Chou HY et al. (2018), SpTransformer proteins from the purple sea urch...

ECB-ART-46316

PLoS One 2018 Jan 01;135:e0196890. doi: 10.1371/journal.pone.0196890.

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

Chou HY , Lun CM , Smith LC .

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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.

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Genes referenced: 185-333 LOC100887844 LOC590297
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	Fig 2. cNi-SpTrf proteins bind to bacteria and yeast.Vibrio diazotrophicus (6.2 X 109 cells), Saccharomyces cerevisiae (2.8 X 105 cells), Bacillus cereus (1.5 X 107 cells), or Bacillus subtilis (1.5 X 107 cells) were incubated with increasing amounts of cNi-SpTrf proteins isolated from four different sea urchins. Cells were washed, and analyzed by Western blot with anti-SpTrf antibodies. cNi-SpTrf proteins from each sea urchin bind to all target cells, but the patterns of the bands are different. Negative controls omitted the cNi-SpTrf proteins (0 μg) showing that neither the anti-SpTrf antibodies nor the secondary antibody bind directly to the bacteria or yeast. SpTrf proteins loaded directly onto the gel without pre-binding to V. diazotrophicus (‒, second lane for sea urchin 17) shows the pattern of bands. The first lane (0 μg/ml) of the blots for animals 18, 19, and 20, was moved closer to the lanes that show binding by the cNi-SpTrf proteins. This was done by digital methods to reduce the size of the figure and to make the comparisons among the lanes more clear. The blots were otherwise unaltered. Protein size standards in kDa are indicated to the right of each blot.
	Fig 3. cNi-SpTrf proteins isolated from a single sea urchin bind variably to Vibrio diazotrophicus, Saccharomyces cerevisiae, Bacillus cereus, and B. subtilis.cNi-SpTrf proteins (μg/ml) were incubated with bacteria and yeast and washed cells were evaluated with multiple Western blot using the identical protocol including imaging timing. Negative controls omitted the cNi-SpTrf proteins (0 μg/ml). The first lane in each image was moved closer to the lanes that show binding by the cNi-SpTrf proteins. This was done by digital methods to reduce the size of the figure and to make the comparisons among the lanes more clear. The blots were otherwise unaltered. Protein size standards in kDa are indicated on the left and right of the blots.
	Fig 4. cNi-SpTrf proteins augment phagocytosis whereas rSpTrf-E1 proteins do not.A. Opsonized Vibrio diazotrophicus was incubated with phagocytes for 70 min followed by fixing, staining, and analysis of the mean percentage of phagocytes (n = 500) that contained V. diazotrophicus. cNi-SpTrf proteins from three different sea urchins were evaluated for opsonization. Phagocytes were obtained from a sea urchin that was different from those that provided the cNi-SpTrf proteins or the cfCF used for opsonization. The percentage of phagocytes with FITC-cNi-SpTrf-Vibrio is intermediate between number of phagocytes with cfCF-Vibrio-FITC (positive control) and aCF-Vibrio-FITC (buffer control) (see Table 1 for definitions of the abbreviations). B. Opsonization of V. diazotrophicus with SpTrf proteins (cfCF-Vibrio-FITC) isolated from three sea urchins results in a greater percentage of phagocytes with bacteria than either FITC-SpTrf-E1-Vibrio or aCF-Vibrio-FITC. C. Vibrio diazotrophicus opsonized with cNi-SpTrf, rSpTrf-E1, cfCF or aCF were incubated with coelomocytes at a ratio of 100 bacteria per phagocyte. Phagocytosis of FITC-cNi-SpTrf-Vibrio results in similar numbers of V. diazotrophicus per phagocyte compared to cfCF-Vibrio-FITC, and both show significantly greater numbers of bacteria per phagocyte than aCF-Vibrio-FITC. Phagocytosis of FITC-SpTrf-E1-Vibrio results in similar numbers of bacteria per phagocyte as that for aCF-Vibrio-FITC. **, statistically significant; ns, not significant.

	Fig 6. cNi-SpTrf proteins are not contaminated with detectible amounts of SpC3.Pelleted coelomocytes were sonicated, the lysate was passed through a nickel column, and an eluted fraction containing cNi-SpTrf proteins was evaluated by Western blot with anti-SpC3-6His [64]. A sample of cfCF that was not subject to nickel affinity was evaluated on the same filter for comparison. The cNi-SpTrf proteins do not contain detectable traces of SpC3 while the cfCF fraction has an autolytic fragment of the SpC3 α chain [70] indicating the presence of an active complement protein homologue.
	Fig 7. SpTrf proteins are secreted from coelomocytes in culture and opsonize Vibrio diazotrophicus.Non-opsonized, heat-killed V. diazotrophicus were incubated with immune-activated coelomocytes for 24 hr at 14°C followed by fixing and staining for fluorescence microscopy. Some of the phagocytosed V. diazotrophicus and almost all of the extracellular V. diazotrophicus are coated with SpTrf proteins. The insert in the lower corner of the merge panel is a magnification of the perinuclear region of the central polygonal phagocyte. The nuclear color of the phagocytes was altered using the confocal microscope imaging program. Some phagocyte nuclei become dissociated from the cells during processing. Scale bar indicates 10 μm.
	Fig 8. SpTrf proteins and rSpTrf-E1 show partial activity to retard bacterial growth.Vibrio diazotrophicus, Bacillus cereus, B. subtilis, or Escherichia coli were grown to OD600 = 1, and 10 μl were incubated with increasing concentrations of cNi-SpTrf proteins isolated from sea urchins A, B, and C, or with increasing concentrations of rSpTrf-E1. Bacterial growth was determined in triplicate by measuring the turbidity by OD600 at 0, 2, and 4 hr. Data are presented as growth or OD600 relative to time in the presence of increasing amounts of SpTrf proteins (μg/200 μl). The mean background OD600 for the blank wells without bacteria was subtracted from the corresponding experimental time points. Student’s one-tailed t test was used to evaluate the significance of change in turbidity in the absence of SpTrf proteins compared to the range of SpTrf protein concentrations added to the cultures. Statistical significance is indicated by *, p ≤ 0.005; , p ≤ 0.05. ns, not significant.
	Fig 9. Putative trafficking and function of SpTrf proteins.The current hypotheses of SpTrf protein function is that they are initially produced and stored in perinuclear vesicles of phagocytes. Note that a single SpTrf gene is expressed in a single phagocyte according to Majeske et al. [47], which would produce a few versions of the SpTrf protein (shown in red) resulting from RNA editing [54] and post translational processing [53]. SpTrf proteins are secreted from vesicles upon bacterial detection but vesicles are also hypothesized to fuse with phagolysosomes and SpTrf proteins may function in the degradation of phagocytosed microbes. After secretion, multiple SpTrf isoforms (indicated in multiple colors) mix in the CF and multimerize upon opsonization of bacteria that augments phagocytosis and may retard bacterial growth. How phagocytes recognize SpTrf proteins on the surface of microbes is not known, however, a speculative receptor (brown) is shown. SpTrf proteins may also bind to phosphatidic acid (PA; green triangles), which has been demonstrated for SpTrf-E1 [56]. The conical structure of PA and its clustering induced by SpTrf binding and multimerization is known to drive membrane curvature, which may augment phagocytosis. ER, endoplasmic reticulum.

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