SpTransformer proteins from the purple sea urchin opsonize bacteria, augment phagocytosis, and retard bacterial growth

Abstract

The purple sea urchin, Strongylocentrotus purpuratus, has a complex and robust immune system that is mediated by a number of multi-gene families including the SpTransformer (SpTrf) gene family (formerly Sp185/333). In response to immune challenge from bacteria and various pathogen-associated molecular patterns, the SpTrf genes are up-regulated in sea urchin phagocytes and express a diverse array of SpTrf proteins. We show here that SpTrf proteins from coelomocytes and isolated by nickel affinity (cNi-SpTrf) bind to Gram-positive and Gram-negative bacteria and to Baker’s yeast, Saccharomyces cerevisiae, with saturable kinetics and specificity. cNi-SpTrf opsonization of the marine bacteria, Vibrio diazotrophicus, augments phagocytosis, however, opsonization by the recombinant protein, rSpTrf-E1, does not. Binding by cNi-SpTrf proteins retards growth rates significantly for several species of bacteria. SpTrf proteins, previously thought to be strictly membrane-associated, are secreted from phagocytes in short term cultures and bind V. diazotrophicus that are located both outside of and within phagocytes. Our results demonstrate anti-microbial activities of native SpTrf proteins and suggest variable functions among different SpTrf isoforms. Multiple isoforms may act synergistically to detect a wide array of pathogens and provide flexible and efficient host immunity.

The Axial Organ and the Pharynx Are Sites of Hematopoiesis in the Sea Urchin

“…Coelomocyte proliferation was stimulated by removing 1.5% or 5% (once or twice) of the estimated BV from adult IQ animals by inserting a needle through the peristomial membrane and into the coelomic cavity and aspirating 0.24–3.45 ml of CF depending on animal size and percentage of the BV to deplete. CF (50 μl) was removed from control animals and diluted into an equal volume of an anticoagulant, calcium- and magnesium-free seawater with EDTA and HEPES [CMFSW-EH; 460 mM NaCl, 10.73 mM KCl, 7.06 mM Na 2 SO 4 , 2.38 mM NaHCO 3 , 70 mM EDTA, 20 mM HEPES; pH 7.4 (10, 18)] for the purposes of cell counts. Proliferation was evaluated based on the incorporation of EdU (25 mM in aCF; ThermoFisher), which was injected on day 0 into each animal through the peristomial membrane for final concentration of 0.18 mM based on BV estimates.…”

“…Coelomocytes (10 5 cells) from IQ sea urchins were centrifuged for 5 min at 1,000 × g at 4°C onto Superfrost Plus slides (Shandon; ThermoScientific) using three-slot chimneys and cytospin slide holders (Eppendorf) as described (10) and cells were fixed for immunofluorescence according to Brockton et al (23). EdU was detected using an Alexa Fluor 555 Click-It EdU Imaging Kit (Invitrogen) according to the manufacturer's protocol.…”

“…Proliferative responses are consistent with a turnover of cells that participate in phagocytosis to clear microbes. In adult sea urchins, phagocytosis is carried out by the phagocyte class of coelomocytes (10) that decrease in concentration during clearance of bacteria injected into the coelom, and subsequently return to pre-challenge levels (11, 12). Coelomocyte proliferation is evident but low in non-challenged sea urchins as measured by 3 H-thymidine incorporation (13) or BrdU incorporation (14).…”

“…The cellular innate immune system of the purple sea urchin, Strongylocentrotus purpuratus , is mediated by four morphologically distinct types of coelomocytes that include phagocytes, colorless, and red spherule cells, and vibratile cells [(15), reviewed in (16, 17)]. Phagocytes respond to immune challenges through phagocytosis (10), encapsulation, syncytia formation (18), and expression of complement components (19) and the SpTransformer ( SpTrf , formerly Sp185/333 ) gene family (20–22), which encodes a large array of secreted, anti-pathogen SpTrf proteins (10, 23–27). In immune challenged larvae, SpTrf gene expression is restricted exclusively to filopodial blastocoelar cells (28) that are likely homologous to adult phagocytes.…”

The role of fasting on spine regeneration and bacteremia in the purple sea urchin Strongylocentrotus purpuratus

“…Matranga et al [19] found elevated red spherula counts in Paracentrotus lividus urchins with spineless lesions covering 10 to 20% of the body whereas mechanical damage is known to increase ceolomocyte number and humoral molecule expression [20]. While many studies have investigated the immune response in sea urchins [21][22][23], few have considered the impact of spine damage on immune function and bacteremia.…”