Reversibly outstanding performances of sodium-oxygen batteries have been realized with the superoxide discharge product (NaO2) even if sodium peroxide (Na2O2) have been also known as the discharge products. Here we report that the Lewis basicity of anions of sodium salts as well as solvent molecules, both quantitatively represented by donor numbers (DNs), determines the superoxide stability and resultantly the reversibility of sodium-oxygen batteries. A DN map of superoxide stability was presented as a selection guide of salt/solvent pair. Based on sodium triflate (CF3SO3−)/dimethyl sulfoxide (DMSO) as a high-DN-pair electrolyte system, sodium ion oxygen batteries were constructed. Pre-sodiated antimony (Sb) was used as an anode during discharge instead of sodium metal because DMSO is reacted with the metal. The superoxide stability supported by the high DN anion/solvent pair (–/DMSO) allowed more reversible operation of the sodium ion oxygen batteries.Stabilizing superoxide (O However, poor rechargeability and low energy efficiency (~60%) due to high polarization during charging are the major concerns of Li-O2 batteries4. Hartmann et al. demonstrated that just substituting metallic Na for Li in metal-air batteries results in extremely low polarization and higher energy efficiency (~90%) even without the use of any catalysts5. Even if the theoretical specific energy of Na-O2 batteries is lower (1605 and 1100 Wh kg−1 respectively for Na2O2 and NaO2 discharge products) than that of Li-O2 batteries, the value is still higher than any other metal-air batteries and lithium-ion batteries3. Further, the capacities of Na-O2 batteries were significantly higher at lower current densities than similarly discharged Li-O2 batteries5. The Na-O2 batteries would have more practical significance as the available Li resources were depleted3. Sodium is the earth’s sixth most abundant element.Lithium metal-oxygen 10 before Hartmann et al.’s work5. Superoxide ion (O2−), which is the first product of oxygen reduction reactions (ORRs), was considered unstable in electrolyte and to be converted readily to peroxide ion (O22−). Electrolytes used in the Na-O2 cells reported to discharge solid peroxide includes: NaPF6 in EC:DMC7  or DME (dimethoxy ethane)8; NaClO4 in TEGDME (tetraethylene glycol dimethyl ether)9; sodium triflate (NaSO3CF3) in an ionic liquid (1-ethyl-3-methyl imidazolium trifluoromethanesulfonate)10.As in Li-Oet al. reported superoxide salt (NaO2) as the solid discharge product of ORR in their Na-O2 cells based on NaSO3CF3 in DEGDME (diethylene glycol dimethyl ether), showing significantly improved performances of Na-O2 batteries5. The superoxide (NaO2) is not thermodynamically more favored relative to peroxide (Na2O2) (E0 (Na2O2) = 2.33 V versus E0 (NaO2) = 2.27 V). Analogous Li-O2 electrochemistry leads only to Li2O2 formation4. Even though the reasons for the differences between Li-O2 and Na-O2 electrochemistry are still ambiguous, Hartmann et al. explained that the superoxide formation is kinetically feasible because smaller number of electron transfer (one electron) is involved in the process than in peroxide formation (two electrons). Later works also confirmed the superoxide discharge product in the same electrolyte15. The main appeal of the Hartmann’s Na-O2 cell was its very low overpotential during charging (less than 200 mV) which was 3–4 times lower than any other reported Li-O2 or Na-O2 cells5. The possible reason for this high performance was the higher electronic conductivity of NaO216. In theoretical calculation, the major discharge product is controversial between superoxide and peroxide17. Superoxide formation might compete with peroxide formation so that operational condition such as discharge currents could determine the composition of discharge products18. NaO2 was more favored at higher current densities while Na2O2 was more favored at lower current densities18. The Na-O2 battery research is still in an infant stage. Therefore, a clear awareness of the factors determining the final discharge product is crucial for the practical development of Na-O2 battery.However, Hartmann 2 electrochemistry, solvents and anions of sodium salts in electrolytes are expected as important factors to determine the final products or superoxide stabilization. Stability of superoxide in non-aqueous electrolytes containing alkali metal cations was explained by using acidity of the alkali metal cations and basicity  of the non-aqueous solvents21. On the basis of Pearson’s hard soft acid base (HSAB) theory, Li+ and Na+ as hard Lewis acids show higher affinity for peroxide (O22−) and oxide anions (O2−) which are hard Lewis bases. However, solvents of high basicity  can solvate these alkali metal cations to produce soft cation complexes. The solvated cation (soft cation) can make a stable ion pair with O2− (superoxide ion as a soft base) in solution. Unlike lithium superoxide which cannot have a stable existence as solid discharge product23, sodium superoxide is stable and it can crystallize on Na-O2 battery cathode18. It is important to note that the works of Hartmann et al. showing the superoxide formation were with a high DN solvent DEGDME (DN = ~24)24. On the contrary, peroxide and/or its hydrated form (Na2O2 and Na2O2∙2H2O) were formed in comparatively lower DN solvents, DME (DN = 20.0) and TEGDME (DN = 16.6), respectively19. Therefore, it looks clear that the solvents affect the superoxide stability and therefore determining discharge products.By analyzing previous works on discharge products of Na-O3CF34.14, On the other hand, the works reporting the superoxide formation have consistently used NaSO3CF3 as the supporting salt13. Therefore, it would be one of easy inferences that anions, in addition to solvent molecules, play a crucial role on stabilizing superoxide ion and forming sodium superoxide as the discharge product.However, the superoxide formation as the discharge product was reported even in the lower DN solvent such as DME in the presence of NaSO25 as well as solvent molecules, in this work, we built superoxide stability phase diagrams by using Gutmanns’ DN for solvents and Linert’s DN for anions as double descriptors to confirm the correlation between DNs and superoxide stability. Based on the finding that high DN pairs of anion/solvent encourage the superoxide stability, we demonstrated that durably rechargeable operation of sodium ion oxygen cells is possible with the NaSO3CF3/DMSO.Focusing on the effects of anions+), a hard Lewis acid, is expected to have high affinity for peroxide (O22−) and oxide (O2−) which are hard Lewis bases21. Also, the stable ion pair formation between solvated Na+ and superoxide (O2−) is expected in solvents of high basicity   of sodium ion after solvation. Therefore, the Gutmann’s DN is understood to describe the environments immediately surrounding sodium ion especially in cases that the contact ion pair formation between the cation and its counter-anions do not perturb the solvation25.Sodium cation (Naue) Fig. 19–21. Th+-O2− formation, which is a lesson from the McCloskey et al.’s and Lutz et al.’s work showing that stable existence of superoxide was guaranteed by a high DN anion (triflate or CF3SO3−) even with a low-DN solvent (DME)14. Abraham et al. suggested that the coordination of anions to lithium ions in the inner solvation sphere by replacing solvent molecules (from Li+(S)n to Li+(S)n-m(A)m with S = solvent and A = anion) softens the cations more significantly than the simple solvation (solvent coordination) does27. In a nuclear magnetic resonance (NMR) study from Popov group, strong concentration-dependency of δ of Na was observed with I− and SCN− in various solvents due to coordination of the anions to sodium ion in the inner solvation sphere, in other words, due to the contact ion pair formation between sodium ion and the anions28. However, ClO4− and tetraphenylborate (BPh4−) did not show any significant changes in chemical shift with the salt concentrations in the most of solvents used in the study. Considering Linert’s DN values of anions29, a parallelism is found between the solvation strength of solvent molecules and the coordination strength of anions in terms that the properties are quantified by DN values. That is to say, high-DN anions favor the coordination (similar to solvation): NMR chemical shift of sodium followed the DN order of BPh4− (DN = 0) < ClO4− (8.44) < I− (28.9) < Br− (33.7)28. Also, it explains why low-DN anions such as BPh4− or ClO4− (not clearly indicated but probably BPh4−) were used to obtain the perfectly linear relationship between δ and DN values of solvents25.In addition to solvents, anions possibly affect the stability of Na27. The DN value of an anion decreases when solvent is changed to high-AN one. The apparent DN values of anions in extremely high-AN solvents , methanol (41.5) and ethanol (37.9)) deviated significantly from the reference value in DCE: e.g. DN of triflate = 16.9 in DCE but -4 in water. However, moderate or low-AN solvents  and ACN (18.9)) do not affect the values significantly: e.g. DN of triflate = 16.9, 15.7 and 15.5 in DCE, ACN and DMSO, respectively. Therefore, we could focus on the cation-solvent and cation-anion interactions if the AN values of solvents are less than 30 or more probably 20. Also, the reference values of Linert’s DN were used as a descriptor for coordination to sodium ion or more forward for superoxide stability in the following DN maps since moderate or low-AN solvents have been used for sodium air cells.When an ionic salt is dissolved in a solvent, three types of interactions are considered: cation-solvent and anion-solvent interactions based on charge-dipole polarization; and cation-anion electrostatic interaction. From a cation’s standpoint, solvent molecules and counter-anions are competitive in coordination to the cation. Therefore, DN values should be compared between solvents and anions. If one were far higher than the other, only one species would occupy or statistically dominate the inner solvation shell over the other. If they had the DN values within a certain range, both of them could be involved together in the nearest coordination shell. Also, the anion-solvent interaction should be considered in addition to the above discussion. The Linert’s DN values of anions are affected by the acceptor numbers (AN) of solvents even if they were quantified in dichloroethane  as a reference solvento) of oxygen/superoxide electrochemistry was contoured on a two dimensional (2D) DN map . Empirically, facile electron transfer kinetics of the forward cathodic reaction of the chemically reversible O2/O2− system resulted in clear existence of anodic peak responsible for its backward reaction. The electrochemical reversibility is interpreted as the superoxide stability because the anodic peaks cannot be found with unstable superoxide generated in the precedent cathodic sweep. According to the 2D DN map, higher DN values of anions as well as solvents stabilized superoxide. Experimental data points (discussed in detail below) were indicated by colored symbols with numbers or X. Circles were used when the anodic peaks responsible for the backward reaction of superoxide formation were observed experimentally. Squares were used for no anodic peak cases, indicating unstable nature of superoxide. Red colors were used for the solvent/anion pairs reported to stabilize superoxide in literatures. The oxygen-to-superoxide reduction was observed irreversible with triflate (SO3CF3−) in DME (0.2 M) and TEGDME (0.5 M) in our cyclic voltammograms even if superoxide was reported to be stable in the same electrolytes in literatures30. However, the comparatively lower concentration of sodium triflate in our study (0.1 M) than that in these literature works (0.2 M in DME and 0.5 M in TEGDME) is relevant while comparing the superoxide stability in these electrolyte systems. The numbers within the symbols indicate the number of electron transfer for peroxide formation usually following superoxide formation. Peroxide is formed via direct 2e reduction of oxygen or via 1e reduction of superoxide generated at more positive potentials. X indicates no or unclear formation of peroxide. The direct reduction of oxygen to peroxide via 2e transfer is another evidence of superoxide stability.In order to consider the interactive effects of solvents and anions on superoxide stability simultaneously, the standard rate constant  that superoxide stability is solvent-independent or superoxide is always stable with triflate as a high DN-anion or unstable with the other two anions (PF6− and ClO4−) as low DN-anions independent of solvents. To test Ho, we used dimethyl sulfoxide  as the solvent, the DN of which is higher than that of DEGDME (DN = 24) that is the highest-DN solvent ever reported for sodium-air cells; and acetonitrile  as the solvent, the DN of which is lower than TEGDME (DN = 16.6) that is the lowest-DN solvent. Three different anions of sodium salts were tested in the solvents for checking superoxide stability: PF6− (DN = 2.50) < ClO4− (8.44) < CF3SO3− (16.90) in the increasing order of DN31. The coordination strength to Na+ is expected to follow the same order.According to the literatures4−, which did not form the superoxide complex in previous reports, successfully produced the superoxide in DMSO. Therefore, the Lewis basicities (or coordination to sodium ion) of anions and solvents should be considered simultaneously for expecting superoxide stability. For guaranteeing rechargeable operation of sodium air batteries, anion/solvent pairs should be selected from the kinetically facile region of superoxide formation in the 2D DN map : the cells based on CF3SO3−/DMSO were superior to those based on Hartmann’s anion/solvent pair (CF3SO3−/DEGDME)5 in terms of reversibility.The superoxide stability results based on electrochemical analysis (discussed below in detail for the DMSO series) rejected the null hypothesis Fig. . Triflat2 electrochemistry, improving rechargeability of Li-O2 cells35. High DN value of DMSO would be an additional benefit for steering the discharge mechanism specifically towards superoxide discharge product. However, DMSO has not been studied so far as a solvent for sodium-oxygen batteries because DMSO reacts with sodium metal36. Three sodium salts, NaPF6, NaClO4 and NaSO3CF3, were compared as supporting electrolytes to study the influence of the anion part of the salt. The results with ClO4−-based electrolyte were found to be exactly similar to that in CF3SO3−-based electrolyte. Therefore, the results of PF6− (DN = 2.5) as the lowest end of DN and CF3SO3− (DN = 16.9) as the highest end are presented in this report (refer to the supporting information for ClO4−). Chemical disproportionation or electrochemical conversion of superoxide to peroxide is more possibly discouraged by adopting higher-DN anions.DMSO is one of the most attractive solvents for lithium-oxygen batteries due to its high polarity, high oxygen solubility and high chemical stability against the intermediates and products of Li-Oirreversibility of oxygen electrochemistry in PF6−-based electrolyte versus reversibility in CF3SO3−-based electrolyte . Three cathodic peaks responsible for ORRs  were identified in both electrolytes  electrolytes. Anodic process a1 that is the counterpart of the first reduction process c1 was not observed with PF6− even when potential was scanned to the value just after the c1 peak and then reversely scanned  with CF3SO3− were evidently reversible at all scan rates . One of the major differences was tes Fig. . Howevertes Figs  and S1b.υ) dependency of cathodic peak currents (jp) of the first ORR step for all anions followed Randles-Sevcik equation (jp ~ υ1/2), indicating that the electrochemical process is diffusion-limited  of oxygen in DMSO at 25 °C was calculated to be 1.65 × 10−5 cm2 s−1 in average (with oxygen concentration at 2.1 mM19). The cathodic Tafel slopes for the first ORR step in all electrolytes were estimated around 120 mV dec−1, indicating an one-electron reduction process n = solvated sodium ion. The reversibility between the first ORR (c1) and its backward reaction (a1) in CF3SO3−-based electrolyte supports the second process (2) indicating stable existence of superoxide ion possibly in a form of an ion pair with solvated sodium cation in electrolyte19. On the other hand, superoxide instability in the presence of PF6− is confirmed by the c1/a1 irreversibility. Any indications of reversibility in CVs were not observed even at higher concentrations  at all scan rates. However, it was observed only at 20 mV s−1 in the PF6−-based electrolyte. The peak c2 at higher scan rates appears to be buried in the first reduction wave around peak c1 of high charging currents37. The lack of peak c2 at slower scan rates (<20 mV s−1) in the PF6−-based electrolyte indicates that the products formed from the previous step at peak c1 are unstable. To clearly reveal the presence of the second cathodic process (c2) in PF6–-containing DMSO at higher scan rates, we used the combined SCV-FTEIS technique by which the transient natures of electrochemical reactions can be kinetically snapshotted while the electrochemistry with potential variation is investigated in real time40  in Warburg admittance (Yw) profile of PF6−-based electrolyte  was partially overlapped with the c1 peak but identifiable at 50 mV s−1 that is the scan rate not showing the clear c2 peak in CV  was ~115 mV larger than the ideal value for completely reversible systems 40. The even larger value of ΔE1/2 at 155 mV for the second Warburg peak (c2 observed at 20 mV s−1) supports that the second ORR step is one-electron reduction of superoxide to peroxide which has more shifts towards the irreversible extreme of electrochemical reduction:A second cathodic peak  because its charge transfer resistance (Rct) was clearly minimized at its potential  is due to the superoxide-to-peroxide conversion -based electrolyte, adsorption of ORR products or intermediates on electrode surface is possibly neglected in impedance interpretation by removing film-related parameters  and its backward reaction (a1) were confirmed quasi-reversible one-electron processes . The standard rate constant (k°) for the ORR to superoxide were calculated to be 7.40 × 10−3 (or 7.05 × 10−3) cm s−1 in CF3SO3− (or ClO4−)-based electrolytes by using their Rct minimum value in i0 = RT/nFRct and k0 = i0/nFko values are more than one order higher than that of superoxide formation in Li+-containing DMSO (k0 = 0.21 × 10−3 cm s−1), being in the same order of magnitude with that of tetrabutylammonium (TBA+)-containing DMSO (k0 = 17 × 10−3 cm s−1)19.Since voltammetric results indicated stable superoxide in CF2) with CF3SO3− (or ClO4−) was estimated to be a two-electron reduction of molecular oxygen to peroxide:Importantly, the second ORR step  while the n value was estimated at 1.9 from scan-rate dependency of peak potential and current -based electrolyte during anodic scans, when considering that the charge under the oxidation peak a2  in DMSO  are of little importance in sodium-air battery electrochemistry.The peak cMSO Figs  and S5 c3CF3 as well as NaClO4 based DMSO clearly show the stability of superoxide in solution phase. However, the superoxide in NaPF6 based electrolyte undergoes immediate disproportionation to the surface adsorbed Na2O2. The potential of the Na-O2 cell remains relatively unaltered at the ORR to superoxide step because oxygen is supplied continuously. Therefore, NaO2 would precipitate as stable discharge product in NaSO3CF3 as well as NaClO4 based Na-O2 cells whereas Na2O2 would be the discharge product in NaPF6 based Na-O2 cell. However, the Na2O2 cannot have a stable existence in electrolyte due to its fast chemical reaction with electrolyte resulting in decomposition products as evident from voltammetric results.The voltammetric and SCV-FTEIS studies in NaSO3SO3−/DMSO) was realized in sodium ion oxygen (Na+/O2) batteries  batteries). Pre-sodiated antimony (Sb:Na) was used as an anode during discharge instead of sodium metal because DMSO is reacted with the metal. The Sb:Na was prepared electrochemically by sodiating Sb nanoparticles in a cell of Sb||Na metal with 1 M NaClO4 in a mixture of ethylene carbonate and propylene carbonate (EC:PC)46–48. The pre-sodiation of Sb and its backward reaction was highly reversible in the electrolyte with the capacity (QdNa) at 1.1 mAh cm−2 or 550 mAh gSb−1  showed higher reversibility than the Hartmann’s anion/solvent pair (CF3SO3−/DEGDME)5 and its low-DN anion control (PF6−/DMSO)  was obtained after 10 cycles with 86% as the coulombic efficiency averaged over ten cycles. The reversibility of the CF3SO3−/DMSO is recognized as a stark improvement when considering only negligible capacities were obtained after two cycles in its counterparts. Even in the most improved cells in literatures, it was difficult to find the sodium oxygen cells delivering capacities over 10 cycles of full charge and discharge46. In the most of works exhibiting durable long-cycle operation of sodium oxygen batteries, the cells were operated only at the low depth of discharge or discharged up to a limited capacity cut-off to prevent the formation of large amount of ORR products from being formed30. The superoxide-stabilizing benefit of the high DN pair (CF3SO3−/DMSO) in the sodium ion oxygen (Na+/O2) batteries was confirmed by identifying products. Sodium superoxide was identified in X-ray diffraction pattern, which was the product formed after full discharge of the CF3SO3−/DMSO-based cells  to its counterparts. The Sb showed serious capacity decay in the cell of Sb||Na with CF3SO3−/DEGDME while the sodiation/de-sodiation of Sb was highly reversible in ClO4−/EC:PC   was significantly less than the desodiation capacity at the previous cycle . However, the coulombic efficiency (η) at each cycle is relatively high . The sodiation/desodiation in Sb||Na cells is kinetically limited to the electrochemical processes of Sb because the deposition/dissolution of Na metal is kinetically fast and highly reversible. When the Na metal was replaced by the air cathode (carbon fiber paper) with oxygen supply in CF3SO3−/DEGDME, the capacity decay was more serious  Figure . The ineion oxygen batteries instead of the conventional sodium metal oxygen batteries. In turn, unfortunately, the Na/Sb alloying reaction in the high DN solvent (DEGDME and probably DMSO even if the DMSO cannot be tested in Sb||Na cells) were not as reversible as in the conventional electrolyte EC + PC for rechargeable batteries. The high DN was favored for the superoxide stability in oxygen electrochemistry but not favored for Na/Sb alloying reaction. Therefore, we are looking for the high DN solvent that is not reactive with sodium metal for sodium metal oxygen cells. Alternatively, novel anode materials guaranteeing high reversibility even in high DN solvent should be found for the novel sodium ion oxygen cells.Even if the DMSO was suggested as the highest DN solvent in this work, it had the serious problem of the reactivity with sodium metal. Therefore, we demonstrated its good reversibility of oxygen electrochemistry by constructing the novel device sodium 3SO3−/DMSO) was proposed as an electrolyte for reversible operation of Na-O2 battery since they were expected to stabilize the superoxide. The electrolyte-dependency of the Na-O2 electrochemistry was investigated from mechanism and reversibility viewpoints by SCV-FTEIS. This study showed for the first time in literature that the nature of discharge products (NaO2 or Na2O2), battery performances and the discharge-charge reversibility greatly depend on the anion part of the sodium salt. The superoxide formed as the first step ORR product in CF3SO3−/DMSO and ClO4−/DMSO was very stable so that the formation of Na2O2 occurred only by a second ORR step of electrochemical two-electron reduction of molecular oxygen to peroxide (not by one-electron reduction of superoxide). In PF6−/DMSO, however, the superoxide immediately disproportionated to the surface adsorbed peroxide. Therefore, the discharge product in Na-O2 cells with CF3SO3− or ClO4−/DMSO was superoxide (NaO2), whereas peroxide (Na2O2) was identified as the discharge product in PF6−/DMSO. The reversibility and the battery performances of CF3SO3−/DMSO-based superoxide cells were promising. This study clearly reveals the importance of a proper selection of anion/solvent pairs of electrolyte for achieving the good reversibility and performance for Na-O2 cells by controlling the nature of discharge product.High-DN pairs of anion/solvent  disk electrodes were used as a working electrode with home-made Ag/AgCl reference electrodes. 0.1 M solutions of different sodium salts in aprotic solvents were used for the electrochemical characterization: anion of salt = hexafluorophosphate (PF6−), perchlorate (ClO4−) or triflate (CF3SO3−); solvent = acetonitrile (ACN), dimethoxy ethane (DME), diethylene glycol dimethyl ether (DEGDME) or dimethyl sulfoxide (DMSO). The SCV-FTEIS  experiments were carried out with a homemade fast-rise potentiostat, a Hewlett-Packard HP 33120 A arbitrary waveform generator and a National Instrument NI-5922 high speed data acquisition system controlled by a computer.All chemical were stored and manipulated in a glove box or a dry room. An air-tight single compartment glass cell was used for cyclic voltammetry (CV) and combined staircase cyclic voltammetry-Fourier transform electrochemical impedance spectroscopy (SCV-FTEIS). High-purity dry O3+ in ethanol by NaBH4 for 3 h (0.6 g NaBH4 in 50 mL ethanol + 1 g SbCl3 in 50 mL ethanol)45. They were pre-sodiated in an electrochemical cell of Sb||Na. 1 M NaClO4 in EC/PC was used, where EC = ethylene carbonate and PC = propylene carbonate. The Sb electrodes of Sb||Na were prepared by coating an aqueous slurry mixture of Sb, carbon black (Super-P) and a binder in a 70:15:15 wt. ratio on a copper foil. PAA (polyacrylic acid)/pullulan was used as the binder. Loading density of Sb was 2.4 mgSb cm−2. The 2032 coin cells were used for the Sb||Na. The capacity of the pre-sodiated Sb electrode (Sb:Na) was measured at 550 mAh g−1. Oxygen cells were constructed by using the Sb:Na as the anode, carbon fiber paper (AvCarb P50) as the cathode and glass fiber separator (Whatman GF/D). Oxygen pressure was controlled at 770 torr by a throttle valve.Sb nanoparticles were synthesized by reducing Sbsupporting information


Arabidopsis. In examples from these organisms, TIR domain self-association is required for signaling function, albeit through distinct interfaces. Here, we investigate these properties in the TIR domain containing resistance protein RPV1 from the wild grapevine Muscadinia rotundifolia. The RPV1 TIR domain, without additional flanking sequence present, is autoactive when transiently expressed in tobacco, demonstrating that the TIR domain alone is capable of cell-death signaling. We determined the crystal structure of the RPV1 TIR domain at 2.3 Å resolution. In the crystals, the RPV1 TIR domain forms a dimer, mediated predominantly through residues in the αA and αE helices (“AE” interface). This interface is shared with the interface discovered in the dimeric complex of the TIR domains from the Arabidopsis RPS4/RRS1 resistance protein pair. We show that surface-exposed residues in the AE interface that mediate the dimer interaction in the crystals are highly conserved among plant TIR domain-containing proteins. While we were unable to demonstrate self-association of the RPV1 TIR domain in solution or using yeast 2-hybrid, mutations of surface-exposed residues in the AE interface prevent the cell-death autoactive phenotype. In addition, mutation of residues known to be important in the cell-death signaling function of the flax L6 TIR domain were also shown to be required for RPV1 TIR domain mediated cell-death. Our data demonstrate that multiple TIR domain surfaces control the cell-death function of the RPV1 TIR domain and we suggest that the conserved AE interface may have a general function in TIR-NLR signaling.The N-terminal Toll/interleukin-1 receptor/resistance protein (TIR) domain has been shown to be both necessary and sufficient for defense signaling in the model plants flax and  To detect pathogens and activate defense responses, plants utilize multi-domain receptor proteins that resemble mammalian innate immunity NLRs [nucleotide-oligomerisation (NOD)-like receptors] . In planThe multi-domain architecture of plant NLRs generally involves a C-terminal leucine-rich repeat (LRR) domain, a central nucleotide-binding (NB) domain and either a coiled-coil (CC) domain or Toll-interleukin receptor (TIR) domain at the N-terminus. The LRR domain was originally defined as the effector recognition domain, but this has only been demonstrated for a handful of NLR-effector pairs . SubsequArabidopsis proteins, and include AtTIR, a protein of unknown function, and the TIR domains from the NLRs RRS1 (RRS1TIR), RPS4 (RPS4TIR) and a heterodimer complex between the two . M. rotundifolia is a wild North American grape species closely related to the cultivated grapevine species Vitis vinifera, and RPV1 confers resistance to the oomycete Plasmopara viticola, the casual agent of downy mildew in cultivated grapevines (TIR20-193) at 2.3 Å resolution. In the crystal structure we observe a molecular interface within the asymmetric unit involving residues within the αA and αE helices that resembles the interface previously observed for AtTIR, RRS1TIR, RPS4TIR and RRS1TIR:RPS4TIR structures. A thorough assessment of sequencing data from the plant Phytozome resource  kindly provided by Dr Maud Bernoux . All constructs were sequenced for verification. All primers used to generate the above constructs are listed in Supplementary Table S1.Truncation constructs coding for the RPV1 TIR domain were prepared by amplification of the corresponding fragments from a plasmid template of gth cDNA  with prigth cDNA  by the igth cDNA . All MrRN. tabacum cv. White Burley) plants were grown in a greenhouse at the Commonwealth Scientific and Industrial Research Organisation (CSIRO), Adelaide, Australia. Agrobacterium (strain EHA105) cells were in grown in yeast extract peptone (YEP) media supplemented with 25 μg/mL rifampicin and 50 μg/mL kanamycin at 28°C for 2 days. Approximately 0.5 ml culture was inoculated into 50 ml of YM+MES (pH 5.6) media supplemented with 20 μM acetosyringone and 50 μg/mL kanamycin and grown at 28°C until the OD600 nm was >0.5. Cells were pelleted and resuspended in infiltration medium  to give a final OD600 nm of 0.5 and incubated at room temperature for 2–3 h. Infiltration of rapidly expanding tobacco leaves was carried out with a blunt needleless syringe. Plants were maintained at a constant temperate of 23°C for 48 h before being transferred to the glasshouse.Tobacco . Yeast cells (AH109) were co-transformed with the prey and bait vectors using a lithium acetate-based protocol. Transformants were first spread on CSM-Trp-Leu plates, and then co-transformed positive colonies were grown on CSM-Trp-Leu-His + 5 mM 3-AT plates to detect activation of the IR29-233  were useg for 10 min at 4°C and the supernatant fraction collected. Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes . Membranes were blocked in 5% skim milk and probed with anti-GFP (Roche) followed by goat anti-mouse antibodies conjugated with horseradish peroxidase (Thermo Scientific). Labeling was detected with a SuperSignal West Pico chemiluminescence kit (Thermo Scientific) following the manufacturer’s instructions. Membranes were stained with Ponceau S staining solution for protein loading.Leaf tissue (∼1 cm diameter disk) was collected from the middle of an infiltrated area 48 h after agroinfiltration. Tissue was ground up in 100 μl of 2X Laemmli extraction buffer, centrifuged at 23,000 x TIR20-193 and RPV1TIR20-193H42A were amplified from plasmid templates described above using the primer combinations RPV1TIR20-F and RPV1TIR193-R (Supplementary Table S1). The PCR product was cloned into the Escherichia coli expression vector pMCSG7 (E. coli BL21 (DE3) cells using the autoinduction method , 300 mM NaCl and 1 mM DTT. The cells were lysed using sonication, clarified by centrifugation and the resulting supernatant was applied to a 5 mL HisTrap FF column . The column was washed with the lysis buffer supplemented with 30 mM imidazole to remove non-specifically bound proteins. The bound protein was eluted using a linear gradient of imidazole from 30 to 250 mM. Fractions containing the protein of interest were pooled, concentrated and buffer-exchanged (to remove imidazole) into 50 mM Tris pH 8.0, 250 mM NaCl, 1 mM DTT and 0.1 mM EDTA for overnight treatment with His-tagged TEV protease at 4°C. The cleaved protein was reapplied to the HisTrap FF column to remove the histidine tag, TEV protease and other contaminants and the flow-through was concentrated and separated further on a Superdex 75 HiLoad 26/60 gel-filtration column  pre-equilibrated with the gel filtration buffer containing 10 mM HEPES (pH 7.5), 150 mM NaCl and 1 mM DTT. The peak fractions were pooled and concentrated to 10 mg/mL. Protein was stored in aliquots at -80°C for future biophysical and crystallization studies.TIR structure (PDB ID 4c6r) as a template. Automated model building was performed in the Phenix package using autobuild, while phenix refine combined with manual inspection and corrections using coot  in a 96-well format. The hanging drop vapor-diffusion method of crystallization was used, and drops consisting of 100 nl protein solution and 100 nl reservoir solution were equilibrated against 80 μl reservoir solution. Eight commercial screens were utilized: Index, PEG/Ion and PEGRx (Hampton Research), Morpheus, ProPlex, JCSG Plus, PACT Premier (Molecular Dimensions) and Precipitant Synergy (Jena Biosciences). A number of single crystals were observed in the screening plates and these were harvested directly from the screens and after cryo-protection with 20% glycerol in well solution the crystals were vitrified in liquid nitrogen. The crystals were subjected to X-ray radiation at the Australian Synchrotron MX1 beamline. X-ray diffraction data to approximately 2.3 Å resolution was obtained from a crystal (approximate dimensions 200 μm × 30 μm × 30 μm) harvested from the PEG/Ion screen, grown in condition 16% PEG3350, 0.1 M Tris pH 8.5 and 2% Tacsimate. Data was collected using a wavelength of 0.9537 Å with a detector distance of 200 mm. The resulting dataset was indexed and integrated with XDS  and scaling coot  were comTIR20-193 in solution. SEC-SAXS was performed at the SAXS/WAXS beamline of the Australian Synchrotron. A Pilatus 1M detector at a sample-to-detector distance of 1.6 m and an energy of 12 keV yielded data over a q-range of 0.007–0.361 Å-1, where q=4π.sin (θ)/λ. 1 mg of both RPV1TIR and RPV1TIR20-193H34A were separated over an inline 3 mL Superdex S200 5/150 GL Increase column  at 16°C, at a flow rate of 0.2 mL/min in 10 mM HEPES (pH 7.5), 150 mM NaCl buffer with 1 mM DTT. Frames were collected in 2 s exposures. Data reduction and subtraction was performed using scatterBrain1. 100 frames immediately preceding each peak were summed and normalized to obtain buffer blanks, which were subtracted from each individual frame across the peak. Values of Rg and I(0) were calculated from each frame via Autorg in the PRIMUS suite (q < 0.3 Å-1 (Multi-angle laser light scattering (MALS) and small-angle x-ray scattering (SAXS) were used in conjunction with size-exclusion chromatography (SEC) to assess the molecular mass of RPV1US suite , and mol 0.3 Å-1 .Supplementary Figure S1). Pfam sequences were mapped to four clades and four individual sequences within Phytozome in order to include representative sequences from a greater number of plant species than present in Pfam . We were able to uncover a larger set of TIR domains by expanding the search from Pfam to Phytozome, except in the cases of Arabidopsis thaliana and V. vinifera, in which mapping to the Phytozome database resulted in fewer proteins than originally identified within Pfam (Supplementary Table S2 and Supplementary Data Sheet 1).To gain insight into whether the AE interface is conserved across a wider sample of plant species, TIR domain-annotated sequences from Pfam version 29.0  were alii.e., missing the αE helix). To address these errors, the full-length protein sequences for each Pfam entry were retrieved from UniProt (The Pfam description of TIR domains (PF01582) does not always account for the entire sequence of the domain, judging from the available structures, with sequences often truncated at the C-terminus of the domain . As TIR-domain autoactivity has been demonstrated for a number of plant NLR proteins . To determine the minimal functional region of RPV1 required for autoactivity, we tested a series of RPV1TIR truncated fragments fused to YFP for autoactivity in tobacco. The addition of the YFP tag had no effect on RPV1TIR1-193 autoactivity . Truncation of the αE helix led to a significant decrease in RPV1TIR autoactivity , in agreement with similar results obtained with L6TIR . These results strongly support the secondary-structure predictions, suggesting that RPV1TIR is located between residues 20–188.The viticola . Based ond L6TIR , the TIRproteins , we testth L6TIR . HoweverTIR20-193) in E. coli and purified it to homogeneity. Crystals of RPV1TIR20-193 diffracted x-rays to 2.3 Å resolution and the structure was solved by molecular replacement . The RPV1TIR20-193 structure resembles closely the AtTIR (PDB ID 3jrn), L6TIR (3ozi) and RPS4TIR  that resembles the heterodimer of the TIR domains from Arabidopsis RRS1 and RPS4 ; we consequently define this protein-protein interface as the AE interface. Analysis of the RPV1TIR structure using the program PISA (2 (∼670 Å2 from each molecule). This is similar to the combined ∼1300 Å2 buried surface in the RRS1TIR:RPS4TIR heterodimer . At the core of the interface, His42 forms an important stacking interaction between the two monomers and hydrogen bonds with Glu170 from the opposing monomer . Hydrogen bonding between Asp41 and Ser171 is also prominent and these equivalent residues were also important in the RPS4TIR:RRS1TIR interaction  and assessed the impact of these mutations on autoactivity. Mutations of residues His42 and Asp41 to alanine within the core of the AE interface, were found to abolish and markedly reduce, RPV1TIR1-193-YFP mediated cell death in tobacco. Mutation of R49 to alanine at the periphery of the interface had no effect on RPV1TIR1-193 autoactivity , while mutation of R36 to alanine, which is also on the periphery of the interface, abolished cell death.Based on the analysis of the crystal structure of RPV1TIR1-193-YFP protein recovered from tobacco-leaf tissue agroinfiltrated for the loss-of-function mutants  appeared higher, 48 h post-infiltration, than from the constructs that displayed strong necrosis at day 5 , potentially due to the impact of cell death on protein yield from agroinfiltrated sectors. These results demonstrate that residues R36, D41, and H42 within the AE interface play a key role in the RPV1TIR signaling.Importantly, these results could not be explained by differences in protein stability. Indeed, levels of RPV1TIR revealed an interface that is spatially distinct to the AE interface, involving the αD and αE helices and the βE strand. In L6TIR, this region was shown to be important in mediating L6TIR self-association, which is a requirement for the cell death signaling function of the L6TIR ; equivalent to P160Y, R164A and G201R in L6TIR, and found that these mutants were compromised in their ability to mediate cell death in tobacco . In the case of L6TIR, cell-death signaling was shown to be compromised when residues outside the L6TIR self-association interface were mutated , consistent with findings for L6. Importantly not all mutations to surface-exposed residues disrupted cell-death signaling, as demonstrated by the fact that mutation of the non-conserved L108 to a valine did not affect the protein . All mutated RPV1TIR1-193YFP proteins were detectable by western-blot analysis , suggesting that these results are not influenced by in planta protein stability.The crystal structure of L6he L6TIR . To inve mutated . We alsoTIR1-193, we did not observe an interaction in Y2H assays  under the conditions tested, despite observing self-association of L6TIR29-233, which was consistent with previous observations . The average SEC-MALS-derived molecular mass of RPV1TIR20-193 was 22.3 kDa, while the theoretical molecular mass of RPV1TIR20-193 is 20.7 kDa. We also tested, by SEC-MALS, the recombinant RPV1TIR20-193 protein carrying an alanine mutation at position H42 (RPV1TIR20-193H42A). The equivalent mutation in RPS4TIR had previously been shown to disrupt RPS4TIR:RRS1TIR dimerisation and inhibited RPS4TIR self-association. This analysis produced a molecular mass of 20.8 kDa, slightly lower than that of RPV1TIR20-193 . Using SEC-SAXS, the averaged molecular masses observed for both proteins were lower than masses determined by SEC-MALS, 19.6 kDa for RPV1TIR20-193 and 18.7 kDa for RPV1TIR20-193H42A, and small shifts in elution time between RPV1TIR20-193 and RPV1TIR20-193H42A were also observed . Molecular masses higher than monomer were previously observed in L6, RPS4, and RRS1 by SEC-MALS : Malvids, containing A. thaliana, A. lyrata, Brassica rapa, and Eutrema salsugineum; Fabales, containing G. max and P. persica; Malpighiales, containing P. trichocarpa and Ricinus communis; and Pentapetalae, that contained all of the three previous sets, plus Solanum lycopersicum, S. tuberosum, and V. vinifera—representing all classified TIR domain sequences from Pfam within the Pentapetalae clade . We also undertook a more specific analysis into five individual species to highlight residue differences occurring at the species level .We previously highlighted the potential conservation of surface-exposed residues in the AE interface after defining its role in RPS4 and RRS1 defense signaling . In lighFigures 5A–C). These residues are mostly buried and appear to assist in stabilizing the positioning of the αA and αE helices. The well-conserved position 26 (RPV1 Ile174) is surface-exposed and contributes to a pocket that in the RPV1TIR20-193 structure is occupied by His42 from the interacting RPV1TIR20-193 protomer. The most important surface-exposed residues in the AE interface are at positions 9, 10, and 22, which form the core of the AE interface . This analysis reveals that at these positions, there is conservation of histidine and glutamate at position 10 and 22, respectively (RPV1 His42 and Glu170). The amino-acid distribution at position 9 (RPV1 Asp41) is more varied across the clades , with evidence of species-specific conservation, for example the conservation of serine in A. thaliana . There is also a general trend for position 23 to contain a small amino-acid such as alanine or serine. While histidine at position 10 is well conserved, there is minor representation of aromatic residues including phenylalanine and tyrosine. Interestingly, in the case of AtTIR, a phenylalanine occupies this position, indicating that phenylalanine is compatible with dimerisation through the AE interface, at least within the context of AtTIR crystals. At the species level, G. max (soybean) appeared to deviate most significantly of the species compared across the important AE interface residues. G. max appears to have a similar preference for histidine and asparagine at position 10  and at position 22, the negatively charged residue glutamate is replaced by aspartate and glutamine in some instances. In general, there is significant variation in other interface interacting residues at both the clade and species levels, with the exception of position 4, which is essentially an invariant arginine.Conservation of residues across a wide range of species in the αA and αE regions showed that the αA region (positions 1–17) was more conserved than the αE region (positions 18–30). A number of hydrophobic residues are highly conserved, including positions 7, 11, 15, 26, 29, and 30 . We previously hypothesized that this interface would have broad functional relevance, beyond signaling in Arabidopsis . We have previously demonstrated that the AE interface is biologically relevant. The AE interface is responsible for the strong heterodimeric interaction between RPS4TIR and RRS1TIR, and also that self-association of the RPS4TIR, suggesting that competition between these two interactions is important for the regulation in the full-length NLR  to be required for self-association and signaling. Interestingly, a similar observation was recently made in studies of the Arabidopsis NLR protein RPP1, whereby residues in both the AE and DE interfaces played were important for RPP1 TIR domain mediated cell death signaling  did not induce this phenotype (in vitro) and cell-death function was observed.An interesting feature of the cell death induced by RPV1 is that the region that encompasses the TIR domain structure is, itself, sufficient to induce cell death. To the best of our knowledge, this is the first demonstration that the TIR domain alone can signal autoactive cell death. In previous studies, amino-acid sequences that extended beyond the structural borders of the TIR domain were included when assaying for autoactivity ll death . Similares 1–235 . Furtherhenotype . In all in vitro) and in planta autoactive cell-death function. Previous studies involving the WsB allele of RPP1 (residues 1–266) demonstrated that the weak dimerisation propensity of the C-terminal GFP tag was required to cause autoactive cell death . It is plausible that RPV1TIR self-association is stabilized by other proteins when overexpressed in N. tabacum; however, it is also possible that RPV1TIR can interact with other TIR-only, TIR-NLR proteins in N. tabacum or a yet to be identified signaling protein in order to initiate cell-death. Regardless of the mechanisms of cell-death activation, the integrity of the AE interface is required.The RPV1 TIR domain represents the first instance of a lack of obvious correlation between self-association , which is at the periphery of the AE interface. Mutation of this residue to in alanine in RPV1TIR prevented TIR domain dependent cell death, which was also observed previously for L6TIR and RPS4TIR , poplar (P. trichocarpa), grape (V. vinifera) and peach (P. persica) but less so in soybean (G. max). It will be interesting to characterize further these divergent residues at conserved positions in terms of TIR domain autoacitivity and NLR function in the future.Our comparison of the AE interfaces across plant species shows that surface-exposed residues that occupy the central region of the interface are highly conserved, while residues further away from the center are generally variable. We suggest that this gives the AE interface a conserved core to facilitate interactions, while the variable exterior residues control the specificity of TIR:TIR domain interactions. An exception to this is the highly conserved arginine at position 4 ( RPS4TIR . While tArabidopsis TIR domains. Our work also confirms that the TIR domain is the minimal region required for cell-death signaling. We suggest the available data build a general paradigm of structure-function relationships in plant TIR domains, where the AE interface and other spatially distinct protein surfaces in plant TIR domains play key roles in the signaling mechanism of plant TIR-NLR proteins.Overall, the data presented here highlights that the AE interface is likely to be functionally important across a broad range of plant species and is not limited to SW, BK, ID, MB, LY, and GF conceived and designed the experiments, SW, LY, GF, LC, MO, and DE performed the experiments, all authors analyzed the data and SW, LY, BK, ID, GF, and LC wrote the paper. All authors reviewed and edited the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Splenogonadal fusion usually presents as a left scrotal mass but rarely is the diagnosis suspected preoperatively. Herein, we present the first report of a left splenogonadal fusion which was suspected preoperatively by doppler ultrasonography in a 2 year old boy.


HH domain, and assessed expression, membrane insertion and surface exposure. Our results show that expression levels differed considerably between the constructs tested. The constructs that included the β-domains of Hbp and IgA protease appeared the most efficient and resulted in expression levels that were detectable on Coomassie-stained SDS-PAGE gels. The VHH domain appeared the most difficult fusion partner to export, probably due to its complex immunoglobulin-like structure with a tertiary structure stabilized by an intramolecular disulfide bond. Overall, the Hbp β-domain compared favorably in exporting the fused recombinant proteins, because it showed in every instance tested a good level of expression, stable membrane insertion and clear surface exposure.Monomeric autotransporters have been extensively used for export of recombinant proteins to the cell surface of Gram-negative bacteria. A bottleneck in the biosynthesis of such constructs is the passage of the outer membrane, which is facilitated by the β-domain at the C terminus of an autotransporter in conjunction with the Bam complex in the outer membrane. We have evaluated eight β-domain constructs for their capacity to secrete fused proteins to the cell surface. These constructs derive from the monomeric autotransporters Hbp, IgA protease, Ag43 and EstA and the trimeric autotransporter Hia, which all were selected because they have been previously used for secretion of recombinant proteins. We fused three different protein domains to the eight β-domain constructs, being a Myc-tag, the Hbp passenger and a nanobody or V Monomeric autotransporters are secreted proteins of the Type V secretion pathway that have been used extensively as a versatile system to export recombinant proteins to the cell surface or medium of Gram-negative bacteria –3. In naPseudomonas aeruginosa. These monomeric autotransporters have passenger domains that fold into an α-helical globular domain and are not cleaved from their β-domains [The type V secretion pathway further includes trimeric autotransporters, two-partner secretion systems and intimin-like proteins . Trimeri-domains .Escherichia coli autotransporter hemoglobin protease (Hbp), which is a member of the SPATE family (for serine protease autotransporters of Enterobacteriaceae) [We have studied the structure and secretion mechanism of the riaceae) , 16–21. riaceae) , 22. Theriaceae) , 23–26. riaceae) , 23, 25.e.g. enzymes, peptide libraries and vaccine antigens) to the cell surface [Various monomeric autotransporters have been used to secrete recombinant proteins . The laE. coli strains TOP10F’ (Invitrogen), MC1061 [degP::S210A [dsbA::Km) [, MC1061 , MC1061dP::S210A , and MC1sbA::Km)  were groRestriction enzymes were from New England Biolab (NEB) or Roche Applied Science (Roche). Alkaline phosphatase & T4-DNA ligase (Rapid DNA Dephos & Ligation Kit), Lumi-Light Western blotting substrate and Proteinase K  were from Roche Applied Science. Phusion High-Fidelity DNA polymerase was obtained from Thermo Scientific. All other reagents and enzymes were from Sigma-Aldrich. The rabbit polyclonal antisera against the Hbp β-domain (α-Hbpβ), the IgAPβ (α-IgAPβ), periplasmic chaperone SurA (α-SurA) and outer membrane protein OmpA (α-OmpA) have been described previously , 12. ThePlasmid pEH3 served as the backbone for all constructs made for this study . The seqSpeI restriction site at nucleotides 3286–3273 of the hbp ORF, which is the region that encodes the junction, of passenger and β-domain. The plasmid was generated using overlap-extension PCR using the mutagenesis primers Hbp SpeI fw and Hbp SpeI rv. To construct plasmids encoding a fusion between the Hbp passenger and the β-domains of Ag43 (Ag43β(700 and Ag43β(710)), IgAP (IgAPβ(1124 and IgAPβ(1245)), EstA (EstAβ) and Hia (Hiaβ), respectively, DNA fragments encoding these β-domains, including their cognate α-helical, were amplified by PCR. All fragments encoded flanking SpeI/EcoRI restriction sites. Fragments encoding Ag43β(700) and Ag43β(710) were generated using forward primers SpeI-Ag43-β-long fw and SpeI-Ag43-β-short fw, respectively. In both cases, Ag43-β rv was used as the reverse primer and E. coli MG1655 genomic DNA was used as a template. To generate IgAPβ(1124) and IgAPβ(1245) encoding fragments forward primers SpeI-IgAP-β-long fw and SpeI-IgAP-β-short fw were used in combination with primer IgAP-β rv as the reverse primer and plasmid pEN440 carrying iga [Pseudomonas aeruginosa PAO1 genomic DNA as a template in combination with the primers SpeI-EstA-β fw and EstA-β rv. The nucleotide fragment encoding the Hia β-domain was generated using Haemophilus Influenzae Rd KW20 genomic DNA as a template. The primers used were SpeI-Hia-β fw and Hia-β rv. To create Hiaβ(3×) a synthetic DNA fragment encoding three interconnected Hia β-domains  and flanking SpeI/EcoRI restriction sites was ordered from GeneArt/LifeTech. The nucleotide and amino acid (AA) sequence of this fragment can be found in SpeI) using the SpeI and EcoRI restriction sites, yielding the following plasmids: pEH3-Hbp-Ag43β(700), pEH3-Hbp-Ag43β(710), pEH3-Hbp-IgAPβ(1124), pEH3-Hbp-IgAPβ(1245), pEH3-Hbp-EstAβ, pEH3-Hbp-Hiaβ and pEH3-Hbp-Hia3β, respectively.First, plasmid pEH3-Hbp(SpeI) was created, which is a derivative of pEH3-Hbp  that incHH) to the Hbpβ, Ag43β(700), Ag43β(710), IgAPβ(1124), IgAPβ(1245), EstAβ, Hiaβ and Hia3β domains all used the N-terminal signal sequence of Hbp and included a myc-tag followed by a Gly-Ser-Gly-Ser-Gly spacer in between VHH and the respective β-domains  as a template in combination with primers Cas/VHH(R2) fw and Cas/Myc rv. The resulting product was cloned into pEH3-HbpD(Δd1) [SacI/BamHI restriction sites in that plasmid, yielding pEH3-Hbp-VHH(R2). In a second PCR, this plasmid and primers pEH-XbaI-Hbp fw and VHH(R2)-GSGSG-SpeI rv were used to generate a XbaI-SpeI fragment encoding a fusion between the Hbp signal sequence and VHH R2. This fragment was subsequently cloned into the pEH3 plasmids that encoded the Hbp passenger β-domain fusions described above to result in plasmids pVHH(R2)-Hbpβ, pVHH(R2)-Ag43(β700), pVHH(R2)-Ag43(β710), pVHH(R2)-IgAP(β1124), pVHH(R2)-IgAP(β1245), pVHH(R2)-EstAβ, pVHH(R2)-Hiaβ and pVHH(R2)-Hia3β, respectively.Plasmids encoding fusions of a nanobody   using thHH domain (HH(R2)-plasmid series described above yielding pSS-myc-Hbpβ, pSS-myc-Ag43(β700), pSS-myc-Ag43(β710), pSS-myc-IgAP(β1124), pSS-myc-IgAP(β1245), pSS-myc-EstAβ, pSS-myc-Hiaβ and pSS-myc-Hia3β, respectively. The constructs all contain the Hbp signal sequence followed by the Myc tag with flexible linker and then the various β-domains .660 of 0.05 and grown to an OD660 of ~0.3 to ~0.5. Cells were induced for protein production by the addition of Isopropyl β-D-1-thiogalactopyranoside (IPTG) (1 mM) or left uninduced (negative control) as indicated. Growth was continued for 2 h, after which culture samples were prepared. Cells and spent medium were separated by low speed centrifugation. Cells were directly resuspended in SDS-sample buffer  to yield whole cell lysates. Medium samples were first trichloroacetic acid (TCA)-precipitated and subsequently resuspended in an equal volume of SDS-sample buffer. All samples were heated for 5–10 min at 96°C prior to analysis by SDS-PAGE and subsequent Coomassie (G-250) staining or Western blotting. The amounts loaded of the whole-cell lysates and medium corresponded to the same amount of culture. Coomassie-stained gels were imaged using a GS800 calibrated densitometer (Biorad). Visualisation of immunoblot signals was carried out using a ChemiDoc XRS+ system. Images were processed and densitometry was performed using Quantity One software (Biorad).Recombinant proteins were expressed from vector pEH3 under control of a lacUV5 promoter  using thUrea extraction assays of isolated membrane fractions were performed as described in . Cells wHeat modifiability assays were performed as described . To halfProteinase K treatment of whole cells was performed as described in . Cells iOne mL of an induced culture was collected by centrifugation, fixed using fomaldehyde and incubated for analysis using immunofluorescence microscopy with α-Myc antiserum as described previously . As secoe.g [i.e. the 12-stranded β-barrel preceded by an α-helical segment; see the Neisseria meningitidis expressing IgAP [The five selected autotransporters for our comparative studies came from various backgrounds, but all have been used previously for the secretion of recombinant protein sequences , 31–37. e.g , 44). Foe.g  selecteding IgAP . This poing IgAP . The staing IgAP . We confing IgAP .Pseudomonas stutzeri A15 [P. aeruginosa, because its crystal structure has been solved [1091 of Hbp. This position is in an α-helical segment and located 10 amino acid residues upstream of the autocatalytic cleavage site that separates the passenger from the β-domain  has been shown to support the export of the passenger domain of monomeric autotransporter Hap of  surface . Other t surface , 47. We,tructure  this segtructure . Hiaβ under control of an IPTG-inducible lac promoter and downstream of a sequence encoding the endogenous Hbp signal sequence for translocation across the inner membrane  . No band . No banE. coli have been shown to be cleaved by outer membrane protease OmpT [The appearance of degradation products for and weak expression of some of the constructs could indicate that these proteins did not fully insert into the outer membrane, making them targets for degradation by periplasmic and outer membrane-based proteases like DegP and OmpT. Our previous work has shown that Hbp mutants that are blocked in outer membrane secretion are degraded by the periplasmic protease/chaperone DegP , 19. As ase OmpT , 49. To ase OmpT , 19. AftThe heat-modifiability assay is based upon the observation that outer membrane β-barrel proteins maintain their structure when incubated in a buffer with a low concentration of SDS (0.4%) at room temperature. Consequently they run at a different, usually lower, position during PAGE when compared to heat-denatured samples . The heaOverall the results suggest that the β-domain constructs, with the exception of EstAβ were inserted into the outer membrane in a folded conformation, although the latter is difficult to assess for the Hia constructs. Furthermore, only the Myc-Hbpβ, -Ag43β and -IgAPβ(1245) reached reasonably high expression levels and outer-membrane insertion, whereas expression and insertion for the other constructs is limited.E. coli autotransporter EspP (EspPβ), which like Hbp belongs to the SPATE family of monomeric autotranporters [E. coli and H. influenza cells as shown by FACS analysis and Hap-mediated binding to cultured human epithelial cell lines [To establish whether the different β-domain constructs can secrete larger cargo than the rather small Myc-tag, we fused them to the Hbp passenger . Previounporters . The Espll lines . Hap is SpeI-fusion site in the hbp sequence ) carries a substitution of Ile1090 for Thr. This substitution did not affect the expression and processing of Hbp, given the appearance of similar levels of the cleaved β-domain in cell samples , Hbp-Ag43β(710), Hbp-IgAPβ(1124), Hbp-IgAPβ(1245), Hbp-EstAβ, Hbp-Hiaβ and Hbp-Hiaβ(3×), respectively , autocatalytic cleavage of the constructs was not expected to occur, because either the required protease domain (IgAPβ or the targeted cleavage site (Ag43β) is missing in the construct or the original autotransporter is not proteolytically processed at all  and Hbp-Hiaβ(3×) . The latstaining . With th samples  at posit samples . Highest samples .degP::S210A, which expresses a mutated version of DegP, which lacks its protease activity through mutation of the active site but retains its activity as a chaperone [degP::S210A affected growth of cultures more than expression in wild-type MC1061, most likely due to periplasmic accumulation of non-secreted proteins that stalled at the outer membrane, as previously suggested for wild-type and mutant Hbp proteins [degP::S210A expressing Hbp(SpeI), indeed, showed slightly reduced levels of secreted Hbp passenger and processed Hbpβ on Coomassie-stained SDS-PAGE gels when compared to expression in wild type MC1061, while levels of unprocessed Hbp increased  construct being the most efficient. The secreted IgA protease passenger itself is a SPATE-like serine protease with a structure very similar to Hbp  and its HH domain [HH domain (PDB 1QD0) revealed an Ig-like domain of ~19 by ~35 Å that includes two cysteine residues that form a disulfide bond that stabilizes the structure [HH domain, led to degradation of the passenger in the periplasm. For example, inclusion of a hairpin of two α-helices stabilized by a disulfide bond blocked secretion of the Hbp passenger [HH domains and other single-chain antibody fragments directly to β-domains including Ag43β(700), IgAPβ(1124) and IgAPβ(1245) were reported to be surface exposed, despite the presence of disulfide bonds [HH domain when fused directly to β-domain constructs.Autotransporters are considered attractive systems for production and export of relevant recombinant proteins , 2. To tH domain . The crytructure  Fig 4)..HH domaiassenger , as did assenger . Insertiassenger , 54. On de bonds , 44, 55.HH and a Myc tag for detection to the eight β-domain constructs and tested expression in E. coli MC1061  construct, which showed a more severely reduced growth. Expression was subsequently analysed on Coomassie-stained gels. The gels showed unprocessed VHH-Hbpβ and VHH-IgAPβ(1245) fusions at positions that matched their calculated molecular weights  constructs appeared similar to wild-type MC1061. Furthermore, clear protein bands could now be detected for the two VHH-Ag43β and the VHH-EstAβ fusions confirming their periplasmic degradation by DegP in wild-type MC1061. Western blots incubated with an anti-Myc antiserum revealed protein bands at the expected positions for unprocessed VHH-β-domain proteins for all fusions , VHH-Ag43β(710) and VHH-IgAPβ(1245). In view of the absence of a natural processing site in these constructs, such a processing could have resulted from an external protease like OmpT. Overall, the results suggested that VHH-Hbpβ and VHH-IgAPβ(1245) were exposed at the cell surface at least to some degree, whereas the two Ag43β-VHH fusions may be exposed too dome, but clearly lower extent.We fused a Vi MC1061 . The ind5 to 2.5  suggesti weights  while th weights . To testned gels . The exp fusions , but witHH domain and subsequent blocking of the translocation across the outer membrane. To test this assumption, the constructs were analyzed in the DsbA knockout strain DHBA and its isogenic wild-type DHB4 (HH-β-domain fusions were not detected in these strains on Coomassie-stained gels except for VHH-Hbpβ and VHH-IgAPβ(1245). However, they could be detected on a blot probed with α-Myc, which yielded a very similar picture to that of MC1061  were expressed at Coomassie-detectable levels, which could indicate stable expression and display of VHH at the cell surface. However, an alternative explanation for the detection of non-processed VHH-Hbpβ fusion could be that translocation across the outer membrane was blocked, since this step precedes and is a prerequisite for the subsequent processing of the wild-type Hbp passenger [HH-Hbpβ and VHH- IgAPβ(1245) constructs. Externally added proteinase K almost fully degraded the Hbpβ-VHH fusion to ~15% of the non-treated sample, as indicated by densitometric analysis, indicating efficient cell surface exposure (HH-IgAPβ(1245) remained detectable and densitometric analysis indicated a decrease of ~45% of the untreated cells. Control bands on the Coomassie-stained gels showed no degradation for the cells expressing the VHH-Hbpβ fusion, and degradation to 75% for the cells expressing the VHH-IgAPβ(1245) fusion. We further assessed the degradation of OmpA and SurA on blot  by in incubating MC1061 cells expressing VHH-Hbpβ and VHH-IgAPβ(1245) with α-Myc antiserum and a secondary antibody conjugated to the fluorescent dye Alexa488 (HH-IgAPβ(1245) fusion, indicating physiological problems resulting from that expression. Apparently, the expression of the fusion interfered with normal cell growth, although its effect was not influencing cell viability per se as judged from the growth curves. In contrast, cells expressing the VHH-Hbpβ fusion  fusion was not detected by the externally added antibodies. The Vhh-IgAPβ(1245) was clearly expressed by the cells, and this result, therefore suggests, that the produced VHH-IgAPβ(1245) accumulates for a large part intracellularly, corroborating the results obtained by the proteinase K experiment.Both Vassenger , 54 and exposure . In cont on blot . DensitoAlexa488 . SurprisHH domains, even over the use of Ag43β and IgAPβ fusions, for which successful secretion has been reported [HH or single chain antibodies fused to the EhaA or BrkA β-domains [HH-IgAPβ fusion in strains lacking OmpT or DsbA improved expression [HH domain using a VHH-Ag43β(700) construct was confirmed by FACS analysis [HH on their cell surface [HH to and secretion to the cell surface is not generally observed for all β-domains studies. The unexpected elongated cell morphology when the Vhh-IgAPβ(1245) construct was expressed has not been found in other studies. The observations could be an anomaly of using strain MC1061, or be a general result of expressing a difficult autotransporter fusion. The result suggests that microscopic analysis could be important in deciding what construct to use for efficient surface display.Overall, the outcome of our comparison favors the use of Hbpβ for export of Vreported , 44, 55.reported , 44 and reported  were tes-domains , 34, 37 pression , 34, 44,analysis , but the surface . Another surface , which a surface , 34, 44.HH domain directly to the Hbpβ domain results in efficient expression and clear cell-surface exposure. Furthermore, a side by side comparison of several β-domain constructs, of which some have been reported to efficiently export VHH-like domains, shows that Hbpβ compares well to these constructs in expression levels and surface display.In the past, we have included various recombinant proteins in the Hbp passenger, replacing the sub-domains that extend from its β-helical stem , 25. NotHH domain may be much smaller than the Hbp passenger but, presumably, it attains a bulky conformation during the secretion process that could interfere with its secretion to the cell surface. Its structure is stabilized by a disulfide bond formed in the bacterial periplasm by the Dsb enzymes. Fusing this domain to the β-domains resulted in reduced expression levels for most β-domain constructs, except for Hbpβ and IgAPβ(1245). The latter two constructs showed reasonably high expression levels, but only the VHH domain fused to the Hbpβ was accessible for degradation by proteinase K and externally added antibodies, indicating surface exposure. Overall, the results indicate that attaining a more bulky secondary structure could interfere with secretion and hamper cell surface display.In this study we compared eight autotransporter β-domain constructs of five different autotransporters for their capacity to secrete fused heterologous sequences varying in size and complexity to the cell surface. We assayed expression levels, assembly in the outer membrane and surface exposure. Our results show that the type of passenger influences the secretion capacity of a β-domain. Structure appears more important than size of the passenger fused, since the long ~110 kDa Hbp passenger, which is structurally related to the endogenous passengers of most of the β-domains used, could be exposed on the bacterial surface by most β-domains, albeit with different efficiencies. The VFor Ag43 and IgAP we have tested two constructs and both configuration have been tested for fusions of heterologous proteins in earlier studies , 36, 44.Overall, our comparative study showed that for all passenger-β-domain fusions tested, the Hbpβ domain yielded the most consistent results. It was able to secrete all fused passengers to levels that were detectable on Coomassie-stained gels. Furthermore, the Hbpβ constructs appeared fully inserted into the outer membrane with clearly cell-surface exposed passengers. Therefore, fusion of recombinant proteins directly to the Hbpβ domain expands the Hbp toolbox for the secretion and cell surface display of heterologous protein sequences.S1 Fig(A) Structural models of the Hbpβ, Hiaβ, Hbp passenger and VHH domain. The PDB codes of the depicted models are given. (B) Nucleotide sequence of the SpeI-EcoRI fragment encoding the Hiaβ construct. Restriction sites used for cloning are indicated. The encoded amino acids are given below the nucleotide sequence with the amino acids constituting the four-stranded β-sheet contributing to the barrel in red. (C) Nucleotide sequence of the SpeI-EcoRI fragment encoding the Hiaβ(3×). The encoded amino acids are given below the nucleotide sequence with the amino acids constituting the three-time repeated segment in red, blue and pink.(PDF)Click here for additional data file.S2 Fig(A) Growth curves of cultures expressing the Hbp-β-domain fusions in MC10161 (left) and MC1061 degP::S210A (right). Expression was induced by adding IPTG at the timepoint indicated by the dotted line. (B) Western blots of cell samples of MC1061 expressing HbpΔβcleavage and Hbp-IgAPβ(1245) incubated with proteinase K either for 60 min on ice (0°) or 30 min at 37 °C (37°). Included are also untreated controls (-). A blot of a single SDS-PAGE gel was cut in two parts after which the top part was incubated with α-SurA antiserumem and the bottom part with α-OmpA antiserum.(PDF)Click here for additional data file.S3 Fig(A) Growth curves of cultures expressing the VHH-β-domain fusions in in MC10161 (top left), MC1061 degP::S210A (top right), DHB4 (bottom left) and DHBA . Expression was induced by adding IPTG at the timepoint indicated by the dotted line. (B) Western blots of cell samples in DHB4 and DHBA (dsbA-) cells. Westernblots incubated with α -Myc of whole cell lysates and culture supernatants of cultures of DHB4 (left panel) and DHBA (dsbA-) (right panel) expressing the VHH-β-domain fusions (*) to assess their expression. (C) Western blot of MC1061 cells expressing VHH-Hbpβ and VHH-IgAPβ(1245) incubated with proteinase K either for 30 min at 37°C (37°). Included are also untreated controls (-). A blot of a single SDS-PAGE gel was cut in two parts after which the top part was incubated with α-SurA antiserum and the bottom part with α-OmpA antiserum. The relevant lanes were cut from a larger image to place them side by side.(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


Leptospira interrogans, LigB. An anti-LigB monoclonal antibody library exhibits cell binding and bactericidal activity with extensive domain coverage complementing the elongated architecture observed in the SAXS structure. Combining antigenic motifs in a single-domain chimeric immunoglobulin-like fold generated a vaccine that greatly enhances leptospiral protection over vaccination with single parent domains. Our study demonstrates how understanding an antigen’s structure and antibody accessible surfaces can guide the design and engineering of improved recombinant antigen-based vaccines.Pathogens rely on proteins embedded on their surface to perform tasks essential for host infection. These obligatory structures exposed to the host immune system provide important targets for rational vaccine design. Here, we use a systematically designed series of multi-domain constructs in combination with small angle X-ray scattering (SAXS) to determine the structure of the main immunoreactive region from a major antigen from  Vaccines encourage the immune system to develop a protection against disease-causing bacteria and viruses. Some types of immune cells release antibodies, which recognize particular proteins on the surface of the invading microbe. A vaccine that contains these surface proteins allows immune cells to develop the antibodies that can help to fight off an infection at a later date. Studying the shape and structure of the surface proteins can reveal how they are detected by our immune systems and can further be used to design more effective vaccines.Leptospirosis is the most common bacterial disease to affect both humans and animals. The symptoms of the disease include fever, muscle pain and bleeding from the lungs. New vaccines against leptospirosis are desperately needed because current ones have severe side effects and do not fully protect against the disease. The most promising new vaccine candidates are the Lig proteins, which are found on the surface of leptospirosis causing bacteria cells, but little was known about their molecular structure.The region of the Lig protein that is recognized by the immune system consists of a series of twelve connected ‘immunoglobulin-like’ domains. Hsieh, Ptak et al. used X-ray scattering to determine the structure of this region and found that the protein is highly elongated. Additional experiments showed that the individual domains provoke immune responses to different extents. Antibodies that can interact more strongly with the Lig protein were also better able to kill the bacteria. Based on this information, Hsieh, Ptak et al. combined parts of the individual domains that bind strongly to antibodies to design a new protein that, when used as a vaccine, protected hamsters against leptospirosis much better than other Lig protein-based vaccines.Further engineering and testing are required to develop an optimized, commercial leptospirosis vaccine, but the work of Hsieh, Ptak et al. shows the effectiveness of structure-based vaccine design methods. In the future, similar methods could be used to develop better vaccines and treatments for other infectious diseases. Leptospira species  protein family  NMR structure informed the generation of chimeras on single Ig-like domain scaffolds capable of eliciting an immune response with either side of the β-sandwich domain. To illustrate the potential for rationally engineered antigens, a vaccine containing the chimera LigB10-B7-B7, which displays identified mAb-interacting surfaces from LigB7 and LigB10, offered greatly improved protection over LigB7 and LigB10 against leptospiral lethal challenge in hamsters. These findings provide a blueprint for combining immunoreactivity mapping from an mAb library and high-resolution structural information from NMR to engineer epitopes and improve the efficacy of LigB vaccines as well as recombinant vaccines from other pathogens.In this study, small angle X-ray scattering (SAXS)  was usedTo determine the architecture of the twelve Ig-like domain stretch in LigB, small angle X-ray scattering (SAXS) was used to generate low-resolution solution structures along a sliding multi-domain window. Because the 5-domain length contains four domain-domain linkers, the 5-domain structure proved to be optimal to define the relative angle of two neighboring domain-domain joints with one additional joint on each end ) is indicative of the length of the 5-domain proteins. The expected length of a fully extended arrangement of five folded LigB Ig-like domains is only slightly longer than the pair distance for most of the eight 5-domain proteins and their corresponding envelopes (All eight possible 5-domain protein constructs (LigB1-5 to LigB8-12) were analyzed with SAXS . Guiniernvelopes . LigB5-9nvelopes  and was Ambiguity assessment using the AMBIMETER program  shows th630 = 1.0  for each of these mAbs was obtained from dose-dependent ELISA curves  exhibit sub-micromolar KD values while only one library V mAb (V10) was able to bind in the sub-micromolar range.For each library, nine mAbs were identified to have moderate to high binding efficiency to LigB antigens during the initial mAb screen. The dissociation constants   Ig-like domain LigB truncates (except LigB5-6) based on the NMR structure  of the seraction . Cells ieraction  or the neraction  failed tproteins , also faproteins . A totaland V33) . The celLeptospira in vitro, the bactericidal activity of the LigB-binding mAbs was tested by incubating live L. interrogans serovar Pomona cells with complement-containing human serum with or without added antibodies. Using dark-field microscopy, motile bacteria were counted to calculate the survival rate at different time points. The PBS only (negative control) condition was unable to effectively kill the live Leptospira cells leaving an 84% survival rate at 120 min post treatment  (To test the role of the mAb libraries in promoting complement-mediated killing of reatment . The ham p<0.05) .lux cassette proteins in another pathogenic L. interrogans serovar Manilae to accurately report the viable cell count  mAb library using these two serovars would be similar. Bioluminescent L. interrogans serovar Manilae were again incubated with complement-containing human serum with or without added antibodies. The survival rate was determined by measuring the luminescence intensity of metabolically active Leptospira relative to that from the spirochetes prior to Ab-incubation. Both incubation time and antibody concentration were varied to obtain time-dependent and dose-dependent curves  between 10 and 30 μg/ml , it can bind to two single domains (LigB4 and LigB6) as well as a double domain in the segment LigB1-3. Further, an additional ELISA assay found that C12 is the only library C mAb that can also bind to LigB7-12 . The abiD . OverallTo provide proof that a single Ig-like domain can act as a scaffold to display multiple protective epitopes, the binding epitope for anti-LigB mAbs was more specifically mapped to a surface within individual Ig-like domains. Chimeric LigB Ig-like domains were designed to differentiate interactions specific to the surface residues of major folding units. The high degree of homology between LigB Ig-like domains  providedAll mAbs showed binding to a subset of the eight proteins with a clear region-specific pattern . Two Lig2 of triple passages of L. interrogans Pomona (Leptospira survived until the end of the experiment (day 21 post infection). The survival rate of the LigB10-B7-B7 group is significantly higher (100%) than either control group (17%) or individual wild-type domain groups (0%) (p<0.05).The ultimate goal of combining structural and immunoreactivity studies of antigens is to explore a strategy for the rational engineering of improved vaccines. A chimeric LigB Ig-like domain was evaluated for the potential to elicit an immune response to multiple regions of LigB and to thereby enhance vaccine protection against leptospiral infection. The chimera LigB10-B7-B7 (β-strands A-C: B10 and β-strands C′-F and G-G′: B7) was chosen as the best candidate for animal studies because the chimeric domain possesses the ability to bind highly bactericidal mAbs specific to each parent domain . Additios Pomona   to identify the , LipL32    at 20 ˚C for 16 hr. After the cells were lysed using a high-pressure cell disruptor, the cell lysates were spun down and the supernatants were purified by Ni-NTA resin (Qiagen). The His-Sumo tagged LigB proteins were eluted with phosphate buffered saline  containing 300 mM imidazole and then digested with Sumo protease Ulp-1 while dialyzing against PBS buffer at 4 ˚C overnight (A series of single (LigB1 to LigB12), double , five-domain (LigB1-5 to LigB8-12), and multiple  Ig-like domains of LigB from vernight . Afterwavernight . Chimerivernight . The thrvernight  and the vernight . After lConstructs corresponding to two, five and eight domains were generated to identify the optimal domain number required to obtain useful SAXS-derived envelopes . The 8-d280. SAXS data for individual samples was measured for 15 scans at 1×, 2/3×, and 1/3 × protein concentrations after dilution with PBS buffer and centrifugation at 14,000 rpm for 10 min. Capillary cells were robotically loaded with 30 μL samples from a 96 well-plate maintained at 4°C (r) plots. Optimal Rmax was determined by screening at 5 Å intervals and the P(r) plots were normalized to the first peak. Profiles best representing the dilute  solution limit were used to generate ab initio models with DAMMAVER and DAMMIF programs from the ATSAS suite (LigB-derived proteins were exchanged into PBS buffer (pH 7.4) and concentrated to 12–20 mg/ml as assessed by absorption at UVd at 4°C . Betweend at 4°C . Backgrod at 4°C . The GNOAS suite . 10 initAS suite . AMBIMETConserved region) and 36 clones of IgG-type mAbs against LigB7-12  were generated. Each library produced 9 mAbs which were found to be of moderate to high binding efficiency and utilized for further characterization. Purification of mAbs was conducted by protein A/G chromatography (Pierce) using the manufacturer-recommended procedure with minor modification  as previously described . Standarfication . Briefly3, pH 9.4) at 4˚C overnight. After blocking with 3% BSA in PBS buffer for 1 hr, hybridoma supernatants were prepared at 1/500 dilutions and individually applied to LigB-coated wells for initial screening. To obtain dissociate constants, selected mAbs with moderate to high binding affinity to LigB were serially diluted in PBS  and then individually applied to LigB-coated wells. Between each step, PBS containing 0.05% Tween-20 (PBS-T) was used to wash the plates for three times. Subsequently, anti-mouse IgG antibody conjugated with HRP  was added to detect the binding of mAbs presented in hybridoma supernatants. Finally, 100 μl of TMB peroxidase substrate (KPL) was applied to each well, the optical density of which was recorded at 630 nm by ELx808 Absorbance Microplate Reader (BioTek). OD630 values represent the mean of three independent trials ± the standard deviation. For each trial, samples were assayed in two replicates.LigB-binding mAbs were selected using an ELISA assay . MicrotiFor examining the domain specificities of mAbs, single domain LigB fragments (LigB1 to LigB12), double domain LigB fragments , positive control , or negative control (BSA) were coated with a fixed concentration (1 μg/well) on the ELISA plates. Each mAb against LigB1-7 or LigB7-12 was then added to corresponding single-domain or double-domain LigB coated wells. For binding analysis of each chimera, the set of six chimeric LigB proteins plus the two parent LigB proteins were coated on the ELISA plates (1 μg/well). Each mAb with activity against either of the two parent LigB proteins was screened for binding activity on the LigB coated wells. For measuring the antibody responses triggered by LigB-based recombinant vaccines, sera from hamsters immunized with LigB7, LigB10 and LigB10-7-7 were collected at different time points . Then, these serum samples (1:500) were added to corresponding LigB Ig-like domain coated wells (1 μg/well), and also applied to LigB12 coated wells (negative control). Finally, anti-hamster IgG or IgM antibody conjugated with HRP  was used to detect the LigB-bound antibodies from hamster sera. All single and double domain ELISA experiments including single chimeric domain experiments were conducted in three trials, the mean ±S.D. of which were shown in bar charts.Leptospira interrogans freshly harvested from hamsters challenged with serovar Pomona or overnight NaCl-treated low passage Leptospira were prepared in PBS buffer containing 5 mM MgCl2 at 108 cells/ml. Various anti-LigB mAbs (100 µg/ml) were individually applied to the bacterial suspension at 1:500 dilution. After a 1 hr incubation at room temperature, the bacteria-mAbs mixtures were spun down at 2,000 g for 7 min and then washed with PBS containing 1% BSA. Subsequently, goat anti-mouse antibodies conjugated with fluorescein (FITC) (1:1000) were used as secondary antibodies for probing the bacteria-bound mAbs. After another PBS-BSA wash, the bacteria-mAbs mixtures were fixed by 0.5% formaldehyde in PBS. The non-pathogenic Leptospira biflex, which does not express LigB, was subjected to the same procedure by incubating with selected mAbs for the control experiments (Leptospira with unrelated mouse IgG (isotypic mouse IgG purchased from ThermoFisher Scientific). Flow cytometry was performed at the Cornell University Flow Cytometry Core facility using a BD LSR-II (BD Biosciences) instrument with the excitation laser at 488 nm and the emission wavelength at 525/575 nm. Unstained Leptospira was identified by forward scatter (FSC) and side scatter (SSC). Selected mAb (C5) treated L. interrogans without secondary antibodies (FITC-conjugated goat anti-mouse IgG) was used as a negative control to set the gating region. The mean FITC-positive (MFI) count was obtained for the gated region of the 525 nm vs. 575 nm scatter plots using BD FACSDiva software. For each sample, at least 20,000 cells were analyzed in two independent trial of two replicates.eriments . Another8 cells/ml of low passage, high virulent L. interrogans seorvar Pomona and Manilae strain M1307 were prepared in PBS buffer containing 2 mM MgCl2 and 1 mM CaCl2, and then mixed with respective mAbs, plus 25% of normal human serum (ImmunoReagents) as a complement source. The mixtures were incubated at 37°C for 1 hr, and the viability of the bacteria were examined using dark-field microscopy. The survival rate of leptospira was calculated as the number of motile  cells in every 100 counts separately by two researchers (blind). The mean value was obtained from the two independent (blind) measurements as a single technical replicate. Three independent trials of two replicates were measured.To examine if the mAbs were effective at killing bacteria in vitro, the serum bactericidal activity (SBA) assay was conducted. 10L. interrogans Manilae strain M1307 (50) and dose (LD50) required to reach 50% lethality were obtained from fitted logistic and dose inhibition curves, respectively (Origin 7.0).The viability of the leptospira was also accessed from the luminescence emitted by metabolically active in M1307 . The samad libitum as previously described  were housed in isolation units approved by the Cornell University Institutional Animal Care and Use Committee (Protocol number: 2015–0133). The Golden Syrian Hamsters used in vaccine trials were allowed to run free in the cage, were fed a commercial ration, and were provided water escribed . Six 5-wescribed . Kidneysescribed . Leptospescribed . The tot, LipL32 , was amp In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.Leptospira antigen offers high bactericidal antibody accessibility amenable to vaccine design" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Wendy Garrett as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Extended structure of a The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Leptospira coat protein, LigB, to rationally design a recombinant multi-epitope antigen that appears to provide superior protection that either the full-length proteins or its individual domains. The present manuscript represents a dizzying amount of work, including comprehensive SAXS analysis of the multi-domain LigB protein and generation and characterization of two mAb libraries against LigB domains. Overall, the manuscript is well-written and easy to parse. Some questions, however, should be clarified.Leptospirosis is a widespread zoonotic bacterial infection that can cause serious disease in both animals and humans and can be refractory to treatment. Prevention is hampered by the fact that current vaccines are only partially effective. The work by Hsieh, et al., uses structural information of the immunoglobulin-like domains of a common Essential revisions:1) While the authors make a point of highlighting the importance of the SAXS data to their design efforts, in reading the paper, it seems that the NMR structures of the individual domains were really the drivers . If the authors are presenting this as a roadmap to others, they might either better explain how the SAXS data really were critical, or change their language a bit.2 for a linear fit? The authors briefly discuss the outlying mAbs – C12 and C23, but it seems that authors missed a huge opportunity in understanding what drives a much worse than expected and much better than expected activity vs. affinity, which has the potential to inform other rationally designed vaccination efforts.2) In 3) While potentially outside of the scope of the present work, it seemed odd that the authors did not compare or even mention differences in memory response when using different vaccination strategies.4) The main text does not state where the 60 antibodies are coming from. Why where there only 60? How many hybridomas have been screened in total? An explanation is only given in the Materials and methods. Additionally, the data for the screening of the antibodies should be presented in its entirety and not buried in the supplement. Only then the reader can fully appreciate how the lead antibodies have been identified. Perhaps a heatmap indicating the binding would be better suited to represent the data.5) The positive control for bactericidal activity is a polyclonal hamster antibody, while the authors test mouse antibodies. Is it possible to discuss the lack of efficiency of the hamster polyclonal in greater detail? Why not use a mouse polyclonal as control?6) When describing in Leptospira than hamsters vaccinated with B10 or B7, respectively. The better control would have been hamsters simultaneously vaccinated with a mixture of B10 and B7. Also, the ELISA in 7) In the vaccination experiment the authors describe that hamsters vaccinated with the chimera B10/7/7 have a better chance of survival when infected with  Essential revisions:1) While the authors make a point of highlighting the importance of the SAXS data to their design efforts, in reading the paper, it seems that the NMR structures of the individual domains were really the drivers . If the authors are presenting this as a roadmap to others, they might either better explain how the SAXS data really were critical, or change their language a bit.We have adjusted our language regarding how the SAXS data was influential in developing the study. Specifically, we have changed the impact statement, the final sentence of the Abstract, and the final sentence of the Introduction. We agree that, in hindsight, the immunoreactivity mapping experiments could have been designed to inform the accessible features of the antigen without the SAXS data. Still, the initial SAXS study yielded a low-resolution structure that suggested generating monoclonal antibodies would be worth the expense for both LigB1-7 and LigB7-12 regions. This is now noted in the Discussion section. If the SAXS study had revealed that some domains were clearly buried, then these regions could have been excluded from the generation of the mAb library. The SAXS data and immunoreactivity data offer complementary views of the overall structural architecture which also creates elements of redundancy.2 for a linear fit? The authors briefly discuss the outlying mAbs – C12 and C23, but it seems that authors missed a huge opportunity in understanding what drives a much worse than expected and much better than expected activity vs. affinity, which has the potential to inform other rationally designed vaccination efforts.2) In 2 is reported for both the total dataset and the dataset without the statistically-determined outliers.For D based on ELISA. While C12 was determined to be a statistical outlier in both the original and new D value of C12 is closer to the expected value from the fitted correlation. We note that C12 binds to two double and two single LigB Ig-like domains within LigB1-7 (not LigB7) and therefore may bind to a conserved region of the domains. To test if C12 was promiscuous enough to also bind to LigB7-12, an additional ELISA was performed to assess the possibility of interactions between library C mAbs and LigB7-12. C12 was the only mAb with significant binding to LigB7-12 suggesting that the ability to bind to several LigB Ig-like domains allows C12 to more effectively kill bacteria. A supplemental figure  While potentially outside of the scope of the present work, it seemed odd that the authors did not compare or even mention differences in memory response when using different vaccination strategies.Leptospira lethal challenge”.We include a figure to illustrate the relative IgG and IgM response to LigB10-B7-B7 immunization . As expe4) The main text does not state where the 60 antibodies are coming from. Why where there only 60? How many hybridomas have been screened in total? An explanation is only given in the Materials and methods. Additionally, the data for the screening of the antibodies should be presented in its entirety and not buried in the supplement. Only then the reader can fully appreciate how the lead antibodies have been identified. Perhaps a heatmap indicating the binding would be better suited to represent the data.630 = 1). The screening data is presented in its entirety. We now provide more detail on how the antibodies were obtained in both the added Results section and the Materials and methods. A heatmap of the data was unable to depict the ELISA cutoffs so we are presenting the data as a bar graph. An additional supplementary figure  The positive control for bactericidal activity is a polyclonal hamster antibody, while the authors test mouse antibodies. Is it possible to discuss the lack of efficiency of the hamster polyclonal in greater detail? Why not use a mouse polyclonal as control?Leptospira than hamster anti-LigB pAb that would be generated using the conventional leptospirosis animal model, we felt the data should be included but we are willing to remove the data if the reviewers would prefer taking it out. There are three possible explanations for the lack of hamster pAb efficiency. First, the pAbs are likely to include both high affinity and low affinity anti-LigB antibodies and also background antibodies with no affinity for LigB. The effectiveness of pAbs are likely to be reduced because the concentration of high affinity anti-LigB antibodies is lower for a pAb solution of the same overall concentration as for a solution the purified mAbs. Second, pAbs contain a mixture of IgG subclasses . The ability of the different hamster-derived IgG subclasses to effectively activate the complement system is unknown; however, different IgG subclasses from both mouse and human exhibit variability in their effectiveness at activating the complement system. Third, while the ability of mouse antibodies to activate the human complement cascade is known, the relative ability of hamster antibodies to activate the same cascade has not been described. Because we do not know if mouse and hamster antibodies are equally efficient at eliciting the killing response, we cannot rule out the possibility that hamster antibodies are less effective in the assay. We now discuss the possible reasons for the lack of hamster pAb efficiency in the Results section of the manuscript .In the serum bactericidal assay, the no antibody and isotypic mouse IgG groups are the true controls for the assay. We now do not refer to the hamster anti-LigB polyclonal antibodies as a control in the manuscript. We agree with the reviewers that the mouse pAb would have been a better control; Unfortunately, mouse pAbs were not retained during the mAb library creation and are therefore not readily available. Hamster anti-LigB pAb were retained from vaccine trials and was easily included in the bactericidal assay. Because the result suggests that the mAbs generated in this study are more effective at killing 6) When describing in D) is a more accurate measurement of mAb-antigen interactions than the ELISA-derived relative antigen binding measurement. We have replaced the relative binding measurements with KD values. KD values for purified mAbs were determined by fitting concentration-dependent ELISA measurements. We have replaced the relative binding measurements with the concentration-dependent ELISA curves and KD values  to suggest bactericidal activity is dependent on binding affinity for most mAbs. The outliers, C12 and C23, were explored further in the three added supplements to We agree with the reviewers that binding affinity  In the vaccination experiment the authors describe that hamsters vaccinated with the chimera B10/7/7 have a better chance of survival when infected with Leptospira than hamsters vaccinated with B10 or B7, respectively. The better control would have been hamsters simultaneously vaccinated with a mixture of B10 and B7. Also, the ELISA in We have added the requested controls for Anti-LigB7 and Anti-LigB10 sera ELISA to There are several reasons why we feel that the benefits of the additional control would not be an ideal control and do not warrant significantly delaying the publication of this manuscript. From a manufacturing standpoint, purification and subsequent mixing of domains would be costlier than purification of a single domain. This control would not generate a useful result in and of itself. Additionally, we show a robust gain in vaccine efficiency and the importance of a positive control would be minimal. Also, since all of the vaccines used in the trial were single domain proteins from a single purification, the inclusion of a mixed protein solution would not be a direct comparison. While a vaccine trial that included another control with a mixture of LigB7 and LigB10 could have been added, the additional vaccine trial would require 3-4 months to complete. Again, we argue that the small benefit of the additional vaccine trial control does not warrant the time commitment and delay that it would require.