Identification of Novel Laminin- and Fibronectin-binding Proteins by Far-Western Blot: Capturing the Adhesins of Streptococcus suis Type 2.
ABSTRACT: Bacterial cell wall (CW) and extracellular (EC) proteins are often involved in interactions with extracellular matrix (ECM) proteins such as laminin (LN) and fibronectin (FN), which play important roles in adhesion and invasion. In this study, an efficient method combining proteomic analysis and Far-Western blotting assays was developed to screen directly for bacterial surface proteins with LN- and FN-binding capacity. With this approach, fifteen potential LN-binding proteins and five potential FN-binding proteins were identified from Streptococcus suis serotype 2 (SS2) CW and EC proteins. Nine newly identified proteins, including oligopeptide-binding protein OppA precursor (OppA), elongation factor Tu (EF-Tu), enolase, lactate dehydrogenase (LDH), fructose-bisphosphate aldolase (FBA), 3-ketoacyl-ACP reductase (KAR), Gly ceraldehyde-3-phosphate dehydrogenase (GAPDH), Inosine 5'-monophosphate dehydrogenase (IMPDH), and amino acid ABC transporter permease (ABC) were cloned, expressed, purified and further confirmed by Far-Western blotting and ELISA. Five proteins (OppA, EF-Tu, enolase, LDH, and FBA) exhibited specifically binding activity to both human LN and human FN. Furthermore, seven important recombinant proteins were selected and identified to have the ability to bind Hep-2 cells by the indirect immunofluorescent assay. In addition, four recombinant proteins, and their corresponding polyclonal antibodies, were observed to decrease SS2 adhesion to Hep-2 cells, which indicates that these proteins contribute to the adherence of SS2 to host cell surface. Collectively, these results show that the approach described here represents a useful tool for investigating the host-pathogen interactions.
Project description:Streptococcus suis serotype 2 (SS2) induced sepsis and meningitis are often accompanied by bacteremia. However, the mechanism whereby it helps S. suis to evade PMN-mediated phagocytosis remain unclear. Because of the central roles of bacteria-human fibrinogen (hFg) interaction in innate immunity, here, a proteomics based Far-western blotting (PBFWB) was developed to identify the fibrinogen-binding surface proteins of S. suis (SsFBPs) on a large-scale. And then thirteen potential SsFBPs were identified by PBFWB and we selected seven potential surface proteins to further confirm their binding ability to hFg, of which the gene mutant strains of MRP displayed significantly decrease in binding to immobilized hFg. Additionally, the polyclonal antibodies against Enolase were found to significantly inhibit the binding of SS2 to hFg. Strikingly, MRP and Enolase were found to improve the antiphagocytic ability of SS2 to PMNs by interacting with hFg and enhance the survival of SS2 in human blood. Taken together, the PBFWB method provides useful clues to the bacteria-host interactions. These studies firstly disclose MRP and Enolase were involved in immune evasion of SS2 at least in part by binding to Fg, which make them potential targets for therapies for SS2 infection.
Project description:The first fully synthetic glycopeptide vaccines against a fungal disease have been used to combat disseminated candidiasis in mice. Six T cell peptides found in Candida albicans cell wall proteins were selected by algorithm peptide epitope searches; each was synthesized and conjugated to the fungal cell wall beta-mannan trisaccharide [beta-(Man)(3)] by novel saccharide-peptide linker chemistry to create glycopeptide conjugates. The six proteins were selected because of expression during human candidiasis and cell wall association and included: fructose-bisphosphate aldolase (Fba); methyltetrahydropteroyltriglutamate (Met6); hyphal wall protein-1 (Hwp1); enolase (Enol); glyceraldehyde-3-phosphate dehydrogenase (Gap1); and phosphoglycerate kinase (Pgk1). By immunization protocols favoring production of protective antibody, the beta-(Man)(3)-Fba, beta-(Man)(3)-Met6 and beta-(Man)(3)-Hwp1 induced protection evidenced by survival and reduced kidney fungal burden, the beta-(Man)(3)-Eno1 and beta-(Man)(3)-Gap1 gave moderate protection, and the beta-(Man)(3)-Pgk1 slightly enhanced disease. For the beta-(Man)(3)-Fba conjugate, protection was uniquely acquired through immunity against the carbohydrate and the Fba peptide. This approach based on fully synthetic chemically defined immunogens should be generally useful in vaccine development.
Project description:Mycoplasma pneumoniae and Mycoplasma genitalium are closely related organisms that cause distinct clinical manifestations and possess different tissue predilections despite their high degree of genome homology. We reported earlier that surface-localized M. pneumoniae elongation factor Tu (EF-Tu(Mp)) mediates binding to the extracellular matrix component fibronectin (Fn) through the carboxyl region of EF-Tu. In this study, we demonstrate that surface-associated M. genitalium EF-Tu (EF-Tu(Mg)), in spite of sharing 96% identity with EF-Tu(Mp), does not bind Fn. We utilized this finding to identify the essential amino acids of EF-Tu(Mp) that mediate Fn interactions by generating modified recombinant EF-Tu proteins with amino acid changes corresponding to those of EF-Tu(Mg). Amino acid changes in serine 343, proline 345, and threonine 357 were sufficient to significantly reduce the Fn binding of EF-Tu(Mp). Synthetic peptides corresponding to this region of EF-Tu(Mp) (EF-Tu(Mp) 340-358) blocked both recombinant EF-Tu(Mp) and radiolabeled M. pneumoniae cell binding to Fn. In contrast, EF-Tu(Mg) 340-358 peptides exhibited minimal blocking activity, reinforcing the specificity of EF-Tu-Fn interactions as mediators of microbial colonization and tissue tropism.
Project description:Differentially expressed and immunogenic spore proteins of the Bacillus cereus group of bacteria, which includes Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis, were identified. Comparative proteomic profiling of their spore proteins distinguished the three species from each other as well as the virulent from the avirulent strains. A total of 458 proteins encoded by 232 open reading frames were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis for all the species. A number of highly expressed proteins, including elongation factor Tu (EF-Tu), elongation factor G, 60-kDa chaperonin, enolase, pyruvate dehydrogenase complex, and others exist as charge variants on two-dimensional gels. These charge variants have similar masses but different isoelectric points. The majority of identified proteins have cellular roles associated with energy production, carbohydrate transport and metabolism, amino acid transport and metabolism, posttranslational modifications, and translation. Novel vaccine candidate proteins were identified using B. anthracis polyclonal antisera from humans postinfected with cutaneous anthrax. Fifteen immunoreactive proteins were identified in B. anthracis spores, whereas 7, 14, and 7 immunoreactive proteins were identified for B. cereus and in the virulent and avirulent strains of B. thuringiensis spores, respectively. Some of the immunodominant antigens include charge variants of EF-Tu, glyceraldehyde-3-phosphate dehydrogenase, dihydrolipoamide acetyltransferase, Delta-1-pyrroline-5-carboxylate dehydrogenase, and a dihydrolipoamide dehydrogenase. Alanine racemase and neutral protease were uniquely immunogenic to B. anthracis. Comparative analysis of the spore immunome will be of significance for further nucleic acid- and immuno-based detection systems as well as next-generation vaccine development.
Project description:The binding and ingestion of Mycobacterium avium subsp. paratuberculosis (MAP) by host cells are fibronectin (FN) dependent. In several species of mycobacteria, a specific family of proteins allows the attachment and internalization of these bacteria by epithelial cells through interaction with FN. Thus, the identification of adhesion molecules is essential to understand the pathogenesis of MAP. The aim of this study was to identify and characterize FN binding cell wall proteins of MAP. We searched for conserved adhesins within a large panel of surface immunogenic proteins of MAP and investigated a possible interaction with FN. For this purpose, a cell wall protein fraction was obtained and resolved by 2D electrophoresis. The immunoreactive spots were identified by MALDI-TOF MS and a homology search was performed. We selected elongation factor Tu (EF-Tu) as candidate for further studies. We demonstrated the FN-binding capability of EF-Tu using a ligand blot assay and also confirmed the interaction with FN in a dose-dependent manner by ELISA. The dissociation constant of EF-Tu was determined by surface plasmon resonance and displayed values within the ?M range. These data support the hypothesis that this protein could be involved in the interaction of MAP with epithelial cells through FN binding.
Project description:NETs constitute a network of DNA and proteins released by neutrophils in response to infectious and immunologic triggers. NET proteins are recognized as autoantigens in ANCA vasculitis; limited knowledge is available in other autoimmune pathologies. The composition of NETs produced ex vivo by resting and Phorbol-myristate acetate (PMA) stimulated neutrophils was analyzed by high-throughput Fusion Orbitrap technology in 16 patients with Systemic Lupus Erythematosus/Lupus nephritis (9 SLE/7 LN) and in 11 controls. Seven-hundred proteins were characterized and specific fingerprints discriminated LN from SLE. We focused on methyl-oxidized ?enolase (methionine sulfoxide 93) that was markedly increased in NETs from LN and was localized in NET filaments in tight connection and outlying DNA. The isotype of anti-?enolase antibodies was IgG2 in LN and IgG4 in other autoimmune glomerulonephritis (Membranous Nephropathy, MN); serum anti-?enolase IgG2 were higher in LN than in SLE and absent in MN. The same IgG2 antibodies recognized 5 epitopes of the protein one containing methionine sulphoxide 93. In conclusion, specific NET protein fingerprints characterize different subsets of SLE; methyl-oxidized ?enolase is over-expressed in LN. Circulating anti-?enolase IgG2 recognize the oxidized epitope and are high in serum of LN patients. Post-translational modified NET proteins contribute to autoimmunity in patients with LN.
Project description:<i>Streptococcus suis</i> (<i>S. suis</i>) serotype 2 (SS2) is the causative agent of swine streptococcosis and can cause severe diseases in both pigs and humans. Although the traditional inactive vaccine can protect pigs from SS2 infection, novel vaccine candidates are needed to overcome its shortcomings. Three infection-associated proteins in <i>S. suis</i>-muramidase-released protein (MRP), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and DLD, a novel putative dihydrolipoamide dehydrogenase-have been previously identified by immunoproteomic assays. In this study, the effective immune protection of the recombinant trivalent protein GAPDH-MRP-DLD (JointS) against SS2, SS7, and SS9 was determined in zebrafish. To improve the immune efficacy of JointS, monophosphoryl lipid A (MPLA) as a TLR4 agonist adjuvant, which induces a strong innate immune response in the immune cells of mice and pigs, was combined with JointS to immunize the mice. The results showed that immunized mice could induce the production of a high titer of anti-<i>S. suis</i> antibodies; as a result, 100% of mice survived after SS2 infection. Furthermore, JointS provides good protection against virulent SS2 strain infections in piglets. Given the above, there is potential to develop JointS as a novel subunit vaccine for piglets to prevent infection by SS2 and other <i>S. suis</i> serotypes.
Project description:Renal targets of autoimmunity in human lupus nephritis (LN) are unknown. We sought to identify autoantibodies and glomerular target antigens in renal biopsy samples from patients with LN and determine whether the same autoantibodies can be detected in circulation. Glomeruli were microdissected from biopsy samples of 20 patients with LN and characterized by proteomic techniques. Serum samples from large cohorts of patients with systemic lupus erythematosus (SLE) with and without LN and other glomerulonephritides were tested. Glomerular IgGs recognized 11 podocyte antigens, with reactivity varying by LN pathology. Notably, IgG2 autoantibodies against ?-enolase and annexin AI were detected in 11 and 10 of the biopsy samples, respectively, and predominated over other autoantibodies. Immunohistochemistry revealed colocalization of ?-enolase or annexin AI with IgG2 in glomeruli. High levels of serum anti-?-enolase (>15 mg/L) IgG2 and/or anti-annexin AI (>2.7 mg/L) IgG2 were detected in most patients with LN but not patients with other glomerulonephritides, and they identified two cohorts: patients with high anti-?-enolase/low anti-annexin AI IgG2 and patients with low anti-?-enolase/high anti-annexin AI IgG2. Serum levels of both autoantibodies decreased significantly after 12 months of therapy for LN. Anti-?-enolase IgG2 recognized specific epitopes of ?-enolase and did not cross-react with dsDNA. Furthermore, nephritogenic monoclonal IgG2 (clone H147) derived from lupus-prone MRL-lpr/lpr mice recognized human ?-enolase, suggesting homology between animal models and human LN. These data show a multiantibody composition in LN, where IgG2 autoantibodies against ?-enolase and annexin AI predominate in the glomerulus and can be detected in serum.
Project description:Degradation of cellular matrix is one of the important processes related to the progression of breast cancer. Tumor cells have the ability to exhibit necessary conditions for growth and survival, promoting degradation processes of extracellular matrix proteins, such as laminin (LN) and fibronectin (FN). In this study, we evaluated whether treatments, based on free rhodium (II) citrate (Rh2(H2cit)4), maghemite nanoparticles coated with citrate (Magh-cit) and maghemite nanoparticles coated with rhodium (II) citrate (Magh-Rh2(H2cit)4), in murine metastatic breast carcinoma models can modulate the expression of laminin and fibronectin proteins. Synthesized nanoparticles were characterized using X-ray diffraction, transmission electron microscopy, energy dispersive spectroscopy and dynamic light scattering. The expression of FN and LN was assessed using immunohistochemistry and western blotting. The gene expression of FN1 and LAMA1 were evaluated using real-time PCR. The FN1 and LAMA1 transcripts from the Magh-Rh2(H2cit)4 treated group were 95% and 94%, respectively, lower than the control group. Significant reduction in tumor volume for animals treated with Magh-Rh2(H2cit)4 was observed, of about 83%. We witnessed statistically significant reductions of FN and LN expression following treatment with Magh-Rh2(H2cit)4. We have demonstrated that the antitumor effects of Magh-Rh2(H2cit)4 and Rh2(H2cit)4 regulate the expression of FN and LN in metastatic breast tumors.
Project description:BACKGROUND: Mycoplasma synoviae is an avian pathogen that can lead to respiratory tract infections and arthritis in chickens and turkeys, resulting in serious economic losses to the poultry industry. Enolase reportedly plays important roles in several bacterial pathogens, but its role in M. synoviae has not been established. Therefore, in this study, the enolase encoding gene (eno) of M. synoviae was amplified from strain WVU1853 and expressed in E. coli BL21 cells. Then the enzymatic activity, immunogenicity and binding activity with chicken plasminogen (Plg) and human fibronectin (Fn) was evaluated. RESULTS: We demonstrated that the recombinant M. synoviae enolase protein (rMsEno) can catalyze the conversion of 2-phosphoglycerate (2-PGA) to phosphoenolpyruvate (PEP), the Km and Vmax values of rMsEno were 1.1 × 10(-3) M and 0.739 ?mol/L/min, respectively. Western blot and immuno-electron microscopy analyses confirmed that enolase was distributed on the surface and within the cytoplasm of M. synoviae cells. The binding assays demonstrated that rMsEno was able to bind to chicken Plg and human Fn proteins. A complement-dependent mycoplasmacidal assay demonstrated that rabbit anti-rMsEno serum had distinct mycoplasmacidal efficacy in the presence of complement, which also confirmed that enolase was distributed on the surface of M. synoviae. An inhibition assay showed that the adherence of M. synoviae to DF-1 cells pre-treated with Plg could be effectively inhibited by treatment with rabbit anti-rMsEno serum. CONCLUSION: These results reveal that M. synoviae enolase has good catalytic activity for conversion of 2-PGA to PEP, and binding activity with chicken Plg and human Fn. Rabbit anti-rMsEno serum displayed an obvious complement-dependent mycoplasmacidal effect and adherent inhibition effect. These results suggested that the M. synoviae enolase plays an important role in M. synoviae metabolism, and could potentially impact M. synoviae infection and immunity.