Downloaded from www.microbiologyresearch.org by IP: 54.145.26.59 On: Sat, 02 Apr 2016 07:34:52 Bacillus cereus Nhe is a pore-forming toxin with structural and functional properties similar to the ClyA (HlyE, SheA) family of haemolysins, able to induce osmotic lysis in epithelia Annette Fagerlund, Toril Lindba ¨ ck, Anne K. Storset, Per Einar Granum and Simon P. Hardy Correspondence Simon P. Hardy simon.hardy@veths.no Department of Food Safety and Infection Biology, Norwegian School of Veterinary Science, PO Box 8146 Dep., N-0033 Oslo, Norway Received 27 October 2007 Revised 13 December 2007 Accepted 13 December 2007 The mechanism by which Bacillus cereus causes diarrhoea is unknown. Three putative enterotoxins have been proposed, haemolysin BL (Hbl), cytotoxin K and non-haemolytic enterotoxin (Nhe). Both Hbl and Nhe are three-component cytotoxins and maximal cytotoxicity of Nhe against epithelia is dependent on all three components. However, little is known of the mechanism of cytotoxicity. Markers of plasma membrane disruption, namely propidium iodide uptake, loss of cellular ATP and release of lactate dehydrogenase (LDH), were observed in epithelia exposed to Nhe from culture supernatants of B. cereus, but not in those exposed to supernatants from a mutant strain lacking NheB and NheC. Consistent with an exogenous cause of membrane damage, purified Nhe components combined to form large conductance pores in planar lipid bilayers. The inhibition of LDH release by osmotic protectants and the increase in cell size caused by Nhe indicate that epithelia lyse following osmotic swelling. Nhe and Hbl show sequence homology, and Hbl component B has remarkable structural similarities to cytolysin A (ClyA), with both structures possessing an a-helix bundle and a unique subdomain containing a hydrophobic b-hairpin. Correspondingly, we show that Nhe has haemolytic activity against erythrocytes from a variety of species. We propose that the common structural and functional properties indicate that the Hbl/Nhe and ClyA families of toxins constitute a superfamily of pore-forming cytotoxins. INTRODUCTION Bacillus cereus is a worldwide cause of food-associated illness in man. Although B. cereus was shown to cause enteritis in humans over 50 years ago (Hauge, 1955), the cellular mechanism of diarrhoea and identity of the responsible enterotoxin(s) remain unknown. Three puta- tive enterotoxins of B. cereus have been proposed: haemolysin BL (Hbl), non-haemolytic enterotoxin (Nhe) and cytotoxin K (CytK), all of which are cytotoxic to epithelia in vitro. CytK is a haemolytic pore-forming toxin with homology to the b-barrel pore-forming toxins, including staphylococcal a-haemolysin (Hardy et al., 2001a; Lund et al., 2000). Hbl and Nhe are tripartite toxins, in which all three components are necessary for maximal cytotoxic activity (Beecher & Macmillan, 1991; Lindba ¨ck et al., 2004). Both are encoded by three genes co- transcribed as operons in which hblCDA encodes Hbl components L 2 ,L 1 and B, and nheABC encodes NheA, NheB and NheC. Sequence homology is apparent both between the three components in each complex and between the proteins of Nhe and Hbl (Granum et al., 1999; Ryan et al., 1997; Table 1). With no sequence homology to other toxins, Nhe and Hbl have remained unclassified as toxins. Hbl has been shown to possess a variety of biological effects, including haemolytic, dermonecrotic and vascular permeability activities and, significantly, possesses enter- otoxigenic ability, as indicated by fluid accumulation in rabbit ligated ileal loops (reviewed by Schoeni & Wong, 2005). Nhe was identified in B. cereus following a large food-poisoning outbreak in Norway (Lund & Granum, 1996). The recovered isolate, NVH 0075/95, lacked both hbl and cytK (Ehling-Schulz et al., 2005) but was still cytotoxic, thus permitting the discovery of Nhe. The initial study indicated that the toxin was non-haemolytic. Although the toxin profile may differ between strains, it appears that Nhe is a dominant cytotoxic component of B. cereus. In a study of over 100 B. cereus strains, cytotoxicity correlated well with the amount of Nhe produced but Abbreviations: EC buffer, extracellular bathing solution; LDH, lactate dehydrogenase; PI, propidium iodide. Microbiology (2008), 154, 693–704 DOI 10.1099/mic.0.2007/014134-0 2007/014134 G 2008 SGM Printed in Great Britain 693