J. Dairy Sci. 86:2276-2282
© American Dairy Science Association, 2003.
Pseudomonas aeruginosa Lectin PA-IIL as a Powerful Probe for Human and Bovine Milk Analysis
E. Lesman-Movshovich and
N. Gilboa-Garber
Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel
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ABSTRACT
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Milk composition exhibits species-specific differences depending on genetic, evolutionary, and environmental factors. In addition, commercial milk preparations are also changed by industrial manipulations, including severe heat processing. Cow milk, used as human food, provides important nutrients but lacks some essential components that are present in raw human milk. The present study, which was aimed at comparing infant breastfeeding to cow-based formula nourishment, shows major differences between the human and the commercial cow milk glycans detectable by the lectins PA-IL (galactose-binding) and PA-IIL (fucose and mannose-binding) isolated from the cells of human pathogen Pseudomonas aeruginosa. More than 40 human milk samples, several cow milks, and bovine milk-based infant formulas, were examined using these two lectins. For purposes of comparison, the plant lectins Concanavalin A (Con A), which binds mannose, and Ulex europaeus 1st lectin (UEA-I), which binds fucose, were also used. The most prominent difference was revealed using PA-IIL, which displayed a unique high sensitivity to the human milk fucosylated compounds. PA-IL and UEA-I also exhibited preferential sensitivity to the human milk but considerably lower than that of PA-IIL. Con A was inhibited by human and the other milk preparations examined to the same extent. These findings indicate the superb applicability of PA-IIL for rapid and reliable comparative investigation of milk glycans from human and cow, indicating which glycans could be added to infant formulas in order to enrich them, as well as for verification and quality control of otherwise improved bovine milk-based infant formulas.
Key Words: human milk • bovine milk • bacterial lectin • milk analysis
Abbreviation key: Con A = Concanavalin A, PA-IL = P. aeruginosa 1st lectin, PA-IIL = P. aeruginosa 2nd lectin, UEA-I = Ulex europaeus 1st lectin
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INTRODUCTION
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Mammalian milk is composed of mammary gland products and serum components that passively diffuse to it (Jensen, 1995). It supplies the newborn with nutrients, enzymes, growth factors, and hormones, as well as protection against infections (Howie et al., 1990; Jensen, 1995; Cesar et al., 1999; Bernt and Walker, 2001). The latter is mainly provided by secretory immunoglobulins, lipids, and oligosaccharides (free and conjugated to macromolecules, including proteins; Wold et al., 1990; Hennart et al., 1991; Hamosh, 1998). The milk carbohydrates may generally act as decoys, competitively inhibiting the microbial adhesion to the target host receptors (present on cell surface or extracellular matrix), which are similar in composition (Newburg, 2001). The variability of both the milk and the host cell receptor saccharides depends on genetic and environmental factors and is related to selective sensitivity to pathogens. Experimental examples for the efficiency of human milk glycosylated molecules to prevent bacterial adhesion to cells were shown both in vitro (Cravioto et al., 1991) and in vivo (Cleary et al., 1983; Newburg et al., 1990), including clinical usage of human milk to cure several human infections (Andersson et al., 1986).
The original aim of the present research was to compare the efficiency of commercial heat-processed bovine milk to raw human milk in blocking the lectin-dependent adhesion of pathogenic bacteria to human cells. For this purpose, we used the lectins of Pseudomonas aeruginosa, which is a widespread, aggressive, opportunistic pathogen involved in severe human infections. Pseudomonas aeruginosa produces several lectins (Gilboa-Garber, 1986; Gilboa-Garber et al., 1997). One of them, P. aeruginosa 1st lectin (PA-IL), binds galactose and its derivatives (Gilboa-Garber, 1972, 1982; Chen et al, 1998), resembling P-fimbriae of Escherichia coli (Kallenius et al., 1980; Leffler and Svanborg-Eden, 1981), and the second one, P. aeruginosa 2nd lectin (PA-IIL), binds fucose, arabinose, and mannose (Gilboa-Garber, 1982; Garber et al., 1987; Gilboa-Garber et al., 2000). In its interaction with mannose, PA-IIL resembles the mannose-sensitive type 1 fimbriae (Duguid et al., 1979; Neeser et al., 1986; Abraham et al., 1988). The production of these P. aeruginosa lectins was shown to be regulated in association with extracellular virulence factors (Gilboa-Garber et al., 1997; Winzer et al., 2000). The scientific advantage of these two lectins over most other microbial lectins is their availability in a purified and stable state (Gilboa-Garber, 1982).
We examined the ability of human and cow milks, as well as bovine milk-based infant formulas, to block the lectins PA-IL and PA-IIL using the hemagglutination inhibition test. For comparison, the plant lectins Concanavalin A (Con A) and Ulex europaeus 1st lectin (UEA-I, which are related to PA-IIL in their sugar specificities (mannose and fucose, respectively), were also used. Because the results indicated a most dramatic differential sensitivity of PA-IIL to inhibition by human milks, we concentrated on exploring its efficiency as a reliable probe for the composition of human, bovine, and bovine-based infant formula glycoprotein glycans, using Western blot analysis.
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MATERIALS AND METHODS
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Lectin Preparations
Purified (single band in SDS-PAGE) PA-IL and PA-IIL were prepared in our laboratory from P. aeruginosa ATCC 33347 cells by disintegration of the cells using ultrasonic vibrations, centrifugation, and separation of the supernatant fluid, heating at 70°C for 15 min, precipitation by 70% saturation of ammonium sulfate at 4°C, collection of the precipitate by centrifugation, dissolving the precipitate in PBS, and affinity chromatography on Sepharose 4B or Sepharose-mannose, respectively, as previously described (Gilboa-Garber, 1982). Con A and UEA-I were purchased from Sigma Chemical Co. (St. Louis, MO). Peroxidase-labeled PA-IL, PA-IIL, and Con A were prepared using glutaraldehyde coupling (Avrameas, 1969; Huet and Garrido, 1972). Peroxidase-labeled UEA-I was purchased from Sigma Chemical Co.
Human Erythrocytes
O-type human bloods of healthy donors (from the Central Blood Bank of Sheba Hospital, Tel Hashomer) were used. They were centrifuged, and the erythrocyte fraction was separated, washed three times, and suspended in PBS (5%, vol/vol). The cells were treated by 0.1% papain at 37°C for 30 min and again washed three times and suspended in saline, as previously described (Gilboa-Garber, 1982).
Milk Preparations
Forty human milk samples were obtained from healthy mothers at various breast-feeding stages. Each sample was centrifuged (3000 x g) at 4°C for 10 min, and the intermediate skim phase was carefully collected. Commercial human milk lactoferrin was purchased from Sigma Chemical Co. Cow and several infant formulas were purchased from supermarkets. A portion of each milk sample was dialyzed at 4°C overnight to remove free oligosaccharides and other low molecular weight glycosylated compounds but not the glycoproteins, using a cellulose membrane with cut-off size of MW 10,000 Da, purchased from Sigma Chemical Co. The protein concentration of the milk samples, verified using the method of Lowry et al. (1951), was ~15 mg•ml-1.
Assay of Lectin Hemagglutinating Activity and its Inhibition by the Milk Preparations
Fifty microliters of each purified lectin solution (containing 1 µg of lectin protein) were serially diluted with 50 µl of saline to a final dilution of 1/256. Then, 50 µl of saline and 50 µl of the erythrocyte suspension were added to each tube. After 30 min at room temperature, the tubes were centrifuged for 30 s (1000 x g), and the hemagglutination intensity was macroscopically examined (Gilboa-Garber, 1982). The highest lectin dilution leading to agglutination of all the erythrocytes in one mass was chosen for the inhibition tests. In the hemagglutination inhibition tests, the milk preparations examined were serially twofold diluted in 50 µl and mixed with 50 µl of the lectin solution in the chosen dilution. Following 30 min of incubation and addition of 50 µl of the erythrocyte suspension, hemagglutination was examined as described above. The inhibition intensity was considered as the number of tubes in which no hemagglutination was observed.
Statistical Evaluation
The results of the hemagglutination inhibition tests were analyzed by Student t-test, and P-value was calculated.
SDS-PAGE and Western Blotting
Fifteen microliters of each human milk sample containing 15 mg of protein/ml (diluted 1:2) or of undiluted cow milk or formula, at the same concentration, as well as commercial human lactoferrin at a 1 mg/ml concentration, were diluted 1:1 with sample buffer [0.1 M Tris-HCl, pH 6.8, containing 4% (wt/vol) sodium dodecyl sulfate (SDS), 20% (vol/vol) glycerol, 0.2 M dithiothreitol (DTT), and 0.2% (wt/vol) bromophenol blue], boiled for 3 min, and applied to the wells in the 10% SDS-PAGE gel. The electrophoresis was carried out (at 140 V) in Mini Protean Cell II Electrophoresis (Bio-Rad, Hercules, CA) at room temperature, according to Laemmli (1970), and stopped when the bromophenol line reached the gel bottom. The gels were analyzed in parallel by Coomassie brilliant blue (CBB) staining (for protein detection) and by Western blotting (for specific glycoprotein-tagging).
Western Blot Analyses
Following SDS-PAGE, proteins were transferred to nitrocellulose (0.45 µm, Bio-Rad) membrane (Towbin et al., 1992) at 4°C for 2 h (85 mA/40–50 V, using the Minitrans-Blot Module, Bio-Rad, Hercules, CA).
The membranes were incubated overnight in blocking buffer (PBS 0.01 M, pH 7.2, containing 3% BSA and 0.05% Tween 20) for prevention of nonspecific lectin binding. They were then exposed to peroxidase-labeled lectin (about 1 µg•ml-1) in blocking buffer (with 0.1% Tween 20), at room temperature for 2 h.
Following thorough washing, the peroxidase reaction was visualized using enhanced chemiluminescence (Amersham International plc, Buckinghamshire, UK), according to the manufacturer’s instructions. The resulting luminescence was recorded onto photographic film. Several exposures were performed for each blot in order to gain greatest sensitivity.
To rule out possible staining of the electroblots by nonspecific lectin binding (which may be due to protein-protein interactions not related to the sugar-binding site), controls with the labeled lectins, each one in presence of 0.3 M of its relevant blocking sugar (mannose for Con A, fucose for both UEA-I and PA-IIL, and galactose for PA-IL), were added.
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RESULTS
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Inhibition of the Lectin Hemagglutinating Activities by Human and Cow Milks and by Infant Formulas
All the lectins examined were inhibited by all 40 human milk samples, but the inhibition of PA-IIL by them was the most pronounced (Figure 1
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Figure 1. Inhibition of Con A, UEA-I, PA-IL, and PA-IIL hemagglutinating activities by human milk (H), cow milk-based infant formula (F), and industrial cow milk (C) before and following dialysis. The details are as described in the Materials and Methods section. The hemagglutination inhibition values represent the number of tubes in which there was no hemagglutination. The data represent means ± SEM of results obtained with about 40 different human milk samples, cow milk samples from two different manufacturers, and samples of two commercial formula types. An asterisk denotes highly significant differences (P < 0.01): one asterisk between each lectin interaction with human milk vs. cow milk preparations, and another asterisk between PA-IIL and the other three lectins. The letters a to g above columns represent results which are not significantly different.
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The four lectins varied in their sensitivity to inhibition by the cow milk and the infant formulas based on cow milk: Con A was inhibited by cow milk and formula (both undialyzed and dialyzed) to the same extent as by human milk. PA-IL was inhibited by undialyzed cow milk, less than by human milk, but that inhibition disappeared following dialysis of the cow milk and formula. In contrast, PA-IIL and UEA-I were not inhibited by any of the cow milk preparations examined, in sharp contrast to their high sensitivity (especially that of PA-IIL) to the human milk (both undialyzed and dialyzed).
SDS-PAGE and Western Blots of the Human and Bovine Milk Proteins
Figure 2 shows CBB-stained SDS-PAGE and lectin-tagged Western blots of commercial human milk lactoferrin, six human milks (chosen to represent the 40 examined), one bovine milk-based infant formula (representing the two types examined), and one cow milk (representing the two different industrial sources). As can be seen in this figure (A to D), the human milk glycoproteins were differentially tagged by the four peroxidase-labeled lectins. Con A most heavily stained lactoferrin (78 kDa) and IgA heavy chain (58 kDa). PA-IL interactions were weaker, and a lower number of glycoproteins were stained by it as compared to Con A, while UEA-I and PA-IIL interacted with most of the human milk glycoproteins.
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Figure 2. SDS-PAGE and Western blotting of commercial human milk lactoferrin (LF), six (1–6) human milks, bovine milk-based infant formula (F) and cow milk (C). Full range Rainbow standards (Amersham) were used as Mr markers. Lanes 1 to 6 contain six diverse human milk samples (obtained from two donors at several different breast feeding stages, including one colostrum, #1) chosen to represent the whole group. Lanes F and C contain representative samples of bovine milk-based infant formula and cow milk. The Western blots were tagged by peroxidase-labeled Con A (A), UEA-I (B), PA-IL (C) and PA-IIL (D). In their controls (A'–D') 0.3 M mannose (for Con A), fucose (for UEA-I and PA-IIL) or galactose (for PA-IL) were included. The SDS-PAGE was stained by CBB (E). The Mr regions noted by the arrows: I around 78 kDa (lactoferrin); II around 67 kDa (albumin); III around 58 kDa Ig(A+G) heavy chains; IV around 46 to 48 kDa lactadherin and CD14; V around 24 to 30 kDa casein + Ig(A+G) light chains; VI around 14 to 15 kDa  -lactalbumin and lysozyme.
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The results obtained comparing human milk vs. cow milk tagging in the Western blot analyses were consistent with those of the hemagglutination inhibition results: Whereas Con A tagged human and cow (including bovine milk-based infant formula) glycoproteins almost to the same extent, PA-IL and especially PA-IIL and UEA-I, scarcely tagged any protein in the bovine milk samples, in contrast to their considerable tagging of the human milk glycoproteins.
The Western blot controls in presence of sugars, as described in the Materials and Methods section, showed that the bulk of the tagging by the peroxidase-labeled lectins was sugar-specific, since only a few faint bands appeared in them (Figure 2A'–D'
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CBB staining of the SDS-PAGE (Figure 2E) showed the pattern of total protein distribution in the same samples as those used for the Western blottings. The cow milk casein types, which were not tagged by the lectins in the Western blots, were most strongly stained by CBB.
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DISCUSSION
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Human milk oligosaccharides were reported to pass largely undigested through the infant intestinal tract and, therefore, have been suggested to act as decoys that hinder pathogen adhesion by competition with their respective receptors on the target human cells (Newburg, 2000). Reports on inhibition of microbial adherence to target cells by such compounds included: Streptococcus pneumoniae (Andersson et al., 1986), enteropathogenic Escherichia coli (Cravioto et al., 1991), and Campylobacter jejuni (Newburg, 2000). The binding of the heat-stable toxin of E. coli to its target cells was also inhibited by human milk oligosaccharides (Cleary et al., 1983; Crane et al., 1994). An additional example to the milk carbohydrate protective function is inhibition of rotavirus infection by the 46-kDa glycoprotein lactadherin, which was shown to block the virus binding to the host intestinal (mucosal) cells (Yolken et al., 1992; Newburg et al., 1998).
The oligosaccharides which blocked the Campylobacter and the E. coli toxin were shown to be fucosylated by use of Ulex europaeus lectin (Newburg et al., 1990; Newburg, 2000). The high efficiency of these fucosylated glycoforms, despite their very low concentration in the milk, has led to the suggestion that the affinity of the toxin to them is very high (Newburg, 2000).
It has been shown that the lectins of P. aeruginosa contribute to its adhesion to target cells (Gilboa-Garber, 1986; Wentworth et al., 1991; Gilboa-Garber et al., 1994) and that fucosylated antigens are receptors for this adhesion, which may be blocked by fucosylated compounds (Scharfman et al., 1999, 2001). Therefore, we examined the inhibition of the P. aeruginosa lectins by human milks, using hemagglutination inhibition test. Because PA-IIL binds both mannose and fucose, its interactions with the milk components were compared not only to PA-IL but also to those of the plant lectins Con A, which binds both mannose (Goldstein and Poretz, 1986) and mannosylated glycoproteins (Shepherd and Montgomery, 1978; Yamashita et al., 1978) and UEA-I that is fucose-specific.
As shown in Figure 1, we found a strong inhibition of PA-IIL by human milk, far exceeding that of Con A, UEA-I, and PA-IL (Figure 1). The hemagglutination inhibition data showed that Con A inhibition by cow and human milks was similar and that most of the inhibitory activities of these two milk types towards that lectin were due to glycoproteins retained following dialysis. The cow milk also inhibited PA-IL, but this inhibition, in contrast to that obtained with the human milk, was mainly due to low molecular weight galactosides, including the milk sugar lactose, removable by dialysis. Different results were obtained with UEA-I and PA-IIL: Both of them were found to be selective for human milk. Their inhibition by the cow milk was negligible, as opposed to that obtained with the human milk, which was retained following dialysis. The dramatic PA-IIL sensitivity to the human milk, much higher than that of UEA-I, is ascribed to its high affinity interactions with fucosylated chains of both H and Lewis types.
Following the findings in the hemagglutination inhibition test, we investigated the efficiency of the same lectins as detectors of the active glycoproteins of the examined milks in Western blots and the efficiency of PA-IIL as a probe for them.
As shown in the SDS-PAGE analysis (Figure 2), CBB clearly stained human milk lactoferrin (region I, 78 kDa), albumin (region II, 67 kDa), Ig(A+G) heavy chain (region III, 58 kDa; Murakami et al., 1998) casein (24–30 kDa; Kunz and Lonnerdal, 1990), and Ig (A+G) light chains (24 kDa) both in region V, as well as -lactalbumin and lysozyme (region VI, 14–15 kDa; Murakami et al., 1998). It also weakly stained the region of lactadherin and CD14 (region IV, 46–48 kDa; Newburg et al., 1998; Labeta et al., 2000). In the cow milk and bovine milk-based infant formula, strongest staining by CBB was located mainly in the casein region (from 24 to 30 kDa) due to the high concentration of these proteins in cow milk (-casein 11.9 mg/ml and ß - 9.8 mg/ml), as reported by Swaisgood (1995). The molecular weights were verified using the standard markers as described in the Materials and Methods section.
The Western blot analyses (Figure 2) showed that Con A tagged most of the above-noted human milk glycoproteins (not albumin and immunoglobulin light chains, which are not glycosylated) and additional ones (not stained by CBB) at the range of 30 to 48 kDa. PA-IIL and UEA-I tagged more human milk glycoproteins than Con A, far surpassing PA-IL, which interacted mainly with lactoferrin (and weakly with few additional bands, including one of around 24 kDa in the colostrum). The interactions of PA-IL and PA-IIL with lactoferrin accord with the report on the presence of terminal galactose and N-acetylgalactosamine residues and fucosylated branches in that molecule (Eda et al., 1997).
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CONCLUSIONS
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The examined lectins, owing to their sugar specificity, are very useful for the study of glycosylated milk components. They may serve for detection of carbohydrate presence, estimation of their levels, and investigations of those, which are crucial for abrogation of bacterial adherence. As shown in the present paper, PA-IIL is superb as a rapid, reliable, and sensitive probe for fucosylated derivatives, which are common in human milk. Moreover, this lectin may be used in the future as a sensitive probe for quality control of infant milk formulas improved by supplying fucosylated derivatives mimicking human milk fucosylated glycans. PA-IIL stability in a purified state and its origin from a ubiquitous antibiotic-resistant pathogenic bacterium, are advantages for its usage as a tool for milk standardization, not only for infant nutrition but also for antiadhesive strategies that are urgently needed for clinical treatment of adult antibiotic-resistant infections.
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ACKNOWLEDGEMENTS
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The authors thank Ms. Sharon Victor for the skillful typing of this manuscript and Ms. Ella Gindy for the graphic presentation. This study, which was supported by the Bar-Ilan University Research and Israel Governmental Estate Property Funds, is part of Efrat Lesman-Movshovich’s Ph.D. thesis.
Corresponding author:
N. Gilboa-Garber; e-mail:
garbern{at}mail.biu.ac.il.
Received for publication October 9, 2002.
Accepted for publication January 6, 2003.
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