Intellectual merit of the research The 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 research The 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.
L. Courtney Smith, PI Intellectual Merit The immune systems in animals and higher plants show a wide range of approaches for detecting and responding to pathogens. The "arms race" between pathogens and hosts results in the diversification of pathogen virulence to overcome, evade or generally outsmart the host immune response, and the diversification of the animal or plant immune response to detect, kill and clear a pathogen. The outcome for animals and plants is that there are a number of mechanisms by which the host generates a large number of proteins that function in detecting and killing pathogens. Examples of highly diverse immune-response proteins include antibodies and T cell receptors in higher vertebrates, variable lymphocyte receptors in lampreys and hagfish, fibrinogen related proteins in snails that fight parasites, and non-self recognition proteins in some tunicates and hydroids that recognize the presence of other individuals and initiates fights to the death for space on which to grow. The purple sea urchin also has a set of diverse but similar proteins that combat pathogens that are encoded by the Sp185/333 gene family. This family is composed of about 50 genes are similar in structure and also show significant sequence diversity (Fig. 1). Six of the Sp185/333 genes are clustered together in a small region of the genome and have an unusual structure with sequence repeats called microsatellites that surround each gene (Fig. 2). These microsatellites may function to drive sequence diversification of the genes by duplication and deletion among other possible mechanisms. When the Sp185/333 genes are expressed messenger RNA is produced, which are edited so that the sequence is changed from that encoded by the genes. This results in a greater diversity of the Sp185/333 proteins and is another level of sequence diversification. An example of this diversity is the detection of about 260 different Sp185/333 proteins produced by a single sea urchin from only about 50 genes. The diversity of the Sp185/333 proteins is an advantage that the sea urchin has for fighting a broad array of pathogens. These proteins bind to certain marine bacteria (Figs. 3), cross-link the bacteria into aggregates, and induce the sea urchin immune cells to take up and kill bacteria (Fig. 4). These proteins have similar effects on inducing immune cells to take up yeast. The sea urchin Sp185/333 gene family and the wide diversity of proteins that it encodes is a unique example of an animal immune system that shows significant diversity that is employed in response to microbial infection. The level of RNA editing, which increases the diversity of the proteins, is surprisingly greater than has been identified in other systems in which RNA editing has been characterized. Although the mechanism for diversifying the genes is not currently known, it is likely to be new to immunology. While the diversification mechanism increases the sequence diversity of the Sp185/333 genes, it surprisingly does not produce genes that do not function properly. This goes against what is known in other families of genes that share sequence. Understanding the structure and diversification of the entire Sp185/333 gene family will be the focus of future research. Broader Impacts During the period of the award, including the no-cost extension years, training in research, writing and teaching was provided to 3 postdocs, 9 grad students, and 5 undergrads, which resulted in 14 publications and 8 honors or Masters Theses plus PhD Dissertations. Eight collaborators were involved in the research. Assistance in finding research opportunities was offered to 123 undergraduates. Two high school students received research opportunities. The PI gave 9 talks at seminars or conferences and students/postdocs presented many talks and posters at conferences at local, national and international meetings.
