We have studied the influence of periplasmic Cu,Zn superoxide dismutase on the intracellular survival of Escherichia coli strains able to invade epithelial cells by the expression of the inv gene from Yersinia pseudotuberculosis but unable to multiply intracellularly. Intracellular viability assays, confirmed by electron microscopy observations, showed that invasive strains of E. coli engineered to increase Cu,Zn superoxide dismutase production are much more resistant to intracellular killing than strains containing only the chromosomal sodC copy. However, we have found only a slight difference in survival within HeLa cells between a sodC-null mutant and its isogenic wild-type strain. Such a small difference in survival correlates with the very low expression of this enzyme in the wild-type strain. We have also observed that acid-and oxidative stress-sensitive E. coli HB101(pRI203) is more rapidly killed in epithelial cells than E. coli GC4468(pRI203). The high mortality of E. coli HB101(pRI203), independent of the acidification of the endosome, is abolished by the overexpression of sodC. Our data suggest that oxyradicals are involved in the mechanisms of bacterial killing within epithelial cells and that high-level production of periplasmic Cu,Zn superoxide dismutase provides bacteria with an effective protection against oxidative damage. We propose that Cu,Zn superoxide dismutase could offer an important selective advantage in survival within host cells to bacteria expressing high levels of this enzyme.Until a few years ago Cu,Zn superoxide dismutase (Cu, ZnSOD) was considered almost exclusively a eukaryotic enzyme, protecting the cytosol and the extracellular environment of higher organisms from damage by oxygen free radicals (1). Recently, Cu,ZnSOD has been identified in the periplasmic space of a wide range of gram-negative bacteria, including Brucella abortus (6), Haemophilus spp., Actinobacillus spp., Pasteurella spp., Neisseria meningitidis (24-26), Escherichia coli K-12 (7), Legionella pneumophila (40), Salmonella spp. (9), and Mycobacterium tuberculosis (45). This enzyme is thought to protect bacteria from toxic oxygen-free radicals generated outside the cell or in the periplasm itself, since superoxide is unable to cross the cytoplasmic membrane (21). Therefore, Cu,ZnSOD has been proposed to be a determinant of virulence in bacteria potentially exposed to toxic free radicals produced by the host in response to bacterial infection. In vivo experiments have demonstrated the role of bacterial Cu,ZnSOD in the virulence and pathogenicity of infecting microorganisms (15,18,19,36,42,43), while in vitro models have provided conflicting data concerning Cu,ZnSOD involvement in bacterial resistance to macrophage killing (19, 42) or survival within nonprofessional phagocytes (42). However, more recent results have shown that this enzyme protects Salmonella enterica serovar Typhimurium (15) and an overproducing strain of E. coli (4) from macrophage killing and that neutropenia restores virulence to an attenuated C...

Bovine lactoferrin (bLf) is known to damage the outer membrane of Gram-negative bacteria by binding to bacterial lipopolysaccharide (LPS). We report that LPS is released from bacterial outer membranes also when apo- or metal-saturated Lf is separated from bacterial cells by a dialysis membrane. This process occurs in phosphate-buffered saline with no added Ca2+ and Mg2+ and is hindered by addition of these cations. The effect of bLf is similar to that induced by EDTA and has been ascribed to chelation of Ca2+. In fact, it may be envisaged that Ca2+-binding sites on LPS have different affinities and that bLf can remove those ions that are more weakly bound. Ca2+ binding does not alter Lf iron-binding properties significantly or its UV and CD spectral features but brings about changes in the FT-IR bands due to carboxylate residues. Ca2+ binding is characterized by an apparent dissociation constant of 6 microM and a stoichiometry of 1.55 Ca2+ per Lf molecule; it enhances bLf stability towards chemical and thermal denaturation. The increase in stability takes place in both the apo- and iron-saturated forms but not in the desialilated protein, indicating that the carboxylate groups of the sialic acid residues present on two of the glycan chains are involved in Ca2+ binding.

The use of adhesive poly(HEMA)-based hydrogels is standard practice in dental restorative procedures. Microorganisms, which potentially can cause oral pathologies, may colonize these polymers. In the present work, bacterial adhesion to polymers prepared with 2-hydroxyethyl methacrylate (HEMA) and to different molar ratios of 2-acrylamido-2-methylpropane-sulfonic acid (AMPS) and/or to 2-methacryloyloxyethyl-tri-methyl-ammonium chloride (METAC) co-monomers were tested. A colorimetric assay system that utilizes the Microbo revelation medium (Microbo srl, Rome, Italy) for microbial counts is shown to be capable of counting the number of adherent bacterial cells without removing them from polymer surfaces. In conditions that mimic those present in the oral cavity, similar bacterial adhesion percentages on the same polymer were observed with the different bacteria belonging to both gram-positive and gram-negative genera, such as Streptococcus sobrinus and Streptococcus oralis (resident microorganisms in the oral cavity) and Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa (transient microorganisms in the oral cavity). It is determined that the physico-chemical characteristics of poly(HEMA)-based hydrogels are the major factors promoting bacterial adhesion, which increased with increasing water content in the swollen polymers, reaching maximal values on the cationic polymers.

Streptococcus mutans, a gram-positive immobile bacterium, is an oral pathogen considered to be the principal etiologic agent of dental caries. Although some researches suggest that trace metals, including iron, can be associated with dental caries, the function of salivary iron and lactoferrin in the human oral cavity remains unclear. The data reported in this study indicates that iron-deprived saliva (Fe3+ < 0.1 microM) increases S. mutans aggregation and biofilm formation in the fluid and adherent phases as compared with saliva (Fe3+ from 0.1 to 1 microM), while iron-loaded saliva (Fe3+ > 1 microM) inhibits both phenomena. Our findings are consistent with the hypothesis that S. mutans aggregation and biofilm formation are negatively iron-modulated as confirmed by the different effect of bovine lactoferrin (bLf), added to saliva at physiological concentration (20 microg/ml) in the apo- or iron-saturated form. Even if saliva itself induces bacterial aggregation, iron binding capability of apo-bLf is responsible for the noticeable increase of bacterial aggregation and biofilm development in the fluid and adherent phases. On the contrary, iron-saturated bLf decreases aggregation and biofilm development by supplying iron to S. mutans. Therefore, the iron-withholding capability of apo-Lf or native Lf is an important signal to which S. mutans counteracts by leaving the planktonic state and entering into a new lifestyle, biofilm, to colonize and persist in the human oral cavity. In addition, another function of bLf, unrelated to its iron binding capability, is responsible for the inhibition of the adhesion of S. mutans free, aggregated or biofilm on abiotic surfaces. Both these activities of lactoferrin, related and unrelated to the iron binding capability, could have a key role in protecting the human oral cavity from S. mutans pathogenicity.

Group A streptococci (GAS) are able to invade cultured epithelial and endothelial cells without evidence of intracellular replication. GAS, like other facultative intracellular bacterial pathogens, evolved such ability to enter and to survive within host cells avoiding the host defences, and bacterial intracellular survival could explain the recurrence of infections. We report here that 1 mg bovine lactoferrin (bLf)/mL significantly hindered the in vitro invasion of cultured epithelial cells by GAS isolated from patients suffering from pharyngitis and completely inhibited the invasiveness of GAS pretreated with subinhibiting concentrations of erythromycin or ampicillin. One milligram of bLf per millilitre was also able to increase the number of epithelial cells undergoing apoptosis following GAS invasion, although the number of intracellular GAS in the presence of bLf decreased by about 10-fold. The ability of bLf to decrease GAS invasion was confirmed by an in vivo trial carried out on 12 children suffering from pharyngitis and already scheduled for tonsillectomy. In tonsil specimens from children treated for 15 days before tonsillectomy with both oral erythromycin (500 mg t.i.d. (three times daily)) and bLf gargles (100 mg t.i.d.), a lower number of intracellular GAS was found in comparison with that retrieved in tonsil specimens from children treated with erythromycin alone (500 mg t.i.d.).