The Complexities of the Sea Urchin Immune System. Dynamic Changes in Coelomocyte Populations and the Evolution of the SpTransformer Genes
Open AccessFor bacteria, fungi, plants, and animals, the ability to recognize self vs. non-self is an essential aspect of survival. The immune system functions by recognizing non-self and mounting a response upon detection of foreign contact. The results presented in this dissertation are the outcome of developing and applying both bioinformatic and wet lab methods to investigate components of the immune system of the California purple sea urchin (Strongylocentrotus purpuratus), focusing on cellular response to pathogen challenge and the SpTransformer (SpTrf) gene family. The SpTrf gene family is upregulated upon immune challenge, inferring the immune effector functions of the encoded proteins. Native SpTrf proteins bind bacteria and yeast, and augment phagocytosis of a marine Vibrio. An in-depth analysis of these genes in the sea urchin genome was conducted to understand the sequence complexities of this family and its genomic structure. SpTrf gene sequences have a series of internal repeats in a mosaic pattern that is characteristic of this gene family. This mosaic pattern and the internal repeats in the coding regions necessitates the insertion of large gaps, which has made sequence alignments computationally difficult. However, the bioinformatic program, PRANK, was developed for sequence alignments that require large gaps and insertions. It was tested for aligning the SpTrf gene family, and while it generated alignments similar to those done manually, there were limits to its ability to generate a robust and expected alignment. Based on to these results, alternative approaches were conducted using the non-coding regions of the SpTrf genes to derive a theoretical evolutionary history. Results suggested that these genes arose from multiple duplications and deletions, ectopic duplication and insertions, indels, and point mutations in the exons, which are consistent with the extant genes and family structure. Additional work using Lipofection methods was conducted in an attempt to identify the regulatory elements controlling expression of the SpTrf gene family in adult coelomocytes along with quantification of coelomocyte response to immune challenge. S. purpuratus possesses seven known distinct populations of coelomocytes in the coelomic fluid, which vary by morphology, size, and cellular complexity. Methods were developed for evaluating sea urchin coelomocytes both for flow cytometry and for lipofection using a commercial lipofection reagent packaged with a range of molecules. While lipofection of coelomocytes failed, a successful gating strategy for flow cytometry was developed, which was employed to quantify the large phagocytes, small phagocytes, red spherule cells, and a mixed population of vibratile cells and colorless spherule cells, and to track their changes over time in response to immune challenges. Fold changes in coelomocytes from immunoquiescent (healthy) sea urchins were evaluated over time. Results showed that sea urchin immune cell populations undergo dynamic changes in vivo in response to injury and show different responses to distinct immune stimuli. In general, the cellular immune responses in S. purpuratus are driven initially by changes in the large phagocyte populations.
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BarelaHudgell_gwu_0075A_15869.pdf | 2022-10-04 | Open Access |
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