- Email: [email protected]
Human milk fractions inhibit the adherence of diffusely adherent Escherichia coli (DAEC) and enteroaggregative E. coli (EAEC) to HeLa cells
FEMS Microbiology Letters 184 (2000) 91^94
www.fems-microbiology.org
Human milk fractions inhibit the adherence of di¡usely adherent Escherichia coli (DAEC) and enteroaggregative E. coli (EAEC) to HeLa cells Andre¨a Nascimento de Arau¨jo, Loreny Gimenes Giugliano * Laborato¨rio de Microbiologia, Departamento de Biologia Celular, Instituto de Biologia, Universidade de Bras|¨lia, 70910-900 Bras|¨lia DF, Brazil Accepted 16 December 1999
Abstract Binding to a specific receptor is an essential step for most enteropathogens to initiate an intestinal infection. We analyzed the inhibitory effect of human milk and its protein components on adhesion of two diarrheagenic Escherichia coli strains, diffusely adherent E. coli (DAEC) and enteroaggregative E. coli (EAEC), to HeLa cells. Defatted milk, whey proteins, immunoglobulin and non-immunoglobulin fractions, in concentrations lower than usually found in whole milk, inhibited both DAEC and EAEC adhesion, indicating that human milk components may contribute to the defense of the infants against enteropathogens. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Diarrhea ; Adherence inhibition; Human milk ; Di¡usely adherent Escherichia coli; Enteroaggregative Escherichia coli
1. Introduction Diarrhea caused by Escherichia coli is an important cause of infantile morbidity and mortality in developing countries. On the basis of distinct virulence properties and syndromes, diarrheagenic E. coli strains have been classi¢ed into six categories: enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), enterohemorragic E. coli, di¡usely adhering E. coli (DAEC) and enteroaggregative E. coli (EAEC) [1]. Diarrheagenic E. coli also have been characterized by their ability to produce three distinctive patterns of adherence when bound to cultured epithelial cells. The localized adherence pattern [2] is characteristic of EPEC which are recognized as an important cause of diarrhea in children aged less than 1 year [3^5]. The di¡use adherence pattern and the aggregative adherence pattern are characteristic of DAEC and EAEC strains, respectively [2,6]. Involvement of DAEC and EAEC in diarrheal illness remains controversial. However, according to several researchers, DAEC strains are signi¢cantly associated with diarrheal disease [7,8], and prospective studies in India, Mexico and Brazil
* Corresponding author. Tel. : +55 (61) 307-2176; Fax: +55 (61) 272-1497; E-mail: [email protected]
have incriminated EAEC as an important agent of persistent diarrhea in infants and young children [9,10,11]. Breast-feeding has been related to protection against intestinal and respiratory infections [12]. Both immunoglobulin and non-immunoglobulin elements of human milk are thought to contribute to protection against diarrheal agents. Silva and Giampaglia [13] have demonstrated that human colostrum and milk inhibited the localized adherence of EPEC strains to cultured cells, indicating that interactions between milk elements and bacteria do occur, preventing the attachment of bacteria to epithelial cells. In 1995, Giugliano and co-workers [14] proposed that human milk glycoproteins also have an important role in the body's defense against pathogens. In the present study, we investigated the e¡ect of defatted human milk, whey proteins, immunoglobulin and nonimmunoglobulin fractions in the adherence of two DAEC and EAEC strains to HeLa cells. 2. Materials and methods 2.1. Bacterial strains The DAEC strain, RS51-1 (F1845+), was kindly provided by Dr. J.P. Nataro (CVD, USA). The EAEC strain,
0378-1097 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 0 ) 0 0 0 2 8 - 8
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A. Nascimento de Arau¨jo, L.G. Giugliano / FEMS Microbiology Letters 184 (2000) 91^94
0431-4 (O64:H4), isolated from a child with diarrhea in Sa¬o Paulo, Brazil, was obtained from Dr. T. Gomes and Dr. B.C. Guth (UNIFESP, Brazil). 2.2. Breast milk samples Human milk samples were obtained from the Human Milk Bank of the Hospital Materno Infantil (Brasilia, Brazil). Aliquots of individual frozen samples were taken from at least 10 lactating mothers up to 2 months after delivery and pooled. 2.3. Fractionation of human milk Defatted milk and concentrated whey were obtained as described previously by Giugliano and co-workers [14]. Concentrated whey was chromatographed on a Sephacryl S-200 HR column (2.6 cmU80 cm) equilibrated with 0.1 M Tris^HCl, pH 7.6, supplemented with 0.5 M NaCl, 1 mM phenylmethanesulfonyl £uoride, NaN3 0.1% and 50 mM O-amino n-caproic acid. Fractions from obtained peaks were pooled and proteins were precipitated with ammonium sulfate to 70% ¢nal saturation and redissolved in bu¡ered saline. After 24-h dialyses against bu¡ered saline, the eluted proteins were evaluated by sodium dodecyl sulfate^polyacrylamide gel electrophoresis (SDS^PAGE) (12.5% polyacrylamide) and Coomassie blue staining. Sample protein concentration was determined by the Bradford method. 2.4. Inhibition of bacterial adherence assay Adhesion tests were carried out as described previously [2] with some modi¢cations. HeLa cells (1.6U105 cells ml31 , 600 Wl) were grown for 48 h at 37³C in 199 medium with 10% fetal bovine serum in 24-well tissue culture plates containing a cover slip. The medium was replaced with 300 Wl of 199 medium supplemented with 2% fetal bovine serum and 1% D-mannose, followed by 100 Wl of a sample of milk, and 75 Wl of an exponential phase bacterial culture (DAEC, 7U107 cells ml31 ; EAEC, 1U108 cells ml31 ) grown in Trypticase Soy Broth. After 30 min at 37³C, wells were washed with phosphate-bu¡ered saline six times, and 500 Wl of 199 medium with 2% fetal bovine serum was added to each well. Plates were incubated for 3 h at 37³C. Cells were then washed three times with phosphate-bu¡ered saline, ¢xed with methanol and stained with May-Gru«nwald and Giemsa as described previously [2]. Adherence assays were repeated at least three times, and 250 or more HeLa cells were observed in each preparation. The e¡ect of defatted milk, whey proteins, immunoglobulin and non-immunoglobulin fractions on the bacterial adherence was determined by calculating the percentage of cells with less than ¢ve attached bacteria in relation to the control, carried out under identical conditions, but in the absence of milk sample.
2.5. Statistical analysis Di¡erences in percentage of bacterial adhesion were compared statistically by the z test of proportions [15]. 3. Results and discussion SDS^PAGE protein separation of defatted milk (data not shown) and whey (Fig. 1, lane 2) showed a pro¢le of proteins described for human milk : the band of V56.3 kDa represented heavy chain of immunoglobulins; the band of V78 kDa represented lactoferrin and secretory component since their molecular masses are similar; serum albumin and K-lactalbumin were represented by the bands of V65.0 and 14.2 kDa ; while caseins varied from 24.0 to 30.0 kDa [16]. Adhesion assays inhibition showed that defatted milk (1.28 mg ml31 ) and whey proteins (0.97 mg ml31 ) inhibited DAEC and EAEC adhesion to HeLa cells (Table 1). Since binding to a speci¢c extracellular receptor is an essential step for many pathogens, these results indicated that human defatted milk is capable of preventing this important event, inhibiting DAEC and EAEC adhesion to host cells. Furthermore, milk proteins seemed to participate in this inhibitory e¡ect. Several studies have shown an important role of sIgA in the protection e¡ect of human milk [17,18]. To verify the e¡ect of the human milk immunoglobulin and non-immunoglobulin fractions in the DAEC and EAEC adhesion, whey proteins were chromatographed. A typical pro¢le of the eluted proteins from Sephacryl S-200 HR column, which has previously been described by Mestecky and Killian [19], is shown in Fig. 2. The ¢rst peak (P1), at 0.325 mg ml31 , inhibited DAEC and EAEC adherence to HeLa cells (Table 1). In Fig. 1, lane 3, it is possible to verify that P1 contained bands of approximately 56.3 and 78.0 kDa corresponding to heavy chain and secretory
Fig. 1. Coomassie blue-stained SDS^PAGE 12.5% of whey, P1, P2 and P3 proteins : lane 1, molecular mass markers (66, 45, 36, 29, 24, 20.1 and 14.2 kDa, Pharmacia Biotech) ; lane 2, whey; lane 3, pooled fractions from ¢rst peak (P1); lane 4, pooled fractions from peak 2 (P2); and lane 5, pooled fractions from third peak (P3).
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Table 1 Inhibition of DAEC and EAEC adherence to HeLa cells by human milk fractions Bacterial strain
DAEC EAEC
Mean inhibition of adherence (%) in the presence of Defatted milk (1.28 mg ml31 )
Whey (0.97 mg ml31 )
P1 (0.325 Wg ml31 )
P2 (0.225 Wg ml31 )
P3 (0.3 mg ml31 )
29.6 29.3
9.4 15.8
12.1 17.2
6.7 3.5
NS NS
All presented results were signi¢cant (P 6 0.05). NS = not signi¢cant. P1, P2 and P3 denote pooled peaks from Sephacryl S-200 HR.
component of immunoglobulin and bands representing caseins [16]. Precipitated casein at 0.3 mg ml31 was assayed and did not inhibit the bacterial adherence (data not shown). Therefore, it was assumed that P1 inhibitory e¡ect was promoted by the human milk immunoglobulins. Likewise, Caªmara and co-workers have shown the inhibitory e¡ect of milk immunoglobulin fraction on EPEC adhesion [20]. SDS^PAGE of second (P2) and third (P3) peaks (Fig. 1, lanes 4 and 5, respectively) did not show the presence of immunoglobulins, since the band that represents the heavy chain was absent [16]. However, P2 (0.225 mg ml31 ) was able to inhibit the adhesion of DAEC and slightly inhibited the EAEC adherence to HeLa cells (Table 1). SDS^ PAGE analysis showed that lactoferrin, secretory component, serum albumin and casein were present in the pooled fractions from P2, indicating that non-immunoglobulin proteins could be involved in the inhibition of adhesion. On the other hand, at 0.3 mg ml31 , P3 caused no inhibitory e¡ect, and its electrophoretic pro¢le showed only one band corresponding to K-lactalbumin molecular mass [16]. Several studies have suggested the role of human milk glycoconjugates, such as glycolipids, glycoproteins, mucins, glycosaminoglycans and the oligosaccharides, in the protection of neonates, acting as cell surface homologues
to inhibit the pathogen binding to host cell receptors. Many examples of glycoconjugate protective components have been shown: oligosaccharides inhibit the adherence of Streptococcus pneumoniae, ETEC, and invasive Campylobater jejuni and also inhibit the toxicity of the heat-stable enterotoxin of E. coli [21]. The glycoproteins, lactoferrin and secretory component, inhibit the ETEC hemagglutination to human enterocytes [14]; and glycolipids inhibit the adhesion of cholera toxin and heat label enterotoxin to the intestinal epithelia [22]. In this study, it was shown that adherence of two DAEC and EAEC strains was inhibited by defatted milk, immunoglobulins and non-immunoglobulin proteins used in concentrations lower than those found in whole milk. It seems that glycoproteins present in P2 may be involved in the inhibitory e¡ect. Also, these results indicate that human milk components may protect infants against intestinal infections. Acknowledgements This study was supported by FAP-DF, Grant no. 193.375/95, and Nestle¨ Indl. Coml. We wish to thank R.A. Harder for revising the manuscript and Dr. E. Miazaki for statistical assistance.
References
Fig. 2. Chromatography of concentrated whey proteins on Sephacryl S200 HR column (2.6 cmU80.0 cm). The peaks were monitored at 280 nm and eluted with 0.1 M Tris^HCl, pH 7.6, supplemented with 0.5 M NaCl, 1 mM phenylmethanesulfonyl £uoride, NaN3 0.1% and 50 mM O-amino n-caproic acid, pH 7.6; the £ow rate was 120 ml h31 and fractions of 5 ml were collected. The horizontal lines under the respective peaks indicate pooled fractions. P1, P2 and P3 denote the pooled peaks.
[1] Nataro, J.P. and Kaper, J.B. (1998) Diarrheagenic Escherichia coli. Clin. Microbiol. Rev. 11 (1), 142. [2] Scaletsky, I.C.A., Silva, M.L.M. and Trabulsi, L.R. (1984) Distinctive patterns of adherence of enteropathogenic Escherichia coli to HeLa cells. Infect. Immun. 45, 534^536. [3] Robins-Browne, R., Still, C.S., Miliotis, M.D., Richardson, N.J., Koornhof, H.J., Frieman, L., Schoub, B.D., Lecatsas, G. and Hartman, E. (1980) Summer diarrhoea in African infants and children. Arch. Dis. Child. 55, 923^928. [4] Gomes, T.A.T., Rassi, V., Macdonald, K.L., Ramos, S.R.T.S., Trabulsi, L.R., Vieira, M.A.M., Guth, B.E.C., Candeias, J.A.N., Ivey, C., Toledo, M.R.F. and Blake, P.A. (1991) Enteropathogens associated with acute diarrheal disease in urban infants in Sa¬o Paulo. Braz. J. Infect. Dis. 164, 331^337. [5] Levine, M.M., Prado, V., Lior, H. and Lagos, R. (1996) Epidemiologic studies of diarrhea associated with enteropathogenic Escherichia coli (EPEC) in infants and young children in Santiago, Chile. Rev. Microbiol. 27, 40^44. [6] Nataro, J.P., Kaper, J.B., Robin-Browse, R., Prado, V., Vial, P. and
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[7]
[8]
[9]
[10]
[11]
[12] [13]
[14]
A. Nascimento de Arau¨jo, L.G. Giugliano / FEMS Microbiology Letters 184 (2000) 91^94 Levine, M.M. (1987) Patterns of adherence of diarrheagenic Escherichia coli to HEp-2 cells. Pediatr. Infect. Dis. 6, 829^831. Giro¨n, J.A., Jones, T., Milla¨n-Velasco, F., Castro-Mun¬oz, E., Za¨rate, L., Fry, J., Frankel, G., Moseley, S.L., Baundry, B., Kaper, J.B., Schoolnik, G.K. and Riley, L.W. (1991) Di¡use-adhering Escherichia coli (DAEC) as a putative cause of diarrhea in Mayan children in Mexico. J. Infect. Dis. 163, 507^513. Jallat, C., Livrelli, V., Darfeuille-Michaud, A., Rich, C. and Joly, B. (1993) Escherichia coli strains involved in diarrhea in France: high prevalence and heterogeneity of di¡usely adhering strains. J. Clin. Microbiol. 31, 2031^2037. Bhan, M.K., Ral, P., Levine, M.M., Kaper, J.B., Bhandary, N., Srivastava, R., Kumar, R. and Sazawal, S. (1989) Enteroaggregative Escherichia coli associated with persistent diarrhea in a cohort of rural children in India. J. Infect. Dis. 159, 1061^1064. Cravioto, A., Tello, A., Navarro, A., Ruiz, J., Villafa¨n, H., Uribe, F. and Eslava, C. (1991) Association of Escherichia coli HEp-2 adherence patterns with type and duration of diarrhoea. Lancet 337, 262^ 264. Wanke, C.A., Schorling, J.B., Barret, L.J., Desouza, M.A. and Guerrant, R.L. (1991) Potential role of adherence traits of Escherichia coli in persistent diarrhea in an urban Brazilian slum. Pediatr. Infect. Dis. 10, 746^751. Hanson, L.A. and Winberg, J. (1972) Breast milk and defense against infection in the newborn. Arch. Dis. Child. 47, 845^853. Silva, M.L.M. and Giampaglia, C.M.S. (1992) Colostrum and human milk inhibit localized adherence of enteropathogenic Escherichia coli to HeLa cells. Acta Paediatr. 81, 266^267. Giugliano, L.G., Ribeiro, S.T.G., Vainstein, M.H. and Ulhoa, C.J.
[15] [16]
[17]
[18]
[19] [20]
[21]
[22]
(1995) Free secretory component and lactoferrin of human milk inhibit the adhesion of enterotoxigenic Escherichia coli. J. Med. Microbiol. 42, 3^9. Glantz, S.A. (1997) Primer of Biostatistics, 4th edn. McGraw-Hill. Rudlo¡, S. and Kunz, C. (1997) Protein and nonprotein nitrogen components in human milk, bovine milk and infant formula : quantitative and qualitative aspects in infant nutrition. J. Pediatr. Gastroenterol. Nutr. 24, 328^344. Carbonare, S.B., Silva, M.L.M., Trabulsi, L.R. and Carneiro-Sampaio, M.M.S. (1995) Inhibition of HEp-2 cell invasion by enteroinvasive Escherichia coli by human colostrum IgA. Int. Arch. Allergy Immunol. 108, 113^118. Cruz, C.R., Gil, L., Cano, F., Caceres, P. and Pareja, G. (1988) Breast milk anti-Escherichia coli heat-labile toxin IgA antibodies protect against toxin-induced infantile diarrhea. Acta Paediatr. Scand. 22, 130^134. Mestecky, J. and Killian, M. (1985) Immunoglobulin A (IgA). Methods Enzymol. 116, 37^75. Caªmara, L.M., Carbonare, S.B., Silva, M.L. and Carneiro-Sampaio, M.M. (1994) Inhibition of enteropathogenic Escherichia coli (EPEC) adhesion to HeLa cells by human colostrum: detection of speci¢c sIgA related to EPEC outer-membrane proteins. Int. Arch. Allergy Immunol. 103, 307^310. Newburg, D.S. (1997) Do the binding properties of oligossacharides in milk protect human from gastrointestinal bacteria ? J. Nutr. 127 (Suppl. 5), 980S^984S. L×greid, A., KolstÖ Otn×ss, A.B. and Fuglesang, J. (1986) Human and bovine milk: comparison of ganglioside composition and enterotoxin-inhibitory activity. Pediatr. Res. 20, 416^420.
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Human milk fractions inhibit the adherence of di¡usely adherent Escherichia coli (DAEC) and enteroaggregative E. coli (EAEC) to HeLa cells Andre¨a Nascimento de Arau¨jo, Loreny Gimenes Giugliano * Laborato¨rio de Microbiologia, Departamento de Biologia Celular, Instituto de Biologia, Universidade de Bras|¨lia, 70910-900 Bras|¨lia DF, Brazil Accepted 16 December 1999
Abstract Binding to a specific receptor is an essential step for most enteropathogens to initiate an intestinal infection. We analyzed the inhibitory effect of human milk and its protein components on adhesion of two diarrheagenic Escherichia coli strains, diffusely adherent E. coli (DAEC) and enteroaggregative E. coli (EAEC), to HeLa cells. Defatted milk, whey proteins, immunoglobulin and non-immunoglobulin fractions, in concentrations lower than usually found in whole milk, inhibited both DAEC and EAEC adhesion, indicating that human milk components may contribute to the defense of the infants against enteropathogens. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Diarrhea ; Adherence inhibition; Human milk ; Di¡usely adherent Escherichia coli; Enteroaggregative Escherichia coli
1. Introduction Diarrhea caused by Escherichia coli is an important cause of infantile morbidity and mortality in developing countries. On the basis of distinct virulence properties and syndromes, diarrheagenic E. coli strains have been classi¢ed into six categories: enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), enterohemorragic E. coli, di¡usely adhering E. coli (DAEC) and enteroaggregative E. coli (EAEC) [1]. Diarrheagenic E. coli also have been characterized by their ability to produce three distinctive patterns of adherence when bound to cultured epithelial cells. The localized adherence pattern [2] is characteristic of EPEC which are recognized as an important cause of diarrhea in children aged less than 1 year [3^5]. The di¡use adherence pattern and the aggregative adherence pattern are characteristic of DAEC and EAEC strains, respectively [2,6]. Involvement of DAEC and EAEC in diarrheal illness remains controversial. However, according to several researchers, DAEC strains are signi¢cantly associated with diarrheal disease [7,8], and prospective studies in India, Mexico and Brazil
* Corresponding author. Tel. : +55 (61) 307-2176; Fax: +55 (61) 272-1497; E-mail: [email protected]
have incriminated EAEC as an important agent of persistent diarrhea in infants and young children [9,10,11]. Breast-feeding has been related to protection against intestinal and respiratory infections [12]. Both immunoglobulin and non-immunoglobulin elements of human milk are thought to contribute to protection against diarrheal agents. Silva and Giampaglia [13] have demonstrated that human colostrum and milk inhibited the localized adherence of EPEC strains to cultured cells, indicating that interactions between milk elements and bacteria do occur, preventing the attachment of bacteria to epithelial cells. In 1995, Giugliano and co-workers [14] proposed that human milk glycoproteins also have an important role in the body's defense against pathogens. In the present study, we investigated the e¡ect of defatted human milk, whey proteins, immunoglobulin and nonimmunoglobulin fractions in the adherence of two DAEC and EAEC strains to HeLa cells. 2. Materials and methods 2.1. Bacterial strains The DAEC strain, RS51-1 (F1845+), was kindly provided by Dr. J.P. Nataro (CVD, USA). The EAEC strain,
0378-1097 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 0 ) 0 0 0 2 8 - 8
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A. Nascimento de Arau¨jo, L.G. Giugliano / FEMS Microbiology Letters 184 (2000) 91^94
0431-4 (O64:H4), isolated from a child with diarrhea in Sa¬o Paulo, Brazil, was obtained from Dr. T. Gomes and Dr. B.C. Guth (UNIFESP, Brazil). 2.2. Breast milk samples Human milk samples were obtained from the Human Milk Bank of the Hospital Materno Infantil (Brasilia, Brazil). Aliquots of individual frozen samples were taken from at least 10 lactating mothers up to 2 months after delivery and pooled. 2.3. Fractionation of human milk Defatted milk and concentrated whey were obtained as described previously by Giugliano and co-workers [14]. Concentrated whey was chromatographed on a Sephacryl S-200 HR column (2.6 cmU80 cm) equilibrated with 0.1 M Tris^HCl, pH 7.6, supplemented with 0.5 M NaCl, 1 mM phenylmethanesulfonyl £uoride, NaN3 0.1% and 50 mM O-amino n-caproic acid. Fractions from obtained peaks were pooled and proteins were precipitated with ammonium sulfate to 70% ¢nal saturation and redissolved in bu¡ered saline. After 24-h dialyses against bu¡ered saline, the eluted proteins were evaluated by sodium dodecyl sulfate^polyacrylamide gel electrophoresis (SDS^PAGE) (12.5% polyacrylamide) and Coomassie blue staining. Sample protein concentration was determined by the Bradford method. 2.4. Inhibition of bacterial adherence assay Adhesion tests were carried out as described previously [2] with some modi¢cations. HeLa cells (1.6U105 cells ml31 , 600 Wl) were grown for 48 h at 37³C in 199 medium with 10% fetal bovine serum in 24-well tissue culture plates containing a cover slip. The medium was replaced with 300 Wl of 199 medium supplemented with 2% fetal bovine serum and 1% D-mannose, followed by 100 Wl of a sample of milk, and 75 Wl of an exponential phase bacterial culture (DAEC, 7U107 cells ml31 ; EAEC, 1U108 cells ml31 ) grown in Trypticase Soy Broth. After 30 min at 37³C, wells were washed with phosphate-bu¡ered saline six times, and 500 Wl of 199 medium with 2% fetal bovine serum was added to each well. Plates were incubated for 3 h at 37³C. Cells were then washed three times with phosphate-bu¡ered saline, ¢xed with methanol and stained with May-Gru«nwald and Giemsa as described previously [2]. Adherence assays were repeated at least three times, and 250 or more HeLa cells were observed in each preparation. The e¡ect of defatted milk, whey proteins, immunoglobulin and non-immunoglobulin fractions on the bacterial adherence was determined by calculating the percentage of cells with less than ¢ve attached bacteria in relation to the control, carried out under identical conditions, but in the absence of milk sample.
2.5. Statistical analysis Di¡erences in percentage of bacterial adhesion were compared statistically by the z test of proportions [15]. 3. Results and discussion SDS^PAGE protein separation of defatted milk (data not shown) and whey (Fig. 1, lane 2) showed a pro¢le of proteins described for human milk : the band of V56.3 kDa represented heavy chain of immunoglobulins; the band of V78 kDa represented lactoferrin and secretory component since their molecular masses are similar; serum albumin and K-lactalbumin were represented by the bands of V65.0 and 14.2 kDa ; while caseins varied from 24.0 to 30.0 kDa [16]. Adhesion assays inhibition showed that defatted milk (1.28 mg ml31 ) and whey proteins (0.97 mg ml31 ) inhibited DAEC and EAEC adhesion to HeLa cells (Table 1). Since binding to a speci¢c extracellular receptor is an essential step for many pathogens, these results indicated that human defatted milk is capable of preventing this important event, inhibiting DAEC and EAEC adhesion to host cells. Furthermore, milk proteins seemed to participate in this inhibitory e¡ect. Several studies have shown an important role of sIgA in the protection e¡ect of human milk [17,18]. To verify the e¡ect of the human milk immunoglobulin and non-immunoglobulin fractions in the DAEC and EAEC adhesion, whey proteins were chromatographed. A typical pro¢le of the eluted proteins from Sephacryl S-200 HR column, which has previously been described by Mestecky and Killian [19], is shown in Fig. 2. The ¢rst peak (P1), at 0.325 mg ml31 , inhibited DAEC and EAEC adherence to HeLa cells (Table 1). In Fig. 1, lane 3, it is possible to verify that P1 contained bands of approximately 56.3 and 78.0 kDa corresponding to heavy chain and secretory
Fig. 1. Coomassie blue-stained SDS^PAGE 12.5% of whey, P1, P2 and P3 proteins : lane 1, molecular mass markers (66, 45, 36, 29, 24, 20.1 and 14.2 kDa, Pharmacia Biotech) ; lane 2, whey; lane 3, pooled fractions from ¢rst peak (P1); lane 4, pooled fractions from peak 2 (P2); and lane 5, pooled fractions from third peak (P3).
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Table 1 Inhibition of DAEC and EAEC adherence to HeLa cells by human milk fractions Bacterial strain
DAEC EAEC
Mean inhibition of adherence (%) in the presence of Defatted milk (1.28 mg ml31 )
Whey (0.97 mg ml31 )
P1 (0.325 Wg ml31 )
P2 (0.225 Wg ml31 )
P3 (0.3 mg ml31 )
29.6 29.3
9.4 15.8
12.1 17.2
6.7 3.5
NS NS
All presented results were signi¢cant (P 6 0.05). NS = not signi¢cant. P1, P2 and P3 denote pooled peaks from Sephacryl S-200 HR.
component of immunoglobulin and bands representing caseins [16]. Precipitated casein at 0.3 mg ml31 was assayed and did not inhibit the bacterial adherence (data not shown). Therefore, it was assumed that P1 inhibitory e¡ect was promoted by the human milk immunoglobulins. Likewise, Caªmara and co-workers have shown the inhibitory e¡ect of milk immunoglobulin fraction on EPEC adhesion [20]. SDS^PAGE of second (P2) and third (P3) peaks (Fig. 1, lanes 4 and 5, respectively) did not show the presence of immunoglobulins, since the band that represents the heavy chain was absent [16]. However, P2 (0.225 mg ml31 ) was able to inhibit the adhesion of DAEC and slightly inhibited the EAEC adherence to HeLa cells (Table 1). SDS^ PAGE analysis showed that lactoferrin, secretory component, serum albumin and casein were present in the pooled fractions from P2, indicating that non-immunoglobulin proteins could be involved in the inhibition of adhesion. On the other hand, at 0.3 mg ml31 , P3 caused no inhibitory e¡ect, and its electrophoretic pro¢le showed only one band corresponding to K-lactalbumin molecular mass [16]. Several studies have suggested the role of human milk glycoconjugates, such as glycolipids, glycoproteins, mucins, glycosaminoglycans and the oligosaccharides, in the protection of neonates, acting as cell surface homologues
to inhibit the pathogen binding to host cell receptors. Many examples of glycoconjugate protective components have been shown: oligosaccharides inhibit the adherence of Streptococcus pneumoniae, ETEC, and invasive Campylobater jejuni and also inhibit the toxicity of the heat-stable enterotoxin of E. coli [21]. The glycoproteins, lactoferrin and secretory component, inhibit the ETEC hemagglutination to human enterocytes [14]; and glycolipids inhibit the adhesion of cholera toxin and heat label enterotoxin to the intestinal epithelia [22]. In this study, it was shown that adherence of two DAEC and EAEC strains was inhibited by defatted milk, immunoglobulins and non-immunoglobulin proteins used in concentrations lower than those found in whole milk. It seems that glycoproteins present in P2 may be involved in the inhibitory e¡ect. Also, these results indicate that human milk components may protect infants against intestinal infections. Acknowledgements This study was supported by FAP-DF, Grant no. 193.375/95, and Nestle¨ Indl. Coml. We wish to thank R.A. Harder for revising the manuscript and Dr. E. Miazaki for statistical assistance.
References
Fig. 2. Chromatography of concentrated whey proteins on Sephacryl S200 HR column (2.6 cmU80.0 cm). The peaks were monitored at 280 nm and eluted with 0.1 M Tris^HCl, pH 7.6, supplemented with 0.5 M NaCl, 1 mM phenylmethanesulfonyl £uoride, NaN3 0.1% and 50 mM O-amino n-caproic acid, pH 7.6; the £ow rate was 120 ml h31 and fractions of 5 ml were collected. The horizontal lines under the respective peaks indicate pooled fractions. P1, P2 and P3 denote the pooled peaks.
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