Inguglia L et al. (2020), Changes in the proteome of sea urchin Paracentr...

ECB-ART-48569

PLoS One 2020 Jan 01;152:e0228893. doi: 10.1371/journal.pone.0228893.

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Changes in the proteome of sea urchin Paracentrotus lividus coelomocytes in response to LPS injection into the body cavity.

Inguglia L , Chiaramonte M , Arizza V , Turiák L , Vékey K , Drahos L , Pitonzo R , Avellone G , Di Stefano V .

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BACKGROUND: The immune system of echinoderm sea urchins is characterised by a high degree of complexity that is not completely understood. The Mediterranean sea urchin Paracentrotus lividus coelomocytes mediate immune responses through phagocytosis, encapsulation of non-self particles, and production of diffusible factors including antimicrobial molecules. Details of these processes, and molecular pathways driving these mechanisms, are still to be fully elucidated. PRINCIPAL FINDINGS: In the present study we treated the sea urchin P. lividus with the bacterial lipopolysaccharide (LPS) and collected coelomocytes at different time-points (1, 3, 6 and 24 hours). We have shown, using label-free quantitative mass spectrometry, how LPS is able to modulate the coelomocyte proteome and to effect cellular pathways, such as endocytosis and phagocytosis, as soon as the immunomodulating agent is injected. The present study has also shown that treatment can modulate various cellular processes such as cytoskeleton reorganisation, and stress and energetic homeostasis. CONCLUSIONS: Our data demonstrates, through mass spectrometry and the following functional annotation bioinformatics analysis, how the bacterial wall constituent is sufficient to set off an immune response inducing cytoskeleton reorganisation, the appearance of clusters of heat shock proteins (Hsp) and histone proteins and the activation of the endocytic and phagocytic pathways. Data are available via ProteomeXchange with identifier PXD008439.

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Genes referenced: LOC100887844 LOC583082

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	Fig 1. Graphical treatment scheme.A total of 40 animals were divided into eight groups of five animals. Four groups of sea urchins received an injection of 2μg/mL of LPS (treated groups) and the remaining four groups received an injection of aCF (control groups). Coelomocytes from animals of treated and control groups were collected at 1, 3, 6 and 24h.
	Fig 2. Protein classes identified by mass spectrometry.Proteins identified by Mass Spectrometry were examined using Panther (Protein Analysis through Evolutionary Relationship, Version 13.1). A total of 137 proteins was recognised and divided into eighteen classes: calcium binding protein, cell adhesion molecule, chaperone, cytoskeletal protein, enzyme modulator, hydrolase, isomerase, ligase, lyase, membrane traffic protein, nucleic acid binding, oxidoreductase, receptor, signaling molecule, transcription factor, transfer/carrier protein, transferase, transporter.
	Fig 3. Analysis of known and predicted protein-protein interaction networks at 1 HPLT.Modulated proteins (Fold change >0.5 and <-0.5) were used as input for STRING analysis to reveal known and predicted protein-protein interaction networks. Proteins are indicated by nodes labeled with the encoding gene symbol. Additional cluster analysis was used to colour the nodes of the interaction networks (k-means = 3). The analysis showed the modulation of a network of interaction that was mainly composed of cytoskeleton and cytoskeleton-related factors (yellow cluster), a network mainly composed of RAS superfamily GTPase members (red cluster), a network mainly composed of heat shock proteins (blue cluster) and a network composed of the histone proteins (green network).
	Fig 4. Analysis of known and predicted protein-protein interaction networks at 3 HPLT.Modulated proteins (Fold change >0.5 and <-0.5) were used as input for STRING analysis to reveal known and predicted protein-protein interaction networks. Proteins are indicated by nodes labeled with the encoding gene symbol. Additional cluster analysis was used to colour the nodes of the interaction networks (k-means = 2). The analysis showed the modulation of a network of interaction that was mainly composed of proteins involved in cytoskeleton and RAS signaling (green cluster) and of a second network mainly composed of proteins involved in energetic homeostasis (red cluster).
	Fig 5. Analysis of known and predicted protein-protein interaction networks at 6 HPLT.Modulated proteins (Fold change >0.5 and <-0.5) were used as input for STRING analysis to reveal known and predicted protein-protein interaction networks. Proteins are indicated by nodes labeled with the encoding gene symbol. Additional cluster analysis was used to colour the nodes of the interaction networks (k-means = 2). The analysis showed the modulation of a network of interaction that was mainly composed of proteins involved in the cytoskeleton organization and RAS GTPases (green cluster), while the latter was composed of RAB proteins specifically involved in vesicles and endosome transport, fusion and recycling.
	Fig 6. Analysis of known and predicted protein-protein interaction networks at 24 HPLT.Modulated proteins (Fold change >0.5 and <-0.5) were used as input for STRING analysis to reveal known and predicted protein-protein interaction networks. Proteins are indicated by nodes labeled with the encoding gene symbol. Additional cluster analysis was used to colour the nodes of the interaction networks (k-means = 2). The analysis showed the modulation of a network of interaction that was mainly composed of proteins related to cellular cytoskeleton (red cluster).
	Fig 7. KEGG Endocytosis pathway.Endocytosis pathway as showed by the KEGG database. Proteins in the pathway are depicted by boxes while arrows depict signaling routes. Red stars correspond to modulated proteins, identified by the MS analysis, at 1HPLT (Hours post LPS treatment), blue stars at 3 HPLT, green stars at 6HPLT and yellow stars at 24HPLT.
	Fig 8. KEGG Phagosome pathway.Phagosome pathway as showed by the KEGG database. Proteins in the pathway are depicted by boxes while arrows depict signaling routes. Red stars correspond to modulated proteins, identified by the MS analysis, at 1HPLT ((Hours post LPS treatment), blue stars at 3 HPLT, green stars at 6HPLT and yellow stars at 24HPLT.
	Fig 9. Modulation of the size of clusters during the LPS treatment.Area chart representing the timing profile of cluster sizes in terms of number of STRING nodes, at 1, 3, 6 and 24HPLT for Cytoskeleton proteins (red), RAB GTPases (lime), Energetic homeostasis (light blue), HSP proteins (yellow), Histones (purple).

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