Yakovenko I et al. (2021), The Diverse Transformer (Trf) Protein Family in...

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Int J Mol Sci 2021 Jun 22;2213:. doi: 10.3390/ijms22136639.

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The Diverse Transformer (Trf) Protein Family in the Sea Urchin Paracentrotus lividus Acts through a Collaboration between Cellular and Humoral Immune Effector Arms.

Yakovenko I , Donnyo A , Ioscovich O , Rosental B , Oren M .

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Sea urchins are long-living marine invertebrates with a complex innate immune system, which includes expanded families of immune receptors. A central immune gene family in sea urchins encodes the Transformer (Trf) proteins. The Trf family has been studied mainly in the purple sea urchin Strongylocentrotus purpuratus. Here, we explore this protein family in the Mediterranean Sea urchin Paracentrotus lividus. The PlTrf genes and predicted proteins are highly diverse and show a typical Trf size range and structure. Coelomocytes and cell-free coelomic fluid from P. lividus contain different PlTrf protein repertoires with a shared subset, that bind specifically to E. coli. Using FACS, we identified five different P. lividus coelomocyte sub-populations with cell surface PlTrf protein expression. The relative abundance of the PlTrf-positive cells increases sharply following immune challenge with E. coli, but not following challenge with LPS or the sea urchin pathogen, Vibrio penaeicida. Phagocytosis of E. coli by P. lividus phagocytes is mediated through the cell-free coelomic fluid and is inhibited by blocking PlTrf activity with anti-SpTrf antibodies. Together, our results suggest a collaboration between cellular and humoral PlTrf-mediated effector arms in the P. lividus specific immune response to pathogens.

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	Figure 1. The PlTrf phylogeny and the structure of the translated protein sequences. (A) An unrooted phylogenetic tree shows separate phylogeny for PlTrf, HeTrf and SpTrf CDS. Only the ML-based tree (Hasegawa–Kishino–Yano model [32], highest log likelihood (−1640.98) (+G, parameter = 2.7099)) is shown. The Trf sequences clustered according to the three echinoid species (P. lividus, S. purpuratus and H. erythrogramma). In P. lividus, sequences clustered into three clades according to the element patterns. The whole genome-based phylogenetic tree (phyloT, NCBI taxonomy) shows evolutionary relationships among echinoderms species. The cidaroid pencil sea urchin, Eucidaris tribuloides, the sea star, Paritia miniata, and the sea cucumber, Cucumaria georgiana, do not contain Trf-like sequences in their genomes. (B) Alignment of 12 full-length translated PlTrf aa sequences shows typical Trf structural features. The sequences were aligned in MEGAX Muscle [33] with manual correction based on element size of three or more aa surrounded by gaps. The 12 sequences were chosen from all publicly available PlTrf sequences to cover all available sequence element combinations. Sequence alignment is shown. Bars underneath the alignment represent the leader (purple) and the 22 sequence elements (in dark and light shades of gray and light pink). The PlTrf protein structure scheme summarizing the alignment is shown at the bottom. Orange triangles indicate glycine residues. Blue triangles indicate histidine residues. Sequence elements are marked as E1–E22.
	Figure 2. Anti-SpTrf antibodies bind to PlTrf proteins (A). Schematic structure of a Trf protein with the locations to which the three SpTrf antibodies bind. The locations and element numbers where the 66, 68, and 71 anti-SpTrf antibodies bind are marked above with black triangles for S. purpuratus (SP) and their equivalent elements in P. lividus (PL) below. (B) WB of native, nickel-bound and unbound proteins shows different subsets of PlTrf proteins. Ni-column PlTrf protein enrichment was performed according to [8], based on binding between the histidine residues and Ni. N, native protein extract from the CF. Ni-b, nickel bound proteins. Ub, unbound proteins that flow through the Ni-column. C. WB of cell-free CF and coelomocytes resulted in similar PlTrf band patterns for each of the three anti-SpTRF antibodies. The WB was made based on a whole protein extract of either CF (H) or coelomocytes (C) of the same sea urchin. Each blot was incubated with either 66, 68 or 71 anti-SpTrf antibodies. Differences are observed between the cellular and humoral fractions of CF.
	Figure 3. Trf protein repertoires of seven P. lividus individuals varies between coelomocytes and cell-free CF. Multiple differences were observed between the cellular and humoral fractions in each of the tested sea urchins, including unique bands in cellular fraction (yellow arrowheads) and unique bands in the cell-free CF fractions (green arrowheads). C. coelomocytes fraction, H. cell-free CF fraction.
	Figure 4. P. lividus Trf-positive coelomocytes. (A) Cell surface Trf expression in live P. lividus coelomocyte of five morphological types. Trf proteins are detected by rabbit anti-SpTrf antibodies and Alexa Fluor 488 secondary goat anti-rabbit-Ig antibody. Trf-positive coelomocytes include: 1. red spherule cells, 2. petaloid or small phagocytes, 3. filopodial phagocytes, 4. colorless spherule cells, and 5. vibratile cells. a, bright field; b, confocal microscope image. Cells in the bright field pictures are not the same cells as in confocal imaging. Scale bars = 10 μm. (B) Trf-positive red spherule cells sorting. The APC-positive cells are the red spherule cells. In the α (anti)-SpTrf antibody-treated sample (left), the APC/FITC double-positive cells (upper right corner) are the Trf-expressing red spherule cells. The FITC-positive/APC-negative cells (upper left corner) are all of other SpTrf-positive cells. In the 2nd Ab only control (right) coelomocytes were incubated with the Alexa Fluor 488 secondary anti-rabbit antibody only. Almost no FITC-positive cells were observed in the control.
	Figure 5. Percent change of Trf-positive cells in response to challenge with different immune elicitors. Due to the high variability in the basal levels of the Trf-positive coelomocytes within the sea urchin population, only the changes in the percentages relative to day 0 are presented. (A) The percent change of Trf-positive coelomocytes in response to injections of heat-killed E. coli, LPS and aCF (control). Sampling and measurements were carried out on days 0, 1, 2, 13 and 43 post-injection. A significant percent increase of Trf-positive coelomocytes was observed following the challenge with heat-killed E. coli over the course of the experiment. (B) The percent change of Trf-positive coelomocytes in response to injections of heat-killed V. penaeicida and aCF (control). Experiment and control groups did not show significant differences.
	Figure 6. Specific WB band pattern of E. coli bound PlTrf proteins. A similar Trf protein band pattern was observed in both coelomocytes (C) and cell-free CF (CF) from three sea urchins that were incubated with live E. coli. The band pattern of the untreated coelomocytes and cell-free CF protein extract counterparts is shown on the right side of the figure. “Native” stands for untreated CF protein extract.
	Figure 7. Phagocytosis of E. coli bacteria by P. lividus phagocytes is inhibited by anti-SpTrf antibodies. (A) P. lividus sea urchin phagocytes with internalized E. coli bacteria. (1a,1b) A petaloid phagocyte with internalized FITC-stained E. coli. (2a,2b) A filopodial phagocyte with multiple mCherry-expressing E. coli in the cytoplasm. Scale bars = 10 μm. (B) Inhibition of phagocytosis by SpTrf antibody. Numbers on Y-axis represent E. coli-containing phagocytes concentration in response to the different treatments. Treatment A. coelomocytes were incubated with bacteria that were pre-incubated with CF. Treatment B. coelomocytes were incubated with untreated bacteria. Treatment C. coelomocytes were pre-incubated with anti-SpTrf antibodies followed by incubation with bacteria that were pre-incubated with CF. Vertical bars indicate standard errors. Differences in E. coli-containing phagocyte concentrations in different treatments were significant by Anova two-factor analysis. Anova single factor analysis indicated that treatment A resulted in significantly higher E. coli-containing phagocytes concentration compared with treatments B and C. * indicates a significant difference (p < 0.05).

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