Binding of flavonoids to staphylococcal enterotoxin B Evgen Benedik a , Mihaela Skrt a ,C ˇ rtomir Podlipnik b , Nataša Poklar Ulrih a,c, * a Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia b Faculty of Chemistry and Chemical Technology, University of Ljubljana, Aškerc ˇeva 5, 1000 Ljubljana, Slovenia c Centre of Excellence for Integrated Approaches in Chemistry and Biology of Proteins (CipKeBiP), Jamova 39, Ljubljana, Slovenia ARTICLE INFO Article history: Received 17 April 2014 Accepted 21 August 2014 Available online 2 September 2014 Keywords: Flavones Catechins Molecular docking Fluorescence emission spectrometry Staphylococcal enterotoxin B A B ST R AC T Staphylococcal enterotoxins are metabolic products of Staphylococcus aureus that are responsible for the second-most-commonly reported type of food poisoning. Polyphenols are known to interact with pro- teins to form complexes, the properties of which depend on the structures of both the polyphenols and the protein. In the present study, we investigated the binding of four flavonoid polyphenols to Staphy- lococcal enterotoxin B (SEB) at pH 7.5 and 25 °C: (-)-epigallocatechin-3-gallate (EGCG), (-)-epigallocatechin (EGC), kaempferol-3-glucoside (KAM-G) and kaempferol (KAM). Fluorescence emission spectrometry and molecular docking were applied to compare experimentally determined binding parameters with mo- lecular modeling. EGCG showed an order of magnitude higher binding constant (1.4 × 10 5 M -1 ) than the other studied polyphenols. Our blind-docking results showed that EGCG and similar polyphenolic ligands is likely to bind to the channel at the surface of SEB that is responsible for the recognition of the T-cell beta chain fragment and influence the adhesion of SEB to T cells. © 2014 Published by Elsevier Ltd. 1. Introduction Intestinal infectious diseases are major contributors to morbid- ity and mortality in tropical countries, and they were estimated to have caused 1.5 million deaths in children in developing countries in 2002 (WHO, 2003). Staphylococcus enterotoxin B (SEB) is one of exotoxins produced by the ubiquitous Gram-positive facultative an- aerobe Staphylococcus aureus which colonizes humans as well as domestic animals, and it is a common opportunistic pathogen. It is estimated that S. aureus persists in 20% of the general popula- tion, while another 60% are intermittent carriers (Kluytmans et al., 1997). The frequency of infections is higher in carriers than in non- carriers (von Eiff et al., 2001). Non-carriers commonly acquire infections through contaminated water or food, such as when food handlers who are carriers contaminate food during its preparation. In humans, the estimated 50% lethal dose (LD50) of SEB is 0.02 μg/ kg and the 50% effective dose (ED50) is 0.0004 μg/kg by aerosol exposure (Gill, 1982; Rusnak et al., 2004). There are no other data on LD50 and ED50 in humans by other routes of exposure. Symp- toms of poisoning with SEB include high fever, nausea, vomiting, abdominal pain, cramps and hypotension, with or without diar- rhea, toxic-shock syndrome, septicemia, and sometimes death. The disease is usually self-resolving; it is rarely lethal, and the elderly are more susceptible (Pinchuk et al., 2010). Some studies have also considered enterotoxins to be a potential biological weapon (Mantis, 2005). There are 23 different enterotoxin types known, and they can be divided into five phylogenetic groups (Argudín et al., 2010; Vasconcelos and de Lourdes Ribeiro de Souza da Cunha, 2010). However, only a few of the enterotoxins have been studied in depth. The most common two of these are Staphylococcus enterotoxin A and SEB. Staphylococcal enterotoxins are similar to the gastroin- testinal toxins, and they are responsible for the second-most- commonly reported type of food poisoning through contaminated water and improperly prepared and stored food, with effects that can appear from a few minutes to 2 hours after ingestion (Argudín et al., 2010). These Staphylococcus enterotoxins have the remark- able ability to resist heat and acid. Therefore, they cannot be completely denaturated by mild cooking of contaminated food. They are pyrogenic and are resistant to inactivation by gastrointestinal proteases, including pepsin, trypsin, rennin and papain (Le Loir et al., 2003). SEB mediates immune activation on intestinal defensins (Dhaliwal et al., 2009). As well as being able to grow over a range of temperatures and pH, S. aureus can grow on a wide assortment of food. Therefore, if food that is contaminated with Staphylococcus- enterotoxin-producing strains is left at temperatures that allow rapid growth of these bacteria (i.e., inadequate refrigeration), they become a common source of food poisoning (Pinchuk et al., 2010). Natural products from plants might be useful, particularly in de- veloping countries where the availability of drugs is limited (Joung * Corresponding author. University of Ljubljana, Biotechnical Faculty, Jamnikarjeva 101, 1000 Ljubljana, Slovenia. Tel.: +386 1 3230780; fax: +386 1 2566296. E-mail address: natasa.poklar@bf.uni-lj.si (N.P. Ulrih). http://dx.doi.org/10.1016/j.fct.2014.08.012 0278-6915/© 2014 Published by Elsevier Ltd. Food and Chemical Toxicology 74 (2014) 1–8 Contents lists available at ScienceDirect Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox