Abstract
Digestive diseases caused by flagellated bacteria are a huge public health problem worldwide and rapid detection methods are needed for contaminated environments. In this study, we propose a method to detect patterns associated with pathogens based on the properties of the innate immune system. Specifically, we use Toll-like receptor 5 (TLR5), a transmembrane protein that specifically recognizes flagellin (the structural protein of bacterial flagella). TLR5, which was obtained by recombinant production in insect cells, was immobilized into liposomes to form TLR5-proteoliposomes. Through surface plasmon resonance (SPR) and competition flow cytometry assays, the sensitivity of proteoliposomes to recognize Escherichia coli and Salmonella typhimurium flagellin was evaluated. In addition, we compared the results obtained by immobilizing anti-flagellin antibodies into liposomes. The results of the flagellin-affinity tests, expressed as an SPR kinetic rate constant ratio in the equilibrium equation K D = k d/k a, showed values of 13.8 × 10−9 and 7.73 × 10−9 M for the TLR5-proteoliposomes and anti-flagellin antibodies, respectively, against S. typhimurium. The anti-flagellin affinity results for E. coli showed K D of 84.1 × 10−8 M for SPR assays and K D of 3.5 × 10−8 M for competitive flow cytometry, which was used as a detection system without the immobilization of proteoliposomes. This research demonstrates the practical possibility of using proteoliposomes as recognition elements in the generation of systems for the rapid detection of flagellated bacteria, which could help avoid consumption of contaminated food by humans and thereby prevent intestinal infections.
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Mead PS, Slutsker L, Dietz V, McCaig LF, Bresee JS, Shapiro C, Griffin PM, Tauxe RV (1999) Food-related illness and death in the United States. Emerg Infect Dis 5(5):607–625
Keusch GT, Fontaine O, Bhargava A, Boschi-Pinto C, Bhutta ZA, Gotuzzo E, Rivera J, Chow J, Shahid-Salles S, Laxminarayan R (2006) In: Jamison T, Breman JG, Measham AR, Alleyne G, Claeson M, Evans DB, Jha P, Mills A, Musgrove P (eds) Disease control priorities in developing countries, 2nd edn. Washington, World Bank
Velusamy V, Arshak K, Korostynska O, Oliwa K, Adley C (2009) An overview of foodborne pathogen detection: in the perspective of biosensors. Biotechnol Adv 28(2):232–254
Arora P, Sindhu A, Dilbaghi N, Chaudhury A (2011) Biosensors as innovative tools for the detection of food borne pathogens. Biosens Bioelectron 28(1):1–12
Leonard P, Hearty S, Brennan J, Dunne L, Quinn J, Chakraborty T, O’Kennedy R (2003) Advances in biosensors for detection of pathogens in food and water. Enzyme Microb Technol 32(1):3–13
Radke SM, Alocilja EC (2005) A high density microelectrode array biosensor for detection of E. coli O157:H7. Biosens Bioelectron 20(8):1662–1667
Jin LQ, Li JW, Wang SQ, Chao FH, Wang XW, Yuan ZQ (2005) Detection and identification of intestinal pathogenic bacteria by hybridization to oligonucleotide microarrays. World J Gastroenterol 11(48):7615–7619
Nayak M, Kotian A, Marathe S, Chakravortty D (2009) Detection of microorganisms using biosensors-a smarter way towards detection techniques. Biosens Bioelectron 25(4):661–667
Nugen SR, Baeumner AJ (2008) Trends and opportunities in food pathogen detection. Anal Bioanal Chem 391(2):451–454
Sanvicens N, Pastells C, Pascual N, Marco MP (2009) Nanoparticle-based biosensors for detection of pathogenic bacteria. Trends Anal Chem 28(11):1243–1252
Zaytseva NV, Goral VN, Montagna RA, Baeumner AJ (2005) Development of a microfluidic biosensor module for pathogen detection. Lab Chip 5(8):805–811
Cooper MA, Singleton VT (2007) A survey of the 2001 to 2005 quartz crystal microbalance biosensor literature: applications of acoustic physics to the analysis of biomolecular interactions. J Mol Recognit 20(3):154–184
Geng T, Morgan MT, Bhunia AK (2004) Detection of low levels of Listeria monocytogenes cells by using a fiber-optic immunosensor. Appl Environ Microbiol 70(10):6138–6146
Lazcka ODJ, Muñoz X (2007) Pathogen detection: a perspective of traditional methods and biosensors. Biosens Bioelectron 22(7):1205–1217
Chambers JP, Arulanandam BP, Matta LL, Weis A, Valdes JJ (2008) Biosensor recognition elements. Curr Issues Mol Biol 10(1–2):1–12
Stephen J (2001) Pathogenesis of infectious diarrhea. Can J Gastroenterol 15(10):669–683
Gay NJ, Gangloff M, Weber AN (2006) Toll-like receptors as molecular switches. Nat Rev Immunol 6(9):693–698
Smith KD, Andersen-Nissen E, Hayashi F, Strobe K, Bergman MA, Barrett SL, Cookson BT, Aderem A (2003) Toll-like receptor 5 recognizes a conserved site on flagellin required for protofilament formation and bacterial motility. Nat Immunol 4(12):1247–1253
Kumar H, Kawai T, Akira S (2009) Toll-like receptors and innate immunity. Biochem Biophys Res Commun 388(4):621–625
Bell JK, Mullen GE, Leifer CA, Mazzoni A, Davies DR, Segal DM (2003) Leucine-rich repeats and pathogen recognition in Toll-like receptors. Trends Immunol 24(10):528–533
Neish AS (2007) TLRS in the gut. II. Flagellin-induced inflammation and antiapoptosis. Am J Physiol Gastrointest Liver Physiol 292(2):G462–466
Elewaut D, DiDonato JA, Kim JM, Truong F, Eckmann L, Kagnoff MF (1999) NF-kappa B is a central regulator of the intestinal epithelial cell innate immune response induced by infection with enteroinvasive bacteria. J Immunol 163(3):1457–1466
Ramos HC, Rumbo M, Sirard JC (2004) Bacterial flagellins: mediators of pathogenicity and host immune responses in mucosa. Trends Microbiol 12(11):509–517
Honko AN, Mizel SB (2005) Effects of flagellin on innate and adaptive immunity. Immunol Res 33(1):83–101
Applequist SE, Rollman E, Wareing MD, Liden M, Rozell B, Hinkula J, Ljunggren HG (2005) Activation of innate immunity, inflammation, and potentiation of DNA vaccination through mammalian expression of the TLR5 agonist flagellin. J Immunol 175(6):3882–3891
Mizel SB, Honko AN, Moors MA, Smith PS, West AP (2003) Induction of macrophage nitric oxide production by Gram-negative flagellin involves signaling via heteromeric Toll-like receptor 5/Toll-like receptor 4 complexes. J Immunol 170(12):6217–6223
Yu Y, Zeng H, Lyons S, Carlson A, Merlin D, Neish AS, Gewirtz AT (2003) TLR5-mediated activation of p38 MAPK regulates epithelial IL-8 expression via posttranscriptional mechanism. Am J Physiol Gastrointest Liver Physiol 285(2):G282–290
Zhou K, Kanai R, Lee P, Wang HW, Modis Y (2011) Toll-like receptor 5 forms asymmetric dimers in the absence of flagellin. J Struct Biol 177(2):402–409
Junge F, Schneider B, Reckel S, Schwarz D, Dotsch V, Bernhard F (2008) Large-scale production of functional membrane proteins. Cell Mol Life Sci 65(11):1729–1755
Basheer S, Samyn D, Hedstrom M, Thakur MS, Persson BL, Mattiasson B (2011) A membrane protein based biosensor: use of a phosphate–H+ symporter membrane protein (Pho84) in the sensing of phosphate ions. Biosens Bioelectron 27(1):58–63
Kreir M, Farre C, Beckler M, George M, Fertig N (2008) Rapid screening of membrane protein activity: electrophysiological analysis of OmpF reconstituted in proteoliposomes. Lab Chip 8(4):587–595
Rigaud JL, Lévi D (2003) Reconstitution of membrane proteins into liposomes. Methods Enzymol 372:65–86
Cooper MA (2004) Advances in membrane receptor screening and analysis. J Mol Recognit 17(4):286–315
Madden DR, Safferling M (2007) Baculoviral expression of an integral membrane protein for structural studies. Methods Mol Biol 363:39–57
Hunte C, von Jagow G, Schagger H (2003) Membrane protein purification and crystallization. A practical guide. 2nd edn. Academic, San Diego
Shi X, Jarvis DL (2007) Protein N-glycosylation in the baculovirus-insect cell system. Curr Drug Targets 8(10):1116–1125
Kubelka V, Altmann F, Kornfeld G, Marz L (1994) Structures of the N-linked oligosaccharides of the membrane glycoproteins from three lepidopteran cell lines (Sf-21, IZD-Mb-0503, Bm-N). Arch Biochem Biophys 308(1):148–157
Bosman GJ, van Oostrum J, Breikers G, Bovee-Geurts PH, Klaassen CH, DeGrip WJ (2003) Functional expression of His-tagged rhodopsin in Sf9 insect cells. Methods Mol Biol 228:73–86
Luckow VA, Lee SC, Barry GF, Olins PO (1993) Efficient generation of infectious recombinant baculoviruses by site-specific transposon-mediated insertion of foreign genes into a baculovirus genome propagated in Escherichia coli. J Virol 67(8):4566–4579
le Maire M, Champeil P, Moller JV (2000) Interaction of membrane proteins and lipids with solubilizing detergents. Biochim Biophys Acta 1508(1–2):86–111
Tanford C, Reynolds JA (1976) Characterization of membrane proteins in detergent solutions. Biochim Biophys Acta 457(2):133–170
Eytan GD (1982) Use of liposomes for reconstitution of biological functions. Biochim Biophys Acta 694(2):185–202
Leonard P, Safsten P, Hearty S, McDonnell B, Finlay W, O'Kennedy R (2007) High throughput ranking of recombinant avian scFv antibody fragments from crude lysates using the Biacore A100. J Immunol Methods 323(2):172–179
Stenlund P, Babcock GJ, Sodroski J, Myszka DG (2003) Capture and reconstitution of G protein-coupled receptors on a biosensor surface. Anal Biochem 316(2):243–250
Jeon JY, Hwang SY, Cho SH, Choo J, Lee EK (2010) Effect of cholesterol content on affinity and stability of factor VIII and annexin V binding to a liposomal bilayer membrane. Chem Phys Lipids 163(4–5):335–340
Heyse S, Stora T, Schmid E, Lakey JH, Vogel H (1998) Emerging techniques for investigating molecular interactions at lipid membranes. Biochim Biophys Acta 1376(3):319–338
Temmerman K, Nickel W (2009) A novel flow cytometric assay to quantify interactions between proteins and membrane lipids. J Lipid Res 50(6):1245–1254
Agola JO, Hong L, Surviladze Z, Ursu O, Waller A, Strouse JJ, Simpson DS, Schroeder CE, Oprea TI, Golden JE, Aube J, Buranda T, Sklar LA, Wandinger-Ness A (2012) A competitive nucleotide binding inhibitor: in vitro characterization of Rab7 GTPase inhibition. ACS Chem Biol 7(6):1095–1108
Rohde KH, Dyer DW (2004) Analysis of haptoglobin and hemoglobin-haptoglobin interactions with the Neisseria meningitidis TonB-dependent receptor HpuAB by flow cytometry. Infect Immun 72(5):2494–2506
Schwartz SL, Tessema M, Buranda T, Pylypenko O, Rak A, Simons PC, Surviladze Z, Sklar LA, Wandinger-Ness A (2008) Flow cytometry for real-time measurement of guanine nucleotide binding and exchange by Ras-like GTPases. Anal Biochem 381(2):258–266
Hara T, Hirasawa A, Sun Q, Koshimizu TA, Itsubo C, Sadakane K, Awaji T, Tsujimoto G (2009) Flow cytometry-based binding assay for GPR40 (FFAR1; free fatty acid receptor 1). Mol Pharmacol 75(1):85–91
Miao Y, Zhang Y, Nakagaki K, Zhao T, Zhao A, Meng Y, Nakagaki M, Park EY, Maenaka K (2006) Expression of spider flagelliform silk protein in Bombyx mori cell line by a novel Bac-to-Bac/BmNPV baculovirus expression system. Appl Microbiol Biotechnol 71(2):192–199
Kim HS, Woo SD, Kim WJ, Choi JY, Kang SK (2000) High-level expression of a foreign gene by a recombinant baculovirus with an expanded host range. Cytotechnology 32(2):87–92
Tian DF, Hong B, Si SY (2005) Development of a new high-throughput screening model for human high density lipoprotein receptor (CLA-1) agonists. Biomed Environ Sci 18(4):265–272
Lichtenberg D (1985) Characterization of the solubilization of lipid bilayers by surfactants. Biochim Biophys Acta 821(3):470–478
Merino-Montero S, Domenech O, Montero MT, Hernandez-Borrell J (2006) Preliminary atomic force microscopy study of two-dimensional crystals of lactose permease from Escherichia coli. Biophys Chem 119(1):78–83
Stewart JC (1980) Colorimetric determination of phospholipids with ammonium ferrothiocyanate. Anal Biochem 104(1):10–14
DuBois M, Gilles KA, Hamilton JK, Rebers PA, Smith F (1956) Colorimetric method for determination of sugars and related substances. Anal Chem 28(3):350–356
Lagutina LS, Shol'ts KF (2002) Turbidimetric method for quantitative determination of triton X-100 with silicotungstic acid. Prikl Biokhim Mikrobiol 38(3):341–344
Baez M, Cabrera R, Guixe V, Babul J (2007) Unfolding pathway of the dimeric and tetrameric forms of phosphofructokinase-2 from Escherichia coli. Biochemistry 46(20):6141–6148
Ibrahim GF, Fleet GH, Lyons MJ, Walker RA (1985) Method for the isolation of highly purified Salmonella flagellins. J Clin Microbiol 22(6):1040–1044
Carrascosa LG, Gomez-Montes S, Avino A, Nadal A, Pla M, Eritja R, Lechuga LM (2012) Sensitive and label-free biosensing of RNA with predicted secondary structures by a triplex affinity capture method. Nucleic Acids Res 40(8):e56
Trevino J, Calle A, Rodriguez-Frade JM, Mellado M, Lechuga LM (2009) Determination of human growth hormone in human serum samples by surface plasmon resonance immunoassay. Talanta 78(3):1011–1016
Treviño J, Calle A, Rodríguez-Frade JM, Mellado M, Lechuga LM (2009) Surface plasmon resonance immunoassay analysis of pituitary hormones in urine and serum samples. Clin Chim Acta 403(1–2):56–62
Hongxia C, Huanga J, Lee J, Hwang S, Koh K (2010) Surface plasmon resonance spectroscopic characterization of antibody orientation and activity on the calixarene monolayer. Sensors Actuators B Chem 147(2):548–553
Cooper MA, Hansson A, Lofas S, Williams DH (2000) A vesicle capture sensor chip for kinetic analysis of interactions with membrane-bound receptors. Anal Biochem 277(2):196–205
Shiming L, Adam LS-Y, Chih-Chen L, Chih-Kung L (2006) Determination of binding constant and stoichiometry for antibody-antigen interaction with surface plasmon resonance. Curr Proteomics 3(4):271–282
O'Shannessy DJ, Brigham-Burke M, Soneson KK, Hensley P, Brooks I (1993) Determination of rate and equilibrium binding constants for macromolecular interactions using surface plasmon resonance: use of nonlinear least squares analysis methods. Anal Biochem 212(2):457–468
Cheng Y, Prusoff WH (1973) Relationship between the inhibition constant (K1) and the concentration of inhibitor that causes 50 percent which inhibitions (I50) of enzymatic reaction. Biochem Pharmacol 22:3099–3108
Neves P, Lopes SC, Sousa I, Garcia S, Eaton P, Gameiro P (2009) Characterization of membrane protein reconstitution in LUVs of different lipid composition by fluorescence anisotropy. J Pharm Biomed Anal 49(2):276–281
de Mol NJ, Fischer MJ (2010) Surface plasmon resonance: a general introduction. Methods Mol Biol 627:1–14
Parmar MM, Edwards K, Madden TD (1999) Incorporation of bacterial membrane proteins into liposomes: factors influencing protein reconstitution. Biochim Biophys Acta 1421:77–90
Zucchi PC, Zick M (2011) Membrane fusion catalyzed by a Rab, SNAREs, and SNARE chaperones is accompanied by enhanced permeability to small molecules and by lysis. Mol Biol Cell 22(23):4635–4646
Yukutake Y, Tsuji S, Hirano Y, Adachi T, Takahashi T, Fujihara K, Agre P, Yasui M, Suematsu M (2008) Mercury chloride decreases the water permeability of aquaporin-4-reconstituted proteoliposomes. Biol Cell 100:355–363
Simao AMS, Yadav MC, Narisawa S, Bolean M, Pizauro JM, Hoylaerts MF, Ciancaglini P, Millan JL (2010) Proteoliposomes harboring alkaline phosphatase and nucleotide pyrophosphatase as matrix vesicle biomimetics. J Biol Chem 285(10):7598–609
Johansen K, Lundstrom I, Liedberg B (2000) Sensitivity deviation: instrumental linearity errors that influence concentration analyses and kinetic evaluation of biomolecular interactions. Biosens Bioelectron 15:503–509
Byrne B, Stack E, Gilmartin N, O’Kennedy R (2009) Antibody-based sensors: principles, problems and potential for detection of pathogens and associated toxins. Sensors 9:4407–4445
Acknowledgments
We thank Silob Chile Ltda., the Universidad Tecnica Federico Santa María (USM), and CONICYT through Fondef Project D07i1057 for funding. We also thank Juan Carlos Espinosa (University of Valparaíso), Valeska Simons (University for Development, Santiago, Chile), and Paz Reyes (Fundación Ciencia para la Vida).
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Olguín, Y., Villalobos, P., Carrascosa, L.G. et al. Detection of flagellin by interaction with human recombinant TLR5 immobilized in liposomes. Anal Bioanal Chem 405, 1267–1281 (2013). https://doi.org/10.1007/s00216-012-6523-4
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DOI: https://doi.org/10.1007/s00216-012-6523-4