Downloaded from www.microbiologyresearch.org by IP: 54.159.222.200 On: Sat, 30 Apr 2016 01:27:04 Enhancement of the cytotoxic activity of Clostridium difficile toxin A by surface-associated antigens Karla Sa ´ nchez-Hurtado and Ian R. Poxton Correspondence Ian R Poxton i.r.poxton@ed.ac.uk Centre for Infectious Diseases, University of Edinburgh College of Medicine and Veterinary Medicine, The Chancellor’s Building, 49 Little France Crescent, Edinburgh EH16 4SB, UK Received 4 October 2007 Accepted 25 January 2008 Cell-surface antigens of Clostridium difficile and LPS from Escherichia coli were investigated for modulating effects on the activity of C. difficile toxin A on Vero and Caco2 cells. The antigens of C. difficile tested comprised: (i) an EDTA extract, which contained several major and minor cell-surface proteins and the membrane-associated lipocarbohydrate (LC); (ii) a guanidine hydrochloride extract, which mainly contained the surface-layer proteins; (iii) an aqueous phenol-extracted, protein-free LC. On their own, none of the antigens had a detrimental effect on the cells, with the EDTA extract and LC having a marginally protective effect. When these antigens were added to suboptimal levels of toxin A, there was significant enhancement of its cytotoxicity by the EDTA and LC preparations on both cell types. LPS showed some enhancement of the effect of toxin on Vero cells at the lowest levels of toxin investigated. It was concluded that this effect seen in vitro may have a role to play in the colon during infection with C. difficile. INTRODUCTION The major virulence factors of most strains of pathogenic Clostridium difficile are accepted to be the two large clostridial toxins: toxin A (TcdA, an enterotoxin) and toxin B (TcdB, a cytotoxin), although certain other cell- associated molecules may have a secondary role in virulence, such as promoting adhesion or evading the immune response (Poxton et al., 2001). Although there is a suggestion that the amount of toxins produced is related to virulence – mainly through the observations that the hypervirulent ribotype 027 strains produce more toxin than other types (Warny et al., 2005) – there is not a strict correlation between the toxigenicity of the strain (the amount of toxin produced in vitro) and the degree of disease (Borriello et al., 1987). The absence of both toxins leads to avirulence of the strain. Strains belonging to the A 2 B + phenotype, which only produce toxin B, are as virulent as those producing both toxins (Drudy et al., 2007). The ADP-ribosylating binary toxin CDT (cytolethal distending toxin), found in a minority of strains of C. difficile, can exert an effect on the cytoskeleton, but has no proven role in pathogenesis (Geric et al., 2006). Other virulence factors, in particular putative adhesins, have been considered and, although the role of these factors is not fully understood, it is believed that they may contribute to pathogenesis (Pe ´chine ´ et al., 2007). However, as far as we are aware, such components, either those belonging to C. difficile cells themselves or other bacterial components found in the colonic lumen, have not been considered as agents capable of modulating the action of toxins. The aim of this study was to test the hypothesis that the cytotoxic effect of the C. difficile toxins can be enhanced by the presence of antigens associated with the cell surface of C. difficile and by LPS, a molecule likely to be found at high concentrations in the gastrointestinal tract. Two different cell lines were selected to test the activity of purified toxin A: the commonly used toxin-sensitive Vero cells and differentiating Caco2 cells. METHODS Production of crude toxin by dialysis culture. An overnight culture (1 ml) of a toxigenic strain of C. difficile (strain MPRL338a) was used to inoculate a sterilized, pre-reduced dialysis bag, containing before sterilization approximately 50 ml 0.85% NaCl, suspended in 500 ml proteose peptone yeast extract (PPY) medium (Deacon et al., 1978), in a stoppered, vented 1 l conical flask. After 4 days of incubation at 37 uC in an anaerobic atmosphere (10 % H 2 , 10 % CO 2 , 80 % N 2 ) in a DWS Mark III workstation (Don Whitley Scientific), the contents of the dialysis bag were harvested (10 000 g, 15 min) and the supernatant was dialysed overnight against Tris-buffered saline [TBS: 0.05 M Tris/HCl (pH 7.0), 0.15 M NaCl] at 4 uC. This supernatant, which contained the extracellular products but none of the culture medium components, was filter-sterilized and stored at 4 uC. Purification of toxin A. This purification was based on methods developed by Krivan & Wilkins (1987) and Kamiya et al. (1989), Abbreviations: GHCl, guanidine hydrochloride; LC, lipocarbohydrate; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; SLP, surface-layer protein. Journal of Medical Microbiology (2008), 57, 739–744 DOI 10.1099/jmm.0.47678-0 47678 G 2008 SGM Printed in Great Britain 739