br Supplementary functional annotations of avian eggshell

Supplementary functional annotations of avian eggshell matrix proteins Potential functions of proteins were determined according to the literature, data annotations and functional domain database and highlighted 77 proteins with potential functions related to a mineralization process [1]. Beside these proteins, we identified 35 proteins with potential antimicrobial functions present at various stages of mineralization (Supplementary Table 3). These antimicrobial proteins were present a quite equally number throughout the different stages of shell formation. The most abundant protein in the shell matrix is lysozyme, which is known to hydrolyze 1–4 beta linkages between N-acetyl muramic gonadotropin releasing hormone receptor and N-acetyl-d-glucosamine residues in peptidoglycans of Gram-positive bacteria [16]. Another notable proteins is OVAX, which exhibits antimicrobial properties against Listeria monocytogenes and Salmonella enteritidis [17]. Avian β defensin 11, belonging to avian β defensin family, is a cationic peptides with three standed β sheet structure connected with β hairpin loop that protect against Gram-positive and Gram-negative bacteria [18]. Ovocledin-17, a C-type lectin protein which exhibits antimicrobial properties against Bacillus subtilis, Staphylococcus aureus and Pseudomonas aeruginosa is also a major eggshell protein [19]. Another group is constituted of antimicrobial proteins by depriving bacteria of essential nutrients. Ovotransferrin, vitellogenin-1 and 2 and MFI2 are antimicrobial by their capacity to chelate iron ions, essential for bacterial growth [20]. GC, a group-specific component and riboflavin protein bind vitamins which are depriving bacteria from vitamin D. We report ovocalyxin-36, BPIFCB and ovoglobulinG2 type AA, which belong to the LBP/BPI/PLUNC family known to bind to the lipid A portion of lipopolysaccharide cell wall in Gram-negative bacteria leading to the death of bacteria [21]. The study also revealed protease inhibitors which can be potentially antimicrobial by their ability to inhibit proteases secreted by some bacteria. Ovoinhibitor, ovomucoid,SPARC,follistatin, follistatin-like 1 and IGFBP7 exhibit Kazal like protease inhibitor domains. Moreover, ovoinhibitor was showed to inhibit Bacillus thuringiensis growth [22]. Ovocalyxin-32 presents homology with latexin, a carboxypeptidase inhibitor, and has been shown to inhibit B. subtilis growth [23]. SERPIND1, SERPINF2, SERPINE2, SERPING1 and SERPINI1 belong to SERPIN family, a known family of serine protease inhibitors [16]. Ovocalyxin-25 contains two inhibitor protease domains, a WAP and a Kunitz-like domain [24]. Cystatin C is a cysteine protease inhibitor, and ovostatin is known to inhibit all four classes of proteases [16]. Finally our study revealed several fragment of immunoglobulin (Ig-light-2, Ig-gamma-heavy, Ig-lambda-1, Ig-lambda-2, Ig-light-1, Ig-mu-heavy and Ig-alpha-heavy). Finally, out of the 175 eggshell matrix proteins showing significant variation of abundance according to the four stages of shell calcification, 81 could not be ascribed to any of these functional groups and were classified as “other or unknown role” (Supplementary Table 4).
Conflict of interest
Acknowledgments This research was funded by the French National Research Agency ANR (ANR-13-BSV6-0007-01, ANR-13-BSV6-0007-02 and ANR-13-BSV6-0007-05). The high resolution mass spectrometer was financed (SMHART project, 35069) by the European Regional Development Fund (ERDF), the Conseil Régional du Centre, the French National Institute for Agricultural Research (INRA) and the French National Institute of Health and Medical Research (Inserm). ARN acknowledges funding through Grants CGL2011-25906 (Ministerio de Economia, Spain).
Specifications tableValue of the data Data, experimental design, materials and methods The data show the lists of proteins identified and quantified in Extracellular Vesicles (EVs) and in the vesicle-free secretome fraction of the wild-type strain Pseudomonas aeruginosa PAO1 and its isogenic crc-deffective derivative FCP001. The analysis was performed by nano-LC ESI-MSMS analysis using a nano-liquid chromatography system coupled to high speed Triple TOF 5600 mass spectrometer with a duo spray ionization source. The proteins were quantified and processed using Analyst® TF 1.5.1 Software (AB SCIEX). Table 1 contains a list of 1058 proteins identified in the P. aeruginosa vesicle-free secretome and Table 2 contains 643 quantified proteins with 2 or more peptides in the same fraction. Table 3 contains a list of 839 proteins identified in the P. aeruginosa EVs fraction. Table 4 list the 489 proteins with 2 or more peptides identified in EVs sample. Of these, 37 proteins were more abundant and 16 were less abundant in the vesicle-free secretome and 50 proteins were more abundant and 14 less abundant in the EVs of the FCP001 mutant defective in crc than they were in the control PAO001 parental strain [1]. All mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository [2] using MIAPE Extractor v3.7.2, with the dataset identifier PXD000687 and DOI 10.6019/PXD000687.