Lactoferrin, a member of the transferrin family of approximately 80 kDa, consists of a single polypeptide chain folded in two symmetric, globular lobes (N- and C-lobes), each able to bind one ferric ion. This glycoprotein, found in physiological fluids of mammals, plays an important role in immune regulation and in defense mechanisms against bacteria, fungi, parasites, and viruses. Although the antiviral activity of lactoferrin is one of the major biological functions of such protein, the mechanism of action is still under debate. We have investigated both the role of tryptic fragments of bovine lactoferrin and the mechanism of lactoferrin antiviral effect toward adenovirus infection in HEp-2 cells. The results obtained demonstrated that the anti-adenovirus activity of lactoferrin is mediated by the N-terminal half of the protein as the N-lobe was able to inhibit adenovirus infection, even if at lower extent than undigested lactoferrin, whereas C-lobe was ineffective. The results also showed that the anti-adenovirus action of lactoferrin and of its N-terminal peptide lactoferricin took place on virus attachment to cell membrane, mainly through competition for common glycosaminoglycan receptors. The data provide evidence that the anti-adenovirus activity of lactoferrin is mediated mainly by the cluster of positive charges at the N-terminus of whole molecule and that the N-terminal peptide lactoferricin alone is sufficient to prevent infection.

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We recently demonstrated that lactoferrin, an antimicrobial glycoprotein, can inhibit adenovirus infection by competing for common glycosaminoglycan receptors. This study further characterizes the antiadenovirus activity of the protein, thus demonstrating that lactoferrin neutralizes infection by binding to adenovirus particles and that its targets are viral III and IIIa structural polypeptides.Lactoferrin is a multifunctional glycoprotein that possesses a variety of physiological roles, such as promotion of iron absorption (26), immunomodulation (16), and inhibiting activity toward different pathogens (14,16,24,26,(32)(33)(34), including viruses (1, 2, 7, 9-13, 17-19, 21, 25, 28-30, 38). For all viruses investigated to date, lactoferrin exerts its antiviral activity during the early phases of infection. Recently, we demonstrated a competition between bovine lactoferrin (bLf) and adenovirus for the attachment to cell membrane glycosaminoglycans, even though a binding between lactoferrin and viral particles could not be ruled out (6). The present study provides deeper insight into the mechanism of the antiadenoviral action of bLf, demonstrating that this glycoprotein also exerts its antiadenoviral activity by a direct interaction with adenovirus particles.Human epidermoid carcinoma larynx (HEp-2) cells, obtained from the American Type Culture Collection (ATCC), and human adenovirus type 2 were grown as previously described (2). Viral particles were purified according to the methods of Wadell and coworkers (35).bLf (Morinaga Milk Industry) and heparin (Sigma, 170 USP U/mg) were dissolved in phosphate-buffered saline (PBS; pH 7.2). bLf was biotinylated according to the manufacturer's instructions (Amersham). Dot blot assays were carried out by applying 50 l of purified adenovirus, heparin, or PBS to nitrocellulose paper and using a Bio-Dot apparatus according to the manufacturer's instructions (Bio-Rad). Air-dried membranes were blocked in block solution, washed with PBS containing 0.1% Tween 20, and then incubated with biotinylated bLf. After washings in PBS containing 0.1% Tween 20, streptavidin-horseradish peroxidase (streptavidin-HRP) conjugate was added to the blot. Blot was revealed by an enhanced chemiluminescence kit (Amersham Biosciences) and the 3,3Ј,5,5Ј-tetramethylbenzidine (TMB) substrate kit for peroxidase (Vector Laboratories) according to the manufacturer's instructions.Neutralization of adenovirus binding to cells was carried out by incubating lactoferrin with adenovirus for 1 h at 37 or 4°C.The suspensions were then added to cells for 1 h at 4°C. Viral antigen synthesis was monitored 24 h after infection (37°C) by immunofluorescence as previously described (2). Statistical analysis was performed using the Student's t test for unpaired data. P values of Ͻ0.05 were considered significant. Adenovirus proteins were resolved by sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis (SDS-PAGE) (15) under denaturing conditions as described by Wadell and coworkers (36). Coxsackievirus proteins were...

It is well known that lactoferrin (Lf) is a potent inhibitor towards several enveloped and naked viruses, such as rotavirus, enterovirus and adenovirus. Lf is resistant to tryptic digestion and breast-fed infants excrete high levels of faecal Lf, so that its effect on viruses replicating in the gastrointestinal tract is of great interest. In this report, we analysed the mechanism of the antiviral action of this protein in three viral models which, despite representing different genoma and replication strategies, share the ability to infect the gut. Concerning the mechanism of action against rotavirus, Lf from bovine milk (BLf) possesses a dual role, preventing virus attachment to intestinal cells by binding to viral particles, and inhibiting a post adsorption step. The BLf effect towards poliovirus is due to the interference with an early infection step but, when the BLf molecule is saturated with Zn+2 ions, it is also capable of inhibiting viral replication after the viral adsorption phase. The anti-adenovirus action of BLf takes place on virus attachment to cell membranes through competition for common glycosaminoglycan receptors and a specific interaction with viral structural polypeptides. Taken together, these findings provide further evidence that Lf is an excellent candidate in the search of natural agents against viral enteric diseases, as it mainly acts by hindering adsorption and internalisation into cells through specific binding to cell receptors and/or viral particles.

In Listeria monocytogenes the acid tolerance response (ATR) takes place through a programmed molecular response which ensures cell survival under unfavorable conditions. Much evidence links ATR with virulence, but the molecular determinants involved in the reactivity to low pHs and the behavior of acid-exposed bacteria within host cells are still poorly understood. We have investigated the effect of acid adaptation on the fate of L. monocytogenes in human macrophages. Expression of genes encoding determinants for cell invasion and intracellular survival was tested for acid-exposed bacteria, and invasive behavior in the human myelomonocytic cell line THP-1 activated with gamma interferon was assessed. Functional approaches demonstrated that preexposure to an acidic pH enhances the survival of L. monocytogenes in activated human macrophages and that this effect is associated with an altered pattern of expression of genes involved in acid resistance and cell invasion. Significantly decreased transcription of the plcA gene, encoding a phospholipase C involved in vacuolar escape and cell-to-cell spread, was observed in acid-adapted bacteria. This effect was due to a reduction in the quantity of the bicistronic plcA-prfA transcript, concomitant with an increase in the level(s) of the monocistronic prfA mRNA(s). The transcriptional shift from distal to proximal prfA promoters resulted in equal levels of the prfA transcript (and, as a consequence, of the inlA, hly, and actA transcripts) under neutral and acidic conditions. In contrast, the sodC and gad genes, encoding a cytoplasmic superoxide dismutase and the glutamate-based acid resistance system, respectively, were positively regulated at a low pH. Morphological approaches confirmed the increased intracellular survival and growth of acid-adapted L. monocytogenes cells both in vacuoles and in the cytoplasm of interferon gamma-activated THP-1 macrophages. Our data indicate that preexposure to a low pH has a positive impact on subsequent challenge of L. monocytogenes with macrophagic cells.