Systemin-responsive phosphorylation of kinases and phosphatases was identified over a time course of 2 to 45 minutes, as well as typical substrate phosphorylation sites. We identified the C-terminal threonine of H HighlightsDefinition of early systemin-responsive phosphorylation time course.Reconstruction of kinase-substrate relationships from phosphorylation time profiles.+-ATPase LHA1 inducing alkalinization of the medium.Phosphatase PLL5 rapidly dephosphorylated HMAP-Kinase MPK2 re-phosphorylated LHA1 after 15 minutes. +-ATPase, rapid phosphorylation of NADPH-oxidase and Ca2+-ATPase. Later responses involved transient phosphorylation of small GTPases, vesicle trafficking proteins and transcription factors. Based on a correlation analysis of systemin-induced phosphorylation profiles, we predicted substrate candidates for 44 early systemin-responsive kinases, which includes receptor kinases and downstream kinases such as MAP kinases, as well as nine phosphatases. We propose a regulatory module in which H+-ATPase LHA1 is rapidly de-phosphorylated at its C-terminal regulatory residue T955 by phosphatase PLL5, resulting in the alkalization of the growth medium within 2 mins of systemin treatment. We found the MAP kinase MPK2 to have increased phosphorylation level at its activating TEY-motif at 15 min post-treatment. The predicted interaction of MPK2 with LHA1 was confirmed by in vitro kinase assays, suggesting that the H+-ATPase LHA1 is re-activated by MPK2 later in the systemin response. Our data set provides a resource of proteomic events involved in systemin signaling that will be valuable for further in-depth functional studies in elucidation of systemin signaling cascades.Systemin is a small peptide with important functions in plant wound response signaling. Although the transcriptional responses of systemin action are well described, the signaling cascades involved in systemin perception and signal transduction at the protein level are poorly understood. Here we used a tomato cell suspension culture system to profile phosphoproteomic responses induced by systemin and its inactive Thr17Ala analog, allowing us to reconstruct a systemin-specific kinase/phosphatase signaling network. Our time-course analysis revealed early phosphorylation events at the plasma membrane, such as dephosphorylation of H A third batch of cells was injected with an equal volume of water as a control treatment for general handling. For each treatment, a batch of cells was harvested after 2, 5, 15, and 45 min (A). At each time point, cells were harvested over a sieve and were immediately frozen in liquid nitrogen  was calculated and divided by the number of data points. (3) Pairwise correlation analysis with a cutoff of r = 0.8 was used when the similarity of patterns between different phosphopeptides was assessed.Batches of cell suspension cultures of d 45 min A. At eacnitrogen . UntreatSolanum peruvianum cell suspension culture was kindly provided by Georg Felix (University of Tübingen). Cells were subcultured weekly by diluting 8 ml of the suspension in 70 ml fresh Murashige-Skoog medium containing Nitsch vitamins  were added from a 200-fold concentrated stock solution in water.The vitamins , 0.34 g/m sucrose, 100 mm KCl, 1 mm EDTA, 50 mm Tris-MES pH 7.5 and fresh 5 mm DTT) in the presence of 1% v/v proteinase inhibitor mixture (Serva) and 0.5% of each phosphatase inhibitor cocktails 2 and 3  in a Dounce Homogenizer. At least 200 strokes were performed. The homogenate was filtered through four layers of miracloth and centrifuged for 15 min at 7500 × g at 4 °C. The supernatant was collected and centrifuged for 75 min at 48,000 × g at 4 °C. The pellet was washed with 5 ml of 100 mm Na2CO3 then again was centrifuged for 75 min at 48,000 × g at 4 °C. The microsomal membrane pellets used for LC-MS/MS were re-suspended in 100 μl UTU . Microsomal membrane pellets used for ATPase assay were resuspended in 100 μl of re-suspension buffer . Protein concentrations were determined using Bradford assay  with BSA as protein standard. Samples were stored at −80 °C until further use.The microsomal fraction was extracted according to  with minm urea 2 m thiourea, pH 8 (Tris-HCl). After 4-fold dilution with 10 mm Tris-HCl (pH 8), samples were digested with 4 μl Sequencing Grade Modified trypsin  overnight at 37 °C. The reaction was stopped by addition of trifluoroacetic acid (TFA) to reach pH ≤ 3 before further processing.Microsomal proteins were predigested for 3 hours with endoproteinase Lys-C  at 37 °C in 6 2, GL Sciences) with some modifications as described  as an HPLC-system and a Quadrupole-Orbitrap hybrid mass spectrometer  as a mass analyzer. Peptides were eluted from a 75 μm x 50 cm C18 analytical column  on a linear gradient running from 4 to 64% acetonitrile in 240 min and sprayed directly into the Q-Exactive mass spectrometer. Proteins were identified by MS/MS using information-dependent acquisition of fragmentation spectra of multiple charged peptides. Up to 12 data-dependent MS/MS spectra were acquired for each full-scan spectrum recorded at 70,000 full-width half-maximum resolution. Fragment spectra were acquired at a resolution of 35,000. Overall cycle time was approximately 1 s.Solanum lycopersicum, ITAG2.4, 34725 entries) using Andromeda (e.g. keratins) and reverse hits identified by MaxQuant were excluded from further analysis. For increasing quantitative coverage in label-free quantitation “match between runs” was selected in MaxQuant with a tolerance of 0.75 min within 20 min time windows throughout the run. Raw files and identified spectra were submitted to ProteomeXChange and are available to the public under the accession number PXD010819. Representative annotated spectra of phosphopeptides are available as supplemental Fig. S2.Protein identification and ion intensity quantitation was carried out by MaxQuant version 1.5.3.8 . Spectrandromeda . Therebym/z) were normalized against the total ion intensities in that sample . Subsequently, each peptide ion species  was scaled against the average normalized intensities of that ion across all treatments. For each peptide, values from three biological replicates were averaged after normalization and scaling. In case of non-phosphopeptides, protein ion intensity sums were calculated from normalized scan scaled ion intensities of all proteotypic peptides. A list of quantified phosphopeptides including normalized ion intensities, standard deviation and number of spectra is available as supplemental Table S1.Reported ion intensity values were used for quantitative data analysis. cRacker  was used+-ATPase activity was determined by resuspending 5 μg of microsomal proteins in 10 μl resuspension buffer and 40 μl of reaction buffer containing 25 mm Tris-HCl pH 6.3, 50 mm KCl, 5 mm MgCl2, 5 mm CaCl2, 0.1 mm ouabain (Thermo Fisher Scientific), in presence of 1 mm NaN3, 100 nm concanamycin A (Sigma-Aldrich), 0.02% Brij58 (Sigma-Aldrich), 0.5 μg/μl BSA and 10 mm DTT. The reaction was initiated by the addition of 1 mm ATP, proceeded for 2 h at room temperature, and stopped with 150 μl of stopping reagent . After 5 min, 150 μl of 2% tri-sodium citrate and 2% acetic acid solution was added and incubated for 15 min at room temperature. The absorbance as a measure of inorganic phosphate was determined at 640 nm using plate reader . ATPase activity was measured with and without 400 μm orthovanadate (Na3VO4), and the difference between these two activities was attributed to the PM H+-ATPase.Plasma membrane (PM) HCCATGGCAGATGGTTCAGCTCCG (forward) and ATATCTCGAGCATGTGCTGGTATTCGGGATT (reverse) with S. lycopersicum leaf cDNA as template. The PCR product was cloned into pCR2.1-TOPO vector (Thermo Scientific) and amplified in E. coli. The MPK2 insert was excised with NcoI and XhoI (underlined in the primer sequence) and ligated to pET21d (+)  to produce 14420HispET21d, which was transferred into E. coli strain BL21 RIL  to be expressed as C-terminally 6xHis-tagged recombinant protein. MPK2 was expressed in unmodified form and as constitutively active mutant (CA) with two amino acid substitutions D216G/E220A (The whole coding sequence of MPK2 (Solyc08g014420.2.1) was amplified by PCR using the primers ATTA6G/E220A .CCATGGCAGATGGTTCAGCTCCG and 14420D216G/E220AR1 (GTCACAACATATGCGGTCATAAAGCCAGTTTCAGAAG); or 14420D216G/E220AF2 (CTTCTGAAACTGGCTTTATGACCGCATATGTTGTGAC) and (ATATCTCGAGCATGTGCTGGTATTCGGGATT).The mutations were introduced using the following primers: ATTACCATGGCACATCACCATCACCATCACTTCAGCAGTGAGTGTAGTTTG and AATTCTCGAGTTATGCACTGGATCTCCATATTC.For PLL5 (Solyc06g076100.2.1), the catalytic domain (residues from 241–708) was amplified by PCR using the primers ATAE. coli strain BL21 RIL to be expressed as N-terminally 6xHis-tagged recombinant protein.The PCR products were cloned into pET21d (+) as described above to produce His76100pET21d, which was transformed into E. coli BL21 RIL cells were grown at 37 °C, 200 rpm to an A600 value of 0.8 and then induced with 1 mm isopropyl-β -d-thiogalactopyranoside (IPTG) for 4 h at 30 °C. Cells were pelleted by centrifugation and lysed in Bugbuster™ protein extraction reagent (Merck) by continuous agitation for 20 min at room temperature. After centrifugation at 12,000 × g for 20 min at 4 °C, the supernatants were collected. The supernatants were subjected to affinity purification on Ni-NTA agarose following the manufacturer's instructions .m HEPES pH 7.0, 10 mm MgCl2, 2 mm DTT and 0.1 mg/ml BSA) with 1 μm ATP at 30 °C. Kinase activity was measured as ATP consumption by addition of 50 μl of Kinase-Glo® Plus Reagents , 10 min incubation at 30 °C, and subsequent recording of the decrease in luminescence (Tecan Spark luminometer). To detect the phosphorylated peptides by mass spectrometry, the reaction was performed as described with 20 μg of peptide substrate. The reaction was stopped by addition of TFA until pH ≤ 3 and reaction products were purified over C18-Stage tips (Recombinant MPK2 (2 μg) was incubated with peptide substrate  overnight in 50 μl of reaction buffer  at room temperature for 30 min. Released phosphate was detected by addition of 150 μl of 8% ascorbic acid, 2.1% HCl and 0.7% ammonium molybdate. After 5 min at room temperature the reaction was stopped by addition of 150 μl 2% tri-sodium citrate and 2% acetic acid  was incubated with the phosphorylated peptide substrate (GLDIETIQQSYT(ph)V; amount as indicated for each experiment) in 50 μl of reaction buffer (20 mtic acid . After iSolanum peruvianum) cell cultures as an alkalization of the culture medium . Systemin-induced medium alkalization is a unique feature of this cell culture system that has been used by us and others to characterize systemin activity . A17 was completely inactive at this concentration. When A17 was added to the systemin treatment, the alkalization response was reduced with increasing concentrations of A17 (B). To explain the antagonistic activity of A17, it was suggested that the peptide may interact with the binding site of the systemin perception system . Systemin-responsive phosphopeptides were defined based on statistical analysis and characterized by over-representation analysis, correlation analysis to predict systemin-responsive kinase-substrate pairs. Finally, selected proteins proposed to be involved in the systemin signaling cascade were characterized in more detail.The aim of this work was to define systemin-induced signaling cascades based on phosphoproteome time-course experiments after systemin treatment. To filter the datasets for changes in phosphorylation that are specifically induced by systemin, two control treatments were used, namely the inactive systemin analogon systemin-A17, as well as mock-treated cell cultures A, 2B. SyC). As described in a thorough meta-analysis of plant phosphorylation sites (D). Quantitative information under at least one treatment condition was obtained for 2960 phosphopeptide species matching 1729 protein groups in at least one biological replicate. Thereby, the majority of phosphopeptides were identified at all time points.A total of 3312 phosphopeptide species were identified which differed in peptide sequence or in the position of their modification sites. Phosphorylation was observed most frequently on serines (89.5%), followed by threonines (10%) and at rather low frequency at tyrosines <1%, C. As deson sites , this amon sites D. QuantiWe studied the dynamics of protein phosphorylation in response to systemin and control  treatments. As shown previously , no signA; supplemental Table S2). A high number of 827 phosphopeptides was rapidly dephosphorylated upon systemin treatment (cluster A). Three clusters were characterized by a transient increase in phosphorylation, with 465 phosphopeptides peaking at 2 min (cluster B), 501 at 5 min (cluster C), and 705 at 15 min after systemin treatment (cluster D). Further 1001 phosphopeptides were classified as not responsive (cluster F). The stimulation of suspension cells with A17 also resulted in characteristic changes in phosphorylation . Interestingly, upon A17 stimulation no phosphopeptides displayed a transient increase in phosphorylation at 5 min, i.e. cluster C is missing. Instead, 1042 phosphopeptides showed an increase in phosphorylation at 45 min (cluster E). The missing cluster C and emergence of a “later” maximum in cluster E in A17 treatment suggests that the control treatment with A17 does trigger some cellular response, but weaker and delayed compared with systemin treatment. Treatment of the suspension cells with water resulted in response groups very similar to the clusters observed upon A17 treatment .Treatment of suspension cell cultures with systemin resulted in characteristic changes of phosphorylation patterns. K-means clustering was used to group these phosphorylation responses . Dephosphorylation of the regulatory threonine residue was associated indeed with a measureable decrease in H+-ATPase activity at 2 to 5 min of systemin treatment (C). In contrast, A17 treatment induced a very transient increase in C-terminal phosphorylation and H+-ATPase activity at 2 min. We conclude that the time profiles presented in +-ATPase, this relationship was verified by activity tests.To substantiate the observed phosphorylation profiles as physiologically relevant, we analyzed the activity of the plasma membrane H+-ATPase  that has+-ATPase . Two pho+-ATPase , 50. In +-ATPase A, 4B. Dereatment C. In conA). For example, 649 out of 727 phosphopeptides in systemin-induced cluster A (de-phosphorylated after 2 min), were classified in a “later” cluster (B to F) upon A17 treatment or were not identified at all (7 phosphopeptides). Thus, these proteins were considered as early systemin-responsive. Only 71 phosphopeptides showed the same rapid de-phosphorylation kinetics  when treated with A17. In contrast, 665 of the 1001 phosphopeptides that were clustered as non-responsive to systemin treatment (cluster F), upon A17 treatment showed a peak phosphorylation in an earlier cluster. A total of 161 proteins were found with equal classification upon A17 and systemin treatment, and 83 phosphopeptides were not identified in the A17 treatment. In total, 1515 phosphopeptides  were classified as systemin-specific.To define precisely which of the phosphorylation events are indeed systemin-specific, we compared for each phosphopeptide the phosphorylation time profile induced by systemin with those induced by A17 A. For exsupplemental Fig. S1) was calculated for each phosphopeptide time profile identified also under A17 treatment and divided by the number of data points. Only phosphopeptides with a minimum of three quantification values were included in this statistical analysis. On average, phosphopeptides classified with a “later” or “earlier” response based on the k-means clustering had significantly higher squared deviations  compared with phosphopeptides with equal time profile classification (B). Phosphopeptides not identified in the A17 treatment were considered systemin-specific . Using a threshold value for the squared deviations of 0.8, less than 5% of the phosphopeptides with “equal” time profiles (A) under systemin and A17 treatment were classified as “systemin-induced.” We considered this an acceptable false classification rate and applied this threshold to all phosphopeptide profiles. As a result, 162 phosphopeptides in systemin-induced cluster A were considered systemin specific, as well as 266, 259, 474, and 672 phosphopeptides in systemin response clusters B, C, D and F, respectively. Based on this classification, a total of 1602 phosphopeptide response profiles were considered as early systemin-responsive. The same analysis was applied also to the systemin-treatment compared with the water responses, yielding a total of 1286 systemin-induced phosphopeptides . Definition of systemin-induced responses firstly compared with A17 and secondly compared with water treatment yielded a high overlap of 1039 systemin-induced phosphopeptide response profiles. The high overlap with phosphopeptides identified as early systemin-responsive using water as control treatment, is consistent with the inability of A17 to trigger the alkalization response . Systemin-responsive phosphopeptides in cluster A mainly related to transport proteins and signaling. Cluster B with transient increase in phosphorylation at 2 min showed highest enrichment of phosphopeptides matching proteins with functions in transport , cellular processes , signaling , and cell wall proteins . Phosphopeptides of proteins in RNA processes (e.g. transcription factors) were mainly enriched in clusters C and D with transient increase in phosphorylation at later time points . Vesicle transport proteins (bin 31.4) were highly abundant in clusters B and C, whereas transcription factors (bin 27.3) and protein post-translational modification (bin 29.4) were overrepresented in all clusters. The phosphopeptides not identified upon A17 treatment were particularly enriched for vesicle transport proteins (bin 31.4), cellulose synthases (bin 10.2), P- and V-ATPases (bin 34.1), and auxin-related processes (bin 17.2.3) . We then derived kinase-substrate relationships between kinases and the putative substrates over-represented among the systemin-responsive phosphopeptides.To better understand the processes affected by systemin-specific phosphorylation at each time point, we carried out an over-representation analysis (Fisher Exact Test) of early systemin-responsive phosphoproteins in clusters A to F C. Systemsupplemental Table S4). Not all these phosphopeptides met the requirement of at least four quantitative values across the time course and were therefore excluded from further analysis. The remaining phosphopeptides matched 56 kinases and 17 phosphatases, and their phosphorylation profiles were used to construct a correlation network of systemin-induced responses. Each systemin-responsive phosphopeptide of a kinase or phosphatase was correlated against other phosphopeptides of categories over-represented in the systemin-induced response clusters (C). A stringent cutoff of r = 0.85 was used to display only proteins that are highly correlated with systemin-responsive kinases and phosphatases. Based on these criteria, phosphopeptides of 44 kinases and of 9 phosphatases showed high correlation with phosphopeptides of 207 potentially interacting proteins . We considered these correlating phosphopeptides as candidate substrates for respective kinases or phosphatases. In the displayed network, each node indicates a kinase or phosphatase with systemin-responsive phosphorylation sites, and each edge describes the correlation between the phosphorylation time profile of a kinase or phosphatase peptide and a potential substrate peptide. For each kinase/phosphatase displayed in the networks, node size reflects its degree, i.e. the number of correlating substrate phosphopeptides.Among the early systemin-induced phosphopeptides, 165 and 29 phosphopeptides matched 67 protein kinases and 17 phosphatases, respectively , a protein phosphatase 1 regulatory subunit (Solyc02g070260), and a protein phosphatase 2C (Solyc10g005640) had highest numbers of systemin-responsive correlation partners . It became apparent that systemin-responsive kinases in clusters B, C, and D, which showed a transient phosphorylation increase, showed a higher degree than kinases without systemin specificity, but kinases in general had higher degree than their target proteins (C). For phosphatases, higher than average degree was observed for nine systemin-responsive phosphatases in cluster A (rapid dephosphorylation response), whereas in cluster F with no systemin-induced phosphorylation response, higher than average degree was observed for nine phosphatases that were not linked to the systemin response. We conclude from the network analysis that kinases showing early systemin-induced phosphorylation, are indeed involved in the phosphorylation events that peak at 2 min (cluster B), 5 min (cluster C) and 15 min (cluster D) of stimulation, whereas early systemin-responsive phosphatases mediate rapid dephosphorylation responses (cluster A). A full list of kinase-target relationships proposed from the correlation analysis is available in supplemental Table S5. Selected candidate targets for systemin-responsive kinases and phosphatases are discussed below. Although our data set reveals correlative relationships between the phosphorylation sites that may well be indicative of kinase/phosphatase-substrate interaction, it must be kept in mind that the data do not directly allow to derive the activity change of the respective proteins without further experiments.Within this kinase-substrate correlation network, we then displayed the degree distribution of phosphopeptides from early systemin-responsive kinases/phosphatases, other kinases/phosphatases not classified as systemin-responsive and the putative targets in systemin response clusters A to F C, 6D. Itproteins C. For phThe MAP-triple kinase (MAPKKK) Solyc11g033270 (degree 210) was identified with two phosphorylation sites outside the kinase domain: S(ph)SNATVEASGLSR (Ser859) showed rapid dephosphorylation (cluster A) and SEEVDGS(ph)SSIR (Ser345) showed transient phosphorylation at 15 min (cluster D). The phosphorylation time profile of the rapidly dephosphorylated MAPKKK peptide S(ph)SNATVEASGLSR showed highest correlation with dephosphorylated peptides of an ubiquitin ligase (LEEGS(ph)SPEQR, Solyc05g007820), a YAK1 homologous protein kinase (TVYSY(ph)IQSR, Solyc03g097350), a RNA recognition motif containing protein (SSDS(ph)QELTTTELK, Solyc02g062290), and a protein phosphatase 2C family protein (LTSS(ph)PEAEIK, Solyc06g065580). The finding that kinases and phosphatases correlate with the same phosphopeptide suggest kinase-phosphatase activation/inactivation loops upon systemin stimulation. The MAPKKK peptide transiently phosphorylated at 15 min of systemin treatment (SEEVDGS(ph)SSIR) showed highest correlation with a transient phosphorylation of a calmodulin domain-containing protein (DYS(ph)ASGYSSR, Solyc06g053530).A). Amon these were protein kinase Solyc02g076780 (AIS(ph)LPS(ph)SPR), chromatin remodeling factor Solyc01g094800 (GGVVDS(ph)DDDLAGK), transcription initiation factor Solyc01g088370 (DGEAS(ph)DEEEEYEAK), kelch-repeat containing protein kinase Solyc11g071920 (LIHPIPPVSS(ph)PENS(ph)PER), and two phosphorylation sites of cell cycle-related protein Solyc10g078180 (ELEDGIELS(ph)AGNEK, ALENM(ox)DDAVGQS(ph)PQEVR). The majority of these putative target peptides contained the expected “(ph)SP” MAPK substrate motif. Interestingly, the orthologs proposed here as substrates of tomato MPK2 have already been confirmed as substrates of MPK6 in Arabidopsis under various conditions (+-ATPase LHA1.For MAP-Kinase MPK2 (Solyc08g014420), which is the tomato homolog of Arabidopsis MPK6, several potential targets were proposed from the correlation network A. Amon tnditions . All of +-ATPases  were correlated with systemin-induced phosphopeptides of distinct kinases and phosphatases (A). Candidate kinases with highest correlation to the C-terminal activation site (T955 in GLDIETIQQSYT(ph)V) of LHA1 were receptor kinase Solyc02g079590 (r = 0.99) and MPK2 (Solyc08g014420) (r = 0.98). This C-terminal LHA1 peptide also was highly correlated with a regulatory phosphorylation site in the N terminus of the homolog of Arabidopsis POLTERGEIST-LIKE 5 phosphatase , a member of the PP2C clade C phosphatases (+-ATPase Solyc07g017780 was found with receptor kinase Solyc11g011020 (r = 0.94).Phosphopeptides encompassing the activating C-terminal phosphorylation site of two different plasma membrane Hphatases A. Candidphatases , 55. Hig+-ATPase Solyc07g017780. The number of data points identified for these two phosphorylation sites was too low to be included in the correlation analysis. Interestingly, the activating site was also identified at 15 min of systemin treatment, but phosphorylation at that site did not qualify as specifically responsive to systemin based on the sum of squares deviation analysis. Interestingly, the phosphopeptide containing the inactivating serine residue was identified at 5 min of systemin treatment, the time point when systemin stimulation resulted in a minimum in measured H+-ATPase activity (C).In addition to the C-terminal phosphorylation site, another activating phosphorylation site (T(ph)LHGLQPPEASNLFNEK, ) and an activity C.+-ATPase LHA1 with candidate kinase MPK2 and candidate phosphatase PLL5 was investigated further by in vitro activity assays . Recombinant MPK2 was expressed either in wild-type form (WT) or as a constitutively active mutant (CA) with D216G and E220A amino acid substitutions (B). Increasing the substrate peptide concentration from 10 μm to 200 μm resulted in increasing kinase activity (C). Phosphorylation of the C-terminal LHA1 peptide substrate at T955 by recombinant MPK2 was confirmed by mass spectrometric analysis . Recombinant PLL5 was able to dephosphorylate synthetic phosphopeptide GLDIETIQQSY(pT)V in vitro (D). PLL5 activity, assayed as release of inorganic phosphate, was observed only in presence of the phosphopeptide substrate, and increased with increasing substrate concentration (E). De-phosphorylation of the LHA1-derived phosphopeptide substrate by PLL5 phosphatase was confirmed by mass spectrometric analysis . Interestingly PLL5 phosphatase was identified in systemin-induced cluster A (rapid dephosphorylation). Thus, the phosphatase may well be involved in dephosphorylation of LHA1 at early systemin stimulation.The interaction of the C-terminal peptide of Hy assays D, 7E. Reitutions . Kinase itutions B. Increaactivity C. Phosphin vitro D. PLL5 antration E. De-phoA) with concomitant increase in H+-ATPase activity  at later time points (15 min) of systemin treatment.MPK2, on the other hand, was identified in systemin-induced cluster D with maximum phosphorylation of peptide VTSETDFM(ox)T(ph)EY(ph)VVTR at 15 min. The identified phosphopeptide contained the typical MAP-Kinase activating motif (pT)E(pY). These data are fully consistent with a previous report showing that MPK2 is transiently activated in response to systemin with maximum activity between 15 and 30 min after treatment . MPK2 thFig. 4r = 0.90; Asupplemental Fig. S4).Another protein with highly systemin-specific phosphorylation response (rapid dephosphorylation) was the respiratory burst oxidase. Phosphopeptides were identified for two isoforms of respiratory burst oxidases (NLSQM(ox)LS(ph)QK, Solyc03g117980; DVFSEPS(ph)QTGR, Solyc06g068680). Interestingly, Solyc03g117980 has previously been shown to be required for the systemic induction of wound-response genes  which isBsupplemental Fig. S4). Systemin-induced phosphorylation changes of cellulose synthase-like proteins were either transient phosphorylation at 2 min (cluster B), or rapid dephosphorylation (cluster A). Peptide S(ph)SEGDLTLLVDGKPK of cellulose synthase-like protein Solyc04g077470 (transiently phosphorylated at 2 min) showed strongest correlation with an uncharacterized receptor kinase (NNTS(ph)SVSPDSVTAK, Solyc12g036330). Rapid dephosphorylation of GLIDSQSLSSS(ph)PVK of cellulose-synthase-like protein (Solyc09g075550) correlated with peptide GQLPS(ph)GQVVAVK matching an uncharacterized receptor-like cytoplasmic kinase (Solyc05g024290). These two peptides also showed a high correlation to a POLTERGEIST-LIKE 1 phosphatase PSQSLR; r = 0.92).Among the target proteins with most significant systemin-induced phosphorylation responses were cellulose synthase proteins. Four phosphopeptides matching cellulose synthase-like proteins (SSS(ph)RLNLSTR Solyc04g077470, SHS(ph)GLMR Solyc08g076320, SSS(ph)ESGLAELNK Solyc09g057640, GLIDSQSLSSS(ph)PVK Solyc09g075550) were identified as potential targets of systemin-specific kinases and phosphatases . Phosphopeptides of small GTPases potentially involved in vesicle budding and fusion also were highly correlated including the RAB-GTPase Solyc09g098170 (AELS(ph)VNR), the RAB GTPase activator Solyc12g009610 (TLS(ph)SLELPR), and the ARF GTPase activator Solyc05g023750 (NS(ph)SELGSGLLSR). Correlating kinases and phosphatases included receptor kinase Solyc07g055130 and PP2C Solyc05g055790 for the RAB-GTPase, as well as receptor kinases Solyc07g056270 and Solyc12g098980 for the RAB and ARF GTPase activators, respectively.In total, 52 kinases and 8 phosphatases were found with high correlations to phosphopeptides of vesicle trafficking proteins. Although phosphopeptides of adaptins and syntaxins were rapidly dephosphorylated (cluster A), most phosphopeptides of vesicle-associated membrane proteins (VAMPs) were transiently phosphorylated at 2 min of systemin stimulation (cluster B) (Four IQ-domain 13-containing proteins (FTLPAGAVS(ph)PR Solyc08g014280, EAS(ph)PKVT(ph)SPR Solyc01g108920, LSFPLTPSS(ph)TGSVK Solyc10g008490, FNSLS(ph)PR Solyc05g014760) were rapidly dephosphorylated upon systemin stimulation (cluster A). These proteins were correlated with phosphopeptides of CBL-interacting kinase (Solyc04g076810), CDPK (Solyc04g009800), MAP-triple-kinases , and two phosphatases (Solyc10g008490 and Solyc01g067500). A phosphopeptide of calcium-ATPase Solyc02g064680 displayed a transient phosphorylation at 5 min (cluster C) and showed high correlation with phosphopeptides of phosphatase 2C (solyc05g055790) and kinases Solyc07g055130 and Solyc01g094920.Among the early systemin-induced phosphorylation/dephosphorylation events were several phosphopeptides of proteins known from other hormone signaling pathways. For example, phosphopeptides of abscisic acid (ABA) response factors (ASSESSSEDEADS(ph)AEEAGSSK, Solyc04g081340; VGS(ph)PGNDFIAR, Solyc01g108080), auxin response factor (INS(ph)PSNLR, Solyc05g047460), EIN4 (GGT(ph)PELVDTR, Solyc05g055070) and PIN3 (PSNFEENCAPGGLVQS(ph)SPR, Solyc05g008060) were rapidly dephosphorylated upon systemin treatment (cluster A). Another phosphorylation site in ABA-response factor Solyc04g081340 (RSS(ph)ASATQVQAEEQAPR), SSS(ph)RLNLSTR of ACC-synthase (Solyc03g007070), and another phosphopeptide of EIN4 (RSS(ph)ASATQVQAEEQAPR) were transiently phosphorylated at 2 min of systemin treatment. A third phosphopeptide of EIN4 (GGS(ph)QTDSSISTSHFGGK) was transiently phosphorylated at 5 min of systemin treatment. Kinases with high correlations to the phosphorylation patterns in cluster A were CTR1 (Solyc09g009090), a receptor kinase (Solyc11g011020), and a CBL-interacting protein kinase (Solyc04g076810).+-ATPase activity (The aim of our work was to (1) identify early events induced by systemin and (2) identify and predict kinases and phosphatases involved in these signaling events. The maturation of the signaling peptide systemin is beginning to be understood , and theactivity , or the activity  have beeAntagonistic and inactive peptides, such as mutant peptide variants, chemically modified peptides or peptide mimetics, are important tools for the characterization of peptide-receptor interaction and to discover endogenous peptide function –61. For +-ATPase , which of NT1.1 . Third,  seconds .Nonetheless, our work identified systemin-specific phosphorylation sites at 56 kinases and 17 phosphatases to be involved in systemin signaling, likely downstream of the primary systemin receptor SYR1 . SubstraThe identified phosphorylation patterns at 2 min may thus represent early responses of the systemin signaling cascade. This point of view is supported by the observation of rapid (de)phosphorylation changes for several cytosolic proteins such as a CBL-interacting kinase, MAP-triple kinase, and CDPKs already at 2 min (clusters A and B), whereas a large number of receptor kinases with systemin specific phosphorylation patterns peak only after 2 min or 5 min. Also observed very rapidly after systemin stimulation were plasma membrane-located proteins that are known to belong to the systemin signaling cascade, including the proton pump that is rapidly dephosphorylated after systemin treatment, and two RBOH isoforms also showing rapid dephosphorylation. In contrast, G-protein signaling or transcription factor phosphorylation was found at later times.Later responses of the systemin signaling cascade included MAP-kinase signaling at 15 min of treatment. Here, phosphorylation of activating TEY motif particularly of MPK2 was observed. MPK2 was experimentally identified as a kinase re-phosphorylating the plasma membrane ATPase and seven additional putative substrates were also described for the Arabidopsis homolog MPK6. Thus, as suggested earlier , the MAPin vitro kinase assays as kinase and phosphatase for reversible phosphorylation of the C-terminal regulatory site of the plasma membrane H+-ATPase. We highlighted several proteomic processes upon systemin stimulation ranging from plasma membrane processes to cytosolic and nuclear signaling, which will provide a valuable resource for further in-depth functional studies in future elucidation of systemin signaling cascades.Our study presents 56 systemin specific kinases and 17 systemin specific phosphatases. Putative substrates were identified by pairwise correlations of systemin induced phosphorylation time profiles. Thereby we identified novel candidates for the systemin signaling pathway. Two of these candidates, MPK2 and PLL5 were confirmed by http://www.ebi.ac.uk/pride) with the dataset identifier PXD010819.The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE  partner


Schistosoma mansoni parasite burden and immune response on allergic reactivity of individuals living in a schistosomiasis endemic area in Brazil. Fecal samples from the participants were collected for extensive parasitological examinations by spontaneous sedimentation, Kato-Katz, Helmintex and Saline Gradient tests and molecular detection of S. mansoni by qPCR. Additionally, the concentrations of cytokines and chemokines, total IgE and IgE-reactivity to common house dust allergens were quantified from serum samples. IgE reactivity to dust allergens was detected in 47 individuals (23.8%), and 140 individuals (54.4%) were diagnosed with S. mansoni infection. Most of the infected population  presented very low parasite burden (≤12 eggs/g of feces). The frequency and intensity (p ≤ 0.03) of allergic reactivity were lower in S. mansoni-infected compared with non-infected individuals. Multivariable logistic regression models adjusted by age revealed that allergic reactivity was positively associated with low IL-10 response  and high concentration of the inflammatory mediators IL-33  or TNF-α  in serum, and inversely associated with S. mansoni infection . Most importantly, the logistic regression demonstrated that the modulatory effects of Schistosoma infection depend on parasite burden, with individuals infected with ≤12 eggs/g of feces showing allergic IgE-reactivity similar to non-infected individuals Altogether, our data show that immunomodulation of allergic reactivity depends on S. mansoni burden, low type-2 inflammatory response, and high level of IL-10.Helminth infections and allergies are characterized by a predominant type-2 immune response. In schistosomiasis, the Th-2 response is usually accompanied by induction of immunoregulatory mechanisms that contribute to worm survival and less severe schistosomiasis. Although helminth-induced immunomodulatory mechanisms seem to affect atopy, epidemiological studies on the relationship between helminths and allergy have been inconsistent, and data suggest that the modulatory effects may be influenced by helminth species, chronicity of infection, and parasite burden. Here we performed a cross-sectional study to investigate the effects of  Allergies are chronic diseases characterized by an intense and uncontrolled type-2 inflammation, with increased expression of typical cytokines such as interleukin (IL)-4, IL-5, IL-9, IL-13, eosinophilia and mast cell activation, elevated reactive immunoglobulin (Ig)E production, and increased mucus production in response to allergen exposition  and assoAllergic inflammation results from a combination of genetic predisposition and environmental exposure and may affect different body parts resulting in clinical manifestations such as asthma, eczema, rhinitis, hay fever, and food allergies –14. The Although helminth infections are characterized by predominant type-2 immune response, they also stimulate an immune regulatory network response with production of anti-inflammatory cytokines, IL-10 and TGF-β, which modulate the immunopathology and facilitate parasite survival –31. PrevSchistosoma mansoni and Trichuris trichiura, were more prone to atopy  and all project details have been registered on the Brazilian Platform for Research with Human Subjects (Plataforma Brasil—protocol number: CAAE#21824513.9.0000.5091). Prior to the commencement of the research activities, the subjects were invited to participate in local meetings or receive house-to-house visits to hear about the aims of the research and any possible risks. All enrolled participants and/or their legal guardians agreed to participate in the research and sign an informed consent form. All the data were anonymized prior to analysis. After the evaluations, the individual test results were sent to each participant and, regardless of participation in the study, patients with confirmed parasite infection received treatment and patients with other diseases were either treated at the local health clinic or directed to specialized treatment. Schistosomiasis cases were treated with praziquantel ; intestinal helminthiases were treated with albendazole (400 mg); and protozoan parasites were treated with metronidazole (250 mg/2x/5 days).The current study was conducted in 2014 with individuals from a rural community near the village of Brejo do Amparo, Municipality of Januária, located in the northern area of the state of Minas Gerais, Brazil (15° 29′ 16″ S 44° 21′ 43″ O). This rural community is historically endemic for schistosomiasis and currently presents around 270 residents. According to the Brazilian Schistosomiasis Control Program and local health authorities, the estimated prevalence of schistosomiasis was around 20% in 2010 , and no We carried out a cross-sectional population-based study including residents of both sexes, aging between 2 and 88 years old who signed the informed consent form. Pregnant women and individuals who could not understand and/or cooperate with the study protocol were excluded. The participants were asked to provide three fecal samples, which were collected on consecutive days and used for the parasitological analysis by different methods. The first fecal sample, containing at least 50 grams of feces, was collected in a 500-ml plastic container and used for complete fecal evacuation. The other two fecal samples were smaller and were collected in 80-ml plastic pots. Participants aging between 6 and 75 years old were also invited to donate a blood sample, which was used for immunological analysis. Five Milliliter of venous blood were collected from each participant in Ethylenediaminetetraacetic acid (EDTA)-coated tubes . Hemogram analyses were performed by a clinical laboratory located in the region and peripheral blood eosinophil counts were used in current study. Additionally, 10–15 ml of blood were collected without anticoagulant, and serum samples were aliquoted and stored at −20°C for subsequent quantification of circulating cytokine and chemokines, total IgE, and the level of IgE reactivity to common domestic dust allergens. The flow diagram in Figure The participants or their legal guardians answered an individual questionnaire providing demographic and occupational information, as well as previous clinical conditions that could be relevant for the research. A family-based questionnaire was also applied to gather information on household construction, water supply, sanitation, and other socio-economical aspects.The parasitological analysis adopted herein has been previously described . BrieflyThe clinical record of cutaneous leishmaniasis, diagnosed and documented by the local health program, was included in the analysis since this protozoan disease is also endemic among the study population.S. mansoni . The amplification reaction was performed in a final volume of 25 μl containing: 12.5 μl of TaqMan® Universal PCR Master Mix , 0.1 μM of each S. mansoni-specific primer, 0.25 μM of probe, BSA 0.1 μg/μL; 4 mM of MgCl2; and 4 μl of 5-fold diluted stool DNA sample. For each run, positive (DNA extracted from adult worms) and negative (no DNA template) controls were performed. The amplification reaction was conducted on the StepOnePlus™ Real-Time PCR System  using the universal cycling program with 45 cycles and annealing temperature of 60°C. A 92 pb fragment of the human β-actin gene was amplified and detected as an internal control. The cut-off for positive and negative samples was defined by a standard curve established with S. mansoni DNA extracted from adult worms. The DNA extraction and amplification procedures were performed by a trained researcher in separate rooms inside a biological safety cabinet using disposable sterile pipette tips with filters.Total DNA was extracted from 500 mg stool samples using the commercial QIAamp® DNA Stool Mini Kit, following the manufacturer's instructions . The primers sense 5′-CCG ACC AAC CGT TCT ATG A-3′ and anti-sense 5′-CAC GCT CTC GCA AAT AAT CTA AA-3′ and the probe 5′-6[FAM]/TCG TTG TAT CTC CGA AAC CAC TGG ACG/[(3BHQ1)] (Integrated DNA Technologies—IDT-USA) were used in the PCR reaction, as previously described . As repoS. mansoni eggs detected in six slides of Kato-Katz  and multiplying the mean value obtained by 24 to determine the number of eggs per gram of feces (EPG). Individuals who had a positive result for S. mansoni infection in qualitative tests , but were negative in the six Kato-Katz slides were classified as infected with parasite burden < 4 EPG.Individuals who presented eggs in any of the parasitological tests or positive PCR-reaction were considered positive. The intensity of infection was calculated by determining the mean number of 2SO4 solution, and absorbance was determined using a 450 nm filter in the ELISA reader . Known concentrations of the recombinant human IgE were used to generate a standard curve, which was used to determine the IgE concentration (ng/mL) in each serum sample based on the optical density (OD) readings.The total IgE concentration in each serum sample was determined by a commercial kit  following the manufacturer's instructions and a previously optimized protocol . Serum sDermatophagoides pteronyssinus, D. farinae, Cladosporium herbarum, Aspergillus fumigatus, and cat and dog fur antigens. Undiluted serum samples from the study population, positive and negative serum controls, and samples containing known concentrations of allergen-reactive IgE for the standard curve were added to the anti-human IgE-sensitized microplates provided by the fabricant. Following washing, 100 μl of the biotin-conjugated allergen mixture (HMx3) was added to the wells containing the serum samples and 100 μl of biotin-conjugated anti-IgE antibody solution was added to the wells with the standard curve. This was followed by the addition of peroxidase (HRP)-conjugated streptavidin solution. The reaction was developed with 100 μl/well of the substrate solution containing Tetra-methyl-benzidine (TMB) and stopped after 20 min by the addition of 100 μl of 4NH2SO4 solution. The color intensity was measured spectrophotometrically at 450 nm (Molecular Devices—Versa Max). The concentrations of reactive-IgE in serum from each patient were estimated based on the reactivity of the standard curve and expressed as International Units (IU). All patients with reactivity above 0.35 IU/ml were considered reactive. The intensity of IgE-reactivity was expressed as total concentration or categorized as Non-reactive (<35 IU/ml), Low (0.35–0.7 IU/ml), Moderate (0.7–17.5 Ul/ml), or High (>17.5 IU/ml).The serum samples were additionally used for quantification of IgE reactivity against common household dust antigens using the commercially available IgE REAST kit  and following the manufacturer's recommendations. For the current analysis, we selected the HMx3 allergenic mixture composed of the common household dust allergens, including Concentrations of pro-inflammatory cytokines, such as IL-1β, IL-6, TNF-α, chemokines and cytokines of type1/17, IL-27, CXCL-10, CCL-3, IL-17, regulatory and type-2, IL-10, IL-5, IL-13, IL-33, CCL-5, CCL-11, CCL-17 were measured in duplicate in serum samples from each participant using a sandwich enzyme-linked immunosorbent assay (ELISA) kit, according to the manufacturer's instructions . Each serum sample was diluted in phosphate buffer (1:2) with 0.1% BSA (PBS/BSA), except for CCL-5, which was diluted 1:20 in PBS/BSA solution. Known concentrations of the recombinant proteins were used to generate a standard curve to determine the concentration (pg/ml) of the samples based on OD readings.x2 test, means were compared using Student's t-test or analysis of variance (ANOVA), and the Kruskal-Wallis test was used to compare medians. Correlation analysis was used to quantify the association between continuous variables. Logistic regression models were used to assess the relationship between IgE-reactivity against common house dust allergens  and the variables analyzed . The strength of the association was assessed using the odds ratio (OR) with a 95% confidence interval (CI). Variables with p-values < 0.25 in the univariate analysis were selected for construction of the multivariate logistic regression models. Variables with low frequency and showing co-linearity were excluded from the multivariate analysis. For the categorical variables, the categories non-responder or non-infected were used as reference values for the logistic regression. The likelihood ratio test was used to define the final model with the best data fit , equally distributed throughout the age groups  participants reported past cutaneous leishmaniasis infection  presented with moderate or high parasite burdens (>100 EPG). The remaining 130 infected individual (93%) showed low parasite burden (≤100 EPG). Noteworthy, among the individuals with low parasite burden, the vast majority (83%) eliminated less than 12 EPG  were reactive (≥0.35 IU/mL). The median intensity of IgE-reactivity in this population was 7 IU/mL (IQR 5.1–9.5), and six individuals showed strong reactivity (≥17 IU/mL) Figure . The ratS. mansoni infection, while up to 60% of non-IgE-reactive individuals  were infected with the parasitic trematode (p = 0.06). Moreover, the intensity of IgE-reactivity against dust allergens was stronger in non-infected individuals in comparison with S. mansoni-infected individuals (p = 0.03)  Figure . Most im) Figure .p = 0.031) and IL-10 (p = 0.001) responders was significantly higher in the IgE-reactive group in comparison with the non-IgE-reactive individuals. A higher percentage of IgE-reactive individuals showed detectable IL-33 (p = 0.071), while a higher percentage of IL-13 responders was observed in the non-IgE-reactive population (p = 0.085)  among IgE-reactive and non-IgE-reactive individuals, independently of the parasitological status. As demonstrated in Table In contrast, almost all the individuals in the study population showed detectable serum levels of the chemokines CCL-3, CCL-5, CXCL-10, CCL-11, and CCL-17, and the cytokine IL-27. Table We also evaluated the total IgE concentration in serum and the number of circulating eosinophils Figure . The medSchistosoma infection status on IgE-reactivity against common house dust allergens. The first model was built using the presence of Schistosoma infection (mono- and co-infected) or absence of infection (negatives and any other parasitic infection) and it was adjusted by individual age. The analysis showed that allergic reactivity was inversely associated with the presence of infection and positively associated with low (≤100 pg/ml) serum concentration of IL-10 and high (>100 pg/ml) concentration of IL-33. Although CXCL-10 was statistically associated with IgE-reactivity  or high (>12 EPG) parasite burden. The analysis demonstrated an inverse correlation between allergic reactivity and Schistosoma infection only in infected individuals eliminating more than 12 EPG. The risk of development of allergic-reactivity was six times lower in these individuals. On the other hand, the protective effect was not observed in infected individuals with very low parasite burden (≤12 EPG). Additionally, allergic reactivity was also positively associated with low (≤100 pg/ml) serum concentration of IL-10 and high (>10 pg/ml) concentration of TNF-α  individuals showed IgE-reactivity to a combination of common household dust allergens, which is similar to the prevalence of atopy observed in other areas of helminth transmission in rural communities or poor neighborhoods of Latin America. In the Province of Esmeraldas, Ecuador, it was reported that 26.5% of the population from an urban area and 10.5% of the individuals from rural communities were IgE-reactive . In BrazSchistosoma-infected individuals, with the latter showing lower allergic reactivity intensity. Moreover, non-IgE-reactive individuals showed higher parasite load compared to IgE-reactive individuals, thus suggesting a modulatory effect of S. mansoni infection on the allergic process. The inverse association between allergic reactivity and S. mansoni infection was confirmed by the multivariable logistic regression model, which showed that individuals infected by S. mansoni were three times less likely to develop allergic reactivity to common dust allergens than non-infected individuals. Since schistosomiasis and allergies are chronic diseases, the regression model was adjusted by age, indicating that the effect of Schistosoma infection, as demonstrated here, was not dependent on host age. A similar outcome was observed for allergic reactivity intensity, with S. mansoni-infected individuals showing lower intensity of IgE-reactivity to dust allergens (data not shown).Our data also showed that the allergic reactivity was more frequent in non-infected than in A. lumbricoides and T. trichiura in rural areas of Ecuador  or allergen-specific IgE (sIgE) in individuals living in helminth highly endemic areas and showed an inverse association between allergen reactivity and the presence of chronic helminth infections, such as  Ecuador , ancylos Ecuador , and S. om Gabon . In cases et al.  demonstrs et al. . Moreovepic risk . Altogetpic risk , 71. In tive IgE , thus suS. mansoni or immunized with schistosome antigens and submitted to ovalbumin-induced asthma  expressed in innate immune cells, such as ILC2, and stimulates the early production of IL-13 and IL-5, which leads to eosinophil infiltration and activation of Th2 response , 77, 78.S. mansoni-infected individuals and should be better evaluated in the human population.The number of circulating eosinophils and the concentration of total IgE in serum was high in individuals living in Brejo do Amparo, independently of the allergic status. Similar results were also reported by Cooper et al.  in areasS. mansoni active infection by a combination of parasitological and molecular tests allowed us to identify a large number of individuals with very low parasite burden. The multivariate logistic regression model confirmed that, while S. mansoni-infected individuals with higher parasite burden (>12 EPG) showed risk of IgE-reactivity 6 times lower, individuals infected with very low parasite burden (≤12 EPG) presented allergic IgE-reactivity similar to non-infected individuals. This is the first time that a minimum threshold of parasite load has been identified as required for modulation of allergic response. The importance of helminth loading for induction of allergic modulation has been suggested by Oliveira et al.  are not enough to trigger modulatory mechanisms and, thus, do not affect the prevalence and intensity of the allergic responses. Even though we do not fully understand how S. mansoni infection modulate the allergic response, the current data indicated that the helminth infection, in a burden dependent fashion, induced anti-inflammatory response, including IL-10 production, that reduced IL-33 and/or TNF-α responses, which are associated with decreased IgE-reactivity. These results represent an important contribution to the understanding of helminthic-induced immunomodulatory mechanisms and should be further explored in the search for novel therapeutic strategies for the treatment of both helminthic infections and allergic diseases.In conclusion, herein we showed that SR, SG, and DN-C: Conceptualization. SR, FM, JR-O, VC, CS, EO, and SG: Performed experiments. SR, FM, EO, MC, and DN-C: Data analysis. MC, SG, and DN-C: Supervision. SG and DN-C: Resources and project administration. SR and DN-C Writing the manuscript. All the authors reviewed and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cryobacterium, Lysinomonas, Pedobacter, and Aeromicrobium phylotypes homologous to psychrotrophic and psychrophilic bacteria from various cold environments were noted in the total community, while the prevalent putative active bacteria belonged to Clostridium, Pseudomonas, Janthinobacterium, Stenotrophomonas, and Massilia genera. Variation in the microbial cell density of ice strata with the dissolved organic carbon (DOC) content and the strong correlation of DOC and silicon concentrations revealed a major impact of depositional processes on microbial abundance throughout the ice block. Post-depositional processes appeared to occur mostly during the 4,000–7,000 years BP interval. A major bacterial composition shift was observed in 4,500–5,000-year-old ice, leading to a high representation of Beta- and Deltaproteobacteria in the potentially active community in response to the increased concentrations of DOC and major chemical elements. Estimated metabolic rates suggested the presence of a viable microbial community within the cave ice block, characterized by a maintenance metabolism in most strata and growth capacity in those ice deposits with high microbial abundance and DOC content. This first survey of microbial distribution in perennial cave ice formed since the Last Glacial period revealed a complex potentially active community, highlighting major shifts in community composition associated with geochemical changes that took place during climatic events that occurred about 5,000 years ago, with putative formation of photosynthetic biofilms.Our understanding of the icy-habitat microbiome is likely limited by a lack of reliable data on microorganisms inhabiting underground ice that has accumulated inside caves. To characterize how environmental variation impacts cave ice microbial community structure, we determined the composition of total and potentially active bacterial communities along a 13,000-year-old ice core from Scarisoara cave (Romania) through 16S rRNA gene Illumina sequencing. An average of 2,546 prokaryotic gDNA operational taxonomic units (OTUs) and 585 cDNA OTUs were identified across the perennial cave ice block and analyzed in relation to the geochemical composition of ice layers. The total microbial community and the putative active fraction displayed dissimilar taxa profiles. The ice-contained microbiome was dominated by Actinobacteria with a variable representation of Proteobacteria, while the putative active microbial community was equally shared between Proteobacteria and Firmicutes. Accordingly, a major presence of  The Earth’s cold biosphere covers a large variety of frozen habitats and supports several unique microbiomes . The divBeyond these developments, little is known so far on the diversity and activity of microbial communities present in perennial ice accumulated in caves . These cScarisoara Ice Cave (Romania), the most extensively studied ice cave , hosts tIn this context, the current investigation represents a comprehensive study of the cave ice microbiome by unraveling the total and potentially active bacterial community structure in a ∼13,000-year-old ice core taken from Scarisoara Ice Cave, based on 16S rRNA gene Illumina sequencing, in relation with the age and geochemical composition of the ice strata. This first report on bacterial temporal distribution in cave ice deposited since the Late Glacial period provides an opportunity to advance our understanding of microbial community resilience and their ecological role in this type of habitat, thereby providing new leads for identification of microbial climatic proxies.3 perennial ice block previously carbon dated to >10,500 years  at 4°C and the microbial biomass was collected by filtration using 0.22 μm sterile MF-membranes  under aseptic conditions, with a vacuum-driven, stainless steel filter system . Filters were placed in 500 μL RNAlater RNA Stabilization Reagent  and stored at -20°C until used for DNA and RNA extraction. All biomass filtering and DNA/RNA purification steps were carried out under sterile conditions using a microbiological biosafety cabinet to avoid contamination. The filtered water was stored at 4°C in 50 mL plastic conical tubes  for chemical analyses.HT Total Organic Carbon Analyzer , and dissolved inorganic carbon (DIC) was calculated by subtraction of DOC from TC values. For chemical measurements, samples were acidified to pH 2 using 60% Ultrapur® nitric acid . Sulfate concentrations were measured by ASTM-D516-07 (ASTM 1995) standard method, using a UV/VIS double beam Lambda 25 spectrophotometer . A SANGAMON-03  certified reference was used to verify the accuracy of measurements. Concentrations of other chemicals were measured using a NexION 300S  ICP-MS system equipped with a S10 Autosampler, following the US-EPA 6020B (2014) standard reference. Concentrations of calcium were estimated in dynamic reaction cell (DRC) mode using ammonia, while those of sodium, potassium, magnesium, manganese, and iron were measured in KED (Kinetic Energy Discrimination) mode, with helium as inert gas. Boron, silicon, total phosphorous and chloride concentrations were determined using the standard mode. Calibration was performed with High-Purity StandardsTM  NIST 1640a, NIST 1643f. The uncertainty of all analytical measurements . Total carbon (TC) and Dissolved Organic Carbon (DOC) contents were measured according to US-EPA 9060A (2004), using a Formacsurements  was esti-1 propidium iodide (PI)  for quantition of dead cells . For assessment of the total microbial community, ice samples (1 mL) were thawed at 4°C under aseptic conditions, incubated with 0.1% Tween 80  for 10 min at 37°C in a Sonorex Digital 10P ultrasonic bath   to disperse cellular aggregates, and labeled with 1 × SYBR Green I (SG) . For the viable cell measurements, 100 μL of freshly thawed ice was incubated for 15 min with 1 μg mLad cells . The denTheoretical metabolic rates were calculated in accordance with Genomic DNA (gDNA) was isolated in triplicate from distinct ice samples of the same age using the DNeasy Blood & Tissue Kit , following the standard protocol modified with an initial lysis step. The cells  were disrupted by incubation for 12 min at 20°C with innuSPEED Lysis Tube X beads  using a SpeedMill PLUS homogenizer  at 50 Hz. Lysates were then processed according to the manufacturer’s procedure.-1) was isolated from a mixture of equal volumes (100 mL each) of the triplicate melted ice samples used for gDNA extraction. After purification, RNA samples were treated with a DNA-free DNA removal kit  to eliminate contaminating DNA. Reverse transcription of 6–15 ng RNA (0.75–1.87 ng⋅μl-1) was carried out based on the Random Hexamer amplification protocol of the Tetro cDNA Synthesis kit , using a Mastercycler proS vapo protect PCR system . DNA, RNA, and cDNA concentrations were measured using a Qubit fluorometer .RNA isolation was performed using the All Prep DNA/RNA kit , following the manufacturer’s protocol modified with the preliminary mechanical lysis step described above. The RNA (12–60 ng at a concentration of 0.24–1.2 ng⋅μl16S rRNA gene and cDNA fragments (V3-V4 variable region) were PCR amplified using the Illumina prokaryotic primer pair 341F/806R  at McGilSRP157726.The raw 16S rRNA gene sequences of the gDNA samples and cDNA samples from Scarisoara ice core were deposited in the Sequence Read Archive (SRA) under the accession number MicrobiomeAnalyst platform    was used Analyst  with the Analyst . Heatmap Analyst . Assigne Analyst  represen-1, with an average of 9.84 ± 4.91 μgg-1. The main component was calcium, as expected for a limestone ice cave, with values ranging from 1.50 to 11.23 μgg-1 (average 6.24 ± 2.39 μgg-1). Sodium, potassium, magnesium, sulfates, and chlorides were the major constituents throughout the ice core, while manganese, iron, boron and phosphorus were present in lower concentrations  between strata  = 13.49, p < 0.0001] between strata. The highest cell abundance was found in the 6,000-year-old ice , followed by ice formed during 11,000–13,000 cal BP  and 700–1,000 cal BP of 1.75 × 104 cells⋅mL-1 (SC700) and 1.49 × 104 cells⋅mL-1 (SC1K). The lowest cell density (0.84 × 103 cells⋅mL-1) occurred in the 5,000-year-old ice (SC5K), closely followed by 100, 4,000, and 10,000-year-old ice containing 1.17 × 103 cells⋅mL-1 (SC10K), 2.02 × 103 cells⋅mL-1 (SC4K), and 3.53 × 103 cells⋅mL-1 (SC100). The cell density of the viable community  = 5.78, p < 0.0001] between strata (4 cells⋅mL-1 range) were found in SCR700, SCR6K, SCR11K, SCR12K, and SCR13K samples, and the lowest abundance (in the 102 cells⋅mL-1 range) in SCR5K and SCR10K. The corresponding cell viability (-1 103 year-1) was recorded in the cave ice deposits from the last 13,000 years  and putative active (cDNA) bacterial communities from the ice core selected samples was assessed by 16S rRNA gene amplicon Illumina sequencing. For the gDNA library, a total of 6,037,525 post quality control filtered reads were obtained, with 402,501 mean reads per sample . The numAnalysis of the bacterial cDNA sequences were able to recover 1,290,207 post quality control filtered reads, with 86,013 mean reads per sample . The numR2: 0.88437; p < 0.25) were observed between the total (SC5K) and the potentially active (SCR5K) bacterial communities contained in the 5,000-year-old cave ice . The potentially active bacterial community (cDNA) was composed of 25 phyla, 58 classes, and 325 genera. In addition, limited archaeal taxa  were detected in both the communities combined.Cryobacterium, Lysinomonas, Pedobacter, and Aeromicrobium genera  microbial community was observed in all ice strata . MeanwhiAt the level of class , ActinobCryobacterium was mostly present in SC6K (61.5%), Lysinimonas in SC11K (52.2%), and Pedobacter in older  ice strata (4.03–24.4%). Both Aeromicrobium and Arthrobacter genera prevailed in SC3K, representing 23.7 and 3.67%, respectively. Moreover, Paenibacillus OTUs had the highest occurrence in SC6K (0.87%) and SC12K (0.79%). The 4,000-year-old ice layer (SC4K) was rich in Escherichia and Shigella OTUs (56.1%). The actinobacterial Iamia (2.6%) and Actinotalea (1.2%) phylotypes were mainly present in the 12,000 and 7000-year-old ice layers, respectively. Among those genera generally recorded from more recently formed ice strata, Sphingomonas and Oryzihumus were dominant in SC100 , Devosia in SC400 (5.9%), SC700 (3.5%), and SC2K (2.3%), and Pusillimonas in the SC700 ice layers (7.5%). A high relative abundance of unidentified species was found in SC5K, SC7K, and SC12K. The dominant genera within the identified cDNA library (Pseudomonas (21.1%), Clostridium sensu stricto 9 (4.6%), Clostridium sensu stricto 13 (41.4%), Janthinobacterium (13.9%), and Stenotrophomonas (1.9%) genera. These taxa also inhabited ice deposits formed during the last four millennia, in particular the SCR3K and SCR4K samples. Massilia (Proteobacteria) species were mostly found in SCR12K (0.89%). Methylobacter, Pseudohongiella, and Propionibacterium species were populating the 400, 5,000 and 12,000-year-old ice viable communities  were present in 2,000 (SCR2K) and 5,000 (SCR5K) year-old samples.The rare (<1‰) archaeal taxa identified in Scarisoara cave ice core  belongedThis characterization of total and potentially active bacterial communities from a 13,000-year-old perennial ice core of Scarisoara Ice Cave revealed a highly diverse microbiome in cave ice strata formed since the Late Glacial period, representing a pioneering study for this type of habitat. A comparable bacterial diversity (median Shannon index of 5.9) of the 400 and 900-year-old ice from the edge of the ice block in Scarisoara Ice Cave  and the A complex potentially active prokaryotic community that preserved 80% of the total microbial community alpha diversity appeared to populate the underground Scarisoara glacier. In comparison with other caves and frozen habitats, Scarisoara perennial ice contained a unique prokaryotic community characterized by a complementary distribution of Actinobacteria and Proteobacteria taxa in most strata. The presence of Bacteroidetes and Firmicutes phyla was also featured in the total (gDNA) communities, with a higher incidence in older ice layers. Actinobacteria, one of the main taxa observed in cryospheric environment microbiomes , was alsRecent studies have shown an elevated proportion of Actinobacteria (86%) in sediments from Hawaiian lava tubes , similarClostridium sensu stricto 9 and 13 (Firmicutes) species, known from soils and as inhabitants of animal intestinal tracts ]. Among the ubiquitous Pseudomonas species, very few are known to thrive in cold environments. The presence of a putative active P. psychrophila in Scarisoara extended the distribution of this cold-adapted species previously found in Arctic fjord water , Desulfuromonas (sulfur-reducing bacteria), and Geobacter (ferric iron-reducing) phylotypes, and aerobic Myxococcus species capable of releasing myxospores under extreme environmental conditions.The abundance of Deltaproteobacteria in the potentially active community from the 5,000-year-old ice deposits revealed a possible climatic-driven switch to a more complex functional diversity of the cave ice microbiome . VariatiCryobacterium sp. are found in most frozen habitats  in both the total and potentially active communities of these strata revealed the possible existence of aerobic anoxygenic phototrophic metabolism in perennial cave ice. Thus, the potentially active microbial community from ice strata formed about five millennia ago including sulfate-reducing bacteria, aerobic and anaerobic anoxygenic phototrophs and methanogenic archaea, might correspond to a microbial mat-like structure that developed in the cave perennial ice block. The formation of such a stable microbial structure at the interface between water and ice sediments in liquid-water veins of ice deposits  of phyla distribution from the total and putative active communities in relation with the geochemical parameters of ice core samples explained 88.7 and 90.5% of the variance for the gDNA and cDNA libraries, respectively . In both2 fixing , the corommunity , could pommunity . The celbacteria .3 cells⋅mL-1–104 cells⋅mL-1) microbial community that varied across the 13,000-year-old strata. Both microbial cell density and the viability of the total and potentially active communities showed a slight increase with the age of the ice layers. However, microbial abundance in the central part of the ice block located in the Great Hall  of total microbial taxa from high and low carbon content strata, explaining 30.4% of the variability , explaining 81.7% of the variation -1 h-1 and 6.22 × 10-5 g C (g C)-1 h-1 corresponding to a maintenance metabolism (-5 g C (g C)-1 h-1 range were calculated for the viable microbial community of the 900-year-old clear ice layer collected from border areas of Scarisoara ice block , suggesting a combined effect of this parameter on both the depositional and post-depositional processes of the potentially active microbiome.The negative correlation between the viable cell density and DOC concentrations in 3,000–7,000-year-old ice strata  could bee layers  but withThe putative active bacterial community found in perennial ice deposited in Scarisoara Ice Cave during the last ∼13,000 years was composed of both autotrophs and heterotrophs. This complex microbial composition suggested that this type of icy habitat was able to support various metabolic processes, shaping a unique microbiome. The carbon content of the ice strata appeared to be a major factor in determining the variability of microbial communities in the ice, in addition to playing an important role in depositional and post-depositional processes. This initial investigation of microbial communities entrapped in perennial cave ice since the Late Glacial through Holocene could help untangling the glacial microbiome response to climate variations. Moreover, the corroborated geochemical signature, cell abundance and diversity profiles of cave ice prokaryotic community along the ice core, in addition to stable isotope data characterizing the climate of the area , suggestVP and CP wrote the manuscript, with significant input from PL, AP, and CD. VP and AP participated to ice sampling. VP performed the sample filtration, DNA/RNA extraction, cDNA synthesis, and quantification experiments. GI and PL carried out the bioinformatics and statistical analyses. CM and AT conducted the geochemical analyses. AP contributed to depth-age model. CP performed the experimental design and coordinated the project. All authors participated in the data interpretation and revision of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Mass drug administration (MDA) for control of schistosomiasis is likely to affect transmission dynamics through a combination of passive vaccination and reduction of local transmission intensity. This is indicated in phenomenological models of immunity and the impact of MDA, yet immunity parameters in these models are not validated by empirical data that reflects protective immunity to reinfection. There is significant empirical evidence supporting the role of IgE in acquired protective immunity. This is proposed to be a form of delayed concomitant immunity, driven at least in part by protective IgE responses to the tegument allergen-like (TAL) family of proteins. Specific questions have arisen from modeling studies regarding the strength and duration of the protective immune response. At present, field studies have not been specifically designed to address these questions. There is therefore a need for field studies that are explicitly designed to capture epidemiological effects of acquired immunity to elucidate these immunological interactions. In doing so, it is important to address the discourse between theoretical modelers and immuno-epidemiologists and develop mechanistic models that empirically define immunity parameters. This is of increasing significance in a climate of potential changing transmission dynamics following long-term implementation of MDA. The age-infection intensity curve for all three major human-infecting schistosomes follows a pattern of increasing intensity through cumulative exposure until adolescence. After this infection intensities drop, irrespective of water exposure patterns . It is tTheoretical models are useful for policy making and can be used in both planning and evaluating the impact of control interventions on a broad epidemiological scale. For theoretical models of transmission to be useful they must have sufficient biological basis. The challenge of developing a schistosomiasis transmission model is in striking the balance between reality and parsimony. Schistosomiasis is highly focal, demonstrating significant spatial heterogeneity both between village and within village. This has been empirically demonstrated when studying the social, behavioural and cultural determinants of transmission on a microgeographical scale . LikewisThe computational predictions of mathematical models are only as good as the data against which they are calibrated and validated. Models are based upon the existing knowledge of biological processes and the data available to describe and quantify these interactions . Recent “A closer collaboration between biometricians and parasitologists, and a better acquaintanceship of each with the methods of the other, is one of the most useful things we can work for today” ,19.When studying the dynamics of infectious diseases interdisciplinary collaboration is essential. This is particularly important in cases of such complex social and ecological interactions as schistosomiasis. It is therefore important to identify specific areas where there is a greater need for quantitative estimates of model parameters. This is especially important with the recent clear shift from a focus on control towards discussion of elimination efforts. Subsequently, a number of recent models have attempted to predict the requirements and impacts of mass drug administration (MDA) in reaching proposed elimination targets ,20,21.It has been argued that models that do not incorporate the dynamics of acquired immunity lack power in reproducing age-intensity profiles ,22 and mInformation on the demographic profile of communities from which immunological cohorts are drawn .Longitudinal studies of age-specific correlations between parasite burden and immune responses, incorporating individual data from a full range of age classes, in addition to comparison of communities with differing overall levels of endemic infection ,26.Better knowledge of patterns of exposure experienced by immuno-epidemiological cohorts , requireSpecific analysis of age-dependent immunological parameters, including immunological responsiveness (strength of protective response) and loss of immunological memory ,20,26, wEffects of inappropriate responses such as blocking antibodies and parasite-induced immunomodulation .The impact of immune responses that affect parasite fecundity and host excretion of eggs ,26.The following gaps in our theoretical understanding and data requirements have been highlighted in phenomenological examination of immune responses, some of which relate to immuno-epidemiological study design, some specifically to immune mechanisms:Mechanisms that confer partial immunity to schistosomes are not fully understood due to reliance on immuno-epidemiological evidence, with the murine experimental model being largely inappropriate for modelling human resistance to infection. However, levels of IgE specific to schistosome antigens, particularly antigens derived from the adult worm (SWA), are negatively associated with reinfection in studies examining all three major human-infecting schistosomes ,28,29.While historically a switch in the balance between SWA-IgE and the blocking antibody SWA-IgG4 was proposed as the key to immunity, with adults having a greater SWA-IgE:IgG4 ratio than children ,31, the Schistosoma mansoni–tegumental allergen-like (SmTALs) protein family. SmTAL1 (previously Sm22.6), the first of the family to be described, is the major IgE antigen detected on western blots probed with sera from endemic populations [This hypothesis of delayed concomitant immunity is best described through the responses to members of the ulations . SmTAL1-ulations ,37. SmTAulations . SmTAL3,ulations ,37; thusResponsiveness to SmTAL2, which is expressed throughout the life cycle, offers an illustrative counterpoint to SmTAL1/3 responses. An IgE response to this protein is measurable, but only amongst the very youngest members of populations in endemic areas, ultimately being regulated by IgG4 ,39. The Immunity via exposure to dying adult worms is not easily reproduced in murine models, with long-term egg deposition altering vasculature, and hence migratory pathways of worms infecting from trickle exposures. Immuno-epidemiological studies suggest a slow-developing IgE-mediated mechanism, rather than sentinel myeloid cell-derived regulatory immune responses, as seen in a multiple (pre-egg laying) cercarial challenge murine model . It is tS. haematobium infection, eosinophils from Gambian individuals with the greatest expansion in eosinophil number have the greatest capability to kill schistosomules [S. mansoni, both eosinophil numbers, and their expression of the tetrameric high-affinity IgE receptor on their surface, are associated with immunity to reinfection [Eosinophil infiltration has also been implicated in protective human IgE responsiveness. In vitro eosinophils can mediate killing of schistosomules in an IgE-mediated manner . In S. hosomules , and in nfection . It is tS. mansoni reinfection. As the number of treatments administered increased, the longer the observed duration to reinfection [The proposed reliance on worm death for development of protective immunity to schistosomes suggests that treatment with praziquantel could provide a ‘passive vaccination’ effect, boosting the development of immunity. This passive vaccination effect is eloquently illustrated in the studies of car-washers conducted in Kisumu, Kenya, in which praziquantel treatment was administered after every detectable nfection . In compnfection . During nfection . The elenfection .S. haematobium infection, the IL-5 levels detected 24 h post-treatment are associated with pre-treatment SWA-IgE and eosinophil levels. Furthermore, post-treatment IL-5 is associated with pre-treatment infection intensity, indicating that both the primed state of the immune system, or prior exposure to dying worms, and the amount of antigen the system is exposed to upon treatment, is crucial in generating this IL-5 response. In turn, 24 h post-treatment levels of IL-5 are positively associated with protective post-treatment SWA-IgE levels and elevated circulating eosinophil numbers [Immunologically, elevated levels of SWA and SmTAL1-, 3- and 5-specific IgE are observed 3–9 weeks post-treatment. These post-treatment IgE levels are better correlates of resistance to reinfection as measured 1–2 years post-treatment, than pre-treatment antibody levels ,38,49. T numbers .What is clear from the above immunological evidence is that treatment with praziquantel can give a boost to proposed elements of a protective immune response; that is, type 2 cytokine responsiveness, levels of specific IgE and eosinophil numbers. Moreover, this could be providing a ‘passive vaccination’ effect, allowing individuals in endemic areas to develop their immunity at an earlier age. While this is encouraging, as stated earlier, when conducting reinfection studies immuno-epidemiologists work on a microgeographical scale and tend to seek out study sites with high transmission, or particularly highly-exposed occupational cohorts, as this allows us to examine correlates of immunity. Little is known about whether a significant vaccination effect occurs if transmission is low or, as is the aim with multifaceted control strategies, driven to be being very low. Successful control programs have, over time, altered the distribution of schistosomiasis. French et al. ,60, noteS. haematobium field studies, but not seen for S. mansoni infections [S. haematobium infection, but does support the hypothesis that MDA may disrupt the build-up of protective immunity. They also proposed that if reduced transmission led to reduced antigen exposure, antibody levels will be strongly influenced by immune decay rates and demonstrated a rebound in egg excretion following cessation of MDA activities [Mitchell et al.  related fections ,62. It wtivities . It is iIn relation to the immune decay rate of IgE, biologically-active IgE may be sustained for months after exposure to the initial stimuli of production due to long-lived IgE plasma cells maintaining IgE production . Yet, whAn important consideration is that numerical analyses are necessarily based upon a restricted set of parameter combinations . These bWe believe, given the field-based evidence, that more mechanistic modelling of immunity in future schistosome transmission models is required. We have provided discussion, from an immunological perspective, on what is known from existing field data, but acknowledge that previous immunology studies, through scale and design, may fail to capture the parameters that modelers identify as important. However, with technological advances, an integrated approach between theoretical modelers and field immunologists is now possible and of utmost importance if transmission models are to truly capture schistosome infection dynamics within endemic areas.


The group II intron and the spliceosome share a common active site architecture and are thought to be evolutionarily related. Here we report the 3.7 Å crystal structure of a eukaryotic group II intron in the lariat-3′ exon form, immediately preceding the second step of splicing, analogous to the spliceosomal P complex. This structure reveals the location of the intact 3′ splice site within the catalytic core of the group II intron. The 3′-OH of the 5′ exon is positioned in close proximity to the 3′ splice site for nucleophilic attack and exon ligation. The active site undergoes conformational rearrangements with the catalytic triplex having different configurations before and after the second step of splicing. We describe a complete model for the second step of group II intron splicing that incorporates a dynamic catalytic triplex being responsible for creating the binding pocket for 3′ splice site capture. Group II introns are large, self-splicing RNAs that catalyze their own excision from pre-mRNA molecules. Here the authors determine the 3.7 Å crystal structure of the group II intron in the stage immediately before the second step of splicing and present a complete model for the second step of group II intron splicing. The spliceosomal U2-U6 snRNA pairing is analogous to domain V in group II introns with these regions forming the active site that binds the catalytic metal ions. Specifically, both U2-U6 and domain V contain a highly conserved AGC catalytic triad, which forms base triples with other nucleotides to form a triple helix known as the “catalytic triplex”. This catalytic triplex forms the binding pocket for the two active site magnesium ions. Furthermore, the spliceosomal branch-site helix contains the bulged adenosine nucleophile that is also found in domain VI of group II introns. The phylogenetic distribution, mechanistic similarities, and structural homology between the two systems suggests that group II introns are ancestral to the eukaryotic spliceosome.Group II introns are self-splicing ribozymes that catalyze two transesterification reactions to excise themselves from pre-messenger RNA. In the first step of splicing, the 2′-OH of a bulged adenosine residue is used as the nucleophile to attack the 5′ splice site Fig. a. This i5. Domain VI  in the lariat-3′ exon form with an intact 3′ splice site docked into the catalytic core. Combined with biochemical data, we compare the structure of the lariat-3′ exon with other group II intron structures to develop a model for the second step of RNA splicing.Here we report the 3.7 Å crystal structure of a eukaryotic group IIB intron from the brown algae Rfree = 25.3%; Rwork = 21.7%) represents the catalytic state of the group II intron immediately preceding the second step of splicing (pre-2s)  was further refined   of the intron. The lack of this interaction accounts for the fact that the 3′ splice site is intact in this structure. This also suggests that γ–γ′ is a transient and dynamic contact that forms during the cleavage of the 3′ splice site (see below). Therefore, further conformational changes are required in order for this active site to engage in the chemistry required for the second step of splicing.A comparison of the pre-2s and post-catalytic structures reveals that the basal stem of domain VI, which contains the branch-point adenosine, undergoes a compression in the transition to the second step. Figure This observed compression of domain VI is likely not the result of crystal packing interactions as this region is relatively free of crystal contacts in the lattice  and domains V and VI (J5/6) are also involved in forming triples at the base of domain V  participating in base triples within the catalytic triplexn V Fig. . This isn V Fig. . We hypoaI5γ intron15.To test the function of the junction sequences, we altered the sequence and length of J2/3, J4/5 and J5/6 ing Fig. . Mappinging Fig. . In contP.li.LSUI2 and O. iheyensis introns and previous biochemical data for interactions in the active site. As a result, each panel in Fig. P.li.LSUI2 crystal structures that transition between the different triplex configurations and 3′ splice site placement. A central feature of this model is a series of conformational rearrangements involving the catalytic triplex that are necessary to create a binding pocket for the γ nucleotide A420. This positions A420 so that it can pair with the 3′ splice site for the second step and brings the scissile phosphate in close proximity to the catalytic metals ions for cleavage. In the first stage, J2/3 residue A422 disengages and then re-engages at a lower level within domain V. This creates an initial opening to allow formation of the γ-binding site. The binding site is completed with rearrangements in J4/5 and J5/6 creating additional base stacking interactions with the incoming γ nucleotide A420. Formation of the γ–γ′ interaction then pulls the 3′ splice site into the catalytic core for exon ligation. These conformational rearrangements can be described as a series of nucleobase flipping events within the active site to prepare for the second step of splicing.The cumulative data are consistent with a model in which dynamic rearrangements of the catalytic triplex are likely responsible for the positioning and removal of exon substrates from the active site in the transition between the two steps of splicing Fig. . This mo19. This construct is a modified version of the wild-type O. iheyensis intron structure previously determined by Toor et al.20. Specifically, this construct is a chimera containing the addition of a truncated domain VI from a IIC intron found in a different bacterial species. As a result, it is lacking both a wild-type J5/6 and an intact 3′ splice site. However, this structure does show the γ–γ′ interaction in an engaged state, which is likely trapped due to the lack of an intact 3′ splice site. Furthermore, the location and conformation of the γ–γ′ interaction in this structure is consistent with our model for the second step of splicing presented above.Recently, the crystal structure of a chimeric IIC intron lariat was determined9 reveals homology in active site architecture and 3′ splice site substrate placement  were mixed with 0.4 mM spermine, 21% 2-methyl-2,4-pentanediol (MPD), 175 mM magnesium acetate tetrahydrate, and 90 mM MES monohydrate (pH 5.6). Crystals appeared within 2 days and were gradually exchanged into 21% MPD, 100 mM magnesium acetate tetrahydrate, 50 mM MES monohydrate (pH 5.6), 3 mM iridium hexammine, 0.5 mM spermine, and 100 mM NaCl, followed by flash freezing in liquid nitrogen. Post-crystallization treatment was altered to exclude iridium hexammine. X-ray data sets were collected at NE-CAT’s 24-ID-C beamline at the Advanced Photon Source . Data was processed using HKL-200023. The post-catalytic structure of P.li.LSUI2 (PDBID 4R0D) was used as a search model for molecular replacement using PHENIX24. The last 10 and 5 nucleotides of the intron and 5′ exon, respectively, were deleted to avoid model bias at the splice sites. RNA nucleotides were modeled using COOT26 and the RCrane plugin28. Structure refinement was done using Buster29, PHENIX, DEN30, and Phenix.Erasser31. Feature-enhanced maps12 were calculated using PHENIX. SBGrid compiled all the software used32.The pre-2s state of the P.li.LSUI2 sequence with the DIV ORF removed and a 250 nt 5′ exon and 75 nt 3′ exon. This construct was cloned into pUC57 using the EcoRV cut site. DNA templates for in vitro transcription were linearized using BamHI. Radiolabeled transcripts were prepared using T7 RNA polymerase, 5 mM MgCl2, 40 mM Tris-HCl pH 7.5, 0.05% Triton X-100, 10 μCi [α-31P]UTP (3000 Ci mmol−1), 0.5 mM UTP, and 1 mM other NTPs. In vitro transcription reactions were done for 1 h at 37 °C followed by purification of the precursor RNA on a denaturing 4% (19:1) polyacrylamide, 8 M urea, 1X TBE gel. Radiolabeled precursor RNA was refolded in 40 mM Tris-HCl pH 7.5 through heating at 90 °C for 1 min followed by incubation in 40 mM Tris-HCl pH 7.5, 10 mM MgCl2 for 15 min. To initiate self-splicing, RNA was mixed with an equal volume of 2X splicing buffer . Reactions were quenched by mixing with an equal volume of 80% formamide, 100 mM EDTA. Splicing products were resolved using denaturing 4% (19:1) polyacrylamide, 8 M urea, 1× TBE gels. All splicing assays were done in triplicate.Constructs used for in vitro self-splicing assays were modifications of the wild-type (WT) Splicing gels were exposed to storage phosphor screens and splicing products were quantitated using Quantity One 1-D Analysis Software. Unequal loading was accounted for by normalizing to an internal control RNA. Band intensity was determined by dividing the background-subtracted intensity of each band by the number of uridine residues in the RNA sequence corresponding to the band. All band intensities were then normalized to an unspliced control to give fractional values of input RNA.3)63+-soaked crystals were rinsed with excess crystallization solution to remove uncrystallized RNA then dissolved in 50 mM HEPES pH 7, 100 mM NaCl, 1 mM DTT, 0.1 μM Entamoeba histolytica Dbr1 debranching enzyme33 and incubated at 37 °C for 30 min to hydrolyze the lariat bond. RNA was phenol-chloroform extracted, ethanol precipitated and resuspended in nuclease free water. One microgram debranched RNA was incubated with 1 mM ATP and 5 units of E. coli poly(A) polymerase from NEB at 37 °C for 30 min to add a poly(A) tail to the RNA. The RNA was phenol-chloroform extracted, ethanol precipitated and resuspended in nuclease free water. 200 ng oligo(dT)18 and 1 μL 10 mM dNTP mix were added to the RNA and the volume was brought to 13 μL with nuclease free water. To anneal the primer, the mixture was heated at 65 °C for 5 min and incubated on ice for 1 min. The RNA was reverse transcribed at 50 °C for 30 min using 200 units SuperScriptTM III RT, 1X first-strand buffer, and 5 mM DTT in a final volume of 20 μL, and the RT was inactivated by heating at 70 °C for 15 min. Two microliter of cDNA was used as a template for PCR using oligo(dT)18 as a reverse primer and a forward primer that anneals within the intron. PCR products containing a short poly(A) tail were gel extracted, cloned into pUC57 using the EcoRV cut site, and transformed into E. coli to sequence single colonies. This 3′ end mapping protocol is derived from that described by Qin et al. (2016)34.Native and Ir using the small RNA workflow of the SHAPE-MaP protocol17. The reverse transcription primer hybridizes to the 3′ exon (lariat-3′ exon) or the 3′ end of the intron . Primers were designed to amplify about 500 nucleotides of the cDNA. Libraries were sequenced on a MiSeq with a 2 × 300 basepair paired-end configuration. Differences in SHAPE reactivities were analyzed with the ΔSHAPE script16. Using the ΔSHAPE program16, the reactivities of the pre-2s mutant were subtracted from the post-catalytic state. Significant differences at a given nucleotide position were determined by applying the following statistical criteria:A second-step deficient Supplementary Information