L. Courtney Smith
- Professor of Biology
- SEH 5690
- [email protected]
Areas of Expertise
Molecular evolution of immune systems; cell and molecular biology of immune responses in echinoderms.
Our interest in Marine Biomedicine is centered on understanding the innate immune system of invertebrates and focused on the purple sea urchin, Strongylocentrotus purpuratus. Annotation of the sea urchin genome and the identification of gene models encoding proteins that function in immunity has demonstrated that innate immune system of this long-lived invertebrate is complex and sophisticated. Many of the immune response genes are expressed in the immune cells, or coelomocytes, that become activated in response to infection or injury. EST analysis has identified a number of genes that are upregulated in activated coelomocytes in response to lipopolysaccharide (LPS), other pathogen associated molecular patterns, and heat killed Vibrio diazotrophicus (a marine bacterium). These include homologues of the complement cascade, a number of lectins, a Tie receptor homologue (a putative growth factor receptor), antimicrobial peptides called SpStrongelocins, and a family of genes called Sp185/333, among other genes.
The Sp185/333 genes are strongly upregulated in response to immune challenge and were identified by suppression subtractive hybridization. BLAST analysis of the resulting ESTs matched to two submissions in GenBank called DD185 and EST333. We combined the numbers for a non-descript name because the deduced amino acid sequences do not have conserved domains and do not show matches to known proteins. The messages are unusual because optimal alignments require the insertion of large gaps that define 25 blocks of sequence called elements. The high level of sequence diversity among the messages is primarily based on the presence and/or absence of elements that generate recognizable element patterns, in addition to small indels and SNPs. Curiously, about half of the messages encode proteins with early stop codons from SNPs and missense sequences resulting from frameshifts.
An evaluation of the sea urchin genome (ver. 0.5) identified a few Sp185/333 genes, which appear tightly clustered, in agreement with amplicon sizes from intergenic PCR. The genes are 2kB or less and are composed of two exons, of which the first encodes a leader sequence and the second encodes the mature protein. The second exon includes the mosaic pattern of elements in addition to a variety of both tandem and interspersed repeats. The presence of repeats enables a second, equally optimal alignment of the sequences wherein the elements are correlated with the repeats. Detailed computational analysis of the repeat and element sequences strongly suggests that the genes undergo frequent recombination, and molecular clock analysis suggests that the extant members of the family are evolutionarily very young. When attempts were made to match messages to the most likely gene from which they had been transcribed, another level of diversification was identified. The messages are known to encode truncated proteins, some with missense sequence, however, the genes have perfect open reading frames encoding full-length proteins. The result of sequence matches between messages and genes shows very poor identity for individual animals, suggesting a mechanism for editing the messages either during or after transcription. The preponderance of C to U substitutions in genes compared to messages suggest that a cytidine deaminase may be involved.
Although the sea urchin genome assembly (ver. 2.6) predicts the presence of only six Sp185/333 genes, estimates suggest that there are 50 (+10) family members, indicating significant assembly difficulties for region(s) of the genome harboring these genes. To clarify the structure this gene family, the assembly of a BAC insert yielded a cluster of six Sp185/333 genes. BAC sequence assembly required bioinformatic and molecular analysis to generate a single insert sequence rather than multiple unconnected fragments, and to verify that no genes had been collapsed or duplicated. The assembly of the region encoding the Sp185/333 genes was challenging for a number of reasons. The genes are tightly clustered with five positioned within 20 kB and the sixth located 14 kB away with the outer flanking genes oriented in the same direction and the four inner genes are oriented in the opposite direction. Each gene and short flanking regions is surrounded by GA microsatellites, and the region between the microsatellites is much more similar to other such regions than are the sequences located outside the microsatellites. There are three segmental duplications within the BAC insert that show 99.7% identity that include three almost identical genes and are surrounded by GAT microsatellites. This genomic region harboring the cluster of Sp185/333 genes suggests significant levels of gene conversion, recombination, local and ectopic duplication, and general genomic instability. Yet, no pseudogenes with altered reading frames have been identified. These results present questions about possible mechanisms for promoting gene diversification while blocking both the formation of pseudogenes and the homogenization of the entire region from gene conversion. It is not clear whether the microsatellites are involved in maintaining the structure and sequence diversity of the region but their presence and positioning is intriguing. The structure of the genomic region is curious and may be required for high rates of diversification of the Sp185/333 gene family, which would be beneficial to the sea urchin as an underlying mechanism to keep pace in the arms race with pathogen diversification.
The Sp185/333 proteins are expressed in two subsets of the phagocyte class of coelomocytes. The polygonal cells are the largest coelomocytes and when spread on glass they have a polygonal shape. The Sp185/333 proteins are located within perinuclear vesicles in some of these cells. Small phagocytes are perpetually filopodial and do not spread into lamellipodial morphology. They also express Sp185/333 proteins which are present in cytoplasmic vesicles and are present on the extracellular surface of the cells, including the filopodia. The number of Sp185/333-positive coelomocytes increases significantly after immune challenge, in agreement with increases in gene expression. In addition to coelomocytes, the major tissues of the sea urchin, including pharynx, esophagus, gut, gonad, and axial organ, also have Sp185/333-positive cells, although these cells may be coelomocytes. It is noteworthy that the axial organ shows a significant increase in Sp185/333-positive cells after challenge with LPS whereas the other organs do not.
Evaluation of recombinant Sp185/333 protein function has been difficult because these proteins are toxic to bacteria when used in expression systems. However, preliminary work suggests that one recombinant Sp185/333 protein binds to bacteria and agglutinates them into large clumps. These opsonization and agglutination activities would be initial steps required prior to phagocytosis and clearance of invading bacteria by coelomocytes.
Immune gene sequence diversity in metazoans is a consequence of the arms race between a host and its pathogens, and is directly related to the ecological niche in which it lives. The array of pathogens in contact with the host, including what the host eats and its mucosal microflora shapes the immune response and the genes that function within it over evolutionary time. Different hosts appear to have quite different means to diversify the proteins that function in pathogen detection and immune defense. Rearrangement of the immunoglobulin class of genes in higher vertebrates is a classic example of how protein sequences of antibodies and T cell receptors are diversified. The assembly of the variable lymphocyte receptors in cyclostomes is another example. The notion of genomic instability for a region that harbors immune genes such as the Sp185/333 genes is an intriguing idea for generating immune gene sequence diversity in animals lacking rearranging capabilities.
B.A., Drake University, 1974
M.S., University of Minnesota, 1976
Ph.D., University of California, Los Angeles, 1985, with William Hildemann
Post-Doctoral Work: California Institute of Technology with Eric Davidson
Golconda P, Buckley KM, Reynolds CR, Romanello JP, Smith, LC. 2019. The axial organ and the pharynx are sites of hematopoiesis in the sea urchin. Frontiers in Immunology 10:870. Supplementary 1.
Chou, H-Y, CM Lun, LC Smith. 2018. The SpTransformer proteins from the California purple sea urchin opsonize bacteria, augment phagocytosis, and retard bacterial growth. PLoS ONE 13(5):e0196890. Supplementary 1.
Lun, CM, R Samuel, SD Gillmor, A Boyd, LC Smith. 2017. SpTransformer, a recombinant Sp185/333 protein, binds to phosphatidic acid and deforms membranes. Frontiers in Immunology, 8:481. Supplementary Video.
Lun, CM, BM Bishop, LC Smith. 2017. Multitasking immune Sp185/333 protein, rSpTransformer-E1, and its recombinant fragments undergo secondary structural transformation upon binding targets. Journal of Immunology, 198(7):2957-2966. Supplementary 1.
Smith, LC, TA Hawley, JH Henson, AJ Majeske, M Oren, B Rosental. 2019. Methods for collection, handling, and analysis of sea urchin coelomocytes. In Methods in Cell Biology (K Foltz, A Hamdoun, eds), Elsevier. Vol 150, Part A, Chapter 16.
Smith, LC, V Arizza, MA Barela Hudgell, G Barone, AG Bodnar, KM Buckley, V Cunsolo, N Dheilly, N Franchi, SD Fugmann, R Furukawa, J Garcia-Arraras, JH Henson, T Hibino, ZH Irons, C Li, CM Lun, AJ Majeske, M Oren, P Pagliara, A Pinsino, DA Raftos, JP Rast, B Samasa, D Schillaci, CS Schrankel, L Stabili, K Stensväg, E Sutton. 2018. Echinodermata: The Complex Immune System in Echinoderms. In “Advances in Comparative Immunology”, EL Cooper, ed. Springer Publisher. Chapter 13, pp 409-501.
Smith, LC, CM Lun. 2017. The SpTransformer gene family (formerly Sp185/333) in the purple sea urchin and the functional diversity of the anti-pathogen rSpTransformer-E1 protein. Frontiers in Immunology 8:725.
LC Smith, MR Coscia. 2016. Tuning the host-pathogen relationship through evolution with a special focus on the echinoid Sp185/333 system. Invertebrate Survival Journal, 13:355-373.
Smith LC, Lun CM. 2016. Research Highlight: Invited Multitasking rSp0032 has anti-pathogen binding activities predicting flexible and effective immune responses in sea urchins mediated by the Sp185/333 system. Pathogens and Infectious Disease 2:e1394.
Oren, M, MA Barela Hudgell, P Golconda, CM Lun, LC Smith. 2016. Genomic instability and shared mechanisms for gene diversification in two distant immune gene families: the plant NBS-LRR genes and the echinoid 185/333 genes. In “The Evolution of the Immune System, Conservation and Diversification” (D Malagoli, ed.). Elsevier Inc. Academic Press, London. pp. 295-310.
Stokes, BA, S Yadav, U Shokal, LC Smith, I Eleftherianos. 2015. Bacterial and fungal pattern recognition receptors in homologous innate signaling pathways of insects and mammals. Frontiers in Microbiology 6:19.
Smith LC (2012) Innate immune complexity in the purple sea urchin: diversity of the Sp185/333 system. Frontiers in Immunology 3: 70.
Ghosh, J, CM Lun, AJ Majeske, S Sacchi, CS Schrankel, LC Smith. 2011. Invertebrate Immune Diversity. Developmental and Comparative Immunology, 35: 959-974.
Smith, L.C., J. Ghosh, K.M. Buckley, L.A. Clow, N.M. Dheilly, T. Haug, J.H. Henson, C. Li, C.M. Lun, A.J. Majeske, V. Matranga, S.V. Nair J.P. Rast, D.A. Raftos, M. Roth, S. Sacchi, C.S. Schrankel, K. Stensvåg. 2010. Echinoderm Immunity. In “Invertebrate Immunity” K. Soderhall, ed. Madame Curie Bioscience Database, Landes Biosciences, Austin TX. Advances in Experimental Medicine and Biology, 708: 260-301.
Smith, LC. 2010. Diversification of innate immune genes: lessons from the purple sea urchin. Disease Models and Mechanisms, 3: 274-279. (electronic publication)
Ghosh J, KM Buckley, SV Nair, DA Raftos, C Miller, AJ Majeske, T Hibino, JP Rast, M Roth, LC Smith. 2010. Sp185/333: A novel family of genes and proteins involved in the purple sea urchin immune response. Developmental and Comparative Immunology, (34: 235-245)
Smith, L.C., J.P. Rast, V. Brockton, D.P. Terwilliger, S.V. Nair, K.M. Buckley & A.J. Majeske. 2006. The sea urchin immune system. Invertebrate Survival Journal, 3: 25-39.
Smith, L.C. 2005. Host responses to bacteria; innate immunity in invertebrates. In The Influence of Bacterial Communities on Host Biology (M. McFall-Ngai, N. Ruby, B. Henderson, eds.). Advances in Molecular and Cellular Microbiology 10: 293-320. Cambridge University Press.
Smith, L.C., L.A. Clow & D.P. Terwilliger. 2001. The ancestral complement system in sea urchins. Immunological Reviews 180: 16-34.
Smith, L.C. 2001. The complement system in sea urchins. In Phylogenetic Perspectives on the Vertebrate Immune Systems (G. Beck, M. Sugumaran, E. Cooper, eds.). Advances in Experimental Medicine and Biology, 363-372. Kluwer Academic/Plenum Publishing Co.New York, NY.
Smith, L.C., K. Azumi & M. Nonaka. 1999. Complement systems in invertebrates. The ancient alternative and lectin pathways. Immunopharmacology, 42: 107-120.
Gross, P.S., W.Z. Al-Sharif, L.A. Clow & L.C. Smith. 1999. Echinoderm immunity and the evolution of the complement system. Developmental and Comparative Immunology, 23: 429-442.
Smith, L.C. & E.H. Davidson. 1994. The echinoderm immune system: characters shared with vertebrate immune systems, and characters arising later in deuterostome phylogeny. In: Primordial Immunity: Foundations for the Vertebrate Immune System. (G. Beck, E.L. Cooper, G.S. Habicht and J.J. Marchalonis, eds.) The New York Academy of Sciences, 712: 213-226.
Smith, L.C. & E.H. Davidson. 1992. The echinoid immune system revisited: reply. Immunology Today, 14: 93-94.
Smith, L.C. & E.H. Davidson. 1992. The echinoid immune system and the phylogenetic occurrence of immune mechanisms in deuterostomes. Immunology Today 13: 356-362.
Additional publications on:
BISC 2202 - Cell Biology
BISC 3212 - Immunology
BISC 6205 - Current Topics in Cell and Molecular Biology
BISC 6218 - Innate Immunity