Abstract
Lactic acid bacteria (LAB) are attractive hosts for the expression of heterologous proteins and can be engineered to deliver therapeutic proteins or peptides to mucosal surfaces. The gastric stable pentadecapeptide BPC-157 is able to prevent and treat gastrointestinal inflammation by reducing the production of reactive oxygen species (ROS). In this study, we used LAB Lactococcus lactis as a vector to deliver BPC-157 by surface display and trypsin shedding or by secretion to the growth medium. Surface display of BPC-157 was achieved by fusing it with basic membrane protein A (BmpA) or with the peptidoglycan binding domain of AcmA and Usp45 secretion signal. While the expression of BmpA-fusion proteins was higher than that of AcmA/Usp45-fusion protein, the surface display ability of BPC-157 was approximately 14-fold higher with AcmA/Usp45-fusion protein. Release of BPC-157 from the bacterial surface or from isolated fusion proteins by trypsinization was demonstrated with anti-BPC-157 antibodies or by mass spectrometry. The concentration of BPC-157 delivered by surface display via AcmA/Usp45-fusion was 30 ng/ml. This increased to 117 ng/ml by Usp45 signal-mediated secretion, making the latter the most effective lactococcal delivery approach for BPC-157. Secreted BPC-157 significantly decreased ROS production in 149BR fibroblast cell model, suggesting its potential benefit in the treatment of intestinal inflammations. Additionally, a comparison of different modes of small peptide delivery by L. lactis, performed in the present study, will facilitate the future use of L. lactis as peptide delivery vehicle.
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References
Amaretti A, di Nunzio M, Pompei A, Raimondi S, Rossi M, Bordoni A (2013) Antioxidant properties of potentially probiotic bacteria: in vitro and in vivo activities. Appl Microbiol Biotechnol 97(2):809–817. https://doi.org/10.1007/s00253-012-4241-7
Ananthakrishnan AN (2015) Epidemiology and risk factors for IBD. Nat Rev Gastroenterol Hepatol 12(4):205–217. https://doi.org/10.1038/nrgastro.2015.34
Berlec A, Zadravec P, Jevnikar Z, Strukelj B (2011) Identification of candidate carrier proteins for surface display on Lactococcus lactis by theoretical and experimental analyses of the surface proteome. Appl Environ Microbiol 77(4):1292–1300. https://doi.org/10.1128/AEM.02102-10
Berlec A, Ravnikar M, Strukelj B (2012) Lactic acid bacteria as oral delivery systems for biomolecules. Pharmazie 67(11):891–898. https://doi.org/10.1691/ph.2012.1705
Berlec A, Skrlec K, Kocjan J, Olenic M, Strukelj B (2018) Single plasmid systems for inducible dual protein expression and for CRISPR-Cas9/CRISPRi gene regulation in lactic acid bacterium Lactococcus lactis. Sci Rep 8(1):1009. https://doi.org/10.1038/s41598-018-19402-1
Bernasconi E, Germond JE, Delley M, Fritsche R, Corthesy B (2002) Lactobacillus bulgaricus proteinase expressed in Lactococcus lactis is a powerful carrier for cell wall-associated and secreted bovine beta-lactoglobulin fusion proteins. Appl Environ Microbiol 68(6):2917–2923. https://doi.org/10.1128/AEM.68.6.2917-2923.2002
Brcic L, Brcic I, Staresinic M, Novinscak T, Sikiric P, Seiwerth S (2009) Modulatory effect of gastric pentadecapeptide BPC 157 on angiogenesis in muscle and tendon healing. J Physiol Pharmacol 60(Suppl 7):191–196
Carvalho RDO, do Carmo FLR, de Oliveira Junior A, Langella P, Chatel JM, Bermudez-Humaran LG, Azevedo V, de Azevedo MS (2017) Use of wild type or recombinant lactic acid bacteria as an alternative treatment for gastrointestinal inflammatory diseases: a focus on inflammatory bowel diseases and mucositis. Front Microbiol 8:800. https://doi.org/10.3389/fmicb.2017.00800
Cerovecki T, Bojanic I, Brcic L, Radic B, Vukoja I, Seiwerth S, Sikiric P (2010) Pentadecapeptide BPC 157 (PL 14736) improves ligament healing in the rat. J Orthop Res 28(9):1155–1161. https://doi.org/10.1002/jor.21107
Chang CH, Tsai WC, Lin MS, Hsu YH, Pang JH (2011) The promoting effect of pentadecapeptide BPC 157 on tendon healing involves tendon outgrowth, cell survival, and cell migration. J Appl Physiol (1985) 110(3):774–780. https://doi.org/10.1152/japplphysiol.00945.2010
Chatel JM, Langella P, Adel-Patient K, Commissaire J, Wal JM, Corthier G (2001) Induction of mucosal immune response after intranasal or oral inoculation of mice with Lactococcus lactis producing bovine beta-lactoglobulin. Clin Diagn Lab Immunol 8(3):545–551. https://doi.org/10.1128/CDLI.8.3.545-551.2001
Chua KJ, Kwok WC, Aggarwal N, Sun T, Chang MW (2017) Designer probiotics for the prevention and treatment of human diseases. Curr Opin Chem Biol 40:8–16. https://doi.org/10.1016/j.cbpa.2017.04.011
Dieye Y, Usai S, Clier F, Gruss A, Piard JC (2001) Design of a protein-targeting system for lactic acid bacteria. J Bacteriol 183(14):4157–4166. https://doi.org/10.1128/JB.183.14.4157-4166.2001
Drmic D, Kolenc D, Ilic S, Bauk L, Sever M, Zenko Sever A, Luetic K, Suran J, Seiwerth S, Sikiric P (2017) Celecoxib-induced gastrointestinal, liver and brain lesions in rats, counteraction by BPC 157 or L-arginine, aggravation by L-NAME. World J Gastroenterol 23(29):5304–5312. https://doi.org/10.3748/wjg.v23.i29.5304
Duzel AVJ, Antunovic M, Malekinusic D, Vrdoljak B, Samara M, Gojkovic S, Krezic I, Vidovic T, Bilic Z, Knezevic M, Sever M, Lojo N, Kokot A, Kolovrat M, Drmic D, Vukojevic J, Kralj T, Kasnik K, Siroglavic M, Seiwerth S, Sikiric P (2017) Stable gastric pentadecapeptide BPC 157 in the treatment of colitis and ischemia and reperfusion in rats: new insights. World J Gastroenterol 23(48):8465–8488. https://doi.org/10.3748/wjg.v23.i48.8465
Filosevic A, Waldowski RA, Vidovic T, Sikiric P, Drmic D (2017) Stable gastric pentadecapeptide BPC 157 antagonizes hydrogen peroxide induced oxidative stress in Drosophila melanogaster. FASEB J 31(1 Supplement):667.14–667.14
Garcia-Fruitos E (2012) Lactic acid Bacteria: a promising alternative for recombinant protein production. Microb Cell Factories 11:157. https://doi.org/10.1186/1475-2859-11-157
Gareau MG, Sherman PM, Walker WA (2010) Probiotics and the gut microbiota in intestinal health and disease. Nat Rev Gastroenterol Hepatol 7(9):503–514. https://doi.org/10.1038/nrgastro.2010.117
Ghouri YA, Richards DM, Rahimi EF, Krill JT, Jelinek KA, DuPont AW (2014) Systematic review of randomized controlled trials of probiotics, prebiotics, and synbiotics in inflammatory bowel disease. Clin Exp Gastroenterol 7:473–487. https://doi.org/10.2147/CEG.S27530
Halasi M, Wang M, Chavan TS, Gaponenko V, Hay N, Gartel AL (2013) ROS inhibitor N-acetyl-L-cysteine antagonizes the activity of proteasome inhibitors. Biochem J 454(2):201–208. https://doi.org/10.1042/BJ20130282
Holo H, Nes IF (1989) High-frequency transformation, by electroporation, of Lactococcus lactis subsp. cremoris grown with glycine in osmotically stabilized media. Appl Environ Microbiol 55(12):3119–3123
Huang T, Zhang K, Sun L, Xue X, Zhang C, Shu Z, Mu N, Gu J, Zhang W, Wang Y, Zhang Y (2015) Body protective compound-157 enhances alkali-burn wound healing in vivo and promotes proliferation, migration, and angiogenesis in vitro. Drug Des Devel Ther 9:2485–2499. https://doi.org/10.2147/DDDT.S82030
Jarc E, Kump A, Malavasic P, Eichmann TO, Zimmermann R, Petan T (2017) Lipid droplets induced by secreted phospholipase A2 and unsaturated fatty acids protect breast cancer cells from nutrient and lipotoxic stress. Biochim Biophys Acta 1863(3):247–265. https://doi.org/10.1016/j.bbalip.2017.12.006
Keremi B, Lohinai Z, Komora P, Duhaj S, Borsi K, Jobbagy-Ovari G, Kallo K, Szekely AD, Fazekas A, Dobo-Nagy C, Sikiric P, Varga G (2009) Antiinflammatory effect of BPC 157 on experimental periodontitis in rats. J Physiol Pharmacol 60(Suppl 7):115–122
Klicek R, Sever M, Radic B, Drmic D, Kocman I, Zoricic I, Vuksic T, Ivica M, Barisic I, Ilic S, Berkopic L, Vrcic H, Brcic L, Blagaic AB, Coric M, Brcic I, Rokotov DS, Anic T, Seiwerth S, Sikiric P (2008) Pentadecapeptide BPC 157, in clinical trials as a therapy for inflammatory bowel disease (PL14736), is effective in the healing of colocutaneous fistulas in rats: role of the nitric oxide-system. J Pharmacol Sci 108(1):7–17. https://doi.org/10.1254/jphs.FP0072161
Kosler S, Strukelj B, Berlec A (2017) Lactic acid bacteria with concomitant IL-17, IL-23 and TNFalpha-binding ability for the treatment of inflammatory bowel disease. Curr Pharm Biotechnol 18(4):318–326. https://doi.org/10.2174/1389201018666170210152218
Le Loir Y, Gruss A, Ehrlich SD, Langella P (1998) A nine-residue synthetic propeptide enhances secretion efficiency of heterologous proteins in Lactococcus lactis. J Bacteriol 180(7):1895–1903
Le Loir Y, Nouaille S, Commissaire J, Bretigny L, Gruss A, Langella P (2001) Signal peptide and propeptide optimization for heterologous protein secretion in Lactococcus lactis. Appl Environ Microbiol 67(9):4119–4127. https://doi.org/10.1128/AEM.67.9.4119-4127.2001
Le Loir Y, Azevedo V, Oliveira SC, Freitas DA, Miyoshi A, Bermudez-Humaran LG, Nouaille S, Ribeiro LA, Leclercq S, Gabriel JE, Guimaraes VD, Oliveira MN, Charlier C, Gautier M, Langella P (2005) Protein secretion in Lactococcus lactis: an efficient way to increase the overall heterologous protein production. Microb Cell Factories 4(1):2. https://doi.org/10.1186/1475-2859-4-2
Lindholm A, Smeds A, Palva A (2004) Receptor binding domain of Escherichia coli F18 fimbrial adhesin FedF can be both efficiently secreted and surface displayed in a functional form in Lactococcus lactis. Appl Environ Microbiol 70(4):2061–2071. https://doi.org/10.1128/AEM.70.4.2061-2071.2004
Luetic K, Sucic M, Vlainic J, Halle ZB, Strinic D, Vidovic T, Luetic F, Marusic M, Gulic S, Pavelic TT, Kokot A, Seiwerth RS, Drmic D, Batelja L, Seiwerth S, Sikiric P (2017) Cyclophosphamide induced stomach and duodenal lesions as a NO-system disturbance in rats: L-NAME, L-arginine, stable gastric pentadecapeptide BPC 157. Inflammopharmacology 25(2):255–264. https://doi.org/10.1007/s10787-017-0330-7
Michon C, Langella P, Eijsink VGH, Mathiesen G, Chatel JM (2016) Display of recombinant proteins at the surface of lactic acid bacteria: strategies and applications. Microb Cell Factories 15:70. https://doi.org/10.1186/s12934-016-0468-9
Mierau I, Kleerebezem M (2005) 10 years of the nisin-controlled gene expression system (NICE) in Lactococcus lactis. Appl Microbiol Biotechnol 68(6):705–717. https://doi.org/10.1007/s00253-005-0107-6
Mittal M, Siddiqui MR, Tran K, Reddy SP, Malik AB (2014) Reactive oxygen species in inflammation and tissue injury. Antioxid Redox Signal 20(7):1126–1167. https://doi.org/10.1089/ars.2012.5149
Molodecky NA, Soon IS, Rabi DM, Ghali WA, Ferris M, Chernoff G, Benchimol EI, Panaccione R, Ghosh S, Barkema HW, Kaplan GG (2012) Increasing incidence and prevalence of the inflammatory bowel diseases with time, based on systematic review. Gastroenterol 142(1):46–54. https://doi.org/10.1053/j.gastro.2011.10.001
Morello E, Bermudez-Humaran LG, Llull D, Sole V, Miraglio N, Langella P, Poquet I (2008) Lactococcus lactis, an efficient cell factory for recombinant protein production and secretion. J Mol Microbiol Biotechnol 14(1–3):48–58. https://doi.org/10.1159/000106082
Ng DT, Sarkar CA (2011) Nisin-inducible secretion of a biologically active single-chain insulin analog by Lactococcus lactis NZ9000. Biotechnol Bioeng 108(8):1987–1996. https://doi.org/10.1002/bit.23130
Pontes DS, de Azevedo MS, Chatel JM, Langella P, Azevedo V, Miyoshi A (2011) Lactococcus lactis as a live vector: heterologous protein production and DNA delivery systems. Protein Expr Purif 79(2):165–175. https://doi.org/10.1016/j.pep.2011.06.005
Ravnikar M, Strukelj B, Obermajer N, Lunder M, Berlec A (2010) Engineered lactic acid bacterium Lactococcus lactis capable of binding antibodies and tumor necrosis factor alpha. Appl Environ Microbiol 76(20):6928–6932. https://doi.org/10.1128/AEM.00190-10
Samazan F, Rokbi B, Seguin D, Telles F, Gautier V, Richarme G, Chevret D, Varela PF, Velours C, Poquet I (2015) Production, secretion and purification of a correctly folded staphylococcal antigen in Lactococcus lactis. Microb Cell Factories 14:104. https://doi.org/10.1186/s12934-015-0271-z
Sandborn WJ (2008) Current directions in IBD therapy: what goals are feasible with biological modifiers? Gastroenterol 135(5):1442–1447. https://doi.org/10.1053/j.gastro.2008.09.053
Sikiric P, Petek M, Rucman R, Seiwerth S, Grabarevic Z, Rotkvic I, Turkovic B, Jagic V, Mildner B, Duvnjak M, Lang N, Danilovic Z, Cviko A, Kolega M, Sallmani A, Djacic S, Bura M, Brkic T, Banic M, Dodig M, Coric V, Simicevic V, Veljaca M, Erceg D, Ježek D, Simunic-Banek L, Skroza N, Bulic K, Buljat G, Hanzevacki M, Orihovac V, Lucinger D, Culig J, Separovic J, Marovic A, Mise S, Suchanek E, Matoz W, Perovic D, Gjurasin M, Mikulandra S, Dernikovic K, Cuk V, Karakas I (1993) A new gastric juice peptide, BPC. An overview of the stomach-stress-organoprotection hypothesis and beneficial effects of BPC. J Physiol Paris 87(5):313–327. https://doi.org/10.1016/0928-4257(93)90038-U
Sikiric P, Seiwerth S, Deskovic S, Grabarevic Z, Marovic A, Rucman R, Petek M, Konjevoda P, Jadrijevic S, Sosa T, Perovic D, Aralica G, Turkovic B (1999) New model of cytoprotection/adaptive cytoprotection in rats: endogenous small irritants, antiulcer agents and indomethacin. Eur J Pharmacol 364(1):23–31. https://doi.org/10.1016/S0014-2999(98)00818-8
Sikiric P, Seiwerth S, Rucman R, Turkovic B, Rokotov DS, Brcic L, Sever M, Klicek R, Radic B, Drmic D, Ilic S, Kolenc D, Stambolija V, Zoricic Z, Vrcic H, Sebecic B (2012) Focus on ulcerative colitis: stable gastric pentadecapeptide BPC 157. Curr Med Chem 19(1):126–132. https://doi.org/10.2174/092986712803414015
Sikiric P, Seiwerth S, Rucman R, Turkovic B, Rokotov DS, Brcic L, Sever M, Klicek R, Radic B, Drmic D, Ilic S, Kolenc D, Aralica G, Safic H, Suran J, Rak D, Dzidic S, Vrcic H, Sebecic B (2013) Toxicity by NSAIDs. Counteraction by stable gastric pentadecapeptide BPC 157. Curr Pharm Des 19(1):76–83. https://doi.org/10.2174/1381612811306010076
Sikiric P, Seiwerth S, Rucman R, Kolenc D, Vuletic LB, Drmic D, Grgic T, Strbe S, Zukanovic G, Crvenkovic D, Madzarac G, Rukavina I, Sucic M, Baric M, Starcevic N, Krstonijevic Z, Bencic ML, Filipcic I, Rokotov DS, Vlainic J (2016) Brain-gut axis and pentadecapeptide BPC 157: theoretical and practical implications. Curr Neuropharmacol 14(8):857–865. https://doi.org/10.2174/1570159X13666160502153022
Skrlec K, Pucer Janez A, Rogelj B, Strukelj B, Berlec A (2017) Evasin-displaying lactic acid bacteria bind different chemokines and neutralize CXCL8 production in Caco-2 cells. Microb Biotechnol 10(6):1732–1743. https://doi.org/10.1111/1751-7915.12781
Song AA, In LLA, Lim SHE, Rahim RA (2017) A review on Lactococcus lactis: from food to factory. Microb Cell Factories 16(1):55. https://doi.org/10.1186/s12934-017-0669-x
Steen A, Buist G, Leenhouts KJ, El Khattabi M, Grijpstra F, Zomer AL, Venema G, Kuipers OP, Kok J (2003) Cell wall attachment of a widely distributed peptidoglycan binding domain is hindered by cell wall constituents. J Biol Chem 278(26):23874–23881. https://doi.org/10.1074/jbc.M211055200
Steidler L, Hans W, Schotte L, Neirynck S, Obermeier F, Falk W, Fiers W, Remaut E (2000) Treatment of murine colitis by Lactococcus lactis secreting interleukin-10. Science 289(5483):1352–1355. https://doi.org/10.1126/science.289.5483.1352
Tian T, Wang Z, Zhang J (2017) Pathomechanisms of oxidative stress in inflammatory bowel disease and potential antioxidant therapies. Oxidative Med Cell Longev 2017:4535194–4535118. https://doi.org/10.1155/2017/4535194
Triantafillidis JK, Merikas E, Georgopoulos F (2011) Current and emerging drugs for the treatment of inflammatory bowel disease. Drug Des Devel Ther 5:185–210. https://doi.org/10.2147/DDDT.S11290
Tuzun A, Erdil A, Inal V, Aydin A, Bagci S, Yesilova Z, Sayal A, Karaeren N, Dagalp K (2002) Oxidative stress and antioxidant capacity in patients with inflammatory bowel disease. Clin Biochem 35(7):569–572. https://doi.org/10.1016/S0009-9120(02)00361-2
van Asseldonk M, Rutten G, Oteman M, Siezen RJ, de Vos WM, Simons G (1990) Cloning of usp45, a gene encoding a secreted protein from Lactococcus lactis subsp. lactis MG1363. Gene 95(1):155–160. https://doi.org/10.1016/0378-1119(90)90428-T
Varma NR, Toosa H, Foo HL, Alitheen NB, Nor Shamsudin M, Arbab AS, Yusoff K, Abdul Rahim R (2013) Display of the viral epitopes on Lactococcus lactis: a model for food grade vaccine against EV71. Biotechnol Res Int 2013:431315. https://doi.org/10.1155/2013/431315
Visweswaran GR, Leenhouts K, van Roosmalen M, Kok J, Buist G (2014) Exploiting the peptidoglycan-binding motif, LysM, for medical and industrial applications. Appl Microbiol Biotechnol 98(10):4331–4345. https://doi.org/10.1007/s00253-014-5633-7
Vuksic T, Zoricic I, Brcic L, Sever M, Klicek R, Radic B, Cesarec V, Berkopic L, Keller N, Blagaic AB, Kokic N, Jelic I, Geber J, Anic T, Seiwerth S, Sikiric P (2007) Stable gastric pentadecapeptide BPC 157 in trials for inflammatory bowel disease (PL-10, PLD-116, PL14736, Pliva, Croatia) heals ileoileal anastomosis in the rat. Surg Today 37(9):768–777. https://doi.org/10.1007/s00595-006-3498-9
Wang Y, Wu Y, Xu H, Mei X, Yu D, Li W (2017) Antioxidant properties of probiotic bacteria. Nutrients 9(5):521. https://doi.org/10.3390/nu9050521
Watterlot L, Rochat T, Sokol H, Cherbuy C, Bouloufa I, Lefevre F, Gratadoux JJ, Honvo-Hueto E, Chilmonczyk S, Blugeon S, Corthier G, Langella P, Bermudez-Humaran LG (2010) Intragastric administration of a superoxide dismutase-producing recombinant Lactobacillus casei BL23 strain attenuates DSS colitis in mice. Int J Food Microbiol 144(1):35–41. https://doi.org/10.1016/j.ijfoodmicro.2010.03.037
Wells JM, Mercenier A (2008) Mucosal delivery of therapeutic and prophylactic molecules using lactic acid bacteria. Nat Rev Microbiol 6:349–362. https://doi.org/10.1038/nrmicro1840
Zadravec P, Mavric A, Bogovic Matijasic B, Strukelj B, Berlec A (2014) Engineering BmpA as a carrier for surface display of IgG-binding domain on Lactococcus lactis. Protein Eng Des Sel 27(1):21–27. https://doi.org/10.1093/protein/gzt059
Zadravec P, Strukelj B, Berlec A (2015) Improvement of LysM-mediated surface display of designed ankyrin repeat proteins (DARPins) in recombinant and nonrecombinant strains of Lactococcus lactis and Lactobacillus Species. Appl Environ Microbiol 81(6):2098–2106. https://doi.org/10.1128/AEM.03694-14
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The authors are grateful to Prof. Roger pain for critical reading of the manuscript.
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This study was funded by the Slovenian Research Agency (grant number P4-0127).
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This article is dedicated in the memory of the late Dr. Rudolf Ručman who made important contributions to this work.
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Škrlec, K., Ručman, R., Jarc, E. et al. Engineering recombinant Lactococcus lactis as a delivery vehicle for BPC-157 peptide with antioxidant activities. Appl Microbiol Biotechnol 102, 10103–10117 (2018). https://doi.org/10.1007/s00253-018-9333-6
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DOI: https://doi.org/10.1007/s00253-018-9333-6