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www.nature.com/scientificreports OPEN The structural and proteomic analysis of Spiroplasma eriocheiris in response to colchicine Received: 14 November 2017 1,2,3 1 1 1 1 1 1 Peng Liu , Jie Du , Jia Zhang , Jian Wang , Wei Gu , Wen Wang & Qingguo Meng Accepted: 23 April 2018 Spiroplasma eriocheiris, a pathogen that causes mass mortality of Chinese mitten crab Eriocheir Published: xx xx xxxx sinensis, is a wall less bacteria and belongs to the Mollicutes. This study was designed to investigate the effects of colchicine on S. eriocheiris growth, cell morphology, and proteins expression. We found that in the presence of colchicine, the spiroplasma cells lost their helicity, and the length of the cells in the experimental group was longer than that of the control. With varying concentrations of the colchicine treatment, the total time to achieve a stationary phase of the spiroplasma was increased, and the cell population was decreased. The virulence ability of S. eriocheiris to E. sinensis was effectively reduced in the presence of colchicine. To expound the toxical mechanism of colchicine on S. eriocheiris, 208 differentially expressed proteins of S. eriocheiris were reliably quantified by iTRAQ analysis, including 77 up-regulated proteins and 131 down-regulated proteins. Especially, FtsY, putative Spiralin, and NADH oxidase were down-regulated. F F ATP synthase subunit delta, ParB, DNABs, and NAD(FAD)- 0 1 dependent dehydrogenase were up-regulated. A qRT-PCR was conducted to detect 7 expressed genes from the iTRAQ results during the incubation. The qRT-PCR results were consistent with the iTRAQ results. All of our results indicate that colchicine have a strong impact on the cell morphology and cellular metabolism of S. eriocheiris. Spiroplasma is a group of bacteria belonging to a class Mollicutes, which includes Mycoplasma, Phytoplasma and 1–3 so on, and featured by small genome sizes and lack of peptidoglycan layer . They have a helical cell morphology 2,4 and swim without flagella when in an appropriate viscous medium . Locomotion is by propagation of kink pairs along the cell body from one pole to another end . Some Spiroplasma cause serious losses in economically important crops and honeybee cultures, and are pathogens of insects and/or plants . Spiroplasma eriocheiris is a novel pathogen causing mass mortality of Chinese mitten crab Eriocheir sinensis, causing disastrous ee ff cts on aquaculture in China in recent years . S. eriocheiris was the first Spiroplasma species isolated from a freshwater crustacean . S. eriocheiris performs chemotaxis without the conventional two-component system, the system commonly found in bacterial chemotaxis. The cells are polarized by a tip structure, a dumbbell-shaped core in the tip that is connected by a flat ribbon forming the internal structure of S . eriocheiris. Sixteen proteins were iden- tified as the components of the internal structure by mass spectrometry, including Fibril protein and four types of MreB proteins . The Spiroplasma genomes reported so far do not have orthologs of other bacterial motility systems, but have one tubulin homolog-FtsZ and five to seven homologs of the protein MreB . MreB is related to actin, which is responsible for many eukaryotic motility systems . 12,13 Colchicine can be extracted naturally from Gloriosa superba L. and Colchicum autumnale L. plants . It is toxic for eukaryotic cells, and is an example of a class of small, tublin-binding molecules . Colchicine is a fat-soluble alkaloid binding to β-tubulin, hindering its polarization with consequent inhibition of neutrophil chemotaxis and reducing expression of adhesion molecules . Moreover, it is known to be a specific mitotic poi- son, inhibiting normal division of the chromosomes and thus causing mutation . Prokaryotic bacterial cells differ from the eukaryotic cells of higher plants and animals . Well-documented electron microscopic studies of Bacillus megaterium have shown that cell morphology was changed by colchicine treatment . Staphylococcus Jiangsu Key Laboratory for Microbes & Functional Genomics and Jiangsu Key Laboratory for Aquatic Crustacean Diseases, College of Life Sciences, Nanjing Normal University, 1 Wenyuan Road, Nanjing, 210023, China. Department of Biology, College of Pharmacy and Biological Sciences, University of South China, Hengyang, 421001, P.R. China. Hunan Province cooperative innovation Center for Molecular Target New Drug Study, Hengyang, 421001, P.R. China. Correspondence and requests for materials should be addressed to Q.M. (email: firstname.lastname@example.org) SCientiFiC RePo R Ts | (2018) 8:8577 | DOI:10.1038/s41598-018-26614-y 1 www.nature.com/scientificreports/ 18,19 showed a temporary variation in cell and colony morphology when grown in the presence of colchicine . But, there is no report about the effect of colchicine on cell morphology and metabolism of wall-less bacteria. In the present study, we evaluated the morphology alterations and proteomics prole o fi f the colchicine-adapted S. eriocheiris cells by using differential interference contrast (DIC) microscope and electronic microscope (EM), iTRAQ and real time PCR (RT-PCR). We attempt to explain and establish a significant correlation between the structural and metabolic profile of the S. eriocheiris undergoing challenge by colchicine. Results Cell morphology. To study the effect of colchicine on S . eriocheiris cell morphology, 3 g/L colchicine was added into the medium as the final concentrations, and until the cells were in exponential phase. Cells of the S. eriocheiris collected from the exponential phase of the growth were examined by DIC microscope and EM in the presence of colchicine. In the first instance, we checked the cells by DIC microscope. With 3 g/L colchicine treatment, the S. eriocheiris cells lost their helical shape, and the length of the cells in the experimental group was longer than that of the control cells (Fig. 1a–d). These results were consistent with EM observations, cells in the colchicine rich medium were longer than those in control conditions, and the treated cells were no longer helically shaped (Fig. 1e,f ). S. eriocheiris cell lengths, when adapted to 3 g/L colchicine, were 13.35 ± 4.03 μm (n = 200) (Fig. 1g). Whereas, under controlled growth conditions, the spiroplasma cell exhibited normal shape, having 5.4 ± 2.08 μm (n = 200) length (Fig. 1h). 3 g/L colchicine-adapted S. eriocheiris cells were approximately 8 μm longer than control cells. Growth kinetics and pathogenic ability. Also, the growth kinetics of Spiroplasma were studied with the selective forces of colchicine, when die ff rent concentrations of colchicine (0, 1, 3, and 5 g/L) were added into the medium as the final concentrations. The cells were cultured for 24 h, until the cells were in exponential phase. A significant difference was noticed between the growth rates of un-adapted control and colchicine-adapted bac- teria (Fig. 2a). Under optimal conditions, the total time taken by the bacteria to achieve a stationary phase was 18 h. The maximum concentration of colchicine acceptable for adaptation of S . eriocheiris cells was determined to be 5 g/L. A gradual shift was noticed in the generation time for colchicine adapted cells. With reference to control un-adapted cells, 5 g/L colchicine-adapted bacterial cells required 60 h to attain the steady state, but the un-adapted cells required only 18 h. At the same time, the virulence ability of S. eriocheiris was investigated. Aer ft 0, 1, 3, and 5 g/L colchicine-adapted cells were washed by PBS, the cells were injected into E. sinensis, and the mor- tality rates of all groups were counted. E. sinensis died 7_days aer t ft he injection of the 0 g/L colchicine-adapted S. eriocheiris, and reached 100% mortality rate after 13 days. When the E . sinensis was injected with the 5 g/L colchicine-adapted cells, the E. sinensis reached 100% mortality rate aer 19 d ft ays’ post S . eriocheiris injection. The results show that the mortality rates were decreasing with the increase of colchicine adaptation of the bacterial cells. It means that the virulence ability of colchicine adapted cells was effectively and gradually reduced with the increase of colchicine concentration (Fig. 2b). iTRAQ quantification. All MS/MS spectra were processed by using Mascot software. As shown in Fig. 3, iTRAQ analysis of S. eriocheiris proteome showed 28057 queries in the database (463,619 sequences), and resulted in 766 identified proteins in Mascot. Gene ontology (GO) analysis of total proteins in S . eriocheiris was based on cellular component (Fig. 4a), molecular function (Fig. 4b), and biological process (Fig. 4c). We analyzed the differential proteins by iTRAQ quantification. Using a 1.2-fold increase or decrease in protein expression as a benchmark for a physiologically significant fold change, 208 differentially expressed proteins were relia- bly quantified by iTRAQ analysis, including 77 up-regulated proteins (Table S1) and 131 down-regulated pro - teins (Table S2) subsequent to colchicine stimulation. In the up-regulated proteins, 5 proteins were involved in energy metabolism processes; 17 proteins were DNA replication and translation related proteins; 6 proteins were related to transport system proteins and transferase; 11 proteins were glycometabolism proteins; 11 proteins were involved in amino acid and protein metabolism processes; 3 proteins were oxidoreductases and 23 proteins were listed as unknown/hypothetical proteins. The up-regulated proteins included: F F ATP synthase subunit delta 0 1 (ATP H) (ACCESSION: AHF57252) with 2.386-fold change, Ribonucleotide-diphosphate reductase beta subunit (ACCESSION: AHF57904) with 3.621-fold change, and Chromosome partitioning protein ParB (ACCESSION: AHF57357) with 1.241-fold change. In addition, a Putative NAD(FAD)-dependent dehydrogenase (ACCESSION: AHF57464) was up regulated with a fold change of 1.274, etc. Of the down-regulated proteins, 12 proteins were grouped within the ribosomal proteins; 7 proteins were involved in energy metabolism processes; 9 proteins were related to carbohydrates, and 13 proteins were involved in amino acid and protein metabolism; 18 proteins were DNA replication and cell division related proteins; 2 were ionic regulation related proteins; 6 proteins were oxidoreductases; 13 proteins were involved in transport systems and served as transferases; 5 proteins were related to lipoprotein and lipid metabolism; and finally, 45 proteins were in the unknown/hypothetical protein category. Especially, the cell division FtsY (ACCESSION: AHF57275) was 0.783-fold down-regulated. FtsY, the prokaryotic signal recognition particle receptor homo- logue, is essential for biogenesis of membrane proteins . Furthermore, other down-regulated proteins included: lipoprotein-putative Spiralin (ACCESSION: AHF58284) with 0.664-fold change and NADH oxidase (ACCESSION: AHF57728) with 0.622-fold change. RT-PCR analysis of the mRNA. In order to provide additional mRNA transcript level information of S. eri- ocheiris and validate the iTRAQ result, we performed qRT-PCR on some selected target gene expressions in both the experimental group and control group aer ft 1, 3, 5, 7, 9, and 11 h incubation using 3 g/L colchicine stimula- tion. In additional, gene expression was investigated when cells were stimulated by using different concentrations (0, 1, 3, and 5 g/L) of colchicine. We measured the mRNA transcription levels of 7 proteins, including 4 genes SCientiFiC RePo R Ts | (2018) 8:8577 | DOI:10.1038/s41598-018-26614-y 2 www.nature.com/scientificreports/ Figure 1. Cell observation by DIC and EM. (a) DIC image of S. eriocheiris cells treated by 3 g/L colchicine. (b) A single cell magnified from the red box of figure A. (c) DIC image of S. eriocheiris cells without colchicine treatment. (d) A single cell magnified from the red box of figure c (Scale bar,10 μm). Two EM images of S. eriocheiris cells treated by 3 g/L colchicine, and those from the control, are presented in figures (e) and (f), respectively (Scale bar: 2 μm). (g) The histogram of S. eriocheiris cell length distribution when treated with 3 g/L colchicine. The average cell length (µm) and measured cell number are shown in the upper right side of the figure. (h) Control. SCientiFiC RePo R Ts | (2018) 8:8577 | DOI:10.1038/s41598-018-26614-y 3 www.nature.com/scientificreports/ Figure 2. e g Th rowth kinetics and pathogenicity of colchicine treated cells. (a) Growth of control (open circle) and colchicine (solid circle-1 g/L, open triangle-3 g/L and solid triangle-5 g/L) adapted S. eriocheiris cells at 30 °C. (b) Mortality rate of E. sinensis aer in ft jection with S. eriocheiris adapted by different concentrations of colchicine. Solid square, hashed square, open square and hatched triangle represent 0, 1, 3, and 5 g/L adapted S. eriocheiris, respectively. Open circle represents control, E. sinensis were injected with PBS. Figure 3. Basic information statistics of proteome resulting from iTRAQ. Total spectra are the secondary mass spectrums, and spectra are the secondary mass spectrums aer q ft uality control. Unique peptide is the identified peptides, which belongs only to a group of proteins, and protein is identified by Mascot 2.3.02 software. of up-regulated proteins: F F ATP synthase subunit delta (ATP H), Chromosome partitioning protein ParB, 0 1 DNABs (ACCESSION: AHF58167), and Putative NAD (FAD)-dependent dehydrogenase and 3 down–regulated proteins: Cell division related protein FtsY, putative Spiralin, and NADH oxidase. As shown in Fig. 5, we investigated the gene expression of 3 down-regulated genes: FtsY, putative Spiralin and NADH oxidase. When stimulated with colchicine, the gene expression of those three genes decreased concom- itant with the increase of colchicine concentration. When the concentration of colchicine was 3 g/L and 5 g/L, the gene expression was significantly lower compared to the 0 g/L treatment (Fig. 5c). During the time course of 3 g/L colchicine stimulation, the three down-regulated genes showed a tendency to decrease over time. The first response gene was putative Spiralin, it showed a significant difference compared to the control group aer 1 ft h of colchicine stimulation (Fig. 5d). But the gene expression of FtsY (Fig. 5b) and NADH oxidase (Fig. 5f ) showed a significant difference with the control group aer 3 a ft nd 7 h of colchicine stimulation, respectively. As shown in Fig. 6, we investigated the gene expressions of 4 up-regulated genes (ATP H, ParB, DNABs and NAD (FAD) dependent dehydrogenase) under the stimulation of colchicine. e g Th enes ATP H (Fig. 6a), DNABs (Fig. 6e) and NAD (FAD) dependent dehydrogenase (Fig. 4g) showed a rapid increased response when the con- centration of colchicine was 1 g/L, and were significantly different from the response with 0 g/L. But, ParB showed a significant die ff rence from the control preparation when the concentration of colchicine was higher than 3 g/L (Fig. 6c). Under the stimulation of 3 g/L colchicine, the 4 up-regulated genes showed an increased response over time ae ft r 3 h of colchicine stimulation. ATP H (Fig. 6b), ParB (Fig. 6d) and NAD (FAD) dependent dehydroge- nase (Fig. 6h) had a significant increase up to 7 h ae ft r the colchicine stimulation when compared with time 0 h. DNABs had a significant increase after 7 h of the colchicine stimulation (Fig. 6f ). To sum up, all the results by RT-PCR analyses were consistent with the iTRAQ analyses. SCientiFiC RePo R Ts | (2018) 8:8577 | DOI:10.1038/s41598-018-26614-y 4 www.nature.com/scientificreports/ Figure 4. Gene ontology (GO) analysis of total proteins in S. eriocheris. (a) Cellular component, (b) molecular function and (c) biological process. Discussion The cell morphology alternated by colchicine. The aim of this work was to evaluate the effect of col- chicine on the S. eriocheiris cell morphological alterations and changes in the proteomic profile. Colchicine is an example of a class of small molecules that bind to tubulin and inhibits its polymerization and thus is toxic to bac- 14,16,17 teria . However, scanty reports are available on structural and metabolic alterations of B. megaterium under 21,22 colchicine treatment . Kashyap Kumar Dubeya demonstrated that the cellular architectural structure of B. megaterium was changed when cells were challenged by colchicine. Colchicine adapted cells lost their helical cell morphology and became longer than control cells . It is well established that the plasma membrane is composed of a phospholipid bilayer, including different types of proteins, and the length of phospholipid chain is almost constant, so the thickness of the plasma membrane may vary depending on the amount of proteins. The reduction in thickness of the bacterial cell membrane is possible due to loosening of weakly immobilized membrane protein components, or inhibition of membrane protein synthesis . According to the iTRAQ results, 3% membrane part and 5% membrane proteins were alliterated (Fig. 4a). Therefore, alterations in cell morphology may be caused by reduction of the amount of membrane proteins, particularly in bacterial cells adapted to high concentrations of colchicine . Even if, no cytoskeleton protein was found from the iTRAQ results, the cytoskeleton of S. eriocheiris may lose the bind point on the membrane. 31% binding proteins in S. eriocheiris cells were changed by colchicine challenge (Fig. 4b). SCientiFiC RePo R Ts | (2018) 8:8577 | DOI:10.1038/s41598-018-26614-y 5 www.nature.com/scientificreports/ Figure 5. qRT-PCR analysis of down-regulated gene expression of challenged S. eriocheiris. (a,c and e) represent FtsY, Spiralin, and NADH oxidase mRNA expression at different concentrations of colchicine challenge; the mRNA expression data under different challenge times (0, 1, 3, 5, 7, 9, and 11 h) are presented in (b,d and f), respectively. Each column represents the mean value with standard error bars based on three samples. Statistical significance (P < 0.05) is indicated with an asterisk (*). The open columns indicate gene expressions when challenged with 3 g/L colchicine and hatched as control. Cell division and energy metabolism process of the S. eriocheiris may be disturbed by colchicine. iTRAQ profiling, a good method for protein measurement, has been shown to be very useful for quantifying changes in plasma proteins in bacteria . For example, Redding et al. used iTRAQ proteomics to study the pro- teomic profile of Desulfovibrio vulgaris under nitrate stress . Evans et al. investigated the regulation and biosyn- thesis of bioactive compounds using iTRAQ and proposed a correlation between phosphate and iron regulation and the biosynthesis of bioactives in Pseudoalteromonas tunicata . In this study, the alteration proteins were reliably quantified by using iTRAQ analysis. Theose proteins are involved in the process of energy metabolism; glycometabolism; protein and amino acid metabolism; DNA transcription and translation or cell division, and other processes. 11,28 The cell morphology and motility of Spiroplasmas are mediated by the internal cytoskeleton . It has been reported that the cytoskeleton of S. eriocheiris contains 16 proteins, including Fibril and four types of Mrebs . But no cytoskeletal proteins were detected by the iTRAQ analysis. The changes of metabolism of S . eriocheiris might have resulted in cell length increase and losing cell helicity as evident from iTRAQ analysis. The colchi- cine adapted cells showed longer cell length than controls. This may be caused by the changes of DNA tran - scription and translation or by changes in cell division proteins. Based on iTRAQ results, there were changes in protein responses, including: cell division related proteins FtsY (0.738-fold down-regulated), ParB (1.24-fold up-regulated), and DnaBs (2.08 -fold up-regulated). FtsY, the prokaryotic signal recognition particle receptor homologue, is essential for biogenesis of membrane proteins and cell division. Also, FtsY is indeed essential for 20,29 expression of integral membrane proteins in E. coli . In unicellular bacteria, ParB is a broadly conserved molec- ular mechanism for plasmid partitioning and chromosome segregation , the ParA and ParB proteins segregate chromosomes and coordinate this process with cell division and chromosome replication . DnaB, a chromosome replication initiation/membrane attachment protein, is an enzyme in bacteria which opens the replication fork SCientiFiC RePo R Ts | (2018) 8:8577 | DOI:10.1038/s41598-018-26614-y 6 www.nature.com/scientificreports/ Figure 6. Analysis of up-regulated gene expressions of challenged S. eriocheiris using qRT-PCR. (a,c,e and g) represent ATP H, ParB, DNABs and NAD(FAD) mRNA expression at different concentrations of colchicine challenge; the mRNA expression under different challenge times (0, 1, 3, 5, 7, 9, and11 h) are presented in (b,d,f and h), respectively. Each column represents the mean value from three samples with the standard error bars. Statistical significance (p < 0.05) is indicated with an asterisk (*). The open columns indicate gene expressions when challenged with 3 g/L colchicine and hatched as control. during DNA replication . Totally, 17 up-regulated proteins and 18 down–regulated proteins were related to bac- terial DNA transcription and translation or cell division. So Spiroplasma cells could not proceed through normal cell division when challenged with colchicine, thus resulting in increased cell length. Pathogenicity and growth speed of the S. eriocheiris was inhibited by colchicine. Under the stimulation of different concentrations of colchicine, pathogenicity and growth kinetics were inversely corre- lated. Probably, these responses were caused by alterations of some energy metabolism proteins and pathogenic factor proteins. According to iTRAQ results, 13 transferase and transport proteins were down-regulated. 7 down-regulated proteins and 4 up-regulated proteins were related to energy metabolism. These energy-related proteins include ATPases, a class of enzymes that catalyze the decomposition of ATP into ADP and a free SCientiFiC RePo R Ts | (2018) 8:8577 | DOI:10.1038/s41598-018-26614-y 7 www.nature.com/scientificreports/ phosphate ion. Transmembrane ATPases import many of the metabolites necessary for cell metabolism and 32,33 export toxins, wastes, and solutes . Therefore, energy transformation in colchicine adapted cell was altered and the growth rate was decreased. At the same time, as the concentration of colchicine was increased, the pathogenic ability decreased. This may be caused by reduction of a pathogenic factor. For example, Spiralin is one of the most thoroughly characterized S. citri membrane lipoprotein proteins . It is important for S. citri GII3 to adhere to and invade insect cells . It might be predicted that the process of responding to colchicine severely ae ff cts the meta- bolic profile and morphology of the S . eriocheiris. er Th e is a correlation between the structural and metabolic pro- file of the cell. This phenomenon is consistent with the changes of B . megaterium cell under colchicine challenge . Conclusion Our results indicate that colchicine may have an effect on the cell morphology and cellular metabolism of S. eriocheiris. There are 208 differentially expressed proteins in the S . eriocheiris proteomics at 24 h post colchicine incubation. Most of the differential proteins are related to changes in intracellular metabolic processes. Based on the evidence of differentially expressed proteins during colchicine treatments, we can speculate about the structural and metabolic changes of S. eriocheiris. In the presence of colchicine, the S. eriocheiris lost its helicity, resulting in a longer cell length than that of the control group. The energy metabolism, cell division process and pathogenicity were interrupted by colchicine. Overall, the novelty of the study is that this is the very first time that the effect of colchicine has been reported on the wall less bacterial cell. Furthermore, our study has shown a correlation between the colchicine-induced structural alterations with the proteomic profile of the bacterium. Methods Bacterial Strains and Culture conditions. e Th strain, S . eriocheiris was isolated from haemolymph of the Chinese mitten crab, E. sinensis . The optimum growth occurs at 30 °C in R2 broth. Pathogenicity ability were estimated by counting the mortality aer t ft he injection of S . eriocheiris cells into E. sinensis. At the initial stages of adaptation, 0.1 g/L colchicine was used as a selective force. Under such conditions the S. eriocheiris cells were grown for about five generations till the specific growth was obtained similar to the control non-adapted bacterial cells. At the end of each generation the bacterial cultures were evaluated. The ability of the bacterial culture to grow in contact with different selective concentrations (0, 1, 3, and 5 g/L) of colchicine was examined when it was added into R2 medium. Cell observation and measurement. 3 g/L colchicine was added into the medium as the final concen- trations, and then the cells were cultured for 24 h, until the cells were in exponential phase. Cultured cells were centrifuged at 11,000 × g, for 30 min at 4 °C and the pellet was resuspended with PBS buffer (75 mM sodium phos- phate (pH 7.3) and 68 mM NaCl) with 0.6% methyl cellulose. The cell was video recorded with a DIC microscope. e v Th ideos were analyzed by Image J ver.1.37 v (http://rsb.info.nih.gov/ij/). 200 cell lengths were measured from videos. As for the EM observation, the cultured cells suspended in PBS equivalent to the original density were placed onto an EM grid, and incubated for 10 min at room temperature (RT). The excess fluid on the EM grid was removed and the preparation was stained with ammonium molybdate for 1 min. Experimental bacterial infection and the growth kinetics evaluation. Experimental E. sinensis (50 ± 3 g) were purchased from a market in Nanjing, China, and cultivated in 10 L tanks. Only healthy E. sin- ensis were selected and randomly divided into five groups. S . eriocheiris cells were washed with PBS two times and adjusted to a final concentration of 10 /ml Spiroplasma cells. The E . sinensis in each group (30 individuals) received an injection of 100 μL washed S. eriocheiris, that were each adapted by treatment with 0, 1, 3, and 5 g/L colchicine, respectively. Thirty E . sinensis, receiving an injection of 100 μL PBS individually, were used as a control group. Aer t ft reatment with different concentrations of colchicine adapted S. eriocheiris, or with PBS as a control, the mortality rates of all groups were determined. As for the growth kinetics evaluation, Spiroplasma cells were cultured in R2 medium containing die ff rent concentrations of colchicine without phenol red stain. The relative cell number was measured by a spectrophotometer at a wavelength of 600 nm. Protein preparation. S. eriocheiris cell samples treated with 0 or 3 g/L colchicine solutions were washed by PBS and centrifuged at 11,000 × g, for 30 min at 4 °C three times. Three different replicates of S . eriocheiris cell samples were mixed as one sample, mixed cell samples were ground into powder in liquid nitrogen, extracted with Lysis buffer A (7 M Urea, 2 M Thiourea, 4% CHAPS, 40 mM Tris-HCl, pH 8.5) containing 1 mM PMSF and 2 mM EDTA. Aer 5 ft min, 10 mM DTT was added. Aer s ft onication and centrifugation, the suspension was mixed well with a 5-fold volume of chilled acetone containing 10% TCA and incubated at −20 °C overnight. Aer ft centrifugation at 4 °C, 30,000 × g, the precipitate was washed with chilled acetone three times. The pellet was air-dried and dissolved in Lysis Buffer B (7 M Urea, 2 M Thiourea, 4% NP40, and 20 mM Tris-HCl, pH 8.5). The suspension was sonicated at 200 W for 15 min and centrifuged at 4 °C, 25,000 × g for 20 min. To reduce disulfide bonds in proteins of the supernatant, 10 mM DTT was added and incubated at 56 °C for 1 h. Subsequently, 55 mM IAM was added to block the cysteines, and incubated for 1 h in the darkroom. The supernatant was mixed well with a 5-fold volume of chilled acetone for 2 h at −20 °C. Aer cen ft trifugation, the pellet was air-dried for 5 min, dissolved in 200 μL of 0.5 M TEAB (Applied Bio-systems, Italy) and sonicated at 200 W for 15 min. Finally, sam- ples were centrifuged at 4 °C, 25,000 × g for 20 min. e Th supernatant was transferred to a new tube and quantified using a 2-D Quant Kit (GE Healthcare). The proteins in the supernatant were kept a −80 °C for further analysis. iTRAQ labeling and SCX fractionation. e iTR Th AQ assays were performed as described previously with minor modification .Total protein (100 μg), taken from each sample solution, was digested with Trypsin Gold (Promega, USA) with the ratio of protein: trypsin = 20: 1 at 37 °C for 4 h. Aer ft trypsin digestion, peptides were dried by vacuum centrifugation. Peptides were reconstituted in 0.5 M TEAB and processed according to the SCientiFiC RePo R Ts | (2018) 8:8577 | DOI:10.1038/s41598-018-26614-y 8 www.nature.com/scientificreports/ Name Sequence (5′–3′) S. eriocheiris FtsY-F CCAAAGTCGCGCCAAAACA S. eriocheiris FtsY-R CGGAAGGTATCACCCGCAA S. eriocheiris putative Spiralin-F ACCGTAACAGTACAGGCTCA S. eriocheiris putative Spiralin-R ACCACCACTTTGAATTGCCG S. eriocheiris NADH oxidase -F TTATTGCGACTGGTGCTCGT S. eriocheiris NADH oxidase -R CCAGCGCCAACAATTACGAC S. eriocheiris ATP H -F AGAACCATTAACGCAAGAGCA S. eriocheiris ATP H -R TTGACCAGCTAATGTTCCATCAA S. eriocheiris dnaBs -F AATTATTGGGCACCAGGCTCA S. eriocheiris dnaBs -R GGGTTCCCTTTTCCCGCTTTA S. eriocheiris ParB -F TTTAGTCGCGGGAGAACGTC S. eriocheiris ParB -R GGCGTTGGCTTCTTCAATGG S. eriocheiris NAD(FAD)-dependent dehydrogenase -F GTTGATGCTTTCCACGCCAA S. eriocheiris NAD(FAD)-dependent dehydrogenase -R TTGCTAAATGCACTCCGGCT Table 1. e p Th rimers used for real-time PCR in the experiment. manufacturer’s protocol for 8-plex iTRAQ reagent (Applied Biosystems). The proteins from the non-treated and treated samples were labeled with 115 and 114, respectively. The labeled peptide mixtures were then pooled and dried by vacuum centrifugation. The labeled samples were pooled and purified using a strong cation exchange chromatography (SCX) column (Phenomenex, USA), and separated by liquid chromatography (LC) using a LC-20AB HPLC Pump system (Shimadzu, Japan). The iTRAQ-labeled peptide mixtures were reconstituted with 4 mL buffer A (25 mM NaH PO in 25% ACN, pH 2.7) and loaded onto a 4.6 × 250 mm Ultremex SCX column 2 4 containing 5 mm particles (Phenomenex). The peptides were eluted at a flow rate of 1 mL/min with a gradient of buffer A for 10 min, 5–60% buffer B (25 mM NaH2PO4, 1 M KCl in 25% ACN, pH 2.7) for 27 min, and 60–100% buffer B for 1 min. The system was then maintained at 100% buffer B for 1 min before equilibrating with buffer A for 10 min prior to the next injection. Elution was monitored by measuring the absorbance at 214 nm, and frac- tions were collected every 1 min. The eluted peptides were pooled into 20 fractions, desalted with a Strata X C18 column (Phenomenex) and vacuum-dried. LC-ESI-MS/MS analysis based on Triple TOF 5600. Each fraction was resuspended in buffer C (5% ACN, 0.1% FA) and centrifuged at 20,000 × g for 10 min, the final concentration of peptide was about 0.5 g/L on average. 10 mL of supernatant was loaded on a LC-20AD nanoHPLC (Shimadzu, Japan) by the autosampler onto a 2 cm C18 trap column. Then, the peptides were eluted onto a 10-cm analytical C18 column packed in-house. The samples were loaded at 8 mL/min for 4 min, then a 35 min gradient was run at 300 nL/min starting from 2 to 35% buffer D (95% ACN, 0.1% FA), followed by 5 min linear gradient to 60%, then followed by a 2 min linear gradient to 80%, and maintenance at 80% buffer D for 4 min, and finally returned to 5% for 1 min. Data acquisition was performed with a Triple TOF 5600 System fitted with a Nanospray III source (AB SCIEX) and a pulled quartz tip as the emitter (New Objectives, MA). Data was acquired using an ion spray voltage of 2.5 kV, curtain gas of 30 psi, nebulizer gas of 15 psi, and an interface heater temperature of 150. The MS was oper - ated with a RP of greater than, or equal to, 30,000 FWHM for TOF MS scans. For IDA, survey scans were acquired in 250 ms and as many as 30 product ion scans were collected if they exceeded a threshold of 120 counts per sec- ond (counts/s). Total cycle time was fixed at 3.3 s. e Q2 Th transmission window was 100 Da for 100%. Four time bins were summed for each scan at a pulser frequency value of 11 kHz through monitoring of the 40 GHz mul- tichannel TDC detector with a four-anode channel detect ion. A sweeping collision energy setting of 35 ± 5 eV , coupled with iTRAQ adjust rolling collision energy, was applied to all precursor ions for collision-induced dis- sociation. Dynamic exclusion was set for 1/2 of peak width (15 s), and then the precursor was refreshed to the exclusion list. Data analysis. Raw data files acquired from the Orbitrap were converted into MGF files using Proteome Discoverer 1.2 (PD 1.2, Thermo) and the MGF files were searched. Protein identification was performed by using Mascot search engine (Matrix Science, UK). The parameters used included: Gln-> pyro-Glu (N-term Q), Oxidation (M), Deamidated (NQ) as the potential variable modifications, and Carbamidomethyl (C), iTRAQ8plex (N-term), and iTRAQ8plex (K) as fixed modifications. The charge states of peptides were set to +2 and +3. Then protein identification was performed using the genome sequence of S . eriocheiris (1242 sequences). To reduce the probability of false peptide identification, only peptides with significance scores (≧ 20) at the 99% confidence interval by a Mascot probability analysis greater than “identity” were counted as identified. And each confident protein identification involved at least one unique peptide. For protein quantization, a protein must have contained at least two unique peptides. The quantitative protein ratios were weighted and normalized by the median ratio in Mascot. We only used ratios with P-values < 0.05, and only changes of >1.2 fold were consid- ered as significant. Functional annotations of the proteins were conducted using Blast2GO program against the non-redundant protein database (NR; NCBI). SCientiFiC RePo R Ts | (2018) 8:8577 | DOI:10.1038/s41598-018-26614-y 9 www.nature.com/scientificreports/ Real-time PCR. The absolute real-time standard curve of focused genes was prepared according to the described method . 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Hunan Province cooperative innovation Center for Molecular Target New Drug Study (No. 0223-0002-0002000-58). Author Contributions P.L. conceived the experiments and wrote the manuscript, J.D. prepared and analyzed iTRAQ quantification, J.W., W.G. and J.Z. did the RT-PCR analysis, W.W. and Q.M. conducted the experiments. All authors reviewed the manuscript. Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-26614-y. Competing Interests: The authors declare no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2018 SCientiFiC RePo R Ts | (2018) 8:8577 | DOI:10.1038/s41598-018-26614-y 11
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