H17 subset of helper T cells clears fungal infections, whereas pathogenic TH17 cells cause inflammation and tissue damage; however, the mechanisms controlling these distinct responses remain unclear. Here we found that fungi sensing by the C-type lectin dectin-1 in human dendritic cells (DCs) directed the polarization of non-pathogenic TH17 cells. Dectin-1 signaling triggered transient and intermediate expression of interferon (IFN)-β in DCs, which was mediated by the opposed activities of transcription factors IRF1 and IRF5. IFN-β-induced signaling led to integrin αvβ8 expression directly and to the release of the active form of the cytokine transforming growth factor (TGF)-β indirectly. Uncontrolled IFN-β responses as a result of IRF1 deficiency induced high expression of the IFN-stimulated gene BST2 in DCs and restrained TGF-β activation. Active TGF-β was required for polarization of non-pathogenic TH17 cells, whereas pathogenic TH17 cells developed in the absence of active TGF-β. Thus, dectin-1-mediated modulation of type I IFN responses allowed TGF-β activation and non-pathogenic TH17 cell development during fungal infections in humans.The non-pathogenic T H17 cells.Gringhuis and colleagues show that the extent of type I IFN responses induced by fungal stimulation in human DCs modulate the activation of TGF-β and the production of pathogenic or non-pathogenic T H) cells for host immunity and tolerance1. Pathogen-recognition receptors (PRRs) expressed by DCs sense invading pathogens and induce cytokine profiles that direct the development of appropriate TH cell subsets2. The C-type lectin receptor (CLR) dectin-1 recognizes the fungal cell wall polysaccharide β-glucan4 and triggers signaling through both Syk-CARD9 and LSP1-Raf-1 to induce interleukin (IL)-12 to instruct TH1 differentiation and IL-1β, IL-23 and IL-6 to induce TH17 differentiation7. IL-17A and IL-17F production by TH17 cells is crucial for clearing fungal infections through the recruitment of neutrophils and the generation of antimicrobial peptides by epithelial cells8. Patients with dectin-1 or CARD9 deficiencies have recurrent and often severe fungal infections due to impaired TH17 responses10, underscoring the importance of dectin-1 signaling for antifungal immunity.DCs orchestrate differentiation of helper T C. albicans by human dectin-1 initiated mostly non-pathogenic TH17 responses. Dectin-1 triggering led to transient and intermediate expression of IFN-β by balancing the activities of the transcription factors IRF1 and IRF5. Type I IFN signaling induced the expression of integrin αvβ8, which allowed the MMP14-mediated cleavage of the latent TGF-β complex to release active TGF-β, which in turn resulted in the development of primarily non-pathogenic TH17 cells. Our data underscore the importance of a tightly controlled type I IFN response, as high amounts of IFN-β inhibited MMP14 through the ISG BST2 and increased pathogenic TH17 polarization. Thus, dectin-1 regulated type I IFN expression to allow the TGF-β-mediated development of non-pathogenic TH17 responses during fungal infections in humans.Here, we demonstrated that recognition of curdlan and IFNB messenger RNA (mRNA) that peaked at 2 h post-stimulation -LyoVec induced higher expression of IFNB mRNA that plateaued 4 h post-stimulation . Silencing of either Syk, CARD9, Bcl10 or Malt1 expression in DCs by transduction of specific short interfering RNAs (siRNAs)  also induced mRNA expression for the ISGs MX1, ISG15, TRIM22, IRF7 and IL27A (encoding IL-27 subunit p28)24  with the dectin-1-specific β-glucan ligand curdlan induced the transient expression of ion Fig. . For comion Fig. . Variousion Fig. . Both cuion Fig. . Dectin-ect Fig. . Curdlan)24 Fig. , which w)24 Fig. . Thus, fH17 differentiation, we co-cultured DCs that were primed for 48 h with curdlan or C. albicans with naive CD45RA+CD4+ T cells and determined the proliferation and intracellular expression of IL-17 and IFN-γ of the cultured T cells. Curdlan- and C. albicans-primed, but not immature DCs, induced strong T cell proliferation as measured by cell count and tracking cell division by carboxyfluorescein succinimidyl ester staining   was partly blocked by neutralizing antibodies against IL-1β (3.4 ± 0.9% and 3.5 ± 0.7%) or IL-23 (2.9 ± 1.0% and 3.0 ± 1.1%), but not IL-12 (6.9 ± 0.8% and 6.7 ± 0.9%)  was not affected by blocking IFN-α/βR antibodies (11 ± 3.4% and 11 ± 2.9%)   was increased by blocking IFN-α/βR antibodies (65 ± 5.5% and 67 ± 4.0%)   Fig. . Develop9%) Fig. . In contly) Fig. , while n0%) Fig. . Supplemly) Fig. .Fig. 2DeC. albicans-primed DCs with memory CD45RO+CD4+ T cells together with Staphylococcus aureus enterotoxin B (SEB), which induces antigen-independent T cell activation, to examine how dectin-1-induced cytokines affect the phenotype of differentiated TH cells. Immature as well as curdlan- and C. albicans-primed DCs induced strong T cell proliferation , but not immature DCs, whereas at day 11–14, when T cells were quiescent, IL-17+ cells represented 6.2 ± 2.9% and 5.9 ± 3.2% of total cells  and IL-23 (1.5 ± 0.7% and 1.5 ± 0.8%), but not IL-12 (6.9 ± 1.8% and 6.9 ± 2.2%) neutralizing antibodies  was reduced by supplementary rhIFN-β (5.1 ± 2.4% and 4.2 ± 1.6%), whereas blocking IFN-α/βR antibodies had no effect (14 ± 6.2% and 13 ± 5.6%)  significantly increased by the addition of blocking IFN-α/βR antibodies (43 ± 4.1% and 45 ± 4.8%) or supplementary rhIFN-β (41 ± 5.0% and 41 ± 4.8%)  Fig. ; however8%) Fig. , suggestH17 cell subtypes through real-time qPCR analyses, including transcription factors c-Maf (encoded by MAF), which regulates the expression of anti-inflammatory cytokine IL-10 and effector molecule prostaglandin D synthase (encoded by PTGDS)25, and T-bet (encoded by TBX21); IL-1R; inflammatory cytokines IFN-γ, IL-22 and GM-CSF (encoded by CSF2); and the effector molecules CXCL3, CCL3 and CCL5, which are expressed by mouse pathogenic TH17 cells28. The expression of these molecular markers was analyzed in peripheral blood IL-17+CD4+ T cells isolated from healthy individuals, to provide a reference point for non-pathogenic TH17 cells, and patients with acute untreated Crohn’s disease (CD), in whom pathogenic TH17 cells are the main mediators of gastrointestinal tract inflammation29. Expression of TBX21, IFNG, IL1R1 and CXCL3 expression was high in IL-17+CD4+ T cells from patients with acute untreated CD and low to moderate in IL-17+CD4+ T cells from healthy individuals  was similar in IL-17+CD4+ T cells from healthy and all CD donors  within its promoter30. IRF5 is involved in dectin-1-mediated IFN-β expression22, while dectin-1 is known to activate IRF131. Curdlan and C. albicans stimulation of DCs led to the nuclear translocation of IRF1 and IRF5 but not IRF3 at 2 h post-stimulation  was reduced by supplementary rhIL-27 (4.6 ± 1.3% and 5.2 ± 0.8%), but did not affect the phenotype of the induced TH17 cells  that is sequestered within the extracellular matrix after secretion. Curdlan or C. albicans stimulation of DCs did not affect the total amount of TGF-β, but resulted in release of active TGF-β, which was inhibited by blocking antibodies against dectin-1  was not affected by the addition of blocking TGF-βR antibodies during co-culture with memory CD45RO+CD4+ T cells (18 ± 4.2% and 18 ± 2.6%); however, the frequency of IFN-γ+IL-17+ T cells  strongly increased by blocking TGF-βR antibodies (43 ± 3.0% and 44 ± 3.1%)  of TH17 cells induced by curdlan- or C. albicans-primed DCs, but reduced the increased development of IFN-γ+IL-17+ T cells after blocking IFN-α/βR signaling (43 ± 4.1% and 45 ± 4.8% of IL-17+ cells) to the frequencies observed with untreated curdlan- or C. albicans-primed DCs (20 ± 3.0% and 16 ± 1.4% IFN-γ+ cells of IL-17+ cells)  increased in the presence of blocking TGF-βR antibodies (63 ± 1.4% and 68 ± 5.1%), whereas supplementary rhTGF-β1 reduced the increase in frequency of IFN-γ+IL-17+ T cells after blocking IFN-α/βR signaling (65 ± 5.5% and 67 ± 4.0%) to the frequencies observed with untreated curdlan- or C. albicans-primed DCs (14 ± 7.4% and 15 ± 7.7% IFN-γ+ cells of IL-17+ cells)  developed when naive CD45RA+CD4+ T cells were co-cultured with DCs that were primed with curdlan or C. albicans for only 3 h, a time point where we could not yet detect release of active TGF-β  developed when rhTGF-β1 was added to these co-cultures . Inhibition of MMP14 activity with the specific inhibitor NSC405020 abrogated the release of active TGF-β by curdlan- or C. albicans-stimulated DCs  is a transmembrane protein that negatively modulates MMP14 activity through interactions between their cytoplasmic domains37. BST2 mRNA and protein expression were induced 4 h and 8 h post-stimulation, respectively, in curdlan- or C. albicans-stimulated DCs and these were completely abrogated by blocking antibodies to dectin-1 or IFN-α/βR or through IRF5 silencing  and human material was obtained in accordance with the AMC Medical Ethics Review Committee  according to the Medical Research Involving Human Subjects Act. Buffy coats obtained after blood donation (Sanquin) are not subjected to informed consent according to the Medical Research Involving Human Subjects Act and the AMC Medical Ethics Review Committee. Blood obtained from healthy volunteers was covered by the BACON protocol. Patients with active CD were recruited at the IBD clinic of Amsterdam UMC, location AMC. After providing written informed consent, an additional blood sample was drawn in addition to routine blood draws. The project was covered by the Future-IBD Biobank protocol. All samples were handled anonymously.−1 IL-4 and 800 U ml−1 GM-CSF (both Invitrogen) and used on day 6 or 7. Donors were routinely screened for dectin-1 single nucleotide polymorphism rs16910526 using TaqMan SNP Genotyping Assays ; only dectin-1 wild-type DCs were used for experiments, unless otherwise indicated. DCs were stimulated with curdlan , Salmonella typhosa LPS , poly(I:C)-LyoVec , heat-killed Candida or Aspergillus species at multiplicity of infection (MOI) 10, rhIFN-β . DCs were preincubated for 2 h with MMP14 inhibitor NSC405020  or blocking antibodies, anti-dectin-1 , anti-IFN-α/βR2 , anti-αvβ1 , anti-αvβ3 , anti-αvβ5 , anti-αvβ6 , anti-αvβ8  or isotype control antibodies, mouse IgG1 , mouse IgG2a  and mouse IgG2b . DCs were transfected with 25 nM siRNA using transfection reagents DF4 (Dharmacon) according to the manufacturer’s instructions and used for experiments 72 h after transfection. SMARTpool siRNAs used were Syk (M-003176-03), CARD9 (M-004400-01), Bcl10 (M-004381-02), Malt1 (M-005936-02), LSP1 (M-012640-00), Raf-1 (M-003601-02), IRF1 (M-011704-01), IRF5 (M-011706-00), IRF7 (M-011810-02), ITGAV (M-004565-03), ITGB8 (M-008014-02), BST2 (M-011817-00) and non-targeting siRNA (D-001206-13) as a control . Silencing of expression was verified by real-time PCR and flow cytometry ; antibodies used for verification were anti-IRF1 , anti-IRF5 , anti-αv , anti-β8 , anti-BST2 , followed by incubation with either PE-conjugated anti-rabbit  or Alexa Fluor 488-conjugated anti-goat . DCs were also transfected with a pCG–BST2–IRES–eGFP expression plasmid (a kind gift from F. Kirchhoff52) coupled to LyoVec (Invivogen), as described by the manufacturer. eGFP− and eGFP+ cells were selected 24–40 h after transfection by sorting on a FACSAria IIu Cell Sorter (BD Biosciences) and used for experiments; BST2 (over)expression was determined in both cell subsets after staining with anti-BST2 and PE-conjugated anti-rabbit (described above) by flow cytometry on a FACS Calibur or Canto (BD Biosciences). All flow cytometry data analysis was performed using FlowJo v.10.8.1 (BD Biosciences).Peripheral blood mononuclear cells were isolated from buffy coats of healthy volunteer blood donors (Sanquin) by a Lymphoprep (Axis-Shield) gradient step and monocytes were subsequently isolated by a Percoll (Amersham) gradient step. Monocytes were differentiated into immature DCs in the presence of 500 U mlCandida strains were grown in Sabouraud dextrose broth and incubated at 25 °C for 3 d, while shaking. A. fumigatus was grown on potato dextrose agar at 37 °C for 3 d. Conidia were dislodged from slants by gentle tapping and then resuspended in PBS/0.1% Tween-80. Hyphal contamination was removed by straining the cell solution through a glass filter. Fungi were heat inactivated at 56 °C for 1 h.Ct value is defined as the number of PCR cycles where the fluorescence signal exceeds the detection threshold value. The normalized amount of target mRNA was calculated from the obtained Ct values for both target and GAPDH mRNA with Nt = 2Ct(GAPDH) − Ct(target). The relative mRNA expression in DCs was obtained by setting Nt in either 2 h (IFNB) or 6 h (ISGs) curdlan-stimulated samples at 1 within one experiment and for each donor, except when donors were used that were homozygous for the minor alleles of rs16910526 where Nt in 2 h LPS-stimulated samples was set at 1.Transcript levels in DCs were quantified with the SYBR green method in an ABI 7500 Fast PCR detection system (Applied Biosystems), after mRNA isolation using the mRNA capture kit (Roche) and cDNA synthesis using the Reverse Transcription System (Promega). Specific primers were designed using Primer Express 2.0  and biotinylated anti-TGF-β1 , followed by streptavidin-HRP (Invitrogen). Supernatants were treated with 0.2 N HCl before ELISA to expose epitopes obscured within the LLC. Bioactive TGF-β levels in DC culture supernatants were measured by transferring supernatant at 4, 8 or 24 h post-stimulation to HEK-Blue TGF-β reporter cells (Invivogen), after which secreted SEAP in the reporter cell culture supernatants was determined 24 h later using the SEAP Reporter Assay kit (Invivogen). HEK-Blue TGF-β reporter cells were also stimulated with a concentration range of rhTGF-β1 (R&D Systems) to generate a standard curve.Secreted IL-1β, IL-12p70, IL-6, IL-23 and IL-27 levels in DC culture supernatants that were collected 28 h post-stimulation were determined by ELISA (Invitrogen). Intracellular IFN-β, αv, β8 and BST2 levels were determined by flow cytometry. Cells were first fixated in 3% paraformaldehyde for 10 min and permeabilized in 90% methanol at 4 °C for 30 min. Staining with anti-αv, anti-β8, anti-BST2, followed by PE-conjugated anti-rabbit was conducted as described above. IFN-β was stained directly with FITC-conjugated anti-IFN-β , while FITC-conjugated IgG1κ mouse antibody  was used as isotype control. Expression was analyzed on a FACS Calibur or Canto (BD Biosciences). All flow cytometry data analyses were performed using FlowJo v.10.8.1 (BD Biosciences). Total TGF-β levels in DC culture supernatants that were collected 24 h post-stimulation were determined by ELISA with anti-pan TGF-β (2 µg mlNuclear and cytoplasmic extracts of DCs were prepared 2 or 6 h post-stimulation using NucBuster protein extraction kit (Novagen) as described by the manufacturer. Then, 20 μg of extract was resolved by SDS–PAGE and cellular localization of IRF1, IRF3, IRF5 or IRF7 was determined by immunoblotting with anti-IRF1 , anti-IRF3 , anti-IRF5  or anti-IRF7 , followed by incubation with HRP-conjugated Clean-Blot IP Detection Reagent  and ECL detection (Pierce). Membranes were also probed with anti-β-actin , followed by HRP-conjugated anti-mouse  to ensure equal protein loading among cytoplasmic and nuclear extracts, respectively. Nuclear IRF1, IRF5 and IRF7 levels were further quantified by ELISA (USCN Life Science). IRF1 and IRF5 localization was also determined by immunofluorescence staining; DCs were fixated with 4% paraformaldehyde 2 h post-stimulation, permeabilized with 0.2% (v/v) Triton X-100 in PBS, stained with anti-IRF1  or anti-IRF5 , followed by incubation with Alexa Fluor 546-conjugated anti-rabbit  or Alexa Fluor 546-conjugated anti-goat , respectively. After staining nuclei with DAPI , cells were preserved in ProLong Diamond Antifade Mountant (Molecular probes) and IRF localization was visualized with a TCS SP8 X confocal microscope (Leica).IFNB promoter was assessed by ChIP assays using the ChIP-IT Express Enzymatic Shearing and ChIP-IT Express HT kits (both from Active Motif). Briefly, cells were fixated with 1% (v/v) paraformaldehyde 2 or 6 h post-stimulation, nuclei were isolated and chromatin DNA fragmented by enzymatic shearing . Protein-DNA complexes were immunoprecipitated using 2 µg anti-IRF1 , anti-IRF5 , anti-IRF7  or negative control IgG  and protein G-coated magnetic beads. DNA was purified after reversal of crosslinks. Quantitative real-time PCR using SYBR green (Applied Biosystems) as described above was performed with a primer set spanning the ISRE of the IFNB promoter  to DCs 4, 8 or 24 h post-stimulation for 30 min at 37 °C. Supernatant containing (cleaved) substrate was then transferred to a black 96-well plate and fluorescence intensity was measured at 320 nm excitation and 420 nm emission wavelength using a Synergy HT reader (Biotek); the detected relative fluorescence units are a measure for MMP14 activity.+ T cells were isolated by negative selection from blood donated by healthy donors or Crohn’s disease patients with the human CD4+ T cell isolation kit II (Miltenyi) and then stimulated for 6 h with 100 ng ml−1 PMA (Sigma) and 1 μg ml−1 ionomycin (Sigma), the last 4 h in the presence of 10 μg ml−1 brefeldin A (Sigma). After fixation in 4% paraformaldehyde for 10 min, permeabilization with 0.5% (v/v) saponin and staining with APC-conjugated anti-IL-17 , IL-17+ cells were selected by sorting on a FACSAria IIu Cell Sorter (BD Biosciences). After reversal of crosslinks by proteinase K (QIAGEN) treatment at 56 °C for 16 h in PKD buffer (QIAGEN), mRNA isolation and complementary DNA synthesis was performed as described above for DCs. Transcript levels in T cells were measured with quantitative real-time PCR using SYBR green as described above. Specific primers were designed using Primer Express 2.0  with the human CD4+ T cell isolation kit II (Miltenyi), combined with staining with PE-conjugated anti-CD45RO , captured on anti-PE beads (Miltenyi). Isolated naive T cells were routinely >99% CD4+  and contained <1% CD45RO+ cells, whereas memory T cells were routinely >99% CD4+ and contained <1% CD45RA+ cells . DCs were either silenced for indicated proteins or preincubated for 2 h with blocking antibodies and then activated for 48 h as indicated, as described above. When DCs were activated for only 3 h before co-culture, this is mentioned explicitly in the text. Primed DCs were co-cultured with either naive CD4+ T cells  for 5 d before restimulation or memory CD4+ T cells  in the presence of 10 pg ml−1 SEB (Sigma) and 10 U ml−1 IL-2 (Chiron) (added after 5 d) until they became quiescent, typically after 11–14 d, before restimulation. During co-culture, cells were treated with either rhIL-27 , rhTGF-β1 , neutralizing antibodies (5 μg ml−1) to IL-1β , IL-23 , IL-12 , IL-27  or TGF-βRII  or normal goat IgG  as a control. Proliferation and survival of co-cultures was monitored by loading naive or memory T cells with 2 μM CFSE (Molecular Probes) and measured every other day in combination with Fixable Viability Dye eFluor 780  staining by flow cytometry on a FACS Canto (BD Biosciences). All flow cytometry data analyses were performed using FlowJo v.10.8.1 (BD Biosciences). Additionally, cell counts were determined every other day using the CASY cell counter and analyzer (OMNI Life Science). Restimulation of T cells occurred for 6 h with PMA and ionomycin in the presence of brefeldin A as described above. After fixation, permeabilization and staining with either APC-conjugated anti-IL-17 or eFluor506-conjugated anti-IL-17  alone or in combination with FITC-conjugated anti-IFN-γ , total IL-17+ or separate IFN-γ-IL-17+ and IFN-γ+IL-17+ cells were measured by flow cytometry and subsequently selected by sorting and used for either mRNA isolation, cDNA synthesis and quantitative real-time PCR as described above. Gating strategies for analyses and sorting are shown in Extended Data Fig. + cells were also subjected to further flow cytometry analyses, after permeabilization using the Foxp3/transcription factor staining kit (eBioscience), by staining with (combinations of) the following antibodies: FITC-conjugated anti-IFN-γ, PE-conjugated anti-IL-10 , Alexa Fluor 647-conjugated anti-RORγt , APC-conjugated anti-T-bet , eFluor660-conjugated anti-c-Maf , APC-conjugated anti-IL-1R1  and APC-conjugated anti-GM-CSF  or matching isotype control antibodies: Alexa Fluor 647-conjugated mouse IgG2b , APC-conjugated mouse IgG1 , eFluor660-conjugated mouse IgG2b , APC-conjugated goat IgG  and APC-conjugated rat IgG2a .For in vitro Tt-test for paired and two-tailed observations using Office Professional Plus 2019, Excel (Microsoft) and GraphPad Prism v.8.3.1 (Dotmatics). Statistical significance was set at P < 0.05.Statistical analyses were performed using the Student’s Further information on research design is available in the Any methods, additional references, Nature Portfolio reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41590-022-01348-2.Supplementary InformationSupplementary InformationReporting SummaryPeer Review File


III atom is coordinated to three S and two N atoms of three different ligands and exhibits a square-pyramidal coordination environment.The Bi 9H6NS2)3], was prepared by reacting BiCl3 and 2-mercapto-4-phenylthiazole (LH) at room temperature in a stoichiometric ratio of 1:4. The molecular structure reveals a slightly distorted square-pyramidal environment around the BiIII atom. Two of the three monoanionic ligands L− coordinate in an N,S-bidentate mode, while one shows a monodentate mode through an S atom. There are no significant intermolecular interactions present in the crystal.The title compound, [Bi(C In the corresponding three bismuth complexes [Bi(L1)4(Cl)2]Cl, [Bi(L2)4Cl2]+[Bi(L2)2Cl4]− and [Bi(L3)2Cl2(μ-Cl)]2  coordination.For general background on this type of bismuth chemistry with S- or -donor ligands, see: Diemer  al. 1995; Stavila al. 2006; Briand  al. 2000. The cooL (LH is 2-mercapto-4-phenyl thiazole) exhibits both monodentate S- and bidentate -coordination modes  while the third one exhibits a monodentate mode via the S5 donor atom, resulting in a slightly distorted square-pyramidal coordination environment. The Bi—N and Bi—S bonds differ in lengths with the Bi—S bonds shorter by ≃ 0.2 Å .In the title compound, the deprotonated ligand es Fig. 1. Two ligÅ Table 1 but the x + 1, y, z) with a distance of 3.473 (1) Å.In the crystal packing Fig. 2, no sign3  and 2-mercapto-4-phenyl thiazole (LH)  in THF at room temperature. After stirring for 4 h, the resulting yellow solution was concentrated, yielding a yellow solid that was separated by decantation and washed with small amounts of THF followed by diethyl ether. The solid was dried and recrystallized from a mixture of THF/pentane (ratio v:v = 1:3). Yellow to orange crystals suitable for X-ray diffraction were obtained by slow evaporation of the THF solution of the complex. Yield 76%; m.p. 507 K. 1H NMR (CDCl3): δ 7.58–7.60 , 7.42–7.49 , 6.78, CH-thiazole ring); 13C NMR (CDCl3): δ 188.5 (C9), 142.5 (C8), 129.9 , 129.4 , 128.1 (C4), 125.9 (C1), 108.9 (C7).The title compound was prepared by reacting BiClCrystal data, data collection and refinement details are summarized in Table 210.1107/S241431462000067X/wm4119sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S241431462000067X/wm4119Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S241431462000067X/wm4119Isup4.cdxSupporting information file. DOI: 1979022CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


H-benzimidazole is described.The crystal structure of the nickel(II) dichloride complex of the ligand 2-(pyridin-2-yl)-1 2(C12H9N3)2]·H2O, a divalent nickel atom is coordinated by two 2-(pyridin-2-yl)-1H-benzimidazole ligands in a slightly distorted octahedral environment defined by four N donors of two N,N′-chelating ligands, along with two cis-oriented anionic chloride donors. The title complex crystallized with a water molecule disordered over two positions. In the crystal, a combination of O—H⋯Cl, O—H.·O and N—H⋯Cl hydrogen bonds, together with C—H⋯O, C—H⋯Cl and C—H⋯π interactions, links the complex molecules and the water molecules to form a supramolecular three-dimensional framework. The title complex is isostructural with the cobalt(II) dichloride complex reported previously .In the title complex, [NiCl al. 2011. Org. Bi The disordered water molecules are linked to the complex molecule by O—H⋯Cl hydrogen bonds, and water H atom H2B is directed to the centroid of the C7–C12 ring  monohydrate  in ethanol (5 ml) was added dropwise to a stirring ethanolic solution of bis(triphenylphosphine)nickel(II) dichloride . The mixture was stirred at room temperature for 24 h. The resulting mixture was concentrated and the product isolated by addition of diethyl ether (5 ml) giving a light-brown solid. Yield: 0.27 g (68%). Analysis calculated for C24H18Cl2N6Ni: C, 55.43; H, 3.49; N, 16.16%. Found: C, 55.23; H, 3.59; N, 16.25%. Light-blue plate-like crystals, suitable for X-ray diffraction analysis, were obtained by slow evaporation of a concentrated ethanol solution.The reaction scheme for the synthesis of the title complex is given in Fig. 5Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2414314620000401/su4175sup1.cifCrystal structure: contains datablock(s) Global, I. DOI: 10.1107/S2414314620000401/su4175Isup2.hklStructure factors: contains datablock(s) I. DOI: 1946553CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Downregulation of NPM1’s Ser-125 phosphorylation was further confirmed using Western blot. Phosphorylation mimicking NPM1 S125D mutant activated Tat-induced HIV-1 transcription and exhibited enhanced NPM1–Tat interaction compared to NPM1 S125A mutant. Inhibition of Aurora A or Aurora B kinases that phosphorylate NPM1 on Ser-125 residue inhibited HIV-1, further supporting the role of NPM1 in HIV-1 infection. Taken together, 1E7-03 reprogrammed PPARα/RXRα and TGF-β pathways that contribute to the inhibition of HIV-1 transcription. Our findings suggest that NPM1 phosphorylation is a plausible target for HIV-1 transcription inhibition.Transcription activation of latent human immunodeficiency virus-1 (HIV-1) occurs due to HIV-1 rebound, the interruption of combination antiretroviral therapy, or development of drug resistance. Thus, novel HIV-1 inhibitors, targeting HIV-1 transcription are needed. We previously developed an HIV-1 transcription inhibitor, 1E7-03, that binds to the noncatalytic RVxF-accommodating site of protein phosphatase 1 and inhibits HIV-1 replication in cultured cells and HIV-1–infected humanized mice by impeding protein phosphatase 1 interaction with HIV-1 Tat protein. However, host proteins and regulatory pathways targeted by 1E7-03 that contribute to its overall HIV-1 inhibitory activity remain to be identified. To address this issue, we performed label-free quantitative proteome and phosphoproteome analyses of noninfected and HIV-1–infected CEM T cells that were untreated or treated with 1E7-03. 1E7-03 significantly reprogramed the phosphorylation profile of proteins including PPARα/RXRα, TGF-β, and PKR pathways. Phosphorylation of nucleophosmin (NPM1) at Ser-125 residue in PPARα/RXRα pathway was significantly reduced  at Ser-125 residue.•Phosphorylation of NPM1 activates Tat-induced HIV-1 transcription.•Inhibition of NPM1 upstream kinases, Aurora A or Aurora B, inhibites HIV-1. Small molecule HIV-1 transcription inhibitor, 1E7-03, that binds to the noncatalytic RVxF accommodating site of protein phosphatase 1 reprogramed protein phosphorylation in the PPARα/RXRα, TGF-β, and PKR pathways. Phosphorylation of nucleophosmin (NPM1) at Ser-125 residue was significantly reduced. NPM1 S125D mutant activated Tat-induced HIV-1 transcription and exhibited enhanced NPM1–HIV-1 Tat interaction. Inhibition of Aurora A or Aurora B kinases that phosphorylate NPM1 inhibited HIV-1. Our findings suggest that NPM1 phosphorylation is a plausible target for HIV-1 transcription inhibition. Human immunodeficiency virus type 1 (HIV-1) remains a global public health threat as it escapes combination antiretroviral therapy (cART) by establishing long-lived stable reservoirs in quiescent memory T cells, tissue-resident macrophages, and hematopoietic stem cells , 3. HIV-35VCF38) that interacts with PP1 and helps to translocate PP1 to the nucleus (b]quinoline scaffold that differs from FDA-approved anti–HIV-1 drugs and known HIV-1 transcription inhibitors (50 ∼ 5 μM) with no toxicity (CC50 ∼ 100 μM) (Protein phosphatase 1 (PP1) is a serine-threonine (Ser/Thr) phosphatase  composed nucleus . The int nucleus  can be t nucleus , 22, 23.hibitors . 1E7-03  100 μM) . HoweverPrevious quantitative phosphoproteome analyses focused on the phosphorylation of host proteins modulated during HIV-1 infection , 27. Her2 columns, were analyzed by tandem liquid chromatography-Fourier transform mass spectrometry (LC-FT/MS) followed by label-free quantitative analysis and Ingenuity Pathway Analysis (IPA). Biological functions and biological pathways were accessed by IPA with cutoff >1.5 fold and p-value <0.05.Noninfected CEM T cells untreated or treated with 1E7-03 were compared to CEM T cells infected with VSV-G–pseudotyped HIV-1 without or with 1E7-03 treatment. The sample size for each condition n = 1. Each sample was divided to three parts for phosphoenrichment. N = 12 samples were processed in instrumental triplicates and that gave N = 36 individual files to analyze. Trypsin-digested protein peptides, either nonenriched or enriched on Fe-NTA or TiO1E7-03 (purity above 98%) was synthesized by Enamine, as previously described . 1E7-03 HIV-1 long terminal repeat–luciferase vector was kindly provided by Dr Manuel López-Cabrera (Unidad de Biología Molecular). FLAG-Tat expression vector was a gift from Dr Patricio Ray . Expression vectors for GFP-nucleophosmin (NPM1) WT, GFP-NPM1 S125A, and GFP-NPM1 S125D were purchased from GenScript.+ T cells (CEM T) and 293T cells were purchased from American Type Culture Collection. CEM T cells were cultured in RPMI media (Invitrogen) containing 10% fetal bovine serum (FBS) and 1% antibiotic solution (penicillin and streptomycin) at 37 °C and 5% CO2. For noninfected group, cells were seeded in 100 mm culture plates (2 × 105 cells/ml) containing 10 ml of RPMI media and treated with 10 μM 1E7-03 or vehicle (DMSO) for 24 h. For HIV-1–infected group, CEM T cells were cultured in 100 ml flask (∼6 × 105 cells/ml) and infected with VSVG pseudotyped pNL4-3.Luc.R-E-virus (HIV-1-LUC-G) at MOI = 0.01 for 24 h. Then, HIV-1–infected cells were seeded in 100 mm culture plates (2 × 105 cells/ml) containing 10 ml of RPMI media and treated with 10 μM 1E7-03 or DMSO for additional 24 h. The 293T cells were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen) containing 10% FBS and 1% antibiotic solution (penicillin and streptomycin).CD4g for 30 min at 4 °C to remove cellular debris. Protein concentration was determined using Bradford assay (Bio-Rad). An aliquot of 500 μg proteins from each sample was mixed with 4-fold volume cold acetone to precipitate proteins. All samples were centrifuged at 13,000g for 5 min. Precipitated proteins were collected and dried in SpeedVac concentrator. The pellet was resuspended in 500 μl of sodium phosphate buffer (pH 8.0), reduced in 10 mM DTT (1 h at 60 °C), alkylated with 30 mM iodoacetamide , and digested with 10 μg trypsin (Promega) at 37 °C on orbital shaker overnight.Cells were collected and washed three times with PBS. To obtain cell lysates, the cells were suspended in 1 ml cold whole cell lysis buffer , supplemented with protease inhibitors  and phosphatase inhibitors . The cytosolic protein fraction was isolated by centrifugation at 13,0004OH (×2), activating with 100 μl of acetonitrile, followed by 100 μl of 1% TFA washing (×2). Peptide mixtures were loaded into columns and incubated for 10 min with periodic vortex mixing. After incubation, each sample was cleaned by 200 μl of 1.0% TFA (×2) and eluted with 100 μl of 0.1% FA in 50% acetonitrile (×3). The elution was gently dried using a SpeedVac concentrator. In the Fe-NTA–enriched group, peptide mixtures were dried, resuspended in 200 μl of binding buffer, added to Fe-NTA spin columns , and incubated for 20 min at room temperature with end-over-end rotation. Samples were in turn washed with 100 μl of wash buffer A (×2), wash buffer B (×2), and ultrapure water. Then, samples were eluted with 50 μl of elution buffer . The elution fractions were pooled together, acidified by adding 200 μl of 2.5% TFA, and purified using Graphite Spin Columns as described above. In TiO2-enriched group, TiO2 columns were prepared by washing with 20 μl of 0.4% TFA in 80% acetonitrile and 20 μl of 25% lactic acid. Peptide mixtures were dried, resuspended in 150 μl of 25% lactic acid, added to TiO2 spin columns , and incubated for 10 min at room temperature with end-over-end rotation (×2). Samples were washed with 20 μl of 25% lactic acid and 20 μl of 25% lactic acid (×2). Then, samples were eluted using 50 μl of 1.5% ammonium hydroxide and 50 μl of 5% pyrrolidine. The elution fractions were combined, acidified by adding 100 μl of 2.5% TFA, and purified using Graphite Spin Columns. Note: (×number) is repeated times of washing and spin speed for centrifuge for each step, please follow manufacturer’s instructions. We used one biological sample for each condition (four), which was processed using three different methods. Twelve samples were analyzed by mass spectrometry, using instrumental triplicates .An aliquot of 150 μl tryptic solution was processed by three different methods following manufacturer’s instructions. In nonenriched group (No ENR), peptide mixture was directly purified by Pierce Graphite Spin Columns . Briefly, columns were prepared by washing with 100 μl of 1 M NHhttps://www.thermofisher.com/order/catalog/product/OPTON-30965). Enriched and/or purified peptides were resuspended in 50 μl of water with 0.1% FA (v/v). A total of 10 μl of sample was loaded and washed for 6 min on a C18-packed precolumn  with a solvent of A:B = 99:1  at a constant flow of 12 μl/min. Peptides were transferred onward to an in house C18-packed analytical column  and separated with a linear gradient of 6–55 min, 2–40% B; 55–62 min, 40–80% B; 62–70 min, 80% B (v/v) at the flow rate of 600 nl/min. Mobile phase A was 0.1% FA in water and mobile phase B was 0.1% FA in acetonitrile. The Orbitrap was operated under data-dependent acquisition mode. The spray voltage, capillary temperature, and capillary voltage were set to 2.0 kV, 200 °C, and 39.5 V, respectively. Full-scan mass spectra were acquired in Orbitrap over 300-2000 m/z with a resolution of 30,000, followed by MSn scans by collision-induced dissociation activation mode. The three most intense ions were selected for fragmentation using collision-induced dissociation in the LTQ . Charge state rejection (charge state 1 was rejected) as well as dynamic exclusion  was enabled.LC-FT/MS analysis was performed on a LC-20AD nano HPLC system (Shimadzu Corporation) coupled to Linear Trap Quadrupole (LTQ) XL Orbitrap mass spectrometer (Thermo Fisher Scientific) with the installed Xcalibur software . Briefly, the chromatographic peaks detected by Orbitrap were aligned and the peptides peaks were detected with a minimum signal intensity of 1 × 105; quantitative frames were determined based on m/z (width: 10 ppm) and retention time (width: 2.5 min). The PD 1.4.–identified phosphopeptides were uploaded as framing seeds. Statistical filters were set to assess the quality of the data. The CV raw MS intensities of the triplicates had to be within 30%. This helped minimizing the effect of run-to-run variability. Nonphosphorylated peptides were filtered out to eliminate their interference on the quantitative result. SIEVE 2.1 generated lists of differentially expressed proteins with a cut off >1.5-fold or <0.66 and p-value<0.05. The raw files and PD1. 4 search files can be accessed at https://massive.ucsd.edu/, The dataset access number is MSV000090850. The public access password is nekhai@1. The download ftp link is ftp://massive.ucsd.edu/MSV000090850/.LC-FT/MS raw data were searched by Proteome Discoverer 1.4 (PD 1.4) using SEQUEST search engine (Thermo Fisher Scientific), against the Uniprot Human database  at a false discovery cut off ≤1%. A maximum of two missed cleavage sites was allowed with trypsin full cleavage. The mass tolerance for the precursor ion was 30 ppm and for the fragment - 0.1 Da. Phosphorylation of serine, threonine, and tyrosine residues were enabled as dynamic modifications, while carbamidomethylation of cysteine was set as fixed modification. Filters were set for peptides with different charges as follows: charge 2 = 1.5 (XCorr score), charge 3 = 2.0, and charges >4 =2.5 for highly confident peptides; charge 2 = 0.5, charge 3 = 0.8, and charges >4 =1 for modestly confident peptides. The label-free quantification of phosphopeptides eluting between 10 min and 70 min was performed with SIEVE 2.1 software  server for a Core analysis to investigate the protein function and biological networks. Canonical pathway analysis was carried out. PPARα/RXRα activation and TGF-β signaling networks were shown using Cytoscape 3.8.0.SIEVE 2.1 generated lists of differential phosphoproteins or expressed proteins with >1.5-fold of upregulation or downregulation and LgBiT-fused PRKA2A and SmBiT-fused PRKACA were obtained from Promega. 293T cells were plated in 96-well white/clear culture plates with 40% confluence and transiently transfected with the indicated constructs (1:1 ratio of interacting pairs) using Lipofectamine 3000 Plus in OPTI-MEM as directed by the manufacturer’s protocol. At 24 h post transfection, cells were treated with serial concentrations of 1E7-03 for the indicated amount of time. Nano-Glo Live Cell Substrate  was added, and luminescence was measured using a GloMax-Multi Detection System (Promega).CEM T cells or HEK293T cells expressing NPM1-EGFP were treated with 10 μM 1E7-03 or DMSO for 3 h or 24 h as indicated. The cells were also treated where indicated with 3 nM or 100 nM okadaic acid (OA) for 2 h. Total proteins were extracted using the whole cell lysis buffer supplemented with protease and phosphatase inhibitors (described above). Equal amounts of total protein were separated by electrophoresis and transferred to polyvinylidene fluoride membrane. The membrane was probed with antibodies for NPM1 , anti–p-S125 NPM1 antibodies , or GFP antibodies .HEK293T cells were cultured in 24-well plates in Dulbecco’s modified Eagle’s medium containing 10% FBS. Cells were transfected at 30% confluence with Lipofectamine 3000 (Invitrogen), according to the manufacturer’s recommendations. Cells were cultured 48 h post-transfection, and luciferase activity was analyzed using Luclite plus Reporter Gene Assay (PerkinElmer) measured by Glo-Max Microplate Multimode reader (Promega).HEK293T cells were transfected with the indicated FLAG-Tat and GFP-NPM1 vectors, as described above. The whole cell extracts were prepared using the whole cell lysis buffer supplemented with protease and phosphatase inhibitors (described above). About 300 μg of whole cell extract supplemented with 8 μg of anti-FLAG antibodies was incubated with 5% bovine serum albumin preblocked protein A/G-agarose beads in TNN buffer  at room temperature for 4 h with rocking. The agarose beads were precipitated and washed with TNN buffer. Proteins were resolved on 10% Bis-Tris SDS-PAGE, transferred to polyvinylidene fluoride membrane, and probed with the indicated antibodies.HEK293T cells were transfected with vectors expressing EGFP-tagged Tat and RFP-tagged NPM1, WT, and mutants. At 24 h post transfection, the cells were photographed on Olympus IX51 using a filter for FITC and Texas Red fluorescence at 400× magnification. Quantification of the colocalization was conducted in Image J using JACoP plug-in that allows Pearson’s correlation analysis. The image colors were split, and threshold parameters were adjusted prior to the correlation analysis.5 cells/ml, 100 μl/well) at 37 °C and 5% CO2. The cells were treated with serial dilutions of 1E7-03, Aurora A inhibitor, Barasertib (Aurora B inhibitor), or vehicle (DMSO). At 24 h posttreatment, 100 μl of reconstituted luciferase buffer  was added to each well, incubated for 10 min, and luminescence was measured using Glo-Max Microplate Multimode reader (Promega).CEM T cells were infected with HIV-1-LUC-G and cultured in 96-well white plates (3 × 105) were grown in 96-well plates and treated overnight with serially diluted compounds or DMSO control. Post treatment, 10 μl of MTT solution (ATCC 30–1010K) was added to each well, and the samples were incubated at 37 °C for 2 h. Then, 100 μl of lysis buffer (0.03% HCl with 10% SDS) was added to each well. The cells were incubated for 20 min in the dark. Absorbance was measured at 490 nm at a microplate reader (Bio-Rad Model 680). Each point was measured in triplicate, and serum-free medium with MTT solution was used as a negative control.Cell viability was measured with MTT assay. CEM T cells (3 × 106 cells) were fixed for 5 min at room temperature in 4% paraformaldehyde and then permeabilized for 15 min in cytofix/cytoperm buffer . Cells were stained with p24-PE antibodies  for 1 h at 4 °C in the dark. After staining, the cells were washed and analyzed in BD FACS Calibur (BD Biosciences) using FlowJo software (https://www.bdbiosciences.com/en-us/products/software/flowjo-v10-software).CEM T cells  and CEM T cells infected with VSV-G–pseudotyped HIV-1 virus expressing luciferase  without or with 1E7-03 treatment for 24 h . The treatment time was chosen based on the effect of 1E7-03 on PP1-dependent interaction of PRKA2a and PRKACA which only showed the effect at 24 h and not at 3 h treatment time (A). HIV-1 infection efficiency determined by FACS analysis of CEM T cells stained with anti-p24 antibodies showed that about 14% of CEM T cells were infected (B). Trypsin-digested protein peptides without enrichment  or enriched on Fe-NTA or TiO2 columns  were analyzed by LC-FT/MS followed by label-free quantitative analysis and IPA . We detected peptides phosphorylated on serine and threonine residues and included in the analysis peptides phosphorylated on tyrosine. We observed approximately 1500, 700, and 600 unique phosphopeptides in the nonenriched, TiO2-, and Fe-NTA–enriched individual groups, respectively . Number of specific serine- and threonine-phosphorylated residues were equal in the nonenriched and Fe-NTA–enriched peptide groups, whereas TiO2-enriched peptide group contained primarily serine phosphorylated peptides .To gain an insight on the host proteins phosphorylation induced by 1E7-03, we compared noninfected CEM T cells, untreated or treated with 1E7-03   . In Fe-Np = 3.23 × 10-3), ACPM , HLA-B/C , EGLN1 , ASL , LSM14A , SMAD7 , TAF4 , and several additional proteins , followed by TGF-β2 , TP53 (p53) , HSP90AA1 , and HSP90AB1  .Proteins within the PPARα/RXRα canonical pathway included HLA-B/C, LSM14A, ASPM whose phosphorylation was increased  included TGF-β2 (A), SMAD7 (B), and PD2 (C). Telomerase signaling pathway includes HSP90AA1, HSP90AB1, MAP2K2, and TP53 proteins , SMAD7 B, and PD and PD2 C. Telomeproteins .in silico screening. Only one protein, MAP kinase kinase kinase 4 (MAP3K4), was found to overlap between the predicted PP1 interactors and the phosphoproteins in 1E7-03–treated CEM T cells (A). We also assessed levels of HIV-1 peptides in the infected CEM cells treated with 1E7-03 which showed overall reduction (B).We compared proteins upphosphorylated and downphosphorylated in CEM T cells infected with HIV-1 and treated with 1E7-03 with the previously set of PP1-interacting proteins  that wer T cells A. We alseduction B.Taken together, modulation of protein phosphorylation in multiple pathways including PPARα/RXRα and TGF-β pathways is likely to contribute to HIV-1 inhibition by 1E7-03.A). Volcano map analysis of total 5311 detected proteins  showed 21 upregulated proteins  and 29 downregulated proteins . Thus, phosphorylation of Hsp90AA1 may be affected indirectly through the changes in its expression level. Taken together, 1E7-03 is likely to achieve antiviral effect by inducing protein phosphorylation and dephosphorylation rather than affecting the expression level of host proteins.We next analyzed the effect of 1E7-03 on host protein expression levels by conducting label-free quantitative proteome analysis in HIV-1–infected CEM T cells with or without 1E7-03 treatment. No changes in overall protein expression levels were observed A. VolcanA). To confirm that PP1 was involved in NPM1 phosphorylation, we analyzed endogenous NPM1 phosphorylation in CEM T cells treated with 10 μM 1E7-03 (24 h) or 100 nM OA (2 h), which served as positive control as OA inhibits PP1 at higher 10 nM concentrations and PP2A at lower 3 nM concentration . We also tested the effect of short-term 3 h treatment with 1E7-03 comparing to 24 h treatment. HEK293T cells were transfected with vectors expressing NPM1-GFP or NPM1 S125A-GFP. While 24 h treatments led to decreased NPM1 phosphorylation, there were no changes in NPM1 phosphorylation at 3 h treatment (D and E), We also analyzed the effect of lower concentration of OA, inhibitory for PP2A, on NPM1 phosphorylation using NPM1-GFP expressed in HEK293T cells. Comparing to the reduced NPM1 phosphorylation at 100 nM OA concentration, there was no effect at 3 nM OA treatments (F and G).We next validated NPM1 phosphorylation and assessed its biological effect. We chose NPM1 as its dephosphorylation was the strongest among the phosphorylation changes that were induced by 1E7-03 A. To conntration . NPM1 exreatment , D and Eeatments , F and GA). Coexpression of NPM1 S125D mutant induced Tat-mediated HIV-1 transcription  compared to the expression of WT NPM1. In contrast, expression of nonphosphorylation–mimicking NPM1 S125A mutant had no effect on Tat-activated HIV-1 transcription . As NPM1 was shown to interact with Tat, we tested their binding by coimmunoprecipitation of GFP-tagged NPM1  and Flag-tagged HIV-1 Tat. NPM1 was migrating as 62-kDa monomeric form and a larger 250-kDa form that might represent an oligomeric form of NPM1 or its covalently modified species . Both protein bands were excised and further analyzed by nano LC-MS/MS that confirmed the presence of NPM1 with 69% coverage (A and B). Phosphorylation of Ser-70 and Ser-125 residues was detected in 250-kDa species (B). Analysis of NPM1 acetylation showed that 62-kDa species was both acetylated and phosphorylated, whereas the 250-kDa species was phosphorylated but not acetylated . In contrast, the 250-kDa species of NPM1 showed equal interaction of WT and S125A mutant with Tat but enhanced interaction of NPM1 125D mutant (F and G). These observations suggest that dephosphorylated and acetylated monomeric form of NPM1 might not bind Tat efficiently unless NPM1 is phosphorylated. In the oligomeric nonacetylated form, NPM1 phosphorylation enhances Tat binding. Taken together, these results show that downregulation of NPM1 Ser-125 phosphorylation by 1E7-03 is likely to contribute to HIV-1 transcription inhibition.Previously, NPM1 was shown to facilitate nuclear localization of HIV-1 Tat, thus enhancing viral transcription . HIV-1 tFig. 6coverage , A and B species B. Analysetylated . Analysi50 = 7.8 μM and IC50= 13.7 μM, respectively) (A). The effects of Aurora kinase inhibitors were less pronounced comparing to the effect of 1E7-03 (IC50 = 5.3 μM) (A). Cell viability analysis showed minimal toxicity for 1E7-03 and Barasertib in CEM T cells , whereas Aurora A inhibitor I showed significant toxicity .NPM1 is phosphorylated on Ser-125 by several kinases including Aurora A, Aurora B, and casein kinase 2 (CK2) . To furtctively) A. The ef 5.3 μM) A. Cell vTaken together, this study shows that HIV-1 inhibition by 1E7-03 might be the outcome of complex attenuation of host protein phosphorylation within the PPARα/RXRα and TGF-β pathways that involves dephosphorylation of NPM1.C). NPM1 Ser-125 phosphorylation is likely to be regulated by various kinases and phosphatases including PP1, Aurora A/B, and CK2. As we showed here, PP1-targeting 1E7-03 blocks HIV-1 replication in part by downregulating NPM1 phosphorylation that may impede NPM1–Tat interaction . Decreased NMP1 phosphorylation in the cells treated with 1E7-03 or high concentration of OA indicate that PP1 is involved in the control of NPM1 phosphorylation. However, the effect of PP1 on NPM1 is indirect as NMP1 phosphorylation decreases upon PP1 inhibition. The NPM1 phosphorylation is mediated by one of the NPM1 kinases, and PP1 is likely affecting an upstream kinase. As NPM1 phosphorylation contributes to the inhibition of HIV-1 transcription, targeting NPM1 phosphorylation, indirectly with 1E7-03 or directly with a kinase inhibitor, such as Barasertib, can serve as a future avenue for HIV-1 transcription inhibition.HIV-1 replication takes advantage of posttranslational modifications of many cellular proteins, including NPM1 , HSP90AA1, TGF-β2, and NPM1. HLA-B∗27, HLA-B∗57, and HLA-B∗58 are well-known protective alleles that contribute to the suppression of HIV-1 replication and prevent progression toward AIDS , through the SMAD signaling pathway .The mass spectrometry raw data and Proteome Discoverer processed files are uploaded to UCSD Massive Server (This article contains The authors declare no competing interests. SN is a US patent holder (US8278326B2) for inhibitors of PP1 that have been shown to slow replication of HIV-1.


Determining how HIV-1 shapes its niche to create a permissive environment is central to informing efforts to limit pathogenesis, disturb reservoirs, and achieve a cure. A key roadblock in understanding HIV-T cell interactions is the requirement to activate T cells in vitro to make them permissive to infection. This dramatically alters T cell biology and virus-host interactions. Here we show that HIV-1 cell-to-cell spread permits efficient, productive infection of resting memory T cells without prior activation. Strikingly, we find that HIV-1 infection primes resting T cells to gain characteristics of tissue-resident memory T cells (TRM), including upregulating key surface markers and the transcription factor Blimp-1 and inducing a transcriptional program overlapping the core TRM transcriptional signature. This reprogramming is driven by Vpr and requires Vpr packaging into virions and manipulation of STAT5. Thus, HIV-1 reprograms resting T cells, with implications for viral replication and persistence.HIV-1 replicates in CD4  •+ T cells permissive to HIV-1 infectionCell-to-cell spread makes resting CD4•Resting memory T cells are productively infected by HIV-1•HIV-1 Vpr drives resting memory T cells to gain characteristics of tissue residency•Vpr induces widespread transcriptional reprogramming of infected T cells in vitro activation. They discover that HIV-1 Vpr reprograms resting T cells phenotypically and transcriptionally to gain characteristics of tissue-resident memory T cells, suggesting that HIV-1 drives changes that may affect viral reservoir establishment and persistence.Reuschl et al. show that HIV-1 cell-to-cell spread promotes productive infection of resting T cells, bypassing the need for  Consistr virus) M and S2Lr virus) M, (2) prr virus) N, and (3r virus) O. Activar virus) M and S2Nr virus) K and S1L+ target T cells infected by cell-to-cell spread maintained their resting phenotype and did not upregulate Ki67 or MCM2, two markers of cell-cycle progression  T cells  cells  cells , consistnduction . Criticaegulated P and S3Q T cells . Inducti Blimp-1   following antigenic-stimulation  mRNA  mRNA E. As exppr virus I. Vpr al T cells F, characRM cells . Importa+ T cells infected with different Vpr mutants revealed that the cell-cycle arrest mutants S79A and R80A behaved similar to WT virus and induced CD69 and CXCR6 upregulation  bind the Cul4A-DDB1 (DCAF1) complex, leading to an interaction with the ubiquitinylation and proteasomal machinery; (2) induce G2/M cell-cycle arrest; and (3) drive apoptosis in infected cells . We abrogulation J. By coneriments J.RM-like phenotype was not inhibited by potently suppressing HIV-1 integration into resting memory CD4+ T cells using the integrase inhibitor raltegravir (+ T cells at approximately one provirus per infected cell (+ T cell infection (PM) cannot package Vpr into virions, but retains an intact vpr gene, allowing for de novo Vpr synthesis from the viral genome following infection, and maintains the capacity for cell-to-cell spread (PM) , and confirmed sufficient Vpr delivery for degradation of a known Vpr target, UNG2, upon spinoculation  E. Secondpresence G and 4H,presence G, of IL-+ T cells infected with HIV-1 WT or ΔVpr virus by cell-to-cell spread in the presence and absence of IL-7. HIV-1 infection alone induced widespread changes in gene expression in resting memory T cells compared with uninfected cells   . HierarcWT virus E. In facWT virus E and 5F.WT virus ; Data S1+ T cells driven largely by Vpr.To gain greater insight into the native effects of HIV-1 and Vpr on T cells, gene set enrichment analysis (GSEA) G and ingRM gene signature was enriched in our transcriptome of HIV-1-infected resting memory T cells. Hierarchical clustering of our data comparing with the core gene signature of TRM cells (CD69+ T cells isolated from human lung and spleen)  that approximated closely with that of bona fide TRM cells   showed tRM cells  and wereRM cells B and wasRM-like phenotype in resting memory T cells and primes cells to become hyperresponsive to IL-7, we hypothesized that Vpr may do so by manipulating JAK-STAT signaling, the pathway downstream of IL-7 signals. In support of this, GSEA of our RNA-seq data revealed increased expression of genes regulated by the transcription factor STAT5   D and 7E P-STAT5) F–7J or uP-STAT5) K, confirAC-4-130 . Strikinnd CXCR6 L and S5Ohenotype I.Figure + T cells can be productively infected by cell-to-cell spread, allowing for viral integration, replication, and dissemination, transforms our ability to determine how T cells respond to and support HIV-1 replication without confounding activation-induced changes. Here, employing our co-culture model, we have revealed that HIV-1 infection of resting memory T cells induces T cell reprogramming, resulting in these cells gaining characteristics that are associated with TRM T cells, a phenotype that we have termed TRM-like. This is evidenced by the upregulation and co-expression of TRM-associated marker proteins on infected cells , induction of a core TRM transcriptional signature, and the gain of functional characteristics associated with TRM cells. HIV-1 establishes cellular and tissue reservoirs, both active and latent, that ultimately prevent cure with anti-retroviral therapy. Importantly, TRM cells are long-lived and are thought to be largely confined to tissue .Further information and requests for resources and reagents should be directed to the lead contact, Professor Clare Jolly (This study did not generate new unique reagents.+ T Cell Isolation kit (Biolegend) according to the manufacturer’s instructions. CD45RA+ naïve and CD45RA- memory populations were further separated after CD4+ T cell isolation with CD45RA MicroBeads (Biolegend). For activated CD4+ T cells, PBMCs were treated with 5 μg/mL PHA (Sigma) and 10 IU/mL IL2  in RPMI1640 with 20% FBS for 72 h prior to CD4+ T cell isolation. Once purified, CD4+ T cells were cultured in RPMI supplemented with 20% FBS and 10 IU/mL IL2. Jurkat T cell lines  were cultured in RPMI with 10% FBS and 100 U/mL penicillin/streptomycin. HEK 293 T/17 cells  were cultured in DMEM with 10% FBS and 100 U/mL penicillin/streptomycin. Tonsil tissue was obtained from an individual with primary HIV infection who underwent routine tonsillectomy (2 months after commencement of ART) or from healthy donors during routine tonsillectomy. As previously described  were isolated from buffy coats from healthy donors (UK NHS Blood and Transplant Service) by density centrifugation using FicollPaque Plus (GE Life Sciences) and cryopreserved in 10% DMSO (Sigma-Aldrich) in 90% FBS (LabTech). Resting CD4+ T cells were isolated from total PBMCs by negative selection using the MojoSort Human CD4escribed , the tonEnv was provided by G. Towers  (PM) and the parental wildtype pNL4-3unc (termed HIV-1 WTPM) were provided by K. Bishop . NL4.3 Vpr Q65R, NL4.3 Vpr S79A, NL4.3 Vpr R80A were generated by site-directed mutagenesis (Promega) using the following primers:NL4.3 VprQ65R fw:GTGGAAGCCATAATAAGAATTCTGCGACAACTGCTGTTTATCCATTTCAGNL4.3 VprQ65R rv:CTGAAATGGATAAACAGCAGTTGTCGCAGAATTCTTATTATGGCTTCCACNL4.3 Vpr S79A fw: GAATTGGGTGTCGACATGCCAGAATAGGCGTTACTCNL4.3 Vpr S79A rv:GAGTAACGCCTATTCTGGCATGTCGACACCCAATTCNL4.3 Vpr R80A fw: GGTGTCGACATAGCGCAATAGGCGTTACTCGNL4.3 Vpr R80A rv: CGAGTAACGCCTATTGCGCTATGTCGACACC.The HIV-1 clone pNL4.3 was obtained from the CFAR, NIBSC (cat# 2006). HIV-1 NL4.3 ΔNef and pNL4.3 ΔVpr were provided by R. Sloan  . NL4.3 ΔUCL, UK) . CCR5 tr6 HEK 293 T cells with 10 μg pNL4.3 ΔVpr and 2 μg Flag-tagged NL4.3 Vpr in pcDNA3.1 . For Env-VLP production, 20 μg p8.91 was co-transfected with 10 μg plasmid encoding the HIV-1 T/F envelope pWEAU_d15_410_5017 HIV-1 envelope  and 2 μg pcDNA3.1 with or without Flag-tagged NL4.3 Vpr. Viral and VLP titres were determined by measuring reverse transcriptase activity by SG-PERT assay (All virus and VLP stocks were produced by plasmid transfection of HEK 293 T cells with Fugene 6 (Promega). Supernatants were harvested at 48 h and 72 h, filtered, DNase treated, purified and concentrated by ultracentrifugation through a 25% sucrose cushion and resuspended in RPMI1640 with 10% FBS. Trans-complementation of HIV-1 ΔVpr was performed by co-transfecting 10RT assay .6 cells of HIV-1 by spinoculation at 1200xg for 2 h at room temperature and incubated in RPMI 20% FBS supplemented with 10 IU/mL IL2 for 72 h. HIV-1+ donor CD4+ T cells were washed with medium, counted and cultured with autologous primary CD4+ target T cells at a 1:1 ratio in RPMI 20% FBS supplemented with 10 IU/mL IL2 for up to 72 h before analysis by flow cytometry or FACS sorting. Uninfected target CD4+ T cells were pre-stained with 1-2 nM CellTrace FarRed dye (Invitrogen) prior to co-culture. For cell-to-cell spread into tonsil-derived lymphocytes, total tonsil lymphocytes were cultured at a 4:1 ratio with HIV-1 infected or uninfected eFluor450-labelled Jurkat T cells. For FACS sorting experiments, donor cells were pre-labeled with cell dye eFluor450 (ThermoFisher). For transwell experiments, HIV-1 infected donor T cells were separated from target T cells by a 0.4 μm transwell insert (Corning). Experiments to quantify cell-to-cell versus cell-free infection in the presence and absence of a transwell were performed in equivalent volumes (600 μL). For some experiments, FACS sorted infected resting CD4+ target T cells were returned into cultured for up to 4 days. Infection levels were measured by intracellular Gag staining and flow cytometry, and virus release into cell culture supernatant determined by SG-PERT , 20 ng/mL IL15 (Peprotech), 20 ng/mL IL12 (Peprotech) or 50 ng/mL TGFβ (Peprotech). The following inhibitors were added 30 min before co-culture at the following concentrations: T20 , Efavirenz , Raltegravir  and Ruxolitinib .For cell-to-cell spread experiments, activated primary CD4+ T cells (donor cells) were infected with 800 mU reverse transcriptase per 10 SG-PERT . At day + T cells were incubated with 200–800 mU of virus or Env-VLPs for 15 min at room temperature and subsequently spinoculated at 1200xg for 2 h at room temperature. Cells were then cultured as described above.For delivery of Vpr by spinoculation, resting CD4+T cells were activated for 4 days with 1 μg/mL plate-bound αCD3 antibody  in the presence of 2 μg/mL soluble αCD28 antibody . RNAi knockdown of DCAF1 was performed as described before . After 48 h, DCAF1 knockdowns were confirmed by western blotting and cells used in cell-to-cell spread experiments as described above.For RNAi knockdown of DCAF1, primary CD4d before  using ON+ T cells after 72 h of cell-to-cell spread, cells were treated throughout the co-culture with IL-7 and treated with Brefeldin A (Biolegend) for 6 h, if not stated differently, before surface staining and fixation. Where indicated, cells were stimulated with 100 ng/mL PMA (Sigma-Aldrich) and 100 ng/mL Ionomycin (Sigma-Aldrich) for the duration of the Brefeldin A treatment. Permeabilisation for intracellular staining was performed with IC perm buffer or FoxP3 Buffer set (Biolegend) according to the manufacturer’s instructions. For detection of intracellular P-STAT5, cells were resuspended in ice cold True-Phos Perm buffer (Biolegend) and permeabilised for 48 h at −20°C. Intracellular P-STAT5 staining was then performed in PBS with wash steps performed at 1800 rpm for 6 min at 4°C.The following antibody clones and fluorochromes were used: CD3 , CD8 , CD4 ; CD45RA ; CD45RO , CD62L , CD69 ; CXCR6/CD186 ; MCM2 ; HLA-DR ; CD25 , CD38 , CD49a ; PD-1 ; Ki67 ; Blimp-1 ; CD101 ; CX3CR1 ; CD103 ; CD127 , IFNγ , Phospho-STAT5 (Clone 47/Stat5 (pY694), BD; PE), Flag-tag  and HIV-1 Gag core antigen . All samples were acquired on either a BD Fortessa X20 or LSR II using BD FACSDiva software and analyzed using FlowJo v10 (Tree Star). Flow cytometry sorting (FACS) was performed with a BD FACSAria III or BD FACSAria IIu Cell Sorter. Cells were either lysed immediately in RLT lysis buffer (Qiagen) with 1% β-mercaptoethanol (Sigma-Aldrich) and stored at −80°C for later RNA extraction or resuspended in RPMI supplemented with 20% FBS and 10 IU/mL IL2 and used immediately.For flow cytometry analysis, cells were washed in PBS and stained with fixable Zombie UV Live/Dead dye, Aqua Live/Dead dye or NIR Live/Dead dye (Biolegend) for 5 min at 37°C. Excess stain was quenched with FBS-complemented RPMI. When tonsil and lymph node lymphocytes were used, Live/Dead staining was quenched using human AB serum (Sigma) in RPMI. Cell surface staining was performed in PBS, complemented with 20% Super Bright Staining Buffer (ThermoFisher) when appropriate, at 4°C for 30 min. Unbound antibody was washed off thoroughly and cells were fixed with 4% FA or PFA before intracellular staining. For intracellular detection of cytokines in infected target CD4Virus-containing supernatants  or 15 μg of total CD4+ T cell protein lysate were separated by SDS-PAGE, transferred onto nitrocellulose and blocked in PBS with 0.05% Tween 20 (v/v) and 5% skimmed milk (w/v). Blots were probed with rabbit antisera raised against HIV-1 Gag p24 (cat# 0432 donated by Dr G. Reid and obtained from the CFAR), Vpr anti-serum , α−P-STAT5 (Tyr694) , α-STAT5 , α-beta-Actin , α-UNG , α−alpha-Tubulin  and α-DCAF1 antibody , followed by goat anti-rabbit or goat anti-mouse IRdye 800CW or 680RD infrared secondary antibody (Abcam) and imaged using an Odyssey Infrared Imager (LI-COR Biosciences) and analysed with Image Studio Lite software.To quantify integration of HIV-1 in resting T cells, nested Alu-gag quantitative PCR was performed as previously described . BrieflyAlu fw: GCCTCCCAAAGTGCTGGGATTACAGHIV-1 Gag rv: GTTCCTGCTATGTCACTTCCRU5 fw: TTAAGCCTCAATAAAGCTTGCCRU5 rv: GTTCGGGCGCCACTGCTAGARU5-WT probe: FAM-CCAGAGTCACACAACAGACGGGCACA-TAMRARU5-degenerate 1 probe: FAM-CCAGAGTCACATAACAGACGGGCACA-TAMRARU5-degenerate 2 probe: FAM-CCAGAGTCACACAACAGATGGGCACA-TAMRA2LTR fw: AACTAGAGATCCCTCAGACCCTTTT2LTR rv: CTTGTCTTCGTTGGGAGTGAAT2LTR probe: FAM-CTAGAGATTTTCCACACTGAC-TAMRAIntegrated DNA was pre-amplified using 100 nM Alu fw primer, 600 nM HIV-1 Gag rv primer, 0.2 mM dNTP, 1 U Phusion Hot Start Flex (Promega), and 45 ng DNA in 50 μL reactions. Cycling conditions were: 94°C for 30 s, followed by 40 cycles of 94°C for 10 s, 55°C for 30 s, and 70°C for 2.5 min. For quantitation of HIV-1 integration, a second round real-time quantitative PCR was performed using the pre-amplified DNA. These samples were run alongside a standard curve of known dilutions of CEM cells containing integrated HIV-1 DNA. Reactions contained 0.25 μM of RU5 fw and rv primers, and 0.2 μM probes, 1× Qiagen Multiplex Mastermix, and 10 μL pre-amplified DNA. Cyclin conditions were: 95°C for 15 min, followed by 50 cycles of 94°C for 60 s and 60°C for 60 s. 2LTR circles were measured by quantitative PCR . Reactio+ T cells with RNeasy Micro Kit (Qiagen) according to the manufacturer’s instructions. cDNA was synthesised using SuperScript IV with random hexamer primers (Invitrogen) and qRT-PCR was performed using Fast SYBR Green Master Mix and 7500 Real-Time PCR System (Applied Biosystems). Gene expression was determined using the 2−ΔΔCt method and normalised to GAPDH expression. The following primers were used:GAPDH fw: ACATCGCTCAGACACCATG, rv: TGTAGTTGAGGTCAATGAAGGG;CXCR6 fw: GACTATGGGTTCAGCAGTTTCA, rv:GGCTCTGCAACTTATGGTAGAAG;PRDM1 fw: ATGCGGATATGACTCTGTGGA, rv: CTGAACCGAAGTACCGCCATC;CD69 fw: ATTGTCCAGGCCAATACACATT, rv: CCTCTCTACCTGCGTATCGTTTT;S1PR1 fw: TCTGCTGGCAAATTCAAGCGA, rv: GTTGTCCCCTTCGTCTTTCTG;KLF2 fw: CTACACCAAGAGTTCGCATCTG; rv: CCGTGTGCTTTCGGTAGTG.RNA was extracted from FACS sorted target memory CD4+ T cells were spinoculated with HIV-1 WT or ΔVpr virus and incubated in the presence or absence of 1 ng/mL IL-7. At 24 h, cells were adhered onto poly-L-lysine tissue-culture treated CellCarrier Ultra plates (Perkin Elmer) for 1 h and subsequently formaldehyde fixed. For staining, a blocking step was carried out for 1 h at room temperature with 10% goat serum/1% BSA in PBS. STAT5 and P-STAT5 detection was performed by primary incubation with rabbit α-P-STAT5 (Tyr694)  or rabbit α-STAT5  for 18 h at 4°C and washed thoroughly in PBS. STAT5 or P-STAT5 staining was followed by incubation with mouse α-Gag  for 18 h at 4°C. Primary antibodies were detected with secondary α-rabbit-AlexaFluor-488 and α-mouse-AlexaFluor-568 conjugates (Jackson Immuno Research) for 1 h at room temperature. All cells were labeled with Hoechst33342 . Images were acquired using the WiScan® Hermes 7-Colour High-Content Imaging System  at magnification 60X/1.2NA. Three channel automated acquisition was carried out sequentially. Images were acquired across a well area density resulting in 350–500 FOV/well and 10–20,000 cells. For image analysis, P-STAT5 and STAT5 channels were pre-processed by applying a batch rolling ball background correction in FIJI ImageJ software package  according to the manufacturer’s instructions. For preparation of RNA-Sequencing libraries, RNA concentration was measured using the Qubit RNA High Sensitivity kit (Life Technologies) and quality checked on the 4200 Tapestation using either the High Sensitivity or standard RNA ScreenTape assay (Agilent Technologies), depending on the measured RNA concentrations. PolyA-tailed mRNA was separated for sequencing during library preparation. Libraries were prepared using KAPA’s mRNA HyperPrep kit (Roche Diagnostics) according to the manufacturer’s instructions using an input of up to 200 ng and a fragmentation incubation time of 8 min at 94°C. Samples were sequenced on Illumina’s NextSeq500 (Illumina Cambridge) using a high output 75 cycle paired-end run. 24 libraries were multiplexed in the same run. Libraries were pooled in equimolar quantities, calculated from concentrations measured using the Qubit dsDNA High Sensitivity kit (Life Technologies) and fragment analysis using the D1000 High Sensitivity assay on the 4200 Tapestation (Agilent Technologies).RNA was extracted from FACS sorted target memory CD4https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) before and after low-quality and adapter trimming using Trimmomatic (http://aug2020.archive.ensembl.org/index.html). Per-transcript counts were imported and aggregated per gene using the TXimport R package  (https://biit.cs.ut.ee/clustvis/)   as descrs (GSEA)  and Ingeustvis/) RM cells (GSE94964)   were sumSE94964) . TPM valg Combat . Expressmbat (RM  were usembat (RM . A transt-tests (when comparing two groups) or one-way ANOVA with Bonferroni or Dunnett’s post-test (when comparing more than two groups). Data show the mean +/− the S.E.M with significance shown on the figures. Where appropriate, the median + IQR is shown and Kruskall-Wallis test was used to compare groups. Significance levels were defined as ∗, p < 0.05; ∗∗, p < 0.01 and ∗∗∗, p < 0.001.Statistical analysis was performed using GraphPad Prism. Normally distributed data was analyzed for statistical significance by two-tailed


A threonine to serine mutation at position 35 (T35S) shifts the interconverting equilibrium to State1 conformation and enables the emergence of such residues in the 2D 1H–15N HSQC spectrum due to gained conformational rigidity. We report here the 1HN, 15N, and 13C backbone resonance assignments for the 19.2 kDa (AA 1–169) protein constructs of KRAS-GppNHp harboring T35S mutation (KRAST35S/C118S-GppNHp) and of its oncogenic counterpart harboring the Q61L mutation (KRAST35S/Q61L/C118S-GppNHp) using heteronuclear, multidimensional NMR spectroscopy at 298 K. High resolution NMR data allowed the unambiguous assignments of 1H–15N correlation cross-peaks for all the residues except for Met1. Furthermore, 2D 1H–15N HSQC overlay of two proteins assisted in determination of Q61L mutation-induced chemical shift perturbations for select residues in the regions of P-loop, Switch-II, and helix α3.RAS proteins cycling between the active-form (GTP-bound) and inactive-form (GDP-bound) play a key role in cell signaling pathways that control cell survival, proliferation, and differentiation. Mutations at codon 12, 13, and 61 in RAS are known to attenuate its GTPase activity favoring the RAS active state and constitutively active downstream signaling. This hyperactivation accounts for various malignancies including pancreatic, lung, and colorectal cancers. Active KRAS is found to exist in equilibrium between two rapidly interconverting conformational states (State1–State2) in solution. Due to this dynamic feature of the protein, the The online version contains supplementary material available at 10.1007/s12104-021-10050-7. RAS is the most frequently mutated oncogene in cancer. RAS proteins exhibit a molecular “on” and “off” cycling mechanism to render RAS in its active form (GTP-bound), and in its inactive form (GDP-bound), respectively. Of the three major isoforms of RAS, mutant KRAS protein plays a causal role in three major human cancers: lung, colorectal, and pancreas , and a C-terminal hypervariable region (comprising ~ 19 AA) anchored to the cell membrane. The canonical RAS members  share highly conserved structural elements within the G domain. These structural elements include the flexible regions of P-loop (AA 10–17) that binds nucleotide phosphates, and Switch-I  and Switch-II  that interact directly with effector proteins, such as Raf, PI3K, and RalGDS, and the guanine nucleotide exchange factors (GEFs). However, the majority of residues from the flexible P-loop, SW-I, and SW-II regions are undetectable in the 2D 1H–15N correlation cross-peaks from these flexible regions in the 2D 1H–15N HSQC spectrum. T35S KRAS-GTP, therefore, could be used for the detection of ligand-binding induced residue perturbations for all the backbone amide signals, and thus, unambiguous determination of ligand binding site(s). Availability of NMR analysis becomes important in the discovery pipeline, especially for those inhibitors that upon binding shift the equilibrium conformation of KRAS-GTP to the “State1 like” conformation. In a G12D KRAS-GTP ligand screening campaign reported recently, the T35S mutation was found important in reducing the likelihood of the protein being constitutively active in the protein-complexes , and of the Q61L oncogenic mutant T35S/Q61L/C118S KRAS4b(1–169) (named KRasT35S/Q61L/C118S-GppNHp hereafter). From these assignments we determine the residues that elicit the Q61L-induced conformational changes in the protein.We applied NMR methods to understand the structural differences between the wildtype (WT) and a Q61 oncogenic mutant of KRAS-GppNHp in solution. We present here, NMR backbone assignments and secondary structures of the T35S/C118S mutant of KRAS4b(1–169)-GppNHp  E. coli strain containing the DE3 lysogen and rare tRNAs  was transformed with the expression plasmids (AmpR).Gateway Entry clones for KRAS4b(1–169) T35S/C118S and KRAS4b(1–169) T35S/Q61L/C118S were generated by standard cloning methods and incorporate an upstream tobacco etch virus (TEV) protease cleavage site (ENLYFQG) followed by the appropriate KRAS sequences. Sequence validated Entry clones were sub-cloned into pDest-566, a Gateway Destination vector containing a His6 and maltose-binding protein (MBP) tag to produce the final 13C glucose as needed, 1 g/L 15N-NH4Cl, 2 mM MgSO4, 100 μM CaCl2, 4 μM ZnSO4, 1 μM MnSO4, 4.7 μM H3BO3 and 0.7 μM CuSO4) was prepared in a 20 L Bioflow IV bioreactor (Eppendorf/NBS). 15N-labeled protein was expressed using 15N-NH4Cl and 12C-glucose as sole N and C sources in the expression medium. When the seed culture reached mid-log phase, the culture was removed, centrifuged at 3000×g for 10 min at 25 °C, the pellet resuspended with 100 mL of ModM9 medium pulled from the bioreactor, and returned to the bioreactor as inoculum. The culture was grown at 37 °C with agitation of 350 RPM. When the OD600 reached 0.4–0.6 (~ 3 h), IPTG was added to a final concentration of 500 μM and the temperature adjusted to 16 °C. After 21 additional hours of growth the cells were collected by centrifugation (5000×g for 10 min at 4 °C). Cell pellets were immediately frozen at − 80 °C.Seed cultures were inoculated from glycerol stocks of the transformed strains into 300 mL of the basal medium and buffer of Studier’s MDAG135 medium in a 2 L baffled shakeflask for 16 h at 37 °C until mid-log growth. In the interim, 15 L of ModM9 medium . Briefly, 30 molar excess of GppNHp solution to protein  and 2 M (NH4)2SO4 solution were prepared in KRAS buffer. (NH4)2SO4 was added to protein sample to achieve 200 mM final concentration. Subsequently a total of 10% of GppNHp stock solution was mixed with protein sample. Alkaline phosphatase on agarose beads  were added to the reaction mixture . Reaction was allowed to occur at RT for 1.5 h. Subsequently, the alkaline phosphatase on agarose beads was removed by filtration using 0.22 µm Millex-GP 33 mm syringe filters . Remaining 90% of stock GppNHp solution was then mixed with agarose free sample of KRAS protein and set for incubation for next 30 min. 0.22 Micron filtered sample was then FPLC purified from low molecular components using 5 × 5 mL (each 16 × 25 mm) Cytiva HiTrap desalting columns . A total of 3 mL sample (a maximum of 15 mg protein per injection) was injected into 5 mL loop for each run. All purified proteins showed apparent purity of > 95% as detected by Coomassie Blue Staining after SDS-PAGE. The GppNHp-bound state of the purified protein was ascertained by HPLC and mass spectrometry. Sample concentration was measured using a NanoDrop One microvolume UV–Vis spectrophotometer .KRAS4b(1–169) proteins were purified as outlined , 50 mM NaCl, 100 mM KCl, 1 mM TCEP-d16, 2 mM MgCl2, 100 µM 2,2-dimethyl-2-silapentanesulfonic acid (DSS) as internal standard, and 0.02% (w/v) NaN3 to avoid any unwanted bacterial growth over time. A total of 320 μL sample volume was used for each sample in a standard Shigemi tube.in-house) and on a Bruker Avance III HD 750 MHz spectrometer equipped with a 5-mm TXI (Z-axis gradient) cryoprobe (NMRFAM). All data were collected at 298 K in the gradient-selected sensitivity-enhanced mode. To accomplish backbone 1HN, 15N, 13Cα, 13Cβ, and 13CO assignments, NMR data of 1D 1H, 2D 1H–15N HSQC, and triple-resonance experiments of HNCACB, CBCA(CO)NH, HNCA, HN(CO)CA, and HNCO were collected. For KRAST35S/Q61L/C118S-GppNHp the HN(CA)CO was also collected. Assignments were further validated in (H)C(CO)NH (2048 × 40 × 96) and 15N-edited TOCSY-HSQC (2048 × 40 × 128) . Spectra were visualized and analyzed using CCPNMR analysis  v 3.0  from the equation: [∆δ = {(∆H)2 + (∆N/5)2}0.5]. A cut-off value of 0.146 ppm  was used in the data analysis. Overlay of 2D 1H–15N HSQC spectra (700 MHz data) was analyzed in CCPNMR and in NMRDraw. Data were collected and processed in an identical manner for both the protein constructs.The T35S/C118S-GppNHp structure was performed with Modeller (v9.24) . The structure model with the lowest DOPE score was chosen as representative structure of K-RasT35S/C118S-GppNHp. Figure The homology modeling of the KRAS1H, 13C, and 15N, chemical shifts assignments of KRAST35S/C118S-GppNHp and KRAST35S/Q61L/C118S-GppNHp have been deposited into BMRB (http://www.bmrb.wisc.edu/). Assigned 2D 1H–15N HSQC spectrum of KRAST35S/C118S-GppNHp and of KRAST35S/Q61L/C118S-GppNHp are shown in Fig. 1H–15N correlation cross-peaks as witnessed in the 2D 1H–15N HSQC spectra of KRAST35S/C118S−GppNHp  were identified and assigned for both the proteins, except for their first methionine residue that could not be visualized presumably due to the exchange of its 1HN proton with the bulk solvent. For KRAST35S/C118S-GppNHp, 13Cα peaks for all 169 residues were assigned. 13Cβ signals were not observed for Ser17, Thr20, and Thr158 (155 out of 158 were observed and assigned), whereas 13C’ (carbonyl) peaks could not be assigned for the 4 residues preceding prolines  and for Lys169 due to unavailability of HN(CA)CO data. KRAST35S/Q61L/C118S-GppNHp also exhibited well resolved spectral data. All 169 resonances of the 13Cα peaks of the Q61L protein were assigned. As noted in non-Q61L counterpart, 13Cβ signals were not detected for residues Ser17, Thr20, and Thr158 (98.1% completion). 13C’ peaks, on the other hand, were assigned for all residues, except for Ile93. Overall, the T35S mutation enables the observation of 1H–15N correlation cross-peak for residues at positions 7, 10, 12, 13, 16, 21, 22, 29, 30–33, 35–42, 54–55, 57–73, 76, 94 in the 2D 1H–15N HSQC spectrum, which are usually absent in the data collected at room temperature and near the solution pH used here.The 1H, 15N, 13Cα, 13Cβ, and 13C’ chemical shifts in the programs Chemical Shift Index (CSI) v 3.0 , α 1 (16–24), β 2 (39–46), β 3 (50–57), α 2 (66–73), β 4 (77–83), α 3 (87–103), β 5 (111–115), α 4 (127–136), β 6 (140–143), α 5 (152–166) , as produced by TALOS-N, represents the conformational rigidity adopted by the particular residue in a protein structure. Such a plot of RCI-S2 of KRAST35S/C118S-GppNHp and of KRAST35S/Q61L/C118S-GppNHp residues is shown in Fig. 2 value of ≥ 0.8 adopted by the above-mentioned structured elements is reflective of a relatively rigid backbone conformation of such residues, in contrast to the flexible loop regions . Notably, the loops in the SW-I and SW-II regions are relatively more flexible than those loops in other parts of protein conformation in both the cases. AA residues 31–38 in SW-I region exhibit relatively lower values for KRAST35S/Q61L/C118S-GppNHp than noted for KRAST35S/C118S-GppNHp , as carried out from the spectral overlay, identifies sixteen residues exhibiting significant perturbations due to the Q61L mutation. As shown in Fig. T35S/C118S-GppNHp  is oriented away from, or packs closer to helix α 3 in solution. These structural investigations will also inform about the conformational variability of the SW-II region attributed to the Q61L mutation, and thus be helpful in characterizing the size(s) of the potential pockets involving the SW-II region  in this oncogenic construct vs that present in the non-Q61L counterpart protein. Moreover, as the residues Gly60-Tyr64 are mostly disordered in the crystal structures of the active form of oncogenic K-RAS (including the Q61L mutant), the solution structure of KRAST35S/Q61L/C118S-GppNHp would provide clues to the conformational packing elicited by these residues.Although it is apparent that Leu61 is structurally proximal to residues Gly60, Glu62, Glu63, and Ala11, the chemical shift differences for other P-loop and switch-2 residues , and α 3 residues are noteworthy. It would be interesting to understand how the local conformations of the Leu61 and other perturbing residues are packed with respect to each other in the three-dimensional solution structure of KRAST35S/C118S-GppNHp and KRAST35S/Q61L/C118S-GppNHp reported here will be helpful to future NMR studies pertaining to drug discovery efforts that involve identification of ligand binding sites for small molecules with detectable affinity regime. Q61L KRAS-GTP exhibits significantly lower rates of intrinsic hydrolysis among the common oncogenic KRAS mutants Below is the link to the electronic supplementary material.


Correction to: Nano-Micro Lett. (2022) 14:197 10.1007/s40820-022-00938-xIn the original publication, the name of third author has been published incorrectly as “Sánchez del Río Saeza, J” and this has corrected as “Sánchez del Río Sáez, J”.The original article has been updated.


Escherichia coli and Salmonella pullorum are two important groups of zoonotic pathogens. At present, the treatment of intestinal pathogenic bacteria infection mainly relies on antibiotics, which directly inhibit or kill the pathogenic bacteria. However, due to long-term irrational, excessive use or abuse, bacteria have developed different degrees of drug resistance. N6, an arenicin-3 derivative isolated from the lugworm, has potent antibacterial activity and is poorly resistant to enzymatic hydrolysis and distribution in vivo. Polyethylene glycol (PEG) is an extensively studied polymer and commonly used in protein or peptide drugs to improve their therapeutic potential. Here, we modified the N-/C-terminal or Cys residue of N6 with liner PEGn of different lengths , and the effects of PEGylation of N6 on the stability, toxicity, bactericidal mechanism, distribution and efficacy were investigated in vitro and in vivo.Enteropathogenic n = 2, N6-COOH-miniPEG) had potent activity against Gram-negative bacteria; PEGylated N6 at the N-terminus and Cys residues showed low or no activity with increasing lengths of PEG. N6-COOH-miniPEG has higher stability in trypsin than the parent peptide-N6. N6-COOH-miniPEG significantly regulated cytokine expression in lipopolysaccharides (LPS)-induced RAW 264.7 cells, and the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-1β were reduced by 31.21%, 65.62% and 44.12%, respectively, lower than those of N6 ; N6-COOH-miniPEG increased the level of IL-10 (37.83%), higher than N6 (-10.21%). The data indicated that N6-COOH-miniPEG has more potent anti-inflammatory and immune-regulatory effect than N6 in LPS-stimulated RAW 264.7 cells. N6-COOH-miniPEG exhibited a much wider biodistribution in mice and prolonged in vivo half-time. FITC-labeled N6-COOH-miniPEG was distributed throughout the body of mice in the range of 0.75 – 2 h after injection, while FITC-labeled N6 only concentrated in the abdominal cavity of mice after injection, and the distribution range was narrow. N6-COOH-miniPEG improved the survival rates of mice challenged with E. coli or S. pullorum, downregulated the levels of TNF-α, IL-6, IL-1β and IL-10 in the serum of LPS-infected mice, and alleviated multiple-organ injuries , superior to antibiotics, but slightly inferior to N6.The antimicrobial activity of the peptide showed that PEGylated N6 at the C-terminus (The antibacterial activity, bactericidal mechanism and cytotoxicity of N6-COOH-miniPEG and N6 were similar. N6-COOH-miniPEG has a higher resistance to trysin than N6. The distribution of N6-COOH-miniPEG in mice was superior to that of N6. In exploring the modulatory effects of antimicrobial peptides on cytokines, N6-COOH-miniPEG had stronger anti-inflammatory and immunomodulatory effects than N6. The results suggested that C-terminal PEGylated N6 may provide an opportunity for the development of effective anti-inflammatory and antibacterial peptides.The online version contains supplementary material available at 10.1186/s12866-022-02534-w. Escherichia coli and Salmonella are common pathogens of bacterial disease infections in livestock and poultry farming, which can cause diarrhea diseases and even sepsis in both animals and humans  with molecular weights (MWs) from 0.4 to 150 kDa; PEG has been used in pharmaceutical fields, consumer products, and cosmetics for decades  elastase , magainielastase  and Bac7elastase  improvedelastase , 30, proelastase , 32, andelastase , 34; theelastase . Noticeaelastase . Howevern = 2, 6, 12, and 24) with MWs from 145 to 1,127 Da to the N-terminus, C-terminus and Cys residues at positions 7 and 16 of a marine AMP-N6 (GFAWNVCVYRNGVRVCHRRAN), an AMP with potent bactericidal activity against Gram-negative bacteria in our previous study, but issues with protease sensitivity  × 100.Erythrocytes from SPF mice were centrifuged at 1,500 rpm for 5 min and washed with 0.9% NaCl three times. 75 μL of 8% (v/v) erythrocyte solution was mixed with 75 μL of N6 or N6-COOH-miniPEG ; the mixture was added into 96-well plates, incubated at 37 °C for 1 h, and centrifuged at 1,500 rpm for 5 min. The supernatants were measured at 540 nm. 0.9% NaCl and 0.1% Triton X-100 were used as the blank  were seeded into a 96-well plate (100 μL/well) and cultured at 37 °C  for 24 h. The medium was then removed and the cells were washed twice with PBS. The peptide solutions (with the concentrations from 1 to 256 μg/mL) were then added to the plates (100 μL/well), incubated for 24 h, and washed twice with PBS. Next, 5 mg/mL MTT (100 μL/well) was added and incubated for 4 h, followed by an addition of dimethyl sulfoxide (DMSO) (150 μL/well). After shaking in a shaker, the crystals in the bottom of wells were completely dissolved, and the absorbance of each well was measured at 570 nm (Apeptide). PBS served as the control (Acontrol). The cell survival rate was calculated by the equation: Survival rate (%) = (Apeptide / Acontrol) × 100.The cytotoxicity of N6 and N6-COOH-miniPEG was evaluated using the 3-- 2, 5-diphenyltetrazolium bromide (MTT) method . RAW 264E. coli CVCC195 was determined by the MIC assay [E. coli CVCC195 were measured as described above [E. coli CVCC195 by the inhibition zone assay [The thermal stability of peptides was determined after treatment for 1 h at different temperatures . The antibacterial activity of peptides against IC assay . To evalne assay , 69. TheE. coli CVCC195 by the inhibition zone assay. N6 or N6-COOH-miniPEG (200 μg/mL) dissolved in PBS was used as a positive control; SGF and SIF were used as the negative controls. To determine the serum of the peptides, N6 or N6-COOH-miniPEG were dissolved in PBS or mice serum at 37 °C to detect their serum stability and samples were removed at different time points to determine their residual activity by the inhibition zone or reverse-phase high-performance liquid chromatography (RP-HPLC) methods [Stability of N6 or N6-COOH-miniPEG in SGF and SIF was carried out as previously described , 70. Bri methods . All ass2O, SDS, and TFE, respectively [2O, 20 mM SDS or 50% TFE and their CD spectra were measured via a MOS-450 spectropolarimeter  using a 1.0 mm path-length cuvette. The spectra of peptides were recorded from 180 to 260 nm at 25 °C at a scanning speed of 100 nm/min with a step resolution of 2.0 nm and an integration time of 2 s. Data were analyzed using CDNN software.The secondary structure of the peptides was determined by CD spectroscopy in ddHectively , 69. N6 E. coli CVCC195 and S. pullorum CVCC533 cells were collected by centrifugation, washed twice, and suspended in n-2-hydroxyethylpiperazine-n-2-ethane sulfonic acid (HEPES) buffer (pH 7.4) to an OD600nm of 0.4. Cell suspension and NPN solutions (10 μM) were added into 96-well black plates, followed by an addition of peptide solutions . Fluorescence intensity was recorded until no further increase with a microplate reader . The cells treated with PBS were used as the negative control; the untreated cells were used as the blank control [The outer membrane permeabilization abilities of peptides were determined using the fluorescent NPN assay. Mid-log phase  control .E. coli CVCC195 and S. pullorum CVCC533 cells (1 × 108 CFU/mL) were incubated with 1 × , 2 × and 4 × MIC peptide solutions at 37 °C for 5, 30, and 120 min, respectively. After incubation, the cells were washed three times with 0.01 M PBS, stained with PI, and analyzed using a flow cytometer . The data were analyzed using CellQuest Pro software  [Mid-log phase BD, USA) .3(5) assay was used to investigate cytoplasmic membrane depolarization [E. coli CVCC195 and S. pullorum CVCC533 cells were washed in 5 mM HEPES buffer (pH 7.2) containing 20 mM glucose and resuspended in buffer  to an OD600 of 0.1. The fluorescence intensity of bacterial cells was monitored for 13 min. N6 and N6-COOH-miniPEG were added and the fluorescence intensity  was measured using a Tecan Infinite M200 PRO microplate reader. PBS was used as the blank control. The experiment was repeated three times.The DiSCrization , 73. MidThe affinity between the peptides and LPS was detected by the fluorescent probe BC . Equal vE. coli CVCC195 and S. pullorum CVCC533 cells (1 × 108 CFU/mL) were incubated with 4 × MIC N6 or N6-COOH-miniPEG for 2 h at 37 °C. After incubation, the cells were washed with 0.01 M PBS for three times and fixed with 2.5% glutaric dialdehyde overnight at 4 °C. The cells were dehydrated with ethanol series  for 15 min/time, dried by CO2, sputtered with platinum, and observed using a QUANTA200 SEM  [Mid-log phase erlands) .4) for 1 h. The cells were dehydrated with a series of graded ethanol solutions  for 15 min/time, transferred to epoxy resins, and stained with 1% uranium acetate; the cells were observed using a JEM1400 TEM  [For TEM observations, the bacterial cells were treated with peptides and fixed as described above. After fixation, bacterial cells were washed with 0.01 M PBS for five times and post-fixed in 1% osmium tetroxide (OsO5 cells/mL) were cultured in 12-well plates. N6 or N6-COOH-miniPEG (100 μg/mL) was mixed with LPS (100 ng/mL) for 30 min and added into the cells. After treatment for 4 h at 37 °C, the supernatant from each well was collected, and the levels of TNF-α, IL-6, IL-1β, and IL-10 were determined by the ELISA [RAW 264.7 cells  were intraperitoneally injected with 10 mg/kg FITC-labeled N6 and N6-COOH-miniPEG, respectively. Free FITC was used as the negative control. Real-time fluorescence in mice was observed at 0.17, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, and 24 h, respectively, with the excitation wavelength at 488 nm using a Maestro 2 IVIS® Spectrum CT  , 77.E. coli CVCC195 or S. pullorum CVCC533 at a concentration of absolute lethal dose (LD100) [E. coli CVCC195 , the mice were treated with N6 (2 and 4 μmol/kg), N6-COOH-miniPEG (2 and 4 μmol/kg) or PMB (0.125 and 0.25 μmol/kg) at 0.5 h and 8 h, respectively.To evaluate in vivo efficacy of N6 and N6-COOH-miniPEG, the 6-week-old female ICR mice, weight 20 ± 2 g (10 mice/group) were intraperitoneally injected with  (LD100) . After tS. pullorum CVCC533 , the mice were treated with N6 (8 and 10 μmol/kg), N6-COOH-miniPEG (8 and 10 μmol/kg) or CIP (0.8 and 1 μmol/kg) at 0.5 h, respectively. The mice injected with antibiotics or PBS served as the positive and blank control (CK), respectively. Survival of the mice was recorded daily for 7 d.Similarly, after the intraperitoneal injection with E. coli CVCC195  or S. pullorum CVCC533  by intraperitoneal injection and treated with N6 (4 and 10 μmol/kg) or N6-COOH-miniPEG (4 and 10 μmol/kg). To evaluate bacterial loads in organs, the livers, spleens, kidneys and lungs were removed from the sacrificed mice at 24 h post treatment and homogenized in sterile PBS for E. coli CVCC195 colony counting; similarly, the livers, spleens, kidneys and lungs were removed from the sacrificed mice at 48 h after treatment and homogenized in sterile PBS for S. pullorum CVCC533 colony counting. The uninfected mice served as the blank control (CK); the infected mice treated with PBS were used as the negative control. The mice were sacrificed by neck dislocation under inhaled isoflurane anesthesia .The mice (10 mice/group) were challenged with E. coli 0111:B4 LPS. Blood was collected in a heparin sodium tube at 2 or 8 h after LPS injection and incubated for 30 min at 37 °C and then overnight at 4 °C. After centrifugation at 3,000 rpm for 10 min at 4 °C, serum was collected from supernatant. The cytokine levels of TNF-α, IL-6, IL-1β and IL-10 in serum were detected by the Jiaxuan Biotech. Co. Ltd.  using an ELISA. RAW 264.7 cells untreated with LPS and antimicrobial drugs were used as a blank control (CK).The mice (10 mice/group) were injected intraperitoneally with N6 and N6-COOH-miniPEG (0.25 μmol/kg), PMB (10 μmol/kg) or CIP (5 μmol/kg) 0.5 h after challenge with 10 mg/kg E. coli CVCC195 for 0.5 and 8 h or S. pullorum CVCC533 for 0.5 h as described above. Livers, spleens, kidneys and lungs were removed from the mice at 24 h and 5 d post-treatment after E. coli CVCC195 injection and at 48 h and 4 d post-treatment after S. pullorum CVCC533 injection, respectively, washed in PBS, fixed in 4% paraformaldehyde at 4 °C for 24 h, and stained with hematoxylin and eosin (HE) for histopathology analysis. Finally, the tissue samples were fixed and observed by the light microscope. The uninfected mice served as the blank control (CK); the infected mice being treated with PBS were used as the negative control.The mice were treated with N6 (4 and 10 μmol/kg) and N6-COOH-miniPEG (4 and 10 μmol/kg) after intraperitoneal injection of p-value of < 0.05 was considered statistically significant.All data were analyzed by Origin 2018pro or GraphPad Prism 7. The results were given as means ± standard error (SE). Statistical analyses between treatments or groups were determined using one-way analysis of variance (ANOVA) models in SAS 9.2 , followed by Dunnett's multiple comparisons test. A Additional file 1: Figure S1. Chemicalstructure of N6. Figure S2. Chemicalstructure of N6-COOH-miniPEG. Figure S3. Chemical structure of N6-NH2-miniPEG.Figure S4. Chemical structure of N6-NH2-PEG6. Figure S5. Chemicalstructure of N6-NH2-PEG12. Figure S6. Chemical structure of N6-NH2-PEG24. Figure S7. Chemicalstructure of N6-Cys7-miniPEG. Figure S8. Chemicalstructure of N6-Cys16-miniPEG. Figure S9. CD of N6 and N6-COOH-miniPEG indifferent solutions. Figure S10. Interaction of N6 and N6-COOH-miniPEG with cell membrane. Outer membrane permeabilization of E. coli CVCC195(A) and S. pullorum CVCC533 (B) cells after treated with N6and N6-COOH-miniPEG. Figure S11. Interactionof N6 and N6-COOH-miniPEGwith cell membrane. Inner membranepermeabilization of E. coli CVCC195 (A) and S. pullorum CVCC533 (B) cells. Bacterial cells were treated with 1×, 2× or 4× MIC for 5, 30 or 120 minand analyzed by flow cytometry. Figure S12. Interactionof N6 and N6-COOH-miniPEGwith cell membrane. (A-D) Effects of N6 and N6-COOH-miniPEG on E. coli CVCC195  and S. pullorum CVCC533  cytoplasmicmembrane potential. Figure S13. Binding affinity of N6 andN6-COOH-miniPEG to LPS. Figure S14. TEM images of E. coli CVCC195 and S. pullorumCVCC533 cells treated with N6 and N6-COOH-miniPEG. After treatment with 4 × MIC N6 or N6-COOH-miniPEGfor 2 h, E. coli CVCC195 and S. pullorum CVCC533 cells weredehydrated, sputtered, and observed on JEM1400 . Figure S15. Effects of N6 and its N6-COOH-miniPEG on organ injury in mice.The mice were infectedintraperitoneally with E. coli CVCC195 and treated with N6 (4 μmol/kg) or N6-COOH-PEG (4 μmol/kg). The livers,spleens, kidneys and lungs were harvested from the mice sacrificed at 5 d afterinfection. Figure S16. Effectsof N6 and its N6-COOH-miniPEG on organ injury in mice. The mice wereinfected intraperitoneally with S. pullorum CVCC533  and treated with N6 (10 μmol/kg) or N6-COOH-PEG (10 μmol/kg).The livers, spleens, kidneys and lungs were harvested from the mice sacrificedat 48 h after infection. Figure S17. Effects ofN6 and N6-COOH-miniPEGon organ injury in mice.Mice were infectedintraperitoneally with S. pullorum CVCC533  and treated with N6 (10 μmol/kg) or N6-COOH-miniPEG (10 μmol/kg). Livers, spleens, kidneys and lungs were harvested from mice sacrificed at 4 dafter infection. CK group: The livers, spleens, kidneys, and lungs were normal; Infected S. pullorum CVCC533 group: Thereis “bridgingnecrosis” around the confluent area of the liver, a marked decrease in the density oflymphocytes in the splenic nodes, diffuse inflammatory cell infiltration in therenal interstitium, “honeycomb” changes throughout the lung tissue, and a large amount of bloody exudate in thealveolar cavity (arrow) ; N6 treatment group: Foci of hepatocyte necrosis are seenin the lobules of the liver, localised reduced lymphocyte density in thesplenic nodules, localised renal tubular degeneration and atrophy, and a smallamount of inflammatory cell infiltration in the interstitial lung (arrow) ; N6-COOH-miniPEG treatment group: Focal foci of hepatocyte necrosis are seen inthe lobules of the liver, localised reduced lymphocyte density in the splenicnodules, localised renal tubular degeneration and atrophy, and a small amountof scattered inflammatory cell infiltration in the interstitial lung ; CIP treatment group: Localized inflammatory cell infiltration in the hepaticsinusoids, reduced lymphocyte density in the splenic nodules, localizedscattered inflammatory cell infiltration in the renal interstitium, markedwidening of the alveolar septa and narrowing of the alveolar cavity . Table S1. Proportion of secondary structure of N6 and N6-COOH-miniPEG in different solutions. Table S2. MIC values (μg/mL) of N6 and N6-COOH-miniPEG against E. coli CVCC195 in differentconditions.Additional file 2. 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