一种水产养殖和野生捕获的食物来源，但相对较少受到关注。最近，在凡纳滨对虾中发现了一类新的抗微生物分子--对虾素类。对虾毒素是在颗粒血细胞中合成的，最初鉴定出3类不同的对虾毒素，其中一类中发现了3种微小的结构变体(对虾毒素3)。初步研究结果表明，对虾毒素至少有4类，每一类在结构上都有很大的差异。本项目的目标是了解对虾类化合物多样性的性质和来源，并确定已确定的新的(第四类)对虾类化合物的抗微生物活性。该项目将通过审查对虾多样性的遗传基础来解决第一个目标。这个假设是，观察到的对虾类化合物的可变性，无论是作为多个类别还是作为每个类别中的变体，都是遗传编码的。将创建重组对虾基因组文库，并对对虾蛋白基因进行克隆、定位和测序。将S的对虾毒素基因的结构与血细胞文库中的对虾毒素克隆的序列进行比较，可以全面分析对虾毒素变异的遗传基础。将定义对虾毒素基因的数量，推断可选的RNA加工途径(可选外显子的使用)的贡献，以及任何其他变异来源对观察到的对虾毒素多样性的贡献。第二个目标将与蒙彼利埃大学的伊夫林·巴切尔博士合作实现。这类新定义的对虾毒素(Penaeidin 4)将在酵母中以重组形式表达，进行纯化，并对其抗微生物功能进行表征。在抗菌肽中，对虾毒素表现出非常高的变异性，即使在单个个体内也是如此。这个项目将有助于我们理解这种变异性的基础，以及从这些多肽的抗微生物活性光谱来看，它对甲壳类免疫的意义。

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.