抗菌蛋白的作用机制

LETTER doi:10.1038/nature12729 Antibacterial membrane attack by a pore-forming intestinal C-type lectin SohiniMukherjee1,Hui Zheng2,MehabawG.Derebe1, KeithM.Callenberg3, Carrie L. Partch4,DarcyRollins1, Daniel C. Propheter1, Josep Rizo5, Michael Grabe3{, Qiu-Xing Jiang2* & Lora V. Hooper1,6* Human body-surface epithelia coexist in close association with complex bacterial communities and are protected by a variety of antibacterial proteins. C-type lectins of the RegIII family are bac- tericidal proteins that limit direct contact between bacteria and the intestinal epithelium and thus promote tolerance to the intestinal microbiota1,2. RegIII lectins recognize their bacterial targets by binding peptidoglycan carbohydrate1,3, but themechanismbywhich they kill bacteria is unknown. Here we elucidate the mechanistic basis for RegIII bactericidal activity. We show that human RegIIIa (also known as HIP/PAP) bindsmembrane phospholipids and kills bacteria by forming a hexameric membrane-permeabilizing oligo- meric pore. We derive a three-dimensional model of the RegIIIa pore by docking the RegIIIa crystal structure into a cryo-electron microscopicmapof theporecomplex, andshowthat themodelaccords with experimentally determined properties of the pore. Lipopoly- saccharide inhibits RegIIIa pore-forming activity, explaining why RegIIIa is bactericidal for Gram-positive but not Gram-negative bacteria. Our findings identify C-type lectins as mediators of mem- brane attack in the mucosal immune system, and provide detailed insight into an antibacterial mechanism that promotes mutualism with the resident microbiota. RegIIIa damages the surfaces of Gram-positive bacteria1, indicating that RegIIIamight target bacterial membranes. We assessed the capa- city of RegIIIa to permeabilize bacterial membranes by quantifying bacterial uptake of a membrane-impermeant fluorescent dye (SYTOX green). RegIIIa increased SYTOX green uptake when added to the Gram-positive species Listeria monocytogenes, indicating damaged membranes (Fig. 1a, b). RegIIIa has an anionic amino-terminal pro- segment that inhibits bactericidal activity (but not peptidoglycan bind- ing) bydocking to theprotein core through charge–charge interactions4. The pro-segment is removed by trypsin on secretion into the intestinal lumen, yielding bactericidally active RegIIIa (ref. 4). Bactericidally inactive pro-RegIIIa did not induce SYTOX green uptake, indicating minimalmembrane permeabilization (Fig. 1a). Thus, RegIIIa permea- bilizes the bacterialmembrane, and the pro-segment inhibits this activity. To test directly for membrane disruption by RegIIIa we used lipo- somes composedof 85%zwitterionicphospholipid (PC) and15%acidic phospholipid (PS). The liposomes encapsulated carboxyfluorescein, a fluorescent dye. RegIIIa induced rapid dye efflux from PC/PS lipo- somes (Fig. 1c),whichwas reducedwhenPC-only liposomeswere used (Fig. 1d, e). This indicates a preference for acidic phospholipids that is consistent with the acidic lipid content of bacterial membranes5 and with the salt sensitivity of RegIIIamembrane toxicity (Extended Data Fig. 2a, b). These findings indicate that RegIIIa interactions with lipid bilayers aremediated by electrostatic interactions. pro-RegIIIa yielded a diminished rate of dye release (Fig. 1f), indicating that the pro-segment inhibits membrane permeabilization. Wenext assessedRegIIIa lipid-binding activity bymeasuring changes in the intrinsic fluorescence of tryptophan residues6.Weobserved increased tryptophan fluorescence intensity only when RegIIIa was added to PS-containing liposomes (Fig. 1g–i), indicating that RegIIIa interacts with acidic phospholipids. Furthermore, we observed fluorescence res- onance energy transfer (FRET) between donor RegIIIa tryptophan residues and dansyl-labelled PC/PS liposomes7 (Fig. 1j, k). FRET was inhibited by the pro-RegIIIaN-terminal pro-segment (Fig. 1j, k), indi- cating that the pro-segment inhibits bactericidal activity by hindering lipid binding. Consistent with its inability to bind lipids, pro-RegIIIa did not inhibit RegIIIa bactericidal activity in mixing experiments (Extended Data Fig. 2c). Several membrane-active toxins destabilize membranes by forming monomeric or multimeric pores8. To test for RegIIIa pores, we per- formed conductance studies in black lipidmembranes, amodel system thatmimics the properties of a cellmembrane9. RegIIIa produced rapid single-channel-like currents at 280mV in the presence of Mg21 ions (Fig. 2a), with no current detected at 0mV. Using the Nernst–Planck equationwe estimated the diameter of thepore at,12–14 A˚ (Extended Data Fig. 3). The calculated pore size agreed with the lack of efflux of fluorescein isothiocyanate-dextran-10 (FD10) or FD4, which have Stokes diameters of ,44 A˚ and ,28 A˚, respectively (Fig. 2b). In con- trast, carboxyfluorescein (,10 A˚) passed readily through the pores (Figs 1c and 2b). These results show that RegIIIa forms functional transmembrane pores and yield an estimate of the inner pore diameter. When visualized by negative-stain electronmicroscopy (EM), numer- ous circular structures of,100 A˚ diameter were observed on liposomes incubated with RegIIIa (Fig. 2c and Extended Data Fig. 4a). Although RegIIIa is a monomer in solution10, the size of the pores suggested that theywereRegIIIamultimers.We therefore treated liposome-associated RegIIIawith a crosslinking agent, solubilized the products in detergent, and separated themby size-exclusion chromatography (Fig. 2d). In addi- tion to a prominent monomer peak we detected a second, liposome- dependent peak at a lower retention volume, indicating the formation of a multimeric complex. Western blotting showed a single RegIIIa species with mobility similar to that predicted for a hexamer (Fig. 2d), suggesting that the pore was a RegIIIa hexamer. After longer incubationswith lipid,RegIIIa formed filaments (Extended Data Fig. 4b) similar to those in pancreatic secretions11. The filaments were ,100 A˚ in diameter, correlating with the dimensions of the RegIIIa pore (Fig. 2c). RegIIIa filamentation required lipid and was dependent on RegIIIa pore formation, as pro-RegIIIa formed neither pores nor filaments (Extended Data Fig. 4b, d). Filamentation partially inhibited the ability of RegIIIa to permeabilize membranes (Extended Data Figs 4c and 5a–c), as observed with other membrane toxic host defence proteins where filamentation traps pore complexes and limits damage to host cells12. These findings indicate that the RegIIIa filaments *These authors contributed equally to this work. 1Department of Immunology, The University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA. 2Department of Cell Biology, The University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA. 3Department of Biological Sciences, University of Pittsburgh, and Joint Carnegie Mellon University-University of Pittsburgh PhD Program in Computational Biology, Pittsburgh, Pennsylvania 15261, USA. 4Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064, USA. 5Department of Biochemistry and Department of Pharmacology, The University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA. 6The Howard Hughes Medical Institute, The University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA. {Present address: Cardiovascular Research Institute, University of California, San Francisco, San Francisco, California 94143, USA. 2 J A N U A R Y 2 0 1 4 | V O L 5 0 5 | N A T U R E | 1 0 3 Macmillan Publishers Limited. All rights reserved©2014 are higher-order assemblies of RegIIIa pore complexes and show that filamentation limits RegIIIa toxicity. Although the ,90-kDa RegIIIa pore complex was too small for structure determination by single-particle cryoelectron microscopy (cryoEM) methods13, the RegIIIa filaments were sufficiently large for such analysis. We therefore reconstructed a three-dimensional map of the RegIIIa filament and extracted the structure of theminimal pore complex (Fig. 3a, b and Extended Data Fig. 6a–f). The nominal resolution of our structure, 9.2 A˚, was limited by symmetry variability and filament bending (Extended Data Fig. 6g–j and Supplementary Information). Consistent with our crosslinking studies (Fig. 2d), the minimal pore was a hexamer formed by three RegIIIa dimers related by helical symmetry. The outer diameter of the pore assembly was 89 A˚, as observed by negative-stain EM (Fig. 2c). The pore height was 55 A˚, sufficient to span a lipid bilayer (35–45 A˚)14. The inner diameter was ,18 A˚, consistent with the pore size predicted by our conductance measurements (Extended Data Fig. 3) and dye release assays (Fig. 2b). RegIIIa, like other epithelial bactericidal proteins such asa-defensins, is constrained by disulphide bonds that prohibit large secondary struc- ture changes onmoving froman aqueous to an apolar environment15,16. This suggested the feasibility of docking the three-dimensional structure of the RegIIIamonomer into the EM density map tomodel the organ- ization of the pore complex further. First, we determined the crystal structure of processed, bactericidally active RegIIIa (Extended Data Fig. 7a) and compared it to the previously determined structure of bactericidally inactive pro-RegIIIa. The two structures were similar, although the amino acid side chains of the loop encompassing residues 93–99 (sequenceKSIGNSY) adopted different orientations in the active RegIIIa structure (Fig. 3c). This was consistent with the conforma- tional flexibility of this loop as indicated by a higher crystallographic B-factor (Extended Data Fig. 7b). The active RegIIIa structure could be docked into the cryo-EM hexameric density map (Fig. 3d and Extended Data Fig. 6k, l), provid- ing good spatial constraints for building a hexameric model. The model indicates that the RegIIIa subunits in the pore assembly are b c 0 50 100 0 2 4 6 8 10 RegIIIα (μM) d 40 20 60 80 100 0 500 1,000 D ye r el ea se (% o f m ax ) Time (s) Liposomes+: No protein RegIIIα OG Protein 0 e f i 40 20 60 80 100 0 PC/PS PC PS Lipid composition *** *** NS *** ** –RegIIIα +RegIIIα RegIIIα Pro-RegIIIα 0 100 200 300 400 0 0.5 1.0 D ye r el ea se r at e (fl uo re sc en ce u ni ts s –1 ) RegIIIα (μM) *** g 0 100 200 300 400 500 0 5,000 10,000 15,000 20,000 Lipid conc. (μM) Fl uo re sc en ce in cr ea se (A U ) Pro-RegIIIα RegIIIα a RegIIIα RegIIIα+PC/PS RegIIIα+PC 320 340 360 380 0 50,000 100,000 150,000 200,000 250,000 300,000 Fl uo re sc en ce (A U ) Wavelength (nm) j h S Y TO X u p ta ke (% o f m ax ) 40 20 60 80 100 0 10 20 30 400 Time (min) Untreated RegIIIα BSA Pro-RegIIIα 0 500 1,000 Time (s) 40 20 60 80 100 0 OG Protein D ye r el ea se (% o f m ax ) PC PC/PS PC+RegIIIα PC/PS+RegIIIα PS PS+RegIIIα Lipid composition: 0 50,000 100,000 150,000 200,000 250,000 320 340 360 380 0 μM Lipid conc.: 50 μM 100 μM 500 μM Wavelength (nm) Fl uo re sc en ce (A U ) 480 500 520 540 560 20,000 Fl uo re sc en ce (A U ) Wavelength (nm) Dansylated liposomes+: Pro-RegIIIα RegIIIα No protein 30,000 40,000 50,000 60,000 k 0 1 2 3 4 5 Protein conc. (μM) 0.0 0.1 0.2 –0.1 FR E T ef fic ie nc y Pro-RegIIIα RegIIIα0.3 S Y TO X u p ta ke (% o f m ax ) D ye r el ea se (% o f m ax ) Figure 1 | RegIIIa permeabilizes the bacterial membrane. a, Listeria monocytogenes was treated with 25mM RegIIIa, pro-RegIIIa, or BSA or left untreated, and bacterial uptake of SYTOX green was measured. Results are representative of three independent experiments, and are expressed as a percentage ofmaximumSYTOXuptake in thepresence of 0.2%SDS. b, SYTOX green uptake by L. monocytogenes in the presence of increasing RegIIIa concentrations. Assays were performed in triplicate. Means6 s.e.m. are plotted. c, Carboxyfluorescein (CF)-loaded liposomes (10mM lipid; 85% PC/ 15% PS) were treated with 1mMRegIIIa. 1.0% octylglucoside (OG) was added towards the end to disrupt remaining liposomes. Dye efflux is expressed as percentage of maximal release by detergent. Results are representative of five independent experiments. d, 10mM RegIIIa was added to carboxyfluorescein- loaded liposomes (100mM lipid; 100% PC, 100% PS or 85% PC:15% PS), and dye efflux was monitored over time. Representative results are shown. e, Averaged results from three independent replicates of the experiment shown in d. NS, not significant; **P, 0.01; ***P, 0.001. f, Initial rate of liposome dye efflux (100mM lipid) as a function of RegIIIa and pro-RegIIIa concentration. Results are representative of three independent experiments. *P, 0.05; **P, 0.01. g, Intrinsic tryptophan fluorescence of 1 mM RegIIIa wasmeasured in the presence of increasing lipid concentrations. h, Tryptophan fluorescence of 1 mM RegIIIa and pro-RegIIIa as a function of lipid concentration. i, Intrinsic tryptophan fluorescence of 1mM RegIIIa was measured in the presence of liposomes (100mM lipid) of varying lipid composition. j, 5.0mMRegIIIa or pro-RegIIIawas added to liposomes (100mM lipid) incorporating 5% dansyl-PE and dansyl fluorescence was monitored. Assays were performed in triplicate. k, FRET efficiency as a function of RegIIIa and pro-RegIIIa concentration. Assays were performed in triplicate. Means6 s.e.m. are plotted. RESEARCH LETTER 1 0 4 | N A T U R E | V O L 5 0 5 | 2 J A N U A R Y 2 0 1 4 Macmillan Publishers Limited. All rights reserved©2014 oriented with the carbohydrate-binding loop pointing towards the central channel, and the loop encompassing residues 93–99 and the N and carboxy (C) termini oriented towards the lipid bilayer (Fig. 3d). The resolution of ourmap did not allow us to extract detailed informa- tion about intermolecular interactions in the pore complex. There was imperfect docking of the carbohydrate-binding loop, the loop encom- passing residues 93–99, and the farN terminus (Fig. 3d), consistentwith the conformational flexibility of these regions (Extended Data Fig. 7b). We used mutagenesis to assess experimentally the orientation of RegIIIa in the pore complex. Ourmodel predicts that the basic residue Lys 93 is oriented towards the lipid bilayer (Fig. 3d) and thus might be involved in interactions with the negatively charged phospholipids required for RegIIIa–liposome interactions (Fig. 1d, e). A Lys93Ala mutation, but not conservative Lys93Arg and Lys93His mutations, reduced the toxicity of RegIIIa for liposomes as well as intact bacteria (Fig. 3e, f and Extended Data Fig. 8a). In contrast, a Glu114Gln muta- tion, which resides in the carbohydrate-binding loop (Fig. 3c)3, did not have an impact on membrane toxicity, consistent with its predicted position near the pore interior (Fig. 3d, e). As expected, the Lys93Ala mutation but not the Glu114Gln mutation inhibited filament forma- tion (ExtendedData Fig. 8b). Finally, the orientation of theN terminus towards the lipid bilayer is consistent with the role of the N-terminal pro-segment in inhibiting RegIIIa interactions with lipid and reducing membrane toxicity (Fig. 1a, f, h, j, k). We next calculated the energetics of pore insertion into a PC-likemem- brane bilayer using physics-based computationalmodelling (Extended Data Fig. 9a–d)17. The model predicts that basic residues are located near the membrane–water interface whereas a strip of hydrophobic and polar residues is buried in the membrane core (Fig. 3g). The complex presents a positive electric field to the membrane (Extended Data Fig. 9e, f), creating an unfavourable electrostatic energy unless negatively charged PS-like lipids are added to the membrane (Fig. 3h). This is consistent with our finding that PS lipids are necessary for RegIIIa toxicity (Fig. 1d, e). Finally, calculations on the Lys93Ala mutant showed reduced stability (Fig. 3h) due to loss of favourable electrostatic interactions between Lys 93 and negatively charged lipids. Thus, the model reveals that charge sequestration is a critical deter- minant of RegIIIa pore stability in the membrane. Furthermore, the model predicts that Arg 166 interacts with the membrane surface (Extended Data Fig. 10a). Consistent with this prediction, an Arg166Ala mutation reduced membrane toxicity of RegIIIa (Extended Data Fig. 10b). In contrast, mutating Arg 39, which is exposed to aqueous solvent in the model, had little effect on RegIIIa membrane toxicity (Extended Data Fig. 10a, b). Thus, our model accurately predicts the experimental behaviour of the RegIIIa pore. RegIIIa selectively targets Gram-positive bacteria1, raising the ques- tion of why RegIIIa cannot kill Gram-negative bacteria by permeabi- lizing the outer membrane. In contrast to PC/PS liposomes, liposomes composed of an Escherichia coli total lipid extract were not disrupted by RegIIIa (Fig. 4a), indicating that a component of the lipid extract inhibitedmembranepermeabilization. Lipopolysaccharide (LPS), amajor constituent of the Gram-negative outer membrane, inhibited RegIIIa- mediated liposome disruption and antibacterial activity (Fig. 4b, c), indicating that LPS is one factor that prevents RegIIIa-mediated per- meabilization of Gram-negative bacteria. Finally, we postulated that the trypsin-cleavable inhibitory N ter- minus of pro-RegIIIa evolved to suppress pore-forming activity and thus minimize cytotoxicity during RegIIIa synthesis and storage in epithelial cells. In support of this idea, RegIIIa was cytotoxic towards cultured intestinal epithelial cells (MODE-K)18, and the pro-segment suppressed this cytotoxicity (Fig. 4d, e). Thus, RegIIIa kills its bacterial targets by oligomerizing on the bac- terial membrane to form a membrane-penetrating pore (Extended Data Fig. 1). Membrane attack by pore formation represents a previ- ously unappreciated biological activity for theC-type lectin family.Our findingsmay provide insight into the evolutionary origins of the lectin- mediated complement pathway, in which recruited complement pro- teins disrupt microbial membranes19. With its intrinsic capacity for membrane attack, RegIIIamay represent a more evolutionarily prim- itivemechanismof lectin-mediated innate immunity.We propose that the lectin-mediated complement pathway could have evolved from a directly bactericidal ancestral lectin, with the bacterial recognition function retained by the descendent C-type lectin(s) and the mem- brane attack function assumed by recruited accessory proteins that assemble into the membrane attack complex. c d 0 mV 0 mV –80 mV 50 p A 5 s a b FD4 Stokes diameter=~28 Å Protein 0 20 40 60 80 100 D ye r el ea se (% ) Protein OG FD10 Stokes diameter=~44 Å 0 2 4 6 8 0 50 100 150 200 250 Elution volume (ml) m A U Dimer Tetramer Hexamer Predicted mobilities: 100 75 50 25 37 20 15 kDa+liposomes –lipo. 10 12 14 16 67 43 13.7 6.5Size-exclusion standards (kDa): Monomer 0 100 200 300 400 –liposomes +liposomes m A U Time (s) Liposomes+: No protein 5 μM RegIIIα 500 1,000 Protein CF Stokes diameter=~10 Å 0 0 20 40 60 80 100 0 20 40 60 80 100 100 Å 100 Å 100 Å 100 Å Figure 2 | RegIIIa forms a transmembrane pore. a, RegIIIa-dependent current flow across a planar lipid bilayer is depicted as a function of time. No current was observed before the application of a voltage across the membrane. Upon the application of 280mV, inward current was observed, and returning the membrane potential to zero diminished the current because the measured reverse potential was 24.0mV. The current trace is representative of multiple independent experiments. b, Liposomes loaded with FITC-Dextran 10 (FD10), FITC-Dextran 4 (FD4), or carboxyfluorescein (CF) were treated with 5.0mM RegIIIa and dye release was monitored ove