Identification of Api88 Binding Partners in <i>Escherichia coli</i> Using a Photoaffinity-Cross-Link Strategy and Label-Free Quantification

Gene-encoded antimicrobial peptides (AMPs) kill bacteria very efficiently by either lytic mechanisms or inhibition of specific bacterial targets. Proline-rich AMPs (PrAMPs), for example, produced in insects and mammals rely on the second mechanism. They bind to the 70 kDa bacterial heat shock protein DnaK and the 60 kDa chaperonin GroEL and interfere with protein folding, but this does not explain their strong bactericidal effects. Thus, we looked for further binding partners of apidaecin 1b, originally identified in honey bees, and two rationally optimized analogues (Api88 and Api137). Because affinity chromatography using Api88 as an immobilized ligand enriched only a few proteins at low levels besides DnaK, we synthesized Api88 analogues substituting Tyr7 with <i>p</i>-benzoyl-phenylalanine (Bpa), which can cross-link the peptide to binding partners after UV irradiation. <i>Escherichia coli</i> was incubated with biotinylated Api88 Tyr7Bpa or the corresponding all-d-peptide, irradiated, and lysed. The protein extract was enriched by streptavidin, separated by SDS-PAGE, digested with trypsin, and analyzed by nanoRP-UPLC-ESI-QqTOF-MS/MS. Among the 41 proteins identified, 34 were detected only in the l-Api88 Tyr7Bpa sample, including five 70S ribosomal proteins, DNA-directed RNA polymerase, and pyruvate dehydrogenase, indicating that PrAMPs might interfere with protein translation and energy metabolism

Additional file 4: of Detection of mammalian orthoreovirus type-3 (Reo-3) infections in mice based on serotype-specific hemagglutination protein sigma-1

Temperature-dependent stability of Strep-rσ-1-His-ELISA. Freshly coated ELISA plates were stored at 4 °C (black) and 37 °C (red), respectively, including all material needed for testing (controls, buffers, conjugate, substrate, sulfuric acid). Positive control (solid line, anti-His antibody) and negative control (dashed line, pool of reovirus type-3 [−] sera) were assayed in replicates of three at a total of nine time points for each storage temperature. Mean values and standard deviations are indicated. (TIF 178 kb

Additional file 5: of Detection of mammalian orthoreovirus type-3 (Reo-3) infections in mice based on serotype-specific hemagglutination protein sigma-1

Excel Sheet Raw data of ELISA validation. Sheet 1: Raw data of repeatability, intermediate precision and calculation of the limit of detection. Sheet 2: Raw data of stability experiments. Sheet 3: Raw data of ROC analysis. (XLSX 21 kb

Additional file 1: of Detection of mammalian orthoreovirus type-3 (Reo-3) infections in mice based on serotype-specific hemagglutination protein sigma-1

Alignment of σ-1 amino acid sequences shows conservation within and diversity across reovirus serotypes. The following GenBank accession numbers were used for the BLASTp alignment: HM159619.1 (T3D), GU589583.1 (T3A), M35964.1 (T2J) and M35963.1 (T1L). Absolute and percentage values of identity, positives and gaps as well as percentage of query cover are provided. Information modified after [36]. (XLSX 11 kb

Identification of Api88 Binding Partners in <i>Escherichia coli</i> Using a Photoaffinity-Cross-Link Strategy and Label-Free Quantification

Gene-encoded antimicrobial peptides (AMPs) kill bacteria very efficiently by either lytic mechanisms or inhibition of specific bacterial targets. Proline-rich AMPs (PrAMPs), for example, produced in insects and mammals rely on the second mechanism. They bind to the 70 kDa bacterial heat shock protein DnaK and the 60 kDa chaperonin GroEL and interfere with protein folding, but this does not explain their strong bactericidal effects. Thus, we looked for further binding partners of apidaecin 1b, originally identified in honey bees, and two rationally optimized analogues (Api88 and Api137). Because affinity chromatography using Api88 as an immobilized ligand enriched only a few proteins at low levels besides DnaK, we synthesized Api88 analogues substituting Tyr7 with <i>p</i>-benzoyl-phenylalanine (Bpa), which can cross-link the peptide to binding partners after UV irradiation. <i>Escherichia coli</i> was incubated with biotinylated Api88 Tyr7Bpa or the corresponding all-d-peptide, irradiated, and lysed. The protein extract was enriched by streptavidin, separated by SDS-PAGE, digested with trypsin, and analyzed by nanoRP-UPLC-ESI-QqTOF-MS/MS. Among the 41 proteins identified, 34 were detected only in the l-Api88 Tyr7Bpa sample, including five 70S ribosomal proteins, DNA-directed RNA polymerase, and pyruvate dehydrogenase, indicating that PrAMPs might interfere with protein translation and energy metabolism

Identification of New Resistance Mechanisms in <i>Escherichia coli</i> against Apidaecin 1b Using Quantitative Gel- and LC–MS-Based Proteomics

Bacteria have acquired resistance mechanisms to overcome antibiotic treatments, triggering major concerns about the return of epidemic infections. Antimicrobial peptides identified in insects, animals, and plants represent a huge pool of promising lead structures that can be further developed for medical applications. Short proline-rich antimicrobial peptides (PrAMPs) have gained much attention due to their clinically interesting activity spectrum, serum protease stability, efficacy in murine infection models, and low adverse effects. Here we induced resistances by incubating <i>Escherichia coli</i> with increasing concentrations of apidaecin 1b, a PrAMP isolated from honeybees, and quantitatively evaluated the proteomes between wild-type and resistant strains. Surprisingly, 2D differential gel electrophoresis did not reveal differences, indicating that the expression levels of dominant proteins were very similar. Reversed-phase chromatography coupled online to a mass spectrometer identified 2131 proteins in the soluble fraction (cytosolic fraction) and 1296 proteins in the nonsolubilized pellet (membrane fraction). Overall 29 proteins showed a statistically significant upregulation in the resistant <i>E. coli</i> strain, whereas 18 proteins were downregulated. Interestingly, periplasmic chaperone FimC, fimbrial protein FimA, and mannose-binding domain protein FimH, which are part of the fimbrial complex, were not detected in the resistant strain that was also unable to form biofilms. Furthermore, the expression of a few other proteins known as virulence factors was downregulated. Additionally, the expression level of isochorismatase hydrolase (YcaC) decreased in the membrane fraction of the resistant strain to 35%, and the corresponding knockout mutant of <i>E. coli</i> BW25113 was eight times less susceptible to apidaecin 1b and the related designer peptide Api88

DataSheet_1_Immunoproteomics enable broad identification of new Aspergillus fumigatus antigens in severe equine asthma.pdf

IntroductionSevere equine asthma (SEA) is a common chronic disease of adult horses with characteristic recurrent airway obstruction and similarities to neutrophilic asthma in humans. As an extrinsic stimulus, hay dust exposure is a major risk factor and induces acute exacerbation in susceptible horses. However, single inducing agents of SEA have hardly been identified on a molecular basis. Aspergillus fumigatus (A. fumigatus) is a common mold species in hay and has been described as a major provoking agent of SEA.MethodsAiming to identify disease-relevant antigens, we analyzed A. fumigatus using an immunoproteomics approach on two-dimensional immunoblots of A. fumigatus protein probed with serum from environmentally matched asthmatic and healthy horses (n=5 pairs). A. fumigatus binding serum immunoglobulins (Pan-Ig), and the isotypes IgG4/7 and IgG3/5 were quantified for each protein spot and then compared between asthmatic and healthy horses.Results and discussionFor 21 out of 289 spots serum immunoglobulin (Ig) binding was different between the two groups for Pan-Ig or the isotypes. If differences were detected, Pan-Ig and IgG4/7 binding to the proteins were lower, while IgG3/5 binding was higher in asthmatic than healthy horse sera. Proteins were extracted from the 21 spots of interest and analyzed by liquid chromatography mass spectrometry. Eight prioritized proteins (candidate antigens) were expressed as recombinant proteins. Some of these have been previously described as major or minor A. fumigatus allergens, alongside other proteins, most with hydrolase activity. Recombinant candidate antigens were tested on 1D immunoblots to confirm their relevance as antigens by serum antibody binding. Four proteins (beta-hexosaminidase, class II aldolase/adducin domain protein, glucoamylase, peptide hydrolase B0XX53) showed different antibody binding characteristics between asthmatic and healthy horses and are likely relevant antigens in SEA. Their identification can provide the basis for innovative diagnostics, prevention, or therapeutic approaches. Additionally, a more profound understanding of SEA and its potential underlying mechanisms can be established. Elevated serum IgG3/5 antibodies correlate with T helper cell 2 responses in other equine pathologies, and the recombinant SEA antigens developed here can become instrumental in analyzing the involvement of SEA-specific T cell responses and Ig responses in future studies.</p

Table_2_Immunoproteomics enable broad identification of new Aspergillus fumigatus antigens in severe equine asthma.xlsx

IntroductionSevere equine asthma (SEA) is a common chronic disease of adult horses with characteristic recurrent airway obstruction and similarities to neutrophilic asthma in humans. As an extrinsic stimulus, hay dust exposure is a major risk factor and induces acute exacerbation in susceptible horses. However, single inducing agents of SEA have hardly been identified on a molecular basis. Aspergillus fumigatus (A. fumigatus) is a common mold species in hay and has been described as a major provoking agent of SEA.MethodsAiming to identify disease-relevant antigens, we analyzed A. fumigatus using an immunoproteomics approach on two-dimensional immunoblots of A. fumigatus protein probed with serum from environmentally matched asthmatic and healthy horses (n=5 pairs). A. fumigatus binding serum immunoglobulins (Pan-Ig), and the isotypes IgG4/7 and IgG3/5 were quantified for each protein spot and then compared between asthmatic and healthy horses.Results and discussionFor 21 out of 289 spots serum immunoglobulin (Ig) binding was different between the two groups for Pan-Ig or the isotypes. If differences were detected, Pan-Ig and IgG4/7 binding to the proteins were lower, while IgG3/5 binding was higher in asthmatic than healthy horse sera. Proteins were extracted from the 21 spots of interest and analyzed by liquid chromatography mass spectrometry. Eight prioritized proteins (candidate antigens) were expressed as recombinant proteins. Some of these have been previously described as major or minor A. fumigatus allergens, alongside other proteins, most with hydrolase activity. Recombinant candidate antigens were tested on 1D immunoblots to confirm their relevance as antigens by serum antibody binding. Four proteins (beta-hexosaminidase, class II aldolase/adducin domain protein, glucoamylase, peptide hydrolase B0XX53) showed different antibody binding characteristics between asthmatic and healthy horses and are likely relevant antigens in SEA. Their identification can provide the basis for innovative diagnostics, prevention, or therapeutic approaches. Additionally, a more profound understanding of SEA and its potential underlying mechanisms can be established. Elevated serum IgG3/5 antibodies correlate with T helper cell 2 responses in other equine pathologies, and the recombinant SEA antigens developed here can become instrumental in analyzing the involvement of SEA-specific T cell responses and Ig responses in future studies.</p

The anti-tumorigenic activity of A2M—A lesson from the naked mole-rat

<div><p>Cancer resistance is a major cause for longevity of the naked mole-rat. Recent liver transcriptome analysis in this animal compared to wild-derived mice revealed higher expression of alpha2-macroglobulin (A2M) and cell adhesion molecules, which contribute to the naked mole-rat’s cancer resistance. Notably, A2M is known to dramatically decrease with age in humans. We hypothesize that this might facilitate tumour development. Here we found that A2M modulates tumour cell adhesion, migration and growth by inhibition of tumour promoting signalling pathways, e.g. PI3K / AKT, SMAD and up-regulated <i>PTEN</i> via down-regulation of miR-21, <i>in vitro</i> and in tumour xenografts. A2M increases the expression of CD29 and CD44 but did not evoke EMT. Transcriptome analysis of A2M-treated tumour cells, xenografts and mouse liver demonstrated a multifaceted regulation of tumour promoting signalling pathways indicating a less tumorigenic environment mediated by A2M. By virtue of these multiple actions the naturally occurring A2M has strong potential as a novel therapeutic agent.</p></div

Effect of A2M* on expression of CD44 and CD29 in different tumour cell lines.

<p><b>(a)</b> 1321N1 and A549 cells were cultured in the absence and presence of increasing concentrations of A2M, lysed and immunoblotted for CD44 and CD29. <b>(b)</b> A549 cells were stimulated by RAP and treated as shown in (a). <b>(c)</b> A549 cells were treated with A2M* and expression of total and cell surface CD44 was analysed by flow cytometry (n = 3), error bars mean ± sem, t-test (*P < 0.05, **P < 0.01). <b>(d)</b> A549 cell were treated with 100 nM A2M* for 12h, fixed by paraformaldehyde and immunestained for CD44 and counterstained with DAPI. <b>(e)</b> Immunoprecipitation of CD44 in A2M*-stimulated and non-stimulated A549 and MDA-MB-231 cells. Cells were treated with 30 nM A2M* for 24h, lysates were precipitated with different antibodies (anti-LRP1, anti-A2M*, anti-GLO1) and Western blotted for CD44 protein. <b>(f)</b> Ligand blot of A2M* for CD44 binding. A2M* was blotted to membranes which were incubated with cell lysate of A549 afore stimulated by 30 nM A2M* for 8h. Binding of CD44 to immobilized A2M* was detected by anti-CD44/HRP-anti-mouse-Ig. a) Immobilized A2M* detected by anti-A2M*/HRP-anti-mouse-Ig and b) Detection of binding of CD44 to immobilized A2M*. <b>(g)</b> Ligand blot demonstrating binding of CD44 to LRP1-associated RAP. Membrane-blotted LRP1 was incubated with A549 cell lysate and bound CD44 was detected by respective antibodies. <b>(h)</b> Recombinant RAP was electro-blotted, followed by incubation with A549 cell lysate. Detection of CD44 bound to immobilized RAP was accomplished by anti-CD44/HRP-anti-mouse-Ig. Immobilized RAP was detected by anti-RAP antibody (blotting control).</p