The serine-rich repeat glycoprotein Srr2 mediates Streptococcus agalactiae interaction with host fibronectin.

BACKGROUND: Group B Streptococcus (GBS) is a commensal of healthy adults and an important pathogen in newborns, the elderly and immunocompromised individuals. GBS displays several virulence factors that promote colonisation and host infection, including the ST-17 strain-specific adhesin Srr2, previously characterised for its binding to fibrinogen. Another common target for bacterial adhesins and for host colonization is fibronectin, a multi-domain glycoprotein found ubiquitously in body fluids, in the extracellular matrix and on the surface of cells. RESULTS: In this study, fibronectin was identified as a novel ligand for the Srr2 adhesin of GBS. A derivative of the ST-17 strain BM110 overexpressing the srr2 gene showed an increased ability to bind fibrinogen and fibronectin, compared to the isogenic wild-type strain. Conversely, the deletion of srr2 impaired bacterial adhesion to both ligands. ELISA assays and surface plasmon resonance studies using the recombinant binding region (BR) form of Srr2 confirmed a direct interaction with fibronectin with an estimated Kd of 92 nM. Srr2-BR variants defective in fibrinogen binding also exhibited no interaction with fibronectin, suggesting that Srr2 binds this ligand through the dock-lock-latch mechanism, previously described for fibrinogen binding. The fibronectin site responsible for recombinant Srr2-BR binding was identified and localised in the central cell-binding domain of the protein. Finally, in the presence of fibronectin, the ability of a Δsrr2 mutant to adhere to human cervico-vaginal epithelial cells was significantly lower than that of the wild-type strain. CONCLUSION: By combining genetic and biochemical approaches, we demonstrate a new role for Srr2, namely interacting with fibronectin. We characterised the molecular mechanism of this interaction and demonstrated that it plays a role in promoting the adhesion of GBS to human cervico-vaginal epithelial cells, further substantiating the role of Srr2 as a factor responsible for the hypervirulence of GBS ST-17 strains. The discovery of the previously undescribed interaction between Srr2 and fibronectin establishes this adhesin as a key factor for GBS colonisation of host tissues.

von Willebrand factor-binding protein (vWbp)-activated factor XIII and transglutaminase 2 (TG2) promote cross-linking between FnBPA from Staphylococcus aureus and fibrinogen.

The secreted von Willebrand factor-binding protein (vWbp) from Staphylococcus aureus interacts with the coagulation factors prothrombin and fibrinogen (Fbg), leading to the non-proteolytic transglutaminase activation of Factor XIII (FXIII). In this study we found that vWbp-activated FXIII catalyses the incorporation of amino-donor dansylcadaverine into region A of fibronectin-binding protein A (FnBPA). Incubation of Fbg with recombinant region A of S. aureus Fbg-binding proteins FnBPA, FnBPB, ClfA or ClfB in presence of vWbp-activated FXIII resulted in the formation of high molecular heteropolymers with FnBPA only, suggesting a specificity of the cross-linking reaction between fibrin(ogen) and the staphylococcal surface. As previously observed, cross-linking sites were mapped to the α-chain and the N1 subdomain of fibrin(ogen) and region A of FnBPA, respectively. Comparable results were obtained when tissue tranglutaminase-2 (TG2) was tested for cross-linking of FnBPA and Fbg. Of note, FnBPA-mediated covalent cross-linking promoted by vWbp-activated FXIII was also observed when bacteria were allowed to attach to fibrin(ogen). Together these findings suggest a novel pathogenetic mechanism by which the transglutaminase action of FXIII and/or TG2 contributes to entrapment and persistence of S. aureus in blood and host tissues.

Structural Insights into the Ligand-LsrK Kinase Binding Mode: A Step Forward in the Discovery of Novel Antimicrobial Agents.

LsrK is a bacterial kinase that triggers the quorum sensing, and it represents a druggable target for the identification of new agents for fighting antimicrobial resistance. Herein, we exploited tryptophan fluorescence spectroscopy (TFS) as a suitable technique for the identification of potential LsrK ligands from an in-house library of chemicals comprising synthetic compounds as well as secondary metabolites. Three secondary metabolites (Hib-ester, Hib-carbaldehyde and (R)-ASME) showed effective binding to LsrK, with KD values in the sub-micromolar range. The conformational changes were confirmed via circular dichroism and molecular docking results further validated the findings and displayed the specific mode of interaction. The activity of the identified compounds on the biofilm formation by some Staphylococcus spp. was investigated. Hib-carbaldehyde and (R)-ASME were able to reduce the production of biofilm, with (R)-ASME resulting in the most effective compound with an EC50 of 14 mg/well. The successful application of TFS highlights its usefulness in searching for promising LsrK inhibitor candidates with inhibitor efficacy against biofilm formation.

CodY Is a Global Transcriptional Regulator Required for Virulence in Group B Streptococcus.

Group B Streptococcus (GBS) is a Gram-positive bacterium able to switch from a harmless commensal of healthy adults to a pathogen responsible for invasive infections in neonates. The signals and regulatory mechanisms governing this transition are still largely unknown. CodY is a highly conserved global transcriptional regulator that links nutrient availability to the regulation of major metabolic and virulence pathways in low-G+C Gram-positive bacteria. In this work, we investigated the role of CodY in BM110, a GBS strain representative of a hypervirulent lineage associated with the majority of neonatal meningitis. Deletion of codY resulted in a reduced ability of the mutant strain to cause infections in neonatal and adult animal models. The observed decreased in vivo lethality was associated with an impaired ability of the mutant to persist in the blood, spread to distant organs, and cross the blood-brain barrier. Notably, the codY null mutant showed reduced adhesion to monolayers of human epithelial cells in vitro and an increased ability to form biofilms, a phenotype associated with strains able to asymptomatically colonize the host. RNA-seq analysis showed that CodY controls about 13% of the genome of GBS, acting mainly as a repressor of genes involved in amino acid transport and metabolism and encoding surface anchored proteins, including the virulence factor Srr2. CodY activity was shown to be dependent on the availability of branched-chain amino acids, which are the universal cofactors of this regulator. These results highlight a key role for CodY in the control of GBS virulence.

Bacillus subtilis as a host for mosquitocidal toxins production.

Aedes albopictus transmits several arboviral infections. In the absence of vaccines, control of mosquito populations is the only strategy to prevent vector-borne diseases. As part of the search for novel, biological and environmentally friendly strategies for vector control, the isolation of new bacterial species with mosquitocidal activity represents a promising approach. However, new bacterial isolates may be difficult to grow and genetically manipulate. To overcome these limits, here we set up a system allowing the expression of mosquitocidal bacterial toxins in the well-known genetic background of Bacillus subtilis. As a proof of this concept, the ability of B. subtilis to express individual or combinations of toxins of Bacillus thuringiensis israelensis (Bti) was studied. Different expression systems in which toxin gene expression was driven by IPTG-inducible, auto-inducible or toxin gene-specific promoters were developed. The larvicidal activity of the resulting B. subtilis strains against second-instar Ae. albopictus larvae allowed studying the activity of individual toxins or the synergistic interaction among Cry and Cyt toxins. The expression systems here presented lay the foundation for a better improved system to be used in the future to characterize the larvicidal activity of toxin genes from new environmental isolates.

The iron-regulated surface determinant B (IsdB) protein from Staphylococcus aureus acts as a receptor for the host protein vitronectin.

Staphylococcus aureus is an important bacterial pathogen that can cause a wide spectrum of diseases in humans and other animals. S. aureus expresses a variety of virulence factors that promote infection with this pathogen. These include cell-surface proteins that mediate adherence of the bacterial cells to host extracellular matrix components, such as fibronectin and fibrinogen. Here, using immunoblotting, ELISA, and surface plasmon resonance analysis, we report that the iron-regulated surface determinant B (IsdB) protein, besides being involved in heme transport, plays a novel role as a receptor for the plasma and extracellular matrix protein vitronectin (Vn). Vn-binding activity was expressed by staphylococcal strains grown under iron starvation conditions when Isd proteins are expressed. Recombinant IsdB bound Vn dose dependently and specifically. Both near-iron transporter motifs NEAT1 and NEAT2 of IsdB individually bound Vn in a saturable manner, with KD values in the range of 16-18 nm Binding of Vn to IsdB was specifically blocked by heparin and reduced at high ionic strength. Furthermore, IsdB-expressing bacterial cells bound significantly higher amounts of Vn from human plasma than did an isdB mutant. Adherence to and invasion of epithelial and endothelial cells by IsdB-expressing S. aureus cells was promoted by Vn, and an αvß3 integrin-blocking mAb or cilengitide inhibited adherence and invasion by staphylococci, suggesting that Vn acts as a bridge between IsdB and host αvß3 integrin.