Ratio of “A-type” to “B-type” Proanthocyanidin Interflavan Bonds Affects Extra-intestinal Pathogenic Escherichia coli Invasion of Gut Epithelial Cells Rodrigo P. Feliciano, †,‡ Jennifer J. Meudt, ‡ Dhanansayan Shanmuganayagam, ‡ Christian G. Krueger, †,§ and Jess D. Reed* ,†,§ † Department of Food Science, University of WisconsinMadison, 1605 Linden Drive, Madison, Wisconsin 53706, United States ‡ Reed Research Group, Department of Animal Sciences, University of WisconsinMadison, 1675 Observatory Drive, Madison, Wisconsin 53706, United States § Complete Phytochemical Solutions LLC, 317 South Street, Cambridge, Wisconsin 53523, United States ABSTRACT: Gut colonization by extra-intestinal pathogenic Escherichia coli (ExPEC) increases the risk of subsequent infections, including urinary tract infection and septicemia. Previous work suggests that cranberry proanthocyanidins (PAC) interact with bacterial surface factors, altering bacterial interaction with host cells. Methods were developed to determine if ratios of “A-type” to “B-type” interflavan bonds in PAC affect ExPEC agglutination and invasion of enterocytes. In cranberries, 94.5% of PAC contain one or more “A-type” bonds, whereas in apples, 88.3% of PAC contain exclusively “B-type” bonds. Results show that cranberry “A-type” PAC have greater bioactivity than apple “B-type” PAC for increasing ExPEC agglutination and decreasing ExPEC epithelial cell invasion. KEYWORDS: cranberry, apple, proanthocyanidins, “A-type” bonds, “B-type” bonds, ExPEC, agglutination, invasion, Caco-2 ■ INTRODUCTION Extra-intestinal pathogenic Escherichia coli (ExPEC) cause many extra-intestinal infections, including urinary tract infections (UTI), bacteremia, septicemia, and neonatal meningitis. ExPEC have a large economic impact on public health, with a conservatively estimated cost of several billion dollars annually in the United States. 1 ExPEC may be part of the gut microbiota in healthy individuals. 2 However, once they get access to niches outside the gut, they are able to efficiently colonize these niches and cause diseases such as UTI. 2,3 Previous research indicates that proanthocyanidins (PAC) from cranberries interact with ExPEC surface virulence factors such as P fimbriae that inhibit adhesion and invasion. In vitro studies demonstrate that cranberry PAC, 4 particularly those with higher degree of polymerization (DP), inhibit the adherence of the pathogen to uroepithelial cells. 5-7 Ironically, PAC, especially those with DP >2, are not absorbed into circulation and do not reach the urinary tract. 8 Although this paradox is readily acknowledged, in vitro mechanistic studies continue to explore the effects of PAC on the interaction between pathogens and uroepithelial and kidney cells. 9 The impact of “A-type” PAC on host cell morphology was described using HeLa cells, a cervical cancer cell line, 10 which are not representative of the polarized cells that line the intestines. On the other hand, Caco-2 cells, which were used in the research reported in this paper, form polarized apical and basolateral surfaces that mimic enterocytes. ExPEC virulence factors that are involved in the colonization of uroepithelial cells are also important in the ability of ExPEC to invade enterocytes. 11 Because the gut is the likely origin of E. coli that cause UTI 12,13 and PAC do not appear in the urine at sufficiently high concentrations to inhibit E. coli adhesion and invasion of uroepithelial cells, we believe a new paradigm is needed to advance the mechanistic and structure-function understanding of how “A-type” PAC affect UTI in vivo by inhibition of transient gut colonization. As part of this paradigm shift, we report on the development of two methods to determine the bioactivity of PAC that are relevant to inhibition of transient gut colonization: ExPEC agglutination and enterocyte cell invasion. We also use a newly developed method for structural elucidation to determine how relative ratios of “A-type” to “B- type” interflavan bonds affect how PAC agglutinate ExPEC and inhibit ExPEC invasion of gut epithelial cells. ■ MATERIALS AND METHODS Chemicals and Reagents. Water, methanol, acetone (HPLC grade), sodium carbonate, sodium chloride, Luria broth (LB) Miller, agar, Dulbecco’s modified Eagle’s medium (DMEM), penicillin/ streptomycin (Pen/Strep) mixture (10000 units of each antibiotic per mL), gentamicin sulfate (50 mg/mL), nonessential amino acid (NEAA) solution (100×), GlutaGRO supplement (200 mM L- alanyl-L-glutamine), and Dulbecco’s phosphate-buffered saline solution 10× (PBS) with calcium and magnesium (PBS + Ca 2+ /Mg 2+ - 0.1 g/L of CaCl 2 and MgCl 2 ) were purchased from Fisher Scientific (Fair Lawn, NJ, USA). Ethanol (200 proof) was obtained from Decon Laboratories Inc. (King of Prussia, PA, USA). Sterilized water, thyamin hydrochloride, 2,5-dihydroxybenzoic acid (DHB), gallic acid, Folin- Ciocalteu reagent, and Triton 100X were purchased from Sigma- Special Issue: 2013 Berry Health Benefits Symposium Received: September 4, 2013 Revised: November 8, 2013 Accepted: November 12, 2013 Article pubs.acs.org/JAFC © XXXX American Chemical Society A dx.doi.org/10.1021/jf403839a | J. Agric. Food Chem. XXXX, XXX, XXX-XXX