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 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

Data_Sheet_1_Evaluation of proline-rich antimicrobial peptides as potential lead structures for novel antimycotics against Cryptococcus neoformans.docx

BackgroundCryptococcosis and cryptococcal meningitis, caused by Cryptococcus neoformans infections, lead to approximately 180,000 deaths per year, primarily in developing countries. Individuals with compromised immune systems, e.g., due to HIV infection (AIDS) or chemotherapy, are particularly vulnerable. Conventional treatment options are often limited and can cause severe side effects. Therefore, this study aimed to investigate the antifungal effect of insect-derived proline-rich antimicrobial peptides (PrAMPs) against C. neoformans. These peptides are known for their low toxicity and their high efficacy in murine infection models, making them a promising alternative for treatment.ResultsA preliminary screening of the minimal inhibitory concentrations (MICs) of 20 AMPs, including the well-known PrAMPs Onc112, Api137, and Chex1Arg20 as well as the cathelicidin CRAMP against the C. neoformans strains 1841, H99, and KN99α revealed promising results, with MICs as low as 1.6 μmol/L. Subsequent investigations of selected peptides, determining their influence on fungal colony-forming units, confirmed their strong activity. The antifungal activity was affected by factors such as peptide net charge and sequence, with stronger effects at higher net charges probably due to better intracellular uptake confirmed by confocal laser scanning microscopy. Inactive scrambled peptides suggest a specific intracellular target, although scanning electron microscopy showed that PrAMPs also damaged the cell exterior for a low proportion of the cells. Possible pore formation could facilitate entry into the cytosol.</p

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