Adhesionblockade effect

E. coli is one of the major etiological agents of gastrointestinal illnesses in humans and animals. Bacterial adherence to intestinal epithelia is an important step in the pathogenesis of this disease. In the colonization process, bacterial adhesins such as fimbriae may recognize various mammalian subepithelial matrix components as receptors.

Substances that interfere in this host-pathogen interaction could be of therapeutic and prophylactic value, and nonimmunoglobulin fractions of milk, ileal mucus and mucin are among such potential inhibitors (Holmgren et al., 1981; Miedzobrodzki et al., 1989; Olusanya & Naidu, 1991; Cravito et al., 1991).

1. Adhesion-blockade of enteric pathogens. Several carbohydrates, such as 0.1% fucose or 0.5% glucose, as well as LPS (10 (ig/ml) isolated from Shigella jlexneri strongly inhibit the adherence of shigellae to guinea pig colonic cells. Fucose-containing peptides from hLF also inhibit the adhesion of S. Jlexneri to colonic epithelial cells (Izhar et al., 1987)

The non-immunoglobulin component of human milk responsible for the inhibition of E. coli cell adhesion (hemagglutination) mediated by colonization factor antigen I (CFA-I) were identified by chromatographic fractionation of human whey proteins (Giugliano et al., 1995). Free secretory component (fSC) and LF were isolated and both compounds inhibited the hemagglutination by E. coli CFA1+. The lowest concentrations of fSC and LF able to inhibit the hemagglutination by E. coli strain TR50/3 CFA1+ were 0.06 mg/ml and 0.1 mg/ml, respectively. Commercial preparations of LF from human milk and TF from human serum also inhibited the hemagglutination, with MIC values of 0.03 mg/ml and 0.4 mg/ml, respectively.

Bovine LF mediated inhibition of hemagglutination activity of type 1 fimbriated E. coli has also been reported (Teraguchi et al., 1996). The agglutination reaction was specifically inhibited by glycopeptides derived from bLF or a-methyl-D-mannoside. These observations indicate that the glycans of bLF could serve as receptors for type 1 fimbrial lectin of E. coli.

The ability of LF to inhibit in vivo colonization of E. coli has been examined (Naidu et al., unpublished). Infection with E. coli strain F18 was established in streptomycin-treated mice by gastric intubation and bacterial excretion was estimated as colony forming units per gram (CFU/g) feces. The excretion of strain F18 in feces reached a steady-state (108 CFU/g) within 7 days, independent of challenge (dose: 8 x 108 or 103 CFU). Oral administration of bLF (20 mg/ml in 20% sucrose solution) caused a 1- to >3-log reduction in CFU/g feces with high and low dosages of strain F18. The bacterial multiplication in vivo was markedly affected during the early 24 hours of infection, reflecting >3-log lower number of bacteria in the feces (2 x 103 CFU/g) than the control group. Oral administration of LF prior to infection reduced fecal excretion of E. coli from mouse intestine. In vitro effects of bLF on the molecular interactions of E. coli with subepithelial matrix proteins were examined. Bovine LF inhibited the binding of 125I-labeled fibronectin, fibrinogen, collagen type-I, collagen type-IV and laminin to bacteria. This inhibitory effect was bLF dose-dependent, and was independent of coexistence (competitive) or préexistence (non-competitive) of bLF with the tissue matrix proteins. In displacement studies with bacteria-matrix protein complexes, bLF dissociated only collagen type-I and laminin interactions. Electron microscopy revealed the loss of type-1, CFA-I and CFA-II fimbria of E. coli grown in broth containing 10 uM LF. The inhibitory affect of LF on fimbrial expression was further confirmed by hemagglutination and yeast cell agglutination. The presence of 10 |_iM LF in the growth media, however, did not affect the P-fimbriation in E.coli. These data suggest a strong influence of LF on adhesion-colonization properties of E.coli.

2. Adhesion-blockade of oral pathogens. The influence of LF, salivary proteins (SP) and BSA on the attachment of Streptococcus ¬°nutans to hydroxyapatite (HA) was reported (Visca et al., 1989). Sorption of LF, SP, and BSA to HA was dependent on the protein concentration and reached the end-point at about 80 mg of proteins per gram of HA. Similarly, the number of streptococci adsorbed to HA was correlated to the amount of cells available up to at least 107 cells per mg of HA. The adsorption of LF, SP and BSA on HA reduced the number of attaching S. mutans cells. In particular, SP reduced the adsorption of S. mutans by 30%, whereas pre-coating of HA with apo- or iron-saturated LF resulted in a three orders of magnitude reduction of S. mutans adsorption to HA. The potent adherence-inhibiting effect of apo-LF together with its antibacterial activity against S. mutans suggests an important biological significance of these phenomena in the oral cavity.

Whole cells of P. intermedia demonstrate a high degree of binding to fibronectin, collagen type I and type IV and laminin, whereas a moderate interaction was detected with fibrinogen. The ability of bLF to affect the interactions of the above proteins with P. intermedia was examined (Alugupalli et al., 1994). In the presence of unlabeled bLF, a dose-dependent inhibition of binding was observed with all five proteins tested. Unlabeled bLF also dissociated the bacterial complexes with these proteins. The complexes with laminin or collagen type I were more effectively dissociated than fibronectin or fibrinogen, whereas the interaction with collagen type IV was affected to a lesser extent. A strain-dependent variation in the effect of bLF was observed.

The ability of hLF and bLF to inhibit adhesion of A actinomycetemcomitans and P. intermedia to monolayers of fibroblasts, HEp-2, KB and HeLa cells was reported (Alugupalli & Kalfas, 1995). The inhibitory effect was dose-dependent in the concentration range 0.5-2500 |ig/ml and not related to the bacterial growth phase. In the presence of LF, decreased association of bacteria with the cell monolayers was also found by microscopic examination of the preparations. These data suggested a possibility that LF could prevent the establishment of bacteria in periodontal tissues through adhesion-counteracting mechanisms in addition to its bacteriostatic and bactericidal properties.

LF also binds to fibroblast monolayers and matrigel, a reconstituted basement membrane, through ionic interactions. The adhesion of A. actinomycetemcomitans to these substrata was mainly dependent on the ionic strength of the environment. P. intermedia and P. nigrescens also adhere to fibroblasts mainly by ionic interactions, while their adhesion to matrigel seems to be mediated by specific mechanisms. Lectin-type interactions were not found involved in the binding of these bacteria to the substrata. Treatment of either A. actinomycetemcomitans or fibroblasts with LF decreased the adhesion in a dose-dependent manner, while LF treatment of matrigel alone had no adhesion-counteracting effect. Adhesion of P. intermedia and P. nigrescens to matrigel was not significantly affected by the ionic strength, but the presence of LF inhibited the adhesion. LF bound to matrigel, P. intermedia and P. nigrescens was rapidly released, while LF bound to A. actinomycetemcomitans and fibroblasts was retained. These findings indicate that LF-dependent adhesion-inhibition of A. actinomycetemcomitans, P. intermedia and P. nigrescens to fibroblasts and matrigel could involve binding of LF to both the bacteria and substrata. The decreased adhesion may be due to blocking of both specific adhesin-ligand as well as non-specific charge-dependent interactions (Alugupalli & Kalfas, 1997).

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Figure 6. LFcin isolation by reverse-phase HPLC. Bovine LF was hydrolyzed with porcine gastric pepsin at pH 4.0 and the hydrolysate was fractionated on a Pep-S column. Shaded peaks are fractions with antimicrobial activity against E.coli H10407 in a microplate assay [Naidu & Erdei, unpublished data]. The amino acid sequence and the primary structures of bLFcin and hLFcin peptides are shown with basic residues encircled and sequence positions numbered [adapted from Bellamy et al., 1992],

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P hLFcin p

p bLFcin s

Figure 6. LFcin isolation by reverse-phase HPLC. Bovine LF was hydrolyzed with porcine gastric pepsin at pH 4.0 and the hydrolysate was fractionated on a Pep-S column. Shaded peaks are fractions with antimicrobial activity against E.coli H10407 in a microplate assay [Naidu & Erdei, unpublished data]. The amino acid sequence and the primary structures of bLFcin and hLFcin peptides are shown with basic residues encircled and sequence positions numbered [adapted from Bellamy et al., 1992],

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