Recent Advances in the Synthesis and Biological Applications of Peptidoglycan Fragments.

The biosynthesis, breakdown, and modification of peptidoglycan (PG) play vital roles in both bacterial viability and in the response of human physiology to bacterial infection. Studies on PG biochemistry are hampered by the fact that PG is an inhomogeneous insoluble macromolecule. Chemical synthesis is therefore an important means to obtain PG fragments that may serve as enzyme substrates and elicitors of the human immune response. This review outlines the recent advances in the synthesis and biochemical studies of PG fragments, PG biosynthetic intermediates (such as Park's nucleotides and PG lipids), and PG breakdown products (such as muramyl dipeptides and anhydro-muramic acid-containing fragments). A rich variety of synthetic approaches has been applied to preparing such compounds since carbohydrate, peptide, and phospholipid chemical methodologies must all be applied.

NMR and MD Analysis of the Bonding Interaction of Vancomycin with Muramyl Pentapeptide.

The article describes an NMR spectroscopy study of interactions between vancomycin and a muramyl pentapeptide in two complexes: vancomycin and a native muramyl pentapeptide ended with D-alanine (MPP-D-Ala), and vancomycin and a modified muramyl pentapeptide ended with D-serine (MPP-D-Ser). The measurements were made in a 9:1 mixture of H2O and D2O. The obtained results confirmed the presence of hydrogen bonds previously described in the literature. At the same time, thanks to the pentapeptide model used, we were able to prove the presence of two more hydrogen bonds formed by the side chain amino group of L-lysine and oxygen atoms from the vancomycin carboxyl and amide groups. This type of interaction has not been described before. The existence of these hydrogen bonds was confirmed by the 1H NMR and molecular modeling. The formation of these bonds incurs additional through-space interactions, visible in the NOESY spectrum, between the protons of the L-lysine amino group and a vancomycin-facing hydrogen atom in the benzylic position. The presence of such interactions was also confirmed by molecular dynamics trajectory analysis.

Selective Monovalent Galectin-8 Ligands Based on 3-Lactoylgalactoside.

Galectin-8 has gained attention as a potential new pharmacological target for the treatment of various diseases, including cancer, inflammation, and disorders associated with bone mass reduction. To that end, new molecular probes are needed in order to better understand its role and its functions. Herein we aimed to improve the affinity and target selectivity of a recently published galectin-8 ligand, 3-O-[1-carboxyethyl]-ß-d-galactopyranoside, by introducing modifications at positions 1 and 3 of the galactose. Affinity data measured by fluorescence polarization show that the most potent compound reached a KD of 12 µM. Furthermore, reasonable selectivity versus other galectins was achieved, making the highlighted compound a promising lead for the development of new selective and potent ligands for galectin-8 as molecular probes to examine the protein's role in cell-based and in vivo studies.

Peptidoglycan Deacetylases in Bacterial Cell Wall Remodeling and Pathogenesis.

The bacterial cell wall peptidoglycan (PG) is a dynamic structure that is constantly synthesized, re-modeled and degraded during bacterial division and growth. Postsynthetic modifications modulate the action of endogenous autolysis during PG lysis and remodeling for growth and sporulation, but also they are a mechanism used by pathogenic bacteria to evade the host innate immune system. Modifications of the glycan backbone are limited to the C-2 amine and C-6 hydroxyl moieties of either GlcNAc or MurNAc residues. This paper reviews the functional roles and properties of peptidoglycan de-Nacetylases (distinct PG GlcNAc and MurNAc deacetylases) and recent progress through genetic studies and biochemical characterization to elucidate their mechanism of action, 3D structures, substrate specificities and biological functions. Since they are virulence factors in pathogenic bacteria, peptidoglycan deacetylases are potential targets for the design of novel antimicrobial agents.

CryoEM structure of the antibacterial target PBP1b at 3.3 Å resolution.

The pathway for the biosynthesis of the bacterial cell wall is one of the most prolific antibiotic targets, exemplified by the widespread use of ß-lactam antibiotics. Despite this, our structural understanding of class A penicillin binding proteins, which perform the last two steps in this pathway, is incomplete due to the inherent difficulty in their crystallization and the complexity of their substrates. Here, we determine the near atomic resolution structure of the 83 kDa class A PBP from Escherichia coli, PBP1b, using cryogenic electron microscopy and a styrene maleic acid anhydride membrane mimetic. PBP1b, in its apo form, is seen to exhibit a distinct conformation in comparison to Moenomycin-bound crystal structures. The work herein paves the way for the use of cryoEM in structure-guided antibiotic development for this notoriously difficult to crystalize class of proteins and their complex substrates.

Utilization of different MurNAc sources by the oral pathogen Tannerella forsythia and role of the inner membrane transporter AmpG.

BACKGROUND: The Gram-negative oral pathogen Tannerella forsythia strictly depends on the external supply of the essential bacterial cell wall sugar N-acetylmuramic acid (MurNAc) for survival because of the lack of the common MurNAc biosynthesis enzymes MurA/MurB. The bacterium thrives in a polymicrobial biofilm consortium and, thus, it is plausible that it procures MurNAc from MurNAc-containing peptidoglycan (PGN) fragments (muropeptides) released from cohabiting bacteria during natural PGN turnover or cell death. There is indirect evidence that in T. forsythia, an AmpG-like permease (Tanf_08365) is involved in cytoplasmic muropeptide uptake. In E. coli, AmpG is specific for the import of N-acetylglucosamine (GlcNAc)-anhydroMurNAc(-peptides) which are common PGN turnover products, with the disaccharide portion as a minimal requirement. Currently, it is unclear which natural, complex MurNAc sources T. forsythia can utilize and which role AmpG plays therein. RESULTS: We performed a screen of various putative MurNAc sources for T. forsythia mimicking the situation in the natural habitat and compared bacterial growth and cell morphology of the wild-type and a mutant lacking AmpG (T. forsythia ΔampG). We showed that supernatants of the oral biofilm bacteria Porphyromonas gingivalis and Fusobacterium nucleatum, and of E. coli ΔampG, as well as isolated PGN and defined PGN fragments obtained after enzymatic digestion, namely GlcNAc-anhydroMurNAc(-peptides) and GlcNAc-MurNAc(-peptides), could sustain growth of T. forsythia wild-type, while T. forsythia ΔampG suffered from growth inhibition. In supernatants of T. forsythia ΔampG, the presence of GlcNAc-anhMurNAc and, unexpectedly, also GlcNAc-MurNAc was revealed by tandem mass spectrometry analysis, indicating that both disaccharides are substrates of AmpG. The importance of AmpG in the utilization of PGN fragments as MurNAc source was substantiated by a significant ampG upregulation in T. forsythia cells cultivated with PGN, as determined by quantitative real-time PCR. Further, our results indicate that PGN-degrading amidase, lytic transglycosylase and muramidase activities in a T. forsythia cell extract are involved in PGN scavenging. CONCLUSION: T. forsythia metabolizes intact PGN as well as muropeptides released from various bacteria and the bacterium's inner membrane transporter AmpG is essential for growth on these MurNAc sources, and, contrary to the situation in E. coli, imports both, GlcNAc-anhMurNAc and GlcNAc-MurNAc fragments.

Synthesis of a meso-Oxa-Diaminopimelic Acid Containing Peptidoglycan Pentapeptide and Coupling to the GlcNAc-anhydro-MurNAc Disaccharide.

The syntheses of peptidoglycan (PG)-derived peptides containing meso-diaminopimelic acid (meso-Dap) are typically quite lengthy due to the need to prepare orthogonally protected meso-Dap. In this work, the preparation of the PG pentapeptide containing the isosteric analog meso-oxa-Dap is described. The synthesis relies on the ring opening of a peptide embedded aziridine via the attack of a serine residue. The pentapeptide was attached to a GlcNAc-anhydro-MurNAc disaccharide, to produce a putative substrate for the AmpG pore protein.

Quantitation of amino sugar stereoisomer and muramic acid biomarkers by hydrophilic interaction liquid chromatography-mass spectrometry.

A rapid separation and quantitation of the stereoisomer amino sugars glucosamine, galactosamine, and mannosamine, along with muramic acid, is needed. These compounds, when their quantities are accurate, can be used to understand the origin and fate of natural organic matter (NOM) in the environment. These target molecules are biomarkers of fungi and bacteria and allow the deconvolution of microbial transformations and degradation of NOM in a wide variety of environmental matrices. Analytical methods applied to this suite of biomarkers are needed to understand carbon and nitrogen biogeochemistry with a changing global climate. Traditional separations of these analytes by gas chromatography require sample derivatization, as does reverse phase liquid chromatography. In contrast, ion chromatography can separate the analytes directly, but requires a separate analytical method to quantify muramic acid. In this work we present a direct analysis of all these molecules using hydrophilic liquid interaction chromatography. Solvent composition, buffer strength, pH, flow rate, and column temperature were optimized. The method can separate these four compounds and the biopolymeric precursor molecule N-acetylglucosamine in a single run in under 8 min with equivalent resolution to the best previously reported separations that did not require derivatization prior to analysis. Detection of the analytes was performed by both tandem and time-of-flight mass spectrometry. The method was assessed for its quantitative capabilities through i) peak area assignment, ii) check standards with ratios of the target analytes likely to be present in real samples, iii) an injection internal standard, and iv) quantitative analysis of real soil hydrolysates by external calibration and standard addition approaches. Across their expected analytical ranges the response for each analyte was highly linear with good accuracy (<25%) and precision (<15%) over three orders of magnitude. Detection limits of 20 µg L-1 were found for galactosamine and 5 µg L-1 for the remainder of the analytes, comparable to the majority of other methods reported in the literature. Overall, this new approach can directly and rapidly quantify amino sugars recovered in environmental hydrolysates.

Metabolic Incorporation of N-Acetyl Muramic Acid Probes into Bacterial Peptidoglycan.

Bacterial cells utilize small carbohydrate building blocks to construct peptidoglycan (PG), a highly conserved mesh-like polymer that serves as a protective coat for the cell. PG production has long been a target for antibiotics, and its breakdown is a source for human immune recognition. A key component of bacterial PG, N-acetyl muramic acid (NAM), is a vital element in many synthetically derived immunostimulatory compounds. However, the exact molecular details of these structures and how they are generated remain unknown due to a lack of chemical probes surrounding the NAM core. A robust synthetic strategy to generate bioorthogonally tagged NAM carbohydrate units is implemented. These molecules serve as precursors for PG biosynthesis and recycling. Escherichia coli cells are metabolically engineered to incorporate the bioorthogonal NAM probes into their PG network. The probes are subsequently modified using copper-catalyzed azide-alkyne cycloaddition to install fluorophores directly into the bacterial PG, as confirmed by super-resolution microscopy and high-resolution mass spectrometry. Here, synthetic notes for key elements of this process to generate the sugar probes as well as streamlined user-friendly metabolic labeling strategies for both microbiology and immunological applications are described. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Synthesis of peracetylated 2-azido glucosamine Basic Protocol 2: Synthesis of 2-azido and 2-alkyne NAM Basic Protocol 3: Synthesis of 3-azido NAM methyl ester Basic Protocol 4: Incorporation of NAM probes into bacterial peptidoglycan Basic Protocol 5: Confirmation of bacterial cell wall remodeling by mass spectrometry.

Structure-function relationships underlying the dual N-acetylmuramic and N-acetylglucosamine specificities of the bacterial peptidoglycan deacetylase PdaC.

Bacillus subtilis PdaC (BsPdaC) is a membrane-bound, multidomain peptidoglycan N-deacetylase acting on N-acetylmuramic acid (MurNAc) residues and conferring lysozyme resistance to modified cell wall peptidoglycans. BsPdaC contains a C-terminal family 4 carbohydrate esterase (CE4) catalytic domain, but unlike other MurNAc deacetylases, BsPdaC also has GlcNAc deacetylase activity on chitooligosaccharides (COSs), characteristic of chitin deacetylases. To uncover the molecular basis of this dual activity, here we determined the X-ray structure of the BsPdaC CE4 domain at 1.54 Å resolution and analyzed its mode of action on COS substrates. We found that the minimal substrate is GlcNAc3 and that activity increases with the degree of glycan polymerization. COS deacetylation kinetics revealed that BsPdaC operates by a multiple-chain mechanism starting at the internal GlcNAc units and leading to deacetylation of all but the reducing-end GlcNAc residues. Interestingly, BsPdaC shares higher sequence similarity with the peptidoglycan GlcNAc deacetylase SpPgdaA than with other MurNAc deacetylases. Therefore, we used ligand-docking simulations to analyze the dual GlcNAc- and MurNAc-binding specificities of BsPdaC and compared them with those of SpPgdA and BsPdaA, representing peptidoglycan deacetylases highly specific for GlcNAc or MurNAc residues, respectively. BsPdaC retains the conserved Asp-His-His metal-binding triad characteristic of CE4 enzymes acting on GlcNAc residues, differing from MurNAc deacetylases that lack the metal-coordinating Asp residue. BsPdaC contains short loops similar to those in SpPgdA, resulting in an open binding cleft that can accommodate polymeric substrates. We propose that PdaC is the first member of a new subclass of peptidoglycan MurNAc deacetylases.

A top-down chemo-enzymatic approach towards N-acetylglucosamine-N-acetylmuramic oligosaccharides: Chitosan as a reliable template.

An unprecedented approach towards oligosaccharides containing N-acetylglucosamine-N-acetylmuramic (NAG-NAM) units was developed. These novel bacterial cell wall surrogates were obtained from chitosan via a top down approach involving both chemical and enzymatic reactions. The chemical modification of chitosan using a molecular clamp based strategy, allowed obtaining N-acetylglucosamine-N-acetylmuramic (NAG-NAM) containing oligomers. Intercalation of NAM residues was confirmed through the analysis of oligosaccharide fragments from enzymatic digestion and it was found that this route affords NAG-NAM containing oligosaccharides in 33% yield. These oligosaccharides mimic the carbohydrate basic skeleton of most bacterial cell surfaces. The oligosaccharides prepared are biologically relevant and will serve as a platform for further molecular recognition studies with different receptors and enzymes of both bacterial cell wall and innate immune system. This strategy combining both chemical modification and enzymatic digestion provides a novel and simple route for an easy access to bacterial cell wall fragments - biologically important targets.

Bacteria's different ways to recycle their own cell wall.

The ability to recover components of their own cell wall is a common feature of bacteria. This was initially recognized in the Gram-negative bacterium Escherichia coli, which recycles about half of the peptidoglycan of its cell wall during one cell doubling. Moreover, E. coli was shown to grow on peptidoglycan components provided as nutrients. A distinguished recycling enzyme of E. coli required for both, recovery of the cell wall sugar N-acetylmuramic acid (MurNAc) of the own cell wall and for growth on external MurNAc, is the MurNAc 6-phosphate (MurNAc 6P) lactyl ether hydrolase MurQ. We revealed however, that most Gram-negative bacteria lack a murQ ortholog and instead harbor a pathway, absent in E. coli, that channels MurNAc directly to peptidoglycan biosynthesis. This "anabolic recycling pathway" bypasses the initial steps of peptidoglycan de novo synthesis, including the target of the antibiotic fosfomycin, thus providing intrinsic resistance to the antibiotic. The Gram-negative oral pathogen Tannerella forsythia is auxotrophic for MurNAc and apparently depends on the anabolic recycling pathway to synthesize its own cell wall by scavenging cell wall debris of other bacteria. In contrast, Gram-positive bacteria lack the anabolic recycling genes, but mostly contain one or two murQ orthologs. Quantification of MurNAc 6P accumulation in murQ mutant cells by mass spectrometry allowed us to demonstrate for the first time that Gram-positive bacteria do recycle their own peptidoglycan. This had been questioned earlier, since peptidoglycan turnover products accumulate in the spent media of Gram-positives. We showed, that these fragments are recovered during nutrient limitation, which prolongs starvation survival of Bacillus subtilis and Staphylococcus aureus. Peptidoglycan recycling in these bacteria however differs, as the cell wall is either cleaved exhaustively and monosaccharide building blocks are taken up (B. subtilis) or disaccharides are released and recycled involving a novel phosphomuramidase (MupG; S.aureus). In B. subtilis also the teichoic acids, covalently bound to the peptidoglycan (wall teichoic acids; WTAs), are recycled. During phosphate limitation, the sn-glycerol-3-phosphate phosphodiesterase GlpQ specifically degrades WTAs of B. subtilis. In S. aureus, in contrast, GlpQ is used to scavenge external teichoic acid sources. Thus, although bacteria generally recover their own cell wall, they apparently apply distinct strategies for breakdown and reutilization of cell wall fragments. This review summarizes our work on this topic funded between 2011 and 2019 by the DFG within the collaborative research center SFB766.

Actinomadura rhizosphaerae sp. nov., isolated from rhizosphere soil of the plant Azadirachta indica.

A novel actinomycete, strain SDA37T, belonging to the genus Actinomadura, was isolated from rhizosphere soil collected from Udon Thani Province, Thailand. The taxonomic position of the strain was characterized using a polyphasic approach. Meso-diaminopimelic acid, glucose, ribose, galactose and madurose were detected in cell-wall and whole-cell hydrolysates. The N-acyl type of muramic acid was acetyl. Menaquinones were MK-9(H6), MK-9(H8) and MK-9(H4). The predominant cellular fatty acids were iso-C16 : 0, C16 : 0, 10-methyl C18 : 0 and iso-C14 : 0. The major polar lipids were phosphatidylglycerol, diphosphatidylglycerol and phosphatidylinositol. blast analysis of the almost-complete 16S rRNA gene sequence showed 98.8 % similarity to Actinomadura oligospora NBRC 104149T, 98.7 % similarity to Actinomadura gamaensis DSM 100815T and 97.2 % similarity to Actinomadura rupiterrae KCTC 19559T. The DNA G+C content was 73.1 mol%. Strain SDA37T showed low DNA-DNA relatedness (44.3±7.3 to 58.5±8.7 %) to A. oligospora NBRC 104149T, Actinomadura gamaensis DSM 100815T and Actinomadura rupiterrae KCTC 19559T. The new strain could also be distinguished from its closely related strains by the differences in the phenotypic characteristics. The results of taxonomic analysis suggested that strain SDA37T represented a novel species of the genus Actinomadura for which the name Actinomadura rhizosphaerae sp. nov. is proposed. The type strain is SDA37T (=KCTC 39965T=NBRC 112909T=TISTR 2523T).

Resuscitation-Promoting Factors Are Required for Mycobacterium smegmatis Biofilm Formation.

Resuscitation-promoting factors (Rpfs) have previously been shown to act as growth-stimulatory molecules via their lysozyme-like activity on peptidoglycan in the bacterial cell wall. In this study, we investigated the ability of Mycobacterium smegmatis strains lacking rpf genes to form biofilms and tested their susceptibilities to cell wall-targeting agents. M. smegmatis contains four distinct rpf homologues, namely, MSMEG_5700 (rpfA), MSMEG_5439 (rpfB), MSMEG_4640 (rpfE2), and MSMEG_4643 (rpfE). During axenic growth of the wild-type strain, all four mRNA transcripts were expressed to various degrees, but the expression of MSMEG_4643 was significantly greater during exponential growth. Similarly, all rpf mRNA transcripts could be detected in biofilms grown for 7, 14, and 28 days, with MSMEG_4643 expressed at the highest abundance after 7 days. In-frame unmarked deletion mutants (single and combinatorial) were generated and displayed altered colony morphologies and the inability to form typical biofilms. Moreover, any strain lacking rpfA and rpfB simultaneously exhibited increased susceptibility to rifampin, vancomycin, and SDS. Exogenous Rpf supplementation in the form of culture filtrate failed to restore biofilm formation. Liquid chromatography-mass spectrometry (LC-MS) analysis of peptidoglycan (PG) suggested a reduction in 4-3 cross-linked PG in the ΔrpfABEE2 mutant strain. In addition, the level of PG-repeat units terminating in 1,6-anhydroMurNAc appeared to be significantly reduced in the quadruple rpf mutant. Collectively, our data have shown that Rpfs play an important role in biofilm formation, possibly through alterations in PG cross-linking and the production of signaling molecules.IMPORTANCE The cell wall of pathogenic mycobacteria is composed of peptidoglycan, arabinogalactan, mycolic acids, and an outer capsule. This inherent complexity renders it resistant to many antibiotics. Consequently, its biosynthesis and remodeling during growth directly impact viability. Resuscitation-promoting factors (Rpfs), enzymes with lytic transglycosylase activity, have been associated with the revival of dormant cells and subsequent resumption of vegetative growth. Mycobacterium smegmatis, a soil saprophyte and close relative of the human pathogen Mycobacterium tuberculosis, encodes four distinct Rpfs. Herein, we assessed the relationship between Rpfs and biofilm formation, which is used as a model to study drug tolerance and bacterial signaling in mycobacteria. We demonstrated that progressive deletion of rpf genes hampered the development of biofilms and reduced drug tolerance. These effects were accompanied by a reduction in muropeptide production and altered peptidoglycan cross-linking. Collectively, these observations point to an important role for Rpfs in mycobacterial communication and drug tolerance.

Actinomadura barringtoniae sp. nov., an endophytic actinomycete isolated from the roots of Barringtonia acutangula (L.) Gaertn.

A novel actinomycete strain, designated GKU 128T, isolated from the roots of an Indian oak tree [Barringtonia acutangula (L.) Gaertn.] at Khao Khitchakut district, Chantaburi province, Thailand, was characterized by using a polyphasic approach. The strain formed a branched substrate and aerial mycelia which differentiated into straight to flexuous chains of smooth-ornamented spores. Analysis of the cell wall revealed the presence of meso-diaminopimelic acid and N-acetylmuramic acid in the peptidoglycan. The whole-cell sugars were glucose, madurose, mannose, rhamnose and ribose. Mycolic acids were absent. The major phospholipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and phosphatidylinositolmannoside. The predominant menaquinones were MK-9(H6), MK-9(H8), MK-9(H0) and MK-9(H4). The major fatty acids were C16 : 0, C18 : 1ω9c and 10-methyl C18 : 0 (tuberculostearic acid). The genomic DNA G+C content was 70.5 mol%. Based on 16S rRNA gene sequence analysis, strain GKU 128T was closely related to the type strains of Actinomadura nitritigenes NBRC 15918T (99.2 % sequence similarity) and Actinomadura fibrosa JCM 9371T (98.7 %). The levels of DNA-DNA relatedness between strain GKU 128T and the closely related type species were less than 19 %. On the basis of phenotypic and genotypic characteristics, strain GKU 128T could be distinguished from its closely related type strains and represents a novel species of the genus Actinomadura, for which the name Actinomadura barringtoniae sp. nov. (=TBRC 7225T=NBRC 113074T) is proposed.

A step-by-step in crystallo guide to bond cleavage and 1,6-anhydro-sugar product synthesis by a peptidoglycan-degrading lytic transglycosylase.

Lytic transglycosylases (LTs) are a class of enzymes important for the recycling and metabolism of peptidoglycan (PG). LTs cleave the ß-1,4-glycosidic bond between N-acetylmuramic acid (MurNAc) and GlcNAc in the PG glycan strand, resulting in the concomitant formation of 1,6-anhydro-N-acetylmuramic acid and GlcNAc. No LTs reported to date have utilized chitins as substrates, despite the fact that chitins are GlcNAc polymers linked via ß-1,4-glycosidic bonds, which are the known site of chemical activity for LTs. Here, we demonstrate enzymatically that LtgA, a non-canonical, substrate-permissive LT from Neisseria meningitidis utilizes chitopentaose ((GlcNAc)5) as a substrate to produce three newly identified sugars: 1,6-anhydro-chitobiose, 1,6-anhydro-chitotriose, and 1,6-anhydro-chitotetraose. Although LTs have been widely studied, their complex reactions have not previously been visualized in the crystalline state because macromolecular PG is insoluble. Here, we visualized the cleavage of the glycosidic bond and the liberation of GlcNAc-derived residues by LtgA, followed by the synthesis of atypical 1,6-anhydro-GlcNAc derivatives. In addition to the newly identified anhydro-chitin products, we identified trapped intermediates, unpredicted substrate rearrangements, sugar distortions, and a conserved crystallographic water molecule bound to the catalytic glutamate of a high-resolution native LT. This study enabled us to propose a revised alternative mechanism for LtgA that could also be applicable to other LTs. Our work contributes to the understanding of the mechanisms of LTs in bacterial cell wall biology.

Metabolic labelling of the carbohydrate core in bacterial peptidoglycan and its applications.

Bacterial cells are surrounded by a polymer known as peptidoglycan (PG), which protects the cell from changes in osmotic pressure and small molecule insults. A component of this material, N-acetyl-muramic acid (NAM), serves as a core structural element for innate immune recognition of PG fragments. We report the synthesis of modifiable NAM carbohydrate derivatives and the installation of these building blocks into the backbone of Gram-positive and Gram-negative bacterial PG utilizing metabolic cell wall recycling and biosynthetic machineries. Whole cells are labelled via click chemistry and visualized using super-resolution microscopy, revealing higher resolution PG structural details and allowing the cell wall biosynthesis, as well as its destruction in immune cells, to be tracked. This study will assist in the future identification of mechanisms that the immune system uses to recognize bacteria, glean information about fundamental cell wall architecture and aid in the design of novel antibiotics.

Synthesis of novel muramic acid derivatives and their interaction with lysozyme: Action of lysozyme revisited.

HYPOTHESIS: The interaction of lysozyme with the N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) unit of peptidoglycan (PGN) polymer of the bacterial cell wall is of immense importance to understand the mechanism of lysozyme on PGN. EXPERIMENTS: The synthesis of three novel NAM derivatives containing fused oxazinone ring to the NAM moiety has been achieved. The synthesized compounds were evaluated for their potential as a glycomimetic acceptor of lysozyme using different biophysical and computational methods such as 1H NMR, STD NMR, DOSY and Molecular docking. FINDINGS: Novel modified muramic acid derivatives have been synthesized in excellent yield containing fused cyclooxazine ring embedded on the muramic acid moiety using a newly developed hydrazinolysis reaction condition. From various biophysical studies, it has been established that the compound containing endo modified muramic acid moiety (compound 1) shows significant binding property for the lysozyme while the other isomer (compound 2) did not bind to the lysozyme. The catalytic residues Glu35 and Asp52 were found to be in the close proximity for the active molecule which justifies the selectivity of this molecule in conjunction to lysozyme enzymatic activity.

Actinocrispum wychmicini gen. nov., sp. nov., a novel member of the family Pseudonocardiaceae, isolated from soil.

A novel actinomycete, designated MI503-A4T, was isolated from soil. Comparative analysis of 16S rRNA gene sequences indicated that MI503-A4T was phylogenetically related to members of the family Pseudonocardiaceae. The most closely related genus was Kibdelosporangium (95.7-96.2 % sequence similarity). Substrate mycelia were branched and pale yellow to pale yellowish-brown. Straight- to zigzag-shaped aerial mycelia were observed, but Sporangium-like structures were absent. The whole-cell hydrolysate contained meso-diaminopimelic acid. The muramic acid residues in the peptidoglycan were N-acetylated. Whole-cell sugars were rhamnose, ribose, arabinose and galactose (cell wall chemotype IV). The predominant menaquinone was MK-9(H4). A small amount of MK-8(H4) was also detected. The DNA G+C content was 70.3-71.1 mol%. Polar lipids contained diphosphatidylglycerol, phosphatidylethanolamine and hydroxyl-phosphatidylethanolamine. Cellular fatty acid analysis of MI503-A4T detected predominantly iso-C14 : 0 (11.5 %), iso-C15 : 0 (13.3 %) and iso-C16 : 0 (35.7 %). Phenotypic and phylogenetic characteristics differentiated MI503-A4T from members of all genera within the family Pseudonocardiaceae with validly published names. Therefore, MI503-A4T is proposed to be a representative of a novel species in a novel genus, Actinocrispum wychmicini gen. nov., sp. nov. The type strain of the type species is MI503-A4T (=NBRC 109632T=DSM 45934T).

Role of the N-Acetylmuramoyl-l-Alanyl Amidase, AmiA, of Helicobacter pylori in Peptidoglycan Metabolism, Daughter Cell Separation, and Virulence.

The human gastric pathogen, Helicobacter pylori, is becoming increasingly resistant to most available antibiotics. Peptidoglycan (PG) metabolism is essential to eubacteria, hence, an excellent target for the development of new therapeutic strategies. However, our knowledge on PG metabolism in H. pylori remains poor. We have further characterized an isogenic mutant of the amiA gene encoding a N-acetylmuramoyl-l-alanyl amidase. The amiA mutant displayed long chains of unseparated cells, an impaired motility despite the presence of intact flagella and a tolerance to amoxicillin. Interestingly, the amiA mutant was impaired in colonizing the mouse stomach suggesting that AmiA is a valid target in H. pylori for the development of new antibiotics. Using reverse phase high-pressure liquid chromatography, we analyzed the PG muropeptide composition and glycan chain length distribution of strain 26695 and its amiA mutant. The analysis showed that H. pylori lacked muropeptides with a degree of cross-linking higher than dimeric muropeptides. The amiA mutant was also characterized by a decrease of muropeptides carrying 1,6-anhydro-N-acetylmuramic acid residues, which represent the ends of the glycan chains. This correlated with an increase of very long glycan strands in the amiA mutant. It is suggested that these longer glycan strands are trademarks of the division site. Taken together, we show that the low redundancy on genes involved in PG maturation supports H. pylori as an actractive alternative model to study PG metabolism and cell shape regulation.