Pathogenesis and toxins Surface proteins from Lactobacillus kefir antagonize in vitro cytotoxic effect of Clostridium difficile toxins Paula Carasi a , Fernando M. Trejo b , Pablo F. Pérez a, b , Graciela L. De Antoni b , María de los Angeles Serradell a, * a Laboratorio de Microbiología, Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata (UNLP), 47 y 115, La Plata (1900), Argentina b Centro de Investigación y Desarrollo en Criotecnología de Alimentos (CIDCA), UNLP-CONICET, 47 y 116, La Plata (1900), Argentina article info Article history: Received 29 July 2011 Received in revised form 24 October 2011 Accepted 14 November 2011 Available online 22 November 2011 Keywords: S-layer protein Lactobacillus kefir Clostridium difficile Bacterial toxins abstract In this work, the ability of S-layer proteins from kefir-isolated Lactobacillus kefir strains to antagonize the cytophatic effects of toxins from Clostridium difficile (TcdA and TcdB) on eukaryotic cells in vitro was tested by cell detachment assay. S-layer proteins from eight different L. kefir strains were able to inhibit the damage induced by C. difficile spent culture supernatant to Vero cells. Besides, same protective effect was observed by F-actin network staining. S-layer proteins from aggregating L. kefir strains (CIDCA 83115, 8321, 8345 and 8348) showed a higher inhibitory ability than those belonging to non-aggregating ones (CIDCA 83111, 83113, JCM 5818 and ATCC 8007), suggesting that differences in the structure could be related to the ability to antagonize the effect of clostridial toxins. Similar results were obtained using purified TcdA and TcdB. Protective effect was not affected by proteases inhibitors or heat treatment, thus indicating that proteolytic activity is not involved. Only preincubation with specific anti-S-layer anti- bodies significantly reduced the inhibitory effect of S-layer proteins, suggesting that this could be attributed to a direct interaction between clostridial toxins and L. kefir S-layer protein. Interestingly, the interaction of toxins with S-layer carrying bacteria was observed by dot blot and fluorescence microscopy with specific anti-TcdA or anti-TcdB antibodies, although L. kefir cells did not show protective effects. We hypothesize that the interaction between clostridial toxins and soluble S-layer molecules is different from the interaction with S-layer on the surface of the bacteria thus leading a different ability to antagonize cytotoxic effect. This is the first report showing the ability of S-layer proteins from kefir lactobacilli to antagonize biological effects of bacterial toxins. These results encourage further research on the role of bacterial surface molecules to the probiotic properties of L. kefir and could contribute to strain selection with potential therapeutic or prophylactic benefits towards CDAD. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction Clostridium difficile is a Gram-positive spore forming bacterium that is responsible for 90e100% of cases of pseudomembranous colitis (PMC), 60e75% of antibiotic-associated colitis and 30e60% of antibiotic-associated diarrhoea (AAD) [1]. Virulence of C. difficile is mainly related to the production of two large protein toxins: TcdA (308 kDa) and TcdB (260 kDa). These toxins act as glycosyl- transferases, modifying host cell small GTPases involved in actin polymerization and cytoskeleton assembly [2,3]. Even though both toxins have the same enzymatic activity, TcdA is mainly an enterotoxin whereas TcdB has citotoxic activity and exerts its biological effect at lower doses than TcdA [4] (Castagliuolo et al., 1998). There is a great interest in using probiotics for the prophylaxis and treatment of different gastrointestinal disorders since it has been demonstrated that they inhibit the growth or invasion of pathogenic bacteria, enhance the intestinal barrier, and modulate the immune system through the regulation of cytokines production [5,6]. Evidence suggests that these agents could constitute an alternative approach to deal with the C. difficile-associated diar- rhoea (CDAD) [7]. In particular, there is evidence that Saccharo- myces boulardii interferes with virulence of C. difficile through neutralization or degradation of toxins [8] and inhibition of inflammatory immune response [9]. In addition, it has been Abbreviations: AAD, Antibiotic-associated diarrhoea; CDAD, Clostridium difficile- associated diarrhoea; SCS, Spent culture supernatant; SCS-Lk, Spent culture supernatant pre-incubated with Lactobacillus kefir cells; SCS-SL, Spent culture supernatant pre-incubated with S-layer proteins from L. kefir. * Corresponding author. Tel.: þ54 221 4250497; fax: þ554 221 4890741. E-mail address: maserr@biol.unlp.edu.ar (M.A. Serradell). Contents lists available at SciVerse ScienceDirect Anaerobe journal homepage: www.elsevier.com/locate/anaerobe 1075-9964/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.anaerobe.2011.11.002 Anaerobe 18 (2012) 135e142