Diversity of the 185/333 System and Innate Immunity in the Purple Sea Urchin

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Intellectual merit of the researchThe research project is an investigation of the 185/333 genes and expressed proteins, which is a large and highly diverse family with putative innate immune functions. One of the surprising results from the sea urchin genome annotation was the discovery that the immune system is very complex. Immune diversification is essential in the arms race between eukaryotic hosts and their pathogens, which, for long-lived hosts, requires molecular mechanisms for gene diversification. Vertebrates employ somatic rearrangement or assembly of immune genes, whereas several novel mechanisms are employed by invertebrates. Understanding the diversity and diversification of the 185/333 system is the central aim of this project. The genes (there are ~50 tightly clustered loci) encode most of the sequence diversity within the second of two exons. No gene sequence is shared among sea urchins, and there are only a few sequence matches between genes and mRNAs from individual animals. There are three types of 5' flanking regions (the putative cis promoters) based on sequence variations. They putatively regulate differential gene expression observed in response to different pathogen associated molecular patterns (PAMPs). Similarly, different suites of 185/333 proteins are expressed in different sea urchins and in response to different PAMPs. Molecular weight and iso-electric point ranges are much broader than predicted from deduced amino acid sequences. Diversification of this system may act on several levels; regulated gene expression, significant gene recombination that increases gene diversity; low fidelity of transcription and/or post transcriptional modifications to the mRNAs, and post translational modifications to the proteins.Dr. Smith will investigate the diversification of the 185/333 system. "Finishing" level genomic sequence of ~280kB encoding up to 55 paralogous 185/333 alleles will provide data to formulate hypotheses on the diversification and/or recombination mechanisms that act on this gene family. Whether the 185/333 genes show any restriction in expression in single coelomocytes using single cell PCR will be investigated. 185/333 protein function will be tested for antimicrobial activity and involvement in cell aggregation and encapsulation, and a gene knockdown system will be established for sea urchin immune cells. Because of the close relationship between echinoderms and chordates, the sea urchin immune system will be important for deducing the evolution of immunity within the deuterostome lineage of animals.Broader impacts resulting from the researchThe new paradigm in the field of invertebrate immunology states that invertebrate immune responses are neither simple nor static. This change has been driven by the identification of complex diversification mechanisms in innate immune responses, including results on the diversity of the 185/333 system published during the prior award period. Future research on this system will provide projects for postdocdtoral fellows, graduate students, undergraduates, technicians, and local high school students. Women and those from underrepresented groups will continue to be recruited to the lab to participate in various aspects of the project. Students will be trained in a multidisciplinary approach that requires familiarity with immunology, molecular biology, cell biology, biochemistry, microbiology, genomics, proteomics, bioinformatics, evolution, systematics and invertebrate zoology including sea urchin development, anatomy and ecology. Integration of research and teaching will include a bioinformatics project for undergraduates enrolled in Cell Biology to participate in annotating the sea urchin genome, of which 56% of the gene models have not been annotated. Results from student projects will be uploaded into the annotation website and will become publically available.