In mammals, studies of mutant or genetically-modified rodents have indicated important roles for the GIMAP GTPases in the development and survival of lymphocytes. No clear picture has yet emerged, however, of the molecular mechanisms by which they perform their function(s). Using biotin tag-affinity purification we identified a major, and highly specific, interaction between the human cytosolic family member GIMAP6 and GABARAPL2, one of the mammalian homologues of the yeast autophagy protein Atg8. Chemical cross-linking studies performed on Jurkat T cells, which express both GIMAP6 and GABARAPL2 endogenously, indicated that the two proteins in these cells readily associate with one another in the cytosol under normal conditions. The GIMAP6-GABARAPL2 interaction was disrupted by deletion of the last 10 amino acids of GIMAP6. The N-terminal region of GIMAP6, however, which includes a putative Atg8-family interacting motif, was not required. Over-expression of GIMAP6 resulted in increased levels of endogenous GABARAPL2 in cells. After culture of cells in starvation medium, GIMAP6 was found to localise in punctate structures with both GABARAPL2 and the autophagosomal marker MAP1LC3B, indicating that GIMAP6 re-locates to autophagosomes on starvation. Consistent with this finding, we have demonstrated that starvation of Jurkat T cells results in the degradation of GIMAP6. Whilst these findings raise the possibility that the GIMAPs play roles in the regulation of autophagy, we have been unable to demonstrate an effect of GIMAP6 over-expression on autophagic flux. The GIMAPs (GTPases of the  The mammalian GIMAP family can be divided into members either with  or without  predicted transmembrane domains near to their carboxy-termini: none of the members expresses known sequence motifs permitting post-translational lipid modifications, such as prenylation or palmitoylation, that might mediate dynamic membrane associations.The GIMAPs are a family of GTPases, occurring sporadically in eukaryotic phyla including molluscs, vertebrates and some protists -4. The fnfection . Sequencnfection . In geneGIMAP genes in autoimmune diseases viz. systemic lupus erythematosus [GIMAP5 in rats [GIMAP5 in mice [GIMAP1 results in severe T lymphopenia; however, in contrast to GIMAP5 mutants in which B cell numbers are relatively normal in young mice , these GIMAP1 conditional knockout mice show a profound B lymphopenia even in young animals [Genetic association studies have implicated ematosus ,7, Behçeematosus  and typeematosus . Consist in rats -13, as w in mice ,15, prod animals . The pro animals  and rats animals .Little is known about the molecular mechanisms by which the GIMAPs influence lymphocyte survival. Findings, indicating that some GIMAP proteins can interact with members of the Bcl-2 protein family  and thatin vivo binding partners for the GIMAPs. Here we present data that (i) identify GABARAPL2 , a mammalian homologue of the yeast autophagy-related gene Atg8, as a major binding partner of GIMAP6 and (ii) demonstrate the relocation of GIMAP6 to autophagosomes in response to cell starvation or mTOR inhibition.In order to extend knowledge of the molecular interactions mediating GIMAP function, we have taken a biochemical approach to identifying Antibodies were sourced from the following companies: anti-MAP1LC3A (product number SAB1408113), anti-MAP1LC3B (L7543), anti-GABARAP (SAB2100873), anti-GABARAPL1 (SAB2103059) anti-SQSTM1 (P0067), and anti-β-ACTIN (A5441) were from Sigma-Aldrich; anti MAP1LC3C (ab150367) was from Abcam; anti-CYCLIN D1 (CC12) was from Calbiochem. Rat monoclonal antibodies to both human GIMAP6 (MAC445) and GABARAPL2 (MAC446) were generated in-house (see below). A rabbit polyclonal antiserum to human GIMAP6 was produced by Harlan Laboratories to an in-house generated antigen (see below).Inhibitors were from the following sources: PP242 was from Cambridge Bioscience UK; emetine and chloroquine were from Sigma-Aldrich; AZD8055 was a gift from Dr Sylvie Guichard, Astrazeneca UK. G418 (Geneticin) and penicillin/streptomycin were from Invitrogen; all other selective antibiotics used in mammalian cell culture were purchased from InvivoGen.AGCTTATGTCCGGCCTGAAGACATCTTCGAGGCTCAGAAAAT) and JP512 (GATCCTTCGTGCCATTCGATTTTCTGAGCCTCGAAGATGTCGTTCAGG) were dissolved in water to final concentrations of 10 µM, mixed 1:1 and heated to 95°C in a heating block for 5 minutes. The block was then switched off and the samples cooled to room temperature to allow annealing. The annealed double-stranded oligonucleotide was then ligated between the HindIII and BamHI sites of plasmid pcDNA3 to generate pcDNA3Biot1. Subsequently, a DNA fragment spanning the ires-BirA region of a BirA expression vector [XhoI and XbaI sites was derived by PCR and cloned between the corresponding sites of pcDNA3Biot1 to derive plasmid pcDNA3Biot1iresBirA. This plasmid was further modified by the insertion of a double-stranded oligonucleotide encoding a 6 x His tag with a downstream stop codon between the NotI and XhoI sites to give plasmid pcDNA3Biot1His6iresBirA.Initially a biotinylation tag recognition sequence (as described in ) was insn vector  with flaEcoRI-XhoI site of pcDNA3Biot1His6iresBirA. Plasmids encoding N-terminally myc-tagged GIMAP proteins or N-terminally HA-tagged proteins were derived by cloning the corresponding cDNA sequences into plasmids pCANmyc1 [ Plasmids biotGIMAP6 and biotGABARAPL2 were derived by PCR-based transfer of human GIMAP6 and GABARAPL2 from full-length cDNA clones BC074744 and BM544477, respectively, into the pCANmyc1 , pCANmycBamHI-EcoRI sites in plasmid pIRES-neo (Clontech). Plasmid biot-GIMAP6-His-pCAG-iPuro was derived by transfer of a PCR-derived DNA fragment spanning the biotinylation tag-GIMAP6-6His sequence in plasmid biotGIMAP6 into the SalI site of plasmid pCAGiPuro.Plasmid pmycBirA-ires-neo was derived by insertion of a PCR-derived myc-tagged BirA fragment between the 7 Jurkat T cells were transfected by electroporation  in RPMI with 20 µg of MluI-linearized pmycBirA-ires-neo. Cells were allowed to recover overnight in complete medium. The following day, the cells were spun down and resuspended in 20 ml of complete medium and plated at 100 µl/well into 96-well plates. The next day an equal volume of complete medium supplemented with 1 mg/ml (active concentration) G418 (Invitrogen) was added to each well. Every three days thereafter, half of the medium was replaced with fresh complete medium containing 500 µg/ml G418. myc-BirA expressing cell lines were identified by Western blotting (see below). A single myc-BirA-Jurkat cell line was selected for subsequent use.Jurkat T cells were routinely maintained in RPMI/10% fetal calf serum/100 units/ml penicillin/100 µg/ml streptomycin (complete medium). For establishment of myc-BirA-Jurkat cells, approximately 107 cells were transfected by electroporation with 20 µg of either PvuI-linearized plasmid biot-GIMAP6-His-pCAG-iPuro or the corresponding vector. Cells were allowed to recover overnight in complete medium containing 500 µg/ml G418. The following day, the cells were spun down and resuspended in 20 ml of complete medium containing 500 µg/ml G418 and plated at 100 µl/well into 96-well plates. The next day an equal volume of complete medium containing 500 µg/ml G418 and 6 µg/ml puromycin was added to each well. Every three days thereafter, half of the medium was replaced with fresh complete medium containing 500 µg/ml G418 and 3 µg/ml puromycin. Cells carrying the parental pCAG-iPuro vector were isolated in parallel, as controls. Biot-GIMAP6-His expressing clones were identified by Western blotting of cell lysates with a horseradish peroxidase-conjugated streptavidin probe. A single vector clone and a single clone carrying Biot-GIMAP6-His (termed the Biot-GIMAP6-His myc-BirA-Jurkat cell line) were maintained for subsequent analysis.The myc-BirA-Jurkat cell line was maintained in complete medium containing 500µg/ml G418. Approximately 10HEK293 cells were plated in a 6-well plate and, 24h later, were transfected with a myc-tagged human GIMAP6-expressing plasmid using lipofectamine (Invitrogen). After a further 24 hours, the cells were trypsinised and re-plated in a 10 cm tissue culture plate. 48 hours after transfection, stably transfected cells were selected by growth in DMEM/10% fetal calf serum/100 units/ml penicillin/100 µg/ml streptomycin/800 µg/ml G418. Single colonies were screened for GIMAP6 expression by Western blotting and immunofluorescence. A cell-line expressing a myc-tagged GIMAP6 lacking the C-terminal 10 amino acids was generated similarly.TM HeLa cell line (Invitrogen) by electroporation. Recombinants were selected in DMEM/10% (v/v) FCS/100 units/ml penicillin/100 µg/ml streptomycin containing 5 µg/ml blasticidin S and 100 µg/ml zeocin. Clones transfected with vector plasmid alone were selected in parallel. As required, myc-tagged GIMAP6 expression was induced by addition of tetracycline to a final concentration of 1-2 µg/ml to the culture medium.An N-terminally myc-tagged GIMAP6-encoding DNA fragment was cloned into plasmid vector pcDNA4.TO and the resulting plasmid transfected into the T-RexBglII and HindIII sites of plasmid pTER[GATCCCGAGTCTAAACTCAGCACCATTCAAGAGATGGTGCTGAGTTTAGACTCTTTTTGGAAA and AGCTTTTCCAAAAAGAGTCTAAACTCAGCACCATCTCTTGAATGGTGCTGAGTTTAGACTCGG (shRNA1) and GATCCCGAACTACAGGAAAGGCAAGTTCAAGAGACTTGCCTTTCCTGTAGTTCTTTTTGGAAA and agcttttccaaaaaGAACTACAGGAAAGGCAAGTCTCTTGAACTTGCCTTTCCTGTAGTTCGG (shRNA2). Resulting plasmid constructs were transfected into the T-RExTM Jurkat cell line (Life Technologies). Recombinants were selected in complete medium containing 10 µg/ml blasticidin S and 200 µg/ml zeocin, and cloned by serial dilution. Cells were treated with 1 µg/ml tetracycline for 4 days to induce shRNA expression prior to analysis.Complementary oligonucleotide pairs encoding two different shRNA GIMAP6 targets were annealed and cloned between the smid pTER. The oliPrimary HUVEC cells purchased from TCS Cellworks were grown in human large vessel endothelial cell basal medium plus human large vessel endothelial cell growth supplement (both from TCS Cellworks) and pencillin/streptomycin.Cells were maintained in DMEM/10% (v/v) fetal calf serum/penicillin/streptomycin. Transfections were performed using either polyethyleneimine  or lipof6 cells/ml. Cells (3-6 x 108) were spun down at 400g for 5 min at 4°C, washed with 2 x 20 ml phosphate-buffered saline (PBS), and then resuspended in 20 ml of the same buffer. Formaldehyde solution (37% (w/v) formaldehyde (product number 252549 – Sigma-Aldrich) was added to a final concentration of 1% (w/v), and the cell suspension placed at 20°C for 1h with occasional agitation. The reaction was terminated by the addition of 1/10th volume of 1.25M glycine and left for a further 10 minutes. The cells were then spun down as before, washed once in PBS, and solubilised into 20ml HEPES RIPA lysis buffer  Triton X-100, 0.1% (w/v) sodium dodecyl sulphate pH 7.5) supplemented with 100 µl mammalian protease inhibitor cocktail (Sigma-Aldrich) at 4°C for 10 minutes. The supernatant was clarified by centrifugation at 20,000 g for 10 minutes at 4°C and passed five times through a streptavidin-agarose affinity column (200 µl packed volume). The column was then washed with 5 x 10 ml of HEPES RIPA buffer and the streptavidin-agarose transferred to a micro-centrifuge tube with 2 x 500 µl of the same buffer. The beads were sedimented at 400 g for 1 minute and then re-suspended in 400 µl of 2 x CSB  sodium dodecyl sulphate, 20% (v/v) glycerol, 200 mM dithiothreitol). The suspension was heated to 100°C for 5 minutes. The beads were then sedimented at 400 g for 1 minute and the supernatant (containing the released proteins) was heated to 100°C to reverse the formaldehyde-induced cross-links.Stably transfected myc-BirA-Jurkat cells carrying either plasmid pCAGiPuro or biot-GIMAP6-His-pCAG-iPuro were grown to approximately 10Protein derived from three separate purifications was pooled, and then precipitated by the addition of trichloroacetic acid to a final concentration of 10% (w/v). Samples were left at 4°C for 20 min and precipitated protein recovered by centrifugation at 20,000 g for 30 min at 4°C. The pellet was washed with 1ml acetone and re-centrifuged as before. The supernatant was aspirated and the pellet dried briefly (30 s) in a 100°C hot block. The pellet was then dissolved in 50 µl 2 x CSB, left at room temperature for 2h and then heated to 100°C for 3 min. Samples were then separated on a 12.5% SDS-PAGE gel and the proteins revealed by staining with Imperial protein stain (Thermo Scientific). Bands of interest were excised and one half of each was reduced, carbamidomethylated and digested overnight with trypsin . Approximately 10% of the resulting tryptic digest was analysed by LC-MS/MS. LC separation was achieved on a reversed-phase column , with an acetonitrile gradient . The column was coupled via a nanospray ion source (Proxeon) to a LTQ Orbitrap Velos mass spectrometer (Thermo Scientific) operated in data-dependent acquisition mode. The acquisition cycle consisted of a high resolution precursor ion spectrum over the m/z range 350 - 1500, followed by up to 5 CID spectra. Mass spectrometric data were processed using Proteome Discoverer (Thermo) and searched against the human entries in Uniprot 15.14 using Mascot software (Matrix Science).6 actively growing HEK293T cells were transfected with plasmids as indicated using polyethyleneimine. 24h later cells were transferred to fresh medium. The following day, dishes were washed in PBS and lysed into 1 ml TX100 lysis buffer  Triton X-100, pH 7.5) supplemented with 100 µl mammalian protease inhibitor cocktail (Sigma-Aldrich) at 4°C for 10 min. The supernatant was clarified by centrifugation at 20,000 g for 10 min at 4°C. An aliquot of the supernatant (50 µl) was removed to an equal volume of 2 x CSB  SDS, 20% (v/v) glycerol, 200 mM dithiothreitol, 0.008% (w/v) bromophenol blue) and boiled to represent a lysate sample. To the remaining supernatant was added either a 50 µl packed volume of streptavidin-agarose (for transfections including pcDNA3Biot1His6iresBirA-based expression plasmids) or 50 µl monoclonal antibody (mAb) 12CA5 (anti-HA) supernatant and a 25 µl packed volume of protein A-Sepharose beads. The suspensions were rotated at 4°C for 4 h, and then centrifuged at 15,000 g for 20 s. The pellets were washed with 8 x 1 ml TX100 lysis buffer and the beads recovered each time by centrifugation at 15,000 g for 20 s. The final washed pellets were resuspended in 100 µl 2 x CSB . A parallel transformation of plasmid pGEX4T-1 was also performed. 20 mL overnight Rosetta cultures in LB medium containing either the GST or the GST-GIMAP6 expression construct were used to inoculate 1 L pre-warmed LB medium containing 200 μg/mL ampicillin and 50 μg/mL chloramphenicol. Cells were grown at 37 °C for 2-3 h to an OD of approximately 0.6. The cultures were then cooled to 16 °C and protein expression induced by the addition of 0.5 mM IPTG. Cultures were then grown for a further 16 h at 16 °C. All subsequent steps were carried out at 4°C. Bacteria were collected by centrifugation at 5000g for 5 min, pellets washed in 20 mL PBS pH 7.3 and centrifuged as described above. The pellets were then re-suspended in 20 mL lysis buffer  Triton-X100, 5mM MgCl2, pH 7.4) per pellet and the cells lysed by sonication. The resulting lysate was centrifuged at 10000 g for 15 min and the supernatant applied to a 125 µl packed volume glutathione Sepharose 4B column previously pre-equilibrated in binding buffer  in 500 μL of binding buffer was added to the column and incubated for 1 h at 4 °C. The column was washed as detailed above, and the bound protein (GST and GST-GIMAP6) was eluted by adding 2 x 100 µl of elution buffer . Eluates were analysed by SDS-PAGE, and were then either stained with Coomassie Brilliant Blue R to assess recovery of GST/GST-GIMAP6 or by Western blotting to assay for the presence of GABARAPL2.A full-length GIMAP6 cDNA fragment was transferred to plasmid pGEX4T-1 and transformed into the Monoclonal rat IgG antibodies raised to full-length human GIMAP6 and GABARAPL2 fused to glutathione-S-transferase were prepared as described previously , except Site-directed mutagenesis was routinely performed using a PCR-based strategy with the Accuzyme DNA polymerase (Bioline). N- and C-terminally truncated fragments were generated using oligonucleotides located at the desired ends of the fragments. Internal site-specific mutations were introduced by generating two overlapping PCR products incorporating the desired mutation within the overlapping region. These were then joined in a second PCR reaction spanning the whole desired fragment. The inserts of all PCR-derived plasmids were sequenced completely to ensure that no unwanted mutations had been introduced.2, 1mM MgCl2, 5.5mM glucose, 20 mM HEPES pH 7.4, 1% (w/v) BSA) and then maintained in the same medium for the duration of the starvation. In some experiments, mTOR inhibitors AZD8055 or PP242 were added directly to cells in complete growth medium to final concentrations of 1 µM or 0.45 µM, respectively, to induce autophagy.Cells to be starved were washed twice with starvation medium  as described by the manufacturer. cDNA synthesis was performed using a Quantitect reverse transcription kit (Qiagen) and then qPCR performed on the resulting cDNA using a SYBR Green PCR master mix (Applied Biosystems) in a Biorad CFX-96 Real Time system with the following oligonucleotide pairs: GAPDH – HEK-293 cells engineered to overexpress myc-GIMAP6, or cells carrying the corresponding vector, were grown on 35 mm dishes. Emetine was added to the medium to a final concentration of 10 µMDishes were incubated in the presence of the inhibitor for various times as indicated. Cells were then washed with 2 ml PBS, lysed into 100 µl CSB and boiled for 3 min. Aliquots (20 µl) were then resolved by SDS gel electrophoresis and analysed by Western blotting. Cells grown on glass coverslips were washed with PBS and then fixed by one of two methods:-a. Cells were fixed in 4% (w/v) paraformaldehyde for 20 min at room temperature. Following fixation, coverslips were rinsed 2-3 times with PBS and left shaking gently for 15 min to remove all traces of paraformaldehyde before subsequent processing.b. Cells were fixed in ice-cold methanol for 1-2 min and then in 1% (w/v) bovine serum albumin in PBS with shaking at room temperature for at least 1 h.For studies on Jurkat T cells, coverslips were first treated with 0.1% (w/v) poly-L-lysine to promote cell attachment prior to cell growth and were then fixed in paraformaldehyde as indicated above.Sections were subsequently processed as described previously  except tin vitro transfected cells  engineered to express both the biotinylating enzyme BirA and a GIMAP construct incorporating a target sequence for this enzyme [r ca. 55,000 compared with ca. 40,000 for biotinylated GIMAP6, suggesting cross-linking of GIMAP6 to a second protein species of approximately 15kD. This 55K band was lost after disruption of cross-links by boiling , we investigated the possibility that induction of autophagy might allow us to detect GIMAP6-GABARAPL2 interaction. However, although starvation of Jurkat-T cells induced autophagy, as assessed by the formation of MAP1LC3B-II from MAP1LC3B-I . After extensive washing, the GST proteins were eluted from the beads with glutathione and the eluates assessed for the presence of associated GABARAPL2. Although GST was expressed at much higher levels than the GST-GIMAP6 fusion protein, whereas no GABARAPL2 was found to elute with GST, it was detected in the GST-GIMAP6 eluates  or a GST-GIMAP6 fusion protein were expressed in  eluates , indicatBoth GIMAP6 and GABARAPL2 are members of multigene families and, in both cases, individual members of these families are predicted to perform related functions. It was therefore important to determine the selectivity of the observed interaction. Using pull-downs from co-transfected HEK293T cell lysates, we first tested the ability of GABARAPL2 to discriminate between mammalian GIMAP family members. Biotin-tagged human GABARAPL2 proved to be highly selective for GIMAP6 amongst a panel of all of the human GIMAP proteins . Next wein vivo situation. Although, as discussed above, endogenous GABARAPL2 could not be co-immunoprecipitated with GIMAP6, preliminary results indicated that endogenous GABARAPL2 could be co-purified with biotinylated GIMAP6 by streptavidin-affinity chromatography from the Biot-GIMAP6-His myc-BirA-Jurkat cell line. We therefore used a panel of antibodies raised to members of the human Atg8 family to determine whether they too could be co-immunoprecipitated. The specificity of all of the antibodies used was first tested against lysates of a panel of HA-tagged Atg8 members transiently over-expressed in HEK293T cells. The antibodies to MAP1LC3A, MAP1LC3C, and GABARAPL2 (the latter prepared in house – see later) reacted only with their expected targets. In contrast, the antibody to MAP1LC3B also reacted with MAP1LC3A, as reported previously [As in this system both the GIMAP and Atg8 family members were over-expressed, we considered the possibility that the weaker interactions observed were being driven by this, rather than reflecting the eviously , and antUsing this panel of antibodies, Western blot analysis of proteins co-purifying with biotinylated GIMAP6 from the Biot-GIMAP6-His myc-BirA-Jurkat cell line indicated that, of the Atg8 family members we could detect in cell lysates , only GABARAPL2 co-purified with GIMAP6 , thus prCommonly, proteins that interact with members of the Atg8 family carry a characteristic Atg8-family interacting motif (AIM), sometimes termed an LC3-interacting region (LIR) see . MoreoveInspection of an alignment of GIMAP6 orthologues from various mammalian species  shows thAlthough these results showed a requirement for the C-terminal region of the protein, when we introduced mutations into the GTPase domain of GIMAP6, we could show that this region too was important. Introduction of mutations into the probable G1 (G50D and S54N), G2 (T76A), or G3 GTPase subdomains (D95A and N98A) prevented GIMAP6-GABARAPL2 interaction , indicatWe then investigated the regions of GABARAPL2 required for its interaction with GIMAP6. Deletion of the N-terminal 10 amino acids of GABARAPL2 was sufficient to prevent the interaction . GABARAPTM-HeLa cell line (Invitrogen) – termed the myc-GIMAP6 T-Rex HeLa cell line. Cells carrying the pcDNA4.TO vector alone expressed no detectable human GIMAP6 and very low levels of GABARAPL2, with or without tetracycline treatment , also expressed higher levels of endogenous GABARAPL2 when compared with cells carrying vector plasmid alone . To addrreatment . Howeverreatment . The increatment , again sreatment .GABARAPL2 mRNA. However, comparison by qPCR of mRNA levels following four days of tetracycline treatment with untreated controls, indicated that whilst GIMAP6 mRNA levels were increased approximately 50-fold, those of GABARAPL2 averaged 1.4-fold  or carrying the corresponding vector. These cells inherently express higher levels of GABARAPL2 than the myc-GIMAP6 T-Rex HeLa cell lines even in the absence of GIMAP6. However, after treatment with emetine for 15 h, no reduction in GABARAPL2 levels was detected even in the absence of GIMAP6 . TreatmeGIMAP6 RNA sequence under the control of a tetracycline-inducible promoter in the mammalian T-RExTM Jurkat cell line (Life Technologies). Of the clones generated, many showed a reduction in the endogenous levels of GIMAP6 protein after treatment with tetracycline for four days  which we have shown fails to interact with GABARAPL2 see . When auAs GIMAP6 is recruited to autophagosomes on starvation, it was of interest to determine whether this recruitment might be associated with GIMAP6 turn-over. We therefore maintained Jurkat T cells in starvation medium for varying lengths of time, and then analysed lysates for levels of GIMAP6, GABARAPL2, MAP1LC3B and β-ACTIN. As expected, starvation stimulated conversion of MAP1LC3B-I to MAP1LC3B-II, indicative of autophagy . The MAPAn unbiased biochemical search for proteins interacting with GIMAP GTPases led us to identify GABARAPL2 as a major binding partner for GIMAP6. GABARAPL2 is a homologue of yeast Atg8, a ubiquitin-like protein involved in cargo recruitment into autophagosomes and in their biogenesis. The best-studied of the mammalian Atg8 homologues is MAP1LC3B and the conversion between its unmodified form (MAP1LC3-I) and its phosphatidylethanolamine-modified form (MAP1LC3-II) constitutes a favoured assay of autophagic activity in cells. in vitro mammalian Golgi transport assay [GABARAPL2 is much less well studied. It was originally reported as a protein involved in intra-Golgi transport . Subsequrt assay , supportThe results we present here show that not only do GIMAP6 and GABARAPL2 interact but also both are recruited to autophagosomes on the induction of autophagy. Among other functions (e.g mediating membrane fusion ) a major These results suggest an important role for the molecular interaction between GIMAP6 and GABARAPL2. However, the precise significance of this molecular relationship remains unclear. One possibility is that GIMAP6 is a passive cargo that GABARAPL2 escorts for degradation via the autophagolysosomal pathway under particular conditions of stress (e.g. starvation), an idea supported by our observation of GIMAP6 degradation during starvation-induced autophagy . Under shttp://biogps.org/?referer=symatlas%20%5C%20goto=genereport&id=121260#goto=welcome), in their expression through T and B cell development [It should be noted that the studies we have reported here suggest that there is no simple role for GIMAP6 in direct modulation of the bulk autophagic pathways. However the findings do indicate a link between the immune system GTPase GIMAP6 and a protein (GABARAPL2) intimately involved in the process of autophagy. This leads one to ask whether engagement in aspects of autophagy or in its regulation might link all of the 7-8 GIMAPs expressed by mammals. Coordinated involvement in a defined intracellular process would be consistent with similarities in the tissue distributions of the GIMAPs  have been ablated conditionally in the T cell lineage. In both cases, intrathymic T cell development is relatively normal while the peripheral T cell population is severely depleted and the remaining T cells [We conclude from this that a more indirect relationship with apoptotic processes could be considered for the GIMAPs. In this respect, autophagic mechanisms are an interesting candidate for their primary functional focus: nutrient salvage via catabolic autophagy clearly plays a pro-survival role in cells, but more specialised roles for autophagy, such as the regulation of mitochondrial number and quality by mitophagy (reviewed by Youle and Narendra ), themse T cells ,48 are u T cells . In the  T cells ,50. Cons T cells , but sho T cells  and it i T cells . The lat T cells .In conclusion we have uncovered a highly specific molecular interaction between a member of the immune-associated GIMAP GTPase family, GIMAP6, and the mammalian Atg8 homologue, GABARAPL2. Further findings demonstrating a re-location of this GTPase to LC3-positive autophagosomes in response to starvation or mTOR inhibition lead us not only to propose a role for this GTPase in autophagy-related processes in lymphocytes but also to speculate more generally on roles in such processes for the other members of the GIMAP family.Figure S1Anti-GIMAP6 monoclonal and polyclonal antibodies are selective for GIMAP6 amongst human GIMAP proteins. Myc-tagged variants of human GIMAP proteins were expressed in HEK293T cells. Lysates were prepared, separated by SDS PAGE and analysed by Western blotting using anti-myc antibody 9E10, or a rat mAb (MAC445) to human GIMAP6 or a rabbit polyclonal antiserum to the same protein.(TIF)Click here for additional data file.Figure S2Characterisation of the specificity of Atg8 family antibodies. Lysates prepared from HEK293-T cells transiently transfected with plasmids encoding N-terminally HA-tagged Atg8 family members were analysed by SDS PAGE and Western blotting, using antibodies either as detailed in the (TIF)Click here for additional data file.Figure S3A 15kDa protein co-purifies with biotinylated GIMAP6 from transiently transfected HEK293Tcells. HEK293T cells were transiently transfected with a plasmid encoding human GIMAP6 in pcDNA3Biot1His6iresBirA or with the corresponding vector as indicated. Lysates were prepared 48 h later and the biotinylated and associated proteins purified using streptavidin-agarose. The purified proteins were separated by SDS PAGE and the gel silver-stained. The electrophoretic mobilities of the purified GIMAP6 and an associated 15kDa protein are indicated. The result shown is representative of two independent experiments.(TIF)Click here for additional data file.Figure S4Endogenous GIMAP6 and GABARAPL2 fail to co-immunoprecipitate. Cell lysates were prepared from Jurkat-T cells which had either been left untreated or starved for 2 h as described in the (TIF)Click here for additional data file.Figure S5Sequence alignment of GIMAP6 protein sequences from various mammalian species. Protein sequences were either taken directly from the NCBI protein database or were deduced from expressed DNA sequence tags or genomic sequences. The conserved AIG1/GTPase domain is boxed in black and the extended C-terminal regions present in most mammals, but absent from mouse, rat and chinese hamster, boxed in red.(TIF)Click here for additional data file.Figure S6Mouse GIMAP6 associates weakly with GABARAPL2. HEK293T cells were transfected with myc-GIMAP6-encoding plasmids with or without GABARAPL2 in pcDNA3Biot1His6iresBirA, as indicated. Lysates were prepared, and biotinylated and associated proteins purified from the lysates using streptavidin-agarose. Western blots of the recovered proteins were probed with HRP-conjugated streptavidin (to show GABARAPL2) or a mouse monoclonal antibody (9E10) to the myc-tag followed by an HRP-conjugated goat anti-mouse IgG (to show GIMAP6). Western blots were developed using Immobilon ECL western blotting substrate. Interpretation of this experiment was complex as mouse GIMAP6 expressed only weakly in our transient assays compared with the human orthologue. However, co-immunoprecipitation of mouse GIMAP6 with GABARAPL2 was demonstrated in two independent experiments.(TIF)Click here for additional data file.Figure S7The mouse anti-myc mAb 9E10 and a rabbit anti-human GIMAP6 polyclonal antiserum show similar intracellular distribution of myc-GIMAP6. Myc-GIMAP6 HEK293 cells were either starved for 90 minutes or left untreated and were then processed for immunocytochemistry, using primary antibodies as indicated. The scale bars indicate 16 µm. The results shown are representative of three independent experiments.(TIF)Click here for additional data file.Figure S8GIMAP6 over-expression does not affect MAP1LC3B-II accumulation. Cell lines  were starved for 2 h (panels A and B) or 1.5h (panel C) or left untreated, with or without treatment with chloroquine as indicated. Cell lysates were prepared and analysed by SDS PAGE and Western blotting with antibodies to MAP1LC3B and ACTIN. Resulting X-ray films were scanned and images analysed using ImageJ software to determine MAP1LC3B-II/ACTIN ratios. Panel D – analysis of MAP1LC3B-II/ACTIN ratios for three experiments on the cells from Panel A. Results are shown as mean ± SD (n=3). The presence of chloroquine is indicated by CQ. One-way ANOVA indicated no significant statistical differences between treatments in the absence or presence of GIMAP6.(TIF)Click here for additional data file.Figure S9GIMAP6 expression does not affect the number of MAP1LC3 or SQSTM1 puncta/cell. myc-GIMAP6 HEK293 cells and the corresponding vector cells were either starved for 90 min or left untreated and subsequently immunocytochemically stained for MAP1LC3B or SQSTM1 as indicated. Typical images are shown in panel A. The scale bars represent 21 µm. Spots were counted . Analysis was performed using Imaris software. Results are presented as spots/cell ± SD – panel B MAP1LC3B; panel C SQSTM1. No significant difference was seen in the number of spots in the absence or presence of GIMAP6. Immunocytochemical results shown are representative of three independent experiments.(TIF)Click here for additional data file.


Danio rerio) as a vertebrate model to analyze estrogen signaling during development. Zebrafish embryos were exposed to 1 µM 17β-estradiol (E2) or vehicle from 3 hours to 4 days post fertilization (dpf), harvested at 1, 2, 3 and 4 dpf, and subjected to RNA extraction for transcriptome analysis using microarrays. Differentially expressed genes by E2-treatment were analyzed with hierarchical clustering followed by biological process and tissue enrichment analysis. Markedly distinct sets of genes were up and down-regulated by E2 at the four different time points. Among these genes, only the well-known estrogenic marker vtg1 was co-regulated at all time points. Despite this, the biological functional categories targeted by E2 were relatively similar throughout zebrafish development. According to knowledge-based tissue enrichment, estrogen responsive genes were clustered mainly in the liver, pancreas and brain. This was in line with the developmental dynamics of estrogen-target tissues that were visualized using transgenic zebrafish containing estrogen responsive elements driving the expression of GFP (Tg(5xERE:GFP)). Finally, the identified embryonic estrogen-responsive genes were compared to already published estrogen-responsive genes identified in male adult zebrafish (Gene Expression Omnibus database). The expressions of a few genes were co-regulated by E2 in both embryonic and adult zebrafish. These could potentially be used as estrogenic biomarkers for exposure to estrogens or estrogenic endocrine disruptors in zebrafish. In conclusion, our data suggests that estrogen effects on early embryonic zebrafish development are stage- and tissue- specific. Estrogen signaling is important for vertebrate embryonic development. Here we have used zebrafish ( Estrogen signaling through its main components cytochrome P450 aromatase CYP19) and the estrogen receptors (ERs), is well conserved through the evolution of vertebrates (reviewed in 9 and theDanio rerio) codes for three estrogen receptors, Esr1, Esr2a and Esr2b  [The genome of the vertebrate zebrafish (ctively) . These rctively) , which eesr2a mRNA is maternally loaded to the oocyte, disappears between 6 and 12 hpf and returns with the start of the zygotic expression after 1 day post fertilization (dpf) [In situ hybridization of esr2a shows that its expression is high in the head/brain region and in proximity to the yolk at 24 to 48 hpf [esr2a mRNA is also expressed during early life stage in the epidermis, pectoral ﬁn buds, hatching gland and is distinctly expressed in neuromast cells of both the anterior and the posterior lateral line [The fetal expression of the different estrogen receptors during zebrafish development is induced at 24 hours post fertilization (hpf) [o 48 hpf ,8. esr2aral line ,9. Althoesr2a efficiently decreases the formation of neuromasts, showing a direct role for esr2a in their development [It is reasonable to surmise that estrogen activity is important for the development of the tissues and organs in which the estrogen receptors are expressed. Consequently, knock down of estrogen receptor expression, or treatment with excess levels of agonists or antagonists would be expected to perturb development of these tissues and organs. In support of this hypothesis, morpholino knock down of elopment . The moselopment  and refeelopment . After pelopment , implicaelopment . Thus, cvtg1 and vtg3), and the brain-specific Aromatase B . These markers have been used to detect estrogenic endocrine disrupters both in laboratory raised zebrafish and in field monitoring of other fish species. To identify additional E2-target genes, studies have been performed in zebrafish using a large-scale transcriptomic approach. In one study, total mRNA from adult male zebrafish was analyzed on a custom-made microarray set containing about 16 K oligonucleotide probes [vtg1, vtg3 and esr1. Three other studies analyzed gene expression changes in liver of adult male zebrafish after E2 treatment using 14-16K microarray platforms [Several biomarkers of estrogenic exposure have been identified in zebrafish, including the liver-produced yolk proteins Vitellogenin 1 and 3  [Tg(ins:mCherry) were used according to the maintenance and experimental protocols approved by the Institutional Animal Care and Use Committee at University of Houston . Adult zebrafish were maintained in 2.5 Liter polyethylene tanks in a Z-MODE holding system from Aquatic Habitat,  or 3.5 liter tanks in a Tecniplast system  supplied continuously with circulating filtered water at 28°C under 14 h of light and 10 h of dark cycle . The fish were fed commercial flake food (Aquatic Habitat) in the morning, baby brine shrimp  at noon and Cyclop-eeze  in the evening during the week. On weekends they were fed baby brine shrimp and Cyclop-eeze.The zebrafish work was conducted according to relevant national and international guidelines. Wild-type strains DZ and TAB14, as well as transgenic fish lines ERE:GFP)  and Tg to obtain 1 μM concentrations. Clutches of zebrafish embryos from several pairs of adult fish were divided and transferred into 6 well plates. 30 embryos were pooled as one biological sample and exposed to 3 ml 1 μM E2 or vehicle (0.1% DMSO) from approximately 3 hpf. Embryo media containing E2 or DMSO were renewed every day. At different time points, 1 dpf (24 hpf), 2 dpf (48 hpf), 3 dpf (72 hpf) and approximately 4 dpf , embryos were collected for analysis. After breeding adult fish from the different fish lines, embryos were collected and allowed to develop in a Petri dish at 28.5°C. 17β-estradiol (E2)  at a 1 mM stock solution in 100% dimethylsulfoxide (DMSO) was diluted in embryo medium  and were maintained at 28°C for 48 h. After the treatment, fish were anesthetized and snap frozen in liquid nitrogen followed by RNA extraction. Total RNA from pooled DZ embryos was extracted using Trizol  and RNeasy spin columns  according to the manufacturer’s protocols. DNase I  digestion was performed to remove remaining DNA. RNA concentrations were measured with NanoDrop 1000 spectrophotometer  and RNA integrity was analyzed with Agilent 2100 Bioanalyzer . cDNA synthesis was carried out using Superscript II reverse transcriptase . Frozen adult fish from E2 treatment and vehicle control (0.1% DMSO) groups were ground to a crude powder using pre-cooled mortars and pestles. Liquid nitrogen was added into the mortars to keep the samples frozen. Crude tissue powder was then transferred to pre-cooled 5 ml sterile centrifuge tubes  containing Trizol . Motorized homogenizer  was used to completely homogenize the fish samples. Total RNA was extracted according to the procedures described above. Agilent zebrafish gene expression microarray v2 (part number G2519F and AMADID (design) 019161) was used for the microarray analysis. Experiments were performed at the Genomic and RNA Profiling Core . The Genomic and RNA Profiling Core first conducted Sample Quality checks using the Nanodrop ND-1000 and Agilent Bioanalyzer Nano chips. For labeling, the Agilent Quick Amp Labeling Kit (for one-color) Protocol Version 6.5 was used. 50ng of total RNA that had passed the quality check was used for the protocol as recommended by Agilent. The Labeling Kit (Agilent p/n 5190-0442) was used along with Agilent’s RNA Spike-In Kit, Agilent’s Hybridization Kit, and Agilent’s Wash Buffers 1 and 2. The RNA Spike-Ins was added to the sample. The sample was simultaneously amplified and Cy3 dye labeled as cRNA was generated using T7 RNA Polymerase. The cRNA was purified using Qiagen RNeasy mini spin columns. Samples were then measured again on the Nanodrop for yield and dye incorporation. The samples were then fragmented and 1.65 µg of sample and hybridization mix was loaded onto each of the 4x44K Expression arrays. The slide was hybridized in Agilent Hybridization Chamber at 65°C at a 10rpm rotation for 17 h. The slide was washed using the Agilent Expression Wash Buffer Set 1 and 2 as per the Agilent protocol. Once dry, the slides were scanned with the Agilent Scanner (G2565BA) using Scanner Version C and Agilent Feature Extraction Software Version 11.0.1.1. Time points 1 and 2 dpf were performed in biological triplicates of independent pools of RNA while time points 3 and 4 dpf were performed in quadruplicates. All biological replicates were prepared from different batches of embryos spawning from different breeding pairs, but from the same fish strain. The microarray results were submitted to Gene Expression Omnibus database (GSE42766).P≤0.05 (*) or P≤0.01 (**).Real-time polymerase chain reaction (PCR) was performed using a 7500 Fast Real-Time PCR  with Fast SYBR Green Master mix . Primer BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) was used to design the primers, which were synthesized by Integrated DNA Technologies, Inc . Primers of 18-22 base pairs (bp) were designed to amplify sequences of 100-300 bp . RelativTg(5×ERE:GFP) embryos were used to follow estrogen responsive tissue development. Hybrid embryos obtained from Tg(5xERE:GFP) crossed with Tg(ins:mCherry) transgenic fish were used to track endocrine pancreas development. Embryo treatments were as described above. Fluorescence in the live embryos was visualized using a Nikon AZ100M microscope equipped with Nikon DS digital camera head and the NIS Elements imaging software . To inhibit pigment formation, embryos and larvae were incubated in 200 μM 1-phenyl-2-thiourea  from 1 dpf. For live imaging, embryos and larvae were anesthetized with 0.04% MS-222 , mounted in 3% methylcellulose on a glass slide and imaged using a 4X objective. Fluorescent images were pseudo-colored, superimposed and adjusted using Adobe Photoshop CS5 . E2 treated Wild type TAB14 embryos were treated with embryo media containing 1 µM E2 or vehicle (0.1% DMSO) using the protocol described above. Embryo media was collected after treatments and E2 levels remaining in the media were analyzed using HPLC (n=3). HPLC analyses were performed on a Binary HPLC pump 1525  equipped with an autosampler 2707 injector and a photodiode array detector (PDA) 2998 set at 280nm (Waters). The HPLC system was based on a Nucleodur C18 column  in the following conditions: mobile phases: A: 20 mM ammonium acetate pH 3.5/acetonitrile , B: 100% acetonitrile; Gradient: 0–2 min, A: 100% isocratic; 2-3 min, linear gradient from A: 100% to A:B 60:40; 3–8 min, A:B 60:40 isocratic; 8–9 min, linear gradient from A:B 60:40 to A:B: 50:50; 9-14 min, A:B 50:50 isocratic; 14–15 min, linear gradient from A:B 50:50 to B 100%; 15–19 min, B: 100% isocratic. In our system, E2 was eluted at a retention time of 11.5 min, and it was quantified by measuring the area under the peak (based on a standard curve previously established). in situ hybridization were performed as described previously [vtg4 (630 bp) was amplified by PCR , 72 °C for 5 min) from cDNA that was prepared from total RNA extracted from 1-month old adult male zebrafish treated with E2 (as described above) using primers forward GATCAATTAACCCTCACTAAAGGCCTATCATCGCCCGTGCTGTT-3’5’- and reverse GATCTAATACGACTCACTATAGGACAGTTCTGCATCAACACATCT-3’5’-  for sense and antisense probes. These primers were designed to contain the T3 (forward primer) and T7 (reverse primer) promoter regions for sense and antisense transcripts, respectively. The promoter regions in the primers are underlined. Because of low PCR yield, the fragment was cloned into pGEM-T-Easy vector  and re-amplified. After PCR amplification, digoxigenin-labeled  antisense and sense transcripts were transcribed using T7  and T3  RNA polymerase, respectively. Following in situ hybridization, embryos were cleared in benzyl alcohol:benzyl benzoate (BABB) 2:1 and mounted in modified GMM mounting media  and photographed on a Nikon AZ100M microscope equipped with a Nikon DS-Fi1 camera.All procedures of whole-mount eviously . Partial2-transformed. Batch effects from the different biological replicates were removed using Partek Genomics Suite v 6.3 (http://www.partek.com/) and residual variance was analyzed by Principal Components Analysis (PCA) (P≤0.01) was used to identify genes altered by treatments at individual developmental stages. Venn diagrams were generated to illustrate the overlapping genes among the four different time points . For the Hierarchical clustering, unsupervised hierarchical clustering was performed using Pearson correlation algorithm for the gene tree and Spearman for the developmental stages (P≤0.005).Raw data from the microarray analysis was mean-centered and quantile-normalized to normalize gene expression distributions across the different samples. The data was then Logis (PCA) . Then thP≤0.01, fold change ≥|±1.4|) were identified using ZFIN (http://zfin.org/) and Ensembl (http://www.ensembl.org). Gene ontology (GO) annotation biological processes enrichment of the estrogen responsive human homologues was performed by using Pathway Studio . Fisher’s Exact test was used to calculate the p-value of each functional categories; P≤0.05 was considered significant. The corresponding human homologues to the differentially expressed zebrafish genes . Fisher’s Exact test was used to calculate the 2-normalized ratios for all samples were downloaded and One-way ANOVA was used for the statistical analysis of the treatment groups and control groups. Benjamini-Hochberg false discovery rate correction (FDR) was applied to the raw p-value. FDR q-value ≤0.01 and fold change ≥|±2.0 | were chosen as a cut off for the differentially expressed genes. Data sets of estrogen-induced gene expression in male adult zebrafish were obtained from the GEO database  (GEO accession # GSE27707). Series matrix.txt files with the logvtg1 and esr1 by qPCR. Wild type zebrafish embryos were treated with E2 at concentrations ranging from 0.01 nM to 1 μM from 3 hpf to 4 dpf with daily media exchange. We have previously shown that 1 μM E2 is the highest concentration that zebrafish embryos can tolerate without showing obvious phenotypic abnormalities [vtg1 and esr1 expression were significantly induced by E2 treatment at 100 nM and maximally induced at 1 μM   (Gene List in ≥|±1.4|) . The expP≤0.005) was performed using the Pearson correlation algorithm. For this clustering we used a P-value cutoff at P≤0.005 at which a clear developmental pattern of the differentially expressed genes was evident  we only detected the up-regulation of vtg3 by RT-qPCR and not by microarray  enrichment analysis was performed. Due to the lack of zebrafish pathway analysis databases, the human homologues to the zebrafish estrogen-responsive genes were identified through the ZFIN (http://zfin.org/) and Ensembl zebrafish Zv9 databases (http://www.ensembl.org/Danio_rerio/), followed by GO enrichment using Pathway Studio mammalian database. Approximately half of the annotated differentially regulated genes were successfully identified with human homologues (64 (47.06%) at 1 dpf, 68 (65.38%) at 2 dpf, 374 (64.93%) at 3 dpf and 112 (54.90%) at 4 dpf). The human homologues to the 15 most up- and down-regulated estrogen-responsive genes at different time points are shown in GO annotation analysis based on the human homologues revealed a significant enrichment in several biological processes after E2 exposure at the four time points . The broin situ hybridization using an anti-sense RNA probe of vtg4, which was the most highly E2-activated gene in the whole microarray, activated 1,218 times at 4 dpf . E2-responsive genes lists were uploaded to NIH DAVID website, followed by tissue enrichment analysis based on previously published tissue-specific gene expression information in ZFIN public database. Enriched tissue categories from the E2-responsive genes represent putative estrogen-responsive tissues. The numbers of genes identified by NIH DAVID and enriched using ZFIN_Anatomy tissue-specific analysis tool are listed in at 4 dpf . Vtg4 RNTg(5xERE:GFP), expressing GFP driven by 5×ERE, to visualize the developmental dynamics of E2-responsive tissues [Tg(5xERE:GFP) fish with wild type DZ fish. The embryos from female Tg(5xERE:GFP) fish crossed with male wild type DZ fish expressed a similar fluorescence signal to the homozygous Tg(5xERE:GFP) embryos (Tg(5xERE:GFP) fish with female DZ fish, no fluorescence was detected in the embryos (Tg(5xERE:GFP) embryos represented maternal load of GFP expression activated by endogenous estrogens in the female fish. At 1 dpf, the maternal GFP fluorescence had faded and the zygotic GFP expression appeared mainly in the head region after E2 treatment but not in untreated embryos (Tg(5xERE:GFP) with Tg(ins:mCherry) transgenic fish, the latter expressing mCherry driven by insulin promoter. The embryos were treated with E2 and fluorescence was observed daily. From 2 dpf to 6dpf, a co-localization of mCherry and GFP fluorescence was observed in the pancreatic islets  transgenic fish after E2 induction  and estrogen responsive genes in male adult fish   , phosphorylation , tissues development , immune system , and synapse function   gene transcript was fkbp5, an immunophilin involved in protein folding and trafficking (cyp19a1b gene showed the most highly activated transcription following E2 treatment (35 fold)  and ubiquitin-related genes (ubn2 and exosc6). The top down-regulated transcripts at 3 dpf were two fatty acid binding proteins (fabp10 and fabp6), and other down-regulated genes encoded proteins involved in transport, including the heme transporter hpx, the ammonium transporters rhcga and rhcgb, and slc6a, which transport amino acids in the kidney. Finally, the most up-regulated transcripts at 4 dpf were clearly the vtgs 4, 3, 1, 2 and 5  (pfkfb4 (9 fold), a hypoxic induced kinase/phosphatase, and upp2 (6 fold), which is a uridine phosphatase. The fold changes of E2-altered genes were much higher at 4 dpf than the other three time points, which might be caused by the higher E2 levels in the embryos  . At 2 dpfficking . At 3 dp35 fold) . Other actively) . The mos embryos . vtg1, vtg3, vtg5, esr1, and cyp19a1b were found to be E2-target genes in our study, confirming that our results are in line with previously published results [vtg1 and vtg3 was not only up-regulated by E2 at embryonic stages but also in male adult fish, as published previously  was highly up-regulated by E2 treatment in 2, 3, 4 dpf embryos as well as male adults (ependymin (epd), encoding a brain extracellular glycoprotein involved in memory and neuronal regeneration (further discussed below), was down-regulated by E2 at three time points. Transcription of anti-Mullerian hormone (amh), encoding a steroidogenic enzyme, was induced at three time points. In previous studies, amh was reported to be induced in both prepubertal and adult rats exposed to methoxychlor, a previously commonly used pesticide, now banned in the US because of its estrogenic disrupting properties [regulator of G-protein signaling 2 (rgs2) and hemopexin (hpx) were also highly altered by E2 treatment, as described above. HPX protein levels are reportedly reduced in estrogen-treated menopausal women [hpx transcripts after E2 treatment in our study. Genes that were regulated at two embryonic time points include ankyrin repeat domain 9 (ankrd9), FK506 binding protein 5 (fkbp5), fatty acid binding protein 10a (fabp10a), purine nucleoside phosphorylase 4b (pnp4b), proopiomelanocortin a (pomca) and dopa decarboxylase (ddc)  . Furtherf13a1a and cyp19a1b, were missed in the microarray comparisons, but identified by RT-qPCR, which could be explained by that the two microarray platforms were designed from different companies and contained different number of probes (16K vs. 44K) [vtg1, vtg3, and esr1, as well as the new markers f13a1a and cpn1, could potentially serve as biomarkers of estrogenic exposure to both embryos and adult fish.Comparison of the E2 target genes between embryonic and adult fish showed that the expression of a few genes were co-regulated, which indicates that the E2-regulated genes identified in our microarray analysis might be development specific. However, some co-regulated genes, such as Tg(5xERE:GFP) transgenic fish (Tg(5xERE:GFP) fish [Putative estrogen target tissues, in which E2-responsive genes have been reported to be expressed, were predicted by using ZFIN anatomy functional analysis at NIH DAVID bioinformatics platform . DiffereFP) fish ,27. Suppfabp10a, pnp4b, hpx, rgs2, and the vtgs, was the liver (Tg(5xERE:GFP) transgenic fish, liver expression of GFP can be detected after 35 hpf, which is in agreement with in situ hybridization of ceruloplasmin of developing liver [Tg [vtgs and esr1. We found that the most highly up-regulated gene at 4 dpf was vtg4, which was predicted to be expressed in the liver at the same time point  . Consistme point . We alsodization . As a melt liver ,30; howeinsulin (ins), ghrelin/obestatin preprohormone (ghrl1) and glucagon a (gcga) were E2-regulated in the pancreas (Tg(5xERE:GFP) transgenic fish (Tg(ins:mCherry)/Tg(5xERE:GFP) embryos after E2 treatment  fish ([-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate (AMPA) type glutamate receptors were identified as E2 target genes. These receptors mediate the majority of fast synaptic glutamate transmissions, which are known to promote neuronal growth, retraction and elongation of glial processes, proliferation and differentiation of retinal progenitors and proliferation of cortical progenitors. Specifically, gria2a, gria3a, gria4a glutamate receptor subunits were regulated by E2. The AMPA receptors have previously been shown to be regulated by estrogen in rats and mice [epd transcript, encoding a protein involved in neuronal plasticity, neurobehavior (memory and aggression) and cold adaptation, and the neurobehavioral disc1 transcript were also regulated by E2 treatment in embryonic zebrafish brain. The disc1 gene has been associated with risk of schizophrenia, bipolar affective disorder and major depression [cyp19a1b (aromatase B) was found to be regulated by estrogen in the brain at 2-4 dpf. Although in the tissue clustering, this gene was designated to several tissues, including liver [cyp19a1b has been reported to be brain-specific in zebrafish while cyp19a1a encodes aromatase in the ovary [cyp19a1b mRNA in adult fish liver extracts by RT-qPCR (results not shown). More specifically, expression of cyp19a1b has been shown to be up-regulated by E2 in zebrafish radial glial cells [Estrogen receptors, in particular ERβ (ESR2), are important for normal brain and behavior development in rodents. Whereas ERα (ESR1) is the predominant ER in the hypothalamus, controlling reproductive cycles, ERβ is expressed in the cerebral cortex, the hippocampus, the cerebellum and the dorsal raphe (reviewed in P) fish , fatty acid binding protein 6 (fabp6), and the solute carrier family members slc2a11l, slc13a1, slc26a6l and slc20a1a. Cyp1b1 has been detected in the developing kidney during early murine development [cyp1b1 dependent [slc2a11l (glucose transporter), slc13a1 (sodium/sulphate transporter), slc26a6l (anion transporter) and slc20a1a (phosphate transporter), one of the biological functional groups that were regulated by E2 was “Transport”, as discussed below.A group of estrogen responsive genes were predicted to be expressed in kidney or pronephric duct, including the cytochrome P450 elopment . It metaelopment . Renal dependent . In lineTg(5xERE:GFP) transgenic fish (data not shown). Normal morphogenesis of ovary and testis does not initiate until 10 dpf [amh may be altered by E2 treatment or play other roles at earlier stages of zebrafish development. In addition, Tg(5xERE:GFP) fish showed weak GFP expression in the heart valves from 4 dpf, which is in agreement with observations from the Tg fish [esr2a for their development [bdnf expression in mammals. In conclusion, estrogen-responsive gene transcripts were predicted to be expressed in various tissues according to knowledge-based tissue enrichment; many of these tissues are in concordance with the ones that have been identified by transgenic estrogen reporter fish [Besides liver, brain, pancreas and kidney, the predicted tissue enrichment in l 10 dpf ,43, but FP) fish ,27. Howeelopment . Expresselopment , was up-ter fish ,27, but Although the estrogen-responsive genes during early zebrafish development were expressed in a time-dependent manner, the biological functional processes were fairly similar across all time points. Analysis by GO-term biological processes enrichment for E2 regulated genes identified metabolic processes, regulation of transcription, transport, signal transduction, phosphorylation, development and immune response to be significantly enriched at all time points . More suCYP19A1, encoding aromatase B (zebrafish homologue cyp19a1b), sulfotransferase SULT1E1 (zebrafish homologue sult2st3) and hydroxysteroid (17β) dehydrogenase HSD17B (zebrafish homologue hsd17b) were all differentially expressed in our study; expression of cyp19a1b and sult2st3 were up-regulated while hsd17b was down-regulated by E2 treatment. For lipid metabolism, lipc (encoding hepatic lipase) was down-regulated, while apolipoprotein A-I (encoded by apoa), which is the major protein component of high density lipoprotein (HDL) in plasma, was up-regulated by E2 treatment. Studies of humans and primates confirm the regulation of LIPC and APOA-1 by E2. First, E2 is known to repress the transcription expression of LIPC in humans, which plays an important role in lowering the plasma level of HDL [ESR1 [apoa1 is induced by E2 in the human hepatoma cell line HepG2 [In line with the liver being one of the major tissue targets for estrogenic signaling, E2-target genes were enriched in the metabolism category. The genes in this category encode both metabolic enzymes in the steroid and hormone pathways, as well as enzymes involved in lipid, nucleic acid, carbohydrate, protein, xenobiotic and energy reserve metabolism . The tral of HDL ,46, a fuDL [ESR1 . Second,ne HepG2 . Furtherne HepG2 . Finallyne HepG2 . slc2a1 (glucose transporter), slc8a1 , slc5a5 (sodium iodide symporter) and slc34a2 (sodium phosphate transporter). Expression of slc2a1 has been shown to be stimulated by E2 to increase glucose uptake in various tissues [slc34a2 mRNA expression is increased by 50% in rat intestine after E2 treatment [slc8a1 was reported to be up-regulated by E2 in hearts [slc5a5 was up-regulated by E2 treatment in mammary gland [Another GO biological category that was enriched in our analysis was transport, including ion transport , lipid transport, protein transport, and carbohydrate transport (including glucose transport)  and S9.  tissues -52 and sreatment . Expressn hearts  and slc5ry gland ,56 and bry gland ,58. HoweESR1 and ESR2 , as well as the membrane bound estrogen receptor (GPER). Signaling through these receptors has been shown to crosstalk with other receptors or transcription factors. The subcategories that were enriched in our GO analysis included steroid hormone mediated signaling pathway, cell surface receptor linked signaling pathway, as well as MAPK pathway (growth hormone releasing hormone receptor (ghrhr), retinoid X receptor α (rxra), esr1, progesterone receptor (pgr), and nuclear receptor subfamily 0b2a, (nr0b2shp), which were up-regulated by E2, and aryl hydrocarbon receptor (ahr), retinoic acid receptor α (rara), nuclear receptor subfamily 5a2 (nr5a2 lrh), luteinizing hormone/choriogonadotropin receptor (lhcgr), glucagon a (gcg), and androgen receptor(ar), which were down-regulated. It has been extensively reported that E2 induces pgr expression, which is in agreement with our data. Both esr1 and esr2 bind to the pgr promoter, and esr1 has been described to induce pgr expression [esr2 has been reported to reduce pgr expression [E2 treatment also regulated gene transcripts that were enriched in the signal transduction and transcription factor categories  and S10, pathway . Expresspression ,60. Howepression  and an ipression . AHR sigpression . Expresspression -66, but pression . Given tpression , the dowcysteine-rich angiogenic inducer 61 (cyr61), B-cell lymphoma 2 (bcl2) and caspase 3 (casp3). Expression of cyr61 has been reported to be induced by estrogen in breast cancer cells [cyr61 expression in our study. Expression of bcl2 was down-regulated in our study, but previous reports have shown both induction and repression of bcl-2 expression by estrogen, potentially in a tissue specific manner [casp3, a death protease activated during apoptosis, was also down-regulated during zebrafish development following E2 treatment. It has been shown that E2 at neuroprotective doses blocks casp3 activation in the hippocampal CA1 of male gerbils [casp3 [cyclin-dependent kinase 5 regulatory subunit 1a  [protein tyrosine phosphatase receptor type C (ptprc) [E2 also altered the expression of genes involved in cell proliferation and apoptosis, such as er cells ,70 and hc manner -75. Expr gerbils . In fetas [casp3 . In accok5, p35)  and prot (ptprc)  was up-rdual specificity phosphatase 3 (dusp3), which has been shown to dephosphorylate and inactivate various MAPKs like ERK and JNK [protein phosphatase 1, regulatory (inhibitor) subunit 1C (ppp1r1c). Finally, the category immune response was also enriched amongst E2-regulated gene transcripts (interferon-gamma (ifng1-1), a vital immunoregulatory cytokine, was up-regulated, which is in agreement with previous studies that reported an increase of IFNG secretion following estrogen treatment in mice and rats [tnfrsf1) was down-regulated in zebrafish embryos after E2 treatment, which is consistent with a study showing that E2 inhibits TNFR1 expression in breast adipose fibroblasts [Our data identified several phosphatase genes that are regulated by E2. Such genes include  and JNK ,80, and nscripts . In our and rats ,82. Bothand rats -85. Tumoroblasts . Tg(5xERE:GFP) transgenic fish. However, Tg(5xERE:GFP) fish cannot mirror all E2-responsive tissues since the GFP expression is ERE driven. Our study revealed E2 responsive genes independently of whether E2 targeted esr1, 2a or 2b or gper. The new target genes may potentially play important roles for estrogen-mediated regulation of development and some may serve as biomarkers to score for endocrine disruption. To further the knowledge of how estrogen regulates embryonic development, or the impact of perturbed estrogen signaling by exposure to estrogen disrupting compounds, it will be necessary to map estrogen receptor type specific responses through selective estrogen modulators and knockdown/knockout of Esr1, 2a, 2b or Gper. In conclusion, our data reveal distinct differences in the cohort of E2-responsive genes across different developmental stages in the zebrafish. Tissue enrichment analysis of E2-responsive genes correlated to tissue-specific GFP expression of Figure S1Principle components analysis of microarray samples. (A) Untreated samples (vehicle only 0.1% DMSO). (B) Samples treated with E2. (TIF)Click here for additional data file.Figure S2Uptake of E2 in embryos at different time points. Thirty wild type zebrafish embryos were pooled and treated with 1 μM E2 (in 0.1% DMSO) from 3 hpf. The media was collected every 24 hours at 1, 2, 3 and 4 dpf, and the amount of E2 remaining in the media was analyzed by UV-HPLC. The data is presented as the percentage of E2 remaining in the water relative to media incubated with E2 but without fish. Statistics were done using Student’s t-test. *P<0.05; **P<0.01; ***P<0.005; ns, not significant. (TIF)Click here for additional data file.Figure S3Correlation analysis of gene expression data from RT-qPCR and microarray experiments. The relative fold change values of each gene at each time point from Figure 4 were used for the correlation analysis. The microarray data (Y axis) were plotted against the RT-qPCR data (X axis).(TIF)Click here for additional data file.Figure S418S rRNA expression of 1-4 dpf embryos and adult fish upon E2 treatment (relative to DMSO treatment).(TIF)Click here for additional data file.Figure S5vtg4 in the liver of 4 dpf DZ zebrafish embryos.E2 up-regulates expression of  Whole-mount ISH was performed with anti-sense vtg4 RNA probes on 4 dpf E2-treated embryos (A) and DMSO-treated embryos (B). ISH of sense vtg4 RNA probes on E2-treated embryos (C) and DMSO-treated embryos were performed as controls. Lateral view; anterior to the left. Arrow (red) indicates expression location of vtg4 in the liver. Scalebars, 200 μm. (TIF)Click here for additional data file.Figure S6Tg(5xERE:GFP) transgenic fish at 5 hpf in the absence of E2.Maternal effect of   Tg(5xERE:GFP) transgenic fish embryos.  Embryos from cross of female Tg(5xERE:GFP) transgenic fish and male wild type DZ fish.  Embryos from cross of male Tg(5xERE:GFP) transgenic fish and female wild type DZ fish.  Wild type DZ fish embryos. A, C, E and G, bright-field images; B, D, F and H corresponding GFP fluorescence images; Scalebars, 500 μm.(TIF)Click here for additional data file.Figure S7Endocrine pancreas is a novel E2 responsive tissue in embryonic zebrafish. Double transgenic Tg(5xERE:GFP)/Tg(ins:mCherry) embryos (4dpf) showing co-localization of GFP and mCherry signals in pancreatic islets upon E2 treatment. (A) GFP fluorescence image; (B) mCherry fluorescence image; (C) merged image of GFP and mCherry; (D) merged image of bright field (BF), GFP and mCherry. Arrows indicate the liver; arrowheads indicate the pancreatic islets. Dorsal view; anterior to the left. Scalebars, 100 μm.(TIF)Click here for additional data file.File S1List of genes with E2-altered expression at 1, 2, 3 and 4 dpf.(XLSX)Click here for additional data file.Table S1Primer sequences used for the RT-qPCR validation of estrogen responsive genes from the microarray.(DOCX)Click here for additional data file.Table S2Number of differentially expressed genes at different developmental stages.(DOCX)Click here for additional data file.Table S3Top 15 up- and down-regulated transcripts at 1 dpf upon E2 treatment (E2 vs control).(DOCX)Click here for additional data file.Table S4Top 15 up- and down-regulated transcripts at 2 dpf upon E2 treatment (E2 vs control).(DOCX)Click here for additional data file.Table S5Top 15 up- and down-regulated transcripts at 3 dpf upon E2 treatment (E2 vs control).(DOCX)Click here for additional data file.Table S6Top 15 up- and down-regulated transcripts at 4 dpf upon E2 treatment (E2 vs control).(DOCX)Click here for additional data file.Table S7Common estrogen responsive genes at 1 dpf, 2 dpf, 3 dpf and 4 dpf upon E2 treatment.(DOCX)Click here for additional data file.Table S8GO terms sub-grouped into the metabolic process category .(DOCX)Click here for additional data file.Table S9GO terms sub-grouped into the transport category .(DOCX)Click here for additional data file.Table S10GO terms sub-grouped into the signaling pathways category .(DOCX)Click here for additional data file.Table S11GO terms sub-grouped into the multicellular organismal development category .(DOCX)Click here for additional data file.Table S12GO terms sub-grouped into the response to chemical stimulus category .(DOCX)Click here for additional data file.Table S13Number of genes enriched by the NIH-DAVID tissue enrichment platform.(DOCX)Click here for additional data file.


Escherichia coli sequence type (ST)131 is an emerging disseminated public health threat implicated in multidrug-resistant extraintestinal infections worldwide. Although the majority of ST131 isolates belong to O25b:H4 serotype, new variants with different serotypes, STs using the discriminative multilocus sequence typing scheme of Pasteur Institute, and virulence-gene profiles (virotypes) have been reported with unknown implications on the pattern of spread, persistence and virulence. The aim of the present study was to compare virulence in a mouse subcutaneous sepsis model of representative ST131 clinical isolates belonging to 2 serotypes  and nine virotypes and subtypes . Fourteen out of the 23 ST131 isolates tested (61%) killed 90 to 100% of mice challenged, and 18 of 23 (78%) at least 50%. Interestingly, different virulence patterns in association with virotypes were observed, from highly rapid lethality (death in less than 24 h) to low final lethality (death at 7 days) but with presence of an acute inflammation. This is the first study to assess virulence of ST131 isolates belonging to serotype O16:H5, which exhibited virotype C. In spite of their low virulence-gene score, O16:H5 isolates did not show significant differences in final lethality compared with highly virulent O25b:H4 isolates of virotypes A, B and C, but killed mice less rapidly. Significant differences were found, however, between virotypes A, B, C  and virotypes D, E. Particularly unexpected was the low lethality of the newly assigned virotype E taking into account that it exhibited high virulence-gene score, and the same clonotype H30 as highly virulent O25b:H4 isolates of virotypes A, B and C. In vivo virulence diversity reported in this study would reflect the genetic variability within ST131 clonal group evidenced by molecular typing. Escherichia coli O25:H4 sequence type (ST)131, associated with the CTX-M-15 extended-spectrum β-lactamase (ESBL), has emerged as a multidrug-resistant pathogen reported internationally Different authors have already described the heterogeneity within the clonal group ST131, not only on the basis of the virulence-gene content, the variety of ESBL enzymes produced, antibiotic resistance and pulsed-field gel electrophoresis (PFGE) profiles, but also for the number of reservoirs from which it has been isolated In vivo studies have reported that the great majority of E. coli isolates belonging to the genetic group B2 are highly virulent in a sepsis mouse model E. coliE. coli clinical isolates belonging to O25b:H4 and O16:H5 serotypes, and representative of nine ST131 virotypes and subtypes .To get more insight into the virulence potential of clonal group ST131, and considering its heterogeneity, we used a mouse subcutaneous sepsis model All animal experimentation was conducted following European  and National (RD 53/2013) regulations for transport, housing and care of laboratory animals. The protocol used was approved by the Animal Welfare Committe of the Veterinary Faculty in Lugo, University of Santiago de Compostela (AE-LU-002/12/INV MED.02/OUTROS 04).ad libitum. All efforts were made to minimize suffering. After inoculation, mice were monitored and clinically inspected several times day and night within 1 week. Surviving mice were euthanatized on day 7 by cervical dislocation.Female RjOrl:Swiss mice  purchased from Janvier Labs  were housed under standard conditions with water and food supplied E. coli isolates used in this study included 23 ST131 Spanish human extraintestinal clinical isolates, mainly recovered from urinary tract infections (UTIs) or bacteremia, plus the commensal derived strain K-12 MG1655 (O16-A-ST98) and the urosepsis strain CFT073 (O6-B2-ST73) as negative and positive controls, respectively, for the murine infection The 25 adk, fumC, gyrB, icd, mdh, purA and recA) (http://mlst.ucc.ie/mlst/dbs/Ecoli), and XbaI-PFGE profiles as referenced previously MT78,fimH, fimAv ISL3-like in fimB, F10 papA, papAH, papC, papEF , sfa/focDE (only positive isolates are tested for sfaS and focG), afa/draBC, afa operon FM955459, iha, bmaE, gafD, sat, cdtB, cnf1, hlyA, iucD, iutA, iroN, fyuA, chuA, kpsM II , kpsM III, cvaC, iss, traT, ibeA, malX (PAI), usp, tsh, and ompT. Isolates were classified as ExPEC if they carried ≥2 of papEF (P fimbriae), sfa/focDE (S/F1C fimbriae), afa/draBC (Afa/Dr adhesins), iutA (aerobactin receptor), and kpsM II (group 2 capsule synthesis) The selection of the 23 representative ST131 isolates was performed based on the virotypes, which had been previously described dinB, icdA, pabB, polB, putP, trpA, trpB and uidA) by use of the protocol and primers specified at the Pasteur Institute website (http://www.pasteur.fr/recherche/genopole/PF8/mlst/EColi.html). The remaining 5 isolates had already been characterized Eighteen of 23 isolates were characterized by gene amplification and sequencing of the eight housekeeping genes  was amplified and sequenced to define clonotypes as described by Weissman et al. A 489-nucleotide internal fragment of PFGE profiles were analyzed with the BioNumerics fingerprinting software . Cluster analysis of the Dice similarity indices based on the unweighted-pair group method using average linkages (UPGMA) was done to generate a dendrogram describing the relationship among the PFGE profiles.8 CFU of log-phase bacteria. Previously, the isolates were isolated in LB agar (Invitrogen) (22 h) and then one colony was incubated with shaking (180 r.p.m.) at 37°C in 10 mL of Luria Broth (Invitrogen) (24 h). Cultures were centrifuged at 3,500 r.p.m for 15 min, washed twice with saline solution, and resuspended in saline solution again. The absorbance of suspensions were measured at 450 nm and adjusted to 1.4. These adjusted suspensions were used to inoculate the mice subcutaneously. After inoculation, the mice (which had free access to food and water) were observed for up to 1 week. Time of death and local presence of lesions (acute inflammation in the region of inoculation) were recorded for each mouse. Surviving mice were euthanatized on day 7 and their status were also recorded. In each lethality assay, two control isolates were included: K-12 MG1655 isolate, which does not kill mice by 7 days postchallenge, and CFT073 isolate which shows a lethality of ≥80% by 7 days postchallenge A mouse sepsis model was used to assess the intrinsic extraintestinal virulence of the 23 ST131 human clinical isolates as described elsewhere P value of <0.05.Comparisons of proportions were tested using Fisheŕs exact test. The criterion for statistical significance was set at a E. coli studied strains and 48 columns corresponding to 48 variables: the serotypes (O25b:H4 and O16:H5), the Pasteur Institute sequence types, the 9 virotypes and subtypes, the lethality in the mouse model of 10 mice in less than 24 hours, the lethality in the mouse model according to 3 categories , the local presence of lesion (acute inflammation) in at least 5 mice, the high (≥18) or low (<17) virulence score, and the ExPEC-associated virulence genes detected in isolates. For each isolate, each variable was coded as present = 1, absent = 0.A FAC was used to describe associations among the data According to the Pasteur Institute MLST scheme, the 23 ST131 isolates showed 7 different sequence types (identified as PST) . O25b:H43-like transposase gene in fimB was present in O25b:H4-ST131 isolates of virotypes A, B, C and E, but in none of virotype D (independently of the subtype). The fimB insertion was not detected in the four O16:H5-ST131 isolates either . The four O16:H5-ST131 isolates of virotype C carried fimH41  than ST131 isolates of serotype O16:H5 . Besides, 3 of 4 O16:H5-ST131 isolates and 1 O25b:H4-ST131 isolate of virotype D4 did not show ExPEC status .XbaI-digested ST131 isolates showed high heterogeneity (<65% identity). Six clusters of similarity >85% grouped O25b-ST131 (2 isolates each) of virotypes A, B, C, D and E. Isolates of subtypes D1 to D5 disaggregated in different branches of the dendrogram. The 4 O16:H5-ST131 isolates remained grouped in the dendrogram with 76.7% identity  versus none of 15 (0%) mice challenged with MG1655.In the mouse sepsis model, within the 7 dayś experiment, each 4 or all 5 mice challenged with positive-control isolate CFT703 died, compared with none of 5 mice challenged with negative control isolate MG1655, which remained completely healthy. The lethality results observed for CFT703 and MG1655 isolates were similar within the 3 test lots in which the 23 ST131 isolates were assayed. Thus, fourteen of 15 (93%) mice challenged with CFT703 died  and the inflammation-causing ability of the isolates. Furthermore, variability was observed even within certain virotypes ; and isolates of virotype E showed the lowest final lethality.Overall, 14 out of the 23 ST131 isolates tested (61%) killed 90 to 100% of mice challenged, and 18 of 23 isolates (78%) at least 50% of mice. Different virulence patterns were observed in the number of total deaths , the rapidity  was shown by both O25b:H4 isolates of virotype A, and 1 O25b:H4 isolate of virotypes B and C, respectively.Rapid lethality  and C (O16:H5) induced an acute inflammatory response in mice, in the neck region. This finding was recorded as number of mice with presence of lesions . And conP<0.01).Although no significant difference in lethality was found between highly virulent ST131 O25b:H4 isolates of virotypes A, B, C and O16:H5 isolates of virotype C, the latter killed mice less rapidly. Significant differences were found, however, between isolates of virotypes A, B, C  and isolates of virotypes D, E  on the positive values with the virotypes E and D4, the PSTs 621 and 527, and the virulence genes papGII, neuC and tsh  and to nine virotypes and subtypes  for extraintestinal virulence in a murine sepsis model, we have found a wide range of mouse virulence patterns with association of certain virotypes.In the assessment of 23 ST131 E. coli concluded that ST131 isolates are neither uniformly virulent nor, as a group, discernibly more virulent than other extraintestinal E. coli isolates. In that study, even 8 out of the 27 ST131 isolates (30%) did not kill any mouse within 7 days  versus 12 of 27 (44%) in the study of Johnson et al. P = 0.01). These discrepancies observed in the mouse sepsis model between the two studies could be due to the differences within both ST131 collections. Thus, only 5 of the 27 ST131 isolates (19%) of Johnson et al. versus 18 of 23 (78%) of the present study (P<0.01).In a preliminary study of virulence in murine sepsis model, Clermont et al. h) in repeated assays (data not shown). In fact, isolates of virotype A killed even more rapidly than positive control CFT073 (60% of mice died within 24h). On the contrary, O25b:H4 isolates of virotype D exhibited variable virulence patterns even within the subtypes D2, D3 and D4, and O25b:H4 isolates of virotype E showed a pattern of slow and low lethality with inflammation-causing ability (presence of lesion). This in vivo virulence diversity within ST131 clonal group would reflect the genetic variability evidenced by molecular typing of the housekeeping genes that defines PSTs, the fimH subtyping that defines clonotypes, and the PFGE profiles obtained from XbaI-digested DNA. Thus, we found that O25b:H4 isolates of virotypes A, B and C showed the same ST (PST43), same fimH30 subtype and their PFGE patterns grouped together in dendrogram (75.8% similarity). O25b:H4 isolates of virotype D belonged to three different STs , two fimH subtypes (22 and 324) and distributed in distant branches of the dendrogram. O25b:H4 isolates of virotype E belonged to PST621, showed fimH30 and grouped with O25b:H4 D4 isolates (72.8% similarity). And finally, O16:H5 isolates of virotype C showed specific STs , specific fimH41 and remained grouped with 76.7% identity.Interestingly, we have observed virulence patterns associated to certain virotypes. ST131 isolates within virotypes A, B and C showed uniform results with high scores of final lethality (≥80%), being both O25b:H4 isolates of virotype A those with more rapid lethality (100% of mice died within 24fumC and fimHTR sequences) is an important tool for the discrimination of sublineages within extraintestinal pathogenic E. coli clones. Furthermore, a recent study reports that specific clonotypes correlate with antimicrobial susceptibility profiles and clinical outcomes fimH subtypes correlated with serotype and virotype: clonotypes H22, H324 (O25b:H4 virotype D), H30  and H41 (O16:H5 virotype C). H30 clonotype, which comprises almost all current fluoroquinolone-resistant ST131 isolates, has expanded more extensively than other ST131 variants fimH30). Surprisingly, O25b:H4 isolates of virotype E belonging to clonotype H30 and with high virulence-gene score showed low lethality in the mouse model, and differently to virotypes A, B and C, O25b:H4 isolates of virotype E show PST621. Therefore, O25b:H4 of virotype E would represent a subclone within clonotype H30.This is the first study to assess virulence of ST131 isolates of serotype O16:H5, which in spite of their low virulence-gene score, did not show significant differences in final lethality with highly virulent O25b:H4 isolates of virotypes A, B and C, but killed less rapidly. High virulence of O16:H5 ST131 isolates in the mouse model would correlate with clinical severity reported by Kudinha et al. Blanco et al. Caenorhabditis elegans and zebrafish embryos). According to our virotype scheme, the 3 O25b:H4 ST131 included in their study belonged to virotypes A, B (both CTX-M-15 isolated from cystitis) and D , respectively. Results obtained in those animal models were very different to ours in mouse. Particularly, ST131-B2 isolates were less virulent than non-ST131 B2 isolates in the nematode model. In zebrafish, the 2 CTX-M-15-producing ST131 UPEC isolates were also less virulent than the non-ST131 B2 strains, but O25b:H4 strain of virotype D (S250) showed higher virulence than the two other O25b:H4 ST131 strains (virotypes A and B). Authors suggested that their findings of low virulence for ST131 isolates could be explained by the absence of several classical virulence factors such as HlyA and Cnf1. Results reported by Lavigne et al. hlyA and cnf1 presence and virulence in mouse model. In fact, O25b:H4 isolates of virotypes D5 and E positive for those genes and exhibiting the highest virulence-gene scores  showed moderate to low lethality .Lavigne et al. i.e. the ST131, virulence is more linked to specific combinations of virulence genes than to the total number of them (The non-correlation of the virulence in mice with the virulence score within the ST131 isolates is different to what has been observed at the species level  of them , which cin vivo virulence patterns associated to specific virotypes.This study has evidenced the high genetic variability within the ST131 clonal group, also reflected in the lethality results of a mouse sepsis model. We have shown that Pasteur Institute MLST scheme, as well as clonotyping, are important molecular tools for the discrimination of sublineages within ST131 clonal group which exhibited


Dromaius ater) of Australia is one of several extinct emu taxa whose taxonomic relationship to the modern Emu (D. novaehollandiae) is unclear. King Island Emu were mainly distinguished by their much smaller size and a reported darker colour compared to modern Emu.The King Island Emu (c oxidase subunit I (COI) region , as well as a region of the melanocortin 1 receptor gene (57 bp) were sequenced using a multiplex PCR approach. The results show that haplotypes for King Island Emu fall within the diversity of modern Emu.We investigated the evolutionary relationships between the King Island and modern Emu by the recovery of both nuclear and mitochondrial DNA sequences from sub-fossil remains. The complete mitochondrial control  and cytochrome These data show the close relationship of these emu when compared to other congeneric bird species and indicate that the King Island and modern Emu share a recent common ancestor. King Island emu possibly underwent insular dwarfism as a result of phenotypic plasticity. The close relationship between the King Island and the modern Emu suggests it is most appropriate that the former should be considered a subspecies of the latter. Although both taxa show a close genetic relationship they differ drastically in size. This study also suggests that rates of morphological and neutral molecular evolution are decoupled. Dromaius novaehollandiae) had several, often smaller relatives living on a number of offshore islands of mainland Australia. These included taxa found on Kangaroo Island (D. baudinianus), King Island (D. ater) and Tasmania (D. n. diemenensis), all of which are now extinct. The smallest taxon, the King Island Emu, was confined to a small island situated in the Bass Strait between Tasmania and Victoria, approximately 100 km from both coasts  as well as part of the nuclear encoded melanocortin 1 receptor (MC1R) gene. In contrast to previous unsuccessful attempts to isolate DNA from King Island Emu To investigate the relationship between the modern Emu and the King Island Emu we characterised the complete mitochondrial control region and cytochrome We recovered nucleotide DNA sequences of the complete mitochondrial control and COI regions  from four King Island Emu specimens (KI01-04), in addition to a MC1R (57 bp) fragment for two of these (KI01-02). A fifth specimen did yield amplification products for the control and COI regions but was excluded from further analyses due to excessive molecular damage including fragmentation and type 2 miscoding lesions The recovered King Island Emu MC1R fragments were identical to those of modern Emu and interestingly did not display a SNP most commonly associated with melanism in birds The control and COI regions recovered for both taxa show very little diversity, only seven and six sites respectively are polymorphic in alignments including the modern Emu mitochondrial genome reference sequence . The seqAlthough the King Island Emu display unique haplotypes for both the control and the COI regions, they fall within the diversity of modern Emu for both regions. This, in combination with the low control region and COI diversity, suggests that future studies may identify King Island Emu specific haplotypes in modern Emu. Hence this study would suggest that research aiming to distinguish both taxa using DNA should not be limited to the control or COI regions. Perhaps more highly variable nuclear sequences, like those often used in population studies (e.g. microsatellites or Major Histocompatibility Complex), may be better able to distinguish these taxa.The sequence data recovered from both mitochondrial DNA regions indicate that the modern and the King Island Emu are very closely related. The control and COI regions of the King Island Emu fall within the diversity of modern Emu, showing the latter is a paraphyletic taxon. The low diversity in the sequences recovered for both taxa however indicates that incomplete lineage sorting is a likely cause for this pattern, in particular the processes involved in divergence of peripheral isolates as a result of founder effects Apteryx sp.) 2.36, 8.22 and 8.65% , Rhea (Rhea sp.) 9.59% . Mean pairwise differences among species from the same genus for the COI region are: Cassowary (Casuarius sp.) 2.40% , Kiwi 1.62, 6.02 and 6.09% , Rhea 7.12% . It is noteworthy that three putative Kiwi  subspecies  show 1.84, 2.37 and 2.76% difference among subspecies for a partial control region  A small number of control and COI regions have been characterised for other ratites, mean pairwise differences among species from the same genus for the control region are : Kiwi , the common Brushtail (Trichosurus vulpecula) and Ringtail (Pseudocheirus peregrinus) Possum and the Black Tiger Snake (Notechis ater) are part of a Tasmania-wide subspecies, whereas the Spotted-tailed Quoll (Dasyurus maculates) and the Blotched Blue-tongued Lizard (Tiliqua nigrolutea) form part of (sub-) species that are also found on mainland Australia Dromaius novaehollandiae ater. Just like the Quagga (Equus quagga quagga), one of the first species to have its ancient DNA sequenced, the King Island Emu proves to be an extinct subspecies on the basis of ancient DNA analyses, despite showing morphological diversification Sphenodon sp.) show a high molecular substitution rate in mitochondrial loci but little morphological diversification over millions of years The King Island Emu and the modern Emu show few morphological differences other than their significant difference in size. Additional traits that supposedly distinguish these taxa have previously been suggested to be plumage colour, the distal foramen of the tarsometatarsus, and the contour of the cranium. However, the distal foramen is known to be variable in the modern Emu showing particular diversity between juvenile and adult forms and is therefore taxonomically insignificant c oxidase subunit I gene and control region, respectively, were independently replicated for KI02 and KI04 at the Alan Wilson Centre for Molecular Ecology and Evolution at Massey University, Albany, New Zealand. Approximately 200 mg of bone was incubated overnight in 3 ml of 0.5 M EDTA with 0.5 mg/ml Proteinase K and 0.1% Triton X-100. The solution was extracted with equal amounts of phenol and phenol/chloroform/isoamyl-alcohol (25∶24∶1) subsequently. The aqueous layer was incubated with 0.5 volumes of 7.5 M ammonium acetate, 2.5 volumes ethanol and 50 µl of linear polyacrylamide (LPA) and incubated at −20°C for 20 min. The DNA/LPA complex was centrifuged at 20,000× g for 15 min and the supernatant was washed with 200 µl of isopropanol and redissolved in 200 µl of ddH2O. Where required, DNeasy Blood & Tissue Kit (Qiagen) and Vivaspin Sample Concentrators  were used according to manufacturer's instructions to remove any PCR-inhibitors. DNA was extracted from the modern Emu blood samples using the former kit and in accordance with the manufacturer's instructions.DNA was extracted from five King Island Emu sub-fossil bones  together with a mock extraction in a dedicated ancient DNA facility at Griffith University, Nathan, Australia. Positions 5457–5636 and 16108–16332 of the cytochrome c oxidase subunit I (COI) gene, a separate PCR amplified a fragment from the nuclear melanocortin 1 receptor (MC1R) gene . The first stage 50 µl multiplex reaction contained 5 µl template, 1× PCR buffer, 1 mg/ml Bovine Serum Albumin, 2.5 mM MgCl2, 0.4 µM of each primer, 0.5 mM of each dNTP and 2 units of Platinum Taq (Invitrogen), thermocycling was as follows: 1 min 94°C, 40× , 5 min 72°C. The odd primer mix contained: 5′-3′ (eCR1F: ACGGTCTGAAAAACCRTCG- eCR1R: GAATATGAGGTAAATATAAGTATGTACG); (eCR3F: TTCAGTGCTGTTACGGTCTAC- eCR3R: AGGAATGACCTCGACTTAGGA); (eCR5F: TAACCTTCAACGTACCCCC- eCR5R: GTGGAAATACCATAACCAGATG); (eCR7F: AACRCATCGTTAACACACATT- eCR7R: CTTCAGTGCCATGCTTTGATG); (eCOI1F: AGGACTACAGCCTAACGCTTA- eCOI1R: TGGTCATCTCCTAGTAGTGTT); (eCOI3F: GTGCTCCAGACATGGCATT- eCOI3R: GATGGAGGAAACACCAGCTA); (eCOI5F: TCCTACTATCGCTCCCAGT- eCOI5R: TTCCCTGCGTAATAAGTCAC); (eCOI7F: TCCGCTACCATAATCATCGC- eCOI7R: GAGAGGACATAATGGAAATGGG); (eCOI9F: ACCTTCTTCCCACAACACTTC- eCOI9R: ATGGATTCACTCAATGTTGGT); The even primer mix contained: 5′-3′ (eCR2F: CATTCAATATACGTACTATACCCAT- eCR2R: ATCCCGATTGACGAGCAG); (eCR4F: CCTGCCCACAACATGGT- eCR4R: TAAATTGTGAGCCTGCTGAC); (eCR6F: CATTCGGRCTCTGATGCAC- eCR6R: TGTAACTCCAGTACTGATGAC); (eCOI2F: CCTACTTATCCGTGCTGAACT- eCOI2R: GGTAAGAGTCAAAAGCTCATGTT); (eCOI4F: GGCTTCTGTAGATCTTGCCAT- eCOI4R: AGGTTTCGGTCTGTGAGGA); (eCOI6F: CCCAGGCTTTGGAATAATCTC- eCOI6R: TAGCTAATCAGCTGAATACCTTA); (eCOI8F: ATCGCCCTACATGATACATACTA- eCOI8R: TGAGTATCGTCGTGGTATTCC); (eCOI10F: AAAGTTGCCCAACCAGAACTA- eCOI10R: GAGGTTCGATTCCTTCCTTTC); keeping overlapping amplicons in separate reactions. The second stage 20 µl multiplex contained 1 µl template, 1× PCR buffer, 1 mg/ml Bovine Serum Albumin, 1.5 mM MgCl2, 0.4 µM of each primer (single pair), 0.2 mM of each dNTP and 1 unit of Platinum Taq, thermocycling was as follows: 1 min 94°C, 40× , 5 min 72°C. The MC1R PCR contained 1 µl template, 1× PCR buffer, 1 mg/ml Bovine Serum Albumin, 1.5 mM MgCl2, 0.4 µM of each primer (5′ eMC1RF: TGCTGCCTGGCCGTCTCC- eMC1RR: TGGATCACCAGCACGCCGTG 3′), 0.2 mM of each dNTP and 1 unit of Platinum Taq, thermocycling was as follows: 1 min 94°C, 50× , 5 min 72°C. DNA from the modern Emu specimens was amplified using the same protocol but with long range amplicons: eCR1F- eCR7R, eCOI1F-eCOI10R.A multiplex PCR approach was used to amplify overlapping fragments of the complete mitochondrial control region (CR) and cytochrome The DNA was either isolated from a gel (in case of unspecific by-products) using the QIAquick Gel Extraction Kit (Qiagen) or cleaned with ExoSAP-IT (USB) according to manufacturer's instructions. The BigDye V3.1 (Applied Biosystems) kit was used according to manufacturer's instructions to sequence the DNA fragments. Sequences showing mononucleotide repeats were re-amplified with Phusion High-Fidelity DNA polymerase (Finnzymes) according to manufacturer's instructions i.e. contamination and molecular damage) and re-sequenced accordingly (at least two independent subsequent PCRs per ambiguous amplicon). The sequences were aligned with publicly available Emu sequences for each region and a concatenated alignment was created for the control and COI regions. The Emu mitochondrial genome reference sequence NC_002784 showed a cytosine deletion at position 15,648 when compared with other emu sequences, this deletion was ignored for analyses as it is likely to be a sequencing error p-distances between the groups using complete deletion for gaps. The haplotype network was constructed using TCS After assembly the DNA sequences were manually screened for errors (