Inhibition of enterotoxigenic Escherichia coli (ETEC) adhesion to Caco-2 cells by human milk and its immunoglobulin and non-immunoglobulin fractions

Brazilian Journal of Microbiology (2007) 38:86-92 ISSN 1517-8283 INHIBITION OF ENTEROTOXIGENIC ESCHERICHIA COLI (ETEC) ADHESION TO CACO-2 CELLS BY HUMAN MILK AND ITS IMMUNOGLOBULIN AND NON-IMMUNOGLOBULIN FRACTIONS Inaiara R. de Oliveira1; Heidi C. Bessler1; Sonia N. Bao2; Renato de L. Lima3; Loreny G. Giugliano*1 1 Laboratório de Microbiologia, Departamento de Biologia Celular, Instituto de Biologia, Universidade de Brasília, Brasília-DF, Brazil; 2Laboratório de Microscopia Eletrônica, Departamento de Biologia Celular, Instituto de Biologia, Universidade de Brasília, Brasília-DF, Brazil; 3Hospital Universitário de Brasília, Departamento de Pediatria, Banco de Leite, Universidade de Brasília, Brasília-DF, Brazil Submitted: January 16, 2006; Returned to authors for corrections: March 23, 2006; Approved: January 18, 2007 ABSTRACT Enterotoxigenic Escherichia coli (ETEC) is the most common cause of diarrhea in children in developing countries and among travelers to ETEC endemic areas. ETEC diarrhea is caused by colonization of the small intestine mediated by colonization factor (CF) antigens, and subsequent elaboration of enterotoxins. Breast feeding has been related to protection against enteric infections. The protective effect of human milk can be ascribed to its immunoglobulin content, specially secretory immunoglobulin A (sIgA), and to nonimmunoglobulin components such as free oligosaccharides, glycoproteins and glycolipids. In this study we investigated the effect of whole human milk and its fractions immunoglobulin and non-immunoglobulin on the adherence of ETEC strains possessing different CFs to Caco-2 cells, as well as the ability of sIgA and free secretory component (fSC) to bind to bacterial superficial proteins. Pooled human milk from three donors were fractionated by gel filtration and analyzed by SDS-PAGE. Our results revealed that whole human milk and its proteins fractions, containing sIgA and fSC, inhibited adhesion ETEC strains harboring different colonization factors antigens. We also verified that sIgA and fSC, using immunoblotting and immunogold labeling assays, bound to some fimbrial proteins and other material present in bacterial surface. Our findings suggest that whole human milk and its fractions may contribute to protection against ETEC infections by blocking bacterial adhesion mediated by different colonization antigens. Keyword: Diarrhea; Adherence inhibition; Escherichia coli enterotoxigenic; Colonization factor antigen; Human milk INTRODUCTION Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrhea among children in developing countries (28,29) and among travelers to ETEC endemic areas (18). Two virulence attributes that characterize ETEC are the colonization of the small intestine surface and the production of enterotoxins. It is well-known that in ETEC strains the colonization of intestinal mucosa is related to the presence of specific fimbrial antigens, called colonization factors (CFs) (10). Twenty one different CFs have been described for human ETEC (12,26). Among the most studied and frequently found CFs are CFA I, CS1, CS2, CS3, CS4, CS5, CS6 and CS8 (12). Most of ETEC isolated in Brazil have been shown to possess CFA I, CFA II and CFA IV (14,17,27). Epidemiological studies of diarrhea have shown that breast feeding protects infants from intestinal infections (20,32). The protective effect of human milk is usually attributed to its high content of immunoglobulin, specially secretory immunoglobulin A (sIgA) (6,16). Some works showed that sIgA inhibit the adhesion of enteropathogenic Escherichia coli to HeLa cells and Hep-2 (5,6,9), and possess effect against ETEC *Corresponding Author. Mailing address: Lab. de Microbiologia - Dep. de Biologia Celular - Instituto de Biologia - Universidade de Brasília. cep 70910900 - Brasília, DF - Brasil. Tel.: +55 (61) 3307-2557; Fax: +55 (61) 3273-4608. E-mail: 86 Inhibition of ETEC by human milk virulence factors (7,8). Recently, some studies of our group demonstrated that sIgA inhibits the adherence of diffusely adherent Escherichia coli (DAEC), enteroaggregative Escherichia coli (EAEC) and Shigella flexneri to HeLa cells (1,31). Protection by human milk can also be ascribed to nonimmunoglobulin components such as free oligosaccharides, glycolipids and glycoproteins (2,6,24). We demonstrated that human milk glycoproteins, such as lactoferrin and free secretory component (fSC), have the ability to inhibit adhesion of ETEC CFA I+ strains to human erytrocytes (15). The secretory component (SC) is found in several secretions complexed with sIgA or as a free glycoprotein (22). It has been shown that fSC is an 80 kDa glycoprotein, consisting of a single polypeptide chain largely glycosilated (20%) (19). There is little information about the role of fSC in secretions, but some workers suggest that it may have a protective role against diarrhea (4,15). Recently, using immunogold labeling, we showed the ability of fSC to interact with CFA I and CFA II (CS1 and CS3) fimbrial adhesins (25). To determine if the interaction of milk proteins to bacterial superficial structures could contribute to inhibition of ETEC adhesion, we investigated the effect of whole human milk, immunoglobulin fraction (IgF) and fSC on the adherence of ETEC strains possessing CFA I, CS1, CS2, CS3, CS4, CS5, CS6 and CS8 to intestinal cells. The binding of IgF and fSC to bacteria superficial proteins also was investigated. MATERIAL AND METHODS Bacterial strains The phenotypes of all human ETEC strains used in this work are shown in Table 1. Breast milk samples Human milk was obtained from the Human Milk Bank of the Hospital Universitário de Brasília (Brasília, Brazil). Samples were obtained from lactating mothers up to one month after delivery and kept frozen -20ºC. Aliquots of individual milk samples were taken from at least three donors and pooled. Fractionation of human milk The fractionation of pooled human milk was performed as described previously by Giugliano et al. (15). Briefly, lipids and casein were removed, and proteins concentrated by adding ammonium sulfate to 70% saturation. The sample obtained was dialyzed and applied to a Sephacryl S-200 HR column (2.6 x 80 cm; Pharmacia Biotech AB, Uppsala, Sweden) eluted with TrisHCl buffer, pH 7.6. Thereafter, the fractions eluted from each peak were pooled and proteins were concentrated, dissolved and dialyzed overnight against Tris-HCl buffer, pH 7.6. This material was used in the adhesion inhibition assays. Table 1. Bacterial samples. Strains c H10407A PB176b E4833b 58R957b E2539C1c 61.1a E11881Ec 118299c B7Ac Serotype CFs Enterotoxins O78:H11 O6:H16 O6:H16 O6:H16 O25:NM O9:H10 ND ND O148:H28 CFA I, longus CS1, CS3 CS2, CS3 CS2 CS3, longus CS8 CS4, CS6, longus CS5, CS6, longus CS6, longus LT/ST LT/ST LT/ST LT LT LT LT/ST ND LT/ST a Strain from our bacterial collection; bStrains kindly provided by Dr. B.C Guth, Universidade Federal de São Paulo, (...truncated)