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

Medical University Of South Carolina, Charleston SC

Investigators

Abstract

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.