Stefanie Wagner

Novel Strategies for the Treatment of Pseudomonas aeruginosa Infections

Infections with Pseudomonas aeruginosa have become a concerning threat in hospital-acquired infections and for cystic fibrosis patients. The major problem leading to high mortality lies in the appearance of drug-resistant strains. Therefore, a vast number of approaches to develop novel anti-infectives is currently pursued. These diverse strategies span from killing (new antibiotics) to disarming (antivirulence) the pathogen. Particular emphasis lies on the development of compounds that inhibit biofilms formed in chronic infections to restore susceptibility toward antibiotics. Numerous promising results are summarized in this perspective. Antibiotics with a novel mode of action will be needed to avoid cross resistance against currently used therapeutic agents. Importantly, antivirulence drugs are expected to yield a significantly reduced rate of resistance development. Most developments are still far from the application. It can however be expected that combination therapies, also containing antivirulence agents, will pave the way toward novel treatment options against P. aeruginosa.

A novel N‐ethyl‐N‐nitrosourea–induced mutation in phospholipase Cγ2 causes inflammatory arthritis, metabolic defects, and male infertility in vitro in a murine model

OBJECTIVE It is difficult to identify a single causative factor for inflammatory arthritis because of the multifactorial nature of the disease. This study was undertaken to dissect the molecular complexity of systemic inflammatory disease, utilizing a combined approach of mutagenesis and systematic phenotype screening in a murine model. METHODS In a large-scale N-ethyl-N-nitrosourea mutagenesis project, the Ali14 mutant mouse strain was established because of dominant inheritance of spontaneous swelling and inflammation of the hind paws. Genetic mapping and subsequent candidate gene sequencing were conducted to find the causative gene, and systematic phenotyping of Ali14/+ mice was performed in the German Mouse Clinic. RESULTS A novel missense mutation in the phospholipase Cγ2 gene (Plcg2) was identified in Ali14/+ mice. Because of the hyperreactive external entry of calcium observed in cultured B cells and other in vitro experiments, the Ali14 mutation is thought to be a novel gain-of-function allele of Plcg2. Findings from systematic screening of Ali14/+ mice demonstrated various phenotypic changes: an abnormally high T cell:B cell ratio, up-regulation of Ig, alterations in body composition, and a reduction in cholesterol and triglyceride levels in peripheral blood. In addition, spermatozoa from Ali14/+ mice failed to fertilize eggs in vitro, despite the normal fertility of the Ali14/+ male mice in vivo. CONCLUSION These results suggest that the Plcg2-mediated pathways play a crucial role in various metabolic and sperm functions, in addition to initiating and maintaining the immune system. These findings may indicate the importance of the Ali14/+ mouse strain as a model for systemic inflammatory diseases and inflammation-related metabolic changes in humans.

Amphiphilic Cationic ß3R3-Peptides: Membrane Active Peptidomimetics and Their Potential as Antimicrobial Agents

We introduce a novel class of membrane active peptidomimetics, the amphiphilic cationic β(3R3)-peptides, and evaluate their potential as antimicrobial agents. The design criteria, the building block and oligomer synthesis as well as a detailed structure-activity relationship (SAR) study are reported. Specifically, infrared reflection absorption spectroscopy (IRRAS) was employed to investigate structural features of amphiphilic cationic β(3R3)-peptide sequences at the hydrophobic/hydrophilic air/liquid interface. Furthermore, Langmuir monolayers of anionic and zwitterionic phospholipids have been used to model the interactions of amphiphilic cationic β(3R3)-peptides with prokaryotic and eukaryotic cellular membranes in order to predict their membrane selectivity and elucidate their mechanism of action. Lastly, antimicrobial activity was tested against Gram-positive M. luteus and S. aureus as well as against Gram-negative E. coli and P. aeruginosa bacteria along with testing hemolytic activity and cytotoxicity. We found that amphiphilic cationic β(3R3)-peptide sequences combine high and selective antimicrobial activity with exceptionally low cytotoxicity in comparison to values reported in the literature. Overall, this study provides further insights into the SAR of antimicrobial peptides and peptidomimetics and indicates that amphiphilic cationic β(3R3)-peptides are strong candidates for further development as antimicrobial agents with high therapeutic index.

Phenotypic and pathomorphological characteristics of a novel mutant mouse model for maturity-onset diabetes of the young type 2 (MODY 2).

Several mutant mouse models for human diseases such as diabetes mellitus have been generated in the large-scale Munich ENU (N-ethyl-N-nitrosourea) mouse mutagenesis project. The aim of this study was to identify the causal mutation of one of these strains and to characterize the resulting diabetic phenotype. Mutants exhibit a T to G transversion mutation at nt 629 in the glucokinase (Gck) gene, leading to an amino acid exchange from methionine to arginine at position 210. Adult Munich Gck(M210R) mutant mice demonstrated a significant reduction of hepatic glucokinase enzyme activity but equal glucokinase mRNA and protein abundances. While homozygous mutant mice exhibited growth retardation and died soon after birth in consequence of severe hyperglycemia, heterozygous mutant mice displayed only slightly elevated blood glucose levels, present from birth, with development of disturbed glucose tolerance and glucose-induced insulin secretion. Additionally, insulin sensitivity and fasting serum insulin levels were slightly reduced in male mutant mice from an age of 90 days onward. While beta-cell mass was unaltered in neonate heterozygous and homozygous mutant mice, the total islet and beta-cell volumes and the total volume of isolated beta-cells were significantly decreased in 210-day-old male, but not female heterozygous mutant mice despite undetectable apoptosis. These findings indicate that reduced total islet and beta-cell volumes of male mutants might emerge from disturbed postnatal islet neogenesis. Considering the lack of knowledge about the pathomorphology of maturity-onset diabetes of the young type 2 (MODY 2), this glucokinase mutant model of reduced total islet and total beta-cell volume provides the opportunity to elucidate the impact of a defective glucokinase on development and maintenance of beta-cell mass and its relevance in MODY 2 patients.

Cinnamide Derivatives of D -Mannose as Inhibitors of the Bacterial Virulence Factor LecB from Pseudomonas aeruginosa

Abstract Pseudomonas aeruginosa is an opportunistic Gram‐negative pathogen with high antibiotic resistance. Its lectin LecB was identified as a virulence factor and is relevant in bacterial adhesion and biofilm formation. Inhibition of LecB with carbohydrate‐based ligands results in a decrease in toxicity and biofilm formation. We recently discovered two classes of potent drug‐like glycomimetic inhibitors, that is, sulfonamides and cinnamides of d‐mannose. Here, we describe the chemical synthesis and biochemical evaluation of more than 20 derivatives with increased potency compared to the unsubstituted cinnamide. The structure–activity relationship (SAR) obtained and the extended biophysical characterization allowed the experimental determination of the binding mode of these cinnamides with LecB. The established surface binding mode now allows future rational structure‐based drug design. Importantly, all glycomimetics tested showed extended receptor residence times with half‐lives in the 5–20 min range, a prerequisite for therapeutic application. Thus, the glycomimetics described here provide an excellent basis for future development of anti‐infectives against this multidrug‐resistant pathogen.

Ciprofloxacin-loaded lipid-core nanocapsules as mucus penetrating drug delivery system intended for the treatment of bacterial infections in cystic fibrosis

Treatment of bacterial airway infections is essential for cystic fibrosis therapy. However, effectiveness of antibacterial treatment is limited as bacteria inside the mucus are protected from antibiotics and immune response. To overcome this biological barrier, ciprofloxacin was loaded into lipid-core nanocapsules (LNC) for high mucus permeability, sustained release and antibacterial activity. Ciprofloxacin-loaded LNC with a mean size of 180nm showed a by 50% increased drug permeation through mucus. In bacterial growth assays, the drug in the LNC had similar minimum inhibitory concentrations as the free drug in P. aeruginosa and S. aureus. Interestingly, formation of biofilm-like aggregates, which were observed for S. aureus treated with free ciprofloxacin, was avoided by exposure to LNC. With the combined advantages over the non-encapsulated drug, ciprofloxacin-loaded LNC represent a promising drug delivery system with the prospect of an improved antibiotic therapy in cystic fibrosis.

Glycomimetic, Orally Bioavailable LecB Inhibitors Block Biofilm Formation of Pseudomonas aeruginosa

The opportunistic Gram-negative bacterium Pseudomonas aeruginosa is a leading pathogen for infections of immuno-compromised patients and those suffering from cystic fibrosis. Its ability to switch from planktonic life to aggregates, forming the so-called biofilms, is a front-line mechanism of antimicrobial resistance. The bacterial carbohydrate-binding protein LecB is an integral component and necessary for biofilm formation. Here, we report a new class of drug-like low molecular weight inhibitors of the lectin LecB with nanomolar affinities and excellent receptor binding kinetics and thermodynamics. This class of glycomimetic inhibitors efficiently blocked biofilm formation of P. aeruginosa in vitro while the natural monovalent carbohydrate ligands failed. Furthermore, excellent selectivity and pharmacokinetic properties were achieved. Notably, two compounds showed good oral bioavailability, and high compound concentrations in plasma and urine were achieved in vivo.

Photorhabdus luminescens lectin A (PllA) - a new probe for detecting α-galactoside-terminating glycoconjugates.

Lectins play important roles in infections by pathogenic bacteria, for example, in host colonization, persistence, and biofilm formation. The Gram-negative entomopathogenic bacterium Photorhabdus luminescens symbiotically lives in insect-infecting Heterorhabditis nematodes and kills the insect host upon invasion by the nematode. The P. luminescens genome harbors the gene plu2096, coding for a novel lectin that we named PllA. We analyzed the binding properties of purified PllA with a glycan array and a binding assay in solution. Both assays revealed a strict specificity of PllA for α-galactoside–terminating glycoconjugates. The crystal structures of apo PllA and complexes with three different ligands revealed the molecular basis for the strict specificity of this lectin. Furthermore, we found that a 90° twist in subunit orientation leads to a peculiar quaternary structure compared with that of its ortholog LecA from Pseudomonas aeruginosa. We also investigated the utility of PllA as a probe for detecting α-galactosides. The α-Gal epitope is present on wild-type pig cells and is the main reason for hyperacute organ rejection in pig to primate xenotransplantation. We noted that PllA specifically recognizes this epitope on the glycan array and demonstrated that PllA can be used as a fluorescent probe to detect this epitope on primary porcine cells in vitro. In summary, our biochemical and structural analyses of the P. luminescens lectin PllA have disclosed the structural basis for PllAs high specificity for α-galactoside–containing ligands, and we show that PllA can be used to visualize the α-Gal epitope on porcine tissues.

Covalent lectin inhibition and application in bacterial biofilm imaging

Abstract Biofilm formation by pathogenic bacteria is a hallmark of chronic infections. In many cases, lectins play key roles in establishing biofilms. The pathogen Pseudomonas aeruginosa often exhibiting various drug resistances employs its lectins LecA and LecB as virulence factors and biofilm building blocks. Therefore, inhibition of the function of these proteins is thought to have potential in developing “pathoblockers” preventing biofilm formation and virulence. A covalent lectin inhibitor specific to a carbohydrate binding site is described for the first time. Its application in the LecA‐specific in vitro imaging of biofilms formed by P. aeruginosa is also reported.

Charakterisierung von ENU-Mausmutanten@@@Characterization of ENU-mutant mice: Tiermodelle für menschliche Erkrankungen mittels morphologischer und molekularer Methoden@@@Animal models for human diseases using morphological and molecular methods

ZusammenfassungNach der Entschlüsselung des Humangenoms besteht die Herausforderung, gezielte Kenntnisse über die genauen Genfunktionen und das Zusammenspiel dieser Gene und Signalwege bei der Entstehung von Krankheiten zu erlangen. Mausmodelle stellen hierfür das Mittel der Wahl dar. Die chemischen Mutagenese mittels N-ethyl-N-nitrosourea (ENU) ermöglicht durch die Erzeugung zufälliger Punktmutationen eine differenzierte Analyse der Auswirkungen eines einzigen Basenaustausches auf den gesamten Organismus. Das Münchner ENU-Mausmutagenese-Projekt hat sich der weltweiten Initiative angeschlossen, Einblicke in die Bedeutung einzelner Genabschnitte zu erhalten. Im Rahmen eines genomweiten systematischen Hochdurchsatz-Screenings wurden Mausmodelle für eine Vielzahl menschlicher Erbkrankheiten entwickelt. Diese Arbeit verdeutlicht, wie die Implementierung des ENU-Mausmutagenese-Projekts und der Genidentifikation im parallelen Hochdurchsatz-Screening, unter der Möglichkeit der engen örtlichen Zusammenarbeit mit erfahrenen Phänotypisierungsgruppen am Helmholtz Zentrum München, zu wesentlichen Fortschritten in der funktionellen Analyse des Säugetiergenoms führen kann.AbstractFollowing sequencing of the human genome there are new challenges to decipher the knowledge concerning gene function and the role of gene interactions and pathways leading to disease. Mouse models have proven to be an ideal tool for this purpose. Point mutations induced by chemical mutagenesis by N-ethyl-N-nitrosourea (ENU) offer possibilities for the analysis of the phenotypic outcome of a single base pair exchange on the entire organism. The Munich ENU mouse mutagenesis project is part of the worldwide efforts to obtain mutations for each gene. The generation of new alleles or allelic series offers relevant insights into the relevance of single gene sections. Various mouse models for human diseases have been generated by a systematic large-scale genome-wide phenotyping screen in the last decade. This work illustrates how the implementation of the ENU mouse mutagenesis project with gene identification and parallel high-throughput screening is taking advantage of local cooperation with experienced phenotyping groups at the Helmholtz Zentrum München, leading to major advances in the functional analysis of the mammalian genome.Following sequencing of the human genome there are new challenges to decipher the knowledge concerning gene function and the role of gene interactions and pathways leading to disease. Mouse models have proven to be an ideal tool for this purpose. Point mutations induced by chemical mutagenesis by N-ethyl-N-nitrosourea (ENU) offer possibilities for the analysis of the phenotypic outcome of a single base pair exchange on the entire organism. The Munich ENU mouse mutagenesis project is part of the worldwide efforts to obtain mutations for each gene. The generation of new alleles or allelic series offers relevant insights into the relevance of single gene sections. Various mouse models for human diseases have been generated by a systematic large-scale genome-wide phenotyping screen in the last decade. This work illustrates how the implementation of the ENU mouse mutagenesis project with gene identification and parallel high-throughput screening is taking advantage of local cooperation with experienced phenotyping groups at the Helmholtz Zentrum München, leading to major advances in the functional analysis of the mammalian genome.