NSF Award
0516279
Invertebrate models have led the way in broadening our understanding of the importance and evolution of innate non-adaptive immunity. Research in arthropods led to the original discovery of antimicrobial peptides (AMPs), which in turn led to the discovery of previously unappreciated signal transduction pathways that regulated these AMPs. These pathways have since been found distributed across diverse phyla, including vertebrates where they regulate important aspects of innate immunity. Crustaceans (and shrimp in particular) are critical components of the marine ecosystem and have only recently received attention as a source of insight into the regulation of immune responses. Expressed in the hemocytes of the Pacific whiteleg shrimp, Litopenaeus vannamei, penaeidins are among the best-studied crustacean AMPs. These AMPs are unique in that they have a two-domain structure with a proline-rich region that bears a conserved Pro-Arg-Pro region seen in other AMPs and a cysteine-rich region with a stabilized a-helix reminiscent of defensins. Penaeidins are also of interest because they exhibit considerable sequence diversity, being divided into three classes with multiple isoforms within each class. Levels of expression differ for each class. Recent research indicates that there is significant diversity of microbial target specificity between classes of penaeidins, which has not been observed previously in a single family of AMPs. The combined observations of significant target specificity and simultaneous expression of all classes with each class expressed at different levels led to the hypothesis that: Penaeidin gene expression is differentially induced by distinct immune challenges, which results in differential resistance to specific infectious agents. The project addresses this idea in several ways, including quantification of message and peptide concentration after different immune challenges (e.g. bacterial, fungal and viral). Specific induction of a particular AMP class by immune challenge would be a unique finding, as it would indicate that hemocytes are specifically responsive to particular immune stimuli. Second, the structure of the penaeidin gene cluster will also be defined in order to begin to understand the underlying mechanism for this induction. Third, the penaeidin-less phenotype will be assessed in an in vivo bioassay system using RNA interference (RNAi) to knockdown the penaeidins. Fourth, this phenotype will be compared with the in vitro specific microbial target range of penaeidin family by standard antimicrobial assays using an expanded panel of microbes to examine and compare specific resistance to particular microbial infection. An improved understanding of the components and mechanisms of disease resistance in shrimp should have broad implications in the understanding of immunity in arthropods and enhance understanding of the evolution of immunity in general. This project will also involve several students at various levels and train them in molecular biology, protein chemistry, and various bioassay techniques.
