CRANBERRY PROANTHOCYANIDINS AS INHIBITORS OF EPITHELIAL CELL INVASION BY ENTHEROPATHOGENIC AND UROPATHOGENIC E. COLI STRAINS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0223796
• Project No.: WIS01519
• Project Start Date: Oct 1, 2010
• Project End Date: Sep 30, 2014

Project Director
Reed, J.

Recipient Organization
UNIV OF WISCONSIN
21 N PARK ST STE 6401
MADISON,WI 53715-1218

Performing Department
Animal Sciences

Non Technical Summary
It is estimated seven million American suffer from urinary tract infections (UTI) each year and the effectiveness of cranberry in easing, or even preventing this disease has to a large extent driven market success. The cranberry industry is marketing products based on health claims such as beneficial to urinary tract health and antioxidant content. Urinary tract infections are caused by E. coli ascending through the urethra, attaching to the walls of the bladder, multiplying and colonizing the epithelium. The microbiologic etiology of UTI usually originates from bowel flora of the host. While virtually every organism has been associated with UTI, the most common cause is E. coli, which accounts for almost 85% of community-acquired infections. Cranberry products taken on a regular basis were clinically proven to prevent UTI and may serve as an alternative to recurrent use of antibiotics. It has been proposed that cranberry products act by inhibiting uropathogenic strains of E. coli from adhering to uroepithelial cells, which is the initial step in development of infection. Cranberry powder and isolated PAC inhibited adhesion of E. coli isolates to primary cultured bladder epithelial cells and vaginal epithelial cells in a linear dose dependent relationship. Cranberry PAC also demonstrated the ability to prevent E. coli from attaching to uroepithelial cells. Naturally occurring chemicals that inhibit microbial adhesion are important alternatives to antibiotics and may help stem the alarming increase in drug resistant bacteria. Laboratory tests can be used to characterize structural changes in cranberry proanthocyanidins (PAC) that result from juice processing. The goals of this proposal are to: 1) Gain a better understanding of how structure of cranberry PAC affects binding to E. coli and inhibition of E. coli invasion of epithelial cells, and 2) How processing of cranberries may be altered to improve PAC inhibition of E. coli. Results of these studies may lead to new fruit processing practices that enrich products in PAC of specific composition and development of value added products from cranberry presscake, the by-product of juice processing.

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
501	1121	1010	10%
502	1121	1010	10%
701	1121	1010	80%

Knowledge Area
501 - New and Improved Food Processing Technologies; 502 - New and Improved Food Products; 701 - Nutrient Composition of Food;

Subject Of Investigation
1121 - Cranberry;

Field Of Science
1010 - Nutrition and metabolism;

Keywords

cranberry

proanthocyanadins

tannins

urinary tract infections

e. coli

Goals / Objectives
The goals of this proposal are to: 1) Gain a better understanding of how structure of cranberry PAC affects binding to E. coli and inhibition of E. coli invasion of epithelial cells, and 2) How processing of cranberries may be altered to improve PAC inhibition of E. coli. Results of these studies may lead to new fruit processing practices that enrich products in PAC of specific composition and development of value added products from cranberry presscake, the by-product of juice processing. As a result of this sponsored research project, companies may adopt new juice processing strategies to enrich the bioactive components (proanthocyanidins) in their beverages. The by-product of the juicing process (press cake) may be re-evaluated as a potential commercial source of proanthocyanidins. Our partnership with the Wisconsin Cranberry Board and the Cranberry Institute will provide an extension mechanism in which results will be shared directly with cranberry growers and handlers. In turn, growers may adopt new varieties and harvest strategies to meet the future needs of the juicing companies. The long term outcome of this research may lead to reduced obesity rates and improved nutrition and health.

Project Methods
We propose to apply LC separation and MALDI-TOF MS characterization methods to produce fractions of cranberry PAC with lower degree of structural heterogeneity for research on agglutination of pathogenic E. coli and inhibition of E. coli invasion of Caco-2 intestinal epithelial cells. Proanthocyanidin fractions will be isolated from cranberry fruit, juice and presscake by chromatography, Sephadex LH-20 and HPLC. Structural features of PAC (interflavan linkage, anthocyanin moieties and degree of polymerization) as determined by MALDI-TOF MS will be related to E. coli structures (O antigens on extracellular LPS, proteins on the outer membrane or adhesins) that influence PAC agglutination. The effect of cranberry PAC structure on E. coli invasion of Caco-2 cells will be determined by slide agglutination tests and inhibition of cellular invasion of epithelial cells by pathogenic E. coli in vitro. To visualize the changes in binding of cranberry PAC to E. coli and E.coli to epithelial cells, fluorescently labeled PAC fractions and E.coli will be incubated with epithelial cells and observed for changes in binding and invasiveness with confocal light microscopy, electron microscopy and flow cytometry. Our partnership with the Wisconsin Cranberry Board and the Cranberry Institute will provide an extension mechanism in which results will be shared directly with cranberry growers and handlers. In turn, growers may adopt new varieties and harvest strategies to meet the future needs of the juicing companies. Information will be disseminated to cranberry juice / fruit consumers directly through scientific publications, oral or poster presentations at relevant meetings or through extension activities of the Wisconsin Cranberry Board and Cranberry Institute.

Progress 10/01/10 to 09/30/14

Outputs
Target Audience: The target audiences are: The Wisconsin Cranberry Board, The Cranberry Institute, cranberry fruit growers, cranberry fruit and juice industries and cranberry product consumers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Rodrigo Feliciano, a PhD candidate, was responsible for conducting the day-to-day research and this project provides a foundation for obtaining his PhD. Jessica Parker -undergraduate research assistant, Nathan Chesmore- undergraduate research assistant, and Kristin Kohlmann-undergraduate research assistant gained experience in methods of cell culture, bacterial culture, separation chemistry and mass spectrometry. How have the results been disseminated to communities of interest? The Cranberry Marketing Committee, The Wisconsin Cranberry Board and The Cranberry Institute are updated annually on the progress of this research project. Results may be used by academic collaborators and potential industry partners to enhance bioactive components of cranberries through development of new cranberry varieties, manipulations of harvest and processing and storage practices. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? 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 contains 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 has a greater bioactivity. Through our interaction with the Cranberry Marketing Committee and Industry partners, we have become aware of new classes of cranberry products entering the market place. These new products are focused on whole berry derivitives (sweetened dried cranberries, whole dried cranberries, cold press cranberry seed oil, residual seed cake, and residual presscake). These products differ significantly in their composition (inclusion of insoluble proanthocyanidins, fiber, protein and fats) from the more classic cranberry products, such as, cranberry juice and spray dried juice. We have developed methods to determine the ability of the proanthocyanidin components of these products to inhibit virulence (invasivity) properties of extra-intestinal pathogenic bacteria were developed and applied. We published the methods is the journal article: Feliciano, R.P., J.J. Meudt, D. Shanmuganayagam, C.G. Krueger and J.D. Reed. Ratio of "A-type" to "B-type" proanthocyanidin interflavan bonds affects extra-intestinal Pathogenic Escherichia coli invasion of gut epithelial cells. J Agric Food Chem. 2013; Web Publication (DOI: 10.1021/jf403839a).

Publications

• Type: Journal Articles Status: Under Review Year Published: 2015 Citation: Krueger, C.G., N. Chesmore, X. Chen, J. Parker, C. Khoo, J. Marais, D. Shanmuganayagam, P. Crump, J.D. Reed. 2015. Multi-operator / multi-day validation of a cranberry (Vaccinium macrocarpon Ait. cv Stevens) proanthocyanidin standard for improved quantitation of proanthocyanidins in cranberry juices and cranberry powders by the 4- (dimethylamino)cinnamaldehyde assay. J Agric. Food Chem.
• Type: Journal Articles Status: Under Review Year Published: 2015 Citation: Feliciano, R.P., C.G. Krueger, J.D. Reed. 2015. Methods to determine relationships between structure and anti-microbial activity of cranberry proanthocyanidins. Molecular Nutrition and Food Research.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Mateos, A.R., D. Vauzour, C.G. Krueger, D. Shanmuganayagam, J.D. Reed, L. Calani, P. Mena, D. Del Rio, A. Crozier. Flavonoids and related compounds, bioavailability and impact on human health: an update. Archives of Toxicology. 2014; 88: 1803-1853.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Feliciano, R.P., J.A. Heintz, C.G. Krueger, M.M. Vestling, J.D. Reed. Fluorescent labeling of cranberry proanthocyanidins with 5-([4,6-dichlorotriazin-2-yl]amino)fluorescein (DTAF). Food Chemistry. 2015; 166:337-345.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Feliciano, R., J. Meudt, D. Shanmuganayagam, B. Metzger, C.G. Krueger, J. D. Reed. 2015. Supercritical fluid extraction, cranberry proanthocyanidins, an enterocyte invasion by extra-intestinal pathogenic Escherichia coli. J Agric. Food Chem. 2014; 62:7730-7737.

Progress 01/01/13 to 09/30/13

Outputs
Target Audience: The Wisconsin Cranberry Board, The Cranberry Institute, Cranberry fruit growers, Cranberry fruit and juice industries and cranberry product consumers. Changes/Problems: . What opportunities for training and professional development has the project provided? Rodrigo Feliciano, PhD Candidate, wasresponsible for conducting the day to day research and this project provides a foundation for obtaining his PhD. How have the results been disseminated to communities of interest? Consultation to The Cranberry Marketing Committe, The Wisconsin Cranberry Board and The CranberryInstitute is provided on the of this research project. Results may be used by academic collaborators andpotential industry partners to enhance bioactive components of cranberries through development of new cranberry varieties andmanipulations of harvest, processing and storage practices. What do you plan to do during the next reporting period to accomplish the goals? Through our interaction with the Cranberry Marketing Committee and Industry partners, we have become aware of new classes of cranberry products entering the market place. These new products are focused on whole berry derivitives (sweetened dried cranberries, whole dried cranberries, cold press cranberry seed oil, residual seed cake, and residual presscake). These products differ significantly in their composition (inclusion of insoluble proanthocyanidins, fiber, protein and fats) from the more classic cranberry products such as cranberry juice and spray dried juice. In the final year of this project we will begin to develop analytic methods aimed at evaluating the ability of the proanthocyanidin components of these products to inhibit virulance (invasivity) properties of extra-intestinal pathogenic bacteria.

Impacts
What was accomplished under these goals? Gut colonization by extra-intestinal pathogenic Escherichia coli (ExPEC) increases the risk of subsequentinfections, 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 ratiosof “A-type” to “B-type” interflavan bonds in PAC affect ExPEC agglutination and invasion of enterocytes. In cranberries, 94.5% ofPAC contain one or more “A-type” bonds, whereas in apples, 88.3% of PAC contain exclusively “B-type” bonds. Results showthat cranberry “A-type” PAC have greater bioactivity than apple “B-type” PAC for increasing ExPEC agglutination and decreasingExPEC epithelial cell invasion.This work provides a new insight on the specificity of theinteraction PAC interflavan bond type with bacterial virulencefactors highlighting the importance of this area of research inthe context of understanding the mechanism by which “A-type”PAC prevent UTI by inhibiting gut colonization by ExPEC.This new understanding of proanthocyanidin structure/bioactivity relationships can be applied to develop a clinicallyrelevant hypothesis of how proanthocyanidin-containing fruits,juices, botanicals, and dietary supplements can be used todevelop therapeutic strategies against ExPEC.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2013 Citation: Krueger, C.G., J.D. Reed, R.P. Feliciano, A.B. Howell. Quantifying and characterizing proanthocyanidins in cranberries in relation to urinary tract health. Analytical and Bioanalytical Chemistry. 2013; 405:4385-4395. PMID: 23397091
• Type: Journal Articles Status: Accepted Year Published: 2013 Citation: Feliciano, R.P., J.J. Meudt, D. Shanmuganayagam, C.G. Krueger and J.D. Reed. Ratio of A-type to B-type proanthocyanidin interflavan bonds affects extra-intestinal Pathogenic Escherichia coli invasion of gut epithelial cells. J Agric Food Chem. 2013; Web Publication (DOI: 10.1021/jf403839a)
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2013 Citation: Shanmuganayagam D, Johnson RE, Meudt JJ, Feliciano RP, Kohlmann KL, Nechyporenko AV, Heinz J, Krueger CG, Reed JD. A-type proanthocyanidins from cranberry inhibit the ability of extraintestinal pathogenic E. coli to invade gut epithelial cells and resist killing by macrophages. Experimental Biology 2013 Meeting, April 2024, Boston, MA.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2013 Citation: Reed, J.D., J.F. Fierre, C.G. Krueger, D. Shanmuganayagam and K.A. Kudsk. Food polyphenols and gut health. Abstracts of Papers of The American Chemical Society. 245; 249-AGFD. April 7, 2013, New Orleans, LA. Oral Presentation.

Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: Activities: Mass spectrometry and high performance liquid chromatography methods were developed to produce fractions of cranberry proanthocyanidins with lower degree of structural heterogeneity. The effect of cranberry proanthocyanidin structure on E. coli invasion of Caco-2 cells was evaluated. Services: Consultation on the phytochemical composition of cranberry fruits and products is provided to the Wisconsin Cranberry Board, the Cranberry Institute and Cranberry Marketing Commission. Results may be used by collaborators and potential partners to enhance bioactive components of cranberries through development of new cranberry varieties and manipulations of harvest, processing and storage practices. PARTICIPANTS: Jess D. Reed - PI, responsible for the overall administration of the project. Walt Hopkins - Co-PI, consultant on issues related to urinary tract health. Christian G. Krueger - Research Program Manager, has oversight of the project aspects involving development of LC and MS methods, Dhanansayan Shanmuganayagam - Scientist, has oversight of the project aspects involving cell culture, Jennifer Meudt - Research Specialist. Rodrigo Feliciano - PhD Candidate, responsible for conducting the day to day research. Kathleen Keene - undergraduate research assistant, Osama Siddiqui - undergraduate research assistant, Kristin Kohlmann - undergraduate research assistant, Ian Padron - undergraduate research assistant, Macaulay Haller - undergraduate research assistant and Anatolly Nechyporenko - undergraduate research assistant. TARGET AUDIENCES: The Wisconsin Cranberry Board, The Cranberry Institute, cranberry fruit growers, cranberry fruit and juice industries. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Change in knowledge: Novel methods of MALDI TOF MS, MALDI-TOF MS/MS and FTICR MS were developed to gain better understanding of the structural variation of proanthocyaindins. Change in Actions: Methods developed here will allow researchers and industry to better address issues of authenticity, standardization and efficacy of cranberry products. Cranberry processing industries may adopt new juice processing strategies to enrich the bioactive components (proanthocyanidin) in their beverages, sweet dried cranberries and cranberry powders. Institutes such as the Wisconsin Cranberry Board and the Cranberry Institute will share the new knowledge with cranberry growers, who may in turn adopt new planting and harvesting strategies to meet the future needs of processors.

Publications

• Pierre, J.F, A.F. Heneghan, R.P. Feliciano, D. Shanmuganayagam, C.G. Krueger, J.D. Reed, K.A. Kudsk. Cranberry proanthocyanidins improve intestinal sIgA during elemental enteral nutrition. Journal of Parenteral and Enteral Nutrition. 2012. Accepted In Press
• Pierre, J.F, A.F. Heneghan, R.P. Feliciano, D. Shanmuganayagam, D.A. Roenneburg, C.G. Krueger, J.D. Reed, K.A. Kudsk. Cranberry proanthocyanidins improve the gut mucous layer morphology and function in mice receiving elemental enteral nutrition. Journal of Parenteral and Enteral Nutrition. 2012. Accepted In Press
• Feliciano, R.P., C.G. Krueger, D. Shanmuganayagam, M. M. Vestling, J. D. Reed. Deconvolution of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry isotope patterns to determine ratios of A-type to B-type interflavan bonds in cranberry proanthocyanidins. Food Chemistry. 2012; 135:1485-1493
• Feliciano, R.P., M.P. Shea, D. Shanmuganayagam, C.G. Krueger, A.B. Howell, J.D. Reed. Comparison of Isolated Cranberry (Vaccinium macrocarpon Ait.) Proanthocyanidins to Catechin and Procyanidins A2 and B2 for Use as Standards in the 4-(Dimethylamino)cinnamaldehyde Assay. J Agric Food Chem. 2012; 60:4578-85. PMID: 22533362
• Krueger, C.G., J.D. Reed and R.P Feliciano. Authentication, standardization andefficacy evaluation of oligomeric polyphenols in foods and botanicals by MALDI-TOF MS. American Society of Mass Spectrometry Asilomar Conference on Mass Spectrometry in Food Safety and Quality, October 5-9, 2012. Abstract & Oral presentation.
• Feliciano. R.P., C.G Krueger, M.M. Vestling and J.D. Reed. Deconvolution of MALDI-TOF MS isotope patterns to determine ratios of cranberry to apple proanthocyanidin extracts in binary mixtures. American Society of Mass Spectrometry Asilomar Conference on Mass Spectrometry in Food Safety and Quality, October 5-9, 2012. Abstract, Poster and Poster Highlight oral presentation.
• Feliciano, R.P., C.G. Krueger, D. Shanmuganayagam, M.M. Vestling and J.D. Reed. Deconvolution of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry isotope patterns to determine ratios of A-type to B-type interflavan bonds in cranberry proanthocyanidins. XXVIth International Conference on Polyphenols. Florence, Italy, July 22-26, 2012. Abstract and Poster Presentation.

Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: Activities: Mass spectrometry and high performance liquid chromatography methods were developed to produce fractions of cranberry proanthocyanidins with lower degree of structural heterogeneity. The effect of cranberry proanthocyanidin structure on E. coli invasion of Caco-2 cells was evaluated. Services: Consultation on the phytochemical composition of cranberry fruits and products is provided to the Wisconsin Cranberry Board, the Cranberry Institute and Cranberry Marketing Commission. Dissemination: The Wisconsin Cranberry Board and the Cranberry Institute are updated annually on the progress of this research project. Results may be used by collaborators and potential partners to enhance bioactive components of cranberries through development of new cranberry varieties and manipulations of harvest, processing and storage practices. PARTICIPANTS: Jess D. Reed, PI, responsible for the overall administration of the project. Walt Hopkins, Co-PI, consultant on issues related to urinary tract health. Christian G. Krueger, Research Program Manager, has oversight of the project aspects involving development of LC and MS methods, Dhanansayan Shanmuganayagam, Scientist, has over site of the project aspects involving cell culture. Rodrigo Feliciany, PhD Candidate, responsible for conducting the day to day research. Kathleen Keene -undergraduate research assistant, Osama Siddiqui- undergraduate research assistant, Kristin Kohlmann-undergraduate research assistant. TARGET AUDIENCES: The Wisconsin Cranberry Board, The Cranberry Institute, Cranberry fruit growers, Cranberry fruit and juice industries. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Change in knowledge: Novel methods of MALDI TOF MS, MALDI-TOF MS/MS and FTICR MS were developed to gain better understanding of the structural variation of proanthocyaindins. Change in Actions: Methods developed here will allow researchers and industry to better address issues of authenticity, standardization and efficacy of cranberry products. Cranberry processing industries may adopt new juice processing strategies to enrich the bioactive components (proanthocyanidin) in their beverages, sweet dried cranberries and cranberry powders. Institutes such as he Wisconsin Cranberry Board and the Cranberry Institute will share the new knowledge with cranberry growers, who may in turn adopt new planting and harvesting strategies to meet the future needs of processors.

Publications

• No publications reported this period