Preliminary Investigation on the Effect of Lactobacillus and Epidermal Growth Factor on Tight Junction Proteins in Experimental Clostridium difficile Infection
Affiliation(s)
Departments of Gastroenterology, Postgraduate Institute of Medical Education and Research, Chandigarh, India. Departments of Gastroenterology, Postgraduate Institute of Medical Education and Research, Chandigarh, India. Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh, India.
Departments of Gastroenterology, Postgraduate Institute of Medical Education and Research, Chandigarh, India. Departments of Gastroenterology, Postgraduate Institute of Medical Education and Research, Chandigarh, India. Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh, India.
ABSTRACT
Clostridium difficile associated disease (CDAD) is the most common hospital acquired infection, due to exposure to various drugs. C. difficile toxins influence barrier function in intestinal epithelium. Biotherapeutic approaches, employing probiotic and epidermal growth factor (EGF) could help in barrier protein protection and aid in CDAD management. A preliminary investigation on the effect of Lactobacillus acidophilus and EGF on tight junction proteins in experimentally induced C. difficile infection was done. BALB/mice were divided into 5 groups. Group 1 was comprised of healthy controls, whereas animals in Groups 2 - 5 were sub-divided into 3 subgroups (a, b and c) each. Animals in Groups 2 - 5 received C. difficile inoculum either on day 1 (Group 2) or after pretreatment with ampicillin (Group 3), cyclosporine (Group 4) or lansoprazole (Group 5). Additionally animals in subgroups “b” and “c” also received L. acidophilus and EGF inocula respectively after C. difficile challenge. All animals were investigated for the presence of tight junction proteins (occludin, α-actinin and zonula occludens) in their colonic segments. Data were analyzed using the SPSS version 10 software. These three proteins were present in significantly less (P < 0.05) number of animals in the drug receiving animals, whereas they were found in significantly more (P < 0.05) number of animals receiving L. acidophilus and EGF after challenge with ampicillin, cyclosporine and lansoprazole, suggesting their role in protecting intestinal barrier function.
Clostridium difficile associated disease (CDAD) is the most common hospital acquired infection, due to exposure to various drugs. C. difficile toxins influence barrier function in intestinal epithelium. Biotherapeutic approaches, employing probiotic and epidermal growth factor (EGF) could help in barrier protein protection and aid in CDAD management. A preliminary investigation on the effect of Lactobacillus acidophilus and EGF on tight junction proteins in experimentally induced C. difficile infection was done. BALB/mice were divided into 5 groups. Group 1 was comprised of healthy controls, whereas animals in Groups 2 - 5 were sub-divided into 3 subgroups (a, b and c) each. Animals in Groups 2 - 5 received C. difficile inoculum either on day 1 (Group 2) or after pretreatment with ampicillin (Group 3), cyclosporine (Group 4) or lansoprazole (Group 5). Additionally animals in subgroups “b” and “c” also received L. acidophilus and EGF inocula respectively after C. difficile challenge. All animals were investigated for the presence of tight junction proteins (occludin, α-actinin and zonula occludens) in their colonic segments. Data were analyzed using the SPSS version 10 software. These three proteins were present in significantly less (P < 0.05) number of animals in the drug receiving animals, whereas they were found in significantly more (P < 0.05) number of animals receiving L. acidophilus and EGF after challenge with ampicillin, cyclosporine and lansoprazole, suggesting their role in protecting intestinal barrier function.
KEYWORDS
Antibiotic, C. difficile, Epidermal Growth Factor, Immunosuppressive Drug, Probiotic, Tight Junction Proteins
Antibiotic, C. difficile, Epidermal Growth Factor, Immunosuppressive Drug, Probiotic, Tight Junction Proteins
Cite this paper
Kaur, S. , Vaishnavi, C. , Ray, P. , Singh, M. and Kochhar, R. (2014) Preliminary Investigation on the Effect of Lactobacillus and Epidermal Growth Factor on Tight Junction Proteins in Experimental Clostridium difficile Infection. Advances in Microbiology, 4, 425-435. doi: 10.4236/aim.2014.48047.
Kaur, S. , Vaishnavi, C. , Ray, P. , Singh, M. and Kochhar, R. (2014) Preliminary Investigation on the Effect of Lactobacillus and Epidermal Growth Factor on Tight Junction Proteins in Experimental Clostridium difficile Infection. Advances in Microbiology, 4, 425-435. doi: 10.4236/aim.2014.48047.
References
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http://dx.doi.org/10.1128/IAI.69.3.1329-1336.2001 [12] Resta-Lenert, R. and Barret, K.E. (2003) Live Probiotics Protect Intestinal Epithelial Cells from the Effects of Infection with Enteroinvasive Escherichia coli (EIEC). Gut, 52, 988-997.
http://dx.doi.org/10.1136/gut.52.7.988 [13] Towbin, J., Staehelin, T. and Gordon, J. (1979) Electrophoresis Transfer of Protein from Polyacrylamide Gels to Nitrocellulose Sheets: Procedure and Some Applications. Proceedings of the National Academy of Sciences, 76, 4350-4354. http://dx.doi.org/10.1073/pnas.76.9.4350 [14] Thalestam, M. and Chaves-Olarte, E. (2000) Cytotoxic Effects of the Clostridium difficile Toxins. Current Topics in Microbiology and Immunology, 250, 85-96. http://dx.doi.org/10.1007/978-3-662-06272-2_4 [15] Yang, K.M., Jiang, Z.Y., Zheng, C.T., Wang, L. and Yang, X.F. (2014) Effect of Lactobacillus plantarum on Diarrhea and Intestinal Barrier Function of Young Piglets Challenged with Enterotoxigenic Escherichia coli K88. Journal of Animal Science, 92, 1496-1503. http://dx.doi.org/10.2527/jas.2013-6619 [16] Miura, M., Kato, H. and Matsushita, O. (2011) Identification of a Novel Virulence Factor in Clostridium difficile That Modulates Toxin Sensitivity of Cultured Epithelial Cells. Infection and Immunity, 79, 3810-3820. http://dx.doi.org/10.1128/IAI.00051-11 [17] Castagluiolo, I., Riegler, M.F., Valenick, L., LaMont, J.T. and Pothoulakis, C. (1999) Saccharomyces boulardii Protease Inhibits the Effects of Clostridium difficile Toxins A and B in Human Colonic Mucosa. Infection and Immunity, 67, 302-307. [18] Mack, D.R., Ahrne, S., Hyde, L., Wei, S. and Hollingsworth, M.A. (2003) Extracellular MUC3 Mucin Secretion Follows Adherence of Lactobacillus Strains to Intestinal Epithelial Cells in Vitro. Gut, 52, 827-833. [19] García-Lafuente, A., Antolin, M., Guarner, F., Crespo, E. and Malagelada, J.R. (2001) Modulation of Colonic Barrier Function by the Composition of the Commensal Flora in the Rat. Gut, 48, 503-507. http://dx.doi.org/10.1136/gut.48.4.503 [20] Hanley, S.C., Assouline-Thomas, B., Makhlin, J. and Rosenberg, L. (2011) Epidermal Growth Factor Induces Adult Human Islet Cell Dedifferentiation. Journal of Endocrinology, 211, 231-239. http://dx.doi.org/10.1530/JOE-11-0213 [21] Gospodarowicz, D. (1981) Epidermal and Nerve Growth Factor in Mammalian Development. Annual Review of Physiology, 43, 251-263. http://dx.doi.org/10.1146/annurev.ph.43.030181.001343 [22] Procaccino, F., Reinshagen, M., Hoffmann, P., Zeeh, J.M., Lakshmanan, J., McRoberts, J.A., Patel, A., French, S. and Eysselein, V.E. (1994) Protective Effect of Epidermal Growth Factor in an Experimental Model of Colitis in Rats. Gastroenterology, 107, 12-17. [23] Banan, A., Zhang, Y., Losurdo, J. and Keshavarzian, A. (2000) Carbonylation and Disassembly of the F-Actin Cyto-skeleton in Oxidant Induced Barrier Dysfunction and Its Prevention by Epidermal Growth Factor and Transforming Growth Factor α in a Human Colonic Cell Line. Gut, 46, 830-837. http://dx.doi.org/10.1136/gut.46.6.830 [24] Nobes, C.D., Hawkins, P., Stephens, L. and Hall, A. (1995) Activation of the Small GTP-Binding Proteins rho and rac by Growth Factor Receptors. Journal of Cell Science, 108, 225-233. [25] Craig, W.S. and Johnson, R.P. (1996) Assembly of Focal Adhesions: Progress, Paradigms, and Portents. Current Opinion in Cell Biology, 8, 74-85. http://dx.doi.org/10.1016/S0955-0674(96)80051-2 [26] Henegouwen, P.M.V.B.E., den Hartigh, J.C., Romeyn, P., Verkleij, A.J. and Boonstra, J. (1992) The Epidermal Growth Factor Receptor Is Associated with Actin Filaments. Experimental Cell Research, 199, 90-97. http://dx.doi.org/10.1016/0014-4827(92)90465-K [27] den Hartigh, J.C., Henegouwen, P.M.V.B.E., Verkleij, A.J. and Boonstra, J. (1992) The EGF Receptor Is an Actin-Binding Protein. The Journal of Cell Biology, 2, 349-355.
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[1] Hossain, M., Crook, T.J. and Keoghane, S.R. (2008) Clostridium difficile in Urology. Annals of the Royal College of Surgeons of England, 90, 36-39. http://dx.doi.org/10.1308/003588408X242358 [2] Tsutsumi, L.S., Owusu, Y.B., Hurdlre, J.G. and Sun, D. (2014) Progress in the Discovery of Treatment for C. difficile infection. A Clinical and Medicinal Chemistry Review. Current Topics in Medicinal Chemistry, 14, 152-175. http://dx.doi.org/10.2174/1568026613666131113154753 [3] Lemeni, D. (2010) Nosocomial Clostridium Difficile Diarrhea—Adverse Effect of Antibiotic Therapy. Bacteriol Virusol Parazitol Epidemiol, 55, 141-144. [4] Crogan, N.L. and Evans, B.C. (2007) Clostridium difficile: An Emerging Epidemic in Nursing Homes. Geriatric Nursing, 28, 161-164. http://dx.doi.org/10.1016/j.gerinurse.2007.04.005 [5] Vaishnavi, C. (2009) Established and Potential Risk Factors for Clostridium difficile Infection. Indian Journal of Medical Microbiology, 27, 291-302. [6] Ulliuwishewa, D., Anderson, R.C., McNabb, W.C., Moughan, P.J., Wells, J.M. and Roy, N.C. (2011) Regulation of Tight Junction Permeability by Intestinal Bacteria and Dietary Components. Journal of Nutrition, 41, 769-776. http://dx.doi.org/10.3945/jn.110.135657 [7] Aktories, K. (1997) Bacterial Toxins that Target Rho Proteins. Journal of Clinical Investigation, 99, 827-829. http://dx.doi.org/10.1172/JCI119245 [8] Riegler, M., Sedivy, R., Sogukoglu, T., Castagliuolo, I., Pothoulakis, C., Cosentini, E., Bischof, G., Hamilton, G., Teleky, B., Feil, W., et al. (1997) Epidermal Growth Factor Attenuates Clostridium difficile Toxin A- and B-Induced Damage of Human Colonic Mucosa. American Journal of Physiology, 273, G1014-G1022. [9] Kaur, S., Vaishnavi, C., Kochhar, R., Prasad, K.K. and Ray, P. (2012) Effect of Biotherapeutics on Antitoxin IgG in Experimentally Induced Clostridium difficile Infection. Indian Journal of Medical Microbiology, 30, 31-36. [10] Buret A., Oslon, M.E., Gall, D.J. and Hardin, J.A. (1998) Effects of Orally Administered Epidermal Growth Factor on Enteropathogenic Escherichia coli Infection in Rabbits. Infection and Immunity, 66, 4917-4923. [11] Nusrat, A., Eichel-Streiber, C., Turner, J.R., Verkade, P., Madara, J.L. and Parkos, C.A. (2001) Clostridium difficile toxins Disrupt Epithelial Barrier Function by Altering Membrane Microdomain Localization of Tight Junction Proteins. Infection and Immunity, 69, 1329-1336.
http://dx.doi.org/10.1128/IAI.69.3.1329-1336.2001 [12] Resta-Lenert, R. and Barret, K.E. (2003) Live Probiotics Protect Intestinal Epithelial Cells from the Effects of Infection with Enteroinvasive Escherichia coli (EIEC). Gut, 52, 988-997.
http://dx.doi.org/10.1136/gut.52.7.988 [13] Towbin, J., Staehelin, T. and Gordon, J. (1979) Electrophoresis Transfer of Protein from Polyacrylamide Gels to Nitrocellulose Sheets: Procedure and Some Applications. Proceedings of the National Academy of Sciences, 76, 4350-4354. http://dx.doi.org/10.1073/pnas.76.9.4350 [14] Thalestam, M. and Chaves-Olarte, E. (2000) Cytotoxic Effects of the Clostridium difficile Toxins. Current Topics in Microbiology and Immunology, 250, 85-96. http://dx.doi.org/10.1007/978-3-662-06272-2_4 [15] Yang, K.M., Jiang, Z.Y., Zheng, C.T., Wang, L. and Yang, X.F. (2014) Effect of Lactobacillus plantarum on Diarrhea and Intestinal Barrier Function of Young Piglets Challenged with Enterotoxigenic Escherichia coli K88. Journal of Animal Science, 92, 1496-1503. http://dx.doi.org/10.2527/jas.2013-6619 [16] Miura, M., Kato, H. and Matsushita, O. (2011) Identification of a Novel Virulence Factor in Clostridium difficile That Modulates Toxin Sensitivity of Cultured Epithelial Cells. Infection and Immunity, 79, 3810-3820. http://dx.doi.org/10.1128/IAI.00051-11 [17] Castagluiolo, I., Riegler, M.F., Valenick, L., LaMont, J.T. and Pothoulakis, C. (1999) Saccharomyces boulardii Protease Inhibits the Effects of Clostridium difficile Toxins A and B in Human Colonic Mucosa. Infection and Immunity, 67, 302-307. [18] Mack, D.R., Ahrne, S., Hyde, L., Wei, S. and Hollingsworth, M.A. (2003) Extracellular MUC3 Mucin Secretion Follows Adherence of Lactobacillus Strains to Intestinal Epithelial Cells in Vitro. Gut, 52, 827-833. [19] García-Lafuente, A., Antolin, M., Guarner, F., Crespo, E. and Malagelada, J.R. (2001) Modulation of Colonic Barrier Function by the Composition of the Commensal Flora in the Rat. Gut, 48, 503-507. http://dx.doi.org/10.1136/gut.48.4.503 [20] Hanley, S.C., Assouline-Thomas, B., Makhlin, J. and Rosenberg, L. (2011) Epidermal Growth Factor Induces Adult Human Islet Cell Dedifferentiation. Journal of Endocrinology, 211, 231-239. http://dx.doi.org/10.1530/JOE-11-0213 [21] Gospodarowicz, D. (1981) Epidermal and Nerve Growth Factor in Mammalian Development. Annual Review of Physiology, 43, 251-263. http://dx.doi.org/10.1146/annurev.ph.43.030181.001343 [22] Procaccino, F., Reinshagen, M., Hoffmann, P., Zeeh, J.M., Lakshmanan, J., McRoberts, J.A., Patel, A., French, S. and Eysselein, V.E. (1994) Protective Effect of Epidermal Growth Factor in an Experimental Model of Colitis in Rats. Gastroenterology, 107, 12-17. [23] Banan, A., Zhang, Y., Losurdo, J. and Keshavarzian, A. (2000) Carbonylation and Disassembly of the F-Actin Cyto-skeleton in Oxidant Induced Barrier Dysfunction and Its Prevention by Epidermal Growth Factor and Transforming Growth Factor α in a Human Colonic Cell Line. Gut, 46, 830-837. http://dx.doi.org/10.1136/gut.46.6.830 [24] Nobes, C.D., Hawkins, P., Stephens, L. and Hall, A. (1995) Activation of the Small GTP-Binding Proteins rho and rac by Growth Factor Receptors. Journal of Cell Science, 108, 225-233. [25] Craig, W.S. and Johnson, R.P. (1996) Assembly of Focal Adhesions: Progress, Paradigms, and Portents. Current Opinion in Cell Biology, 8, 74-85. http://dx.doi.org/10.1016/S0955-0674(96)80051-2 [26] Henegouwen, P.M.V.B.E., den Hartigh, J.C., Romeyn, P., Verkleij, A.J. and Boonstra, J. (1992) The Epidermal Growth Factor Receptor Is Associated with Actin Filaments. Experimental Cell Research, 199, 90-97. http://dx.doi.org/10.1016/0014-4827(92)90465-K [27] den Hartigh, J.C., Henegouwen, P.M.V.B.E., Verkleij, A.J. and Boonstra, J. (1992) The EGF Receptor Is an Actin-Binding Protein. The Journal of Cell Biology, 2, 349-355.
http://dx.doi.org/10.1083/jcb.119.2.349 [28] Hotchin, N.A. and Hall, A. (1995) The Assembly of Integrin Adhesion Complexes Require Both Extracellular Matrix and Intracellular rho/rac GTPases. The Journal of Cell Biology, 131, 1857-1865. http://dx.doi.org/10.1083/jcb.131.6.1857 [29] Parsons, J.T. (1996) Integrin-Mediated Signaling: Regulation by Tyrosine Kinases and Small GTP-Binding Proteins. Current Opinion in Cell Biology, 8, 148-152. http://dx.doi.org/10.1016/S0955-0674(96)80059-7