Applied Microbiology and Biotechnology

, Volume 98, Issue 15, pp 6689–6700 | Cite as

Generation of food-grade recombinant Lactobacillus casei delivering Myxococcus xanthus prolyl endopeptidase

  • Patricia Alvarez-Sieiro
  • Maria Cruz Martin
  • Begoña Redruello
  • Beatriz del Rio
  • Victor Ladero
  • Brad A. Palanski
  • Chaitan Khosla
  • Maria Fernandez
  • Miguel A. Alvarez
Applied genetics and molecular biotechnology


Prolyl endopeptidases (PEP) (EC, a family of serine proteases with the ability to hydrolyze the peptide bond on the carboxyl side of an internal proline residue, are able to degrade immunotoxic peptides responsible for celiac disease (CD), such as a 33-residue gluten peptide (33-mer). Oral administration of PEP has been suggested as a potential therapeutic approach for CD, although delivery of the enzyme to the small intestine requires intrinsic gastric stability or advanced formulation technologies. We have engineered two food-grade Lactobacillus casei strains to deliver PEP in an in vitro model of small intestine environment. One strain secretes PEP into the extracellular medium, whereas the other retains PEP in the intracellular environment. The strain that secretes PEP into the extracellular medium is the most effective to degrade the 33-mer and is resistant to simulated gastrointestinal stress. Our results suggest that in the future, after more studies and clinical trials, an engineered food-grade Lactobacillus strain may be useful as a vector for in situ production of PEP in the upper small intestine of CD patients.


Celiac disease Gluten Prolyl endopeptidase Myxococcus xanthus Heterologous expression Lactobacillus casei 



The authors are grateful to Isabel Cuesta and Jorge R. Álvarez-Buylla for their technical assistance. This research was supported by project 201370E094 from the CSIC. P.A. is the recipient of a fellowship from FICYT (BP09093) and B.D.R. is a beneficiary of a JAE-DOC contract (CSIC). C.K. is supported by a grant from the NIH (R01 DK 063158).


  1. Alvarez MA, Herrero M, Suárez JE (1998) The site-specific recombination system of the Lactobacillus species bacteriophage A2 integrates in gram-positive and gram-negative bacteria. Virology 250(1):185–193. doi: 10.1006/viro.1998.9353 PubMedCrossRefGoogle Scholar
  2. Axelsson L (1998) Lactic acid bacteria: Classification and physiology. In: Salminen SVWA (ed) Lactic acid bacteria: Microbiological and functional aspects, 2nd edn. CRC, Boca Raton, pp 1–66Google Scholar
  3. Bethune MT, Khosla C (2012) Oral enzyme therapy for celiac sprue. Methods Enzymol 502:241–271PubMedCentralPubMedCrossRefGoogle Scholar
  4. Carr FJ, Chill D, Maida N (2002) The lactic acid bacteria: a literature survey. Crit Rev Microbiol 28(4):281–370. doi: 10.1080/1040-840291046759 PubMedCrossRefGoogle Scholar
  5. Charteris, Kelly, Morelli, Collins (1998) Development and application of an in vitro methodology to determine the transit tolerance of potentially probiotic Lactobacillus and Bifidobacterium species in the upper human gastrointestinal tract. J Appl Microbiol 84(5):759–768. doi: 10.1046/j.1365-2672.1998.00407.x PubMedCrossRefGoogle Scholar
  6. Clarke G, Cryan JF, Dinan TG, Quigley EM (2012) Review article: probiotics for the treatment of irritable bowel syndrome—focus on lactic acid bacteria. Aliment Pharmacol Ther 35(4):403–413. doi: 10.1111/j.1365-2036.2011.04965.x PubMedCrossRefGoogle Scholar
  7. Comino I, Real A, Vivas S, Síglez MÁ, Caminero A, Nistal E, Casqueiro J, Rodríguez-Herrera A, Cebolla Á, Sousa C (2012) Monitoring of gluten-free diet compliance in celiac patients by assessment of gliadin 33-mer equivalent epitopes in feces. Am J Clin Nutr 95(3):670–677. doi: 10.3945/ajcn.111.026708 PubMedCentralPubMedCrossRefGoogle Scholar
  8. Conway PL, Gorbach SL, Goldin BR (1987) Survival of lactic acid bacteria in the human stomach and adhesion to intestinal cells. J Dairy Sci 70(1):1–12. doi: 10.3168/jds.S0022-0302(87)79974-3 PubMedCrossRefGoogle Scholar
  9. Corrao G, Corazza GR, Bagnardi V, Brusco G, Ciacci C, Cottone M, Sategna Guidetti C, Usai P, Cesari P, Pelli MA, Loperfido S, Volta U, Calabro A, Certo M (2001) Mortality in patients with coeliac disease and their relatives: a cohort study. Lancet 358(9279):356–361. doi: PubMedCrossRefGoogle Scholar
  10. Daniel C, Roussel Y, Kleerebezem M, Pot B (2011) Recombinant lactic acid bacteria as mucosal biotherapeutic agents. Trends Biotechnol 29(10):499–508. doi: 10.1016/j.tibtech.2011.05.002 PubMedCrossRefGoogle Scholar
  11. D'Arienzo R, Maurano F, Luongo D, Mazzarella G, Stefanile R, Troncone R, Auricchio S, Ricca E, David C, Rossi M (2008) Adjuvant effect of Lactobacillus casei in a mouse model of gluten sensitivity. Immunol Lett 119(1–2):78–83 doi: 10.1016/j.imlet.200804.006 Google Scholar
  12. D'Arienzo R, Stefanile R, Maurano F, Mazzarella G, Ricca E, Troncone R, Auricchio S, Rossi M (2011) Immunomodulatory effects of Lactobacillus casei administration in a mouse model of gliadin-sensitive enteropathy. Scand J Immunol 74(4):335–341. doi: 10.1111/j.1365-3083.2011.02582.x PubMedCrossRefGoogle Scholar
  13. del Rio B, Dattwyler RJ, Aroso M, Neves V, Meirelles L, Seegers JFML, Gomes-Solecki M (2008) Oral immunization with recombinant Lactobacillus plantarum induces a protective immune response in mice with Lyme disease. Clin Vaccine Immunol 15(9):1429–1435. doi: 10.1128/cvi.00169-08 PubMedCentralPubMedCrossRefGoogle Scholar
  14. del Rio B, Fuente JL, Neves V, Dattwyler R, Seegers JFML, Gomes-Solecki M (2010) Platform technology to deliver prophylactic molecules orally: an example using the class A select agent Yersinia pestis. Vaccine 28(41):6714–6722. doi: 10.1016/j.vaccine.2010.07.084 PubMedCentralPubMedCrossRefGoogle Scholar
  15. Fernández de Palencia P, López P, Corbí AL, Peláez C, Requena T (2008) Probiotic strains: survival under simulated gastrointestinal conditions, in vitro adhesion to Caco-2 cells and effect on cytokine secretion. Eur Food Res Technol 227(5):1475–1484. doi: 10.1007/s00217-008-0870-6 CrossRefGoogle Scholar
  16. Gass J, Ehren J, Strohmeier G, Isaacs I, Khosla C (2005) Fermentation, purification, formulation, and pharmacological evaluation of a prolyl endopeptidase from Myxococcus xanthus: implications for Celiac Sprue therapy. Biotechnol Bioeng 92(6):674–684. doi: 10.1002/bit.20643 PubMedCrossRefGoogle Scholar
  17. Green PH, Jabri B (2003) Coeliac disease. Lancet 362(9381):383–391PubMedCrossRefGoogle Scholar
  18. Gujral N, Freeman HJ, Thomson AB (2012) Celiac disease: prevalence, diagnosis, pathogenesis and treatment. World J Gastroenterol 18(42):6036–6059. doi: 10.3748/wjg.v18.i42.6036 PubMedCentralPubMedCrossRefGoogle Scholar
  19. Hausch F, Shan L, Santiago NA, Gray GM, Khosla C (2002) Intestinal digestive resistance of immunodominant gliadin peptides. Am J Physiol Gastrointest Liver Physiol 283(4):G996–G1003. doi: 10.1152/ajpgi.00136.2002 PubMedGoogle Scholar
  20. Jankovic I, Ventura M, Meylan V, Rouvet M, Elli M, Zink R (2003) Contribution of aggregation-promoting factor to maintenance of cell shape in Lactobacillus gasseri 4B2. J Bacteriol 185(11):3288–3296. doi: 10.1128/jb.185.11.3288-3296.2003 PubMedCentralPubMedCrossRefGoogle Scholar
  21. Janssen GR, Bibb MJ (1993) Derivatives of pUC18 that have BgfII sites flanking a modified multiple cloning site and that retain the ability to identify recombinant clones by visual screening of Escherichia coli colonies. Gene 124(1):133–134. doi: 10.1016/0378-1119(93)90774-W PubMedCrossRefGoogle Scholar
  22. Kabashima T, Fujii M, Meng Y, Ito K, Yoshimoto T (1998) Prolyl endopeptidase from Sphingomonas capsulata: isolation and characterization of the enzyme and nucleotide sequence of the gene. Arch Biochem Biophys 358(1):141–148. doi: 10.1006/abbi.1998.0836 PubMedCrossRefGoogle Scholar
  23. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685PubMedCrossRefGoogle Scholar
  24. Marcotte H, Ferrari S, Cesena C, Hammarström L, Morelli L, Pozzi G, Oggioni MR (2004) The aggregation-promoting factor of Lactobacillus crispatus M247 and its genetic locus. J Appl Microbiol 97(4):749–756. doi: 10.1111/j.1365-2672.2004.02364.x PubMedCrossRefGoogle Scholar
  25. Marietta EV, Murray JA (2012) Animal models to study gluten sensitivity. Semin Immunopathol 34(4):497–511. doi: 10.1007/s00281-012-0315-y PubMedCentralPubMedCrossRefGoogle Scholar
  26. Marteau P, Minekus M, Havenaar R, Huisin’t Veld JHJ (1997) Survival of lactic acid bacteria in a dynamic model of the stomach and small intestine: validation and the effects of bile. J Dairy Sci 80(6):1031–1037. doi: 10.3168/jds.S0022-0302(97)76027-2 PubMedCrossRefGoogle Scholar
  27. Marti T, Molberg O, Li Q, Gray GM, Khosla C, Sollid LM (2005) Prolyl endopeptidase-mediated destruction of T cell epitopes in whole gluten: chemical and immunological characterization. J Pharmacol Exp Ther 312(1):19–26. doi: 10.1124/jpet.104.073312 PubMedCrossRefGoogle Scholar
  28. Martin MC, Alonso JC, Suarez JE, Alvarez MA (2000) Generation of food-grade recombinant lactic acid bacterium strains by site-specific recombination. Appl Environ Microbiol 66(6):2599–2604PubMedCentralPubMedCrossRefGoogle Scholar
  29. Martin MC, Fernandez M, Martin-Alonso JM, Parra F, Boga JA, Alvarez MA (2004) Nisin-controlled expression of Norwalk virus VP60 protein in Lactobacillus casei. FEMS Microbiol Lett 237(2):385–391. doi: 10.1016/j.femsle.2004.07.002 PubMedCrossRefGoogle Scholar
  30. Martin MC, Pant N, Ladero V, Gunaydin G, Andersen KK, Alvarez B, Martinez N, Alvarez MA, Hammarstrom L, Marcotte H (2011) Integrative expression system for delivery of antibody fragments by lactobacilli. Appl Environ Microbiol 77(6):2174–2179. doi: 10.1128/AEM.02690-10 Google Scholar
  31. Mazé A, Boël G, Zúñiga M, Bourand A, Loux V, Yebra MJ, Monedero V, Correia K, Jacques N, Beaufils S, Poncet S, Joyet P, Milohanic E, Casarégola S, Auffray Y, Pérez-Martínez G, Gibrat J-F, Zagorec M, Francke C, Hartke A, Deutscher J (2010) Complete genome sequence of the probiotic Lactobacillus casei strain BL23. J Bacteriol 192(10):2647–2648. doi: 10.1128/jb.00076-10 PubMedCentralPubMedCrossRefGoogle Scholar
  32. Nyanga-Koumou AP, Ouoba LII, Kobawila SC, Louembe D (2012) Response mechanisms of lactic acid bacteria to alkaline environments: a review. Crit Rev Microbiol 38(3):185–190. doi: 10.3109/1040841X.2011.640978 PubMedCrossRefGoogle Scholar
  33. Parish JH (1986) Genetic manipulation of Streptomyces—a laboratory manual: by D A Hopwood, M J Bibb, K F Chatter; T Kieser CJ Bruton, H M Kieser, D J Lydiate, C P Smith, J M Ward and H Schrempf. pp 356. The John Innes Foundation, Norwich, UK and Cold Spring Harbour Laboratory. 1985. ISBN 0-7084-0336-0. Biochem Educ 14(4):196–196CrossRefGoogle Scholar
  34. Piper JL, Gray GM, Khosla C (2004) Effect of prolyl endopeptidase on digestive-resistant gliadin peptides in vivo. J Pharmacol Exp Ther 311(1):213–219. doi: 10.1124/jpet.104.068429 PubMedCrossRefGoogle Scholar
  35. Pyle GG, Paaso B, Anderson BE, Allen DD, Marti T, Li Q, Siegel M, Khosla C, Gray GM (2005) Effect of pretreatment of food gluten with prolyl endopeptidase on gluten-induced malabsorption in celiac sprue. Clin Gastroenterol Hepatol 3(7):687–694PubMedCrossRefGoogle Scholar
  36. Qiao SW, Bergseng E, Molberg O, Xia J, Fleckenstein B, Khosla C, Sollid LM (2004) Antigen presentation to celiac lesion-derived T cells of a 33-mer gliadin peptide naturally formed by gastrointestinal digestion. J Immunol 173(3):1757–1762PubMedCrossRefGoogle Scholar
  37. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: A laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  38. Sánchez B, Schmitter JM, Urdaci MC (2009) Identification of novel proteins secreted by Lactobacillus rhamnosus GG grown in de Mann–Rogosa–Sharpe broth. Lett Appl Microbiol 48(5):618–622. doi: 10.1111/j.1472-765X.2009.02579.x PubMedCrossRefGoogle Scholar
  39. Savijoki K, Ingmer H, Varmanen P (2006) Proteolytic systems of lactic acid bacteria. Appl Microbiol Biotechnol 71(4):394–406. doi: 10.1007/s00253-006-0427-1 PubMedCrossRefGoogle Scholar
  40. Scheirlinck T, Mahillon J, Joos H, Dhaese P, Michiels F (1989) Integration and expression of alpha-amylase and endoglucanase genes in the Lactobacillus plantarum chromosome. Appl Environ Microbiol 55(9):2130–2137PubMedCentralPubMedGoogle Scholar
  41. Schuppan D, Junker Y, Barisani D (2009) Celiac disease: from pathogenesis to novel therapies. Gastroenterology 137(6):1912–1933. doi: 10.1053/j.gastro.2009.09.008 Google Scholar
  42. Sealey-Voyksner JA, Khosla C, Voyksner RD, Jorgenson JW (2010) Novel aspects of quantitation of immunogenic wheat gluten peptides by liquid chromatography–mass spectrometry/mass spectrometry. J Chromatogr A 1217(25):4167–4183. doi: 10.1016/j.chroma.2010.01.067 Google Scholar
  43. Shan L, Molberg O, Parrot I, Hausch F, Filiz F, Gray GM, Sollid LM, Khosla C (2002) Structural basis for gluten intolerance in celiac sprue. Science 297(5590):2275–2279. doi: 10.1126/science.1074129 PubMedCrossRefGoogle Scholar
  44. Shan L, Marti T, Sollid LM, Gray GM, Khosla C (2004) Comparative biochemical analysis of three bacterial prolyl endopeptidases: implications for coeliac sprue. Biochem J 383(Pt 2):311–318. doi: 10.1042/BJ20040907 PubMedCentralPubMedGoogle Scholar
  45. Stoven S, Murray JA, Marietta EV (2013) Latest in vitro and in vivo models of celiac disease. Expert Opin Drug Discov 8(4):445–457. doi: 10.1517/17460441.2013.761203 PubMedCentralPubMedCrossRefGoogle Scholar
  46. Tuohy KM, Probert HM, Smejkal CW, Gibson GR (2003) Using probiotics and prebiotics to improve gut health. Drug Discov Today 8(15):692–700. doi: 10.1016/S1359-6446(03)02746-6 PubMedCrossRefGoogle Scholar
  47. Turroni F, Ventura M, Buttó LF, Duranti S, O'Toole PW, Motherway MOC, Sinderen D (2013) Molecular dialogue between the human gut microbiota and the host: a Lactobacillus and Bifidobacterium perspective. Cell Mol Life Sci 1–21. doi:  10.1007/s00018-013-1318-0
  48. van Overbeek FM, Uil-Dieterman IG, Mol IW, Kohler-Brands L, Heymans HS, Mulder CJ (1997) The daily gluten intake in relatives of patients with coeliac disease compared with that of the general Dutch population. Eur J Gastroenterol Hepatol 9(11):1097–1099PubMedCrossRefGoogle Scholar
  49. Ventura M, Jankovic I, Walker DC, Pridmore RD, Zink R (2002) Identification and characterization of novel surface proteins in Lactobacillus johnsonii and Lactobacillus gasseri. Appl Environ Microbiol 68(12):6172–6181. doi: 10.1128/aem.68.12.6172-6181.2002 PubMedCentralPubMedCrossRefGoogle Scholar
  50. Vieira J, Messing J (1991) New pUC-derived cloning vectors with different selectable markers and DNA replication origins. Gene 100:189–194PubMedCrossRefGoogle Scholar
  51. Wang J, Zhong Z, Zhang W, Bao Q, Wei A, Meng H, Zhang H (2012) Comparative analysis of the gene expression profile of probiotic Lactobacillus casei Zhang with and without fermented milk as a vehicle during transit in a simulated gastrointestinal tract. Res Microbiol 163(5):357–365. doi: 10.1016/j.resmic.2012.04.002 Google Scholar
  52. Wei M-Q, Rush CM, Norman JM, Hafner LM, Epping RJ, Timms P (1995) An improved method for the transformation of Lactobacillus strains using electroporation. J Microbiol Methods 21(1):97–109. doi: 10.1016/0167-7012(94)00038-9 CrossRefGoogle Scholar
  53. Wells J, Mercenier A (2008) Mucosal delivery of therapeutic and prophylactic molecules using lactic acid bacteria. Nat Rev Microbiol 6(5):349–362. doi: 10.1038/nrmicro1040 Google Scholar
  54. Yoshimoto T, Walter R, Tsuru D (1980) Proline-specific endopeptidase from Flavobacterium. Purification and properties. J Biol Chem 255(10):4786–4792PubMedGoogle Scholar
  55. Yoshimoto T, Kanatani A, Shimoda T, Inaoka T, Kokubo T, Tsuru D (1991) Prolyl endopeptidase from Flavobacterium meningosepticum: cloning and sequencing of the enzyme gene. J Biochem 110(6):873–878PubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Patricia Alvarez-Sieiro
    • 1
  • Maria Cruz Martin
    • 1
  • Begoña Redruello
    • 1
  • Beatriz del Rio
    • 1
  • Victor Ladero
    • 1
  • Brad A. Palanski
    • 2
    • 3
  • Chaitan Khosla
    • 2
    • 3
  • Maria Fernandez
    • 1
  • Miguel A. Alvarez
    • 1
  1. 1.Instituto de Productos Lácteos de Asturias (IPLA-CSIC)VillaviciosaSpain
  2. 2.Department of ChemistryStanford UniversityStanfordUSA
  3. 3.Department of Chemical EngineeringStanford UniversityStanfordUSA

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