Robert Unckless
Assistant Professor, Department of Molecular Biosciences and Center for Bioinformatics
University of Kansas

 

 

A functional dissection of the maintenance genetic variation in immune genes (2017-18)

Balancing selection is responsible for the maintenance of several disease-related traits in human populations, including the classic case of sickle-cell anemia. This proposal aims to understand the forces that maintain different immune gene alleles through balancing selection using a model system approach. The use of antimicrobial peptides as a focus of study may also provide for the development of new therapeutics for resistant pathogens.

Insects lack an adaptive immune system and so defense against pathogens is achieved by the innate immune response. An important component of innate immunity is the complement of antimicrobial peptides (AMPs) that are produced and secreted by cells upon infection. Variation in the genes encoding these AMPs is often maintained by balancing selection, the process by which multiple alleles are maintained at the same locus through various mechanisms. While instances of balancing selection are being reported more and more frequently, we lack a comprehensive understanding of the mechanistic basis of balancing selection in most examples. The ability to connect broad scale patterns of DNA sequence diversity to mechanistic differences in protein function would provide a comprehensive view of balancing selection. Furthermore, the identification of particular amino acid polymorphisms that are maintained by balancing selection facilitates the mechanistic study of balancing selection because the presumptive causative mutations are known a priori.

This project focuses on AMPs in the genetic model species, Drosophila. The use of Drosophila as a model system also allows for study of AMP variation in vivo in a way that is much more cost effective than several other model systems, while allowing the flexibility to move between in vitro and whole organism in vivo study. These peptides are ideal for the functional study of balancing selection because a) genetic variation in several peptides is maintained by balancing selection, providing a natural replication across genes, b) AMPs are effectors and thus interact directly with pathogens allowing for a more direct cause and effect relationship and c) AMPs are small and can be easily expressed or synthesized and studied in vitro. Aim 1 involves expression of AMPs in cell culture and subsequent pathogen inhibition assays. Two variants each of several different AMPs will be synthesized and tested against a panel of gram positive, gram negative and fungal pathogens. This will test the hypothesis that the AMP variants will show differential efficacy against some pathogens. Aim 2 will determine whether AMP variants exhibit different structural properties or differ in the way that they interact with pathogen molecules. Because of parallelism in the design of the proposal with multiple AMPs, it is expected that the results will allow for the development of a full R01 proposal to investigate the interaction between AMPs and pathogens both in vitro and in vivo