Bovine Lactoferrin Decreases Cholera-Toxin-Induced Intestinal Fluid Accumulation in Mice by Ganglioside Interaction Fulton P. Rivera 1,2 *, Anicia M. Medina 1 , Sandra Bezada 3 , Roberto Valencia 4 , Marı´a Bernal 5 , Rina Meza 5 , Ryan C. Maves 5,6 , Theresa J. Ochoa 1,7 1 Instituto de Medicina Tropical Alexander von Humboldt, Universidad Peruana Cayetano Heredia, Lima, Peru, 2 Laboratorio de Fisiopatogenia, Departamento de Fisiologı ´a, Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina, 3 Laboratorio de Farmacologı ´a, Facultad de Farmacia y Bioquı ´mica, Universidad Nacional Mayor de San Marcos, Lima, Peru, 4 Facultad de Veterinaria y Zootecnia, Universidad Peruana Cayetano Heredia, Lima, Peru, 5 Department of Bacteriology, U.S. Naval Medical Research Unit SIX, Lima, Peru, 6 Division of Infectious Diseases, Naval Medical Center San Diego, San Diego, California, United States of America, 7 Center for Infectious Diseases, University of Texas School of Public Health, Houston, Texas, United States of America Abstract Secretory diarrhea caused by cholera toxin (CT) is initiated by binding of CT’s B subunit (CTB) to GM1-ganglioside on the surface of intestinal cells. Lactoferrin, a breast milk glycoprotein, has shown protective effect against several enteropathogens. The aims of this study were to determine the effect of bovine-lactoferrin (bLF) on CT-induced intestinal fluid accumulation in mice, and the interaction between bLF and CT/CTB with the GM1-ganglioside receptor. Fluid accumulation induced by CT was evaluated in the mouse ileal loop model using 56 BALB/c mice, with and without bLF added before, after or at the same time of CT administration. The effect of bLF in the interaction of CT and CTB with GM1- ganglioside was evaluated by a GM1-enzyme-linked immunosorbent assay. bLF decreased CT-induced fluid accumulation in the ileal loop of mice. The greatest effect was when bLF was added before CT (median, 0.066 vs. 0.166 g/cm, with and without bLF respectively, p,0.01). We conclude that bLF decreases binding of CT and CTB to GM1-ganglioside, suggesting that bLF suppresses CT-induced fluid accumulation by blocking the binding of CTB to GM1-ganglioside. bLF may be effective as adjunctive therapy for treatment of cholera diarrhea. Citation: Rivera FP, Medina AM, Bezada S, Valencia R, Bernal M, et al. (2013) Bovine Lactoferrin Decreases Cholera-Toxin-Induced Intestinal Fluid Accumulation in Mice by Ganglioside Interaction. PLoS ONE 8(4): e59253. doi:10.1371/journal.pone.0059253 Editor: Min Wu, University of North Dakota, United States of America Received September 15, 2012; Accepted February 13, 2013; Published April 8, 2013 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: This work has been funded by the Institutional Research Fund (Fondo Concursable) of Universidad Peruana Cayetano Heredia (F.P.R) and by the United States Military Infectious Disease Research Program (MIDRP) (work unit number 60000.000.0.B0017). The views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the Department of the Navy, Department of Defense, the U.S. Government, nor that of the National Institutes of Health or other funding institutions. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Fulton.Rivera@upch.pe Introduction Diarrheal disease due to enteric infection is a major cause of death among children under five years of age, especially in developing countries. Enterotoxigenic bacteria are responsible for a large proportion of these diseases. Among the enterotoxin- producing bacteria, Vibrio cholerae causes the most severe disease, while enterotoxigenic Escherichia coli (ETEC) is responsible for the largest number cases. ETEC is also a major cause of diarrhea in adult travelers from industrialized countries to the developing world. The major virulence factors of these bacteria are their homologous enterotoxins, cholera toxin (CT) and heat-labile enterotoxin (LT), respectively. These toxins are examples of bacterial AB 5 toxins, consisting of one enzymatically active A subunit (CTA or LTA) that assemble with five B subunits (CTB or LTB) which are responsible for the toxins’ binding properties [1]. Breastfeeding has been identified as one of the most effective interventions to prevent pediatric diarrhea as well as all-cause mortality [2]. Lactoferrin is a major nonimmune milk factor that has been thought to be important in protecting infants from intestinal infections. It is an iron-binding 78-kDa glycoprotein that is resistant to proteolytic enzymes [3,4,5,6]. Lactoferrin is pro- duced not only in breast milk but also in other mucosal secretions and phagocytic cells. Both human and bovine lactoferrin may protect against Gram-negative bacteria in a variety of ways [7]. It was originally thought that it impaired bacterial multiplication due to its ability to decrease availability of iron required for growth [8,9]. However, the antibacterial activity of lactoferrin is not due solely to its bacteriostatic iron-binding capacity [10]. A pepsin- derived fragment of lactoferrin has iron independent bactericidal activity that is associated with release of lipopolysaccharide (LPS). However, undigested human and bovine lactoferrin are able to interact with LPS thus inducing bactericidal effect due to release of LPS [11,12,13]. Lactoferrin kills or slows bacterial growth synergistically with other factors that may be present in mucosal secretions, including immunoglobulins, complement, and lyso- zyme [7]. The mechanism of the diarrhea induced by both CT and LT is initiated by the binding of B subunits to the GM1-ganglioside present on the surface of intestinal epithelial cells [14,15]. This PLOS ONE | www.plosone.org 1 April 2013 | Volume 8 | Issue 4 | e59253