Differential contribution of sodC1 and sodC2 to intracellular survival and pathogenicity of Salmonella enterica serovar Choleraesuis.

Several of the most virulent Salmonella enterica strains possess two genes encoding periplasmic Cu,Zn superoxide dismutase, sodC1 and sodC2, located on a lambdoid prophage and on the chromosome, respectively. These genes contribute to Salmonella virulence by protecting bacteria from superoxide generated by the host's phagocytes. To investigate the respective contributions of sodC1 and sodC2 to the virulence of a clinical isolate of Salmonella enterica serovar Choleraesuis (S. choleraesuis), we have analyzed both the intracellular survival of wild type and sodC mutant strains within J774 macrophages and Caco-2 cells, and their ability to proliferate in intraperitoneally-infected mice in competition assays. In agreement with previous studies, mutant strains lacking one or both sodC genes were equally impaired in their ability to survive within activated macrophages. However, when macrophage killing experiments were carried out with non-opsonized bacteria, sodC2 contributed to intracellular survival more than sodC1, indicating that changes in the pathways of bacterial uptake can modify the relative role of the two sodC genes. More unexpectedly, we have found that the ability of S. choleraesuis to survive within Caco-2 cells was severely affected by inactivation of sodC genes, sodC2 being more important than sodC1. As Caco-2 cells actively produce superoxide, this suggests that oxygen radical production by colonic cells has a role in controlling proliferation of facultative intracellular bacteria. Mouse infection studies confirmed that, in the S. choleraesuis strain under investigation, both sodC genes are required to confer full virulence, sodC2 contributing slightly more than sodC1 to Salmonella pathogenesis. Our findings contrast with the results of other studies carried out in S. enterica serovar Typhimurium and suggest that the relative contributions of sodC1 and sodC2 to host-pathogen interactive biology may vary depending on the Salmonella serovar or strain.

Both lactoferrin and iron influence aggregation and biofilm formation in Streptococcus mutans.

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.

Quantitative evaluation of bacteria adherent to polyelectrolyte HEMA-based hydrogels.

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.

Anti-invasive activity of bovine lactoferrin towards group A streptococci.

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.).

Ca2+ binding to bovine lactoferrin enhances protein stability and influences the release of bacterial lipopolysaccharide.

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.