Teredinibacter turnerae T7901 was recently sequenced and in addition to the potential for cellulolytic enzymes and diazotrophy, the genome also revealed a rich potential for secondary metabolites. With nine distinct biosynthetic gene clusters, nearly 7% of the genome is dedicated to secondary metabolites. Bioinformatic analyses predict that one of the gene clusters is responsible for the production of a catecholate siderophore. Here we describe this gene cluster in detail and present the siderophore product from this cluster. Genes similar to the entCEBA genes of enterobactin biosynthesis involved in the production and activation of dihydroxybenzoic acid (DHB) are present in this cluster, as well as a two-module non-ribosomal peptide synthetase (NRPS). A novel triscatecholate siderophore, turnerbactin, was isolated from the supernatant of iron-limited T. turnerae T7901 cultures. Turnerbactin is a trimer of N--L-Orn-L-Ser with the three monomeric units linked by Ser ester linkages. A monomer, dimer, dehydrated dimer, and dehydrated trimer of 2,3-DHB-L-Orn-L-Ser were also found in the supernatant. A link between the gene cluster and siderophore product was made by constructing a NRPS mutant, TtAH03. Siderophores could not be detected in cultures of TtAH03 by HPLC analysis and Fe-binding activity of culture supernatant was significantly reduced. Regulation of the pathway by iron is supported by identification of putative Fur box sequences and observation of increased Fe-binding activity under iron restriction. Evidence of a turnerbactin fragment was found in shipworm extracts, suggesting the production of turnerbactin in the symbiosis.Shipworms are marine bivalve mollusks (Family Teredinidae) that use wood for shelter and food. They harbor a group of closely related, yet phylogenetically distinct, bacterial endosymbionts in bacteriocytes located in the gills. This endosymbiotic community is believed to support the host's nutrition in multiple ways, through the production of cellulolytic enzymes and the fixation of nitrogen. The genome of the shipworm endosymbiont  Iron is required by all but a few organisms and serves as an important cofactor for many essential enzymes. The insolubility of Fe(III) at physiological pH and aerobic conditions severely limits its bioavailability. In the world's oceans, 99% of the Fe(III) is bound by uncharacterized organic ligands f = 1049, f = 1047.6, Dickeya chrysanthemi. Streptobactin, a cyclic trimeric ester of 2,3-DHB-L-Arg-L-Thr, was recently reported from the marine-derived actinomycete Streptomyces sp. YM5-799. In addition to the cyclic triscatecholate siderophores, a linear (non-cyclized) triscatecholate siderophore, trivanchrobactin, was reported from the marine isolate Vibrio campbelli DS40M4 The class of siderophores that possess the highest stability constants measured to date are the triscatecholate siderophores, including enterobactin Teredinibacter turnerae has been found in numerous genera and species of teredinid bivalves from across the globe T. turnerae T7901 was recently sequenced T. turnerae is an intracellular symbiont, its genome does not show the typical modifications experienced by obligate endosymbionts, most notably reduced genome size, higher A+T content, and loss of DNA repair and transcriptional regulatory genes T. turnerae is a facultative symbiont, or is part of a recently established symbiosis. To date, T. turnerae has not been detected outside of the shipworm host. Analysis of T. turnerae's genome also reveals nine gene clusters predicted to code for secondary metabolites, constituting nearly 7% of its genome. One of the nine gene clusters was recently reported to synthesize an antibacterial compound in the tartrolon family The shipworm endosymbiont T. turnerae T7901, a cluster that was previously proposed to encode a siderophore biosynthesis gene cluster N--L-Orn-L-Ser, named turnerbactin. The gene cluster responsible for the biosynthesis of turnerbactin is analyzed and a model for its biosynthesis is proposed. The biosynthetic genes involved in turnerbactin production are herein referred to as tnb, for turnerbactin biosynthesis. Turnerbactin represents the first described siderophore from a shipworm symbiont.Here we report on another secondary metabolite gene cluster from Teredinibacter turnerae T7901 was originally isolated from the shipworm Bankia gouldi collected in the wild near Duke University Marine Lab, Beaufort, NC T. turnerae T7901 was grown in a low-iron, modified version of shipworm basal medium (SBM) 2PO4, 0.094 mM Na2CO3, 0.01 mM Na2MoO4•2H2O, 0.5% (w/v) sucrose, 5 mM NH4Cl, 20 mM HEPES buffer (pH 8.0), 0.1 μM EDTA-chelated ferric iron (Sigma), and 1 ml A5+Co trace metal mix T. turnerae was prepared by inoculating an iron-replete (10 μM EDTA-chelated ferric iron) SBM liquid culture with a single colony from an iron-replete SBM agar plate. The 2 L cultures were inoculated with 1 mL of overnight-grown pre-culture. Cultures were grown for 2 days, when Fe(III)-binding activity of culture supernatants reached maximum activity as measured by the chrome azurol sulfonate (CAS) assay T. turnerae T7901 were grown in 100 mL flasks with the same conditions described above, containing either 10 μM EDTA-chelated ferric iron (iron-replete) or 0.1 μM EDTA-chelated ferric iron (iron-limited). After growth for approximately 38 hours at which point cultures were at late log phase, 1 mL aliquots of all cultures were collected and centrifuged at 10000 rpm for 3 min. Supernatants were tested for CAS activity. Uninoculated iron-replete media showed no CAS activity, indicating that the EDTA from the iron source did not interfere with the assay.To determine CAS activity of iron-replete and iron-limited cultures, duplicate 50 mL SBM cultures of sample/Abblank) ×100.All growth and CAS activity measurements were recorded on a SpectraMax M2 Multidetection Reader (Molecular Devices) with absorbance measured at 600 nm and 630 nm, respectively. CAS activity was calculated as (1-(Abin vacuo. The siderophores were purified by reverse-phase high–performance liquid chromatography (RP-HPLC) on a semi-preparative C18 column  using water and acetonitrile (ACN) as solvents at a flow-rate of 3 mL/min at 25°C. Both solvents contained 0.05% trifluoroacetic acid (TFA). The mobile phases consisted of a gradient of 10% ACN in water to 15% ACN from 0–10 min, followed by an isocratic step of 15% ACN from 10–15 min, then a gradient from 15% to 25% ACN from 15–35 min, then a final gradient from 25% to 100% ACN from 35–40 min. The eluent was continuously monitored at 215 nm and 320 nm. Fractions were collected manually and immediately tested for CAS activity and then concentrated under vacuum. Collected and pooled fractions were further purified on an analytical RP-Amide-C16 column  using the same method as above at a flow-rate of 1 mL/min. Pure siderophores were lyophilized. The approximate yields of siderophores from a 4 L culture are 1.2 mg DHB-Orn-Ser, 2.1 mg (DHB-Orn-Ser)2, 2.7 mg turnerbactin, 2.4 mg dehydro-(DHB-Orn-Ser)2, and 1.2 mg dehydro-(DHB-Orn-Ser)3.Culture supernatant was cleared of cells and debris by centrifugation at 10000 rpm for 25 min. Decanted supernatant was incubated with ∼20 g/L Dianion HP20 resin in 2.8 L Fernbach flasks at 4°C on an orbital shaker (110 rpm) for 4 hrs. The resin was collected and washed sequentially with MilliQ water followed by 25%, 50%, and finally 100% isopropanol (IPA). The siderophores eluted with the 25% IPA fraction as detected by the CAS assay. The 25% IPA fraction was concentrated by rotary evaporation T. turnerae T7901. Cultures of Dickeya chrysanthemi were grown and siderophores were purified as described by Sandy and Butler T. turnerae T7901 were purified concurrently and in the same manner. The identity of siderophores from both cultures was confirmed by HPLC retention time, high-resolution electrospray ionization mass spectrometry (HRESIMS), and 1H-NMR.The triscatecholate siderophores that have been reported in the literature have not contained dehydrated amino acid constituents. Additionally, with the exception of linear trivanchrobactin, the triscatecholate siderophores have been found in the cyclic form. A control experiment was carried out to rule out the possibility that the dehydrated siderophores and the lack of a cyclic trimer were the result of the purification method. A side-by-side purification of the structurally similar siderophore cyclic trichrysobactin d4-methanol .Mass spectra were obtained on a ThermoElectron LTQ-Orbitrap high-resolution mass spectrometer. Samples were dissolved in 50% methanol and MS analysis was performed in positive mode using electrospray ionization (ESI). All 1-D and 2-D NMR experiments were carried out on a Bruker Avance II Ultrashield Plus 800 MHz instrument with a cryoprobe in UV-visible absorption spectrum of purified turnerbactin in water was collected on a Varian Cary 50 spectrophometer.2O, to which 200 μL of a 1% (w:v) solution of Marfey's reagent  3 were added to derivatize the primary amines of the amino acids. The reaction was heated for 1 hour at 40°C, after which 20 μL 2 M HCl was added to stop the reaction. The derivatized sample was analyzed by HPLC on an analytical YMC ODS-AQ C18 column  using a linear gradient from 90% water with 0.1% TFA/10% CH3CN to 60% water with 0.1% TFA/40% CH3CN over 45 min. The eluent was continuously monitored on a Waters UV-visible detector at 340 nm. The derivatized sample was compared to chiral amino acid standards prepared the same way. Peaks were collected and verified by MS.A dried preparation of purified turnerbactin (∼1 mg) was hydrolyzed in 200 μL 6 M HCl for 17 hours at 110°C. The solution was brought to room temperature and evaporated to dryness. The dried, hydrolyzed sample was redissolved in 100 μL HLyrodus pedicellatus maintained at the Ocean Genome Legacy was sent to Oregon Health & Science University and stored at room temperature in an aerated aquarium until further processed. L. pedicellatus specimens were removed from wood using pliers, slowly peeling away layers of wood until shipworms were exposed, careful to not damage the shipworms. Six L. pedicellatus shipworms were removed from the wood, rinsed in filter-seawater, pooled, and lyophilized. The resulting dry weight was 85.4 mg. The tissue was then homogenized with a plastic pestle. The homogenized tissue was extracted three times with three volumes of methanol on a rotary mixer for one hour. The methanolic extract was separated from the tissue by centrifugation at 15,000 rpm for 3 min and subsequently dried. Approximately 1 mg of the crude methanolic extract was dissolved in water and injected onto a ThermoElectron LTQ-Orbitrap high-resolution mass spectrometer with an Accela HPLC system using a C18 column . Mass spectra were recorded in positive mode using ESI. The following gradient was used at a flow rate of 200 ul/min: 10% methanol in water to 50% methanol over 30 min, followed by an increase to 100% methanol over 5 min. Both solvents contained 0.1% formic acid. Pure siderophores (approximately 10 μg) were used as standards using the same method. The L. pedicellatus extract was analyzed first, followed by washing of the column, a blank injection, and then the pure siderophore standard.A laboratory culture of http://hmmer.org/. Alignments for Profile Hidden Markov Models (pHMMs) were downloaded from either the Pfam database Module identification and domain organization of the NRPS was carried out using the online tools NRPS-PKS Adenylation (A) domain: Protein sequences of A domains in TnbF were retrieved using the PKS/NRPS analysis website Condensation (C) domain: Protein sequences of C domains in TnbF were retrieved using the PKS/NRPS analysis website tnbF was amplified with specific PCR primers TnbF851F and TnbF1193R  Sigmacell cellulose Type 101 (Sigma) as the sole carbon source and 10 μg/ml chlorampenicol (Cm). The location of integration by pDMtnbF into the chromosome of T7901 was confirmed by PCR and DNA sequencing with vector-specific forward primers pNQ705 and pDM4CAT189 and the T7901 chromosomal-specific reverse primer TnbF1604R  Sigmacell cellulose Type 101, with 10 μg/ml Cm in the case of TtAH03. Spent supernatant was extracted with HP20 resin. HP20 resin was washed with MilliQ water, 50% IPA, and 100% IPA. The 50% IPA fraction from each culture was dried by lyophilization and approximately 0.1 mg of each extract was used for HPLC analysis using the same method as described for compound isolation. Pure DHB was purchased from Sigma.Teredinibacter turnerae T7901 revealed a gene cluster with similarity to siderophore biosynthesis and iron transport genes tnbCEBA are homologous to the entCEBA genes, responsible for the biosynthesis and activation of 2,3-dihydroxybenzoate (DHB) via the shikimate pathway Annotation of the genome of tnbF codes for a 2399 amino acid, two module NRPS. Each module contains a C, A, and peptidyl-carrier protein (PCP) domain, followed by a C-terminus thioesterase (TE) domain  domain . A phospEscherichia colifes. In E. coli, once ferric enterobactin has entered the cell, iron is removed from the siderophore by hydrolyzing the ester bonds of the siderophore's backbone with the fes gene product In addition to biosynthesis genes, a number of other genes are also found in this cluster that are likely to assist in siderophore transport and uptake. TERTU_4066 codes for an MbtH-like protein Bacillus subtilis in tnbC-TERTU_4068. Support for the role of iron in regulating expression of this gene cluster was provided by assaying CAS activity of iron-limited and iron-replete cultures  box sequences , suggestcultures . The higThe crystal structure of the Phe-activating A domain of gramicidin synthetase A (GrsA) by Conti et al. The online tool NRPSpredictor2 DCL functional domains and this placement is strongly supported, evidenced by the high bootstrap values at the base of the Dual E/C and DCL groups. In most cases, DCL domains are preceded by an epimerase (E) domain, catalyzing the epimerization of amino acids from L to D configuration. However, an E domain was not detected in TnbF through pHMM analysis and an external racemase was not detected in the gene cluster. In conjunction with the chemical analysis of the siderophore product (below), this suggests that the M2 C domain of TnbF exhibits LCL activity as opposed to its predicted DCL activity.In addition to A domain specificity prediction, C domains can also be subject to bioinformatic prediction. Rausch et al. T. turnerae T7901 are shown in T. turnerae were purified from iron-deficient liquid SBM cultures by adsorbing siderophores from culture supernatants on an HP20 column, followed by purification by RP-HPLC. The CAS assay was used to track the siderophores throughout the purification process. RP-HPLC revealed five peaks displaying CAS activity (+: DHB-Orn-Ser (1), m/z 356.1454, corresponding to a molecular formula of C15H22N3O7 ; (DHB-Orn-Ser)2 (2), m/z 693.2736, corresponding to a molecular formula of C30H41N6O13 ; turnerbactin (3), m/z 1030.4003, corresponding to a molecular formula of C45H60N9O19 ; dehydro-(DHB-Orn-Ser)2 (4), m/z 675.2626, corresponding to a molecular formula of C30H39N6O12 ; dehydro-(DHB-Orn-Ser)3 (5), m/z 1012.3905, corresponding to a molecular formula of C45H58N9O18 .The structures of the siderophores isolated from activity . High reESIMS/MS analysis of each of these compounds is summarized in 1H and 13C NMR assignments of 1 were confirmed by 2–D 1H-1H TOCSY, HSQC, and HMBC experiments (13C NMR spectrum shows 15 distinct C resonances corresponding to three carbonyl carbons (δ 169.98 to 172.12), four methylene carbons , two methine carbons , and six aromatic carbons (δ 116.01 to 147.81). The 1H NMR spectrum shows 12 distinct resonances corresponding to three aromatic protons (δ 6.77 to 7.36), seven methylene protons , and two methine protons . The aromatic splitting pattern in the 1H NMR spectrum is indicative of a 2,3-DHB moiety. A HMBC correlation between the α-proton of ornithine and the carbonyl carbon of DHB indicates that DHB is attached to the α-amine group of ornithine. A HMBC correlation between the α-proton of serine and the carbonyl carbon of ornithine confirms the ornithine-serine peptide bond.The 1–D eriments -S14. The2 and 3 were inferred using MS, 1H NMR, and 2–D NMR experiments HSQC and HMBC  compared to the serine methylene protons of 1 , indicating the presence of serine ester linkages in 2 and 3.The structures of BC 2: 3: -S26. The5 suggested a cyclic trimer as the result of an additional serine ester bond, leading to a triserine lactone backbone. However, 1H and 13C NMR in addition to 2-D experiments 1H-1H TOCSY, HSQC, and HMBC revealed a dehydrated analogue of the linear trimer  resistance cassette into the M1 C domain of tnbF by a single crossover recombination (In order to correlate the bination . The restnbF resulted in a significant reduction in Fe(III)-binding activity as measured by the CAS assay compared to wild-type strain T7901 3-O-TE intermediate, which is then hydrolyzed by water to yield the linear trimer (DHB-Orn-Ser)3, turnerbactin (3). It is uncertain whether the other compounds  discovered in this study are byproducts from regulated biosynthesis by the NRPS, incomplete biosynthesis by the NRPS, enzymatic degradation by an esterase, or hydrolysis products from either the purification process or culture conditions.The proposed biosynthesis is shown in T. turnerae and its shipworm host was examined. Detection of the compound in the shipworm host would provide evidence that the compound is produced and utilized in the symbiosis. The presence of turnerbactin and the related DHB-Orn-Ser products in the shipworm host was investigated using MS. The shipworm, Lyrodus pedicellatus, is known to contain the symbiont T. turneraeL. pedicellatus tissue samples was analyzed by LC-HRESIMS/MS and compared to pure siderophores isolated from T. turnerae T7901 as standards. The method used in this analysis shows pure DHB-Orn-Ser eluting from the column over the span of approximately 7.5–8.5 min , distantly located from the turnerbactin biosynthetic gene cluster. However, since this enzyme is essential for serine biosynthesis, it is difficult to implicate this enzyme in the dehydration of Ser in turnerbactin. More broadly, a dehydrated amino acid in a siderophore has been previously reported in the loihichelins Halomonas sp. LOB-5 and contain dehydroamino-2-butyric acid. However, a mechanism for this dehydration has not been reported. Alternatively, the dehydration of Ser in turnerbactin may occur through a non-enzymatic means, though as noted above, the control experiment with cyclic trichrysobactin suggests that this does not occur during the purification process.A Dha residue is not unprecedented in other biosynthesized compounds. The formation of Dha residues has been previously reported in ribosomally-produced (RP) compounds such as lantibiotics tnbF's catalytic domains suggest these domains elude accurate prediction. Predictive analysis of the M1 A domain failed to suggest a specificity with reasonable cut-off scores. C domain analysis suggests a DCL acting domain, while chemical data suggest a LCL acting domain. Taken together, the addition of tnbF to the databases of NRPS domains may help to improve the functional prediction of as-yet-undiscovered NRPS domains.The bioinformatic analyses of LCL domains while clustering in the DCL group of C domains in phylogenetic analysis, similar to the M2 C domain of TnbF  compared to its linearized form, the linear trimer of DHB-serine (1043) T. turnerae T7901 cultures in response to iron-limited conditions.The recent reports of trivanchrobactin 1), was detected in extracts of the shipworm L. pedicellatus. The compound detected in the shipworm shared the same LC retention time, high-resolution molecular ion mass, and daughter ions in tandem MS fragmentation as a pure DHB-Orn-Ser standard. The detection of this monomer unit in a shipworm sample suggests that turnerbactin is produced and utilized in some capacity in the symbiosis. Since whole animal tissue was used in these experiments, it is not known whether these siderophores are confined to the immediate vicinity of the symbionts in the gills, or if they appear in other locations of the shipworm. The exact role of endosymbiont-derived siderophores in the host is currently unclear. The caecum of L. pedicellatus is largely devoid of microbes T. turnerae may be translocated to the caecum and contribute to suppression of microbial growth A fragment of turnerbactin, DHB-Orn-Ser Click here for additional data file.Figure S2HPLC trace of HP20 extract from T. turnerae T7901 culture supernatant, recorded at 215 nm.(TIF)Click here for additional data file.Figure S3HPLC trace of derivatized hydrolysate of turnerbactin and chiral amino acid standards, recorded at 340 nm. (A) hydrolysate of turnerbactin, (B) L-ornithine, (C) DL-ornithine, (D) L-serine, (1) D-ornithine, (2) L-ornithine, (3) L-serine, (4) Marfey's reagent (FDAA).(TIF)Click here for additional data file.Figure S41H NMR spectrum (800 MHz) in CD3OD.1 (TIF)Click here for additional data file.Figure S513C NMR spectrum (800 MHz) in CD3OD.1 (TIF)Click here for additional data file.Figure S61H-13C HSQC spectrum (800 MHz) in CD3OD.1 (TIF)Click here for additional data file.Figure S71H-13C HMBC spectrum (800 MHz) in CD3OD.1 (TIF)Click here for additional data file.Figure S81H-13C HMBC spectrum (800 MHz) in CD3OD, expanded region.1 (TIF)Click here for additional data file.Figure S91H-13C HMBC spectrum (800 MHz) in CD3OD, expanded region.1 (TIF)Click here for additional data file.Figure S101H-13C HMBC spectrum (800 MHz) in CD3OD, expanded region.1 (TIF)Click here for additional data file.Figure S111H-1H TOCSY spectrum (800 MHz) in CD3OD.1 (TIF)Click here for additional data file.Figure S121H-1H TOCSY spectrum (800 MHz) in CD3OD, expanded region.1 (TIF)Click here for additional data file.Figure S131H-1H TOCSY spectrum (800 MHz) in CD3OD, expanded region.1 (TIF)Click here for additional data file.Figure S141H-1H TOCSY spectrum (800 MHz) in CD3OD, expanded region.1 (TIF)Click here for additional data file.Figure S151H NMR spectrum (800 MHz) in CD3OD.2 (TIF)Click here for additional data file.Figure S161H-13C HSQC spectrum (800 MHz) in CD3OD.2 (TIF)Click here for additional data file.Figure S171H-13C HMBC spectrum (800 MHz) in CD3OD.2 (TIF)Click here for additional data file.Figure S181H-13C HMBC spectrum (800 MHz) in CD3OD, expanded region.2 (TIF)Click here for additional data file.Figure S191H-13C HMBC spectrum (800 MHz) in CD3OD, expanded region.2 (TIF)Click here for additional data file.Figure S201H-13C HMBC spectrum (800 MHz) in CD3OD, expanded region.2 (TIF)Click here for additional data file.Figure S211H NMR spectrum (800 MHz) in CD3OD.3 (TIF)Click here for additional data file.Figure S221H-13C HSQC spectrum (800 MHz) in CD3OD.3 (TIF)Click here for additional data file.Figure S231H-13C HMBC spectrum (800 MHz) in CD3OD.3 (TIF)Click here for additional data file.Figure S241H-13C HMBC spectrum (800 MHz) in CD3OD, expanded region.3 (TIF)Click here for additional data file.Figure S251H-13C HMBC spectrum (800 MHz) in CD3OD, expanded region.3 (TIF)Click here for additional data file.Figure S261H-13C HMBC spectrum (800 MHz) in CD3OD, expanded region.3 (TIF)Click here for additional data file.Figure S271H NMR spectrum (800 MHz) in CD3OD.4 (TIF)Click here for additional data file.Figure S2813C NMR spectrum (800 MHz) in CD3OD.4 (TIF)Click here for additional data file.Figure S291H-13C HMBC spectrum (800 MHz) in CD3OD.4 (TIF)Click here for additional data file.Figure S301H-13C HMBC spectrum (800 MHz) in CD3OD, expanded region.4 (TIF)Click here for additional data file.Figure S311H-13C HMBC spectrum (800 MHz) in CD3OD, expanded region.4 (TIF)Click here for additional data file.Figure S321H-13C HMBC spectrum (800 MHz) in CD3OD, expanded region.4 (TIF)Click here for additional data file.Figure S331H-1H TOCSY spectrum (800 MHz) in CD3OD.4 (TIF)Click here for additional data file.Figure S341H-1H TOCSY spectrum (800 MHz) in CD3OD, expanded region.4 (TIF)Click here for additional data file.Figure S351H-1H TOCSY spectrum (800 MHz) in CD3OD, expanded region.4 (TIF)Click here for additional data file.Figure S361H-1H TOCSY spectrum (800 MHz) in CD3OD, expanded region.4 (TIF)Click here for additional data file.Figure S371H NMR spectrum (800 MHz) in CD3OD.5 (TIF)Click here for additional data file.Figure S3813C NMR spectrum (800 MHz) in CD3OD.5 (TIF)Click here for additional data file.Figure S391H-13C HMBC spectrum (800 MHz) in CD3OD.5 (TIF)Click here for additional data file.Figure S401H-13C HMBC spectrum (800 MHz) in CD3OD, expanded region.5 (TIF)Click here for additional data file.Figure S411H-13C HMBC spectrum (800 MHz) in CD3OD, expanded region.5 (TIF)Click here for additional data file.Figure S421H-13C HMBC spectrum (800 MHz) in CD3OD, expanded region.5 (TIF)Click here for additional data file.Figure S431H-1H TOCSY spectrum (800 MHz) in CD3OD.5 (TIF)Click here for additional data file.Figure S441H-1H TOCSY spectrum (800 MHz) in CD3OD, expanded region.5 (TIF)Click here for additional data file.Figure S451H-1H TOCSY spectrum (800 MHz) in CD3OD, expanded region.5 (TIF)Click here for additional data file.Figure S461H-1H TOCSY spectrum (800 MHz) in CD3OD, expanded region.5 (TIF)Click here for additional data file.Table S12 (2), Dehydrated (DHB-Orn-Ser)2 (4), and Dehydrated (DHB-Orn-Ser)3 (5) (800 MHz) in CD3OD.NMR data for (DHB-Orn-Ser)(PDF)Click here for additional data file.


Recent studies have shown that glucosamine inhibits the proliferation of various human cancer cell lines and downregulates the activity of COX-2, HIF-1α, p70S6K, and transglutaminase 2. Because the IGF-1R/Akt pathway is a common upstream regulator of p70S6K, HIF-1α, and COX-2, we hypothesized that glucosamine inhibits cancer cell proliferation through this pathway.in vitro assays including flow cytometry assays, small interfering RNA (siRNA) transfection, western blot analysis, MTT -2,5-diphenyltetrazolium bromide) assays, reverse transcription-polymerase chain reaction, and in vivo xenograft mouse model to confirm anticancer activities of glucosamine and to investigate the molecular mechanism.We used various in vivo xenograft model, we confirmed that glucosamine prohibits primary tumor growth through reducing IGF-1R signalling and increasing ER-stress.We found that glucosamine inhibited the growth of human non-small cell lung cancer (NSCLC) cells and negatively regulated the expression of IGF-1R and phosphorylation of Akt. Glucosamine decreased the stability of IGF-1R and induced its proteasomal degradation by increasing the levels of abnormal glycosylation on IGF-1R. Moreover, picropodophyllin, a selective inhibitor of IGF-1R, and the IGF-1R blocking antibody IMC-A12 induced significant cell growth inhibition in glucosamine-sensitive, but not glucosamine-resistant cell lines. Using Taken together, our results suggest that targeting the IGF-1R/Akt pathway with glucosamine may be an effective therapeutic strategy for treating some type of cancer. Resulectively ,9.The PI3K/Akt pathway is often overactivated in various types of cancer cells. PI3K/Akt can transmit signals from RTKs and G-protein-coupled receptors that are activated by growth factors or cytokines; therefore, the PI3K/Akt signal transduction pathway regulates multiple cellular functions, including transcription, translation, and cell proliferation, cell cycle progression, and survival -12. AlthRecent studies have demonstrated that target proteins of glucosamine may exist in cancer cells -16. GlucHuman NSCLC cell lines A549, H226B, H1299, and H460 were purchased from the American Type Culture Collection .The HA-Akt1 (T308D/S473D) expression vector was kindly provided by Dr. Gordon Mills (The University of Texas MD Anderson Cancer Center). The H226B-Babe cells were produced by infecting H226B NSCLC cells with a pBabe retroviral control vector. The H226B-Akt1-DD cells that possess a constitutively active form of Akt were produced by infecting H226B with a pBabe-HA-Akt1-DD construct harboring mutations that change Ser473 and Thr308 to aspartic acids. The H226B-Akt2-DD and The H226B-Akt3-DD cells were kindly provided by Dr. Ho-Young Lee .D-(+)-Glucosamine hydrochloride, MG132, and tunicamycin (TN) were purchased from Sigma-Aldrich . Antibodies against pIGF-1R, pAkt, pERK1/2, Akt, PTEN, PARP, PDI, IRE1α, ATF4, GRP78, CHOP, and a/β-tubulin were purchased from Cell Signaling Technology . Antibodies against IGF-1R, COX-2, CDK2, CDK4, and β- ACTIN were purchased from Santa Cruz Biotechnology, Inc. , and the antibody against TGase 2 was obtained from Thermo Fisher Scientific, Inc. .6 cells) were subcutaneously injected into flank region of BALB/c nude mice. After cancer cell injection, glucosamine (500 mg/kg body weight/day) was administered intrapenitorially to immuoncompromised mice. Tumor volume was measured using caliper and calculated according to the formula (L x W2)/2. All of the mice were sacrificed on Day 71, and tumor tissues were isolated from them. The results were represented as the mean of tumor volumes (n = 10) with SEM.All animal experimental procedures were approved by Institutional Animal Care and Use Committee (IACUC) of National Cancer Center in Republic of Korea. To confirm antitumor effect of glucosamine in animal, we used xenograft tumor model. A549 cells (5 x 10IGF-1R (5′-CUG ACA UGG GCC UUU AAG A-3′), and a scrambled non-targeting siRNA were synthesized by Bionner . Cells were transfected with siRNAs using Lipofectamine reagent  according to the manufacturer’s protocol.For RNA interference, A549, H1299, and H460 cells transfected with 40 nM siRNA. Double-stranded siRNAs designed to target IGF-1R, COX-2, XBP1, GRP78, CHOP, ATF4, GAPDH, and β-ACTIN . RT-PCR was carried out with gene-specific primers for Preparation of whole-cell lysates from cancer cells, electrophoresis, and membrane transfer were performed as previously described . The memTo determine the effects of glucosamine on the proliferation of various cancer cell lines, cells were seeded in 96-well plates . On the following day, the medium was replaced with medium containing glucosamine, picropodophyllin, and A12 at the desired concentrations. After incubation for an additional 2 days, MTT assay was performed according to standard procedures. The bars represent SD of results.For the cell cycle analysis, three human NSCLC cell lines were treated with the indicated concentration of glucosamine. Floating and attached cells were fixed in 70% ethanol for 1 hour at 4°C. After centrifugation, the cell pellet was washed twice with phosphate-buffered saline (PBS) and stained with propidium iodide (PI) containing RNase A (40 μg/ml) for 30 minutes at 4°C in the dark. The total cellular DNA content of each cancer cell line was quantified by flow cytometry.To analyze the number of apoptotic cells after 2 days of glucosamine treatment, A549, H1299, and H460 cells were harvested and washed twice with PBS on ice. The cells were resuspended in 1 X binding buffer containing 5 μl fluorescein isothiocyanate (FITC)-conjugated Annexin V and 5 μl PI. Apoptotic events were detected by flow cytometry at 488 nm and 633 nm using the FITC Annexin V apoptosis detection kit I . All procedures were carried out according to the manufacturer’s instructions.Primary tumors from PBS or glucosamine treated animals were embedded in paraffin depending on the application. The 5 μm tumor tissue sections were prepared for immunohistochemistry. Paraffin sections were incubated overnight at 4°C with primary antibody against anti-phospho-Akt  and then processed for avidin – biotin immunohistochemistry according to the manufacturer’s instructions . These sections counterstained with hematoxylin and eosin-Y (H&E). Immunohistochemical analysis were performed as previously described .Previous studies have reported that glucosamine inhibits cell growth ,16 and cIn addition, expression of cleaved poly-(ADP-ribose) polymerase (PARP), a marker for apoptosis, was high in A549 and H226B cells and low in H460 cells Figure D. TreatmWe investigated the expression levels of TGase 2 and COX-2 proteins that were previously identified as major targets of glucosamine. Expression of TGase 2 was markedly higher in A549 and H1299 cells than in H460 and H226B cells. We also found that A549 and H460 cell lines showed a high basal level of COX-2 expression, whereas COX-2 expression was not detected in H1299 cells  ,28. Oncever rate . In thisAlthough our results suggest that glucosamine effectively inhibits cell proliferation and tumor growth in A549, some of NSCLCs such as H460 relatively show glucosamine-resistant phenotype. Because the IGF-1R/Akt signaling axis includes many signal regulators, such as PI3K, PTEN, and p53, that can influence the pAkt level, -35 reducIn summary, our results indicate that glucosamine is an effective inhibitor of the IGF-1R/Akt pathway. The findings of the present study provide evidence supporting the value of glucosamine as an effective and non-toxic IGF-1R blocking agent for cancer therapeutics.The authors declare that they have no competing interests.All authors participated in design of the study. K-HS, J-HK, and H-YM performed the experimental work and wrote the manuscript. J-KW, J-SN, H-YL, and S-YK contributed to data analysis and interpretation. S-HO conceived of the study, participated in the experimental design, and helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/14/31/prepubDifferential effect of glucosamine-induced apoptosis in A549 and H1299 cells. A549 and H1299 cells were treated with 1mM glucosamine for 48 hrs. Cells were stained with Annexin V-FITC and PI and then analyzed by flow cytometry. Results shown are representative of three independent experiments.Click here for fileRelationship between glucosamine-induced growth inhibition and TGase 2 expression in NSCLC cells. NSCLC cells were grown in RPMI medium 1640 containing 10% FBS for 2 days. Western blot analysis of the expression of TGase 2 and COX-2 was performed using cell lysates from harvested cells.Click here for fileGlucosamine induce ER stress- and unfolded protein response. A549 cells were treated with glucosamine and tunicamycin for 6 hours. RT-PCR analysis was conducted with indicated gene specific primers.Click here for file


Mevalonate kinase deficiency (MKD) is an early-onset autosomal recessive autoinflammatory fever syndrome lacking specific treatment options. Current data point to protein misfolding as underlying molecular mechanism.To characterize the molecular pathophysiology of eight MK variants associated with a hyperimmunoglobulinemia D with periodic fever syndrome (HIDS)  and/or mevalonic aciduria (MA)  phenotype.E. coli. Following affinity purification and size exclusion chromatography, the MBP-MK fusion (fMK) or cleaved MK (cMK) proteins, respectively, were characterized with regard to oligomerization, thermal unfolding monitored by differential scanning fluorimetry, thermal aggregation analyzed by right angle light scattering, and enzyme activity. These data were correlated with the disease phenotype and MK activities measured in blood cells and/or fibroblasts of 101 homozygous or compound heterozygous MKD patients, respectively, that were collected retrospectively from our center and the literature.Recombinant wild-type (WT) and variant MK proteins N-terminally fused to a maltose binding protein (MBP) were expressed in MK dimer assembly was impaired in all analyzed MK variants. Oligomerization profiles of V377I and V203A were most similar to that of WT, A334T showed reduced amounts of fMK and cMK dimers, while H20P and I268T revealed only small peaks of fMK dimers, and W188X, H380R, and L264F lacked any dimers, thus pointing to altered protein conformation of various degree. This was confirmed by partial unfolding in the native state, and variably accelerated thermal unfolding of all MK variants. Furthermore, thermal aggregation kinetics investigated in cMK V203A, A334T, and V377I, respectively, were altered. Catalytic function varied from high residual activity in cMK V377I (100% of WT), to moderately decreased activity in cMK V203A (70%) and fMK I268T (80%), markedly decreased activity in cMK A334T (3.1%), and almost no activity in fMK H20P (0.4%), as well as W188X (0.7%), L264F (0.9%), and H380R (1.5%). Consistently, MKD patients carrying at least one V377I mutation presented the HIDS phenotype, while those being compound heterozygous or homozygous for H20P, L264F, I268T, and/or A334T mutations were associated with MA. While patient MK activities were highest among V377I homozygous or V377I/I268T and V377I/H20P genotypes, there was a considerable variability of MK activities for other genotypes analyzed being associated with HIDS or MA.These results support the hypothesis of protein misfolding with loss of function being the molecular basis in MKD, and thus may assist the development of novel targeted therapeutic strategies.None declared.


Xenopus, and human is required for accumulation of Aurora B on the centromere, and the subsequent activation of Aurora B kinase activity for accurate chromosome alignment and segregation. Although extensive analyses of Haspin have been carried out in yeast and animals, the function of Haspin in organogenesis remains unclear.Haspin kinases are mitotic kinases that are well-conserved from yeast to human. Human Haspin is a histone H3 Thr3 kinase that has important roles in chromosome cohesion during mitosis. Moreover, phosphorylation of histone H3 at Thr3 by Haspin in fission yeast, Arabidopsis thaliana. The purified AtHaspin phosphorylated histone H3 at both Thr3 and Thr11 in vitro. Live imaging of AtHaspin-tdTomato and GFP-α-tubulin in BY-2 cells showed that AtHaspin-tdTomato localized on chromosomes during prometaphase and metaphase, and around the cell plate during cytokinesis. This localization of AtHaspin overlapped with that of phosphorylated Thr3 and Thr11 of histone H3 in BY-2 cells. AtHaspin-GFP driven by the native promoter was expressed in root meristems, shoot meristems, floral meristems, and throughout the whole embryo at stages of high cell division. Overexpression of a kinase domain mutant of AtHaspin decreased the size of the root meristem, which delayed root growth.Here, we identified a Haspin kinase, designated AtHaspin, in A. thaliana. AtHaspin phosphorylated histone H3 at both Thr3 and Thr11 in vitro. The expression and dominant-negative analysis showed that AtHaspin may have a role in mitotic cell division during plant growth. Further analysis of coordinated mechanisms involving Haspin and Aurora kinases will shed new light on the regulation of chromosome segregation in cell division during plant growth and development.Our results indicated that the Haspin kinase is a histone H3 threonine kinase in  The mitotic phase, which comprises mitosis and cytokinesis, is a fundamental process for faithful transmission of genetic information from one cell generation to the next. The main purpose of mitosis is to segregate sister chromatids into two daughter cells. The regulation of mitotic progression relies mainly on two post-translational mechanisms; protein phosphorylation and proteolysis. Cell division is regulated by mitotic kinases, such as the cyclin-dependent kinase 1 (CDK1), the Polo family, the NIMA (never in mitosis A), and the Aurora family, as well as kinases implicated in mitotic checkpoints, mitotic exit and cytokinesis [Arabidopsis thaliana Aurora kinase3) phosphorylates histone H3 at Ser10 and Ser28 in vitro [Arabidopsis suspension cells [Post-translational modifications of core histones play a crucial role in chromatin structure and gene expression . Althougin vitro . Inhibiton cells . Thus, AH3T3 and H3T11 are also phosphorylated, but their distribution patterns differ from those of H3S10ph and H3S28ph during mitosis. In mammalian cells, H3T3ph and H3T11ph occur preferentially at the centromere from prophase to anaphase ,14. In cSaccharomyces cerevisiae, Xenopus, and human revealed the novel cascade leading to the recruitment of mitotic kinases to the centromere [S. cerevisiae, Haspin interacts with cohesin, and the cohesin-associated Haspin phosphorylates histone H3 at Thr3 on the inner centromere [Haspin (haploid germ cell-specific nuclear protein kinase) was first identified as a testis-specific gene in mice ,18. Althntromere -24. In Sntromere . The phontromere -24. ThusA. thaliana Haspin, characterized its kinase activity, and determined its localization during mitosis. Expression of a kinase domain mutant of AtHaspin inhibited root growth, suggesting that Haspin is involved in cell division during mitosis.Analyses of Haspin were first carried out in yeast and animals, and although it is clear that this protein has roles in mitosis and cell division, the function of Haspin in organogenesis remains unclear. In this study, we identified Caenorhabditis elegans and S. cerevisiae, most organisms have one Haspin kinase gene. In a BLAST search of the A. thaliana genome, one Haspin candidate gene showed high similarity  to human Haspin kinase in the kinase domain. The second hit gene showed lower similarity . One Haspin candidate gene has been identified in some plant species, including ferns, mosses, and algae  is designated as AtHaspin . The C-terminal regions of Haspin proteins have a conserved kinase domain [2+ ion-binding sites were conserved between human and A. thaliana  (K309A) with or without ATP. Phosphorylated proteins were detected by ProQ Diamond Phosphoprotein Stain. As expected, GST-AtHaspin-KD was not autophosphorylated even in the presence of ATP , and plant histone H3 as the substrate. The positive control, GST-AtAUR3, is a histone H3 Ser10 and Ser28 kinase [in vitro. Surprisingly, GST-AtHaspin also phosphorylated histone H3 at Thr11 in vitro . Using anti-H3T3ph antibodies, bands were detected in the case of normal histone H3 and S10A, T11A, and G12A mutants, but not in the case of R2A, T3A, or K4A mutants  cultured cells with GFP-fused AtHaspin and observed tobacco BY-2 cells stably expressing AtHaspin-GFP with DNA stained by Hoechst 33342 . AtHaspin-GFP was expressed in meristems and primordia of root tips, lateral roots  using the estradiol-inducible XVE system . When gr Figures  and 6B.  Figures  and 7B.  Figures . These r Figures , and 7D. Figures . These rA. thaliana. Haspin kinases are conserved from yeast to human. Most organisms have one Haspin kinase gene, except for C. elegans and S. cerevisiae. Some plant species, including A. thaliana, have one Haspin candidate gene  and Ser28 (A25R26K27S28) [1R2T3K4) differs from that around Thr11 (K9S10T11G12). Point mutational analysis of human Haspin revealed the important residues for histone H3 phosphorylation in the catalytic cleft [A. thaliana . The human Haspin mutant D709N shows reduced affinity for the histone H3 tail and impaired ability to phosphorylate H3 [in vitro [in vivo. However, the centromeric localization of Dlk/ZIP during mitosis suggests that this kinase is responsible for H3T11ph. The A. thaliana genome contains no Dlk/ZIP kinase orthologues, and thus, AtHaspin has an additional role as a H3 Thr11 kinase in A. thaliana.As expected, GST-AtHaspin phosphorylated histone H3 at Thr3 in vitro ,22,24. I6K27S28) . Howeveric cleft . These ra Figure , green, ylate H3 . Althougin vitro , but theMitotic phosphorylation of histone H3 at Ser10, Ser28, Thr3, and Thr11 is highly conserved among eukaryotes. Although phosphorylation of histone H3 was first observed more than 30 years ago, the functions of this modification remain unclear . There iA. thaliana  was localized in the cytoplasm during interphase until NEBD. The interphase localization of AtHaspin differs from that of human Haspin, which localizes in the nucleus during interphase . After Nin vitro kinase assay revealed that the antibodies against H3T3ph and H3T11ph did not show cross-reactivity with other histone H3 phosphorylations  [Arabidopsis and BY-2 cells  . MAPs has Figure  and 4. Ts Figure . Furthers Figure . AlthougA. thaliana was identified as a mitotic histone H3 threonine kinase. The expression and dominant-negative analysis showed that AtHaspin may have a role in cell division during mitosis. The functions of H3 threonine phosphorylation remain obscure in animals and plants. Recent studies have shown that H3T3ph by S. cerevisiae, Xenopus and human Haspin is required for the accumulation of Aurora B on the centromere, and for subsequent activation of Aurora B kinase activity [A. thaliana [In this study, the Haspin kinase in activity -24. Theractivity ,41, homoactivity -24. The thaliana ,10-12,42A. thaliana (ecotype Columbia) seeds were grown on plates containing half-strength Murashige and Skoog salts, Gamborg B5 vitamins, 0.05% MES-KOH (pH 5.8), and 1% agar in a growth chamber at 22°C under continuous light. Tobacco 'Bright Yellow-2' (BY-2) cells were maintained as previously described by Kurihara et al. [a et al. .AtHaspin was cloned into the pDEST15 expression vector by Gateway technology (Invitrogen). Site-directed mutations of GST-AtHaspin for K309A and of the GST-H3 tail for R2A, T3A, K4A, S10A, T11A, and G12A were generated by PCR-based mutagenesis from the GST-AtHaspin and GST-H3 tail expression vectors, as described elsewhere [A full-length cDNA for the candidate Haspin kinase (RAFL15-11-J01) was obtained from the RIKEN BioResource Center . Site-dilsewhere .AtHaspin cDNA was cloned into the spUC-GFP vector, which contains the CaMV 35S promoter. spUC-AtHaspin-GFP was digested and ligated with pEBis-kH2 as described by Fujimoto et al. [o et al. .AtHaspin cDNA was cloned into the spUC-tdTomato vector [For inducible expression constructs, o vector . For Veno vector . PCR frao vector .AtHaspin native promoter, the promoter and genomic regions were amplified from A. thaliana genomic DNA. PCR products were cloned into pENTR using a pENTR/D-TOPO cloning kit (Invitrogen), and then into the pGWB4 expression vector by the LR reaction of the Gateway Cloning System (Invitrogen).For construction of the GFP expression vector driven by the in vitro kinase assay was performed with purified GST-AtHaspin or GST-AtHaspin-KD, the GST-histone H3 tail, and mutants as substrates as previously described by Kurihara et al. [The a et al. .Agrobacterium-mediated methods as described previously [Transformation of BY-2 cells and BY-GT16 cells  was carreviously . Stable eviously .Agrobacterium-mediated Arabidopsis transformation [Arabidopsis plants expressing AtHaspin-GFP driven by the native promoter were observed using an upright fluorescence microscope , or FluoView FV1000 (Olympus). Cell walls were stained with 10 μg/ml propidium iodide (PI).The floral-dip method was used for ormation . TransgeFor induction of AtHaspin-Venus and AtHaspin-KD-Venus, 10 μM 17-β-estradiol (Sigma) was added to MS medium  to induce expression during seed germination in sterile conditions. The root length of vertically grown seedlings was measured with MBF ImageJ software.The size of root meristems was determined by the position of the first endoduplicated cells, which were identified on the basis of their DNA content. Roots were fixed with 4% paraformaldehyde and 0.1% Triton X-100 for 40 min and stained with CyStain staining solution containing DAPI (Partec) for 5 min. Images were acquired using the IX-81 fluorescence microscope with a 0.5-μm section at the Z axis. These 2D images were used to construct 3D deconvolution images using AutoQuant X (Media Cybernetics). Cell ploidy was calculated using Metamorph (Molecular Devices). When three cells with more than 4C DNA content were aligned continuously, the top cell was defined as the first endoduplicated cell. Cell wall patterning was analyzed using the mPS-PI method . PI-staiANOVA: analysis of variance; BLAST: basic local alignment search tool; BY-2: Bright Yellow-2; CPC: chromosomal passenger complex; DAPI: 4',6-diamidino-2-phenylindole; EST: expressed sequence tag; GFP: green fluorescent protein; GST: glutathione S-transferase; H3S10ph: phosphorylation of histone H3 at Ser10; H3S28ph: phosphorylation of histone H3 at Ser28; H3T3ph: phosphorylation of histone H3 at Thr3; H3T11ph: phosphorylation of histone H3 at Thr11; KD: kinase dead; MAP: microtubule-associated protein; NEBD: nuclear envelope breakdown; PFA: paraformaldehyde; PPB: pre-prophase band; QC: quiescent center.DK conceived and designed the study, carried out the experiments, performed the statistical analyses, and wrote the manuscript. SM participated in the design of the study, carried out the ploidy analysis and the observations of AtHaspin-KD overexpression in root tips, and revised the draft of the manuscript. TO carried out the phenotypic analyses of plants overexpressing AtHaspin-KD. TH and KF participated in the coordination of the study. All authors read and approved the final manuscript.Dynamics of AtHaspin-tdTomato and GFP-α-tubulin in tobacco BY-2 cells. Dynamics of AtHaspin-tdTomato (magenta) and GFP-α-tubulin (green) from late G2 phase to early G1 phase in BY-2 cells after 48-h induction with 10 μM 17-β-estradiol. Images were acquired at 30-s intervals under a 40 × objective lens, and movie is displayed at 15 frames per second (fps). Numbers indicate time (h: min).Click here for fileArabidopsis root tipsDynamics of AtHaspin-GFP in . Images were acquired at 1-min intervals under a 40 × objective lens, and movie is displayed at 15 fps. Numbers indicate time (h: min). Arrowheads indicate mitotic cells.Click here for fileArabidopsis torpedo embryoDynamics of AtHaspin-GFP in . Images were acquired at 1-min intervals under a 40 × objective lens, and movie is displayed at 10 fps. Numbers indicate time (h: min). Arrowheads indicate mitotic cells.Click here for fileArabidopsis root tipsDynamics of AtHaspin-GFP in . Images were acquired at 90-s intervals under a 100 × objective lens, and movie is displayed at 5 fps. Numbers indicate time (h: min). Arrowheads indicate mitotic cells.Click here for fileAbnormality of cell orientation in AtHaspin-KD plants. At 6 days after imbibition, Col. plants and transformants with inducible AtHaspin-KD-Venus vectors with or without induction were analyzed using the mPS-PI method. In AtHaspin-KD plants, arrows indicate abnormalities in orientations of cell walls between transverse neighboring cells. Scale bar: 50 μm.Click here for file


Bordetella pertussis is the primary etiologic agent of the disease pertussis. Universal immunization programs have contributed to a significant reduction in morbidity and mortality of pertussis; however, incidence of the disease, especially in adolescents and adults, has increased in several countries despite high vaccination coverage. During the last three decades, strains of Bordetella pertussis in circulation have shifted from the vaccine-type to the nonvaccine-type in many countries. A comparative proteomic analysis of the strains was performed to identify protein(s) involved in the type shift.Proteomic analysis identified one differentially expressed protein in the B. pertussis strains: the type III cytotoxic effector protein BteA, which is responsible for host cell death in Bordetella bronchiseptica infections. Immunoblot analysis confirmed the prominent expression of BteA protein in the nonvaccine-type strains but not in the vaccine-type strains. Sequence analysis of the vaccine-type strains revealed an IS481 insertion in the 5′ untranslated region of bteA, −136 bp upstream of the bteA start codon. A high level of bteA transcripts from the IS481 promoter was detected in the vaccine-type strains, indicating that the transcript might be an untranslatable form. Furthermore, BteA mutant studies demonstrated that BteA expression in the vaccine-type strains is down-regulated by the IS481 insertion.The cytotoxic effector BteA protein is expressed at higher levels in B. pertussis nonvaccine-type strains than in vaccine-type strains. This type-dependent expression is due to an insertion of IS481 in B. pertussis clinical strains, suggesting that augmented expression of BteA protein might play a key role in the type shift of B. pertussis. Bordetella pertussis is a human-specific pathogen that is the etiologic agent of whooping cough, an acute respiratory disease that is often particularly severe in infants B. pertussis vaccine strains and circulating clinical strains in many countries B. pertussis virulence factors: pertussis toxin S1 subunit (ptxA), pertactin (prn) and fimbriae 3 (fim3). Among circulating B. pertussis strains, vaccine-type alleles  have been replaced mainly with nonvaccine-type alleles . It has been speculated that adaptation of the bacterial population to vaccine-induced immunity has produced this genetic shift, and is one possible explanation for the resurgence of pertussis B. pertussis expresses various virulence factors, including adhesins and toxins, which function to establish and maintain host infection. Several virulence factors such as filamentous haemagglutinin (FHA) and pertussis toxin (PT) are expressed under the control of the BvgAS two-component regulatory system B. pertussis, T3SS plays a role in subverting the protective innate and adaptive immunity of the host. Three T3SS-secreted proteins, BopN, BopD and Bsp22, have been identified so far Bordetella bronchiseptica, BopN is involved in the up-regulation of cytokine IL-10 B. pertussis clinical isolates but not in Tohama and Wellcome 28, the common laboratory-adapted vaccine strains B. pertussis clinical strains and the vaccine strain Tohama have been investigated. The comparative genomics profiling revealed that the genome of B. pertussis Tohama differs from clinical isolates in four regions (RD11 to RD14) 481 insertion sequence elements has been observed among recently circulating strains of B. pertussis isolates 481 is present in multiple copies on the B. pertussis chromosome, and it plays a critical role in B. pertussis evolution through genomic rearrangement.Genomic differences between Bordetella spp. facilitates comparative proteomic approaches to the investigation of B. pertussis clinical strains B. pertussis strains. The protein profile analyses identified one differentially expressed protein, the T3SS effector BteA  B. bronchiseptica but not in the B. pertussis human pathogen. Here we studied the differential expression of BteA protein in B. pertussis clinical strains and identified a specific IS481 insertion in the 5′ untranslated region (5′-UTR) of bteA in vaccine-type strains.Proteomic analysis has been widely applied to comparisons of protein expression among different strains, and information accumulated from genomic studies of B. pertussis strains from vaccine-type to nonvaccine-type. B. pertussis hypothetical protein BP0500 (NCBI accession: NP_879352). Hypothetical protein BP0500 was identified as the T3SS effector BteA, since the sequence is highly conserved with 98% amino acid identity to the BteA (BopC) of B. bronchisepticaA comparative proteomic analysis of two clinical strains was performed to investigate the shift of B. pertussis clinical strains using anti-BteA antiserum detected high levels of a protein of ∼68 kDa in all nonvaccine-type clinical strains , whereas BteA expression was greatly reduced in the vaccine-type clinical strains . Additional products of >200 kDa were also detected in the nonvaccine-type clinical strains. These high molecular mass signals appear to be the protein bands that have been reported as a multimeric complex of BteA in B. bronchisepticaBordetella spp. virulence factor, was detected at similar levels in both strain types.Immunoblots of B. pertussis strains, BteA polypeptide in the culture supernatants (CS) was subjected to immunoblot analysis. BteA was detected in secreted proteins from the nonvaccine-type clinical strain BP159 at 12, 24 and 48 h, whereas the signal was very low in the vaccine-type clinical strain BP155 over the 48-h time period  and vaccine-type  clinical strains  showed an average bteA transcript level of 0.146  in nonvaccine-type strains and 0.095  in vaccine-type clinical strains, a difference that was not statistically significant (P = 0.11) ( strains . Similar = 0.11) .bteA 5′-UTR of the five vaccine-type strains , revealed a 1,043-bp insertion sequence (IS481) −136 bp upstream of the bteA start codon , one single nucleotide polymorphism (A→G) was observed at 207 bp upstream of the bteA translation start site  produced amplicons of ∼3.1 kb, a size indicative of an IS481 insertion in the bteA 5′-UTR. One strain (BP121) had a product of ∼2.1 kb, corresponding to the predicted size of the native 5′-UTR (data not shown). Of the 23 nonvaccine-type strains examined, all generated ∼2.1 kb amplicons, confirming the absence of the IS481 insertion.The bteA transcription start site in vaccine-type strain BP155. Nucleotide sequences of the 5′-RACE PCR products revealed two transcription start sites, P1 and P2, located −68 and −147 bp from the bteA translation start codon  was located within IS481a. Only the P1 start site was also found in the nonvaccine-type strain BP159  in B. pertussiskatA transcription start site. The transcription start site of btcA, also determined by 5′-RACE, was mapped to a T residue 31 bp upstream of the btcA translation start codon in both the vaccine-type and nonvaccine-type strains  in B. pertussis vaccine-type strain BP155 was analyzed by qRT-PCR with TaqMan probes (bteA (P1 + P2) transcripts were determined individually and the ratio of P2 transcript to total bteA transcript was calculated. Based on four independent experiments, the ratio (P2 transcript/P1 + P2 transcripts) was estimated to be 0.88 , indicating that the P2 transcript is the major bteA transcript in the vaccine-type strain (data not shown).Expression of the ISn probes . The P2 481 insertion on BteA expression, four BteA mutants  were constructed from B. pertussis BP155 (vaccine-type) and BP157 (nonvaccine-type) by homologous recombination  replaced the native bteA 5′-UTR + IS481a gene. In contrast, +IS481-BP157 mutant had a 3.2-kb insertion containing a bteA 5′-UTR + IS481a (derived from B. pertussis BP155) instead of its own bteA 5′-UTR. Consequently, ΔIS481-BP155 had an IS481a deletion from the bteA 5′-UTR, whereas the +IS481-BP157 mutant had an IS481a insertion in the bteA 5′-UTR. The btcA−bteA region of the mutants was verified by DNA sequence analysis.To clarify the effect of the ISbination . The Δbt481-BP155 bacterial cells and CS, BteA polypeptide(s) corresponding to ∼68 kDa and >200 kDa were detected at the same level as was observed in the BP157 wild-type strain. In contrast, the signals of BteA polypeptide(s) from +IS481-BP157 mutant were very low in both bacterial cells and CS. Similarly, BteA polypeptide(s) were not detected in either ΔbteA-BP155 or ΔbteA-BP157. These results clearly showed that BteA protein expression is down-regulated by the IS481 insertion in B. pertussis, and that the anti-BteA antiserum is highly specific to BteA.BteA expression in the bacterial cells and CS after 24 h in culture was analyzed by immunoblot with anti-BteA antiserum . In ΔIS4B. bronchiseptica infections, and is thought to play a pivotal role in T3SS-mediated cell death Bordetella spp. In B. pertussis, low-passage clinical strains have an ability to express a functionally active T3SS; however, BteA protein had not been detected in the clinical and common laboratory-adapted strains by MALDI-TOF mass spectrometry B. pertussis nonvaccine-type strains but not in the vaccine-type strains, and that BteA protein expression is down-regulated by IS481a insertion in the vaccine-type strains. We provide the first evidence that BteA protein expression is type-dependent due to the IS481a insertion in B. pertussis clinical strains.The BteA effector  is required for the induction of necrotic cell death during B. pertussis circulating strains began to change from vaccine-type to nonvaccine-type in the mid-1990s B. pertussis circulating strains has also been observed in many other countries. A possible explanation for the genetic divergence is that the type shift is a result of vaccine-driven evolution B. pertussis strains with increased PT production has contributed to the resurgence of pertussis in the Netherlands. Here we showed prominent expression of the T3SS effector protein BteA in the nonvaccine-type strains, and that PT and ACT (important virulence factors of B. pertussis) are expressed at the same level in both the nonvaccine and vaccine-type strains. Besides vaccine-driven evolution, our findings could provide another possible explanation for the type shift from vaccine-type to nonvaccine-type, i.e., the augmented expression of BteA protein in B. pertussis nonvaccine-type strains may be involved in the type shift.In Japan, B. bronchiseptica BteA has in vitro cytotoxic activity against cultured mammalian cells B. pertussis BteA mutants by measuring the release of lactate dehydrogenase (LDH) from L2 rat lung epithelial cells, J774 mouse macrophage-like cells, or HeLa cells. However, even BteA-expressing strains (ΔIS481-BP155 and wild-type BP157) showed low cytotoxicity (<10%), and consequently no statistically significant differences in cytotoxicity were observed among the wild-type and mutant strains. B. pertussis is known to have a lower in vitro cytotoxicity than B. bronchisepticaB. pertussis as compared to B. bronchiseptica  compared with its own promoter (P1). However, the vaccine-type strains showed a low level of BteA protein expression, suggesting that insertion of IS481a represses P1 promoter activity, and that P2 transcript has a low translational efficiency from the additional nucleotide sequence (79 nucleotides) at its 5′ end. Use of a cell-free coupled transcription-translation system revealed that the additional nucleotide sequence is involved in down-regulation of transcription and/or translation  the P2 transcript is translated into BteA under certain environmental conditions, and (ii) the P2 transcript is translated into another novel protein by translational frameshifting. BteA is known to be regulated by the BvgAS system and an extracytoplasmic function (ECF) sigma factor BtrS in B. bronchisepticaB. pertussis, it has been suggested that expression of the T3SS translocon Bsp22 is blocked by post-transcriptional regulation B. pertussis vaccine-type clinical strains.In this study, the 5′-UTRs of five B. pertussis vaccine-type strains have been replaced with the nonvaccine-type strains in many countries, and the resurgence of pertussis has been observed in several nations. In Japanese B. pertussis clinical strains, the T3SS effector BteA is highly expressed in nonvaccine-type strains as compared with the vaccine-type strains. Our findings indicate that augmented expression of BteA protein in B. pertussis circulating strains could play a key role in the type shift. However, it is unclear whether BteA protein is implicated in the resurgence of pertussis. Further studies are needed to determine the expression of BteA protein in B. pertussis circulating strains on a global scale.In conclusion, B. pertussis clinical strains were selected from the laboratory collection of the National Institute of Infectious Diseases, Tokyo, Japan. The selection criteria included the time and geographic location of isolation, and their ptxA and prn alleles. A total of 10 clinical strains from 2002 to 2004 in Japan were included. Of the 10 clinical strains, 5 harbored ptxA1 and prn2 alleles , while the others carried ptxA2 and prn1 . All strains were cultured on Bordet-Gengou agar  supplemented with 1% glycerol and 15% defibrinated horse blood or in modified Stainer-Scholte (SS) medium B. pertussis clinical strains grown on BG agar plates were resuspended in casamino acid solution . Bacterial cells were precipitated by centrifugation  and resuspended in SDS-lysis buffer  by sonication. Total protein was extracted by boiling for 3 min, followed by centrifugation. A portion  of the protein solution was mixed with 20 µl of sample buffer , and applied to an isoelectric focusing tube gel (2.0 mm inside diameter by 12.0 cm) containing 4% polyacrylamide, 8.5 M urea, 2% Nonidet P-40, and 2% Ampholine (pH 5 to 7 and pH 3.5 to 10 in a ratio of 1∶4). Proteins were focused at 10°C for 17 h  with 10 mM H3PO4 (anolyte) and 20 mM NaOH (catholyte). In the second dimension, the electrofocused tube gel was electrophoresed in 12% SDS-PAGE. The separated polypeptides were visualized by silver staining and analyzed with the PDQuest 2-D Analysis Software . The Lowry assay was used to measure protein concentrations in a trichloroacetic acid (TCA) pellet (resuspended in 1 N NaOH) using bovine serum albumin as a standard.2D-PAGE was performed based on the O'Farrell method TM Deca XP ion trap mass spectrometer  with electrospray ionization (ESI) interface using the Nanosprayer FS . To identify peptides, data files were generated from the MS/MS scans by Bioworks 3.0 using the SEQUEST algorithm  and searched against the complete amino acid database derived from the B. pertussis Tohama genome database.2D-PAGE gels were stained with silver nitrate without glutaraldehyde fixation B. pertussis Tohama DNA using BteA-F and BteA-R primers, and cloned into the XmnI/HindIII sites of pMal-c2X  to generate a maltose binding protein (MBP) fusion with BteA. Production of this fusion protein was induced in E. coli BL21 with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) and subsequently purified using amylose resin (New England Biolabs) and Resource Q  columns. A two-step PCR was carried out to amplify recombinant BtcA (NCBI accession: NP_879351). The first PCR was performed using the BtcA-BteA-F3 and BtcA-BteA-R3 primers (B. pertussis Tohama genome (GenBank accession: BX640412). In the second PCR, btcA was amplified from the first PCR product with the 5-BtcA and 3-BtcA primers . A recombinant catalytic domain of B. pertussis adenylate cyclase toxin (ACT) was a gift from Mineo Watanabe.The BteA gene (NCBI accession: NP_879352) was amplified by PCR from  primers , which a primers  and clonAntibodies against MBP-BteA, BtcA and ACT were generated in mice at Nippon Biotest Laboratories, Inc. . The MBP-BteA antiserum was pre-absorbed with MBP2 protein (New England BioLabs) and the resulting antiserum was used.B. pertussis clinical strains were inoculated in modified SS medium with a starting optical density of 0.2 at 600 nm, and further cultured with shaking at 36°C. Total protein was extracted with SDS-lysis buffer, and culture supernatant (CS) proteins were precipitated with 10% TCA. Protein samples were subjected to SDS-PAGE, transferred to nitrocellulose membranes (Bio-Rad) and incubated with anti-BteA, anti-BtcA, anti-BopD btcA and bteA gene corresponding to positions 165122 to 168021 of B. pertussis Tohama (GenBank accession: BX640412) was amplified in vaccine-type and nonvaccine-type clinical strains with the appropriate primers and sequenced. Sequencing reactions were carried out with the BigDye Terminator v3.1 Cycle Sequencing Kit , and the products were sequenced on an ABI PRISM 3130xl Genetic Analyzer (Applied Biosystems).The region between the bteA and btcA amplification, respectively  and treated with RNase-free DNase (Promega) to degrade contaminating DNA. Reverse transcriptase-PCR (RT-PCR) was performed with bteA RT-R and btcA RT-R primers  using thectively . ProductbteA and recA transcripts were determined using TaqMan probes  with the ABI PRISM 7500 Sequence Detection System (Applied Biosystems). The qRT-PCR conditions were 30 s at 95°C, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The expression of recA was used as an internal control bteA transcript (P1 + P2 transcripts) was normalized to the recA transcript for each sample. The bteA IS481a-promoter transcript (P2 transcript) was determined using a TaqMan probe (IS481-bteA probe). The qRT-PCR conditions were 30 s at 95°C, followed by 40 cycles of 95°C for 15 s and 55°C for 1 min. The ratio of P2 transcript to total bteA transcript (P2 transcript/P1 + P2 transcripts) was estimated from four independent experiments. The regions amplified by qRT-PCR are shown in For quantitative RT-PCR (qRT-PCR), 5 µg of RNA was reverse transcribed into cDNA using the SuperScript First-Strand Synthesis System  with random hexamer primers. Relative levels of total E. coli XL1-Blue, which were plated on LB agar plates. Several clones were sequenced. The transcription start site of btcA was located using 5′-RACE with five primers, btcA-RT (5′ phosphorylated primer), btcA-S1 (S1), btcA-A1 (A1), btcA-S2 (S2) and btcA-A2 (A2) (5′ rapid amplification of cDNA ends (5′-RACE) was performed using 5′-Full RACE Core Set (TAKARA Bio Inc.) according to the manufacturer's instructions. Reverse transcription was executed at 55°C using a 5′ phosphorylated RT primer bteA-RT, . The fir-A2 (A2) .bteA-BP155, ΔbteA-BP157, ΔIS481-BP155 and +IS1481-BP157, were constructed by homologous recombination as described previously with minor modifications Four BteA mutants, ΔBteA-deficient mutants: A 2.2-kbp DNA fragment containing a 5′ portion of the bteA gene was amplified by PCR with the B1-bteA and B2-bteA primers (B. pertussis Tohama genomic DNA as the template. The PCR product was cloned into the pDONR221 vector (Invitrogen) to obtain pDONR-bteA by means of adaptor PCR and site-specific recombination techniques with the Gateway Cloning System (Invitrogen). Inverse PCR was then carried out with R1-bteA and R2-bteA primers -resistant B. pertussis BP155 (vaccine-type) and BP157 (nonvaccine-type) clinical strains. The resultant mutant strains were designated ΔbteA-BP155 and ΔbteA-BP157. primers  using th primers  using ciIS481-deletion mutant: pABB-bteA was constructed from pDONR-bteA. pABB-bteA was introduced into E. coli SM10λpir and transconjugated into SM-resistant B. pertussis vaccine-type BP155. The resultant mutant strain was designated ΔIS481-BP155.IS481-insertion mutant: a 3.2-kbp DNA fragment (bteA+IS481) containing the bteA 5′-UTR and IS481a was amplified with the B1-bteA and B2-bteA primers , B. pertussis BP155 (vaccine-type) and BP157 (nonvaccine-type) were cultured in modified SS medium for 24 h. Total protein extracted from the bacterial cells (Cell) and culture supernatants (CS) was separated by SDS-PAGE followed by silver staining (left panel). Immunoblots were incubated with anti-BteA, anti-BtcA or anti-BopD antiserum (right panel). For BteA detection, 0.5 µg of total protein (for Cell) and 5 µl of CS were loaded in the indicated lanes. The amount of total protein loaded was one-twentieth of that in (TIF)Click here for additional data file.Figure S2In vitro transcription-translation analysis of a bteA 5′-UTR deletion series. (A) bteA 5′-UTR deletion genes were PCR-amplified using B. pertussis BP155 (vaccine-type) as the template. Proteins were synthesized using the WakoPURE System . The 5′-UTR deletion genes harbored the T7 promoter at their 5′ end. (B) Expression of BteA protein in an in vitro transcription-translation system (WakoPURE System). The synthesized product was analyzed with immunoblots using anti-BteA antiserum. NC, negative control. (C) A predicted stem-loop structure in the 5′-UTR of bteA mRNA (P2 transcript). The RNA secondary structure was analyzed by CentroidFold (http://www.ncrna.org/centroidfold). The schematic shows a simplified map. TIR, translation initiation region.(TIF)Click here for additional data file.Table S1Primers and probes in this study.(XLS)Click here for additional data file.