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1.
Angew Chem Int Ed Engl ; : e202408360, 2024 Aug 08.
Article in English | MEDLINE | ID: mdl-39113573

ABSTRACT

The use of highly potent but very toxic antibiotics such as colistin has become inevitable due to the rise of antimicrobial resistance. We aimed for a chemically-triggered, controlled release of colistin at the infection site to lower its systemic toxicity by harnessing the power of click-to-release reactions. Kinetic experiments with nine tetrazines and three dienophiles demonstrated a fast release via an inverse-electron-demand Diels-Alder reaction between trans-cyclooctenes (TCO) and the amine-functionalized tetrazine Tz7. The antibiotic activity of colistin against Escherichia coli was masked by TCO units, but restored upon reaction with d-Ubi-Tz, a tetrazine functionalised with the bacterial binding peptide d-Ubi29-41. While standard TCO did not improve toxicity against human proximal tubular kidney HK-2 cells, the installation of an aspartic acid-modified TCO masking group reduced the overall charge of the peptide and entry to the kidney cells, thereby dramatically lowering its toxicity. The analog Col-(TCO-Asp)1 had favourable pharmacokinetic properties in mice and was successfully activated locally in the lung by d-Ubi-Tz in an in vivo infection model, whereas it remained inactive and non-harmful without the chemical trigger. This study constitutes the first example of a systemically acting two-component antibiotic with improved drug tolerability.

2.
Infection ; 52(1): 59-71, 2024 Feb.
Article in English | MEDLINE | ID: mdl-37253816

ABSTRACT

PURPOSE: Human Borna disease virus (BoDV-1) encephalitis is an emerging disease in Germany. This study investigates the spectrum of human BoDV-1 infection, characterizes anti-BoDV-1-antibodies and kinetics, and compares laboratory test performances. METHODS: Three hundred four encephalitis cases, 308 nation-wide neuropsychiatric conditions, 127 well-defined psychiatric cases from Borna disease-endemic areas, and 20 persons with contact to BoDV-1 encephalitis patients or animals were tested for BoDV-1 infections by serology and PCR. RESULTS: BoDV-1 infections were only found in encephalitis patients with residence in, or recent travel to, virus-endemic areas. Antibodies were detected as early as 12 days after symptom onset. Serum antibody levels correlated with disease duration. Serology was ordered after 50% of the disease duration had elapsed, reflecting low awareness. BoDV-1-antibodies were of IgG1 subclass, and the epitope on BoDV-1 antigens was determined. Specificity of the indirect immunofluorescence antibody test (IFAT) and lineblot (LB) from serum and cerebrospinal fluid (CSF), as well as PCR testing from CSF, was 100%. Sensitivity, depending on first or all samples, reached 75-86% in serum and 92-94% in CSF for the IFAT, and 33-57% in serum and 18-24% in CSF for the LB. Sensitivity for PCR in CSF was 25-67%. Positive predictive values were 100% each, while negative predictive values were 99% (IFAT), 91-97% (LB), and 90% (PCR). CONCLUSIONS: There is no hint that BoDV-1 causes other diseases than encephalitis in humans. Awareness has to be increased in virus-endemic areas. Tests are robust but lack sensitivity. Detection of IgG1 against specific peptides may facilitate diagnosis. Screening of healthy individuals is likely not beneficial.


Subject(s)
Borna disease virus , Bornaviridae , Encephalitis , Viruses , Animals , Humans , Borna disease virus/genetics , Bornaviridae/genetics , Correlation of Data , Viruses/genetics , Antibodies, Viral , RNA, Viral/genetics , Immunoglobulin G
3.
Chembiochem ; 24(16): e202300369, 2023 08 15.
Article in English | MEDLINE | ID: mdl-37435861

ABSTRACT

Polymicrobial infections involving various combinations of microorganisms, such as Escherichia, Pseudomonas, or Yersinia, can lead to acute and chronic diseases in for example the gastrointestinal and respiratory tracts. Our aim is to modulate microbial communities by targeting the posttranscriptional regulator system called carbon storage regulator A (CsrA) (or also repressor of secondary metabolites (RsmA)). In previous studies, we identified easily accessible CsrA binding scaffolds and macrocyclic CsrA binding peptides through biophysical screening and phage display technology. However, due to the lack of an appropriate in bacterio assay to evaluate the cellular effects of these inhibitor hits, the focus of the present study is to establish an in bacterio assay capable of probing and quantifying the impact on CsrA-regulated cellular mechanisms. We have successfully developed an assay based on a luciferase reporter gene assay, which in combination with a qPCR expression gene assay, allows for the monitoring of expression levels of different downstream targets of CsrA. The chaperone protein CesT was used as a suitable positive control for the assay, and in time-dependent experiments, we observed a CesT-mediated increase in bioluminescence over time. By this means, the cellular on-target effects of non-bactericidal/non-bacteriostatic virulence modulating compounds targeting CsrA/RsmA can be evaluated.


Subject(s)
Escherichia coli Proteins , Escherichia coli Proteins/metabolism , Carbon/metabolism , RNA-Binding Proteins/chemistry , Gene Expression , Genes, Reporter , Gene Expression Regulation, Bacterial , Bacterial Proteins/metabolism
4.
Bioorg Chem ; 131: 106331, 2023 02.
Article in English | MEDLINE | ID: mdl-36587505

ABSTRACT

In order to develop novel inhibitors of the bacterial deacetylase LpxC bearing a substituent to target the UDP binding site of the enzyme, a series of aldotetronic acid-based hydroxamic acids was accessed in chiral pool syntheses starting from 4,6-O-benzylidene-d-glucose and l-arabinitol. The synthesized hydroxamic acids were tested for LpxC inhibitory activity in vitro, revealing benzyl ether 17a ((2S,3S)-4-(benzyloxy)-N,3-dihydroxy-2-[(4-{[4-(morpholinomethyl)phenyl]ethynyl}benzyl)oxy]butanamide) as the most potent LpxC inhibitor. This compound was additionally tested for antibacterial activity against a panel of clinically relevant Gram-negative bacteria, bacterial uptake, and susceptibility to efflux pumps. Molecular docking studies were performed to rationalize the observed structure-activity relationships.


Subject(s)
Amidohydrolases , Anti-Bacterial Agents , Enzyme Inhibitors , Escherichia coli , Amidohydrolases/antagonists & inhibitors , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Bacteria/metabolism , Binding Sites , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Hydroxamic Acids/pharmacology , Hydroxamic Acids/chemistry , Molecular Docking Simulation , Structure-Activity Relationship
5.
ACS Infect Dis ; 9(2): 330-341, 2023 02 10.
Article in English | MEDLINE | ID: mdl-36719860

ABSTRACT

The rise of antimicrobial resistance, especially in Gram-negative bacteria, calls for novel diagnostics and antibiotics. To efficiently penetrate their double-layered cell membrane, we conjugated the potent antibiotics daptomycin, vancomycin, and sorangicin A to catechol siderophores, which are actively internalized by the bacterial iron uptake machinery. LC-MS/MS uptake measurements of sorangicin derivatives verified that the conjugation led to a 100- to 525-fold enhanced uptake into bacteria compared to the free drug. However, the transfer to the cytosol was insufficient, which explains their lack of antibiotic efficacy. Potent antimicrobial effects were observed for the daptomycin conjugate 7 (∼1 µM) against multidrug-resistant Acinetobacter baumannii. A cyanin-7 label aside the daptomycin warhead furnished the theranostic 13 that retained its antibiotic activity and was also able to label ESKAPE bacteria, as demonstrated by microscopy and fluorescence assays. 13 and the cyanin-7 imaging conjugate 14 were stable in human plasma and had low plasma protein binding and cytotoxicity.


Subject(s)
Daptomycin , Humans , Daptomycin/chemistry , Chromatography, Liquid , Tandem Mass Spectrometry , Anti-Bacterial Agents/chemistry , Bacteria/metabolism
6.
Nat Commun ; 13(1): 7402, 2022 12 01.
Article in English | MEDLINE | ID: mdl-36456567

ABSTRACT

Pseudomonas aeruginosa is a major cause of nosocomial infections and also leads to severe exacerbations in cystic fibrosis or chronic obstructive pulmonary disease. Three intertwined quorum sensing systems control virulence of P. aeruginosa, with the rhl circuit playing the leading role in late and chronic infections. The majority of traits controlled by rhl transcription factor RhlR depend on PqsE, a dispensable thioesterase in Pseudomonas Quinolone Signal (PQS) biosynthesis that interferes with RhlR through an enigmatic mechanism likely involving direct interaction of both proteins. Here we show that PqsE and RhlR form a 2:2 protein complex that, together with RhlR agonist N-butanoyl-L-homoserine lactone (C4-HSL), solubilizes RhlR and thereby renders the otherwise insoluble transcription factor active. We determine crystal structures of the complex and identify residues essential for the interaction. To corroborate the chaperone-like activity of PqsE, we design stability-optimized variants of RhlR that bypass the need for C4-HSL and PqsE in activating PqsE/RhlR-controlled processes of P. aeruginosa. Together, our data provide insight into the unique regulatory role of PqsE and lay groundwork for developing new P. aeruginosa-specific pharmaceuticals.


Subject(s)
Protein Folding , Pseudomonas aeruginosa , Virulence , Pseudomonas aeruginosa/genetics , Transcription Factors
7.
mSphere ; 7(5): e0030222, 2022 Oct 26.
Article in English | MEDLINE | ID: mdl-35993700

ABSTRACT

Amidochelocardin is a broad-spectrum antibiotic with activity against many Gram-positive and Gram-negative bacteria. According to recent data, the antibiotic effect of this atypical tetracycline is directed against the cytoplasmic membrane, which is associated with the dissipation of the membrane potential. Here, we investigated the effect of amidochelocardin on the proteome of Clostridioides difficile to gain insight into the membrane stress physiology of this important anaerobic pathogen. For the first time, the membrane-directed action of amidochelocardin was confirmed in an anaerobic pathogen. More importantly, our results revealed that aromatic compounds potentially play an important role in C. difficile upon dissipation of its membrane potential. More precisely, a simultaneously increased production of enzymes required for the synthesis of chorismate and two putative phenazine biosynthesis proteins point to the production of a hitherto unknown compound in response to membrane depolarization. Finally, increased levels of the ClnAB efflux system and its transcriptional regulator ClnR were found, which were previously found in response to cationic antimicrobial peptides like LL-37. Therefore, our data provide a starting point for a more detailed understanding of C. difficile's way to counteract membrane-active compounds. IMPORTANCE C. difficile is an important anaerobe pathogen causing mild to severe infections of the gastrointestinal tract. To avoid relapse of the infection following antibiotic therapy, antibiotics are needed that efficiently eradicate C. difficile from the intestinal tract. Since C. difficile was shown to be substantially sensitive to membrane-active antibiotics, it has been proposed that membrane-active antibiotics might be promising for the therapy of C. difficile infections. Therefore, we studied the response of C. difficile to amidochelocardin, a membrane-active antibiotic dissipating the membrane potential. Interestingly, C. difficile's response to amidochelocardin indicates a role of aromatic metabolites in mediating stress caused by dissipation of the membrane potential.


Subject(s)
Clostridioides difficile , Clostridioides , Gram-Negative Bacteria , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Gram-Positive Bacteria , Proteome , Tetracyclines/pharmacology , Phenazines/pharmacology
8.
Viruses ; 14(6)2022 06 17.
Article in English | MEDLINE | ID: mdl-35746797

ABSTRACT

The development of antibody therapies against SARS-CoV-2 remains a challenging task during the ongoing COVID-19 pandemic. All approved therapeutic antibodies are directed against the receptor binding domain (RBD) of the spike, and therefore lose neutralization efficacy against emerging SARS-CoV-2 variants, which frequently mutate in the RBD region. Previously, phage display has been used to identify epitopes of antibody responses against several diseases. Such epitopes have been applied to design vaccines or neutralize antibodies. Here, we constructed an ORFeome phage display library for the SARS-CoV-2 genome. Open reading frames (ORFs) representing the SARS-CoV-2 genome were displayed on the surface of phage particles in order to identify enriched immunogenic epitopes from COVID-19 patients. Library quality was assessed by both NGS and epitope mapping of a monoclonal antibody with a known binding site. The most prominent epitope captured represented parts of the fusion peptide (FP) of the spike. It is associated with the cell entry mechanism of SARS-CoV-2 into the host cell; the serine protease TMPRSS2 cleaves the spike within this sequence. Blocking this mechanism could be a potential target for non-RBD binding therapeutic anti-SARS-CoV-2 antibodies. As mutations within the FP amino acid sequence have been rather rare among SARS-CoV-2 variants so far, this may provide an advantage in the fight against future virus variants.


Subject(s)
Bacteriophages , COVID-19 , Antibodies, Neutralizing , Antibodies, Viral , Antibody Formation , Bacteriophages/metabolism , Epitopes , Humans , Pandemics , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus
9.
Emerg Microbes Infect ; 11(1): 1037-1048, 2022 Dec.
Article in English | MEDLINE | ID: mdl-35320064

ABSTRACT

The coronavirus SARS-CoV-2 is the causative agent for the disease COVID-19. To capture the IgA, IgG, and IgM antibody response of patients infected with SARS-CoV-2 at individual epitope resolution, we constructed planar microarrays of 648 overlapping peptides that cover the four major structural proteins S(pike), N(ucleocapsid), M(embrane), and E(nvelope). The arrays were incubated with sera of 67 SARS-CoV-2 positive and 22 negative control samples. Specific responses to SARS-CoV-2 were detectable, and nine peptides were associated with a more severe course of the disease. A random forest model disclosed that antibody binding to 21 peptides, mostly localized in the S protein, was associated with higher neutralization values in cellular anti-SARS-CoV-2 assays. For antibodies addressing the N-terminus of M, or peptides close to the fusion region of S, protective effects were proven by antibody depletion and neutralization assays. The study pinpoints unusual viral binding epitopes that might be suited as vaccine candidates.


Subject(s)
COVID-19 , SARS-CoV-2 , Antibodies, Neutralizing , Antibodies, Viral , Antibody Formation , Epitopes , Humans , Machine Learning , Peptides , Spike Glycoprotein, Coronavirus
10.
Viruses ; 14(2)2022 01 20.
Article in English | MEDLINE | ID: mdl-35215792

ABSTRACT

Vaccinia virus (VACV) belongs to the genus Orthopoxvirus of the family Poxviridae. There are four different forms of infectious virus particles: intracellular mature virus (IMV), intracellular en-veloped virus (IEV), cell-associated enveloped virus (CEV) and extracellular enveloped virus (EEV). The F13 protein occupies the inner side of the CEV- and EEV-membranes and the outer side of the IEV-membranes. It plays an important role in wrapping progress and EEV production. We constructed a human single-chain fragment variable (scFv) library with a diversity of ≥4 × 108 independent colonies using peripheral blood from four vaccinated donors. One anti-F13 scFv was isolated and characterised after three rounds of panning. In Western blotting assays, the scFv 3E2 reacted with the recombinant F13VACV protein with a reduction of binding under denatured and reduced conditions. Two antigenic binding sites (139-GSIHTIKTLGVYSDY-153 and 169-AFNSAKNSWLNL-188) of scFv 3E2 were mapped using a cellulose membrane encompassing 372 15-mere peptides with 12 overlaps covering the whole F13 protein. No neutralisation capa-bilities were observed either in the presence or absence of complement. In conclusion, the con-struction of recombinant immunoglobulin libraries is a promising strategy to isolate specific scFvs to enable the study of the host-pathogen interaction.


Subject(s)
Antibodies, Viral/immunology , Single-Chain Antibodies/immunology , Vaccinia virus/immunology , Amino Acid Sequence , Antibodies, Viral/chemistry , Antibodies, Viral/genetics , Epitope Mapping , Gene Library , Humans , Neutralization Tests , Single-Chain Antibodies/chemistry , Single-Chain Antibodies/genetics , Vaccinia virus/chemistry , Vaccinia virus/genetics
11.
EBioMedicine ; 73: 103652, 2021 Nov.
Article in English | MEDLINE | ID: mdl-34740109

ABSTRACT

BACKGROUND: The clinical-stage drug candidate EBL-1003 (apramycin) represents a distinct new subclass of aminoglycoside antibiotics for the treatment of drug-resistant infections. It has demonstrated best-in-class coverage of resistant isolates, and preclinical efficacy in lung infection models. However, preclinical evidence for its utility in other disease indications has yet to be provided. Here we studied the therapeutic potential of EBL-1003 in the treatment of complicated urinary tract infection and acute pyelonephritis (cUTI/AP). METHODS: A combination of data-base mining, antimicrobial susceptibility testing, time-kill experiments, and four murine infection models was used in a comprehensive assessment of the microbiological coverage and efficacy of EBL-1003 against Gram-negative uropathogens. The pharmacokinetics and renal toxicology of EBL-1003 in rats was studied to assess the therapeutic window of EBL-1003 in the treatment of cUTI/AP. FINDINGS: EBL-1003 demonstrated broad-spectrum activity and rapid multi-log CFU reduction against a phenotypic variety of bacterial uropathogens including aminoglycoside-resistant clinical isolates. The basicity of amines in the apramycin molecule suggested a higher increase in positive charge at urinary pH when compared to gentamicin or amikacin, resulting in sustained drug uptake and bactericidal activity, and consequently in potent efficacy in mouse infection models. Renal pharmacokinetics, biomarkers for toxicity, and kidney histopathology in adult rats all indicated a significantly lower nephrotoxicity of EBL-1003 than of gentamicin. INTERPRETATION: This study provides preclinical proof-of-concept for the efficacy of EBL-1003 in cUTI/AP. Similar efficacy but lower nephrotoxicity of EBL-1003 in comparison to gentamicin may thus translate into a higher safety margin and a wider therapeutic window in the treatment of cUTI/API. FUNDING: A full list of funding bodies that contributed to this study can be found in the Acknowledgements section.


Subject(s)
Anti-Bacterial Agents/therapeutic use , Hydrogen-Ion Concentration , Nebramycin/analogs & derivatives , Pyelonephritis/drug therapy , Urinary Tract Infections/drug therapy , Animals , Anti-Bacterial Agents/pharmacology , Bacterial Infections/drug therapy , Bacterial Infections/microbiology , Disease Models, Animal , Dose-Response Relationship, Drug , Drug Resistance, Multiple, Bacterial/drug effects , Humans , Mice , Microbial Sensitivity Tests , Nebramycin/pharmacology , Nebramycin/therapeutic use , Pyelonephritis/etiology , Rats , Treatment Outcome , Urinary Tract Infections/etiology
12.
J Med Chem ; 64(20): 15440-15460, 2021 10 28.
Article in English | MEDLINE | ID: mdl-34619959

ABSTRACT

The development of novel drugs against Gram-negative bacteria represents an urgent medical need. To overcome their outer cell membrane, we synthesized conjugates of antibiotics and artificial siderophores based on the MECAM core, which are imported by bacterial iron uptake systems. Structures, spin states, and iron binding properties were predicted in silico using density functional theory. The capability of MECAM to function as an effective artificial siderophore in Escherichia coli was proven in microbiological growth recovery and bioanalytical assays. Following a linker optimization focused on transport efficiency, five ß-lactam and one daptomycin conjugates were prepared. The most potent conjugate 27 showed growth inhibition of Gram-positive and Gram-negative multidrug-resistant pathogens at nanomolar concentrations. The uptake pathway of MECAMs was deciphered by knockout mutants and highlighted the relevance of FepA, CirA, and Fiu. Resistance against 27 was mediated by a mutation in the gene encoding ExbB, which is involved in siderophore transport.


Subject(s)
Anti-Bacterial Agents/pharmacology , Benzamides/chemistry , Benzamides/pharmacology , Gram-Negative Bacteria/drug effects , Gram-Positive Bacteria/drug effects , Hydroxybenzoates/chemistry , Hydroxybenzoates/pharmacology , Siderophores/pharmacology , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/chemistry , Dose-Response Relationship, Drug , Microbial Sensitivity Tests , Molecular Structure , Siderophores/chemical synthesis , Siderophores/chemistry , Structure-Activity Relationship
13.
Bioorg Chem ; 107: 104603, 2021 02.
Article in English | MEDLINE | ID: mdl-33429229

ABSTRACT

LpxC inhibitors represent a promising class of novel antibiotics selectively combating Gram-negative bacteria. In chiral pool syntheses starting from D- and L-xylose, a series of four 2r,3c,4t-configured C-furanosidic LpxC inhibitors was obtained. The synthesized hydroxamic acids were tested for antibacterial and LpxC inhibitory activity, the acquired biological data were compared with those of previously synthesized C-furanosides, and molecular docking studies were performed to rationalize the observed structure-activity relationships. Additionally, bacterial uptake and susceptibility to efflux pump systems were investigated for the most promising stereoisomers.


Subject(s)
Amidohydrolases/antagonists & inhibitors , Anti-Bacterial Agents/pharmacology , Enzyme Inhibitors/pharmacology , Molecular Docking Simulation , Xylose/pharmacology , Amidohydrolases/metabolism , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/chemistry , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Molecular Structure , Structure-Activity Relationship , Xylose/chemical synthesis , Xylose/chemistry
14.
J Virol ; 94(2)2020 01 06.
Article in English | MEDLINE | ID: mdl-31666384

ABSTRACT

To counteract the serious health threat posed by known and novel viral pathogens, drugs that target a variety of viruses through a common mechanism have attracted recent attention due to their potential in treating (re)emerging infections, for which direct-acting antivirals are not available. We found that labyrinthopeptins A1 and A2, the prototype congeners of carbacyclic lanthipeptides, inhibit the proliferation of diverse enveloped viruses, including dengue virus, Zika virus, West Nile virus, hepatitis C virus, chikungunya virus, Kaposi's sarcoma-associated herpesvirus, cytomegalovirus, and herpes simplex virus, in the low micromolar to nanomolar range. Mechanistic studies on viral particles revealed that labyrinthopeptins induce a virolytic effect through binding to the viral membrane lipid phosphatidylethanolamine (PE). These effects are enhanced by a combined equimolar application of both labyrinthopeptins, and a clear synergism was observed across a concentration range corresponding to 10% to 90% inhibitory concentrations of the compounds. Time-resolved experiments with large unilamellar vesicles (LUVs) reveal that membrane lipid raft compositions (phosphatidylcholine [PC]/PE/cholesterol/sphingomyelin at 17:10:33:40) are particularly sensitive to labyrinthopeptins in comparison to PC/PE (90:10) LUVs, even though the overall PE amount remains constant. Labyrinthopeptins exhibited low cytotoxicity and had favorable pharmacokinetic properties in mice (half-life [t1/2] = 10.0 h), which designates them promising antiviral compounds acting by an unusual viral lipid targeting mechanism.IMPORTANCE For many viral infections, current treatment options are insufficient. Because the development of each antiviral drug is time-consuming and expensive, the prospect of finding broad-spectrum antivirals that can fight multiple, diverse viruses-well-known viruses as well as (re)emerging species-has gained attention, especially for the treatment of viral coinfections. While most known broad-spectrum agents address processes in the host cell, we found that targeting lipids of the free virus outside the host cell with the natural products labyrinthopeptin A1 and A2 is a viable strategy to inhibit the proliferation of a broad range of viruses from different families, including chikungunya virus, dengue virus, Zika virus, Kaposi's sarcoma-associated herpesvirus, and cytomegalovirus. Labyrinthopeptins bind to viral phosphatidylethanolamine and induce virolysis without exerting cytotoxicity on host cells. This represents a novel and unusual mechanism to tackle medically relevant viral infections.


Subject(s)
Bacteriocins/pharmacology , Membrane Microdomains/metabolism , Virus Diseases/metabolism , Viruses/metabolism , Aedes , Animals , Cell Line , Membrane Microdomains/virology , Phosphatidylethanolamines/metabolism , Virus Diseases/drug therapy
15.
Anal Chem ; 91(17): 11030-11037, 2019 09 03.
Article in English | MEDLINE | ID: mdl-31365232

ABSTRACT

Macacine herpesvirus or B Virus (BV) is a zoonotic agent that leads to high mortality rates in humans if transmitted and untreated. Here, BV is used as a test case to establish a two-step procedure for developing high throughput serological assays based on synthetic peptides. In step 1, peptide microarray analysis of 42 monkey sera (30 of them tested BV positive by ELISA) revealed 1148 responses against 369 different peptides. The latter could be grouped into 142 different antibody target regions (ATRs) in six different glycoproteins (gB, gC, gD, gG, gH, and gL) of BV. The high number of newly detected ATRs was made possible inter alia by a new preanalytical protocol that reduced unspecific binding of serum components to the cellulose-based matrix of the microarray. In step 2, soluble peptides corresponding to eight ATRs of particularly high antigenicity were synthesized and coupled to fluorescently labeled beads, which were subsequently employed in immunochemical bead flow assays. Their outcome mirrored the ELISA results used as reference. Hence, convenient, fast, and economical screening of arbitrarily large macaque colonies for BV infection is now possible. The study demonstrates that a technology platform switch from two-dimensional high-resolution peptide arrays used for epitope discovery to a readily available bead array platform for serology applications is feasible.


Subject(s)
Antibodies, Viral/blood , Epitopes/blood , Herpesviridae Infections/veterinary , Herpesvirus 1, Cercopithecine/immunology , Primate Diseases/diagnosis , Viral Proteins/blood , Animals , Binding Sites , Epitopes/chemistry , Herpesviridae Infections/diagnosis , Herpesviridae Infections/immunology , Herpesviridae Infections/virology , Herpesvirus 1, Cercopithecine/genetics , Humans , Immune Sera/chemistry , Immunoconjugates/chemistry , Macaca mulatta/immunology , Macaca mulatta/virology , Models, Molecular , Primate Diseases/immunology , Primate Diseases/virology , Protein Array Analysis/instrumentation , Protein Array Analysis/methods , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Viral Proteins/chemistry
16.
Viruses ; 11(6)2019 05 29.
Article in English | MEDLINE | ID: mdl-31146446

ABSTRACT

The vaccinia virus (VACV) A27 protein and its homologs, which are found in a large number of members of the genus Orthopoxvirus (OPXV), are targets of viral neutralization by host antibodies. We have mapped six binding sites (epitopes #1A: aa 32-39, #1B: aa 28-33, #1C: aa 26-31, #1D: 28-34, #4: aa 9-14, and #5: aa 68-71) of A27 specific monoclonal antibodies (mAbs) using peptide arrays. MAbs recognizing epitopes #1A-D and #4 neutralized VACV Elstree in a complement dependent way (50% plaque-reduction: 12.5-200 µg/mL). Fusion of VACV at low pH was blocked through inhibition of epitope #1A. To determine the sequence variability of the six antigenic sites, 391 sequences of A27 protein homologs available were compared. Epitopes #4 and #5 were conserved among most of the OPXVs, while the sequential epitope complex #1A-D was more variable and, therefore, responsible for species-specific epitope characteristics. The accurate and reliable mapping of defined epitopes on immuno-protective proteins such as the A27 of VACV enables phylogenetic studies and insights into OPXV evolution as well as to pave the way to the development of safer vaccines and chemical or biological antivirals.


Subject(s)
Antigens, Viral/genetics , Epitopes/immunology , Membrane Proteins/genetics , Vaccinia virus/genetics , Vaccinia/virology , Viral Fusion Proteins/genetics , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Antigens, Viral/immunology , Binding Sites, Antibody , Cross Reactions , Epitope Mapping , Epitopes/genetics , Hydrogen-Ion Concentration , Membrane Proteins/immunology , Mutation , Neutralization Tests , Phylogeny , Species Specificity , Vaccinia virus/immunology , Viral Fusion Proteins/immunology
17.
Sci Rep ; 9(1): 3648, 2019 03 06.
Article in English | MEDLINE | ID: mdl-30842564

ABSTRACT

Zika virus (ZIKV) is a mosquito-borne flavivirus. Homologous proteins of different flaviviruses display high degrees of sequence identity, especially within subgroups. This leads to extensive immunological cross-reactivity and corresponding problems for developing a ZIKV-specific serological assay. In this study, peptide microarrays were employed to identify individual ZIKV antibody targets with promise in differential diagnosis. A total of 1643 overlapping oligopeptides were synthesized and printed onto glass slides. Together, they encompass the full amino acid sequences of ZIKV proteomes of African, Brazilian, USA, and French Polynesian origins. The resulting ZIKV scanning microarray chips were used to screen three pools of sera from recent Zika outbreaks in Senegal and Cape Verde, in Brazil, and from overseas travelers returning to the EU. Together with a mixed pool of well characterized, archived sera of patients suffering from infections by dengue, yellow fever, tick-borne encephalitis, and West Nile viruses, a total of 42 sera went into the study. Sixty-eight antibody target regions were identified. Most of which were hitherto unknown. Alignments and sequence comparisons revealed 13 of which could be classified as bona fide ZIKV-specific. These identified antibody target regions constitute a founding set of analytical tools for serological discrimination of ZIKV from other flaviviruses.


Subject(s)
Antibodies, Viral/chemistry , Antigens, Viral/metabolism , Peptides/immunology , Zika Virus Infection/diagnosis , Zika Virus/classification , Brazil , Cabo Verde , Cross Reactions , Diagnosis, Differential , Disease Outbreaks , Flavivirus/classification , Flavivirus/immunology , Flavivirus/isolation & purification , Humans , Protein Array Analysis , Senegal , Species Specificity , Zika Virus/immunology , Zika Virus/isolation & purification , Zika Virus Infection/immunology
18.
Anal Chem ; 91(3): 1863-1872, 2019 02 05.
Article in English | MEDLINE | ID: mdl-30485749

ABSTRACT

Infections by Gram-negative pathogens represent a major health care issue of growing concern due to a striking lack of novel antibacterial agents over the course of the last decades. The main scientific problem behind the rational optimization of novel antibiotics is our limited understanding of small molecule translocation into, and their export from, the target compartments of Gram-negative species. To address this issue, a versatile, label-free assay to determine the intracellular localization and concentration of a given compound has been developed for Escherichia coli and its efflux-impaired ΔTolC mutant. The assay applies a fractionation procedure to antibiotic-treated bacterial cells to obtain periplasm, cytoplasm, and membrane fractions of high purity, as demonstrated by Western Blots of compartment-specific marker proteins. This is followed by an LC-MS/MS-based quantification of antibiotic content in each compartment. Antibiotic amounts could be converted to antibiotic concentrations by assuming that an E. coli cell is a cylinder flanked by two half spheres and calculating the volumes of bacterial compartments. The quantification of antibiotics from different classes, namely ciprofloxacin, tetracycline, trimethoprim, and erythromycin, demonstrated pronounced differences in uptake quantities and distribution patterns across the compartments. For example, in the case of ciprofloxacin, a higher amount of compound was located in the cytoplasm than in the periplasm (592 ± 50 pg vs 277 ± 13 pg per 3.9 × 109 cells), but owing to the smaller volume of the periplasmic compartment, its concentration in the cytoplasm was much lower (37 ± 3 vs 221 ± 10 pg/µL for the periplasm). For erythromycin and tetracycline, differences in MICs between WT and ΔTolC mutant strains were not reflected by equal differences in uptake, illustrating that additional experimental data are needed to predict antibiotic efficacy. We believe that our assay, providing the antibiotic concentration at the compartment in which the drug target is expressed, constitutes an essential piece of information for a more rational optimization of novel antibiotics against Gram-negative infections.


Subject(s)
Escherichia coli/isolation & purification , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Ciprofloxacin/chemistry , Ciprofloxacin/pharmacology , Erythromycin/chemistry , Erythromycin/pharmacology , Escherichia coli/drug effects , Escherichia coli/metabolism , Escherichia coli Infections/drug therapy , Escherichia coli Infections/metabolism , Microbial Sensitivity Tests , Tetracycline/chemistry , Tetracycline/pharmacology
19.
Emerg Infect Dis ; 24(6): 978-987, 2018 06.
Article in English | MEDLINE | ID: mdl-29774846

ABSTRACT

Limbic encephalitis is commonly regarded as an autoimmune-mediated disease. However, after the recent detection of zoonotic variegated squirrel bornavirus 1 in a Prevost's squirrel (Callosciurus prevostii) in a zoo in northern Germany, we retrospectively investigated a fatal case in an autoantibody-seronegative animal caretaker who had worked at that zoo. The virus had been discovered in 2015 as the cause of a cluster of cases of fatal encephalitis among breeders of variegated squirrels (Sciurus variegatoides) in eastern Germany. Molecular assays and immunohistochemistry detected a limbic distribution of the virus in brain tissue of the animal caretaker. Phylogenetic analyses demonstrated a spillover infection from the Prevost's squirrel. Antibodies against bornaviruses were detected in the patient's cerebrospinal fluid by immunofluorescence and newly developed ELISAs and immunoblot. The putative antigenic epitope was identified on the viral nucleoprotein. Other zoo workers were not infected; however, avoidance of direct contact with exotic squirrels and screening of squirrels are recommended.


Subject(s)
Bornaviridae/physiology , Limbic Encephalitis/epidemiology , Limbic Encephalitis/etiology , Mononegavirales Infections/complications , Occupational Exposure/adverse effects , Animals , Bornaviridae/classification , Epitope Mapping , Female , Germany/epidemiology , History, 21st Century , Humans , Immunohistochemistry , Limbic Encephalitis/diagnosis , Limbic Encephalitis/history , Magnetic Resonance Imaging , Middle Aged , Mononegavirales Infections/virology , Phylogeny , RNA, Viral , Sciuridae/virology , Serologic Tests , Structure-Activity Relationship , Viral Proteins/chemistry , Viral Proteins/metabolism , Whole Genome Sequencing , Zoonoses
20.
PLoS Pathog ; 13(9): e1006639, 2017 Sep.
Article in English | MEDLINE | ID: mdl-28938025

ABSTRACT

Kaposi's sarcoma-associated herpesvirus (KSHV) is the infectious cause of the highly vascularized tumor Kaposi's sarcoma (KS), which is characterized by proliferating spindle cells of endothelial origin, extensive neo-angiogenesis and inflammatory infiltrates. The KSHV K15 protein contributes to the angiogenic and invasive properties of KSHV-infected endothelial cells. Here, we asked whether K15 could also play a role in KSHV lytic replication. Deletion of the K15 gene from the viral genome or its depletion by siRNA lead to reduced virus reactivation, as evidenced by the decreased expression levels of KSHV lytic proteins RTA, K-bZIP, ORF 45 and K8.1 as well as reduced release of infectious virus. Similar results were found for a K1 deletion virus. Deleting either K15 or K1 from the viral genome also compromised the ability of KSHV to activate PLCγ1, Erk1/2 and Akt1. In infected primary lymphatic endothelial (LEC-rKSHV) cells, which have previously been shown to spontaneously display a viral lytic transcription pattern, transfection of siRNA against K15, but not K1, abolished viral lytic replication as well as KSHV-induced spindle cell formation. Using a newly generated monoclonal antibody to K15, we found an abundant K15 protein expression in KS tumor biopsies obtained from HIV positive patients, emphasizing the physiological relevance of our findings. Finally, we used a dominant negative inhibitor of the K15-PLCγ1 interaction to establish proof of principle that pharmacological intervention with K15-dependent pathways may represent a novel approach to block KSHV reactivation and thereby its pathogenesis.


Subject(s)
Herpesvirus 8, Human/physiology , Sarcoma, Kaposi/virology , Viral Proteins/metabolism , Virus Replication/physiology , Blotting, Western , Fluorescent Antibody Technique , Gene Knockdown Techniques , Humans , Sarcoma, Kaposi/metabolism , Virus Activation/physiology , Virus Latency/physiology
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