RESUMO
The proline-rich antimicrobial peptide (PrAMP) Drosocin (Dro) from fruit flies shows sequence similarity to other PrAMPs that bind to the ribosome and inhibit protein synthesis by varying mechanisms. The target and mechanism of action of Dro, however, remain unknown. Here we show that Dro arrests ribosomes at stop codons, probably sequestering class 1 release factors associated with the ribosome. This mode of action is comparable to that of apidaecin (Api) from honeybees, making Dro the second member of the type II PrAMP class. Nonetheless, analysis of a comprehensive library of endogenously expressed Dro mutants shows that the interactions of Dro and Api with the target are markedly distinct. While only a few C-terminal amino acids of Api are critical for binding, the interaction of Dro with the ribosome relies on multiple amino acid residues distributed throughout the PrAMP. Single-residue substitutions can substantially enhance the on-target activity of Dro.
Assuntos
Peptídeos Antimicrobianos , Biossíntese de Proteínas , Animais , Escherichia coli/metabolismo , Glicopeptídeos/química , Drosophila/química , Drosophila/metabolismoRESUMO
The global spread of multi- and pan-resistant bacteria has triggered research to identify novel strategies to fight these pathogens, such as antimicrobial peptides and, more recently, bacteriophages. In a proof-of-concept study, we have genetically modified lytic T7Select phages targeting Escherichia coli Rosetta by integrating DNA sequences derived from the proline-rich antimicrobial peptide, apidaecin. This allowed testing of our hypothesis that apidaecins and bacteriophages can synergistically act on phage-sensitive and phage-resistant E. coli cells and overcome the excessive cost of peptide drugs by using infected cells to express apidaecins before cell lysis. Indeed, the addition of the highly active synthetic apidaecin analogs, Api802 and Api806, to T7Select phage-infected E. coli Rosetta cultures prevented or delayed the growth of potentially phage-resistant E. coli Rosetta strains. However, high concentrations of Api802 also reduced the T7Select phage fitness. Additionally, plasmids encoding Api802, Api806, and Api810 sequences transformed into E. coli Rosetta allowed the production of satisfactory peptide quantities. When these sequences were integrated into the T7Select phage genome carrying an N-terminal green fluorescent protein (GFP-) tag to monitor the expression in infected E. coli Rosetta cells, the GFP-apidaecin analogs were produced in reasonable quantities. However, when Api802, Api806 and Api810 sequences were integrated into the T7Select phage genome, expression was below detection limits and an effect on the growth of potentially phage-resistant E. coli Rosetta strains was not observed for Api802 and Api806. In conclusion, we were able to show that apidaecins can be integrated into the T7Select phage genome to induce their expression in host cells, but further research is required to optimize the engineered T7Select phages for higher expression levels of apidaecins to achieve the expected synergistic effects that were visible when the T7Select phages and synthetic Api802 and Api806 were added to E. coli Rosetta cultures.
RESUMO
BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered the worldwide coronavirus disease 2019 (COVID-19) pandemic. Serological assays for the detection of SARS-CoV-2 infections are important to understand the immune response in patients and to obtain epidemiological data about the number of infected people, especially to identify asymptomatic persons not aware of a past infection. METHODS: We recombinantly produced SARS-CoV-2 nucleocapsid (N)-protein in Escherichia coli. We used the purified protein to develop an indirect enzyme-linked immunosorbent assay (ELISA) for the detection of SARS-CoV-2 specific antibodies. This ELISA method was optimized and validated with serum samples collected from 113 patients with RT-PCR-confirmed SARS-CoV-2 infections including hospitalized COVID-19 patients and 1500 control sera mostly collected before 2015 with different clinical background. RESULTS: The optimized N-protein-ELISA provided a sensitivity of 89.7% (n = 68) for samples collected from patients with confirmed SARS-CoV-2 infections and mild to severe symptoms more than 14 days after symptom onset or a positive PCR test. The antibody levels remained low for serum samples collected in the first six days (n = 23) and increased in the second week (n = 22) post symptom onset or PCR confirmation. At this early phase, the ELISA provided a sensitivity of 39.1% and 86.4%, respectively, reflecting the time of an IgG immune response against pathogens. The assay specificity was 99.3% (n = 1500; 95% CI 0.995-0.999). Serum samples from persons with confirmed antibody titers against human immunodeficiency viruses 1/2, parvovirus B19, hepatitis A/B virus, cytomegalovirus, Epstein Barr virus, and herpes simplex virus were tested negative. CONCLUSIONS: We conclude that the N-protein-based ELISA developed here is well suited for the sensitive and specific serological detection of SARS-CoV-2 specific IgG antibodies in human serum for symptomatic infections. It may also prove useful to identify previous SARS-CoV-2 infections in vaccinated people, as all currently approved vaccines rely on the SARS-CoV-2 spike (S-) protein.
Assuntos
COVID-19 , Infecções por Vírus Epstein-Barr , COVID-19/diagnóstico , Ensaio de Imunoadsorção Enzimática , Herpesvirus Humano 4 , Humanos , Proteínas do Nucleocapsídeo , SARS-CoV-2RESUMO
Proline-rich antimicrobial peptides (PrAMPs) are promising candidates to treat bacterial infections. The designer peptide ARV-1502 exhibits strong antimicrobial effects against Enterobacteriaceae both in vitro and in vivo. Since the inhibitory effects of ARV-1502 reported for the 70 kDa heat-shock protein DnaK do not fully explain the antimicrobial activity of its 176 substituted analogs, we further studied their effect on the bacterial 70S ribosome of Escherichia coli, a known target of PrAMPs. ARV-1502 analogues, substituted in positions 3, 4, and 8 to 12 (underlined) of the binding motif D3KPRPYLPRP12 with aspartic acid, lysine, serine, phenylalanine or leucine, were tested in a competitive fluorescence polarization (FP) binding screening assay using 5(6)-carboxyfluorescein-labeled (Cf-) ARV-1502 and the 70S ribosome isolated from E. coli BW25113. While their effect on ribosomal protein expression was studied for green fluorescent protein (GFP) in a cell-free expression system (in vitro translation), the importance of known PrAMP transporters SbmA and MdtM was investigated using E. coli BW25113 and the corresponding knockout mutants. The dissociation constant (Kd) of 201 ± 16 nmol/L obtained for Cf-ARV-1502 suggests strong binding to the E. coli 70S ribosome. An inhibitory binding assay indicated that the binding site overlaps with those of other PrAMPs including Onc112 and pyrrhocoricin as well as the non-peptidic antibiotics erythromycin and chloramphenicol. All these drugs and drug candidates bind to the exit-tunnel of the 70S ribosome. Substitutions of the C-terminal fragment of the binding motif YLPRP reduced binding. At the same time, inhibition of GFP expression increased with net peptide charge. Interestingly, the MIC values of wild-type and ΔsbmA and ΔmdtM knockout mutants indicated that substitutions in the ribosomal binding motif altered also the bacterial uptake, which was generally improved by incorporation of hydrophobic residues. In conclusion, most substituted ARV-1502 analogs bound weaker to the 70S ribosome than ARV-1502 underlining the importance of the YLPRP binding motif. The weaker ribosomal binding correlated well with decreased antimicrobial activity in vitro. Substituted ARV-1502 analogs with a higher level of hydrophobicity or positive net charge improved the ribosome binding, inhibition of translation, and bacterial uptake.
Assuntos
Anti-Infecciosos , Escherichia coli , Antibacterianos/química , Anti-Infecciosos/metabolismo , Peptídeos Antimicrobianos , Sítios de Ligação , Escherichia coli/metabolismo , Prolina/metabolismo , Ribossomos/metabolismoRESUMO
Fluorescent tagging of bioactive molecules is a powerful tool to study cellular uptake kinetics and is considered as an attractive alternative to radioligands. In this study, we developed fluorescent histone deacetylase (HDAC) inhibitors and investigated their biological activity and cellular uptake kinetics. Our approach was to introduce a dansyl group as a fluorophore in the solvent-exposed cap region of the HDAC inhibitor pharmacophore model. Three novel fluorescent HDAC inhibitors were synthesized utilizing efficient submonomer protocols followed by the introduction of a hydroxamic acid or 2-aminoanilide moiety as zinc-binding group. All compounds were tested for their inhibition of selected HDAC isoforms, and docking studies were subsequently performed to rationalize the observed selectivity profiles. All HDAC inhibitors were further screened in proliferation assays in the esophageal adenocarcinoma cell lines OE33 and OE19. Compound 2, 6-((N-(2-(benzylamino)-2-oxoethyl)-5-(dimethylamino)naphthalene)-1-sulfonamido)-N-hydroxyhexanamide, displayed the highest HDAC inhibitory capacity as well as the strongest anti-proliferative activity. Fluorescence microscopy studies revealed that compound 2 showed the fastest uptake kinetic and reached the highest absolute fluorescence intensity of all compounds. Hence, the rapid and increased cellular uptake of 2 might contribute to its potent anti-proliferative properties.
Assuntos
Compostos de Dansil/farmacologia , Corantes Fluorescentes/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Peptoides/farmacologia , Acetilação/efeitos dos fármacos , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Compostos de Dansil/síntese química , Compostos de Dansil/metabolismo , Compostos de Dansil/farmacocinética , Desenho de Fármacos , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/metabolismo , Corantes Fluorescentes/farmacocinética , Histona Desacetilase 1/metabolismo , Desacetilase 6 de Histona/metabolismo , Inibidores de Histona Desacetilases/síntese química , Inibidores de Histona Desacetilases/metabolismo , Inibidores de Histona Desacetilases/farmacocinética , Histonas/química , Histonas/metabolismo , Humanos , Microscopia Confocal , Microscopia de Fluorescência , Simulação de Acoplamento Molecular , Peptoides/síntese química , Peptoides/metabolismo , Peptoides/farmacocinética , Ligação ProteicaRESUMO
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 Escherichia coli 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 E. coli 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 E. coli BW25113 was eight times less susceptible to apidaecin 1b and the related designer peptide Api88.
Assuntos
Peptídeos Catiônicos Antimicrobianos , Farmacorresistência Bacteriana , Escherichia coli/química , Proteoma/efeitos dos fármacos , Proteômica/métodos , Animais , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos/farmacologia , Abelhas/química , Cromatografia em Gel , Cromatografia de Fase Reversa , Farmacorresistência Bacteriana/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/fisiologia , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Espectrometria de MassasRESUMO
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 p-benzoyl-phenylalanine (Bpa), which can cross-link the peptide to binding partners after UV irradiation. Escherichia coli 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.
Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Insetos/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/farmacologia , Benzofenonas/metabolismo , Western Blotting , Cromatografia de Afinidade , Cromatografia Líquida de Alta Pressão/métodos , Reagentes de Ligações Cruzadas/química , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Insetos/genética , Proteínas de Insetos/farmacologia , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Fenilalanina/análogos & derivados , Fenilalanina/genética , Fenilalanina/metabolismo , Ligação Proteica/efeitos da radiação , Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas , Proteômica/métodos , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Tirosina/genética , Tirosina/metabolismo , Raios UltravioletaRESUMO
In view of increasing health threats from multiresistant pathogens, antimicrobial peptides (AMPs) and, specifically, proline-rich AMPs (PrAMPs) have been investigated in animal models. PrAMPs enter bacteria via the ABC transporter SbmA and inhibit intracellular targets. We used phage transduction (Tn10 insertion) to screen by random mutagenesis for alternative uptake mechanisms for analogs of apidaecin 1b, a honeybee-derived PrAMP. All 24 apidaecin-resistant mutants had the Tn10 insertion in the sbmA gene. These sbmA::Tn10 insertion mutants and the Escherichia coli BW25113 ΔsbmA (JW0368) strain were still susceptible to the bactenecin PrAMP Bac7(1-35) and oncocin PrAMPs Onc18 and Onc112, as well as to Chex1-Arg20, despite significantly reduced internalizations. In a second round of random mutagenesis, the remaining susceptibility was linked to the yjiL-mdtM gene cluster. E. coli BW25113 and its ΔyjiL null mutant (JW5785) were equally susceptible to all PrAMPs tested, whereas the BW25113 ΔmdtM mutant was less susceptible to oncocins. The JW0368 yjiL::Tn10 transposon mutant (BS2) was resistant to all short PrAMPs and susceptible only to full-length Bac7 and A3-APO. Interestingly, PrAMPs appear to enter bacteria via MdtM, a multidrug resistance transporter (drug/H(+) antiporter) of the major facilitator superfamily (MFS) that can efflux antibiotics, biocides, and bile salts. In conclusion, PrAMPs enter bacteria via ABC and MFS transporters that efflux antibiotics and cytotoxic compounds from the cytoplasm to the periplasm.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Peptídeos Catiônicos Antimicrobianos/farmacologia , Antiporters/genética , Farmacorresistência Bacteriana/efeitos dos fármacos , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana Transportadoras/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Antiporters/metabolismo , Abelhas/química , Abelhas/fisiologia , Transporte Biológico , Colífagos/genética , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Elementos de DNA Transponíveis , Farmacorresistência Bacteriana/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/deficiência , Família Multigênica , Mutação , Peptídeos Cíclicos/isolamento & purificação , Peptídeos Cíclicos/farmacologia , Periplasma/efeitos dos fármacos , Periplasma/metabolismo , Domínios Proteicos Ricos em Prolina , Transdução GenéticaRESUMO
Short proline-rich antimicrobial peptides (PrAMPs) are a promising class of antibiotics that use novel mechanisms, thus offering the potential to overcome the health threat of multiresistant pathogens. The peptides bind to the bacterial 70S ribosome and can inhibit protein translation. We report that PrAMPs can be divided into two classes, with each class binding to a different site, and thus use different lethal mechanisms. Oncocin-type peptides inhibit protein translation in Escherichia coli by binding to the exit tunnel of the 70S ribosome with half maximal inhibitory concentrations (IC50 values) of around 2 to 6 µmol L(-1), whereas apidaecin-type peptides block the assembly of the large (50S) subunit of the ribosome, resulting in similar IC50 values. The revealed mechanisms should allow the design of new antibiotics to overcome current bacterial resistance mechanisms.
Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Prolina/metabolismo , Subunidades Ribossômicas Maiores de Bactérias/metabolismo , Sequência de Aminoácidos , Peptídeos Catiônicos Antimicrobianos/química , Escherichia coli/metabolismo , Cinética , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Prolina/química , Ligação Proteica , Subunidades Ribossômicas Maiores de Bactérias/químicaRESUMO
Proline-rich antimicrobial peptides (PrAMPs) have been investigated and optimized by several research groups and companies as promising lead compounds to treat systemic infections caused by Gram-negative bacteria. PrAMPs, such as apidaecins and oncocins, enter the bacteria and kill them apparently through inhibition of specific targets without a lytic effect on the membranes. Both apidaecins and oncocins were shown to bind with nanomolar dissociation constants to the 70S ribosome. In apidaecins, at least the two C-terminal residues (Arg17 and Leu18) interact strongly with the 70S ribosome, whereas residues Lys3, Tyr6, Leu7, and Arg11 are the major interaction sites in oncocins. Oncocins inhibited protein biosynthesis very efficiently inâ vitro with half maximal inhibitory concentrations (IC50 values) of 150 to 240â nmol L(-1). The chaperone DnaK is most likely not the main target of PrAMPs but it binds them with lower affinity.
Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bactérias/efeitos dos fármacos , Proteínas de Bactérias/genética , Prolina/análise , Biossíntese de Proteínas/efeitos dos fármacos , Ribossomos/genética , Animais , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/química , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Insetos , Testes de Sensibilidade Microbiana , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can infect human cells by first attaching to the ACE-2 receptor via its receptor-binding domain (RBD) in the spike protein. Here, we report the influence of N-glycosylation sites of the RBD and the membrane (M) protein on IgG antibody binding in serum samples from patients infected with the original SARS-CoV-2 strain in Germany. The RBDs of the wildtype, alpha, beta, gamma, and kappa variants expressed in HEK293S GnTI- cells were all N-glycosylated at Asn331, Asn334, Asn343, and Asn360 or Asn370, whereas the M-protein was glycosylated at Asn5. An ELISA using a coated RBD and probed with anti-RBD IgG antibodies gave a sensitivity of 96.3% and a specificity of 100% for the wildtype RBD, while the sensitivity decreased by 5% to 10% for the variants of concern, essentially in the order of appearance. Deglycosylation of the wildtype RBD strongly reduced antibody recognition by ~20%, considering the mean of the absorbances recorded for the ELISA. This effect was even stronger for the unglycosylated RBD expressed in Escherichia coli, suggesting structural changes affecting epitope recognition. Interestingly, the N-glycosylated M-protein expressed in HEK293S GnTI- cells gave good sensitivity (95%), which also decreased to 65% after deglycosylation, and selectivity (100%). In conclusion, N-glycosylation of the M-protein, the RBD, and most likely the spike protein are important for proper antibody binding and immunological assays, whereas the type of N-glycosylation is less relevant.
RESUMO
Introduction: Severe 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. Methods: Aiming 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 discussion: For 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.
Assuntos
Asma , Doença Pulmonar Obstrutiva Crônica , Humanos , Adulto , Animais , Cavalos , Aspergillus fumigatus , Asma/veterinária , Antígenos de Fungos , Imunoglobulina GRESUMO
Proline-rich antimicrobial peptides (PrAMPs) inhibit bacterial protein biosynthesis by binding to the polypeptide exit tunnel (PET) near the peptidyl transferase center. Api137, an optimized derivative of honeybee PrAMP apidaecin, inhibits protein expression by trapping release factors (RFs), which interact with stop codons on ribosomes to terminate translation. This study uses cryo-EM, functional assays and molecular dynamic (MD) simulations to show that Api137 additionally occupies a second binding site near the exit of the PET and can repress translation independently of RF-trapping. Api88, a C-terminally amidated (-CONH2) analog of Api137 (-COOH), binds to the same sites, occupies a third binding pocket and interferes with the translation process presumably without RF-trapping. In conclusion, apidaecin-derived PrAMPs inhibit bacterial ribosomes by multimodal mechanisms caused by minor structural changes and thus represent a promising pool for drug development efforts.
Assuntos
Peptídeos Catiônicos Antimicrobianos , Simulação de Dinâmica Molecular , Ribossomos , Ribossomos/metabolismo , Peptídeos Catiônicos Antimicrobianos/metabolismo , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Biossíntese de Proteínas , Sítios de Ligação , Microscopia Crioeletrônica , Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Fatores de Terminação de Peptídeos/metabolismo , Fatores de Terminação de Peptídeos/química , Fatores de Terminação de Peptídeos/genética , Ligação Proteica , Peptídeos Antimicrobianos/química , Peptídeos Antimicrobianos/metabolismo , Peptídeos Antimicrobianos/farmacologiaRESUMO
The favorable properties of antimicrobial peptides (AMPs) to rapidly kill pathogens are often limited by unfavorable pharmacokinetics due to fast degradation and renal clearance rates. Here, a prodrug strategy linking proline-rich AMP Onc72 to polyethylene glycol (PEGs) with average molecular weights of 5 and 20 kDa via a peptide linker containing a protease cleavage site is tested for the first time in vivo. Onc72 is released from these 5k- and 20k-prodrugs in mouse serum with half-life times (t1/2 ) of 8 and 14 h, respectively. Importantly, PEGylation protects Onc72 from proteolytic degradation providing a prolonged release of Onc72, balancing the degradation of free Onc72, and leading to relatively stable Onc72 concentrations and high antibacterial activities. The prodrugs are not hemolytic on human erythrocytes and show only slight cytotoxic effects on human cell lines indicating promising safety margins. When administered subcutaneously to female CD-1 mice, the prodrugs elimination t1/2 are 66 min and ≈5.5 h, respectively, compared to 43 min of free Onc72. The maximal Onc72 plasma levels are obtained ≈1 and ≈8 h postadministration, respectively. In conclusion, the prodrugs provide extended elimination t1/2 and a constant release of Onc72 in mice, potentially limiting adverse effects and increasing efficacy.
Assuntos
Antineoplásicos , Pró-Fármacos , Camundongos , Feminino , Humanos , Animais , Pró-Fármacos/química , Peptídeos , Polietilenoglicóis/química , AntibacterianosRESUMO
Background: Cryptococcosis 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. Results: A 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.
RESUMO
In view of the global spread of multiresistant bacteria and the occurrence of panresistant bacteria, there is an urgent need for antimicrobials with novel modes of action. A promising class is antimicrobial peptides (AMPs), including them proline-rich AMPs (PrAMPs), which target the 70S ribosome to inhibit protein translation. Here, we present a new designer peptide, Api805, combining the N- and C-terminal sequences of PrAMPs Api137 and drosocin, respectively. Api805 was similarly active against two Escherichia coli B strains but was inactive against E. coli K12 strain BW25113. These different activities could not be explained by the dissociation constants measured for 70S ribosome preparations from E. coli K12 and B strains. Mutations in the SbmA transporter that PrAMPs use to pass the inner membrane or proteolytic degradation of Api805 by lysate proteases could not explain this either. Interestingly, Api805 seems not to bind to the known binding sites of PrAMPs at the 70S ribosome and inhibited in vitro protein translation, independent of release factors, most likely using a "multimodal effect". Interestingly, Api805 entered the E. coli B strain Rosetta faster and at larger quantities than the E. coli K-12 strain BW25113, which may be related to the different LPS core structure. In conclusion, slight structural changes in PrAMPs significantly altered their binding sites and mechanisms of action, allowing for the design of different antibiotic classes.
RESUMO
The rapid development, approval, and production of vaccines against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in less than 1 year after the first reports of a new infectious disease was a real game changer, providing 80%-90% efficacy in preventing severe etiopathologies of the coronavirus disease 2019 (COVID-19). These vaccines induce an immune response against the SARS-CoV-2 spike (S) protein located on the surface of the virus particle. Antibodies (Abs) recognizing the S-protein can inhibit binding of the virus via the S-protein to the angiotensin-converting enzyme-2 (ACE-2) receptor expressed on different human cells, especially when these Abs bind to the interaction site, the so-called receptor-binding domain (RBD). We have expressed the RBDs of wild-type SARS-CoV-2 and five variants of concern (VOCs) to test the immune response in people before vaccination with mRNA vaccines BNT162b2 and mRNA-1273 and after up to three vaccinations using in-house ELISA and inhibition assays. The methods of both assays are provided. Both vaccines initiated similarly high IgG titers after two vaccinations against the wild-type and even two VOC-RBDs (alpha and delta) and strongly inhibited the corresponding RBD-ACE-2 binding. The IgG titers and inhibition of ACE-2 binding were lower for beta and gamma RBDs and much lower for omicron RBD. The third vaccination after 6 months strongly increased both the IgG titers and the neutralizing effect against all variants, especially for omicron, leading to 63% ± 13% neutralization potential. Importantly, neutralization linearly increased with the IgG titers.
Assuntos
COVID-19 , SARS-CoV-2 , Vacina BNT162 , COVID-19/prevenção & controle , Humanos , Imunoglobulina G , RNA MensageiroRESUMO
This study investigated the IgG and IgA antibody response against recombinant S1 and receptor binding domains (RBD) of the spike (S-) protein and the membrane (M-) protein using a set of 115 serum samples collected from patients infected with SARS-CoV-2 in Germany before April 2021 using protein and peptide ELISA. As S1- and RBD-proteins expressed in Escherichia coli provided poor sensitivities in ELISA, they were replaced by proteins expressed in HEK cells. The RBD-ELISA provided a sensitivity of 90.6% (N = 85) for samples collected from patients with confirmed SARS-CoV-2 infections more than 14 days after symptom onset or a positive PCR test. In population-based controls, the specificity was 97.9% (N = 94). In contrast, the sensitivities were only 41.2% and 72.6% for M- and N-proteins, respectively, while the specificities were 88.5% and 100%, respectively. Considering also 20 samples collected during the first two weeks of symptom onset or PCR confirmation, the sensitivity of RBD- and N-protein ELISA decreased to 82.6% and 72.6%, respectively. The combination of two data sets, i.e., N- and RBD-, N- and M-, or RBD- and M-proteins increased the sensitivity to 85.8%, 77.9%, and 87.8%, respectively. Peptide mapping mostly confirmed epitopes previously reported for S1- and M-proteins, but they were only recognized by a few samples already tested positive in the corresponding protein ELISA indicating that peptide-based assays will not improve the diagnostic sensitivity.
RESUMO
Cryptococcus neoformans, an opportunistic fungal pathogen ubiquitously present in the environment, causes cryptococcal meningitis (CM) mainly in immunocompromised patients, such as AIDS patients. We aimed to identify disease-associated cryptococcal protein antigens targeted by the human humoral immune response. Therefore, we used sera from Colombian CM patients, with or without HIV infection, and from healthy individuals living in the same region. Serological analysis revealed increased titers of anti-cryptococcal IgG in HIV-negative CM patients, but not HIV-positive CM patients, compared to healthy controls. In contrast, titers of anti-cryptococcal IgM were not affected by CM. Furthermore, we detected pre-existing IgG and IgM antibodies even in sera from healthy individuals. The observed induction of anti-cryptococcal IgG but not IgM during CM was supported by analysis of sera from C. neoformans-infected mice. Stronger increase in IgG was found in wild type mice with high lung fungal burden compared to IL-4Rα-deficient mice showing low lung fungal burden. To identify the proteins targeted by human anti-cryptococcal IgG antibodies, we applied a quantitative 2D immunoproteome approach identifying cryptococcal protein spots preferentially recognized by sera from CM patients or healthy individuals followed by mass spectrometry analysis. Twenty-three cryptococcal proteins were recombinantly expressed and confirmed to be immunoreactive with human sera. Fourteen of them were newly described as immunoreactive proteins. Twelve proteins were classified as disease-associated antigens, based on significantly stronger immunoreactivity with sera from CM patients compared to healthy individuals. The proteins identified in our screen significantly expand the pool of cryptococcal proteins with potential for (i) development of novel anti-cryptococcal agents based on implications in cryptococcal virulence or survival, or (ii) development of an anti-cryptococcal vaccine, as several candidates lack homology to human proteins and are localized extracellularly. Furthermore, this study defines pre-existing anti-cryptococcal immunoreactivity in healthy individuals at a molecular level, identifying target antigens recognized by sera from healthy control persons.