Antonella Di Poto

The effect of photodynamic treatment combined with antibiotic action or host defence mechanisms on Staphylococcus aureus biofilms

Staphylococcus aureus is one of the most important etiological agents of infections associated with medical devices. This is in part due to the ability of the organism to form biofilm, which provides a microenvironment that protects from attack by the hosts immune system and by antibiotics. In this study we examined the structure of polysaccharide intercellular adhesin (PIA)-dependent or protein-based S. aureus biofilms. We defined new strategies aimed at treatment of mature established biofilms using photodynamic treatment (PDT) combined with chemotherapy or phagocytosis. Significant inactivation of bacteria was observed when structurally distinct biofilms were exposed to the cationic porphyrin, tetra-substituted N-methyl-pyridyl-porphine (TMP), and simultaneously to visible light. Moreover, PDT-treated biofilms exposed to vancomycin or subjected to the phagocytic action of whole blood resulted in their almost complete eradication. The drastic reduction in staphylococcal survival and the disruption of biofilms were confirmed by confocal laser scanning microscopy and scanning electron microscopy. The results suggest that PDT combined with vancomycin and the host defences may be a useful approach for the inactivation of staphylococcal biofilms adhering to medical implant surfaces.

Structural and functional role of Staphylococcus aureus surface components recognizing adhesive matrix molecules of the host

Staphylococcus aureus is a versatile and harmful human pathogen in both hospital- and community-acquired infections. S. aureus can initiate host infection by adhering to components of the extracellular matrix. Adherence is mediated by a variety of protein adhesins of the microbial surface component recognizing adhesive matrix molecule (MSCRAMM) family. In this article, we describe these MSCRAMMs in terms of structural organization and ligand-binding capacity and discuss their role as a possible target for immunotherapy.

Toll-like receptors (TLRs) in innate immune defense against Staphylococcus aureus

Toll-like receptors (TLRs) are the most important class of innate pattern recognition receptors (PRRs) by which host immune and non-immune cells are able to recognize pathogen-associated molecular patterns (PAMPs). Most mammalian species have 10 to 15 types of TLRs. TLRs are believed to function as homo- or hetero-dimers. TLR2, which plays a crucial role in recognizing PAMPs from Staphylococcus aureus, forms heterodimers with TLR1 or TLR6 and each dimer has a different ligand specificity. Staphylococcal lipoproteins, Panton-Valentine toxin and Phenol Soluble Modulins have been identified as potent TLR2 ligands. Conversely, the ligand function attributed to peptidoglycan and LTA remains controversial. TLR2 uses a MyD88-dependent signaling pathway that results in NF-κB translocation into the nucleus and activation of the expression of pro-inflammatory cytokine genes. Recognition rouses both an inflammatory response, culminating in the phagocytosis of bacteria, and an adaptive immune response, with the presentation of resulting bacterial compounds to T cells. Here, recent advances on the recognition of S. aureus by TLRs are presented and discussed, as well as the new therapeutic opportunities deriving from this new knowledge.

Purification of human plasma fibronectin using immobilized gelatin and Arg affinity chromatography

This protocol describes a method for purification of fibronectin (Fn) from human plasma based on a combination of gel filtration and affinity chromatography steps. Clarified plasma is first loaded onto a Sepharose CL-4B column and unbound material is sequentially purified on columns containing covalently coupled gelatin and Arg. The elution conditions are optimized to obtain a homogeneous preparation of Fn on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Although the Fn yield is expected to be lower than that obtained using other methods, affinity adsorbents based on gelatin and Arg and gentle elution steps offer advantages including a high purity of the preparation and a correctly folded protein. The preparation can be useful for interaction studies and analysis of biological and immunological activities of Fn.

The A domain of fibronectin-binding protein B of Staphylococcus aureus contains a novel fibronectin binding site.

The fibronectin‐binding proteins FnBPA and FnBPB are multifunctional adhesins than can also bind to fibrinogen and elastin. In this study, the N2N3 subdomains of region A of FnBPB were shown to bind fibrinogen with a similar affinity to those of FnBPA (2 μm). The binding site for FnBPB in fibrinogen was localized to the C‐terminus of the γ‐chain. Like clumping factor A, region A of FnBPB bound to the γ‐chain of fibrinogen in a Ca2+‐inhibitable manner. The deletion of 17 residues from the C‐terminus of domain N3 and the substitution of two residues in equivalent positions for crucial residues for fibrinogen binding in clumping factor A and FnBPA eliminated fibrinogen binding by FnBPB. This indicates that FnBPB binds fibrinogen by the dock–lock–latch mechanism. In contrast, the A domain of FnBPB bound fibronectin with KD = 2.5 μm despite lacking any of the known fibronectin‐binding tandem repeats. A truncate lacking the C‐terminal 17 residues (latching peptide) bound fibronectin with the same affinity, suggesting that the FnBPB A domain binds fibronectin by a novel mechanism. The substitution of the two residues required for fibrinogen binding also resulted in a loss of fibronectin binding. This, combined with the observation that purified subdomain N3 bound fibronectin with a measurable, but reduced, KD of 20 μm, indicates that the type I modules of fibronectin bind to both the N2 and N3 subdomains. The fibronectin‐binding ability of the FnBPB A domain was also functional when the protein was expressed on and anchored to the surface of staphylococcal cells, showing that it is not an artifact of recombinant protein expression.

IsdC from Staphylococcus lugdunensis Induces Biofilm Formation under Low-Iron Growth Conditions

ABSTRACT Staphylococcus lugdunensis is a coagulase-negative staphylococcus that is a commensal of humans and an opportunistic pathogen. It can cause a spectrum of infections, including those that are associated with the ability to form biofilm, such as occurs with endocarditis or indwelling medical devices. The genome sequences of two strains revealed the presence of orthologues of the ica genes that are responsible for synthesis of poly-N-acetylglucosamine (PNAG) that is commonly associated with biofilm in other staphylococci. However, we discovered that biofilm formed by a panel of S. lugdunensis isolates growing in iron-restricted medium was susceptible to degradation by proteases and not by metaperiodate, suggesting that the biofilm matrix comprised proteins and not PNAG. When the iron concentration was raised to 1 mM biofilm formation by all strains tested was greatly reduced. A mutant of strain N920143 lacking the entire locus that encodes iron-regulated surface determinant (Isd) proteins was defective in biofilm formation under iron-limited conditions. An IsdC-null mutant was defective, whereas IsdK, IsdJ, and IsdB mutants formed biofilm to the same level as the parental strain. Expression of IsdC was required both for the primary attachment to unconditioned polystyrene and for the accumulation phase of biofilm involving cell-cell interactions. Purified recombinant IsdC protein formed dimers in solution and Lactococcus lactis cells expressing only IsdC adhered to immobilized recombinant IsdC but not to IsdJ, IsdK, or IsdB. This is consistent with a specific homophilic interaction between IsdC molecules on neighboring cells contributing to accumulation of S. lugdunensis biofilm in vivo.

Molecular Characterization of the Multiple Interactions of SpsD, a Surface Protein from Staphylococcus pseudintermedius, with Host Extracellular Matrix Proteins

Staphylococcus pseudintermedius, a commensal and pathogen of dogs and occasionally of humans, expresses surface proteins potentially involved in host colonization and pathogenesis. Here, we describe the cloning and characterization of SpsD, a surface protein of S. pseudintermedius reported as interacting with extracellular matrix proteins and corneocytes. A ligand screen and Western immunoblotting revealed that the N-terminal A domain of SpsD bound fibrinogen, fibronectin, elastin and cytokeratin 10. SpsD also interfered with thrombin-induced fibrinogen coagulation and blocked ADP-induced platelet aggregation. The binding site for SpsD was mapped to residues 395–411 in the fibrinogen γ-chain, while binding sites in fibronectin were localized to the N- and C-terminal regions. SpsD also bound to glycine- and serine-rich omega loops within the C-terminal tail region of cytokeratin 10. Ligand binding studies using SpsD variants lacking the C-terminal segment or containing an amino-acid substitution in the putative ligand binding site provided insights into interaction mechanism of SpsD with the different ligands. Together these data demonstrate the multi-ligand binding properties of SpsD and illustrate some interesting differences in the variety of ligands bound by SpsD and related proteins from S. aureus.

Merocyanine-540 Mediated Photodynamic Effects on Staphylococcus epidermidis Biofilms

Staphylococci are important causes of nosocomial and medical-device-related infections. Their virulence is attributed to the elaboration of biofilms that protect the organisms from immune system clearance and to increased resistance to phagocytosis and antibiotics. Photodynamic treatment (PDT) has been proposed as an alternative approach for the inactivation of bacteria in biofilms. In this study, we evaluated the antimicrobial activity of merocyanine 540 (MC 540), a photosensitizing dye that is used for purging malignant cells from autologous bone marrow grafts, against Staphylococcus epidermidis biofilms. We evaluated the effect of the combined photodynamic action of MC 540 and 532 nm laser on the viability and structure of biofilms of two Staphylococcus epidermidis strains. Significant inactivation of cells was observed in the biofilms treated with MC-540 and then exposed to laser radiation. Furthermore we found that the PDT effect, on both types of cells, was significantly dependent on both the light-dose and on the impinging lightintensity. Disruption of PDT-treated biofilm was confirmed by scanning electron microscopy (SEM).