Fibronectin-binding Protein A Research Articles

Targeting the Enterococcal Fibronectin Binding Protein- A of Enterococcus faecalis with Bioactive Compounds from Aegle Marmelos: An In-vitro and In-silico Pilot Study

Introduction: Enterococcus faecalis (E. faecalis) is a significant endodontic pathogen, and its virulence has been shown to be significantly influenced by Enterococcal fibronectin binding protein-A (EfbA). Targeting this virulent protein using alternative strategies would be a novel idea to combat the complications of E. faecalis in dental healthcare settings. Thus, the rationale of this investigation is to identify potent bioactive compounds from Aegle marmelos for their antimicrobial properties against EfbA of E. faecalis. Aim: To assess the frequency of EfbA among clinical isolates of E. faecalis and evaluate the antibacterial activity of essential bioactive compounds from Aegle marmelos. Materials and Methods: An in-vitro and in-silico pilot study was conducted from April 2022 to June 2022 in the Department of Microbiology, Saveetha Dental College and Hospitals, Chennai, Tamil Nadu, India to detect the frequency of EfbA in E. faecalis. Patients with typical root caries were included. Microbiological processing of 20 root caries samples was performed to characterise E. faecalis and detect the EfbA gene using Polymerase Chain Reaction (PCR) amplification. Crude methanol extract was obtained from A. marmelos and assessed for its antimicrobial effect against the isolated strains of E. faecalis. Furthermore, several biomolecules from A. marmelos were evaluated for their inhibitory properties through in-silico docking analysis. Results: E. faecalis was identified in 12 (60%) out of 20 root caries samples, and three isolates (25%) were identified as Multidrug-Resistant (MDR) strains based on the antibiogram susceptibility profile, showing resistance to more than three different tested antibiotic groups. EfbA was detected in two of the three MDR strains (66.7%). The crude extract of A. marmelos exhibited promising antibacterial activity. In-silico analysis of the essential oil compounds from A. marmelos revealed that Aegeline had a high interaction with low docking energy and a high number of hydrogen bonds. Conclusion: The current study highlights the potential of aegeline from A. marmelos as an antibacterial agent against resistant strains of E. faecalis. However, additional in-vivo research must be conducted to experimentally validate these findings.

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.

The Two-Track Investigation of Fibronectin Binding Protein A of Staphylococcus aureus from Bovine Mastitis as a Potential Candidate for Immunodiagnosis: A Pilot Study

Bovine mastitis is the most common disease affecting dairy cattle worldwide and it generates substantial losses for cattle breeders. One of the most common pathogens identified in infected milk samples is Staphylococcus aureus. Currently, there is no fast test for recognizing bacteria species on the market. The aim of this study was to bioinformatically and laboratory detect and characterize the fibronectin binding protein A (FnBPA) of S. aureus (SA) in milk samples obtained from cows diagnosed with mastitis. More than 90,000,000 amino acid sequences were subjected to bioinformatic detection in the search for a potential biomarker for bovine SA. The analysis of FnBPA included the detection of signal peptides and nonclassical proteins, antigenicity, and the prediction of epitopes. To confirm the presence of the fnbA gene in four SA isolates, amplification with specific primers was performed. FnBPA was detected by immunoblotting. The immunoreactivity and selectivity were performed with monoclonal anti-FnBPA antibodies and SA-negative serum. The bioinformatic analysis showed that FnBPA is a surface, conservative, immunoreactive, and species-specific protein with antigenic potential. Its presence was confirmed in all of the SA isolates we studied. Immunoblotting proved its immunoreactivity and specificity. Thus, it can be considered a potential biomarker in mastitis immunodiagnostics.

Oral vaccination with invasive Lactobacillus plantarum delivered nucleic acid vaccine co-expressing SS1 and murine interleukin-4 elicits protective immunity against Trichinella spiralis in BALB/c mice

Trichinellosis is a foodborne zoonosis caused by Trichinella spiralis (T. spiralis) that not only causes considerable economic losses for the global pig breeding and food industries, but also seriously threats the health of human. Therefore, it is very necessary to develop an effective vaccine to prevent trichinellosis. In this study, the invasive Lactobacillus plantarum (L. plantarum) expressing fibronectin-binding protein A (FnBPA) was served as a live bacterial vector to deliver DNA to the host to produce a novel oral DNA vaccine. Co-expressing T. spiralis SS1 and murine interleukin-4 (mIL-4) of DNA vaccine were constructed and subsequently delivered to intestinal epithelial cells via invasive L. plantarum. At 10 days after the third immunization, the experimental mice were challenged with 350 T. spiralis infective larvae. The results found that the mice orally vaccinated with invasive L. plantarum harboring pValac-SS1/pSIP409-FnBPA not only stimulated the production of anti-SS1-specific IgG, Th1/Th2 cell cytokines, and secreted(s) IgA but also decreased worm burden and intestinal damage. However, the mice inoculated with invasive L. plantarum co-expressing SS1 and mIL-4 (pValac-SS1-IL-4/pSIP409-FnBPA) induced the highest protective immune response against T. spiralis infection. The DNA vaccine delivered by invasive L. plantarum provides a novel idea for the prevention of T. spiralis infection.

Analyzing the adhesion mechanism of FnBPA, a surface adhesin from Staphylococcus aureus on its interaction with nanoparticle

Staphylococcus aureus expresses many Microbial Surface Recognizing Adhesive Matrix Molecules (MSCRAMM's) to recognize host extracellular matrix (ECM) molecules to initiate colonization. The MSCRAMM, fibronectin binding protein A (FnBPA), is an important adhesin for S. aureus infection. FnBPA also binds with fibrinogen (Fg) by using a unique ligand binding mechanism called dock, lock and latch. Nanoparticles, especially nanosilver particles have been widely used in a variety of biomedical applications which includes disease diagnosis and treatment, drug delivery and implanted medical device coating. In a biological system, when protein molecules encounter nanoparticle, they can be absorbed onto its surface which results in the formation of protein corona. In the present study, we have analysed the fibrinogen binding ability of rFnBPA(189-512) in the presence of silver nanoparticles by employing techniques like gel shift assay, Western blot, size exclusion chromatography, enzyme-linked immunosorbent assay, bio-layer interferometry and circular dichroism spectroscopy. The results indicate that rFnBPA(189-512) is unable to bind to Fg in the presence of a nanoparticle. This could be due to the inaccessibility of the Fg binding site and conformational change in rFnBPA(189-512). With nanoparticles, rFnBPA(189-512) undergoes significant structural changes as the β-sheet content has drastically reduced to 10% from the initial 60% at higher concentration of the nanoparticle. Pathogenic bacteria interact with its surrounding environment through their surface molecules which includes MSCRAMMs. Therefore MSCRAMMs play an important role when bacteria encounter nanoparticles. The results of the present study suggest that the orientation of the protein during the absorption on the surface of a nanoparticle as well as the concentration of the nanoparticle, will dictate the function of the absorbed protein and in this case the Fg binding property of rFnBPA(189-512).

Novel peptides screened by phage display peptide library can mimic epitopes of the FnBPA-A protein and induce protective immunity against Staphylococcus aureus in mice.

Fibronectin‐binding protein A (FnBPA) is a key adhesin of Staphylococcus aureus, and the protein binding to fibrinogen and elastin is mediated by its N‐terminal A domain. Thus, FnBPA‐A has been considered a potential vaccine candidate, but the relevant epitopes are not fully understood. Here, purified rabbit anti‐FnBPA‐A antibodies were produced and used to screen for peptides corresponding to or mimicking the epitope of native FnBPA‐A protein by using a phage random 12‐mer peptide library. After four rounds of panning, 25 randomly selected phage clones were detected by phage‐ELISA and competition‐inhibition ELISA. Then, eight anti‐rFnBPA‐A antibody‐binding phage clones were selected for sequencing, and six different 12‐mer peptides were displayed by these phages. Although these displayed peptides shared no more than three consecutive amino acid residues identical to the sequence of FnBPA‐A, they could be recognized by the FnBPA‐A‐specific antibodies in vitro and could induce specific antibodies against FnBPA‐A in vivo, suggesting that these displayed peptides were mimotopes of FnBPA‐A. Finally, the protective efficiencies of these mimotopes were investigated by mouse vaccination and challenge experiments. Compared with that of control group mice, the relative percent survival of mice immunized with phage clones displaying a mimotope was 13.33% (C2 or C15), 0% (C8), 6.67% (C10), 26.67% (C19 or 1:2 mixture of C23 and C19), 53.33% (C23), 33.33% (1:1 mixture of C23 and C19), and 66.67% (2:1 mixture of C23 and C19). Overall, five peptides mimicking FnBPA‐A protein epitopes were obtained, and a partially protective immunity against S. aureus infection could be stimulated by these mimotope peptides in mice.

Fibrinogen binding is affected by amino acid substitutions in C-terminal repeat region of fibronectin binding protein A

Fibronectin-binding protein A (FnBPA), a protein displayed on the outer surface of Staphylococcus aureus, has a structured A-domain that binds fibrinogen (Fg) and a disordered repeat-region that binds fibronectin (Fn). Amino acid substitutions in Fn-binding repeats (FnBRs) have previously been linked to cardiovascular infection in humans. Here we used microtiter and atomic force microscopy (AFM) to investigate adhesion by variants of full-length FnBPA covalently anchored in the outer cell wall of Lactococcus lactis, a Gram-positive surrogate that otherwise lacks adhesins to mammalian ligands. Fn adhesion increased in five of seven FnBPA variants under static conditions. The bond targeting Fn increased its strength with load under mechanical dissociation. Substitutions extended bond lifetime (1/koff) up to 2.1 times for FnBPA-Fn. Weaker adhesion was observed for Fg in all FnBPA variants tested with microtiter. However, mechanical dissociation with AFM showed significantly increased tensile strength for Fg interacting with the E652D/H782Q variant. This is consistent with a force-induced mechanism and suggests that the dock, lock, and latch (DLL) mechanism is favored for Fg-binding under mechanical stress. Collectively, these experiments reveal that FnBPA exhibits bimodal, ligand-dependent adhesive behavior. Amino acid substitutions in the repeat-region of FnBPA impact binding to both ligands. This was unexpected for Fg since all variants have the same A-domain sequence, and the Fg-binding site is distant from the repeat region. This indicates that FnBRs may fold back on the A-domain in a way that impacts the DLL binding mechanism for Fg.

Recombinant invasive Lactobacillus plantarum expressing fibronectin binding protein A induce specific humoral immune response by stimulating differentiation of dendritic cells.

Recombinant lactic acid bacteria (LAB), especially Lactococcus lactis, have been genetically engineered to express heterogeneous invasion proteins, such as the fibronectin binding protein A (FnBPA) from Staphylococcus aureus, to increase the invasion ability of the host strains, indicating a promising approach for DNA vaccine delivery. The presence of FnBPA has been also shown to be an adjuvant for co-delivered antigens, however, the underlying mechanisms are still not clear. To explore the above underlying mechanisms, in this study, we constructed a novel Lactobacillus plantarum strain with surface displayed FnBPA, which could significantly improve the adhesion and invasion ratios of L. plantarum strain on a porcine intestinal epithelial cell line (IPEC-J2) about two-fold compared with the empty vector. At the same time, the presence of FnBPA significantly stimulated the differentiation of bone marrow-derived dendritic cells (DCs) and increased the secretion of interleukin (IL)-6 and mRNA level of IL-6 gene, which were proved by flow cytometry, enzyme-linked immunosorbent assay (ELISA) and quantitative reverse transcription PCR (qRT-PCR). With regard to in vivo study, the presence of FnBPA significantly stimulated the differentiation of DCs in the Peyer's patch (PP) and the percentages of IL-4+ and IL-17A+ T helper (Th) cells of splenocytes in flow cytometry assay. In consistent with these results, the levels of IL-4 and IL-17A in serum as measured via ELISA also increased in mice treated with FnBPA+ L. plantarum. Finally, the FnBPA strain increased the production of B220+ B cells in mesenteric lymph node (MLN) and PP and the levels of FnBPA-specific IgG and sIgA antibodies, indicating the its possible application in vaccine field. This study demonstrated that the invasive L. plantarum with surface displayed FnBPA could modulate host immune response by stimulating the differentiation of DCs and Th cells which could possibly be responsive for the adjuvant effects of FnBPA.

Liposomal and CpG-ODN formulation elicits strong humoral immune responses to recombinant Staphylococcus aureus antigens in heifer calves

Bovine mastitis caused by Staphylococcus aureus is a serious problem in dairy production and effective immunoprophylaxis is an unmet goal so far. The objective of this work was to assess the humoral immune response of heifer calves against two recombinant S. aureus antigens: Clumping factor A (ClfA) and Fibronectin Binding Protein A (FnBPA), formulated with a novel adjuvant based on cationic liposomes (Lip) and CpG oligodeoxynucleotides (CpG-ODN). Six groups of 6–8 months old heifer calves received three doses biweekly of antigens, formulated with Al(OH)3, liposomes, CpG-ODN or Lip + CpG-ODN. Animals also received a fourth dose after a year (day 410) and a booster before calving. The administration of Al(OH)3+FnBPA/ClfA and Lip + FnBPA/ClfA + CpG-ODN induced the highest specific IgG levels, after the first 3 doses and induced a fast increase of antibodies after the fourth dose. All the formulations stimulated the production of specific IgG1, after the third and fourth dose. Specific IgG2 for both proteins was only stimulated after the fourth dose by Lip + FnBPA/ClfA + CpG-ODN. Pre-calving immunisation with Lip + FnBPA/ClfA + CpG-ODN led to the highest IgG levels during the calving period and to the production of the IgG2 subclass. The formulation was also able to stimulate the highest antibody levels in milk, 30 and 45 days after pre-calving booster. The combination of liposomes and CpG-ODN as adjuvant for a subunit vaccine, together with the immunisation schedule described, induced a strong humoral immune response with production of specific IgG2. The formulation demonstrated to induce immune memory allowing the application of a single pre-calving booster to maintain high antibody levels throughout the period of increased susceptibility to intramammary infections.

Human Immunoglobulin G Cannot Inhibit Fibrinogen Binding by the Genetically Diverse A Domain of Staphylococcus aureus Fibronectin-Binding Protein A.

The fibronectin-binding protein A (FnBPA) is a cell surface-associated protein of Staphylococcus aureus which mediates adherence to the host extracellular matrix and is important for bacterial virulence. Previously, substantial sequence diversity was found among strains in the fibrinogen-binding A domain of this protein, and 7 different isotypes were described. The effect of this sequence diversity on the human antibody response, in terms of both antibody production and antibody function, remains unclear. In this study, we identify five different FnBPA A domain isotypes based on the sequence results of 22 clinical S.aureus isolates, obtained from the same number of patients suffering from bacteremia. Using a bead-based Luminex technique, we measure the patients' total immunoglobulin G (IgG) against the 7 FnBPA isotypes at the onset and during the time course of bacteremia (median of 10 serum samples per patient over a median of 35days). A significant increase in IgG against the FnBPA A domain, including the isotype carried by the infecting strain, is observed in only three out of 22 patients (14%) after the onset of bacteremia. Using a Luminex-based FnBPA-fibrinogen-binding assay, we find that preincubation of recombinant FnBPA isotypes with IgG from diverse patients does not interfere with binding to fibrinogen. This observation is confirmed using an alternative Luminex-based assay and enzyme-linked immunosorbent assay (ELISA). IMPORTANCE Despite the many in vitro and murine in vivo studies involving FnBPA, the actual presence of this virulence factor during human infection is less well established. Furthermore, it is currently unknown to what extent sequence variation in such a virulence factor affects the human antibody response and the ability of antibodies to interfere with FnBPA function. This study sheds new light on these issues. First, the uniform presence of a patient's IgG against FnBPA indicates the presence and importance of this virulence factor during S.aureus pathogenesis. Second, the absence of an increase in antibody production in most patients following bacteremia indicates the complexity of S.aureus-host interactions, possibly involving immune evasion or lack of expression of FnBPA during invasive infection. Finally, we provide new insights into the inability of human antibodies to interfere with FnBPA-fibrinogen binding. These observations should be taken into account during the development of novel vaccination approaches.

Identification of a novel linear B-cell epitope as a vaccine candidate in the N2N3 subdomain of Staphylococcus aureus fibronectin-binding protein A.

To explore an epitope-based vaccine against Staphylococcus aureus, we screened the epitopes in the N2N3 subdomain of fibronectin-binding protein A (FnBPA) as a surface component of S. aureus. We expressed N2N3 proteins and prepared monoclonal antibodies (mAbs) against N2N3 by the hybridoma technique, before screening the B-cell epitopes in N2N3 using a phage-displayed random 12-mer peptide library with these mAbs against N2N3. Finally, we analysed the characters of the screened epitopes using immunofluorescence and an S. aureus infection assay. In this paper, we identified a linear B-cell epitope in N2N3 through screening a phage-displayed peptide library with a 3C3 mAb against the N2N3. The 3C3 mAb recognized the 159IETFNKANNRFSH171 sequence of the N2N3 subdomain. Subsequently, site-directed mutagenic analysis demonstrated that residues F162, K164, N167, R168 and F169 formed the core of 159IETFNKANNRFSH171, and this core motif was the minimal determinant of the B-cell epitope recognized by the 3C3 mAb. The epitope 159IETFNKANNRFSH171 showed high homology among different S. aureus strains. Moreover, this epitope was exposed on the surface of the S. aureus by using an enzyme-linked immunosorbent assay (ELISA) assay and an indirect immunofluorescence assay. As expected, the epitope peptide evoked a protective immune response against S. aureus infection in immunized mice. We identified a novel linear B-cell epitope, 159IETFNKANNRFSH171, in the N2N3 subdomain of S. aureus fibronectin-binding protein A that is recognized by 3C3 mAb, which will contribute to the further study of an epitope-based vaccine candidate against S. aureus.

A novel bivalent fusion vaccine induces broad immunoprotection against Staphylococcus aureus infection in different murine models

With more and more drug-resistant Staphylococcus aureus strains emerging in hospitals, there is an urgent need to develop an effective vaccine to combat S. aureus infection. In this study, we constructed a novel bivalent fusion vaccine, SpA-DKKAA-FnBPA37-507 (SF), based on the D domain of staphylococcal protein A (SpA) and the A domain of fibronectin-binding protein A (FnBPA). Immunisation with SF induced a more ideal protective effect compared with the single components alone in a sepsis model. It also showed broad immunoprotection against seven FnBPA isotypes. Vaccination with SF induced strong antibodies responses and Th1/Th17 polarized cellular responses. Further we demonstrated the protective effect of antibodies by the opsonophagocytic assay (OPA) and passive immunisation. Moreover, vaccination with SF showed protective efficacy in a murine pneumonia model and skin abscess model. These results suggest that SF can be regarded as a promising vaccine candidate for the prevention of S. aureus infections.

Quantitative Expression Analysis of SpA, FnbA and Rsp Genes in Staphylococcus aureus: Actively Associated in the Formation of Biofilms.

In Staphylococcus aureus, adherence and secretory proteins play chief role in the formation of biofilms. This mode of growth exhibits resistance to a variety of antibiotics and spreads its infections. In the present study, secretary and adherence proteins, Protein-A, Fibronectin-binding protein-A (FnbA) and Rsp (a transcription regulator encoding proteolytic property) expression levels were evaluated at different stages of growth in S. aureus ATCC12600 a drug-sensitive strain and multidrug-resistant strains of S. aureus. Initially, the SpA, FnbA and Rsp genes of S. aureus ATCC12600 were cloned, sequenced, expressed and characterized. The proteolytic property of recombinant Rsp was conspicuously shown when this pathogen was grown in aerobic conditions correlating with reduced biofilm units. In anaerobic mode of growth, S. aureus exhibited a higher expression of SpA and FnbA in early and mid adherence phases and finally stabilized at 48h of incubation. This expression was more pronounced in methicillin-resistant strains (LMV1-8 and D1-4) of S. aureus. In all these stages, Rsp gene expression was at the lowest level and these results concur with the increased biofilm units. The results of the present study explain proteins chiefly contribute in the formation of biofilms.

Amino acid polymorphisms in the fibronectin-binding repeats of fibronectin-binding protein A affect bond strength and fibronectin conformation

The Staphylococcus aureus cell surface contains cell wall-anchored proteins such as fibronectin-binding protein A (FnBPA) that bind to host ligands (e.g. fibronectin; Fn) present in the extracellular matrix of tissue or coatings on cardiac implants. Recent clinical studies have found a correlation between cardiovascular infections caused by S. aureus and nonsynonymous SNPs in FnBPA. Atomic force microscopy (AFM), surface plasmon resonance (SPR), and molecular simulations were used to investigate interactions between Fn and each of eight 20-mer peptide variants containing amino acids Ala, Asn, Gln, His, Ile, and Lys at positions equivalent to 782 and/or 786 in Fn-binding repeat-9 of FnBPA. Experimentally measured bond lifetimes (1/koff) and dissociation constants (Kd = koff/kon), determined by mechanically dissociating the Fn·peptide complex at loading rates relevant to the cardiovascular system, varied from the lowest-affinity H782A/K786A peptide (0.011 s, 747 μm) to the highest-affinity H782Q/K786N peptide (0.192 s, 15.7 μm). These atomic force microscopy results tracked remarkably well to metadynamics simulations in which peptide detachment was defined solely by the free-energy landscape. Simulations and SPR experiments suggested that an Fn conformational change may enhance the stability of the binding complex for peptides with K786I or H782Q/K786I (Kdapp = 0.2-0.5 μm, as determined by SPR) compared with the lowest-affinity double-alanine peptide (Kdapp = 3.8 μm). Together, these findings demonstrate that amino acid substitutions in Fn-binding repeat-9 can significantly affect bond strength and influence the conformation of Fn upon binding. They provide a mechanistic explanation for the observation of nonsynonymous SNPs in fnbA among clinical isolates of S. aureus that cause endovascular infections.

IL-17A plays an important role in protection induced by vaccination with fibronectin-binding domain of fibronectin-binding protein A against Staphylococcus aureus infection

Fibronectin-binding protein A (FnBPA) of Staphylococcus aureus is a microbial surface component recognizing adhesive matrix molecules and has been known as one of the most important virulence factors involved in the initiation step of S. aureus infection. Therefore, it has been considered as a potential vaccine candidate. Previous studies have reported that vaccination with FnBPA protects animals against S. aureus infection. In this study, we demonstrated that vaccination with fibronectin-binding domain of FnBPA (FnBPA541-870) protects wild-type mice but not interleukin-17A (IL-17A)-deficient mice against S. aureus infection. Moderate levels of antigen-specific immunoglobulins were produced in the sera of vaccinated wild-type and IL-17A-deficient mice. The spleen cells of vaccinated mice produced IL-17A by stimulation with the antigen, and IL-17A mRNA expression was increased in the spleens and livers of vaccinated mice after infection. CXCL1 and CXCL2 mRNA expression was increased in the spleens, and myeloperoxidase (MPO) activity in the spleens and livers was increased in the vaccinated mice after infection. These results suggest that vaccination with FnBPA541-870 induces the IL-17A-producing cells and that IL-17A-mediated cellular immunity is involved in the protective effect on S. aureus infection.

Expression of fibronectin binding protein A (FnBPA) from Staphylococcus aureus at the cell surface of Lactococcus lactis improves its immunomodulatory properties when used as protein delivery vector

A recombinant strain of Lactococcus lactis displaying a cell-surface anchored fibronectin binding protein A (FnBPA) from Staphylococcus aureus (LL-FnBPA) had been shown to be more efficient in delivering plasmid than its wild-type counterpart both in vitro and in vivo, and have the ability to orientate the immune response toward a Th2 profile in a context of a DNA vaccination. The aim of this work was to test whether this LL-FnBPA strain could shape the immune response after mucosal administration in mice. For this, we used a mouse model of human papilloma virus (HPV)-induced cancer and a L. lactis strain displaying at its cell surface both HPV-16-E7 antigen (LL-E7) and FnBPA (LL-E7+FnBPA). Our results revealed a more efficient systemic Th1 immune response with recombinant LL-E7+FnBPA. Furthermore, mice vaccinated with LL-E7+FnBPA were better protected when challenged with HPV-16-induced tumors. Altogether, the results suggest that FnBPA displays adjuvant properties when used in the context of mucosal delivery using L. lactis as a live vector.

Construction of a new shuttle vector for DNA delivery into mammalian cells using non-invasive Lactococcus lactis

Use of food grade Lactococcus lactis (L. lactis) is fast emerging as a safe alternative for delivery of DNA vaccine. To attain efficient DNA delivery, L. lactis, a non-invasive bacterium is converted to invasive strain either by expressing proteins like Internalin A (InlA) or Fibronectin binding protein A (FnBPA) or through chemical treatments. However the safety status of invasive L. lactis is questionable. In the present report, we have shown that non-invasive L. lactis efficiently delivered the newly constructed reporter plasmid pPERDBY to mammalian cells without any chemical enhancers. The salient features of the vector are; I) Ability to replicate in two different hosts; Escherichia coli (E. coli) and Lactic Acid Bacteria (LAB), II) One of the smallest reporter plasmid for DNA vaccine, III) Enhanced Green Fluorescence Protein (EGFP) linked to Multiple Cloning Site (MCS), IV) Immunostimulatory CpG motifs functioning as an adjuvant. Expression of EGFP in pPERDBY transfected CHO-K1 and Caco-2 cells demonstrates its functionality. Non-invasive r-L. lactis was found efficient in delivering pPERDBY to Caco-2 cells. The in vitro data presented in this article supports the hypothesis that in the absence of invasive proteins or relevant chemical treatment, L. lactis was found efficient in delivering DNA to mammalian cells.

The potential of bacteriophage cocktail in eliminating Methicillin-resistant Staphylococcus aureus biofilms in terms of different extracellular matrices expressed by PIA, ciaA-D and FnBPA genes.

BackgroundThis study assessed novel approach of using highly lytic phages against methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA) biofilms with and without biofilm extracellular matrix- disrupting chemical.MethodThe resultant phage-based control was assessed in relation to the type of biofilm extracellular matrix namely, polysaccharide intercellular adhesion (PIA) or proteinacious fibronectin-binding protein A (FnBPA). The biofilms were formed in vitro by 24 h incubation of bacteria in 96 wells microtiter plates at room temperature. The formed biofilms were assessed by tissue culture plate (TCP). Moreover, the nature of the biofilm was assessed by scanning electron microscopy (SEM) and PCR assay for detecting PIA genes, ciaA-D and FnBPA genes.Resultsthis study showed that applied phages with 0.08 % benezenthonium chloride, for PIA biofilms, and 0.06 % ethanol, for proteinacious FnBPA biofilms, exerted 100 % eradication for MSSA biofilms and about 78 % of MRSA biofilms. The phage-based control of biofilms with chemical adjuvant showed significantly higher efficiency than that without adjuvant (P < 0.05). Moreover, FnBPA biofilms were more common in MRSA than in MSSA while PIA biofilms were more common in MSSA than in MRSA. And the most resistant type of biofilms to phage-based control was FnBPA in MRSA where 50 % of biofilms were reduced but not eradicated completely.ConclusionsIt is concluded that PIA-disturbing agent and protein denaturing alcohol can increase the efficiency of attacking phages in accessing host cell walls and lysing them which in turn lead to much more efficient MRSA and MSSA biofilm treatment and prevention.

Staphylococcus aureus Fibronectin-Binding Protein A Mediates Cell-Cell Adhesion through Low-Affinity Homophilic Bonds.

ABSTRACTStaphylococcus aureus is an important opportunistic pathogen which is a leading cause of biofilm-associated infections on indwelling medical devices. The cell surface-located fibronectin-binding protein A (FnBPA) plays an important role in the accumulation phase of biofilm formation by methicillin-resistant S. aureus (MRSA), but the underlying molecular interactions are not yet established. Here, we use single-cell and single-molecule atomic force microscopy to unravel the mechanism by which FnBPA mediates intercellular adhesion. We show that FnBPA is responsible for specific cell-cell interactions that involve the FnBPA A domain and cause microscale cell aggregation. We demonstrate that the strength of FnBPA-mediated adhesion originates from multiple low-affinity homophilic interactions between FnBPA A domains on neighboring cells. Low-affinity binding by means of FnBPA may be important for biofilm dynamics. These results provide a molecular basis for the ability of FnBPA to promote cell accumulation during S. aureus biofilm formation. We speculate that homophilic interactions may represent a generic strategy among staphylococcal cell surface proteins for guiding intercellular adhesion. As biofilm formation by MRSA strains depends on proteins rather than polysaccharides, our approach offers exciting prospects for the design of drugs or vaccines to inhibit protein-dependent intercellular interactions in MRSA biofilms.

Construction of scFv that bind both fibronectin-binding protein A and clumping factor A of Stapylococcus aureus

Bovine mastitis (BM) causes significant losses to the dairy industry. Vaccines against the causative agent of BM, Staphylococcus aureus, do not confer adequate protection. Because passive immunization with antibodies permits disease prevention, we constructed a recombinant single-chain antibody (scFv) against fibronectin-binding protein A (FnBPA) and clumping factor A (ClfA), two important virulence factors in S. aureus infection. The DNA coding sequences of the variable heavy (VH) and variable light (VL) domains of antibodies produced in the peripheral blood lymphocytes of cows with S. aureus-induced mastitis were obtained using reverse transcription and polymerase chain reaction, and the VH and VL cDNAs were assembled in-tandem using a DNA sequence encoding a (Gly4Ser)3 peptide linker. The scFv cDNAs were cloned into the pOPE101 plasmid for the expression of soluble scFv protein in Escherichia coli. The binding of the scFvs to both FnBPA and ClfA was confirmed using an indirect ELISA and Western blotting. The DNA sequences of the framework regions of the VH and VL domains were highly conserved, and the complementarity-determining regions displayed significant diversity, especially in CDR3 of the VH domain. These novel bovine antibody fragments may be useful as a therapeutic candidate for the prevention and treatment of S. aureus-induced bovine mastitis.