Structural insights into the regulatory mechanism of the Pseudomonas aeruginosa YfiBNR system

Abstract

YfiBNR is a recently identified bis-(3’-5’)-cyclic dimeric GMP (c-di-GMP) signaling system in opportunistic pathogens. It is a key regulator of biofilm formation, which is correlated with prolonged persistence of infection and antibiotic drug resistance. In response to cell stress, YfiB in the outer membrane can sequester the periplasmic protein YfiR, releasing its inhibition of YfiN on the inner membrane and thus provoking the diguanylate cyclase activity of YfiN to induce c-di-GMP production. However, the detailed regulatory mechanism remains elusive. Here, we report the crystal structures of YfiB alone and of an active mutant YfiB^L43P complexed with YfiR with 2:2 stoichiometry. Structural analyses revealed that in contrast to the compact conformation of the dimeric YfiB alone, YfiB^L43P adopts a stretched conformation allowing activated YfiB to penetrate the peptidoglycan (PG) layer and access YfiR. YfiB^L43P shows a more compact PG-binding pocket and much higher PG binding affinity than wild-type YfiB, suggesting a tight correlation between PG binding and YfiB activation. In addition, our crystallographic analyses revealed that YfiR binds Vitamin B6 (VB6) or L-Trp at a YfiB-binding site and that both VB6 and L-Trp are able to reduce YfiB^L43P-induced biofilm formation. Based on the structural and biochemical data, we propose an updated regulatory model of the YfiBNR system.

Keywords

the YfiBNR system / c-di-GMP / Vitamin B6 / L-Trp / peptidoglycan layer / bioflim formation

References

[1] Adams PD, Afonine PV, Bunkoczi G, Chen VB, Davis IW, Echols N, Headd JJ, Hung LW, Kapral GJ, Grosse-Kunstleve RW (2010) PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr 66:213–221

[2] Afonine PV, Grosse-Kunstleve RW, Echols N, Headd JJ, Moriarty NW, Mustyakimov M, Terwilliger TC, Urzhumtsev A, Zwart PH, Adams PD (2012) Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr D Biol Crystallogr 68:352–367

[3] Beaumont HJ, Gallie J, Kost C, Ferguson GC, Rainey PB (2009) Experimental evolution of bet hedging. Nature 462:90–93

[4] Boehm A, Kaiser M, Li H, Spangler C, Kasper CA, Ackermann M, Kaever V, Sourjik V, Roth V, Jenal U (2010) Second messengermediated adjustment of bacterial swimming velocity. Cell 141:107–116

[5] Caly DL, Bellini D, Walsh MA, Dow JM, Ryan RP (2015) Targeting cyclic di-GMP signalling: a strategy to control biofilm formation ? Curr Pharm Des 21:12–24

[6] Camilli A, Bassler BL (2006) Bacterial small-molecule signaling pathways. Science 311:1113–1116

[7] Choi KH, Kumar A, Schweizer HP (2006) A 10-min method for preparation of highly electrocompetent Pseudomonas aeruginosa cells: application for DNA fragment transfer between chromosomes and plasmid transformation. J Microbiol Methods 64:391–397

[8] Desmarais SM, Cava F, de Pedro MA, Huang KC (2014) Isolation and preparation of bacterial cell walls for compositional analysis by ultra performance liquid chromatography. J Vis Exp 83:e51183

[9] Duerig A, Abel S, Folcher M, Nicollier M, Schwede T, Amiot N, Giese B, Jenal U (2009) Second messenger-mediated spatiotemporal control of protein degradation regulates bacterial cell cycle progression. Genes Dev 23:93–104

[10] Emsley P, Lohkamp B, Scott WG, Cowtan K (2010) Features and development of Coot. Acta Crystallogr D Biol Crystallogr 66:486–501

[11] Evans TJ (2015) Small colony variants of Pseudomonas aeruginosa in chronic bacterial infection of the lung in cystic fibrosis. Future Microbiol 10:231–239

[12] Giardina G, Paiardini A, Fernicola S, Franceschini S, Rinaldo S, Stelitano V, Cutruzzola F (2013) Investigating the allosteric regulation of YfiN from Pseudomonas aeruginosa: clues from the structure of the catalytic domain. PLoS One 8:e81324

[13] Giddens SR, Jackson RW, Moon CD, Jacobs MA, Zhang XX, Gehrig SM, Rainey PB (2007) Mutational activation of niche-specific genes provides insight into regulatory networks and bacterial function in a complex environment. Proc Natl Acad Sci USA 104:18247–18252

[14] Govan JR, Deretic V (1996) Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia. Microbiol Rev 60:539–574

[15] Grubman A, Phillips A, Thibonnier M, Kaparakis-Liaskos M, Johnson C, Thiberge JM, Radcliff FJ, Ecobichon C, Labigne A, de Reuse H. (2010) Vitamin B6 is required for full motility and virulence in Helicobacter pylori. MBio 1

[16] Ha DG, O’Toole GA (2015) c-di-GMP and its effects on biofilm formation and dispersion: a Pseudomonas aeruginosa review. Microbiol Spectr 3, MB-0003-2014

[17] Haussler S, Tummler B, Weissbrodt H, Rohde M, Steinmetz I (1999) Small-colony variants of Pseudomonas aeruginosa in cystic fibrosis. Clin Infect Dis 29:621–625

[18] Haussler S, Ziegler I, Lottel A, von Gotz F, Rohde M, Wehmhohner D, Saravanamuthu S, Tummler B, Steinmetz I (2003) Highly adherent small-colony variants of Pseudomonas aeruginosa in cystic fibrosis lung infection. J Med Microbiol 52:295–301

[19] Hengge R (2009) Principles of c-di-GMP signalling in bacteria. Nat Rev Microbiol 7:263–273

[20] Hickman JW, Tifrea DF, Harwood CS (2005) A chemosensory system that regulates biofilm formation through modulation of cyclic diguanylate levels. Proc Natl Acad Sci USA 102:14422–14427

[21] Hmelo LR, Borlee BR, Almblad H, Love ME, Randall TE, Tseng BS, Lin C, Irie Y, Storek KM, Yang JJ (2015) Precisionengineering the Pseudomonas aeruginosa genome with two-step allelic exchange. Nat Protoc 10:1820–1841

[22] Holm L, Rosenstrom P (2010) Dali server: conservation mapping in 3D. Nucleic Acids Res 38:W545–W549

[23] Huertas MG, Zarate L, Acosta IC, Posada L, Cruz DP, Lozano M, Zambrano MM (2014) Klebsiella pneumoniae yfiRNB operon affects biofilm formation, polysaccharide production and drug susceptibility. Microbiology 160:2595–2606

[24] Hufnagel DA, DePas WH, Chapman MR (2014) The disulfide bonding system suppresses CsgD-independent cellulose production in Escherichia coli. J Bacteriol 196:3690–3699

[25] Jenal U (2004) Cyclic di-guanosine-monophosphate comes of age: a novel secondary messenger involved in modulating cell surface structures in bacteria ? Curr Opin Microbiol 7:185–191

[26] Kirillina O, Fetherston JD, Bobrov AG, Abney J, Perry RD (2004) HmsP, a putative phosphodiesterase, and HmsT, a putative diguanylate cyclase, control Hms-dependent biofilm formation in Yersinia pestis. Mol Microbiol 54:75–88

[27] Kirisits MJ, Prost L, Starkey M, Parsek MR (2005) Characterization of colony morphology variants isolated from Pseudomonas aeruginosa biofilms. Appl Environ Microbiol 71:4809–4821

[28] Kulasakara H, Lee V, Brencic A, Liberati N, Urbach J, Miyata S, Lee DG, Neely AN, Hyodo M, Hayakawa Y (2006) Analysis of Pseudomonas aeruginosa diguanylate cyclases and phosphodiesterases reveals a role for bis-(3′-5′)-cyclic-GMP in virulence. Proc Natl Acad Sci USA 103:2839–2844

[29] Laskowski RA, MacArthur MW, Moss DS, Thornton JM (1993) PROCHECK: a program to check the stereochemical quality of protein structures. J Appl Crystallogr 26:283–291

[30] Lebedev AA, Vagin AA, Murshudov GN (2008) Model preparation in MOLREP and examples of model improvement using X-ray data. Acta Crystallogr D Biol Crystallogr 64:33–39

[31] Li S, Li T, Xu Y, Zhang Q, Zhang W, Che S, Liu R, Wang Y, Bartlam M (2015) Structural insights into YfiR sequestering by YfiB in Pseudomonas aeruginosa PAO1. Sci Rep 5:16915

[32] Malone JG (2015) Role of small colony variants in persistence of Pseudomonas aeruginosa infections in cystic fibrosis lungs. Infect Drug Resist 8:237–247

[33] Malone JG, Jaeger T, Spangler C, Ritz D, Spang A, Arrieumerlou C, Kaever V, Landmann R, Jenal U (2010) YfiBNR mediates cyclic di-GMP dependent small colony variant formation and persistence in Pseudomonas aeruginosa. PLoS Pathog 6:e1000804

[34] Malone JG, Jaeger T, Manfredi P, Dotsch A, Blanka A, Bos R, Cornelis GR, Haussler S, Jenal U (2012) The YfiBNR signal transduction mechanism reveals novel targets for the evolution of persistent Pseudomonas aeruginosa in cystic fibrosis airways. PLoS Pathog 8:e1002760

[35] Matias VR, Al-Amoudi A, Dubochet J, Beveridge TJ (2003) Cryotransmission electron microscopy of frozen-hydrated sections of Escherichia coli and Pseudomonas aeruginosa. J Bacteriol 185:6112–6118

[36] Meroueh SO, Bencze KZ, Hesek D, Lee M, Fisher JF, Stemmler TL, Mobashery S (2006) Three-dimensional structure of the bacterial cell wall peptidoglycan. Proc Natl Acad Sci USA 103:4404–4409

[37] Navarro MV, Newell PD, Krasteva PV, Chatterjee D, Madden DR, O’Toole GA, Sondermann H (2011) Structural basis for c-di-GMPmediated inside-out signaling controlling periplasmic proteolysis. PLoS Biol 9:e1000588

[38] Otwinowski Z, Minor W (1997) Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol 276:307–326

[39] Parsek MR, Singh PK (2003) Bacterial biofilms: an emerging link to disease pathogenesis. Annu Rev Microbiol 57:677–701

[40] Parsons LM, Lin F, Orban J (2006) Peptidoglycan recognition by Pal, an outer membrane lipoprotein . Biochemistry 45:2122–2128

[41] Pesavento C, Hengge R (2009) Bacterial nucleotide-based second messengers. Curr Opin Microbiol 12:170–176

[42] Raterman EL, Shapiro DD, Stevens DJ, Schwartz KJ, Welch RA (2013) Genetic analysis of the role of yfiR in the ability of Escherichia coli CFT073 to control cellular cyclic dimeric GMP levels and to persist in the urinary tract. Infect Immun 81:3089–3098

[43] Reinhardt A, Kohler T, Wood P, Rohner P, Dumas JL, Ricou B, van Delden C (2007) Development and persistence of antimicrobial resistance in Pseudomonas aeruginosa: a longitudinal observation in mechanically ventilated patients. Antimicrob Agents Chemother 51:1341–1350

[44] Ren GX, Yan HQ, Zhu H, Guo XP, Sun YC (2014) HmsC, a periplasmic protein, controls biofilm formation via repression of HmsD, a diguanylate cyclase in Yersinia pestis. Environ Microbiol 16:1202–1216

[45] Romling U, Galperin MY, Gomelsky M (2013) Cyclic di-GMP: the first 25 years of a universal bacterial second messenger. Microbiol Mol Biol Rev 77:1–52

[46] Ross P, Weinhouse H, Aloni Y, Michaeli D, Weinberger-Ohana P, Mayer R, Braun S, de Vroom E, van der Marel GA, van Boom JH (1987) Regulation of cellulose synthesis in Acetobacter xylinum by cyclic diguanylic acid. Nature 325:279–281

[47] Ross P, Mayer R, Benziman M (1991) Cellulose biosynthesis and function in bacteria. Microbiol Rev 55:35–58

[48] Sanchez-Torres V, Hu H, Wood TK (2011) GGDEF proteins YeaI, YedQ, and YfiN reduce early biofilm formation and swimming motility in Escherichia coli. Appl Microbiol Biotechnol 90:651–658

[49] Schirmer T, Jenal U (2009) Structural and mechanistic determinants of c-di-GMP signalling. Nat Rev Microbiol 7:724–735

[50] Schuck P (2000) Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling. Biophys J 78:1606–1619

[51] Shimazaki J, Furukawa S, Ogihara H, Morinaga Y (2012) L-Tryptophan prevents Escherichia coli biofilm formation and triggers biofilm degradation. Biochem Biophys Res Commun 419:715–718

[52] Smith EE, Buckley DG, Wu Z, Saenphimmachak C, Hoffman LR, D’Argenio DA, Miller SI, Ramsey BW, Speert DP, Moskowitz SM (2006) Genetic adaptation by Pseudomonas aeruginosa to the airways of cystic fibrosis patients. Proc Natl Acad Sci USA 103:8487–8492

[53] Tamayo R, Tischler AD, Camilli A (2005) The EAL domain protein V<?Pub Caret?>ieA is a cyclic diguanylate phosphodiesterase. J Biol Chem 280:33324–33330

[54] Ueda A, Wood TK (2009) Connecting quorum sensing, c-di-GMP, pel polysaccharide, and biofilm formation in Pseudomonas aeruginosa through tyrosine phosphatase TpbA (PA3885). PLoS Pathog 5:e1000483

[55] Xu K, Li S, Yang W, Li K, Bai Y, Xu Y, Jin J, Wang Y, Bartlam M (2015) Structural and biochemical analysis of tyrosine phosphatase related to biofilm formation A (TpbA) from the opportunistic pathogen Pseudomonas aeruginosa PAO1. PLoS One 10:e0124330

[56] Yang X, Yang XA, Xu M, Zhou L, Fan Z, Jiang T (2015) Crystal structures of YfiR from Pseudomonas aeruginosa in two redox states. Biochem Biophys Res Commun 461:14–20

RIGHTS & PERMISSIONS

2016 The Author(s) 2016. This article is published with open access at Springerlink.com and journal.hep.com.cn