Concerted Action of Sphingomyelinase and Non-Hemolytic Enterotoxin in Pathogenic Bacillus cereus

Viktoria M Doll,Monika Ehling-Schulz,Roger Vogelmann,Nancy E Freitag

doi:10.1371/journal.pone.0061404

Abstract

Bacillus cereus causes food poisoning and serious non-gastrointestinal-tract infections. Non-hemolytic enterotoxin (Nhe), which is present in most B. cereus strains, is considered to be one of the main virulence factors. However, a B. cereus ΔnheBC mutant strain lacking Nhe is still cytotoxic to intestinal epithelial cells. In a screen for additional cytotoxic factors using an in vitro model for polarized colon epithelial cells we identified B. cereus sphingomyelinase (SMase) as a strong inducer of epithelial cell death. Using single and double deletion mutants of sph, the gene encoding for SMase, and nheBC in B. cereus we demonstrated that SMase is an important factor for B. cereus cytotoxicity in vitro and pathogenicity in vivo. SMase substantially complemented Nhe induced cytotoxicity in vitro. In addition, SMase but not Nhe contributed significantly to the mortality rate of larvae in vivo in the insect model Galleria mellonella. Our study suggests that the role of B. cereus SMase as a secreted virulence factor for in vivo pathogenesis has been underestimated and that Nhe and SMase complement each other significantly to cause full B. cereus virulence hence disease formation.

Highlights

Bacillus cereus is a spore forming Gram-positive bacterium that is found in natural habitats like soil and plants
Even two B. cereus strains isolated from two different probiotic formulas used in humans and piglet feeding [40,41,42,43], B. cereus var. toyoi and IP5832, revealed similar cytotoxicity compared to B. cereus NVH 0391-98 isolated from a food borne outbreak associated with high patient mortality [1] or the nhe reference strain B. cereus NVH 0075-95 (Fig. 1)
Cellular cytotoxicity was mediated by a secreted B. cereus factor as rapid rounding and detachment in intestinal epithelial cell (IEC) was induced by bacterial supernatants alone. (Fig. S1)

Summary

Introduction

Bacillus cereus is a spore forming Gram-positive bacterium that is found in natural habitats like soil and plants. Their spores can enter the food processing chain due to heat and dryness resistance. B. cereus is commonly accepted as a food borne pathogen causing mostly mild food borne gastroenteritis. Fatal outbreaks of B. cereus food poisoning [1,2] and local and systemic non-gastrointestinal-tract infections (e.g. endophthalmitis, pneumonia, sepsis) in humans have been reported [3]. Ingestion of the emetic toxin cereulide, a cyclic dodecadepsipeptide, causes nausea and vomiting, whereas the diarrheal syndrome has been associated mainly with the enterotoxins cytotoxin K (CytK), hemolysin BL (Hbl) and non-hemolytic enterotoxin (Nhe) [4,5]

Methods

Results

Conclusion