Blastomyces adhesin-1 (BAD-1) is a 120-kD surface protein on B. dermatitidis yeast. We show here that BAD-1 contains 41 tandem repeats and that deleting even half of them impairs fungal pathogenicity. According to NMR, the repeats form tightly folded 17-amino acid loops constrained by a disulfide bond linking conserved cysteines. Each loop contains a highly conserved WxxWxxW motif found in thrombospondin-1 (TSP-1) type 1 heparin-binding repeats. BAD-1 binds heparin specifically and saturably, and is competitively inhibited by soluble heparin, but not related glycosaminoglycans. According to SPR analysis, the affinity of BAD-1 for heparin is 33 nM±14 nM. Putative heparin-binding motifs are found both at the N-terminus and within each tandem repeat loop. Like TSP-1, BAD-1 blocks activation of T cells in a manner requiring the heparan sulfate-modified surface molecule CD47, and impairs effector functions. The tandem repeats of BAD-1 thus confer pathogenicity, harbor motifs that bind heparin, and suppress T-cell activation via a CD47-dependent mechanism, mimicking mammalian TSP-1. Blastomyces dermatitidis harbors an essential virulence factor BAD-1. We describe new structural and functional features of BAD-1 that account for fungus' ability to cause disease. BAD-1 harbors a repetitive domain – a tandem repeat - that is present in up to 41 copies in the protein and essential in pathogenesis. We report an example of mimicry between this BAD-1 repeat and mammalian thrombospondin type-1 (TSP-1), a molecule that helps regulate human immunity. BAD-1 tandem repeats shares a tryptophan-rich sequence with TSP-1 that binds to tissue matrix and circulating immune cells. Like TSP-1, BAD-1 binds to this matrix. Through this property, BAD-1 binds and retards the action of white blood cells that fight fungal infection. Thus, we describe how fungi have evolved the means to mimic molecules that mammals commonly use to dampen unwanted immune responses, thus promoting pathogen survival.Work on fungi is of worldwide importance due to the increasing burden of diseases caused by these agents in humans, plants and animals globally and throughout our ecosystem. The human pathogen  Blastomyces dermatitidis is endemic to the Ohio and Mississippi river valleys, where it is the causative agent of blastomycosis. Blastomycosis is one of the principal systemic mycoses of humans and animals worldwide, and results from the inhalation of spores and/or hyphal fragments released into the air by this soil-dwelling fungus. Pulmonary infections that go undiagnosed or untreated may progress and disseminate, leading to substantial morbidity and mortality even in immunocompetent hosts.The dimorphic fungus Blastomyces adhesin-1 (BAD-1) is a 120-kDa protein of B. dermatitidis that mediates multiple functions including adhesion, modulation of pro-inflammatory immune responses and virulence B. dermatitidis mold to yeast B. dermatitidis strains BAD-1 is composed of a short N-terminal region that harbors a secretion signal, an extensive core of tandem repeats that are responsible for adhesion B. dermatitidis infection. NMR structural analysis demonstrated that the repeats adopt a tightly folded 17-amino acid loop conformation, constrained by a disulfide bond between conserved cysteines. We found no evidence for a conformational shift upon interaction of the tandem repeats with divalent cations, nor evidence for an EF-hand structure. Rather, each tandem repeat loop contains a conserved WxxWxxW motif found in TSP-1 type 1 heparin-binding repeats. BAD-1 was confirmed to bind to heparin specifically, saturably and with high affinity. A novel BAD-1 action involved TSP-1-like suppression of T lymphocyte activation and effector function in a manner similarly dependent on heparan sulfate decoration of CD47 on T cells. Our work sheds new light on the structure and TSP-1-like function of BAD-1 in virulence, and offers a striking example of molecular mimicry likely contributing to the pathogenesis of this fungal disease.We sought here to elucidate the 3-D structure of BAD-1 by NMR to gain deeper insight into the function of its tandem repeats in the pathogenesis of B. dermatitidis ATCC strain 26199 identified 30 tandem repeats based on their adherence to a consensus sequence Our earlier descriptions of BAD-1 in B. dermatitidis (ATCC 26199) from which native BAD-1 had been deleted. Two independently engineered strains (TrepeatΔ20-Y and TrepeatΔ20-AE) were selected for their capacity to display amounts of surface BAD-1 similar to strains expressing the full-length protein (BAD1-6H-J and -AC)  . Each ofThe transformed strains expressing the truncated forms of BAD-1 (TrepeatΔ20) were significantly less virulent than each strain expressing BAD-1 with the full complement of 41 repeats . In contE. coli . The shortest recombinant protein that we could express in quantity contained four tandem repeats (TR4). Initially, TR4 displayed a random assortment of disulfide linkages, determined by variations in mobility by non-reducing PAGE . These results suggest that TR4 does not adopt a unique tertiary fold in solution and precluded resolution of the molecule as a single homogenous structure. As an alternative, we calculated the 3-D structure for a single, representative BAD-1 repeat. To this end, the NOESY data derived from the residues of one tandem repeat were identified, compiled separately and submitted for algorithmic derivation of distance constraints and structural calculation. To accommodate inconsistencies in NOE peak intensities derived from partially overlapping peaks, NOE-derived distance restraints  were relaxed, marginally increasing average distance limits. Nevertheless, this approach yielded a consistent, tightly-folded 17 amino acid loop structure constrained at the base by a disulfide bond between the two conserved cysteines . The onlThe 3-D structure determined for the tandem repeat is inconsistent with the calcium-binding EF-hand structure previously hypothesized Dynamic light scattering (DLS) data from two independent samples of BAD-1 showed polydispersity, but a relatively homogeneous population with a hydrodynamic radius of 7.2±0.5 nm accounted for ∼70% of the scattering intensity. This hydrodynamic radius contains contributions from the protein, bound water of hydration and shape factors  NMR data suggests a 5 amino-acid flexible “hinge” between each tandem repeat “loop”, like beads on a string. Given the results of DLS, it is likely that BAD-1 is elongated with little in the way of tertiary structure. The hinge regions would afford limited flexibility. Energy minimization of the hinge regions of this model, constrained by the steric limitations of the tandem repeat “beads”, supports a  extended, helical conformation .Each tandem repeat loop of BAD-1 contains a significant number of tryptophans (4 of 24 amino acids - 17%). Because of this, BAD-1 absorbs UV wavelengths exceptionally well and may be detected readily by its characteristic OD280. A 1 mg/ml preparation of BAD-1 has an OD280 of over 6.6 Blastomyces yeast bound Matrigel  by studying the adherence of yeast to Matrigel, which contains heparan sulfate and other ECM proteins such as laminin, collagen IV and nidogen. Matrigel . Anti-BAMatrigel , suggestWe investigated whether native BAD-1 protein could bind heparin. Initially, purified BAD-1 was incubated with a heparin-coated agarose resin. Due to the strong UV absorbance of BAD-1, the percentage of BAD-1 that bound to resin could be assayed spectrophotometrically by measuring the OD280 of the aqueous phase before and after incubation. Heparin-agarose resin pulled BAD-1 (100 µl of 0.1 mg/ml) out of solution, with a 20 µl volume of resin absorbing nearly 100% of the soluble BAD-1. Binding was dependent upon resin volume (0.5–1.5 µg BAD-1/µl resin) and saturable . While bWe assessed the specificity of the interaction between BAD-1 and immobilized heparin using soluble competitors . Pre-incTo measure the affinity of BAD-1 for heparin, biotinylated heparin was immobilized to several densities on a neutravidin NLC chip. Dose-response curves were generated with BAD-1 in concentrations ranging from 1.5 µM to 94 nM. The kinetic constants for the dose-response data above were calculated using the Langmuir 1∶1 binding model in the instrument software. For the curves shown in in vivo effects on interaction with the immune system (below), the Trepeat20 strain has only half the molar equivalent of repeats of the parental strain.SPR analysis of the binding of truncated Trepeat20 BAD-1 to heparin showed little variation in affinity compared to full length BAD-1 . The redThe interaction between heparin and TSP-1 involves an ionic component and may be inhibited by concentrations of NaCl above 350 mM The interaction between TSP-1 and heparin is inhibited by peptides containing a WxxW motif We also tested whether TR4 could be used to block BAD-1 binding to heparin agarose, as each repeat bears a WxxWxxW motif. Native TR4, however, bound poorly to immobilized heparin . BAD-1 lOnce we established that DTT-unfolded TR4 binds heparin agarose upon oxidation, we investigated whether this domain – harboring a WxxWxxW motif – could interfere with BAD-1 binding to heparin. As with the synthetic WxxW peptide, reduced TR4 failed to inhibit binding and instead augmented BAD-1 binding to heparin agarose in a concentration-dependent manner . This reMolecules with heparin-binding motifs can modulate the activation of immune cells such as T cells via interaction with GAG-modified cell surface proteins. The heparin-binding protein, TSP-1, inhibits the activation of T cells via interaction with the surface protein CD47 through a GAG-modified serine at position 64 We next tested the role of GAG modification of CD47 in BAD-1 suppression of T cell activation. We transfected CD47-deficient JinB8 T cells with a plasmid encoding wild-type CD47 or a mutant CD47 containing a serine to alanine substitution that precludes GAG-modification (CD47-S64A). Transfections led to re-expression of CD47 on JinB8 cells. Re-expression of wild-type CD47, but not CD47-S64A, in JinB8 cells enabled TSP-1 to significantly inhibit CD69 induction by 35.1% . BAD-1 a+ T cells that respond to Blastomcyes in an antigen-specific manner and mediate immunity during infection Blastomyces reactive CD4+ T cells + T cells from these mice, analyzing their ability to become activated and differentiate into cytokine producing cells in vitro in response to co-culture with BAD-1 null Blastomyces yeast and dendritic cells (DC). Upon culture with yeast, 1807 cells became activated as measured by CD69 expression in ∼30% of the cells  Candida albicans reacts to mechanochemical pressure as it is stretched, evolving new conformations that propagate Als5p adhesive nanodomains on the fungal surface, while also drawing the host and pathogen together C. albicansYersinia sp.The tandem repeats of BAD-1 contribute to its adhesive functions Because the tandem repeats are necessary for virulence, we sought further insight into their structure and function. One notable aspect of their sequence is an exceptionally high tryptophan content. In an average protein perhaps one residue in a hundred will be tryptophan, but BAD-1 surpasses this ratio 17-fold. In nearly every repeat, three tryptophans are arranged in a highly conserved WxxWxxW pattern, a motif proven to mediate heparin binding by TSP-1. Short peptides bearing this motif are capable of binding to GAGs B. dermatitidis pathogenesis. BAD-1 is known to alter host innate immune responses, suppressing TNF-α Our work discloses a previously unrecognized capacity of BAD-1 to bind heparin. The activity is saturable, specific and high-affinity, and constitutes an advance in our understanding of BAD-1 and Candida albicans expresses an integrin-like protein Int1p Histoplasma capsulatum CBP1, a virulence factor, is similar in its 3-D NMR structure to mammalian saposin B Despite the evolutionary similarity between fungi and mammals, molecular mimicry is infrequently reported in pathogenic fungi. There are additional implications stemming from the finding that BAD-1 binds heparin. Binding of mammalian cell surface GAGs is a mechanism used by many pathogens. Viruses, bacteria, and parasites exploit host cell-surface GAGs, mediating attachment with adhesins to impede clearance Our observation that the TR4 repeats did not share the heparin-binding activity of BAD-1 was at first surprising because NMR analysis established structural identity between TR4 and the native BAD-1 repeats. Perhaps the need for an activation trigger in a critical pathogenicity factor like BAD-1 makes sense. Forty-one tandem repeats constitute enormous potential avidity, and non-specific adherence may disadvantage the pathogen. Given that a segment of the repeat loop lies across the putative heparin-binding motif, it was perhaps to be expected that our “model” tandem repeat would bind heparin poorly. Our observation that relaxing TR4's structure via disulfide reduction brings its heparin-binding up to parity with BAD-1 suggests that a reconfiguration of the loop structure may be requisite for heparin binding. Enzymatic reduction of disulfide bonds in the extra-cellular environment is one means of achieving this There are other means of exchanging disulfide bonds. Cell-surface adhesins exposed to significant mechanochemical stress (stretching) are subject to accelerated disulfide cleavage and reorganization after initial ligation of target molecules Our effort to solve the NMR structure of TR4 complexed with heparin was unsuccessful. The 3-D structure of the tandem repeat motif that binds heparin therefore remains uncertain and is the subject of ongoing work. B. dermatitidis yeast adherence to host cells and ECM via binding to heparan sulfate. In binding heparin, BAD-1 mimics TSP-1 in similarly down-regulating the activation of T cells through its interaction with a GAG-modified serine of CD47. These findings shed new light on the structure of BAD-1, its diverse functions, and novel mechanisms through which it may promote fungal pathogenicity. BAD-1 is a rare, but stunning example of molecular mimicry among fungal pathogens.In conclusion, we describe here structural features of BAD-1 tandem repeats, as determined by NMR, and a novel heparin-binding function associated with this essential virulence domain. This activity may govern All animal procedures were performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Care was taken to minimize animal suffering. The work was done with the approval of the IACUC of the University of Wisconsin-Madison.Complete Mini protease inhibitor tablets (EDTA free) were from Roche . Heparin was from Sagant pharmaceuticals . Unless noted otherwise, chemicals were from Sigma.B. dermatitidis, a wild-type, virulent isolate originally obtained from a human patient, was used in this study, together with the isogenic attenuated, BAD-1 knockout strain #55 B. dermatitidis were maintained in the yeast form on Middlebrook 7H10 agar slants with oleic acid-albumin complex, grown at 39°C. Liquid cultures of yeast were grown in Histoplasma macrophage media (HMM) American Type culture Collection (ATCC) strain 26199 of Magneportha grisea, generously provided by Dr. James Sweigard ) An expression cassette for a truncated form of BAD-1 in which 20 of the tandem repeats were deleted was created by digesting the deletion construct pBAD1-6H B. dermatitidis yeasts as previously described 4 yeast were thus administered. All animal procedures were performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Care was taken to minimize animal suffering. The work was done with the approval of the IACUC of the University of Wisconsin-Madison.Male BALB/c mice ∼5–6 wk of age (Harlan Sprague Dawley) were infected intra-nasally with Complementary oligos RS33 and RS34 were annealed and ligated into pUC18 digested with BamH1/EcoR1 to make pUC18/33-34.GATCCGAAGACGACCCTACAACTGTGACTGGGACAAGTCCCATGAGAAGTATGATTGGGAGCTCTGGGATAAGTGGTGCAAGGACG,(RS33-AATTCGTCCTTGCACCACTTATCCCAGAGCTCCCAATCATACTTCTCATGGGACTTGTCCCAGTCACAGTTGTAGGGTCGTCTTCG)RS34-pUC18/33-34 contains DNA coding for one tandem repeat, with a Bbs I site and a BamHI site just upstream. The annealed complementary oligos RS35 and RS36 were ligated into these sites, creating a new set of BbsI/BamHI sites for the next digestion/ligation cycle. After each cycle, the newly cloned plasmid was opened by Bbs I/BamHI and purified from an agarose gel .GATCCGAAGACGACCCTACAACTGTGACTGGGACAGTCCCATGAGAAGTATGATTGGGAACTCTGGGATAAGTGGTGCAAGGAC,(RS35- AGGGGTCCTTGCACCACTTATCCCAGAGTTCCCAATCATACTTCTCATGGGACTTGTCCCAGTCACAGTTGTAGGGTCGTCTTCG)RS36- E. coli with a 6-histidine tag (TR4).This process was repeated until the plasmid contained four repeats. The final construct was cut out of pUC18 with BamH1/EcoRI (EcoR1 site blunted) and ligated into pQE32 cut with BamH1/HindIII (HindIII site blunted) creating plasmid pQETR4 for expression of the 4-repeat model protein in TR4 MRGSHHHHHHGIRRRPYNCDWDKSHEKYDWELWDKWCKDPYNCDWDKSHEKYDWELWDKWCKDPYNCDWDKSHEKYDWELWDKWCKDPYNCDWDKSHEKYDWELWDKWCKDELAE.coli (grown at 30°C in LB medium) by induction with IPTG and isolated from cell lysates using an NiNTA column . Protein was refolded while immobilized on NiNTA resin by subjecting it to a gradient from 100% buffer A  to 100% buffer B  over the course of three hours. Refolded protein was then eluted with 250 mM Imidizole, which was removed by dialysis.TR4 was expressed in 4HCO3 (pH 8.5) was reduced with DTT  for 30 minutes at 55°C. Another 5 µg sample was left untreated as a control. Samples were spun through Pierce detergent removal columns (Thermo Scientific) to remove DTT and subsequently digested with trypsin solution (Trypsin Gold from Promega Corp.). Peptides were loaded on LC/MSD TOF (Agilent Technologies) and analyzed by MALDI TOF/TOF (AB SCIEX). “In Liquid” digestion and mass spectrometric analysis was done at the Mass Spectrometry Facility . In short, 5 µg of purified protein in 125 mM NHThe hydrodynamic radius of BAD-1 was measured by dynamic light scattering (DLS) collected at the 90 degree angle using a Beckman-Coulter N4 Plus instrument with the sampling time and prescaling as optimized by the instrument. Two samples of BAD-1 at 7 µM in 70 mM NaCl, 40 mM Tricine pH 7.0 were measured. For each sample ten autocorrelation functions were recorded and analyzed using both the unimodal and the size distribution software supplied with the instrument. The averages reported exclude repetitions with >1% baseline error in unimodal analysis or >5% dust fraction in the size distribution analysis. The refractive index of water at 15°C, 1.333, from instrument's database was used. The contribution of 40 mM tricine to the solvent viscosity was approximated by linear interpolation between increments reported for 20 and 100 mM tricine Three samples of BAD-1 prepared by serial dilution were analyzed by sedimentation equilibrium to ascertain the association state. The buffer was 10 mM sodium phosphate, 100 mM NaCl at pH 7.6 with a computed density of 1.005 g/mL 2O and then purified on a metal-chelate resin (NiNTA). Due to its divalent cation-binding property, BAD-1 protein could be purified on NiNTA resin regardless of whether it included a 6-histidine tag. The stringency of the wash buffer was reduced by eliminating imidazole and reducing NaCl to 150 mM. Homogeneity of purified BAD-1 was analyzed by SDS-PAGE, Sypro Ruby stain (Invitrogen), and Western blot using anti-BAD-1 antibody (DD5-CB4) BAD-1 was purified as described Blastomyces yeast were labeled with Na51CrO4 for 105 minutes at 37°C, washed and diluted to 2×107/ml in HBSS+0.1%BSA. 50 µl was added to each well and incubated for 60 minutes, then washed with HBSS. Well contents were subjected to scintillation counting (compared to controls with known numbers of labeled yeast) to quantify bound yeast. To block BAD-1-mediated binding, rabbit anti-BAD-1 immune serum Matrigel  was diluted to 5 mg/ml, 0.5 mg/ml and 0.05 mg/ml in RPMI. 30 µl was put into wells of a 96-well plate and allowed to gel at 37°C overnight. Heparin-agarose resin was obtained from Sigma as were control agarose beads and agarose beads coated with BSA, hemoglobin, and mannan. 100 µl of 0.1 mg/ml BAD-1 was incubated with agarose resins (5 µl bed volume) in 20 mM tricine, pH 7, 50 mM NaCl for 30 min at 25°C with agitation. Resin beads were pelleted by centrifugation in a microfuge at 7000xG. Concentration of BAD-1 before and after incubation with resin was monitored by A280 via Nanodrop Spectrophotometer . Binding was calculated by comparing the A280 of supernates to that of starting material. Binding inhibition studies were done with soluble medical grade heparin purchased from Elkins-Sinn Inc , dermatan sulfate (chondroitin sulfate B)(Sigma), chondroitin sulfate A (Sigma) and hyaluronan (Sigma). Baseline absorbance was corrected to account for absorbance of added GAG inhibitors. During optimization studies, heparin resin bed volume was varied from 1 µl to 20 µl, NaCl concentrations were varied from 40 mM to 2000 mM and alternative buffers were tested . Reduction of BAD-1 and TR4 was accomplished with 10 mM DTT and heating at 100°C for three minutes. Re-oxidation of thiols was accomplished by diluting or dialyzing away DTT followed by exposure to air at room temperature.BAD-1 binding to heparin and control resins, and binding inhibition studies also were performed using BAD-1 labeled with the eFluor605NC kit from eBioscience . BAD-1 was fluorescently labeled following the manufacturer's instructions. BAD-1 (eFluor605) was incubated with heparin-coated agarose beads or control beads in 20 mM tricine buffer, pH 7, 50 mM NaCl and washed three times with the same buffer before quantification. Association of BAD-1 (eFluor605) with resins was verified visually using an Olympus BX60 fluorescent microscope. For binding and inhibition studies, BAD-1 (eFluor605) was quantitated using a FilterMax F5 multi-mode microplate reader  and opaque 96 well plates . BAD-1 binding to heparin also was reproduced at higher ionic strengths of 100 mM NaCl and 150 mM NaCl, as observed in alveolar mucus and plasma, respectively E. coli strain XL-1 Blue transformed with pQE-TR4 was grown in defined M9 medium supplemented with 15N ammonium chloride (1 g/L) and 13C dextrose (4 g/L)  for 20 hrs at 30°C, under ampicillin selection (100 µg/ml) followed by induction with IPTG and 4 hours of further incubation. -labeled TR4 was purified and refolded as described above. 15N labeled native BAD-1 was produced by growing 26199 B. dermatitidis yeast at 37°C for five days in M9 medium supplemented with 15N ammonium chloride (1 g/L) and purifying as described above. Purity of isolated proteins was verified by PAGE prior to NMR analysis.13C,15N]-labeled TR4 dissolved in NMR buffer with 10 mM phosphate, pH 8.0, 95% H2O, 5% D2O 1H-15N HSQC and 3D HNCO data sets were used to identify the number of spin systems, and these identifications plus 3D HNCACB and 3D CBCA(CO)NH data sets were used as input to the PINE server to determine sequence specific backbone resonance assignments 15N-edited 1H-1H NOESY spectrum. To assign side chain and HB and HA resonances 2D 1H-13C aliphatic HSQC, 3D HBHA(CO)NH, 3D HC(CO)NH, 3D C(CO)NH and 3D H(C)CH TOCSY experiments were used. Furthermore, a 2D 1H-13C aromatic HSQC spectrum together with a 3D 13C aromatic-edited 1H-1H NOESY were used to assign resonances from aromatic side chains. Finally, a 3D 15N-edited 1H-1H NOESY (100 ms mixing time) spectrum, a 3D 13C aliphatic-edited 1H-1H NOESY (100 ms) spectrum and a 3D 13C aromatic-edited 1H-1H NOESY (100 ms) spectrum were acquired and used to derive the distance constraints to determine the three dimensional structure of the protein.All NMR spectra were recorded at the National Magnetic Resonance Facility at Madison (NMRFAM) on Varian VNMRS (600 MHz and 900 MHz) spectrometers equipped with triple-resonance cryogenic probes. The temperature of the sample was regulated at 25°C for experiments. For sequence specific backbone resonance assignments, a series of two-dimensional (2D) and three-dimensional (3D) heteronuclear NMR spectra were collected on a sample containing 0.1 mM of the [15N resolved 1H-1H 3D NOESY and 13C resolved 1H-1H 3D NOESY spectra were used to derive 1H-1H distance restraints. Backbone dihedral angle restraints φ and ψ were obtained from 1H, 15N, 13CA, 13CB, 13C′ using TALOS+ software A Biorad Proteon XPR36 instrument was used for surface plasmon resonance studies. To prepare a heparin surface, medical grade heparin was biotinylated with EZ-link Biocytin Hydrazide (Thermo Scientific) and 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) following manufacturer's instructions. Unreacted biotin was removed by precipitating biotinylated heparin with 66% methanol, which was then applied in binding buffer  (0.1 mg/ml) to a Biorad neutravidin (Proteon Sensor Chip NLC) according to the manufacturer's protocol. Subsequently, single and multiple injections of varying lengths of time and with varied concentrations of biotinylated-heparin were made onto chip in its vertical orientation. After immobilization, the surface was washed with several injections of 1 M NaCl in 50 mM NaOH. Final immobilization levels of biotinylated heparin were: 0, 5, 30, 59 and 96 RU. Analysis of BAD-1 binding utilized the horizontal orientation of the chip; length of injection and dissociation time varied, but the flow rate was a constant 30 µL/min. BAD-1 remaining bound at the end of the dissociation period was removed by a 1-minute pulse at 30 µL/min of 1 M NaCl, 50 mM NaOH. The buffer used in the binding studies was 20 mM Hepes pH 7,100 mM NaCl, and 0.1% Tween20. To demonstrate specificity of the binding, a mixture of BAD-1 and heparin were co-injected across the surfaces.Baseline and injection alignments used software supplied with the instrument. BAD-1 did bind to surfaces lacking heparin, but to a much lesser degree than to modified surfaces; therefore, the inter-spot data was used as a reference to correct for this non-specific binding. The kinetic analysis of the corrected sensorgrams utilized the 1∶1 Langmuir binding model in the software. The fitted curves shown in The UW-biotechnology center synthesized a peptide of SHWSPWSS based on the published sequence known to bind to heparin and inhibit binding by TSP-1 Heparin resin was washed with binding buffer  three times. Binding competitors  were added to 4 cubic mm of resin and incubated with agitation at room temp for 20 min. Resin was washed once with binding buffer before addition of fluorescent BAD-1 (eFluor605) and incubation continued for another 20 min. Resin was washed three times with binding buffer and binding was quantified by fluorescence on a FilterMax F5 multi-mode microplate reader as above.T cell inhibition was assessed as described with minor modifications Blastomyces-reactive 1807 TCR transgenic mice + T cells were purified with magnetic beads  according to the manufacturer's instructions. Purified 1807 cells (3×105/well) were added to co-cultures of B. dermatitidis yeast strain #55 (3×105/well) and bone-marrow derived DCs (3×105/well). After 96 hours of co-culture, supernate was harvested and tested for levels of IL-17A or IFN-γPrimary T cells were obtained from according to manufacturer's instructions , and T cells were analyzed by FACscan flow cytometry (BD Biosciences) for activation as measured by surface display of CD69, CD25, CD44, and CD62L . 1807 cells were detected with antibody against the surface Thy1.1 marker specific for the T cells. In some experiments of T cell suppression, 1807 cells were pre-incubated with BAD-1 in varied amounts in PBS/0.5% BSA for 90 minutes at 37°C, and the cells were washed to remove free BAD-1 before addition into the assay. BAD-1 was also tested for suppression of T cell function by adding the protein directly into the co-culture of yeast, DC and T cells.CD47-deficient JinB8 T cells were transiently transfected with plasmids encoding either CD47 or CD47 with a serine-to-alanine mutation at position 64 (CD47-S64A)(a generous gift of Dr. David Roberts) using Lipofectamine Plus (Invitrogen). Transfections were performed overnight prior to initiation of experiments. To verify re-expression, untransfected and transfected JinB8 cells were incubated with PE-conjugated anti-CD47 antibody (BD Bioscience) and analyzed by flow cytometry. T cell inhibition studies were done, as above, using these transfected cells.www.rosettacommons.org). The sequence WCKDPYNCD produced several models of the disulfide region, with the best-fit model used to link the two chains. All the models were energy minimized using the Sybyl suite of Tripos software and the Tripos force field until convergence. All modeling was performed in Sybyl.The disulfide loop was created using the Rosetta suite of protein structure prediction software  with error bars representing simple SEM.Kaplan Meier The coordinates and structure factors have been deposited at the Protein Data Bank (PDB) with the following accession codes. PDB: 2LWP; BMRB: 18618.Supplementary Methods and Figures are available and appended.Figure S1Recombinant B. dermatitidis yeast displaying BAD-1 derivatives. Yeast were probed with primary anti-BAD-1 monocolonal antibody DD5-CB4 and a secondary GAM-FITC antibody (Sigma) to quantify surface BAD-1 using a FACscan flow cytometer (Becton Dickenson). B. dermatitidis yeast transformed to produce truncated forms of BAD-1  displayed as much or more BAD-1 on their surfaces as yeast transformed to produce full-length BAD-1 (BAD1-6H J and –AC). Truncated and full-length forms both included a 6-histidine tag for purification. MFI = mean fluorescence intensity.(EPS)Click here for additional data file.Figure S2Refolding and analysis of TR4 expressed and purified from E.coli. TR4 migrated at varied Mr under non-reducing conditions (Non-reduced), and chiefly at 10 kD under reducing conditions (Reduced TR4). Refolding conditions and glutathione gradient parameters were adjusted until TR4 eluted from the NiNTA column as a single predominant band (Refolded). This band migrated at 14 kD and was subjected to NMR analysis to confirm that the residues in refolded TR4 and the residues of native BAD-1 existed in identical environments.(EPS)Click here for additional data file.Figure S3BAD-1 binding to heparin agarose BAD-1 Heparin binding was quantified by comparing the initial A280 of the purified, soluble BAD-1 to the A280 of BAD-1 in the unbound aqueous phase. (A) Binding of BAD-1 to heparin and alternative resins. Heparin agarose resin pulled down the majority of BAD-1 in this assay (right column). The relative binding of BAD-1 to no-agarose control, uncoated agarose resin and resins coated with BSA, hemoglobin, ConA and mannan was measured for comparison. BAD-1 bound better to heparin agarose than control resins  and binding to control resins was insignificant (p>0.05). (B) Inhibition of BAD-1 binding to heparin resin by soluble heparin. BAD-1 was pre-incubated with increasing amounts of soluble heparin prior to exposure to heparin-agarose resin. (C) Inhibition of BAD-1 binding to heparin agarose by alternate GAGs. 0.1 mg/ml BAD-1was pre-incubated with heparin, chondroitin sulfate A, or hyaluronan for 20 min, followed by exposure to heparin-agarose for an additional 30 min. The A280 of the starting BAD-1 solution was 0.8±0.04 and the A280 of the positive binding control was 0.4±0.04 (∼50% binding). Heparin inhibited binding significantly better than controls , while inhibition by chondroitin and hyaluronan were not significant (p>0.05).(EPS)Click here for additional data file.Figure S4SPR of BAD-1 lacking 20 copies of the tandem repeat (TrepeatΔ20). Interaction of TrepeatΔ20 with heparin measured by surface plasmon resonance (SPR). TrepeatΔ20 binding was monitored using a Biorad Proteon XPR36. TrepeatΔ20 at the indicated concentrations was injected onto Biorad NLC neutravidin surface with biotinylated heparin immobilized to levels of 5 (circles) and 30 (squares) RUs. For clarity, only every 15th data point is shown. The solid lines are fits to the Langmuir binding model. On and off rates were fit to each sensogram, but maximal response was fit to a single value for each immobilization level.(EPS)Click here for additional data file.Figure S5Effect of salt and pH on binding of BAD-1 to heparin. Effect of NaCl (A) and pH (B) on binding of BAD-1 to immobilized heparin. A known concentration of BAD-1 was incubated with heparin agarose. The percentage of BAD-1 bound was quantified by measuring A280 of supernate after incubation, compared to that of the starting material. Results are the mean ± SEM of two independent experiments.(EPS)Click here for additional data file.Figure S63-D representation of a model of the tandem repeat heparin-binding domain. (A) The “distal conformation” has the tryptophans of the WxxWxxW motif intercalating with the basic residues of the BxBxB motif distal to the disulfide bond, in contrast to the proximal model. (B) In the “hairpin conformation”, the tryptophans and basic residues intercalate both proximally and distally to the disulfide bond, but instead of forming a repeating, anti-parallel β-sheet, repeats in this configuration would be expected to form extended hairpin structures. Please see (EPS)Click here for additional data file.Text S1Supplementary Materials.(DOCX)Click here for additional data file.


Here, we show that microRNA-mediated upregulation of Rictor, an mTORC2-specific component, contributes to tumor progression. Rictor is upregulated via the repression of the miR-424/503 cluster in human prostate and colon cancer cell lines that harbor c-Src upregulation and in Src-transformed cells. The tumorigenicity and invasive activity of these cells were suppressed by re-expression of miR-424/503. Rictor upregulation promotes formation of mTORC2 and induces activation of mTORC2, resulting in promotion of tumor growth and invasion. Furthermore, downregulation of miR-424/503 is associated with Rictor upregulation in colon cancer tissues. These findings suggest that the miR-424/503–Rictor pathway plays a crucial role in tumor progression. The evolutionarily conserved Ser/Thr kinase mTOR  plays pivotal roles in regulating cell growth, proliferation, and survival ,2. DysreThe mTORC2 complex consists of mTOR, Rictor, mLST8, mSin1, Protor, and Deptor ,14. OverRecently, potential roles have been proposed for microRNAs (miRNAs) in the regulation of mTORC2 components. miRNAs are non-coding small RNA molecules that control diverse cellular functions, such as cell proliferation and differentiation, by regulating expression of target genes. Because dysregulation of miRNA expression is associated with a variety of human cancers, specific miRNAs can be considered to act as oncogenes or tumor suppressors ,19. For Csk-/- cells), which can be transformed by wild-type c-Src, as a model system [The tyrosine kinase c-Src is upregulated in various human cancers and plays a crucial role in tumor progression -29. Oncel system . In prevl system . Earlierl system ,34, but To elucidate the molecular link between the activation of the Src-related oncogenic pathway and mTOR-mediated tumor progression, we investigated the expression of mTOR complex components and the activity of downstream signaling molecules in human colon and prostate cancer cells in which the Src pathway is activated. In cancer cells and Src-transformed fibroblasts, we found that Rictor is upregulated via the repression of the miR-424/503 cluster, resulting in promotion of mTORC2 formation and activation implicated in cell proliferation and migration. Furthermore, the significant correlation of downregulation of miR-424/503 and Rictor upregulation in human colon cancer cells strongly suggests that the upregulation of the miR-424/503–Rictor pathway is crucial for promoting growth and invasive potential of various human cancers. Csk-/- cells), which can be transformed by wild-type c-Src [We first investigated the expression of distinctive mTOR complex components in several lines of human colon and prostate cancer cells in which Src is upregulated. Western-blot analysis of whole-cell lysates revealed significant (P<0.05) upregulation of Rictor in all colon and prostate cancer cell lines tested . By contpe c-Src . Using t-/- MEFs were relatively resistant to v-Src–induced transformation, as compared to Rictor+/+ cells  knockdown of Rictor on Src transformation. Rictor knockdown efficiently suppressed anchorage-independent growth in c-Src transformed cells  and S1C./+ cells  and S1D.The effect of Rictor upregulation on the formation of the mTOR complex was confirmed by immunoprecipitation assays. mTOR was co-precipitated with Rictor in quantities that paralleled the levels of Rictor expression. By contrast, Raptor was not co-precipitated with Rictor, and the interaction of mTOR with Raptor  was not affected by Rictor expression . These oTo address the mechanism for Rictor upregulation, we focused on our previous observations in c-Src–transformed cells, where c-Src upregulation induced selective downregulation of a set of potentially tumor-suppressive miRNAs . The bioRICTOR mRNA contains two potential miR-424 binding sequences , the latter of which (3’-UTR-2) partly overlaps with a potential miR-503 binding sequence. However, a luciferase reporter assay in c-Src–transformed cells revealed that miR-424 and miR-503 selectively target 3’-UTR-1 and 3’-UTR-2, respectively  and thus comprise the miR-424/503 gene cluster . The matditively . AlthougPreviously, we identified several tumor-suppressive miRNAs that are repressed in c-Src–transformed cells: miR-99a targets mTOR to suppress tumor progression, and miR-542-3p targets ILK to suppress tumor malignancy ,38. miR-Here, we provided the first evidence that Rictor is substantially upregulated in a subset of human colon and prostate cancer cell lines. Furthermore, it is upregulated in human colon cancer tissues at a very high incidence . To confirm the significance of Rictor upregulation in cancers, we examined human cancer mRNA profiles available at the Gene Expression Omnibus. Rictor is significantly upregulated in endometrial (GSE17025), adrenocortical (GSE12368), and pancreatic carcinoma (GSE15481) relative to non-cancerous tissues. These observations suggest that Rictor upregulation deserves further investigation in a wide variety of human cancers. Together with our previous observation that mTOR kinase is frequently upregulated via activation of oncogenic signaling , these fThese findings suggest that inhibition of mTOR kinase activity within mTORC2 would be valuable in treatment of cancers. This idea is supported by our observations that downregulation of Rictor suppressed tumorigenicity of cancer cells without any effects on normal cells, whereas downregulation of either Raptor, a component of mTORC1, or mTOR itself strongly suppressed the growth of normal cells . Taken tIn summary, we have demonstrated a critical role for the miR-424/503–Rictor pathway in controlling cancer progression. Considering that this novel axis is activated in both human colon and prostate cancer cells and transformed mouse fibroblasts, it is likely to represent a fundamental mechanism underlying mTOR signaling in cancers. Therefore, study of this pathway should be valuable in the investigation of many types of human cancers. Our findings reveal new avenues for anticancer therapeutic intervention targeted on this pathway.Csk-/- mouse embryonic fibroblasts (Csk-/- MEFs) were a kind gift from Dr. Akira Imamoto [Rictor-/- MEFs and Rictor+/+ MEFs were kind gifts from Dr. David M Sabatini [Snap-frozen colon tissues were divided visually into tumor (T) and non-cancerous (N) regions that were then confirmed histologically (see Immunohistochemistry). The research protocol for the collection of human samples was approved by the ethical review board of the Graduate School of Medicine, Osaka University, Japan. Informed consent was obtained from all patients in writing before enrollment in the study.  Imamoto . RictorSabatini . Human c4 cells were plated in 6-well culture dishes in 1.5 ml of DMEM containing 10% FCS and 0.36% agar on a layer of 2.5 ml of the same medium containing 0.7% agar. Colonies were stained with 3-- 2,5-diphenyltetrazolium bromide  7-14 days after plating, and micrographs of the stained colonies were used to count the numbers of colonies.Soft-agar colony-formation assay was performed as described . Briefly6 cells suspended in 200 μl of serum-free DMEM at one location. Tumors were monitored every 2 or 3 days and the tumor volume was estimated using the following formula: 0.5 × L × W2. At least three mice were used in each experiment. The mice used for this study were housed in environmentally-controlled rooms of the animal experimentation facility at Osaka University and sacrificed under deep anesthesia with isoflurane. All experiments were conducted under the applicable laws and guidelines for the care and use of laboratory animals in the Research Institute for Microbial Diseases, Osaka University, approved by the Animal Experiment Committee of the Research Institute for Microbial Disease, Osaka University.Immunodeficient mice  were injected subcutaneously (s.c.) as described . Briefly5 cells) in serum-free medium was added to the inserts and each insert was placed in the lower chamber, which contained NIH3T3 cell-conditioned medium. After 48 h of incubation, invasiveness was evaluated by staining the cells that migrated through the extracellular matrix layer. Numbers of invading cells were counted for five microscopic fields per well at a magnification of 100 ×, and the extent of invasion was expressed as the average number of cells per mm2.Invasion assays were performed as described . Briefly5 cells were seeded onto 6-well plates. Different concentrations  of precursor and 30 nM of inhibitor, as well as the negative control, were transfected using Lipofectamine RNAiMAX in 16 μl per 6-well plate according to the manufacturer’s instructions . Using this approach, 90% of cells were transfected as judged by comparison to FAM-labeled controls .miR-503 precursor (PM10378), miR-424 (PM10306), antisense miR-503 (AM10378), and anti-miR-424 (AM10306) were purchased from Applied Biosystems. miRNA transfection was performed as described previously . The dayCells were lysed in n-octyl-β-D-glucoside (ODG) buffer , and immunoblotting was performed as described previously . ImmunocHistologic specimens were fixed in 10% formalin and routinely processed for paraffin embedding. Histological sections 4-μm thick were stained with hematoxylin and eosin and reviewed by two pathologists (JI and EM) to define the cancerous and corresponding normal tissues. An immunoperoxidase procedure was performed on the paraffin-embedded sections as described previously . After aLuciferase assay was performed as described previously . Two setRictor 3’UTR-1-F:5’- AGCTTAGCAGATAAGGGCTGTAAGCTGCTGCTTATGTTGAAAAGTGGTTCTTA -3’;Rictor 3’UTR-1-R:5’- CTAGTAAGAACCACTTTTCAACATAAGCAGCAGCTTACAGCCCTTATCTGCTA -3’;Rictor 3’UTR-2-F:5’- AGCTTTTCTTTTTTATTTAGTAATACGCTGCTACATATTTGGAGGTTCTGGTGA -3’;Rictor 3’UTR-2-R:5’- CTAGTCACCAGAACCTCCAAATATGTAGCAGCGTATTACTAAATAAAAAAGAAA -3’;Rictor-mt 3’UTR-1-F:5’- AGCTTAGCAGATAAGGGCTGTAAGCGCGGCGGTATGTTGAAAAGTGGTTCTTA-3’;Rictor-mt 3’UTR-1-R:5’- CTAGTAAGAACCACTTTTCAACATACCGCCGCGCTTACAGCCCTTATCTGCTA-3’;Rictor-mt 3’UTR-2-F:5’- AGCTTTTCTTTTTTATTTAGTAATAGCGGCGGCATATTTGGAGGTTCTGGTGA-3’; andRictor-mt 3’UTR-2-R:5’- CTAGTCACCAGAACCTCCAAATATGCCGCCGCTATTACTAAATAAAAAAGAAA-3’.Retroviral gene transfer was performed as described previously . RetroviLentiviral vectors, empty and carrying human mTOR (ID: NM_004958.2-8344s1c1) were purchased from Sigma. Lentiviral vectors carrying human Rictor, mouse Rictor, and human Raptor were kind gifts from Dr. David M Sabatini. The production of lentiviruses and the infection of cells were performed according to the manufacturer’s instructions.Expression of mature miRNA was assayed using TaqMan MicroRNA assays (Applied Biosystems) as described previously . miRNA eTotal RNA was prepared using Sepasol  and reverse transcribed using SuperScript II reverse transcriptase (Invitrogen). The expression of housekeeping gene gapdh was used to normalize the amount of total RNA. Real-time quantitative PCR analysis was performed as described . SpecifiFigure S1Role of Rictor on tumor growth of c-Src–transformed cells. (A) Whole-cell lysates from MEFs were stimulated with or without 10 ng/ml EGF for 4 days and immunoblotted with the indicated antibodies. (B) Whole-cell lysates from Csk-/-/c-Src cells expressing control (sh-cont) or Rictor shRNA  were immunoblotted with the indicated antibodies. The relative expression levels of Rictor are shown. (C) Soft-agar colony-formation assay for the cells indicated in (B). The mean number of colonies ± S.D. was obtained from three independent experiments. *, p < 0.05 by Student’s t-test. (D) MEFs (Rictor+/+) and Rictor-deficient MEFs (Rictor-/-) were infected with retrovirus expressing empty vector (Mock) or v-Src. Cell lysates were immunoblotted with the indicated antibodies. (E) Cell lysates from Csk-/- cells (mock) and Csk-/-/c-Src cells expressing control (sh-cont) or Rictor shRNA #1 (sh-Rictor) were subjected to immunoprecipitation (IP) with anti-Rictor or anti-mTOR, followed by immunoblotting with the indicated antibodies. The relative expression levels of mTOR and Rictor are shown at the bottom of the panels.(TIF)Click here for additional data file.Figure S2miR-424/503 cluster as a tumor suppressor of tumor growth.(A) LNCaP cells were treated with 30 nM of miR-503, miR-424, or cont-miR and subjected to the soft-agar colony-formation assay for 8 days. (B) Colony-forming activity of LNCaP cells expressing control (sh-cont) or Rictor shRNA. Colonies were scored 8 days after plating. The mean number of colonies ± S.D. was obtained from three independent experiments. *, p < 0.05 and **, p < 0.01 by Student’s t test.(TIF)Click here for additional data file.Figure S3Rictor downregulation does not affect cell proliferation of human normal epithelial cells. (A) Whole-cell lysates from HaCaT cells expressing control, mTOR, Raptor or Rictor shRNA  were immunoblotted with the indicated antibodies. (B) Cell proliferation of the indicated HaCaT cells indicated in (A) was examined by an invitro proliferation assay using WST-1. Mean values ± S.D. were obtained from three independent experiments.(TIF)Click here for additional data file.


Coxiella burnetii, the etiologic agent of human Q fever, is a Gram-negative and naturally obligate intracellular bacterium. The O-specific polysaccharide chain (O-PS) of the lipopolysaccharide (LPS) of C. burnetii is considered a heteropolymer of the two unusual sugars β-D-virenose and dihydrohydroxystreptose and mannose. We hypothesize that GDP-D-mannose is a metabolic intermediate to GDP-β-D-virenose. GDP-D-mannose is synthesized from fructose-6-phosphate in 3 successive reactions; Isomerization to mannose-6-phosphate catalyzed by a phosphomannose isomerase (PMI), followed by conversion to mannose-1-phosphate mediated by a phosphomannomutase (PMM) and addition of GDP by a GDP-mannose pyrophosphorylase (GMP). GDP-D-mannose is then likely converted to GDP-6-deoxy-D-lyxo-hex-4-ulopyranose (GDP-Sug), a virenose intermediate, by a GDP-mannose-4,6-dehydratase (GMD). To test the validity of this pathway in C. burnetii, three open reading frames  annotated as bifunctional type II PMI, as PMM or GMD were functionally characterized by complementation of corresponding E. coli mutant strains and in enzymatic assays. CBU0671, failed to complement an Escherichia coli manA (PMM) mutant strain. However, complementation of an E. coli manC (GMP) mutant strain restored capsular polysaccharide biosynthesis. CBU0294 complemented a Pseudomonas aeruginosa algC (GMP) mutant strain and showed phosphoglucomutase activity (PGM) in a pgm E. coli mutant strain. Despite the inability to complement a manA mutant, recombinant C. burnetii PMI protein showed PMM enzymatic activity in biochemical assays. CBU0689 showed dehydratase activity and determined kinetic parameters were consistent with previously reported data from other organisms. These results show the biological function of three C. burnetii LPS biosynthesis enzymes required for the formation of GDP-D-mannose and GDP-Sug. A fundamental understanding of C. burnetii genes that encode PMI, PMM and GMP is critical to fully understand the biosynthesic pathway of GDP-β-D-virenose and LPS structure in C. burnetii. Lipopolysaccharide (LPS) is a complex molecule and represents the major component of the outer leaflet of the outer membrane of Gram-negative bacteria. The LPS molecule consists of three structural domains: (1) lipid A, which represent the hydrophobic anchor of the LPS molecule and is responsible for the endotoxic properties, (2) a short non-repeating inner and outer core oligosaccharide, which is attached to lipid A and extends outwardly and (3) the O-specific polysaccharide chain (O-PS), which is composed of repeating sugar units and determines the serological heterogeneity among bacterial isolates. The primary function of LPS is to serve as a permeability barrier against external agents such as hydrophobic antibiotics and to maintain the structural integrity of the Gram-negative cell wall C. burnetii, a Gram-negative small pleomorphic coccobacillus, is the causative agent of the zoonosis Q fever. Q fever manifests in humans generally as an acute, debilitating flu-like illness or less common as chronic Q fever, which develops mainly as endocarditis or hepatitis. C. burnetii is a naturally obligate intracellular bacterium and so far no method for generation of specific mutants has been established. C. burnetii is considered a potential biological weapon because it consistently causes disability, can be manufactured on a large scale, remains stable under various conditions and can be efficiently disseminated C. burnetii as a category B bioterrorism agent. There is no licensed vaccine for C. burnetii infection in the U.S. because of adverse reactions to killed whole cell vaccination. Therefore, the understanding of C. burnetii physiology and vaccine development remains an important public health and U.S. national security objective C. burnetii undergoes a shortening of its LPS, traditionally referred to as phase variation in Enterobacteriaceae. Phase variation of C. burnetii is characterized by a non-reversible switch from virulent phase I smooth LPS (S-LPS), which has a full length O-polysaccharide (O-PS) chain to an avirulent phase II rough-LPS (R-LPS). The R-LPS variant is missing the O-PS chain and unknown sugar residues located within the outer core oligosaccharide C. burnetii occurred during phase variation and the only characterized difference between virulent phase I and avirulent phase II isolates is LPS C. burnetii challenge while mice vaccinated with whole cell phase II bacteria were not protected C. burnetii LPS.Upon serial passage in an immune-incompetent host, virulent C. burnetii LPS glycero-D-manno-heptose  and 3-deoxy-α-D-manno-oct-2-ulopyranoside (Kdo), in the molar ratio 2∶2∶3, comparable to the enterobacterial inner core region C. burnetii LPS. Subsequent studies resolved the structure of virenose, while linkage and chemical compositional analysis indicated that C. burnetii O-PS is likely a heteropolymer of 1?4 linked β-D-virenose, dihydrohydroxystreptose and mannose wzm (CBU0703) and wzt (CBU0704) are located in a genomic region associated with O-PS synthesis Structural and compositional studies revealed several unique characteristics of the LPS molecule of C. burnetii is accompanied by the deletion of a large chromosomal fragment which contains glycosyl transferases and sugar processing genes required to complete β-D-virenose biosynthesis, O-PS chain elongation and inner membrane transport C. burnetii Nine Mile strain RSA439 C. burnetii phase II variant, the in C. burnetii enzymes for the biosynthesis of GDP-D-mannose and examine the initial steps of GDP-β-D-virenose biosynthesis. The presented data provide fundamental knowledge necessary to further characterize the formation of GDP-β-D-virenose, a novel saccharide, and may help develop potential vaccine candidates such as in vivo and in vitro generated glycoconjugates.Phase variation in C. burnetii enzymes predicted to be responsible for GDP-D-mannose biosynthesis (C. burnetii CBU0671 has the bioinformatic signatures of a type II phosphomannose isomerase (PMI), a small but growing class of PMIs identified in Gram-negative bacteria C. burnetii CBU0671 appeared to be unrelated to a type I PMI from E. coli EDL933, but contains the conserved PMI active site, which is characteristic of the type II PMIs C. burnetii CBU0294 is predicted to catalyze the second step in the GDP-mannose biosynthesis pathway, the conversion of D-mannose 6-phosphate to D-mannose 1-phosphate. Amino acid sequence alignment indicated a high degree of identity to P. aeruginosa AlgC, which was shown to be bifunctional and exhibits phosphoglucomutase (PGM) as well as phosphomannomutase (PMM) activity lyxo-hex-4-ulopyranose (GDP-Sug) by conversion of GDP-D-mannose. Further CBU0671 and CBU0689 are located within a genomic region associated with O-PS biosynthesis Bioinformatic analysis was carried out on the ynthesis . Amino aC. burnetii CBU0671, this protein was expressed in its native form and used for complementation of E. coli manA and manC mutant strains, defective for O-PS or CPS synthesis, respectively. To test for PMI activity the CBU0671 containing plasmid pCN606_2 was introduced into the manA mutant strain E. coli CWG634 and O-PS patterns compared to wild type E. coli CWG28 O9a. Inactivation of manA in E. coli CWG364 was shown to abolish synthesis of mannose-6-phosphate, the precursor of GDP-D-mannose and resulted in a R-LPS phenotype E. coli CWG634 with CBU0671 did not result in restoration of an S-LPS phenotype. Analysis of LPS from wild type, mutant and complemented strains using silver stained SDS-PAGE detected only revealed R-LPS chemotypes (data not shown). However, complementation of E. coli CWG634 with E. coli DH5α manA (pCN601a_5), which encoded a type I PMI, resulted in a smooth LPS phenotype (data not shown).In order to characterize the enzymatic activities of E. coli manC mutant strain CWG152 and the CPS pattern was compared to wild type E. coli CWG44 K30 manC from E. coli DH5α was cloned and expressed in its native form and the resulting plasmid, pCN603_1, was introduced into CWG152. CPS isolated from wild-type strain E. coli CWG44, manC mutant strain E. coli CWG152 and complemented strains E. coli CWG152/pCN606-2 and E. coli CWG152/ pCN603_1 were analyzed using silver-stained SDS-PAGE and immunoblot with anti-K30 antiserum. Complementation of E. coli CWG152 with CBU0671 or E. coli DH5α manC resulted in typical high and low molecular mass CPS bands as detected for the wild type strain . These data indicate that CBU0294 might encode a bifunctional enzyme, which exhibits PMM as well as PGM activity. Thus, CBU0294 is likely to catalyze the second step in the GDP-mannose biosynthesis pathway of C. burnetii.To evaluate the PGM activity of C. burnetii CBU0671 and CBU0294 were expressed as His-tagged proteins in E. coli DH5α and isolated to near homogeneity. Kinetic analyses of purified enzymes were carried out by measuring initial enzyme specific activity relative to varying concentrations of substrate. Km and Vmax values were determined by Lineweaver-Burk Plot analysis with a regression coefficient greater than 0.99  in alkaline conditions. GMD activity for CBU0689 was determined as 14 NKat/mg. Taken together these data clearly show that all three C. burnetii open reading frames (orf's) exhibit the necessary enzymatic activities for formation of GDP-D-mannose and GDP-Sug as intermediates for virenose synthesis.GMD activity of C. burnetii, which were bioinformatically predicted as the initial steps of GDP-β-D-virenose biosynthesis. Structural evidence of β-D-virenose isolated from the virulent phase I C. burnetii RSA493 O-PS further supports this hypothesis C. burnetii CBU0671, a predicted bifunctional type II PMI, failed to complement an E. coli manA mutant strain (PMI), it did complement an E. coli manC mutant strain (GMP). An exhaustive bioinformatic search of the annotated genome failed to reveal an alternative C. burnetii PMI. Clustal analysis showed that CBU0671 contained the signature sites observed in other type II PMIs, such as WbpW or AlgA of P. aeruginosa; pyrophosphorylase signature, GMP active site, nucleotidyl transferase domain, mannose-6-phosphate isomerase domain, zinc binding motif and PMI active site C. burnetii failed to complement a manA mutation in E. coli in vitro assays using natively formed and His-tagged C. burnetii CBU0671 showed specific activities, Km and Vmax values, comparable to previously reported values for PMI and GMP manA deficient background or in vitro observed enzymatic activities, respectively. Both, type I and type II PMI's possess a highly conserved motif within the active side, but other proteins have lost the specific catalytic function despite the shared motif The goal of this work was to characterize the enzymatic steps responsible for formation of GDP-D-mannose in C. burnetii CBU0294 successfully complemented a P. aeruginosa O5 algC mutant strain and restored expression of a smooth LPS. Previous studies showed that algC of P. aeruginosa O-serotype O5 is involved in formation of D-mannose and D-glucose-1-phosphate, both necessary intermediates for synthesis of the O5 O-specific chain and core oligosaccharide. Therefore inactivation of algC leads to a deep rough phenotype in P. aeruginosa O5 C. burnetii CBU0294 simultaneously carried out PGM and PMM activities in this strain. To further demonstrate that C. burnetii CBU0294 also exhibits phosphoglucomutase activity, a pgm E. coli mutant strain, W1485 pgm::tet, was successfully complemented with CBU0294. This finding supports the notation C. burnetii CBU0294 is bifunctional and carries out PGM and PMM activities as described for algC.E. coli GMD, CBU0689 was 52% identical, 69% similar on the amino acid level with an Evalue of 5×10−12C. burnetii GMD in crude extracts and its gene location within the LPS associated genome region in C. burnetii supports its bioinformatic assignment C. burnetii genome C. burnetii with the exception of a single report in which rhamnose was identified by GC-MS in the C. burnetii LPS outer core Bioinformatic analysis indicats that CBU0689 encodes a GDP-mannose 4,6-dehydratase (GMD). When compared to C. burnetii RSA439 genomic deletion C. burnetii fucose synthase indicated that it bears the characteristic Ser-Tyr-Lys catalytic triad necessary to catalyze three reactions within a single active site; epimerization at both C3″ and C5″ and NADPH dependent reduction of the ketone at C4 Since the characterization of the avirulent C. burnetii LPS biosynthesis and its structure are lacking. The intracellular nature of C. burnetii, lack of genetic tools and its status as a select agent has made elucidating these basic physiological mechanism challenging. This study establishes the foundation necessary to fully characterize the GDP-β-D-virenose biosynthesis pathway and ultimately the formation of C. burnetii O-PS, which is the only known virulence factor of C. burnetii.A fundamental understanding of P. aeruginosa strains were selected on carbenicillin (500 µg/mL) and tetracycline (100 µg/mL), as required.Bacterial strains and plasmids used in this study are described in E. coli and P. aeruginosa cells were prepared as described elsewhere DNA isolation and manipulations were carried out in according to Sambrook and Russel (2001) C. burnetii RSA 439 with CBU0672FNcoI and CBU0671R and cloned into pBAD for native protein expression. The resulting plasmid, pCN606_2, was used for complementation LPS manA or CPS manC E. coli mutant strains CWG634 and CWG152. As positive controls E. coli DH5α manA (EcmanAF/EcmanAR) and manC (EccpsBNcoIF/EccpsBR) were cloned into pBAD and the resulting plasmids, pCN601a_5 and pCN603_1, used for native protein expression in the corresponding manA and manB mutant strains. All generated plasmids were verified by sequencing. Complemented strains were grown in LB broth supplemented with 0.4% glucose, to avoid uptake of exogenous mannose, and protein expression induced with 0.2% arabinose over night E. coli CWG28 (serotype O9a) and E. coli CWG44 (serotype K30), respectively. Lysates were prepared as described elsewhere CBU071, including the native stop codon, was amplified from chromosomal DNA of C. burnetii RSA 439 DNA and obtained DNA fragment was digested with ScaI and XbaI. CBU0294 was subsequently cloned into the ScaI, XbaI treated shuttle vector pUCP20 to generate pCN620 P aeruginosa PAO1 algC:tet was transformed with pCN620 and additionally with a P. aeruginosa algC containing shuttle vector, pLPS188 P aeruginosa PAO1 algC:tet harboring pCN620 or pLPS188 were analyzed as described under 4.3 and compared to wild type LPS from P.aeruginosa PAO1. PAO1 specific antiserum (1∶1000) was used for detection of OPS expression and traced with horse radish peroxidase-conjugated goat anti-rabbit IgG(γ) monoclonal antibody (1∶5000).CBU0294 was amplified (CBU0294FScaI/CBU0294RXbaI) from chromosomal E. coli W1485pgm::tet. Phosphoglucomutase positive wild type E. coli W1485 and complemented mutant strains, were then distinguished from pgm negative strains using MacConkey agar (Difco Laboratories) supplemented with 1% (w/v) galactose and 0.2% (w/v) arabinose as previously described CBU0294 was amplified (CBU0294F/CBU0294R) and cloned into pBAD for native protein expression. The resulting plasmid, pCN607a_3, was sequenced and used for transformation of C. burnetii CBU0671 (CBU0671F/CBU0671Rpoly-His) and CBU0294 (CBU0294Fpoly-His/CBU0294Rpoly-His) were amplified and subsequently cloned into pBAD for expression of His-tagged proteins. The generated plasmids pCN606c_E1 and pCN607z_A2 were sequenced for correct insertion of target genes and used for transformation of E. coli DH5α. As controls E. coli DH5α manA and cpsB (manC) genes were amplified with EcmanAFpoly-His and EcmanARpoly-His or EccpsBFpoly-His and EccpsBFpoly-His and cloned into pBAD. The resulting plasmids pCN601c_A1 and pCN603a_A4 were sequenced for correct insertion of target genes and used for transformation of E. coli DH5α. Expression was induced with 0.2% arabinose for 4 to 8 h at an OD600 of 0.6. Bacteria were harvested , resuspended in 10 mL binding buffer  with DNase (10 µg/mL), RNase (10 µg/mL) and lysozyme (10 µg/mL) and incubated for 30 minutes on ice. Cells were lysed using French press and cell debris separated by centrifugation . His-tagged proteins were isolated from supernatants (crudes extracts) using the ProBond purification system as described by the supplier (Invitrogen). Purified proteins were analyzed for purity and size by SDS-PAGE and silver staining or immunoblot analysis with 6×His monoclonal antibody . PMI, PMM or GMD activity was also determined in bacterial crude extracts that contained natively formed C. burnetii proteins.+ at 340 nm (εM = 6.22 mM−1 cm−1) et al.2, 1.0 µmol of NADP+, 1 unit phosphoglucose isomerase, 1 unit glucose-6-phoshphate dehydrogenase, 1.1 µmol of D-mannose-6-phosphate in 50 mM tris HCl buffer pH 7.55. MgCl2, NADP+ and D-mannose-6-phosphate were dissolved in 50 mM tris HCl buffer pH 7.55 prior to adding them to the reaction mixture. The reaction mixture was equilibrated for 5 min at 25°C and the reaction initiated by adding 50 to 200 µL of crude extract that contained natively formed PMI or 7–15 µg of purified His-tagged PMI (CBU0671).PMI enzyme activity was determined by monitoring the reduction of NADP+ at 340 nm (4.6.1.) as described by Sa-Correia et al.2, 1.0 µmol of NADP+, 1 unit phosphoglucose isomerase, 1 unit glucose-6-phoshphate dehydrogenase, 5 units of purchased PMI (Sigma), 0.25 µmol of D-glucose-1,6-diphosphate (ADGD) and 5.5 µmol of D-mannose-1-phosphate in 50 mM tris HCl buffer pH 7.55. C. burnetii CBU0294 is annotated as a bifunctional phosphomannomutase (PMM) and phosphoglucomutase (PGM). Therefore, the addition of ADGD moved the kinetics of the reaction towards the formation of D-gluconate-6-phosphate. The reaction mixture was equilibrated for 5 minutes at 25°C and the reaction initiated by adding 50–200 µl of crude extract that contained natively formed PMM or of 7–15 µg of purified His-tagged PMM (CBU0294).PMM activity was determined by monitoring the reduction of NADPet al.+ (4.6.1.). The reaction mixture in a 1 mL total volume contained 10 µmol of MgCl2, 1.0 µmol of NADP+, 0.1 µmol of ADP, 2 µmol of PPi, 5.0 µmol of NaF, 1 unit of hexokinase, 1 unit of nucleoside kinase, 1 unit of glucose-6-phoshphate dehydrogenase, 0.8 µmol of glucose, 5.5 µmol of GDP-D-mannose in 50 mM tris HCl buffer pH 7.55. The reaction mixture was equilibrated for 5 minutes at 25°C, and initiated by adding 50–200 µl of crude extract. Endogenous activity in crude extracts of E. coli DH5α carrying the empty pBAD vector were subtracted from the test samples.GMD activity was determined using a modified protocol described by Munch-Peterson E. coli DH5α. Crude extract GMD activity was determined by a modified protocol described by Albermann et al.+ and 5.5 µmol of GDP-D-mannose in 50 mM tris HCl buffer pH 7.55. After equilibrating for 5 minutes at 37°C, the reaction was initiated by adding 60 µL of prewarmed crude extract. Aliquots of 50 µL were taken every 10 min and added to 950 µL of 37°C 100 mM NaOH. The reaction was incubated for an additional 20 minutes. The formation of GDP- 4-keto-6-deoxy-D-mannose was measured directly at OD320 (εM = 2.2 mM−1 cm−1) CBU0689 was amplified (CBU0689FNcoI/CBU0689R) and cloned into pBAD, retaining the native stop codon. The resulting plasmid, pCN608c_1, was used for transformation of Figure S1Clustal analysis of C. burnetii fucose synthase CBU0688 (GFS). The C. burnetii GFS has the characteristic “Catalytic Triad,” Ser (S) 107-Tyr (Y) 136-Lys (K) 140 boxed in black, observed in SDR family enzymes. Additionally, boxed in red are active sites implicated as the acid/bases involved in promoting the epimerization reactions.(TIF)Click here for additional data file.Table S1Oligonucleotides used in this study. *Introduced endonuclease restriction sites are underlined.(DOC)Click here for additional data file.


Cercopithecus denti (C. denti) whose genome code for a Vpu gene. We have compared the activity of HIV-1 Vpu and of SIVden Vpu on tetherin proteins from humans, from C. denti and from Cercopithecus neglectus (C. neglectus), a monkey species that is naturally infected by SIVdeb, a virus closely related to SIVden but which does not encode a Vpu protein. Here, we demonstrate that SIVden Vpu, is active against C. denti tetherin, but not against human tetherin. Interestingly, C. neglectus tetherin was more sensitive to SIVden Vpu than to HIV-1 Vpu. We also identify residues in the tetherin transmembrane domains that are responsible for the species-specific Vpu effect. Simultaneous mutation (P40L and T45I) of human tetherin conferred sensitivity to SIVden Vpu, while abolishing its sensitivity to HIV-1 Vpu. We next analyzed the anti-tetherin activity of the Nef proteins from HIV-1, SIVden and SIVdeb. All three Nef proteins were unable to rescue virus release in the presence of human or C. denti tetherin. Conversely, SIVdeb Nef enhanced virus release in the presence of C. neglectus tetherin, suggesting that SIVdeb relies on Nef in its natural host. Finally, while HIV-1 Vpu not only removed human tetherin from the cell surface but also directed it for degradation, SIVden Vpu only induced the redistribution of both C. denti and C. neglectus tetherins, resulting in a predominantly perinuclear localization.In different primate lentiviruses, three proteins  have been shown to have anti-tetherin activities. SIVden is a primate lentivirus harbored by a  Evolution of mammalian species, and of primate species in particular, has been strongly affected by retroviral infections Primate lentiviruses principally use two viral proteins, Nef and Vpu, to down-regulate surface expression of tetherin in vitro and in vivo was reported recently Between these two extremes, a few viral lineages express both Nef and Vpu. Some of these viruses, such as SIVcpz, a group of chimpanzee viruses from which HIV-1 was transmitted to humans, rely essentially on Nef to counteract tetherin expression, in spite of the presence of a Vpu protein C. denti, which expresses a Vpu protein C. neglectus, a virus that belongs to a lineage phylogenetically close to SIVden but that does not express a Vpu protein C. denti and C. neglectus tetherins to SIVden Vpu and HIV-1 Vpu. Counteraction of tetherin antiviral activity showed a species-specific pattern, and some key residues in Vpu involved in antagonizing BST2 were identified by mutagenesis. We then analyzed the effect of Nef proteins from HIV-1, SIVden and SIVdeb on tetherin from the three host species. Significant counteraction of tetherin activity was observed only when Nef from SIVdeb (which does not encode a vpu gene) was used against tetherin from its natural host, C neglectus. These findings are discussed from the perspective of the evolutionary forces acting on the viruses and their hosts.To gain further insight into primate lentiviral regulation of tetherin proteins, we have focused our attention on two closely related monkey viruses, SIVden from C. denti and C. neglectus reveals a number of important differences. The first such difference is the deletion of 5 amino acids in the intracytoplasmic domain of human tetherin, the cause of its insensitivity to Nef proteins As shown on C. denti and C. neglectus tetherin sequences tagged with a hemagglutinin (HA) epitope at their N-terminus. Human epithelial 293T cells, a cell line that does not express human tetherin unless treated with type I interferon C. denti tetherin or of C. neglectus tetherin, a phenomenon essentially explained by the higher level of stable expression of human tetherin in 293T cells, as it can be seen in C. denti tetherin or of C. neglectus tetherin expression plasmid reduced pNL4.3-ΔVpuΔNef virion release to levels that represented 10–15% of those obtained in absence of tetherin. A comparable level of inhibition was measured when only 0.1 microgram of human tetherin expression plasmid was transfected  and 43 (L vs. P) in C. denti tetherin numbering. Comparison of the transmembrane domains of human and C. denti tetherins revealed eight differences at positions 23, 24, 30, 33, 36, 40 and 45 (in human tetherin numbering) and 2 amino acid indel positions 25–26.Although the species-specificity of tetherin antagonism by Vpu is known to be determined by sequences in the transmembrane region of tetherin, the role of particular residues in this region remains unclear. Comparison of the transmembrane domains of C. denti/C. neglectus tetherins is the presence of a 2 amino acid insertion (glycine-isoleucine) in human tetherin at positions 25–26. These 2 amino acid are deleted in tetherins proteins from Old World monkeys, but presents in hominoids and New World Monkeys C. denti tetherin produced a moderate drop in susceptibility to SIVden Vpu and no change in susceptibility to HIV-1 Vpu  because of the divergence between C. denti and C. neglectus and on residue 45T/48I/48I that was shown to be required for the antagonism by Vpu We then focused our study on the residue 40P/43L/43P  and SIVrcm (a monkey virus without a vpu gene) These observations and assumptions, however, do not fully explain why, earlier in primate lentiviral evolution, some monkey viruses have acquired a Vpu protein while others have solely relied on their Nef protein for counteracting tetherin.C. denti, which expresses a Vpu protein, and SIVdeb from C. neglectus, which does not have a Vpu gene. The tetherin proteins from these two monkey species are closely related, diverging only by a few polymorphisms in their cellular and transmembrane domains, which are known to determine tetherin sensitivity to Vpu and Nef, respectively.The ability of SIVden Vpu protein to antagonize tetherins was already addressed C. denti and C. neglectus tetherins to SIVden Vpu and HIV-1 Vpu , and reminiscent of observations by Lim et al. with SIVagm from African green monkeys C. neglectus, infection by SIVdeb may induce only limited induction of interferon type I production, thereby limiting the amount of tetherin to which the virus is confronted during in vivo replication. Similarly, the impact of SIVden Vpu on C. denti tetherin is clearly partial (60%) compared to that observed with HIV-1 and human tetherin. Again, such partial activity is suggestive of the fact that C. denti may not need as much neutralization of tetherin as that required for human infection by HIV-1. Nonetheless, the fact that SIVden relies essentially on Vpu for activity on tetherin, and the fact that this effect on C. denti tetherin is clearly stronger than that of SIVdeb Nef on C. neglectus tetherin, together suggest that during infection of C. denti, SIVden may be confronted to higher levels of tetherin expression than those in SIVdeb-infected C. neglectus monkeys.In this study, we also examined susceptibility of the three tetherins to the Nef proteins from HIV-1, SIVden and SIVdeb. Consistent with previous studies, the antiviral activity of human tetherin was resistant to all three Nef proteins. Clearly, These results do not exclude the possibility that the Env protein from SIVdeb may also participate in antagonizing the antiviral activity of tetherin, as has been observed for HIV-2 C. denti and C. neglectus tetherins, only resulted in redistribution of these proteins, with a predominantly perinuclear expression. Although it is conceivable that this effect is related to differences in intrinsic recycling and degradation pathway targeting properties between HIV-1 and SIVden Vpu proteins, it is also possible that redistribution and perinuclear expression without degradation is related to the partial activity of SIVden Vpu on C. denti and C. neglectus tetherins. In this case the incomplete activity of SIVden Vpu would be sufficient for redirecting tetherins to the perinuclear compartment but not quite sufficient for achieving full targeting to a degradation compartment.Another interesting finding of our study, pertaining to the particularly strong activity of HIV-1 Vpu on human tetherin, was the striking difference observed on the pattern of cellular expression of the three tetherins tested here in response to the two Vpu proteins. As shown on Our findings endorse the assumption that Vpu and Nef-mediated tetherin antagonism are original properties of primate lentiviruses, and the resistance of human tetherin to primate lentivirus Vpus and Nefs seems to represent a significant barrier to zoonotic transmissions The Vpu region of the plasmid deleted of Nef pNL4-3-lucR C. denti and C. neglectus uncultured peripheral blood mononuclear cell (PBMC) DNA. A unique forward primer was used (5′-TGGAACTTGCTATTGGTCAG-3′). Two reverse primers were synthesized, one for human and C. denti tetherins (5′-CCTTCTCACTGGATTCTCC-3′) and one for C. neglectus (5′-CCTTCTCACTGGATTCTCA-3′).Tetherin genomic regions were PCR amplified from HeLa cells and from Splice-overlap-extension PCR was used to splice the introns. The integrity of all PCR derived cDNAs was confirmed by sequence analysis.N-terminal HA-tagged tetherins were obtained by PCR using a N-terminal primer containing the HA sequence. These products were cloned into the KpnI-Xho1 sites of pcDNA3.1/CT-GFP-TOPO (Invitrogen).The pNG227 plasmid containing four copies of Mason-Pfizer monkey virus constitutive transport element was a gift of Michael H. Malim. The two Vpu proteins and the three Nef proteins were cloned in pNG227 using the XbaI-SalI sites.5′-CCGGTACCATGGCATCTACTTCGTATGA-3′ (forward) and 5′-AGTCTGGGACGTCGTATGGGTATGCGGCGTAGTACTTCTTGTCCGCGATTCTCAC –3′ (reverse), and PCR-B primers 5′- CATACCCATACGACGTCCCAGACTACGCTGCCCCCAGCTCCCAGGACTCCAGCTC –3′ (forward) and 5′- GTCTCGAGTCACTGCAGCAGAGCGCTTAGG –3′ (reverse). For the monkey tetherins, the HA epitope was inserted at a position orthologous to nucleotide 463 using primers C. denti tetherin PCR-A 5′- CCGGTACCATGGCACCTATTTTGTATGACT -3′ (forward), 5′- GGCAGCGTAGTCTGGGACGTCGTATGGGTATGCGGCGGAGTTCGTGTCCGCGATT-3′ (reverse), PCR-B 5′- TACCCATACGACGTCCCAGACTACGCTGCCGCCAGCCCCCAGGACTCCAGCTGCG-3′ (forward), and 5′- GGCTCGAGTCACAGCAGCAGAGCGCCCAAG-3′ (reverse), respectively. C. neglectus tetherin primers PCR-A 5′- CCGGTACCATGGCACCTATTTTGTATGACT-3′ (forward), 5′- GCGTAGTCTGGGACGTCGTATGGGTATGCGGCGGAGTTCGTGTCCGCGATTCTCA-3′ (reverse), PCR-B 5′- TACCCATACGACGTCCCAGACTACGCTGCCAGCCCCCAGGACTCCAGCTGCGCGG-3′ (forward), and 5′- GTCTCGAGTCACAGCAGCAGAGCGCCCAAG-3′ (reverse), respectively. PCR fragments were cloned into pcDNA3 vector by inserting a KpnI-XhoI restriction site. All plasmids were confirmed by nucleotide sequence analyses.Tetherin-HA for confocal microscopy was obtained by inserting a single HA epitope tag to the ectodomain of the three BST2 proteins by primer-directed mutagenesis using PCR The human cell line 293T were obtained from the American Type Culture Collection (ATCC Cat No. CRL-11268) and cultured according to the instructions provided.C. denti (1000 ng) and C. neglectus (1000 ng)], 250 ng µg HIV-1 Vpu or SIVden Vpu expression plasmids and 600 ng of Nef expressing plasmids. A pcDNA3 vector (Invitrogen) was used to equalize the DNA concentration. Transfection were made using jetPEI transfection reagent (PolyPlus transfection) according to manufacturer's recommendations. At 42 hours post-transfection supernatants were harvested, clarified by centrifugation at 800 g and analyzed for p24 antigen by an enzyme-linked immunosorbent assay (ELISA) kit .To determine the capability of Vpu and Nef to antagonize tetherin, 293T cells were seeded in a 12-well plate and transfected with 500 ng of the pNL4.3-ΔVpuΔNef proviral construct, tetherin expression vectors , proviral constructs pNL4.3-ΔVpuΔNef-Renilla (500 ng) and HIV-1 Vpu (250 ng) or SIVden Vpu (250 ng) plasmids. The transfections were made as described above. One day post-transfection cells were washed once with PBS and lysed in the sample buffer . Proteins were solubilized by heating for 10 min at 95°C. Residual insoluble material was removed through centrifugation (10 min at 11000× g). Cell lysates were separated on SDS-PAGE and transferred to Nitrocellulose membranes and probed with anti-HA Rat antibody . Specific antibody-antigen complexes were identified using horseradish peroxidase-labeled goat anti-rat IgG secondary antibodies  and then incubated with the chemiluminescent substrate . Densitometry quantification was achieved using ImageJ software.To monitor tetherin expression, 293T cells were cotransfected with the indicated amount of N-terminally HA-tagged tetherin expression constructs [(human (100 ng), For internal controls, blots were re-incubated with mouse antibodies specific for HIV1 p24  and actin . Subsequently, blots were probed with anti-mouse or anti-rabbit IRDye antibodies  and proteins revealed using a LI-COR Odyssey scanner.Subconfluent monolayer of adherent human 293T cells grown on coverslips in 6-well plates were cotransfected with tetherin plasmids carrying HA-tag in the ectodomain (1000 ng), with a proviral constructs pNL4.3-ΔVpuΔNef (1000 ng) and HIV-1 Vpu or SIVden Vpu (500 ng), and with appropriate amounts of the empty pcDNA3 vector used as carrier plasmid DNA to normalize the quantity of DNA transfected per well. The transfections were made as described above. After 24 hours, cells were fixed with 3% formaldehyde in PBS for 15 min at room temperature, washed three times with 50 mM ammonium chloride in PBS. The cells were incubated with 1% bovine serum albumin in PBS. For labeling the cell surface, the cells were incubated with anti-HA rat primary antibodies  for 1 h. For permeabilization, cells were treated for 5 min at room temperature with 0.1% Triton X-100 buffered with PBS. After washing three times with PBS, non-specific binding was blocked with 1% bovine serum albumin in PBS for 1 h at room temperature. Cells were then incubated for 1 h at room temperature with an anti-HA Rat primary antibodies . Cells were washed for 20 min in PBS, and incubated with anti-mouse immunoglobulin G-Alexa Fluor  for 1 h at room temperature. Finally, cells were washed again for 20 min in PBS and then mounted using Vectashield+DAPI . Cells were then examined using a LSM ZEISS 510 confocal microscope.C. denti (HE680870), C. neglectus (HE680871) tetherins and SIVneg Nef gene (HE680872).The sequences have been submitted to the EMBL database: Others accession number are: Nef pNL4.3 (M19921), SIVden Nef (AJ580407) and SIVden Vpu (CAE46404).All statistical calculations were performed with a one-way analysis of variances (ANOVA) using GraphPad PrismVersion 4.0. Post test comparisons, performed only if p<0.05, were made using Bonferroni's Multiple Comparison Test.


Escherichia coli (E. coli) cells expressing heterologous proteins. Previously several independent groups have found that active protein aggregates or pseudo inclusion bodies can be induced by a fusion partner such as a cellulose binding domain from Clostridium cellulovorans (CBDclos) when expressed in E. coli. More recently we further showed that a short amphipathic helical octadecapeptide 18A (EWLKAFYEKVLEKLKELF) and a short beta structure peptide ELK16 (LELELKLKLELELKLK) have a similar property.Inactive protein inclusion bodies occur commonly in 6KD, L6K2, DKL6) were fused to the carboxyl termini of model proteins including Aspergillus fumigatus amadoriase II , Bacillus subtilis lipase A , Bacillus pumilus xylosidase (XynB), and green fluorescent protein (GFP), and expressed in E. coli. All fusions were found to predominantly accumulate in the insoluble fractions, with specific activities ranging from 25% to 92% of the native counterparts. Transmission electron microscopic (TEM) and confocal fluorescence microscopic analyses confirmed the formation of protein aggregates in the cell. Furthermore, binding assays with amyloid-specific dyes (thioflavin T and Cong red) to the AMA-L6KD aggregate and the TEM analysis of the aggregate following digestion with protease K suggested that the AMA-L6KD aggregate may contain structures reminiscent of amyloids, including a fibril-like structure core.In this work, we explored a third type of peptides, surfactant-like peptides, for performing such a "pulling-down" function. One or more of three such peptides (L6KD and it derivatives can act as a pull-down handler for converting soluble proteins into active aggregates, much like 18A and ELK16. These peptide-mediated protein aggregations might have important implications for protein aggregation in vivo, and can be explored for production of functional biopolymers with detergent or other interfacial activities.This study shows that the surfactant-like peptides L E. coli [E. coli, which were found to be biologically active [Trigonopsis variabilis (TvDAO) fused with a cellulose binding domain from Clostridium cellulovorans (CBDclos) yielded an enzyme aggregate retaining high specific activity [Inactive inclusion bodies are commonly formed during the overexpression of heterologous proteins in recombinant hosts such as E. coli . Only a E. coli . It has E. coli . One noty active . In recey active -7. For eactivity . Similaractivity ,7. Theseactivity .E. coli. The fourier transform infrared (FTIR) spectra of the 18A peptide induced protein aggregates revealed enhanced α helical secondary structures, suggesting it was the assoication of the 18A peptide that led to the formation of pseudo inclusion bodies. Since amphipathic alpha peptides are abundent in protein strucutres [In our previous studies ,9, we unrucutres , this ob6KD, L6K2, DKL6) to the carboxyl termini of model proteins, and expressed the fusions in E. coli. Most of the fusion proteins were indeed found to be largely insoluble but retained high biological activity, showing that terminally attached surfactant-like peptides can also drive proteins into biologically active pseudo inclusion boides in E. coil.To study the generality of this short-peptide induced protein aggregation, in this work we set out to test a third type of small peptides, i.e., surfactant-like peptides that were designed to mimic surfactants and normally do not resemble either α-helical or β-sheet structure ,12. Thes6K2, L6KD, and DKL6 based on the literature  via a PT linker PTPPTTPTPPTTPTPTP . All three fusion proteins in the insoluble fractions were found to be active. For L6K2 fusion, the activity of the insoluble fraction accounted for about 36.8% of the total activity, and for L6KD and DKL6 fusions, it was 60.5% , and Bacillus pumilus xylosidase (XynB)  Figure  and 2C. 6KD. From the TEM images .To study the intracellular locations of these pseudo inclusion bodies, transmission electron microscopic (TEM) analyses were performed for recombinant cells expressing the fusion protein AMA-Ls Figure  and 3B, 6KD fusion, and subjected the E. coli cells expressing GFP-L6KD to confocal fluorescence microscopic analyses. As shown in Figure To further confirm the cellular locations of the active protein aggregates, we similarly constructed GFP-LE. coli may contain amyloidal structures [6KD aggregate by using thioflavin T and Congo red. Thioflavin T will exhibit a significantly enhanced fluorescence at 480 nm relative to free dye upon binding to amyloid fibrils [6KD clearly resulted in a 24-fold increase in the fluorescence at 480 nm  or ELK16 (16 residues in length). Judging from the activities of the insoluble fractions for their fusions with AMA as the model protein, it seems that a hydrophilic head with a neutral charge (as in the case of AMA-L6KD or AMA-DKL6) is better at promoting active protein aggregation than a hydrophilic head with a charge (as in the case of AMA-L6K2). Notably however, the position of the hydrophilic head in relation to the target protein in the fusion, i.e., between the target protein and the hydrophobic tail (as in the case of AMA-DKL6) or toward the C-terminal end of the hydrophobic tail (as in the case of AMA-L6KD), makes much less of a difference.This study demonstrates that terminally attached surfactant-like peptides like L6KD aggregate following digestion of protease K revealed fibril-like structures  [in vivo, and for protein aggregation-related diseases. The active protein aggregation induced by these peptides has potential biotechnological applications. For example, it can be used to design a facile expression and purification scheme for protein [Recent studies have shown that, in addition to aggregation-prone domain such as the cellulose binding domain CBDclos , several42F19D) , a modif42(F19D) , and theD , a mod protein ,23, or t protein ,25.6KD) as aggregation tags, in terms of both pulling down efficiency (PDE) and specific enzyme activity relative to native enzyme (SArN). As shown in Table 6KD. L6KD is generally comparable with 18A both in terms of PDE and SArN, except that the SArN for LipA-18A fusion aggregate (84%) is much higher than that for LipA-L6KD (30.2%). It thus seems that L6KD has no superiority in terms of PDE and SArN compared with ELK16 or 18A, except for its smallest length which in some cases may confer advantage for fusion construction albeit in the cost of pulling down efficiency. Additionally, given its unique surfactant property, it is worthwhile to explore the possible use of this tag for design and mass-production of functional biopolymers with detergent or other interfacial activities via microorganism.Along this line, it is interesting to compare the efficiency of these three short peptides  were used to amplify the gene encoding AMA-L6KD from the previously constructed plasmid pET30a-AMA-C18 [NdeI and XhoI, and inserted into similarly digested plasmid pET30a (+) (Novagen) to yield pET30a-AMA-L6KD. Plasmids pET30a-AMA-L6K2 and pET30a-AMA-DKL6 were similarly constructed using primers AMA-L6K2-down (5'-ATGAACTCGAGTCATTTTTTCAGCAGCAGCAGCAGCAGCGGCGTCGGGGTTGGGGT-3') and AMA-DKL6-down (5'-ATGAACTCGAGTCACAGCAGCAGCAGCAGCAGTTTATCCGGCGTCGGGGTTGGGGTG-3'). Plasmids pET30a-Lip A-L6KD, pET30a-XynB-L6KD and pET30a-GFP-L6KD were obtained by replacing the sequence encoding AMA in pET30a-AMA-L6KD with that of Lip A, XynB and GFP, respectively.To construct plasmid pET30a-AMA-LE. coli BL21 (DE3) (Novagen) was used for all the experiments. The recombinant strains harboring the plasmids were cultured in Luria-Bertani (LB) medium supplemented with 50 mg/l kanamycin at 37°C. Isopropyl β-D-1-thiogalactopyranoside  was added to the culture medium at 30°C to induce fusion protein expression, when the cell optical density at 600 nm (OD600) reached 0.4-0.6. After 6 h, cells were harvested by centrifugation, and cell pellets were resuspended in lysis buffer  with a final concentration of 10 OD600 per ml. The resuspended cells were lysed by ultrasonication on ice, and the lysates separated by centrifugation. The insoluble fractions were washed twice with 1 ml of lysis buffer, and resuspended again in a same volume of lysis buffer. The amounts of target proteins in all samples were determined densitometrically by denaturing polyacrylamide gel electrophoresis  using bovine serum albumin (BSA) as standard, followed by staining with Coomassie Brilliant Blue G-250. The values of target protein amounts were calculated with Quantity One software .2/mmol) at 37°C. 5 ml of enzyme was added to 175 ml of reaction mixture (100 mM potassium phosphate buffer (pH 8.0), 2.7 purpurogallin units of peroxidase, 0.45 mM 4-aminoantipyrine, 0.5 mM N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine (TOOS), and 5.0 mM D-fructosyl-glycine). One unit of amadoriase was defined as the amount of enzyme that produced 1 nmol H2O2 per min. LipA activity [p-nitrophenol at 405 nm (e = 18.7 cm2/mmol) at 37°C. For lipase, 5 ml of diluted enzyme was added to 175 ml of reaction mixture . For β-xylosidase, 5 ml of enzyme was added to 175 ml of reaction mixture . One unit of lipase was defined as the amount of enzyme producing 1 μmol of p-nitrophenol (pNP) per min while one unit of xylosidase was defined as the amount of enzyme producing 1 nmol of pNP per min.The enzyme activities in both the soluble and insoluble fractions were assayed with a SPECTRAMAX M2 microplate reader . The amadoriase activity  was measactivity  and XynBactivity  were mea6KD were cultivated at 23°C for 22 h after induction with 0.2 mM IPTG. Cells were harvested and washed twice with phosphate buffered saline (PBS). The cell pellets were then fixed with 4% paraformaldehyde and photographed at 488 nm using a Zeiss LSM 710 confocal microscope .The cells expressing fusion protein GFP-L4. After that, the protein aggregates were washed with 0.5% Triton-X solution and then PBS. The protein aggregates (50 μg/ml) were then digested using protease K (20 μg/ml) at 37°C for 30 min in PBS, and then washed with PBS. The insoluble fractions were resuspended in a same volume of deionized distilled water, and spotted on copper grids for 5 min. The grids were washed with water, and stained with 1% (w/v) aqueous uranyl formate solution. The prepared samples were then analyzed with a Hitachi H-7650B transmission electron microscope at an accelerating voltage of 75 kV.TEM was used to analyze the intracellular location and the morphology of protein aggregates. For the intracellular analyses, recombinant cells were harvested after expression 6 h at 30°C and fixed with fixing solution . The fixed cells were dehydrated through a graded-ethanol serial dehydration step, and embedded in epoxy resins. The embedded cells were then sectioned into ultrathin slices, stained by stain solution (containing uranyl acetate solution and lead citrate), and observed with a Hitachi H-7650B  transmission electron microscope at an accelerating voltage of 80 kV. For the morphology analyses , extractThioflavin T (ThT) fluorescence assays were measured with a SPECTRAMAX M2 microplate reader  with an excitation wavelength of 445 nm and an emission range from 475 nm to 570 nm at 37°C ,29. The The authors declare that they have no competing interests.BZ designed part of the experiments, performed most of the experiments, and prepared the manuscript draft. LX and WW participated in the enzymatic assays and instrumental analyses. ZL and XZ conceived the study, designed and supervised the experiments, and revised the manuscript. All authors read and approved the final manuscript.