Light-Activatable, 2,5-Disubstituted Tetrazoles for the Proteome-Wide Profiling of Aspartates and Glutamates in Living Bacteria

2019 ◽  
Author(s):  
Kathrin Bach ◽  
Bert L. H. Beerkens ◽  
Patrick R. A. Zanon ◽  
Stephan M. Hacker
Keyword(s):  
Amino Acids ◽  
Binding Sites ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Protein Ligands ◽  
New Class ◽  
Covalent Inhibitors ◽  
Hydrazonyl Chlorides ◽  
Living Bacteria ◽  
Excellent Selectivity

Covalent inhibitors have recently seen a resurgence of interest in drug development. Nevertheless, compounds, that do not rely on an enzymatic activity, have almost exclusively been developed to target cysteines. Expanding the scope to other amino acids would be largely facilitated by the ability to globally monitor their engagement by covalent inhibitors. Here, we present the use of light-activatable 2,5-disubstituted tetrazoles that allow quantifying 8971 aspartates and glutamates in the bacterial proteome with excellent selectivity. Using these probes, we competitively map the binding sites of two isoxazolium salts and introduce hydrazonyl chlorides as a new class of carboxylic acid-directed covalent protein ligands. As the probes are unreactive prior to activation, they allow global profiling even in living Gram-positive and Gram-negative bacteria. Taken together, this method to monitor aspartates and glutamates proteome-wide will lay the foundation to efficiently develop covalent inhibitors targeting these amino acids

Light-Activatable, 2,5-Disubstituted Tetrazoles for the Proteome-Wide Profiling of Aspartates and Glutamates in Living Bacteria

2019 ◽  
Author(s):  
Kathrin Bach ◽  
Bert L. H. Beerkens ◽  
Patrick R. A. Zanon ◽  
Stephan M. Hacker
Keyword(s):  
Amino Acids ◽  
Binding Sites ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Protein Ligands ◽  
New Class ◽  
Covalent Inhibitors ◽  
Hydrazonyl Chlorides ◽  
Living Bacteria ◽  
Excellent Selectivity

Covalent inhibitors have recently seen a resurgence of interest in drug development. Nevertheless, compounds, that do not rely on an enzymatic activity, have almost exclusively been developed to target cysteines. Expanding the scope to other amino acids would be largely facilitated by the ability to globally monitor their engagement by covalent inhibitors. Here, we present the use of light-activatable 2,5-disubstituted tetrazoles that allow quantifying 8971 aspartates and glutamates in the bacterial proteome with excellent selectivity. Using these probes, we competitively map the binding sites of two isoxazolium salts and introduce hydrazonyl chlorides as a new class of carboxylic acid-directed covalent protein ligands. As the probes are unreactive prior to activation, they allow global profiling even in living Gram-positive and Gram-negative bacteria. Taken together, this method to monitor aspartates and glutamates proteome-wide will lay the foundation to efficiently develop covalent inhibitors targeting these amino acids

scholarly journals The antimicrobial potential of cannabidiol

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Mark A. T. Blaskovich ◽  
Angela M. Kavanagh ◽  
Alysha G. Elliott ◽  
Bing Zhang ◽  
Soumya Ramu ◽  
...  
Keyword(s):  
Bacterial Infections ◽  
Modern Medicine ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
New Class ◽  
Clostridioides Difficile ◽  
Excellent Activity ◽  
Induce Resistance ◽  
First Time

AbstractAntimicrobial resistance threatens the viability of modern medicine, which is largely dependent on the successful prevention and treatment of bacterial infections. Unfortunately, there are few new therapeutics in the clinical pipeline, particularly for Gram-negative bacteria. We now present a detailed evaluation of the antimicrobial activity of cannabidiol, the main non-psychoactive component of cannabis. We confirm previous reports of Gram-positive activity and expand the breadth of pathogens tested, including highly resistant Staphylococcus aureus, Streptococcus pneumoniae, and Clostridioides difficile. Our results demonstrate that cannabidiol has excellent activity against biofilms, little propensity to induce resistance, and topical in vivo efficacy. Multiple mode-of-action studies point to membrane disruption as cannabidiol’s primary mechanism. More importantly, we now report for the first time that cannabidiol can selectively kill a subset of Gram-negative bacteria that includes the ‘urgent threat’ pathogen Neisseria gonorrhoeae. Structure-activity relationship studies demonstrate the potential to advance cannabidiol analogs as a much-needed new class of antibiotics.

Val-Geninthiocin: Structure Elucidation and MSn Fragmentation of Thiopeptide Antibiotics Produced by Streptomyces sp. RSF18

2008 ◽  
Vol 63 (10) ◽  
pp. 1223-1230 ◽  
Author(s):  
Imran Sajid ◽  
Khaled A. Shaaban ◽  
Holm Frauendorf ◽  
Shahida Hasnain ◽  
Hartmut Laatscha
Keyword(s):  
Amino Acids ◽  
Biological Activity ◽  
Antifungal Activity ◽  
Structure Elucidation ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Unusual Amino Acids ◽  
Streptomyces Sp ◽  
Gram Positive Bacteria ◽  
Thiopeptide Antibiotics

AbstractVal-Geninthiocin (2), a new member of thiopeptide antibiotics, was isolated from the mycelium of Streptomyces sp. RSF18, along with the closely related geninthiocin (1) and the macrolide, chalcomycin. By intensive NMR and MS studies, Val-geninthiocin (2) was identified as desoxygeninthiocin, a thiopeptide, containing several oxazole and thiazole units and a number of unusual amino acids. Compound 2 shows potent activity against Gram-positive bacteria and minor antifungal activity, while it is not effective against Gram-negative bacteria or microalgae. Here we describe the fermentation, isolation and structure elucidation as well as the biological activity of 2.

scholarly journals β-Aminopeptidases: Insight into Enzymes without a Known Natural Substrate

2019 ◽  
Vol 85 (15) ◽  
Author(s):  
Marietta John-White ◽  
James Gardiner ◽  
Priscilla Johanesen ◽  
Dena Lyras ◽  
Geoffrey Dumsday
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Gram Negative Bacteria ◽  
Diverse Range ◽  
Α Subunit ◽  
Gram Negative ◽  
Content Type ◽  
Proteolytic Resistance ◽  
Insight Into ◽  
Unique Capability

ABSTRACT β-Aminopeptidases have the unique capability to hydrolyze N-terminal β-amino acids, with varied preferences for the nature of β-amino acid side chains. This unique capability makes them useful as biocatalysts for synthesis of β-peptides and to kinetically resolve β-peptides and amides for the production of enantiopure β-amino acids. To date, six β-aminopeptidases have been discovered and functionally characterized, five from Gram-negative bacteria and one from a fungus, Aspergillus. Here we report on the purification and characterization of an additional four β-aminopeptidases, one from a Gram-positive bacterium, Mycolicibacterium smegmatis (BapAMs), one from a yeast, Yarrowia lipolytica (BapAYlip), and two from Gram-negative bacteria isolated from activated sludge identified as Burkholderia spp. (BapABcA5 and BapABcC1). The genes encoding β-aminopeptidases were cloned, expressed in Escherichia coli, and purified. The β-aminopeptidases were produced as inactive preproteins that underwent self-cleavage to form active enzymes comprised of two different subunits. The subunits, designated α and β, appeared to be tightly associated, as the active enzyme was recovered after immobilized-metal affinity chromatography (IMAC) purification, even though only the α-subunit was 6-histidine tagged. The enzymes were shown to hydrolyze chromogenic substrates with the N-terminal l-configurations β-homo-Gly (βhGly) and β3-homo-Leu (β3hLeu) with high activities. These enzymes displayed higher activity with H-βhGly-p-nitroanilide (H-βhGly-pNA) than previously characterized enzymes from other microorganisms. These data indicate that the new β-aminopeptidases are fully functional, adding to the toolbox of enzymes that could be used to produce β-peptides. Overexpression studies in Pseudomonas aeruginosa also showed that the β-aminopeptidases may play a role in some cellular functions. IMPORTANCE β-Aminopeptidases are unique enzymes found in a diverse range of microorganisms that can utilize synthetic β-peptides as a sole carbon source. Six β-aminopeptidases have been previously characterized with preferences for different β-amino acid substrates and have demonstrated the capability to catalyze not only the degradation of synthetic β-peptides but also the synthesis of short β-peptides. Identification of other β-aminopeptidases adds to this toolbox of enzymes with differing β-amino acid substrate preferences and kinetics. These enzymes have the potential to be utilized in the sustainable manufacture of β-amino acid derivatives and β-peptides for use in biomedical and biomaterial applications. This is important, because β-amino acids and β-peptides confer increased proteolytic resistance to bioactive compounds and form novel structures as well as structures similar to α-peptides. The discovery of new enzymes will also provide insight into the biological importance of these enzymes in nature.

scholarly journals Curative Treatment of Severe Gram-Negative Bacterial Infections by a New Class of Antibiotics Targeting LpxC

mBio ◽  
2017 ◽  
Vol 8 (4) ◽  
Author(s):  
Nadine Lemaître ◽  
Xiaofei Liang ◽  
Javaria Najeeb ◽  
Chul-Jin Lee ◽  
Marie Titecat ◽  
...  
Keyword(s):  
Bacterial Infections ◽  
Biosynthetic Pathway ◽  
Lipid A ◽  
Multidrug Resistant ◽  
Gram Negative Bacteria ◽  
Drug Resistant ◽  
Bubonic Plague ◽  
Gram Negative ◽  
New Class

ABSTRACT The infectious diseases caused by multidrug-resistant bacteria pose serious threats to humankind. It has been suggested that an antibiotic targeting LpxC of the lipid A biosynthetic pathway in Gram-negative bacteria is a promising strategy for curing Gram-negative bacterial infections. However, experimental proof of this concept is lacking. Here, we describe our discovery and characterization of a biphenylacetylene-based inhibitor of LpxC, an essential enzyme in the biosynthesis of the lipid A component of the outer membrane of Gram-negative bacteria. The compound LPC-069 has no known adverse effects in mice and is effective in vitro against a broad panel of Gram-negative clinical isolates, including several multiresistant and extremely drug-resistant strains involved in nosocomial infections. Furthermore, LPC-069 is curative in a murine model of one of the most severe human diseases, bubonic plague, which is caused by the Gram-negative bacterium Yersinia pestis. Our results demonstrate the safety and efficacy of LpxC inhibitors as a new class of antibiotic against fatal infections caused by extremely virulent pathogens. The present findings also highlight the potential of LpxC inhibitors for clinical development as therapeutics for infections caused by multidrug-resistant bacteria. IMPORTANCE The rapid spread of antimicrobial resistance among Gram-negative bacilli highlights the urgent need for new antibiotics. Here, we describe a new class of antibiotics lacking cross-resistance with conventional antibiotics. The compounds inhibit LpxC, a key enzyme in the lipid A biosynthetic pathway in Gram-negative bacteria, and are active in vitro against a broad panel of clinical isolates of Gram-negative bacilli involved in nosocomial and community infections. The present study also constitutes the first demonstration of the curative treatment of bubonic plague by a novel, broad-spectrum antibiotic targeting LpxC. Hence, the data highlight the therapeutic potential of LpxC inhibitors against a wide variety of Gram-negative bacterial infections, including the most severe ones caused by Y. pestis and by multidrug-resistant and extensively drug-resistant carbapenemase-producing strains. IMPORTANCE The rapid spread of antimicrobial resistance among Gram-negative bacilli highlights the urgent need for new antibiotics. Here, we describe a new class of antibiotics lacking cross-resistance with conventional antibiotics. The compounds inhibit LpxC, a key enzyme in the lipid A biosynthetic pathway in Gram-negative bacteria, and are active in vitro against a broad panel of clinical isolates of Gram-negative bacilli involved in nosocomial and community infections. The present study also constitutes the first demonstration of the curative treatment of bubonic plague by a novel, broad-spectrum antibiotic targeting LpxC. Hence, the data highlight the therapeutic potential of LpxC inhibitors against a wide variety of Gram-negative bacterial infections, including the most severe ones caused by Y. pestis and by multidrug-resistant and extensively drug-resistant carbapenemase-producing strains.

scholarly journals Functional and structural characterization of Serratia marcescens ExbB: determinants of the interaction with HasB/TonB

2021 ◽  
Author(s):  
Valérie Biou ◽  
Ricardo Jorge D Adaixo ◽  
Mohamed Chami ◽  
Pierre-Damien Coureux ◽  
Benoist Laurent ◽  
...  
Keyword(s):  
Serratia Marcescens ◽  
Molecular Motor ◽  
Opportunistic Pathogen ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
E Coli ◽  
New Class ◽  
Heme Acquisition ◽  
Shed Light

ExbBD is part of a cytoplasmic membrane molecular motor driven by the proton-motive force. It belongs to the larger family of motors involved in nutriment import across the outer membrane of Gram-negative bacteria (ExbBD), flagellar rotation (MotAB) or late steps of cell division in Gram-negative bacteria (TolQR). ExbB and ExbD are integral membrane proteins with three (ExbB) or one (ExbD) transmembrane segment. Here we have solved by single-particle cryo-EM the structures of ExbB alone and of the ExbB-ExbD complex of the opportunistic pathogen Serratia marcescens. ExbBSm alone behaves as a stable pentamer, and the complex displays the ExbB5-ExbD2 stoichiometry. This is similar to what has been observed for ExbB-ExbD complexes from Escherichia coli and Pseudomonas savastanoi as well as MotAB complexes from various species. We identified residues located in the first TM of ExbBSm and ExbBEc that are likely involved in the interaction with TonB/HasB and that are essential for function. ExbBSm has a ca. 40 residues long periplasmic extension absent in E. coli. Such long ExbBs are found in some Gammaproteobacteria, and several genera of Alphaproteobacteria. We show that this extension interacts with HasB, a dedicated TonB paralog from the heme acquisition system (Has) from S. marcescens. We also show that it is involved in heme acquisition via the Has system from S. marcescens. ExbBSm represents thus a new class of ExbB protein and our results shed light on the specificity determinants between the ExbB-ExbD complex and their associated TonB partners.

scholarly journals Synthesis, Characterization and Antibacterial Activity of 2-Phenylsulphonamide Derivatives of Some Amino Acids

2016 ◽  
Vol 70 ◽  
pp. 42-47
Author(s):  
Chinyere Benardette Chinaka Ikpa ◽  
Uchechukwu C. Okoro ◽  
Collins I. Ubochi ◽  
Kieran O. Nwanorh
Keyword(s):  
Amino Acids ◽  
Inhibitory Concentration ◽  
Gram Negative Bacteria ◽  
Standard Drug ◽  
Gram Negative ◽  
E Coli ◽  
Gram Positive Bacteria ◽  
Simple Substitution ◽  
Ft Ir ◽  
Derivatives Of

The 2-phenylsulphonamide derivatives of amino acids were synthesis by simple substitution of benzenesulphonylchloride (6) with amino acids (1-5) containing pharmacological active functionalities. Structures of the synthesised compounds (7a-7e) were characterised using FT-IR, NMR(1H,13C) and elemental analysis. The anti bacterial activities of the synthesised compounds were evaluated against gram positive bacteria: Staph and Streptococcus, gram negative bacteria: E-coli, Klebsiella, Proteus, and pseudomonas using 200 µl of 10 mg/ml and minimum inhibitory concentration (MIC) were also determined. The compounds exhibited effective anti bacterial properties though some are not more active than the standard drug ciprofloxacin.

scholarly journals A novel class of polymorphic toxins in Bacteroidetes

2020 ◽  
Vol 3 (4) ◽  
pp. e201900631
Author(s):  
Biswanath Jana ◽  
Dor Salomon ◽  
Eran Bosis
Keyword(s):  
Gut Microbiota ◽  
Unknown Function ◽  
Secretion System ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
New Class ◽  
Terminal Domain ◽  
Domain Of Unknown Function ◽  
Type Ix Secretion System ◽  
Encoding Gene

Bacteroidetes are Gram-negative bacteria that are abundant in the environment as well as in the gut microbiota of animals. Many bacteroidetes encode large proteins containing an N-terminal domain of unknown function, named TANFOR. In this work, we show that TANFOR-containing proteins carry polymorphic C-terminal toxin domains with predicted antibacterial and anti-eukaryotic activities. We also show that a C-terminal domain that is prevalent in TANFOR-containing proteins represents a novel family of antibacterial DNase toxins, which we named BaCT (Bacteroidetes C-terminal Toxin). Finally, we discover that TANFOR-encoding gene neighborhoods are enriched with genes that encode substrates of the type IX secretion system (T9SS), which is involved in exporting proteins from the periplasm across the outer membrane. Based on these findings, we conclude that TANFOR-containing proteins are a new class of polymorphic toxins, and we hypothesize that they are T9SS substrates.

scholarly journals An OregonGreen488-labelled d-amino acid for visualizing peptidoglycan by super-resolution STED nanoscopy

Microbiology ◽  
2020 ◽  
Vol 166 (12) ◽  
pp. 1129-1135 ◽  
Author(s):  
Bill Söderström ◽  
Alessandro Ruda ◽  
Göran Widmalm ◽  
Daniel O. Daley
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Chemical Synthesis ◽  
Super Resolution ◽  
Molecular Probes ◽  
Stimulated Emission ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Peptidoglycan Synthesis ◽  
Peptidoglycan Layer

Fluorescent d-amino acids (FDAAs) are molecular probes that are widely used for labelling the peptidoglycan layer of bacteria. When added to growing cells they are incorporated into the stem peptide by a transpeptidase reaction, allowing the timing and localization of peptidoglycan synthesis to be determined by fluorescence microscopy. Herein we describe the chemical synthesis of an OregonGreen488-labelled FDAA (OGDA). We also demonstrate that OGDA can be efficiently incorporated into the PG of Gram-positive and some Gram-negative bacteria, and imaged by super-resolution stimulated emission depletion (STED) nanoscopy at a resolution well below 100 nm.

Evaluating H2O2 in Living Bacteria by Ratiometric Fluorescent Probe

2022 ◽  
Vol 905 ◽  
pp. 210-217
Author(s):  
Qian Qian Chen
Keyword(s):  
Fluorescent Probe ◽  
Living Cells ◽  
Gram Negative Bacteria ◽  
Signal Molecule ◽  
Gram Negative ◽  
Key Factor ◽  
Live Bacteria ◽  
First Time ◽  
Living Bacteria ◽  
Ratiometric Fluorescent Probe

Hydrogen peroxide (H2O2) is a significant signal molecule in physiological and pathological processes. Levels of H2O2 in bacteria are proved to be a key factor in immune response. To sum up, detection of H2O2 levels in living bacteria is remarkable for further study of its physiological and pathological effects. Herein, we propose a novel ratiometric fluorescent probe (Nahp) to detect H2O2 in living cells and bacteria. In addition, based on boronate, Nahp has satisfactory selectivity and sensitivity toward H2O2 (LOD = 0.158 μM). Furthermore, with excellent detection performance to H2O2, Nahp is successfully used for fluorescent bioimaging of H2O2 and measuring H2O2 accumulation in bacteria. Most importantly, the probe was also used to image H2O2 in three Gram-negative bacteria, clearly revealing for the first time significant differences in H2O2 expression levels in live bacteria.

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