Quantification of Bacterial Metabolic Activities in the Gut by d-Amino Acid-Based In Vivo Labeling

Herein, we propose a metabolic d -amino acid-based labeling and in situ hybridization-facilitated (MeDabLISH) strategy for the quantitative analysis of the indigenous metabolic status of gut bacteria. The fluorescent d -amino acid (FDAA)-based labeling intensities of bacteria were found to highly correlate with their temporal and steady-state metabolic status. Then, after taxonomic identification of bacterial genera in the in vivo FDAA-labeled mouse gut microbiota, by corresponding fluorescence in situ hybridization (FISH) probes, the metabolic activities of different gut bacterial genera are quantified by flow cytometry, using FISH signals to differentiate genera and FDAA signals to indicate their basal metabolic levels. It was found that Gram-negative genera in the mouse microbiota have stronger metabolic activities during the daytime, and Gram-positive genera have higher activities at the night. Our strategy will be instrumental in deepening our understanding of the highly complex microbiota.     

Metabolic Labeling of Peptidoglycan with NIR‐II Dye Enables In Vivo Imaging of Gut Microbiota

Deepening our understanding of mammalian gut microbiota has been greatly hampered by the lack of a facile, real‐time, and in vivo bacterial imaging method. To address this unmet need in microbial visualization, we herein report the development of a second near‐infrared (NIR‐II)‐based method for in vivo imaging of gut bacteria. Using d ‐propargylglycine in gavage and then click reaction with an azide‐containing NIR‐II dye, gut microbiota of a donor mouse was strongly labeled with NIR‐II fluorescence on their peptidoglycan. The bacteria could be readily visualized in recipient mouse gut with high spatial resolution and deep tissue penetration under NIR irradiation. The NIR‐II‐based metabolic labeling strategy reported herein, provides, to the best of our knowledge, the first protocol for facile in vivo visualization of gut microbiota within deep tissues, and offers an instrumental tool for deciphering the complex biology of these gut “dark matters”.     

Metabolic Labeling of Peptidoglycan with NIR-II Dye Enables In Vivo Imaging of Gut Microbiota

Deepening our understanding of mammalian gut microbiota has been greatly hampered by the lack of a facile, real-time, and in vivo bacterial imaging method. To address this unmet need in microbial visualization, we herein report the development of a second near-infrared (NIR-II)-based method for in vivo imaging of gut bacteria. Using d -propargylglycine in gavage and then click reaction with an azide-containing NIR-II dye, gut microbiota of a donor mouse was strongly labeled with NIR-II fluorescence on their peptidoglycan. The bacteria could be readily visualized in recipient mouse gut with high spatial resolution and deep tissue penetration under NIR irradiation. The NIR-II-based metabolic labeling strategy reported herein, provides, to the best of our knowledge, the first protocol for facile in vivo visualization of gut microbiota within deep tissues, and offers an instrumental tool for deciphering the complex biology of these gut “dark matters”.     

Visualization and In Situ Ablation of Intracellular Bacterial Pathogens through Metabolic Labeling

Protected by the host cells, the hidden intracellular bacteria are typically difficult to kill by common antibiotics and cannot be visualized without complex cellular pretreatments. Herein, we successfully developed a bacteria‐metabolizable dual‐functional probe TPEPy‐d ‐Ala, which is based on d ‐alanine and a photosensitizer with aggregation‐induced emission for fluorescence turn‐on imaging of intracellular bacteria in living host cells and photodynamic ablation in situ. Once metabolically incorporated into bacterial peptidoglycan, the intramolecular motions of TPEPy‐d ‐Ala are inhibited, leading to an enhanced fluorescent signal, which allows the clear visualization of the intracellular bacteria. Moreover, TPEPy‐d ‐Ala can effectively ablate the labeled intracellular bacteria in situ owing to covalent ligation to peptidoglycan, yielding a low intracellular minimum inhibitory concentration (MIC) of 20±0.5 μg mL^?1, much more efficient than that of a commonly used antibiotic, vancomycin.     

Chemical Synthesis of Burkholderia Lipid A Modified with Glycosyl Phosphodiester‐Linked 4‐Amino‐4‐deoxy‐β‐L‐arabinose and Its Immunomodulatory Potential

Modification of the Lipid A phosphates by positively charged appendages is a part of the survival strategy of numerous opportunistic Gram‐negative bacteria. The phosphate groups of the cystic fibrosis adapted Burkholderia Lipid A are abundantly esterified by 4‐amino‐4‐deoxy‐β‐L ‐arabinose (β‐L ‐Ara4N), which imposes resistance to antibiotic treatment and contributes to bacterial virulence. To establish structural features accounting for the unique pro‐inflammatory activity of Burkholderia LPS we have synthesised Lipid A substituted by β‐L ‐Ara4N at the anomeric phosphate and its Ara4N‐free counterpart. The double glycosyl phosphodiester was assembled by triazolyl‐tris‐(pyrrolidinyl)phosphonium‐assisted coupling of the β‐L ‐Ara4N H‐phosphonate to α‐lactol of β(1→6) diglucosamine, pentaacylated with (R)‐(3)‐acyloxyacyl‐ and Alloc‐protected (R)‐(3)‐hydroxyacyl residues. The intermediate 1,1′‐glycosyl‐H‐phosphonate diester was oxidised in anhydrous conditions to provide, after total deprotection, β‐L ‐Ara4N‐substituted Burkholderia Lipid A. The β‐L ‐Ara4N modification significantly enhanced the pro‐inflammatory innate immune signaling of otherwise non‐endotoxic Burkholderia Lipid A.     

Metabolic Profiling of Bacteria by Unnatural C‐terminated D‐Amino Acids

Bacterial peptidoglycan is a mesh‐like network comprised of sugars and oligopeptides. Transpeptidases cross‐link peptidoglycan oligopeptides to provide vital cell wall rigidity and structural support. It was recently discovered that the same transpeptidases catalyze the metabolic incorporation of exogenous D ‐amino acids onto bacterial cell surfaces with vast promiscuity for the side‐chain identity. It is now shown that this enzymatic promiscuity is not exclusive to side chains, but that C‐terminus variations can also be accommodated across a diverse range of bacteria. Atomic force microscopy analysis revealed that the incorporation of C‐terminus amidated D ‐amino acids onto bacterial surfaces substantially reduced the cell wall stiffness. We exploited the promiscuity of bacterial transpeptidases to develop a novel assay for profiling different bacterial species.     

Development of a Universal Fluorescent Probe for Gram‐Positive Bacteria

The rapid and sensitive classification of bacteria is the first step of bacterial community research and the treatment of infection. Herein, a fluorescent probe BacGO is presented, which shows the best universal selectivity for Gram‐positive bacteria among known probes with a minimum staining procedure for sample detection and enrichment of the live bacteria. BacGO could also be used to assess of the Gram status in the bacterial community from wastewater sludge. Furthermore, BacGO could sensitively and selectively detect a Gram‐positive bacterial infection, not only in vitro but also using an in vivo keratitis mouse model. BacGO provides an unprecedented research tool for the study of dynamic bacterial communities and for clinical application.     

Targeting Bacterial Membranes: NMR Spectroscopy Characterization of Substrate Recognition and Binding Requirements of D‐Arabinose‐5‐Phosphate Isomerase

Lipopolysaccharide (LPS) is an essential component of the outer membrane of Gram‐negative bacteria and consists of three elements: lipid A, the core oligosaccharide, and the O‐antigen. The inner‐core region is highly conserved and contains at least one residue of 3‐deoxy‐D ‐manno‐octulosonate (Kdo). Arabinose‐5‐phosphate isomerase (API) is an aldo–keto isomerase catalyzing the reversible isomerization of D ‐ribulose‐5‐phosphate (Ru5P) to D ‐arabinose‐5‐phosphate (A5P), the first step of Kdo biosynthesis. By exploiting saturation transfer difference (STD) NMR spectroscopy, the structural requirements necessary for API substrate recognition and binding were identified, with the aim of designing new API inhibitors. In addition, simple experimental conditions for the STD experiments to perform a fast, robust, and efficient screening of small libraries of potential API inhibitors, allowing the identification of new potential leads, were set up. Due to the essential role of API enzymes in LPS biosynthesis and Gram‐negative bacteria survival, by exploiting these data, a new generation of potent antibacterial drugs could be developed.     

Total Synthesis and Functional Evaluation of Fourteen Derivatives of Lysocin E: Importance of Cationic,Hydrophobic, and Aromatic Moieties for Antibacterial Activity

Lysocin E ( 1 ) is a structurally complex 37‐membered depsipeptide comprising 12 amino‐acid residues with an N‐methylated amide and an ester linkage. Compound 1 binds to menaquinone (MK) in the bacterial membrane to exert its potent bactericidal activity. To decipher the biologically important functionalities within this unique antibiotic, we performed a comprehensive structure‐activity relationship (SAR) study by systematically changing the side‐chain structures of l ‐Thr‐1, d ‐Arg‐2, N‐Me‐d ‐Phe‐5, d ‐Arg‐7, l ‐Glu‐8, and d ‐Trp‐10. First, we achieved total synthesis of the 14 new side‐chain analogues of 1 by employing a solid‐phase strategy. We then evaluated the MK‐dependent liposomal disruption and antimicrobial activity against Staphylococcus aureus by 1 and its analogues. Correlating data between the liposome and bacteria experiments revealed that membrane lysis was mainly responsible for the antibacterial functions. Altering the cationic guanidine moiety of d ‐Arg‐2/7 to a neutral amide, and the C7‐acyl group of l ‐Thr‐1 to the C2 or C11 counterpart decreased the antimicrobial activities four‐ or eight‐fold. More drastically, chemical mutation of d ‐Trp‐10 to d ‐Ala‐10 totally abolished the bioactivities. These important findings led us to propose the biological roles of the side‐chain functionalities.     

Highly Efficient One‐Pot Three‐Component Synthesis and Antimicrobial Activity of 2‐Amino‐4H‐chromene Derivatives

A remarkably efficient three‐component reaction to synthesize 2‐amino‐4H‐chromenes derivatives from malononitrile, various aromatic aldehydes, and orcinol was described at room temperature in CH₂Cl₂ in the presence of catalytic amount of triethylamine. In a facile one‐pot procedure, excellent yields of products were achieved in less than 1 h. Some of the synthesized 2‐amino‐4H‐chromenes derivatives demonstrated potent antibacterial activities against Gram‐positive bacteria including Staphylococcus aureus and Bacillus anthracis, indicated by disk method, minimum inhibitory concentration, and minimum bactericidal concentration approaches. However, none of the tested compounds expressed any antibacterial activities against Enterococcus faecalis and Gram‐negative bacteria.     

Enantioseparation of aromatic amino acids using CEC monolith with novel chiral selector,N‐methacryloyl‐l‐histidine methyl ester

A new type of polymethacrylate‐based monolithic column with chiral stationary phase was prepared for the enantioseparation of aromatic amino acids, namely d ,l ‐phenylalanine, d ,l ‐tyrosine, and d ,l ‐tryptophan by CEC. The monolithic column was prepared by in situ polymerization of butyl methacrylate (BMA), N‐methacryloyl‐l ‐histidine methyl ester (MAH), and ethylene dimethacrylate (EDMA) in the presence of porogens. The porogen mixture included DMF and phosphate buffer. MAH was used as a chiral selector. FTIR spectrum of the polymethacrylate‐based monolith showed that MAH was incorporated into the polymeric structure via in situ polymerization. Some experimental parameters including pH, concentration of the mobile phase, and MAH concentration with regard to the chiral CEC separation were investigated. Single enantiomers and enantiomer mixtures of the amino acids were separately injected into the monolithic column. It was observed that l ‐enantiomers of aromatic amino acids migrated before d ‐enantiomers. The reversal enantiomer migration order for tryptophan was observed upon changing of pH. Using the chiral monolithic column (100 μm id and 375 μm od), the best chiral separation was performed in 35:65% ACN/phosphate buffer (pH 8.0, 10 mM) with an applied voltage of 12 kV in CEC. SEM images showed that the chiral monolithic column has a continuous polymeric skeleton and large through‐pore structure.     

Synthesis and Antimicrobial Activity of Some New N‐substituted‐5‐arylidene‐thiazolidine‐2,4‐diones

A total of 17 new N‐substituted derivatives ( 2b , 2c , 2d , 2e , 2f , 2g , 2h , 2i , 2j , 2k and 3b , 3c , 3d , 3e , 3f , 3g , 3h ) of 5‐((2‐phenylthiazol‐4‐yl)methylene) thiazolidine‐2,4‐dione ( 2a ) and 5‐(2,6‐dichloro‐ benzylidene)thiazolidine‐2,4‐dione ( 3a ) were synthesized. The structural elucidation of the newly synthesized compounds was based on elemental analysis and spectroscopic data (MS, ¹H NMR, ¹³C NMR), and their antimicrobial activities were assessed in vitro against several strains of Gram‐positive and Gram‐negative bacteria and one fungal strain (Candida albicans) as growth inhibition diameter. Some of them showed modest to good antibacterial activity against Gram‐negative Escherichia coli and Salmonella typhimurium and Gram‐positive Staphylococcus aureus, Bacillus cereus, and Enterococcus fecalis bacterial strains, whereas almost all the compounds were inactive against Listeria monocytogenes. All of the synthesized compounds showed moderate to very good activity against C. albicans.     

Biopolymer Skeleton Produced by Rhizobium radiobacter: Stoichiometric Alternation of Glycosidic and Amidic Bonds in the Lipopolysaccharide O‐Antigen

The lipopolysaccharide (LPS) O‐antigen structure of the plant pathogen Rhizobium radiobacter strain TT9 and its possible role in a plant‐microbe interaction was investigated. The analyses disclosed the presence of two O‐antigens, named Poly1 and Poly2. The repetitive unit of Poly2 constitutes a 4‐α‐l ‐rhamnose linked to a 3‐α‐d ‐fucose residue. Surprisingly, Poly1 turned out to be a novel type of biopolymer in which the repeating unit is formed by a monosaccharide and an amino‐acid derivative, so that the polymer has alternating glycosidic and amidic bonds joining the two units: 4‐amino‐4‐deoxy‐3‐O‐methyl‐d ‐fucose and (2′R,3′R,4′S)‐N‐methyl‐3′,4′‐dihydroxy‐3′‐methyl‐5′‐oxoproline). Differently from the O‐antigens of LPSs from other pathogenic Gram‐negative bacteria, these two O‐antigens do not activate the oxidative burst, an early innate immune response in the model plant Arabidopsis thaliana, explaining at least in part the ability of this R. radiobacter strain to avoid host defenses during a plant infection process.     

Analysis of the human breast milk microbiome and bacterial extracellular vesicles in healthy mothers

The microbiota of human breast milk (HBM) contribute to infant gut colonization; however, whether bacterial extracellular vesicles (EVs) are present in HBM or might contribute to this process remains unknown. In this study, we characterized the HBM microbiota of healthy Korean mothers and measured the key bacteria likely affecting infant gut colonization by analyzing both the microbiota and bacterial EVs. A total of 22 HBM samples were collected from lactating mothers. The DNA of bacteria and bacteria-derived EVs was extracted from each sample. In alpha-diversity analyses, bacterial samples showed higher richness and evenness than bacterial EV samples, and beta-diversity analyses showed significant differences between bacteria and bacterial EVs within identical individual samples. Firmicutes accounted for the largest proportion among the phyla, followed by Proteobacteria, Bacteroidetes, and Actinobacteria, in both bacteria and bacterial EV samples. At the genus level, Streptococcus (25.1%) and Staphylococcus (10.7%) were predominant in bacterial samples, whereas Bacteroides (9.1%), Acinetobacter (6.9%), and Lactobacillaceae(f) (5.5%) were prevalent in bacterial EV samples. Several genera, including Bifidobacterium, were significantly positively correlated between the two samples. This study revealed the diverse bacterial communities in the HBM of healthy lactating mothers, and found that gut-associated genera accounted for a high proportion in bacterial EV samples. Our findings suggest the existence of key bacteria with metabolic activity that are independent of the major bacterial populations that inhabit HBM, and the possibility that EVs derived from these bacteria are involved in the vertical transfer of gut microbiota.Subject terms: Immunogenetics, Immunization     

Enantioseparation of dansyl amino acids by ligand‐exchange capillary electrophoresis with zinc(II)‐L‐phenylalaninamide complex

A novel method of chiral ligand‐exchange CE was developed with L ‐amino acylamides as a chiral ligand and zinc(II) as a central ion. It has been demonstrated that these chiral complexes, such as Zn(II)‐L ‐alaninamide, Zn(II)‐L ‐prolinamide, and Zn(II)‐L ‐phenylalaninamide, are suitable for use as chiral selectors for the enantioseparation of either individual pair of or mixed dansyl amino acids. The optimal separation running buffer consisted of 5 mM ammonium acetate, 100 mM boric acid, 4 mM ZnSO₄·7 H₂O, and 8 mM L ‐amino acylamides at pH 8.2. The experiments showed that apart from the effect of the concentration of the complexes on the resolution and the migration time, the buffer pH also had a sharp influence on resolution. The employed chiral ligands exhibited different enantioselectivities and enantiomer migration orders. D ‐Amino acids migrate faster than L ‐amino acids when Zn(II)‐L ‐alaninamide and Zn(II)‐L ‐phenylalaninamide are used as chiral selectors, but it was observed that the migration order is reversed when Zn(II)‐L ‐prolinamide is used as the chiral selector. Furthermore, the amount of dansylated amino acids is found to be highly dependent on the labeling temperature.     

Gramicidin S Derivatives Containing cis‐ and trans‐Morpholine Amino Acids (MAAs) as Turn Mimetics

The cyclic decapeptide gramicidin S (GS) was used as a model for the evaluation of four turn mimetics. For this purpose, one of the D ‐Phe‐Pro two‐residue turn motifs in the rigid cyclic β‐hairpin structure of GS was replaced with morpholine amino acids (MAA 2 – 5 ), differing in stereochemistry and length of the side‐chain. The conformational properties of the thus obtained GS analogues ( 6 – 9 ) was assessed by using NMR spectroscopy and X‐ray crystallography, and correlated with their biological properties (antimicrobial and hemolytic activity). We show that compound 8 , containing the dipeptide isostere trans‐MAA 4 , has an apparent high structural resemblance with GS and that its antibacterial activity against a panel of Gram positive and ‐negative bacterial strains is better than the derivatives 6 , 7 and 9 .     

4‐Quinolone Derivatives and Their Activities Against Gram‐negative Pathogens

Gram‐negative pathogens represent a significant global health threat, while the emergency and widespread of drug resistance make the situation even worse. As “privileged building blocks,” 4‐quinolones including fluoroquinolones are mainstays of chemotherapy against various bacterial infections. However, as other antibiotics, the resistance of Gram‐negative bacteria to 4‐quinolones develops rapidly and spreads widely throughout the world. To overcome the resistance and improve the potency, a number of 4‐quinolone derivatives were designed, synthesized, and screened for their in vitro and in vivo activities against representative Gram‐negative pathogens. This review aims to summarize the recent advances made towards the discovery of 4‐quinolone derivatives as anti‐Gram‐negative agents as well as their structure–activity relationship. The enriched structure–activity relationship paves the way to the further rational development of 4‐quinolones with excellent potency against both drug‐susceptible and drug‐resistant Gram‐negative pathogens.     

Tetracyanoethene and 1‐Amino‐1,2,2‐ethenetricarbonitrile in the Synthesis of Heterocycles of Prospective Antioxidant and Antibacterial

Reaction of 1,1,2,2‐ethenetetracarbonitrile (TCNE) with aroyl thioureas in dioxane catalyzed by few drops of piperidine, led to the corresponding 1,2,4‐oxathiazoles. Under the same reaction condition, thiosemicarbazide or thiosemicarbazone reacted with either TCNE or 1‐amino‐1,2,2‐ethene‐tricarbonitrile. The structures of the products were elucidated via NMR, IR, mass spectra, and elemental analyses. The mechanism of formation was discussed. Biological (against Gram‐positive and Gram‐negative bacteria) and antioxidant activities were tested. Some of these heterocyclic compounds showed high activity as antioxidant and antibacterial compounds. The structure–activity relationship was investigated.     

Butelase‐Mediated Macrocyclization of d‐Amino‐Acid‐Containing Peptides

Macrocyclic compounds have received increasing attention in recent years. With their large surface area, they hold promise for inhibiting protein–protein interactions, a chemical space that was thought to be undruggable. Although many chemical methods have been developed for peptide macrocyclization, enzymatic methods have emerged as a promising new economical approach. Thus far, most enzymes have been shown to act on l ‐peptides; their ability to cyclize d ‐amino‐acid‐containing peptides has rarely been documented. Herein we show that macrocycles consisting of d ‐amino acids, except for the Asn residue at the ligating site, were efficiently synthesized by butelase 1, an Asn/Asp‐specific ligase. Furthermore, by using a peptide‐library approach, we show that butelase 1 tolerates most of the d ‐amino acid residues at the P1′′ and P2′′ positions.     

Synthesis,crystal structure,electronic spectroscopy,electrochemistry and biological studies of carbohydrate containing ferrocene amides

A series of four new ferrocene–carbohydrate amides was prepared from pentose and hexose sugar derivatives. These include (5‐amino‐5‐deoxy‐1,2‐O‐isopropylidene‐α‐d ‐xylofuranose)‐1‐ferrocene carboxamide (2a), (5‐amino‐3‐O‐benzyl‐5‐deoxy‐1,2‐O‐isopropylidene‐α‐d ‐xylofuranose)‐1‐ferrocene carboxamide (2b), (methyl‐6‐amino‐6‐deoxy‐2,3‐O‐isopropylidene‐β‐d ‐ribofuranoside)‐1‐ferrocene carboxamide (2c) derived from furanose sugars and (6‐amino‐6‐deoxy‐1,2:3,4‐di‐O‐isopropylidene‐α‐d ‐galactopyranose)‐1‐ferrocene carboxamide (2d) derived from pyranose sugar. The compounds were characterized by spectroscopic means and the structure of amide derived from α‐d ‐xylofuranose (2a) was determined by X‐ray crystallography. The electronic and optical properties of the compounds were studied by means of cyclic voltammetry and absorption spectroscopy. The UV and electrochemical studies of these compounds, performed in aqueous solutions under physiological conditions (at pH 7.4), confirmed their stability. These results indicated that the compounds were suitable for conducting biological studies. The CD spectral analysis displays the effect of sugar substituents on the compounds. The cytotoxicity and antimicrobial activity of these conjugates were investigated on different cancer cell lines and microbes respectively. The degree of inhibition varied over a broad spectrum of Gram‐ positive and Gram‐negative bacteria. In addition, the compounds also exhibited antioxidant properties. Copyright © 2012 John Wiley & Sons, Ltd.