Effect of the vitamin B12-binding protein haptocorrin present in human milk on a panel of commensal and pathogenic bacteria

Jensen et al. BMC Research Notes 2011, 4:208 http://www.biomedcentral.com/1756-0500/4/208 SHORT REPORT Open Access Effect of the vitamin B12-binding protein haptocorrin present in human milk on a panel of commensal and pathogenic bacteria Henrik R Jensen1†, Martin F Laursen1†, Dorte L Lildballe2, Jens B Andersen1, Ebba Nexø2 and Tine R Licht1* Abstract Background: Haptocorrin is a vitamin B12-binding protein present in high amounts in different body fluids including human milk. Haptocorrin has previously been shown to inhibit the growth of specific E. coli strains, and the aim of the present study was to elucidate whether the antibacterial properties of this protein may exert a general defense against pathogens and/or affect the composition of the developing microbiota in the gastrointestinal tracts of breastfed infants. Findings: The present work was the first systematic study of the effect of haptocorrin on bacterial growth, and included 34 commensal and pathogenic bacteria to which infants are likely to be exposed. Well-diffusion assays addressing antibacterial effects were performed with human milk, haptocorrin-free human milk, porcine holohaptocorrin (saturated with B-12) and human apo-haptocorrin (unsaturated). Human milk inhibited the growth of S. thermophilus and the pathogenic strains L. monocytogenes LO28, L. monocytogenes 4446 and L. monocytogenes 7291, but the inhibition could not be ascribed to haptocorrin. Human apo-haptocorrin inhibited the growth of only a single bacterial strain (Bifidobacterium breve), while porcine holo-haptocorrin did not show any inhibitory effect. Conclusions: Our results suggest that haptocorrin does not have a general antibacterial activity, and thereby contradict the existing hypothesis implicating such an effect. The study contributes to the knowledge on the potential impact of breastfeeding on the establishment of a healthy microbiota in infants. Findings Background and hypothesis Haptocorrin (previously named transcobalamin I and III, and R-binder) is a glycoprotein, which is present in human milk in relatively large amounts [1,2]. It is characterized by its ability to bind vitamin B12 and other corrinoids [3], and exists in two forms in human milk; apohaptocorrin, which is unsaturated with vitamin B12, and holo-haptocorrin, which is saturated with vitamin B12 [4]. Human milk contains a much higher concentration of apo-haptocorrin than of holo-haptocorrin [1,2], but the role for the high amount of apo-haptocorrin in human milk is unknown. Furthermore, the function of haptocorrin in different body fluids, i.e. secreted from * Correspondence: † Contributed equally 1 National Food Institute, Mørkhøj Bygade 19, Technical University of Denmark, DK-2860 Søborg, Denmark Full list of author information is available at the end of the article exocrine glands or released by erythrocytes and leucocytes, remains unknown. However, the current concept is that haptocorrin in adults is responsible for transporting dietary vitamin B12 through the stomach to the intestine. Haptocorrin is believed to be degraded by the intestinal enzymes and the vitamin B12 released from the protein is subsequently bound to intrinsic factor and absorbed in the distal part of ileum [5]. It has been proposed that haptocorrin contributes to the transport and absorption of vitamin B12 in infants while the gastric secretion of IF is not sufficiently developed [6]. On the other hand, the high amount of apo-haptocorrin in human milk has lead to suggestions that haptocorrin, by withholding vitamin B12, exhibits antimicrobial effects in the intestine and thereby potentially protects the infant against pathogenic bacteria [1,7]. Additionally, we speculate that an antibacterial (protective) function of haptocorrin could explain its presence in various body fluids. © 2011 Licht et al; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Jensen et al. BMC Research Notes 2011, 4:208 http://www.biomedcentral.com/1756-0500/4/208 The possible effect of haptocorrin on bacteria has been discussed since the late 1970’s [1,7-9]. However, only a very limited number of studies addressing such an effect have been performed, and these studies have focused on the effect of haptocorrin on E. coli [8-10]. Samson et al demonstrated that apo-haptocorrin, derived from human milk, inhibits the growth of the vitamin B12-dependent E. coli N.C.I.B. 8134 [8]. This effect was shown to be diminished by pretreatment with trypsin [9]. The gastrointestinal tract of infants differs from the adult gut in terms of higher pH and lower trypsin activity [11-14], thus haptocorrin may still be functional in the upper part of the intestine during infancy. Indeed, it has been demonstrated that porcine haptocorrin is not degraded in vitro when subjected to an environment simulating the gastrointestinal tract of infants [10]. Previous studies have shown that both apo-and holohaptocorrin inhibits the growth of a single enteropathogenic E. coli O127 strain (EPEC) [10], thereby suggesting that the haptocorrin moity rather than its ability to bind cobalamin inhibits the growth of pathogens [15]. However, to date no study has systematically investigated the effect of haptocorrin (saturated or unsaturated) on a panel of bacteria. Recently, the importance of the first colonizing bacteria in infants for the development of a healthy immune system has been highlighted [16,17], and it is known that the microbiota of breastfed infants differ from that of formula fed infants [18]. We hypothesized that haptocorrin from human milk, by inhibiting the growth of certain bacteria and thereby favoring the growth of other bacteria, may influence the initial colonization in breastfed infants. We therefore performed the first systematical study of the effect of haptocorrin on a panel of 34 commensal and pathogenic bacteria to which infants may be exposed, and conclude that human unsaturated haptocorrin had no general antibacterial effect, but was seen to inhibit the growth of Bifidobacterium breve, which is commonly found in the intestinal tract of neonates. Materials and methods Biochemical methods The concentration of vitamin B12, in BHI and MRS media, was analyzed employing the Cobas 6000 E immunoassay system and the analytical kit supplied by the manufacturer (Roche). Unsaturated vitamin B12 binding capacity (UB12BC) in BHI and MRS was measured as described previously [19]. The vitamin B12 content and the UB12BC of Clostridium difficile media were not measured due to lack of the liquid form of the specific agar media used in the well-diffusion assay. Page 2 of 6 Human milk Human donor milk, collected at Randers Hospital, Denmark was centrifuged at 10,000 g, 4°C, 15 min. 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