Cryo-EM structure of the EspA filament from enteropathogenic Escherichia coli: Revealing the mechanism of effector translocation in the T3SS

•Structure of native EspA adopts a unique fold compared with other T3SS tip proteins•Native different conformation from the chaperone-bound state•The structure reveals an electropositive pattern within its lumen•In vivo studies show that electrostatics play crucial role in function The type III secretion system (T3SS) is virulence mechanism employed by Gram-negative pathogens. forms proteinaceous channel projects needle into extracellular medium where it interacts host cell to deliver factors. Enteropathogenic Escherichia coli (EPEC) adopting extension T3SS—a filament formed EspA—which absolutely required for efficient colonization gut. Here, we describe cryoelectron microscopy filaments EPEC at 3.6-Å resolution. Within filament, positively charged residues adjacent hydrophobic groove line lumen spiral manner, suggesting substrate translocation mediated via electrostatics. Using structure-guided mutagenesis, corroborate these and function. high-resolution could aid drug design antivirulence therapeutics. syringe-like multimembrane-spanning nanomachine often essential pathogenicity, but not viability, many clinically relevant bacteria, such as enteropathogenic (EPEC), Salmonella enterica, Pseudomonas aeruginosa. pathogens directly responsible downstream development illnesses whooping cough, sexually transmitted chlamydial disease, hospital-acquired pneumonia (Lyons Strynadka, 2019Lyons B.J.E. Strynadka N.C.J. 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Krämer Deibel Guzmán Chakraborty Initial binding Shiga toxin-producing cells subsequent induction actin rearrangements depend on filamentous EspA- containing surface appendages.Mol. 1998; 30: 147-161Crossref (137) knockouts exhibit drastic decrease virulence, loss consequent inability form characteristic T3SS-mediated A/E lesion infected surface. has also been promising target prophylactic immunotherapies prevent EHEC cattle, causative agent agriculture water contamination (Allen 2011Allen K.J. Rogan Brett Finlay Potter A.A. Asper D.J. Vaccination leads decreased shedding calves after experimental infection O157.Can. Vet. Res. 2011; 75: 98-105PubMed 2018Schmidt Barth Frahm Dänicke Geue Menge Decreased STEC cattle following passive active vaccination based recombinant toxoids.Vet. 49: 1-15Crossref (10) Wadamori 2017Wadamori Y. Gooneratne R. Hussain Outbreaks influencing microbiological fresh produce.J. Food Agric. 2017; 97: 1396-1403Crossref (95) Preventing assembly adherent therefore avenue pursue inhibition EPEC/EHEC human diseases they inflict. present (cryo-EM) harvested strain. greatly enhances our current understanding this component provides scaffold inhibitors. architectural features protomer reveal significant differences between orthologous may allow extended nature passage partially unfolded effectors cell. We complement mutagenic guided structural knowledge, illustrating key are functional importance. switching or removing charge luminal-facing lysine residue severely diminishes ability lesions HeLa cells. purified natively sheared O127:H6 strain E2348/69 methods previously described (Wang 2006Wang Y.A. X. Yip N.C. Egelman E.H. 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Bacteriol. 2015; 197: 1263-1275Crossref (16) Compared filament-forming proteins, shares ?80% identity rodentium homologs 1G) range much sequence 10%–12% common T3SS-reliant shown require T3SS-specific cognate chaperone, CesA, maintain unpolymerized state (Yip 2005Yip C.K. characterization chaperone.Nat. 2005; 12: 75-81Crossref previous crystal (PDB: 1XOU) bound CesA 1:1 allowed tracing only N-terminal 31–59) 148–190) ? helices EspA. segment coiled-coil motif, complimented coiled coil chaperone When comparing assembled (EspACHAP; root-mean-square deviation [RMSD] 68 C? atoms = 3.1 Å), EspACHAP relaxed having little terminal 2A), closest distance being 9.3 Å. Figure 2B depicts conformational changes (EspACHAP) Remarkably, blocks oligomerization now make most extensive interactions 2C). thus proposed effectively premature protomers delivery platform. Larzábal 2019Larzábal Baldoni H.A. Suvire F.D. Curto L.M. Gomez G.E. Da Silva W.M. Giudicessi S.L. Camperi Delfino Cataldi al.An inhibitory action peptides against three Pept. 25: e3149Crossref Scholar had recently developed optimized peptide inhibitors mimic guide inhibitor (Larzábal 2013Larzábal Zotta Ibarra Rabinovitz B.C. Vilte D.A. Mercado E.C. Á. Effect system-dependent activity O157: H7 rodentium.Int. Med. 2013; 303: 9-15Crossref (17) Scholar, These showed promise reduction assembly; however, perhaps surprising given here, mutation found N- did markedly improve affinity. As show, distinct EspACHAP. Thus, should optimization Previously, vaccine ruminant animals, asymptomatic carrier contributor global food crisis contained mixture T3SS-secreted effector Tir antigens. formulation protected propagation when challenged. addition, immunotherapy needle-tip aeruginosa, ortholog, pseudomonal (Le 2018Le H.N. Quetz Tran V.G. Le V.T.M. Aguiar-Alves Pinheiro M.G. Sellman B.R. Stover al.MEDI3902 correlates protection severe rabbit acute model.Antimicrob. 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Notably, region outlined electronegative both PrgI structure. 2018Hu Caveney T3S open secretin.Nat. 9: 3840Crossref provide evidence use proton-motive force generation ATP-driven release (Hu dependence enterocolitica (Wilharm 2004Wilharm Lehmann Krauss Lehnert Richter Ruckdeschel Heesemann Trülzsch depends proton motive flagellar motor components MotA MotB.Infect. Immun. 72: 4004-4009Crossref (87) Following parameters assembles rise 4.3 Å twist 64.3° 4A), nearly identical 63.3° low-resolution study multiple variations dynamic breathing potentially propulsion. During processing class (4,300 particles 28% total particle set 3D classification) 4.7 63.8°, again possibility. Further population yield secondary structure, future collections possibly be parse out populations potential variants. kinked coil, connected well-ordered 4B). Two segments overall helices, ?3-?4. runs parallel 4C); ?3-?4 makes majority protomer, along represents bottom (relative outer membrane). ?3 ?4 ordered region, short helix-turn segment. Adjacent pack distal coil-loop-coil buried area 1,500 Å2, PISA (Krissinel Henrick, 2007Krissinel Henrick Inference crystalline state.J. 372: 774-797Crossref (6322) 1–29) ?1 kink Gly (? 94.5°, ? 178.8°), presumably disordered probably (based prediction) decorates filament. 2-nm indicates during translocation, substrate—or even helix—has plenty room traverse 4D). murine pathogen T3SSs. lacks domain: Yersinia, globular domain predicted docking whereas systems hairpin acts intrinsic (Johnson 2007Johnson Roversi Espina Olive Deane Birket Field W.D. Blocker Galyov E.E. al.Self-chaperoning BipD.J. 282: 4035-4044Scopus (119) does contain intramolecular domain, uses separate (CesA) Each non-filament-forming contains mixed ?/? domain. For small ?-turn connects coil. feature difference allowing formation over ring seen species, although no oligomeric atomic any known currently verify this. hallmark protein-protein adjoining itself, translocon. Of domains alike LcrV (RMSD 2.3 80 atoms; PDB: 1R6F), SipD 96 3NZZ) straight 5) (Chatterjee 2011Chatterjee Zhong Nordhues Battaile K.P. Lovell De Guzman R.N. structures deoxycholate chenodeoxycholate.Protein 20: 75-86Crossref (53) Chaudhury 2013Chaudhury Plano G.V. Structure 1.65 resolution.Acta F Cryst. F69: 477-481Crossref (18) T3SS, flagellin organizes itself motifs. alignment FliC 4.6 134 5WK5) illustrated 5B 2017Wang Burrage A.M. Postel Clark Orlova Sundberg E.J. Kearns D.B. A across species.Nat. 8: 960Crossref (54) hypothesized T3SSs, (monomeric) would follow same and, therefore, overlaid aligned coordinates single (as 5C), followed successively assembly. However, must adopt (and needle), superposition sits continuing likely block 5D). discussed above, suggest One important note themselves early substrates, so mutagenesis if delivery, expected so-called middle EspB EspD components. Residues appear include K192 well K59, K62, D63, R71 needle. chromosomal mutants, espAK192E escFR71E, were generated homologous recombination suicide vector pRE112. mutants behaved similarly deletion complemented mutated genes pACYC184; (EspAK192A, EspAR183A, EspAR183E, EscFK59A, EscFK59E, EscFK62A, EscFD63A, EscFR71A) produced complementation respective mutant gene pACYC184 corresponding knockout (?espA ?escF). sought test hypothesis employing site-directed coupled SDS-PAGE western blotting 6), actin-rich pedestals 7), virulence. 6A R71E displayed secretion, R71A exhibiting similar phenotype. EscFK62E secretion; EscFK62A moderately affected, K62 more charge-charge stabilization neighboring D63; thus, substituting appeared lead abolished EscFK59A/E effect secretion. It noted amount EspC, dependent, reduced some 6A). due fact efficiency EspC precipitation 10% trichloroacetic acid correlated culture supernatant, act carriers process. Since vast DMEM too precipitated seems less escF knockout, defective (escFK62E escFR71A/E).Figure 7Pedestal mutantsShow full captionHeLa espA strains various then processed immunofluorescent fluorescent staining DNA/bacteria (blue), (red), (green).(A) ?espA.(B) ?espA/pespAWT.(C) ?espA/pespAR183A.(D) ?espA/pespAK192A.(E) ?espA/pespAR183E.(F) Number per induced mutants. numbers quantified 100 randomly selected microscopic fields. Significance one-way ANOVA Tukey's comparison test. ???p < 0.001; ????p 0.0001.View Large Image ViewerDownload Hi-res image Download (PPT)Table 1Cryo-EM collection, refinement, validation statisticsData processingMagnification47,000Voltage (kV)300Electron exposure (e?/Å2)0.475Defocus (?m)?2 ?4.5Pixel size (Å)1.75Symmetry imposedHelicalInitial images (n)146,137Final (n)15,523Map (Å) FSCaFourier correlation. threshold0.143RefinementModel building/refinement softwareCoot/Phenix/RosettaResolution (Å)3.56RMSbRoot-mean-square. deviations Bond lengths (Å)0.009 angles (°)0.916Validation MolProbity score2.4 Clashscore5.42 Poor rotamers (%)1.4Ramachandran plot Favored (%)93.5 Allowed (%)6.5 Outliers (%)0a correlation.b Root-mean-square. Open table new tab (green). (A) ?espA. (B) ?espA/pespAWT. (C) ?espA/pespAR183A. (D) ?espA/pespAK192A. (E) ?espA/pespAR183E. (F) 0.0001. To analyze used sets strains. nalidixic acid-resistant (NalR) derivative earlier (Kenny 1996Kenny Lai L.C. Donnenberg M.S. coli, induce epithelial cells.Mol. 1996; 313-323Crossref (227) Scholar) wild-type R183A/E K192A expressed plasmid pACYC184, recombined chromosome replace streptomycin-resistant (SmR) 6B, level NalR ?espA espAWT, espAR183A/E, espAK192A under tetracycline-resistance promoter than parental control SmR strains, promoter. espAWT exhibited still pedestal 7). Interestingly, though unable capable levels components, EspD, able independent 6B). K192A/E studied here display attenuated observation supports collaborative formation, indicating preferably point EspAK192E background 6B), blocking slowing down EspAK192A EspAWT, assembling filaments, EM S2). EspAR183A/E tested All producing experiments average eight 7F). EspAR183E significantly opposite change (R E) impact Tir, critical formation. plays food- water-borne EHEC. requirement adherence, transported reach Previous have already exemplified pathogenic coli. Our solid foundation progressing established toward offering alternative growing antibiotic resistance crisis.