Single Sea Urchin Phagocytes Express Messages of a Single Sequence from the DiverseSp185/333Gene Family in Response to Bacterial Challenge

Abstract

Immune systems in animals rely on fast and efficient responses to a wide variety of pathogens. The Sp185/333 gene family in the purple sea urchin, Strongylocentrotus purpuratus, consists of an estimated 50 (±10) members per genome that share a basic gene structure but show high sequence diversity, primarily due to the mosaic appearance of short blocks of sequence called elements. The genes show significantly elevated expression in three subpopulations of phagocytes responding to marine bacteria. The encoded Sp185/333 proteins are highly diverse and have central effector functions in the immune system. In this study we report the Sp185/333 gene expression in single sea urchin phagocytes. Sea urchins challenged with heat-killed marine bacteria resulted in a typical increase in coelomocyte concentration within 24 h, which included an increased proportion of phagocytes expressing Sp185/333 proteins. Phagocyte fractions enriched from coelomocytes were used in limiting dilutions to obtain samples of single cells that were evaluated for Sp185/333 gene expression by nested RT-PCR. Amplicon sequences showed identical or nearly identical Sp185/333 amplicon sequences in single phagocytes with matches to six known Sp185/333 element patterns, including both common and rare element patterns. This suggested that single phagocytes show restricted expression from the Sp185/333 gene family and infers a diverse, flexible, and efficient response to pathogens. This type of expression pattern from a family of immune response genes in single cells has not been identified previously in other invertebrates.

The SpTransformer Gene Family (Formerly Sp185/333) in the Purple Sea Urchin and the Functional Diversity of the Anti-Pathogen rSpTransformer-E1 Protein

“…There are four major morphotypes of coelomocytes in S. purpuratus that include phagocytes, red and colorless spherule cells, and vibratile cells (24), and only some of the phagocyte class of coelomocytes express the SpTrf proteins (41, 43). Surprisingly, the cells with the highest SpTrf expression are the small phagocytes in which the proteins are localized to cytoplasmic vesicles and the cell surface (Figure 6B).…”

“…This would be advantageous in responding to infections and to protect the host from being overwhelmed by and succumbing to a pathogen. Surprisingly, when single phagocytes are evaluated for SpTrf transcripts, not only do most of the individual cells yield SpTrf amplicons of the same size (Figure 7) but the amplicon sequences from single cells are the same (41). This implies that one gene from the SpTrf family is expressed per individual phagocyte.…”

“…We propose an overall model for SpTrf protein function in response to bacterial challenge that attempts to include the results described in this review (Figure 12). Individual phagocytes appear to express a single SpTrf gene and produce a single SpTrf protein (41), given minor changes from mRNA editing (30). SpTrf proteins are stored in perinuclear vesicles of phagocytes (Figures 7B–D) (41, 43) and are speculated to be inactive with regard to binding and multimerization.…”

The Recombinant Sea Urchin Immune Effector Protein, rSpTransformer-E1, Binds to Phosphatidic Acid and Deforms Membranes

“…Native SpTrf and HeTrf proteins are found in all morphotypes of sea urchin phagocytes, on the surface of small phagocytes in association with the plasma membrane and with the membranes of cytoplasmic vesicles (14, 19–21). But rather than integrated into membranes via transmembrane regions or associated through GPI linkages, rSpTrf-E1 and its rGly-rich and rHis-rich fragments may associate with membranes, at least in part, by binding directly to PA or other lipids with exposed phosphate groups.…”

“…Native SpTrf proteins are present within the perinuclear vesicles of all types of phagocytes and are present on the surface of small phagocytes, although the percentage of cells that express the proteins is variable among animals (14, 19). HeTransformer proteins (HeTrf, formerly He185/333) have also been noted in association with vesicle membranes and with plasma membranes of gut-associated amoebocytes (an alternative term for phagocytes) from another sea urchin species, Heliocidaris erythrogramma (20, 21).…”