Inhibition of Streptococcus pneumoniae growth by masarimycin.

Despite renewed interest, development of chemical biology methods to study peptidoglycan metabolism has lagged in comparison to the glycobiology field in general. To address this, a panel of diamides were screened against the Gram-positive bacterium Streptococcus pneumoniae to identify inhibitors of bacterial growth. The screen identified the diamide masarimycin as a bacteriostatic inhibitor of S. pneumoniae growth with an MIC of 8 µM. The diamide inhibited detergent-induced autolysis in a concentration-dependent manner, indicating perturbation of peptidoglycan degradation as the mode-of-action. Cell based screening of masarimycin against a panel of autolysin mutants, identified a higher MIC against a ΔlytB strain lacking an endo-N-acetylglucosaminidase involved in cell division. Subsequent biochemical and phenotypic analyses suggested that the higher MIC was due to an indirect interaction with LytB. Further analysis of changes to the cell surface in masarimycin treated cells identified the overexpression of several moonlighting proteins, including elongation factor Tu which is implicated in regulating cell shape. Checkerboard assays using masarimycin in concert with additional antibiotics identified an antagonistic relationship with the cell wall targeting antibiotic fosfomycin, which further supports a cell wall mode-of-action.

Synthesis of Masarimycin, a Small Molecule Inhibitor of Gram-Positive Bacterial Growth.

Peptidoglycan (PG) in the cell wall of bacteria is a unique macromolecular structure that confers shape, and protection from the surrounding environment. Central to understanding cell growth and division is the knowledge of how PG degradation influences biosynthesis and cell wall assembly. Recently, the metabolic labeling of PG through the introduction of modified sugars or amino acids has been reported. While chemical interrogation of biosynthetic steps with small molecule inhibitors is possible, chemical biology tools to study PG degradation by autolysins are underdeveloped. Bacterial autolysins are a broad class of enzymes that are involved in the tightly coordinated degradation of PG. Here, a detailed protocol is presented for preparing a small molecule probe, masarimycin, which is an inhibitor of N-acetylglucosaminidase LytG in Bacillus subtilis, and cell wall metabolism in Streptococcus pneumoniae. Preparation of the inhibitor via microwave-assisted and classical organic synthesis is provided. Its applicability as a tool to study Gram-positive physiology in biological assays is presented.

Trophic upgrading and mobilization of wax esters in microzooplankton.

Heterotrophic protists play pivotal roles in aquatic ecosystems by transferring matter and energy, including lipids, from primary producers to higher trophic predators. Using Oxyrrhis marina as a model organism, changes to the non-saponifiable protist lipids were investigated under satiation and starvation conditions. During active feeding on the alga Cryptomonas sp., the O. marina hexane soluble non-saponifiable fraction lipid profile reflected its food source with the observed presence of long chain mono-unsaturated fatty alcohols up to C25:1. Evidence of trophic upgrading in O. marina was observed with long chain mono-unsaturated fatty alcohol accumulation of up to C35:1. To the best of our knowledge, this is the first evidence that heterotrophic dinoflagellates are capable of producing ester derived alcohols and that dinoflagellates like O. marina are capable of synthesizing fatty alcohols up to C35. Additionally, we show evidence of trophic upgrading of lipids. During a 20-day resource deprivation, the lipid profile remained constant. During starvation, the mobilization of wax esters as energy stores was observed with long chain fatty alcohols mobilized first. Changes in lipid class profile and utilization of wax esters in O. marina provides insight into the types of lipids available for energy demand, the transfer of lipids through the base of marine food webs, and the catabolic response induced by resource deprivation.

RNA-Seq reveals a central role for lectin, C1q and von Willebrand factor A domains in the defensive glue of a terrestrial slug.

The tough, hydrogel glue produced by the slug Arion subfuscus achieves impressive performance through metal-based, protein cross-links. The primary sequence of these proteins was determined through transcriptome sequencing and proteome analysis by tandem mass spectrometry. The main proteins that correlate with adhesive function are a group of 11 small, highly abundant lectin-like proteins. These proteins matched the ligand-binding C-lectin, C1q or H-lectin domains. The variety of different lectin-like proteins and their potential for oligomerization suggests that they function as versatile and potent cross-linkers. In addition, the glue contains five matrilin-like proteins that are rich in von Willebrand factor A (VWA) and EGF domains. Both C-lectins and VWA domains are known to bind to ligands using divalent cations. These findings are consistent with the double network mechanism proposed for slug glue, with divalent ions serving as sacrificial bonds to dissipate energy.

Diamide Inhibitors of the Bacillus subtilis N-Acetylglucosaminidase LytG That Exhibit Antibacterial Activity.

N-Acetylglucosaminidases (GlcNAcases) play an important role in the remodeling and recycling of bacterial peptidoglycan by degrading the polysaccharide backbone. Genetic deletions of autolysins can impair cell division and growth, suggesting an opportunity for using small molecule autolysin inhibitors both as tools for studying the chemical biology of autolysins and also as antibacterial agents. We report here the synthesis and evaluation of a panel of diamides that inhibit the growth of Bacillus subtilis. Two compounds, fgkc (21) and fgka (5), were found to be potent inhibitors (MIC 3.8 ± 1.0 and 21.3 ± 0.1 µM, respectively). These compounds inhibit the B. subtilis family 73 glycosyl hydrolase LytG, an exo GlcNAcase. Phenotypic analysis of fgkc (21)-treated cells demonstrates a propensity for cells to form linked chains, suggesting impaired cell growth and division.

Small-molecule inhibitors of the pseudaminic acid biosynthetic pathway: targeting motility as a key bacterial virulence factor.

Helicobacter pylori is motile by means of polar flagella, and this motility has been shown to play a critical role in pathogenicity. The major structural flagellin proteins have been shown to be glycosylated with the nonulosonate sugar, pseudaminic acid (Pse). This glycan is unique to microorganisms, and the process of flagellin glycosylation is required for H. pylori flagellar assembly and consequent motility. As such, the Pse biosynthetic pathway offers considerable potential as an antivirulence drug target, especially since motility is required for H. pylori colonization and persistence in the host. This report describes screening the five Pse biosynthetic enzymes for small-molecule inhibitors using both high-throughput screening (HTS) and in silico (virtual screening [VS]) approaches. Using a 100,000-compound library, 1,773 hits that exhibited a 40% threshold inhibition at a 10 µM concentration were identified by HTS. In addition, VS efforts using a 1.6-million compound library directed at two pathway enzymes identified 80 hits, 4 of which exhibited reasonable inhibition at a 10 µM concentration in vitro. Further secondary screening which identified 320 unique molecular structures or validated hits was performed. Following kinetic studies and structure-activity relationship (SAR) analysis of selected inhibitors from our refined list of 320 compounds, we demonstrated that three inhibitors with 50% inhibitory concentrations (IC50s) of approximately 14 µM, which belonged to a distinct chemical cluster, were able to penetrate the Gram-negative cell membrane and prevent formation of flagella.

The effects of fungal volatile organic compounds on bone marrow stromal cells.

Evidence has shown that individuals exposed to indoor toxic molds for extended periods of time have elevated risk of developing numerous respiratory illnesses. It is not clear at the cellular level what impact mold exposure has on the immune system. Herein, we show that 2 fungal volatiles (E)-2-octenal and oct-1-en-3-ol have cytotoxic effects on murine bone marrow stromal cells. To further analyze alterations to the cell, we evaluated the impact these volatile organic compounds have on membrane composition and hence fluidity. Both (E)-2-octenal and oct-1-en-3-ol exposure caused a shift to unsaturated fatty acids and lower cholesterol levels in the membrane. This indicates that the volatile organic compounds under investigation increased membrane fluidity. These vast changes to the cell membrane are known to contribute to the breakdown of normal cell function and possibly lead to death. Since bone marrow stromal cells are vital for the appropriate development and activation of immune cells, this study provides the foundation for understanding the mechanism at a cellular level for how mold exposure can lead to immune-related disease conditions.

Functional analysis of SleC from Clostridium difficile: an essential lytic transglycosylase involved in spore germination.

Clostridium difficile is the most common cause of enteric disease and presents a major burden on healthcare systems globally due in part to the observed rapid rise in antibiotic resistance. The ability of C. difficile to form endospores is a key feature in the organism's pathogenesis and transmission, and contributes greatly to its resilient nature. Endospores are highly resistant to disinfection, allowing them to persist on hospital surfaces. In order for the organism to cause disease, the spores must germinate and revert to a vegetative form. While spore germination in Bacillus spp. is well understood, very little is known about this process in Clostridia. Here we report the characterization of SleC (CD0551) from C. difficile 630. Bioinformatic analysis of SleC indicated a multi-domained protein possessing a peptidoglycan-binding (PGB) domain, a SpoIID/LytB domain and an undefined N-terminal region. We have confirmed that SleC is an exo-acting lytic transglycosylase with the catalytic activity localized to the N-terminal region. Additionally, we have shown that both the N-terminal catalytic domain and the C-terminal PGB domain require muramyl-δ-lactam for substrate binding. As with carbohydrate-binding modules from cellulases and xylanases, the PGB domain may be responsible for increasing the processivity of SleC by concentrating the enzyme at the surface of the substrate.

Structural characterization of surface glycans from Clostridium difficile.

Whole-cell high-resolution magic angle spinning (HR-MAS) NMR was employed to survey the surface polysaccharides of a group of clinical and environmental isolates of Clostridium difficile. Results indicated that a highly conserved surface polysaccharide profile among all strains studied. Multiple additional peaks in the anomeric region were also observed which prompted further investigation. Structural characterization of the isolated surface polysaccharides from two strains confirmed the presence of the conserved water soluble polysaccharide originally described by Ganeshapillai et al. which was composed of a hexaglycosyl phosphate repeat consisting of [→6)-ß-D-Glcp-(1-3)-ß-D-GalpNAc-(1-4)-α-D-Glcp-(1-4)-[ß-D-Glcp(1-3]-ß-D-GalpNAc-(1-3)-α-D-Manp-(1-P→]. In addition, analysis of phenol soluble polysaccharides revealed a similarly conserved lipoteichoic acid (LTA) which could be detected on whole cells by HR-MAS NMR. Conventional NMR and mass spectrometry analysis indicated that the structure of this LTA consisted of the repeat unit [→6)-α-D-GlcpNAc-(1-3)-[→P-6]-α-D-GlcpNAc-(1-2)-D-GroA] where GroA is glyceric acid. The repeating units were linked by a phosphodiester bridge between C-6 of the two GlcNAc residues (6-P-6). A minor component consisted of GlcpN-(1-3) instead of GlcpNAc-(1-3) in the repeat unit. Through a 6-6 phosphodiester bridge this polymer was linked to →6)-ß-D-Glcp-(1-6)-ß-D-Glcp-(1-6)-ß-D-Glcp-(1-1)-Gro, with glycerol (Gro) substituted by fatty acids. This is the first report of the utility of HR-MAS NMR in the examination of surface carbohydrates of Gram positive bacteria and identification of a novel LTA structure from Clostridium difficile.

Never take candy from a stranger: the role of the bacterial glycome in host-pathogen interactions.

With the comprehensive study and complete sequencing of the Haemophilus influenzae genome in 1995 came the term 'genomics' and the beginning of the 'omics' era. Since this time, several analogous fields, such as transcriptomics and proteomics, have emerged. While growth and advancement in these fields have increased understanding of microbial virulence, the study of bacterial glycomes is still in its infancy and little is known concerning their role in host-pathogen interactions. Bacterial glycomics is challenging owing to the diversity of glyco-conjugate molecules, vast array of unusual sugars and limited number of analytical approaches available. However, recent advances in glycomics technologies offer the potential for exploration and characterization of both the structures and functions of components of bacterial glycomes in a systematic manner. Such characterization is a prerequisite for discerning the role of bacterial glycans in the interaction between host defences and bacterial virulence factors.

Characterization of lipid-linked oligosaccharides by mass spectrometry.

N- Glycosylation of proteins is recognized as one of the most common post-translational modifications. Until recently it was believed that N-glycosylation occurred exclusively in eukaryotes until the discovery of the general protein glycosylation pathway (Pgl) in Campylobacter jejuni. We have developed a new glycomics strategy based on lectin-affinity capture of lipid-linked oligosaccharides (LLOs) coupled to capillary electrophoresis mass spectrometry. The LLO intermediates of the C. jejuni Pgl pathway were used to validate the methodology and to better characterize the bacterial model system for protein N-glycosylation. This method provides a rapid, non-radioactive approach for the characterization of intermediates in polysaccharide biosynthesis and is a useful tool for glycoengineering efforts in bacteria.

Analysis of bacterial lipid-linked oligosaccharide intermediates using porous graphitic carbon liquid chromatography-electrospray ionization mass spectrometry: heterogeneity in the polyisoprenyl carrier revealed.

N-glycosylation of proteins is recognized as one of the most common post-translational modifications. It was believed that N-glycosylation occurred exclusively in eukaryotes until the recent discovery of the general protein glycosylation pathway (Pgl) in Campylobacter jejuni, which has similarities to the eukaryotic system and adds proteins en bloc from a lipid carrier to a protein acceptor. In addition to N-linked glycans, a number of pathogenic bacteria such as Pseudomonas aeruginosa and Neisseria species have been shown to O-glycosylate their proteins through polyisoprene-linked intermediates. To date, most techniques to analyze lipid-linked oligosaccharides (LLOs) of these pathways involve the use of radiolabels and chromatographic separation. With the increasing frequency of reports of bacterial protein glycosylation that proceed through lipid-mediated steps, there is a need for technologies capable of characterizing these newly described bacterial systems as well as eukaryotic pathways from biologically relevant samples in an accurate, rapid, and cost-effective manner. In this paper, a new glycomics strategy based on porous graphite carbon (PGC) liquid chromatography mass spectrometry (LC-MS) was devised and validated on the C. jejuni N-glycan pathway. Lipid-linked oligosaccharide intermediates of the Pgl pathway from crude lipid extracts were separated using online chromatography on a capillary PGC column with a chloroform gradient. By exploiting the retention properties of hydrophobic and polar analytes on PGC, baseline separation of LLOs with minor changes in oligosaccharide structure and polyisoprene chain length was obtained. This method is capable of analyzing low levels of LLOs (from approximately 10(6) bacterial cells) and distinguishing the LLOs that differ by as little as one monosaccharide or polyisoprene unit. Furthermore, we have demonstrated for the first time that oligosaccharides of the C. jejuni Pgl pathway are assembled on different polyisoprenes, e. g. C(45), C(60), and apparent hydroxylated forms, in addition to those previously reported (i.e., C(50) and C(55)). The hydroxylated forms of the LLOs are believed to be an intermediate in the degradation of accumulated LLOs for polyisoprene carrier recycling.

Motility and flagellar glycosylation in Clostridium difficile.

In this study, intact flagellin proteins were purified from strains of Clostridium difficile and analyzed using quadrupole time of flight and linear ion trap mass spectrometers. Top-down studies showed the flagellin proteins to have a mass greater than that predicted from the corresponding gene sequence. These top-down studies revealed marker ions characteristic of glycan modifications. Additionally, diversity in the observed masses of glycan modifications was seen between strains. Electron transfer dissociation mass spectrometry was used to demonstrate that the glycan was attached to the flagellin protein backbone in O linkage via a HexNAc residue in all strains examined. Bioinformatic analysis of C. difficile genomes revealed diversity with respect to glycan biosynthesis gene content within the flagellar biosynthesis locus, likely reflected by the observed flagellar glycan diversity. In C. difficile strain 630, insertional inactivation of a glycosyltransferase gene (CD0240) present in all sequenced genomes resulted in an inability to produce flagellar filaments at the cell surface and only minor amounts of unmodified flagellin protein.

Affinity-capture tandem mass spectrometric characterization of polyprenyl-linked oligosaccharides: tool to study protein N-glycosylation pathways.

N-glycosylation of proteins is recognized as one of the most common post-translational modifications. Until recently it was believed that N-glycosylation occurred exclusively in eukaryotes before the discovery of the general protein glycosylation pathway (Pgl) in Campylobacter jejuni. To date, most techniques to analyze lipid-linked oligosaccharides (LLOs) of these pathways involve the use of radiolabels and chromatographic separation. Technologies capable of characterizing eukaryotic and the newly described bacterial N-glycosylation systems from biologically relevant samples in a quick, accurate, and cost-effective manner are needed. In this paper a new glycomics strategy based on lectin-affinity capture was devised and validated on the C. jejuni N-glycan pathway and the engineered Escherichia coli strains expressing the functional C. jejuni pathway. The lipid-linked oligosaccharide intermediates of the Pgl pathway were then enriched using SBA-agarose affinity-capture and examined by capillary electrophoresis-mass spectrometry (CE-MS). We demonstrate that this method is capable of detecting low levels of LLOs, the sugars are indeed assembled on undecaprenylpyrophosphate, and structural information for expected and unexpected LLOs can be obtained without further sample manipulation. Furthermore, CE-MS analyses of C. jejuni and the E. coli "glyco-factories" showed striking differences in the assembly and control of N-glycan biosynthesis.

Polyisoprenol specificity in the Campylobacter jejuni N-linked glycosylation pathway.

Campylobacter jejuni contains a general N-linked glycosylation pathway in which a heptasaccharide is sequentially assembled onto a polyisoprenyl diphosphate carrier and subsequently transferred to the asparagine side chain of an acceptor protein. The enzymes in the pathway function at a membrane interface and have in common amphiphilic membrane-bound polyisoprenyl-linked substrates. Herein, we examine the potential role of the polyisoprene component of the substrates by investigating the relative substrate efficiencies of polyisoprene-modified analogues in individual steps of the pathway. Chemically defined substrates for PglC, PglJ, and PglB are prepared via semisynthetic approaches. The substrates included polyisoprenols of varying length, double bond geometry, and degree of saturation for probing the role of the hydrophobic polyisoprene in substrate specificity. Kinetic analysis reveals that all three enzymes exhibit distinct preferences for the polyisoprenyl carrier whereby cis-double bond geometry and alpha-unsaturation of the native substrate are important features, while the precise polyisoprene length may be less critical. These findings suggest that the polyisoprenyl carrier plays a specific role in the function of these enzymes beyond a purely physical role as a membrane anchor. These studies underscore the potential of the C. jejuni N-linked glycosylation pathway as a system for investigating the biochemical and biophysical roles of polyisoprenyl carriers common to prokaryotic and eukaryotic glycosylation.

Role of Ser216 in the mechanism of action of membrane-bound lytic transglycosylase B: further evidence for substrate-assisted catalysis.

Lytic transglycosylases cleave the beta-(1-->4)-glycosidic bond in the bacterial cell wall heteropolymer peptidoglycan between the N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) residues with the concomitant formation of a 1,6-anhydromuramoyl residue. Based on sequence alignments, Ser216 in Pseudomonas aeruginosa membrane-bound lytic transglycosylase B (MltB) was targeted for replacement with alanine to delineate its role in the enzyme's mechanism of action. The specific activity of the Ser216-->Ala MltB derivative was less than 12% of that for the wild-type enzyme, while its substrate binding affinity remained virtually unaltered. These data are in agreement with a role of Ser216 in orienting the N-acetyl group on MurNAc at the -1 subsite of MltB for its participation in a substrate-assisted mechanism of action.

Role of arginine residues in the active site of the membrane-bound lytic transglycosylase B from Pseudomonas aeruginosa.

Lytic transglycosylases cleave the beta-(1-->4)-glycosidic bond in the bacterial cell wall heteropolymer peptidoglycan between the N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) residues with the concomitant formation of a 1,6-anhydromuramoyl residue. On the basis of both sequence alignments with and structural considerations of soluble lytic transglycosylase Slt35 from Escherichia coli, four residues were predicted to be involved in substrate binding at the -1 subsite in the soluble derivative of Pseudomonas aeruginosa membrane-bound lytic transglycosylase MltB. These residues were targeted for site-specific replacement, and the effect on substrate binding and catalysis was determined. The residues Arg187 and Arg188, believed to be involved in binding the stem peptide on MurNAc, were shown to play an important role in substrate binding, as evidenced by peptidoglycan affinity assays and SUPREX analysis using MurNAc-dipeptide as ligand. The Michaelis-Menten parameters were determined for the respective mutants using insoluble peptidoglycan as substrate. In addition to affecting the steady-state binding of ligand to enzyme, as indicated by increases in K(M) values, significant decreases in k(cat) values suggested that replacement of either Arg187 and Arg188 with alanine perturbed the stabilization of both the transition state(s) and reaction intermediate. Thus, it appears that Arg187 and Arg188 are vital for proper orientation of the substrate in the active site, and furthermore this supports the proposed role of the stem peptide at binding subsite -2 in catalysis. Replacement of Gln100, a residue that would appear to interact with the N-acetyl group on MurNAc, did not show any changes in substrate affinity or activity.

Substrate binding affinity of Pseudomonas aeruginosa membrane-bound lytic transglycosylase B by hydrogen-deuterium exchange MALDI MS.

Lytic transglycosylases cleave the beta-(1-->4)-glycosidic bond in the bacterial cell wall heteropolymer, peptidoglycan, between the N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) residues with the concomitant formation of a 1,6-anhydromuramoyl residue. With 72% amino acid sequence identity between the enzymes, the theoretical structure of the membrane-bound lytic transglycosylase B (MltB) from Psuedomonas aeruginosa was modeled on the known crystal structure of Escherichia coli Slt35, the soluble derivative of its MltB. Of the twelve residues in Slt35 known to make contacts with peptidoglycan derivatives in Slt35, nine exist in the same position in the P. aeruginosa homologue, with two others only slightly displaced. To probe the binding properties of an engineered soluble form of the P. aeruginosa MltB, a SUPREX method involving hydrogen/deuterium exchange coupled with MALDI mass spectrometry detection was developed. Dissociation constants were calculated for a series of peptidoglycan components and compared to those obtained by difference UV absorption spectroscopy. These data indicated that GlcNAc alone does not bind to MltB with any measurable affinity but it does contribute to the binding of GlcNAc-MurNAc-dipeptide. With the MurNAc series of ligands, significant binding contributions are made through both the N-acetyl and C-3 lactyl moieties of the aminosugar with additional contributions to binding provided by associated peptides.

The effect of NAG-thiazoline on morphology and surface hydrophobicity of Escherichia coli.

The beta-hexosaminidase inhibitor and structural analog of the putative oxazolium reaction intermediate of lytic transglycosylases, N-acetylglucosamine thiazoline (NAG-thiazoline), was synthesized in 46% overall yield and tested as an inhibitor of Escherichia coli growth. NAG-thiazoline, at concentrations up to 1 mg/ml, was not found to affect the viability of E. coli DH5alpha. However, the compound did induce morphological changes to the cells. Growth of cells in the presence of NAG-thiazoline caused an apparent inhibition of the biosynthesis of the cylindrical regions of the cells such that they became much shorter in length. The surface of these shorter cells was found to be much less hydrophobic compared to untreated cells as determined by the bacterial adhesion to hydrocarbon (BATH) assay. In addition, the co-administration of NAG-thiazoline with 1.7 x MIC concentrations of ampicillin prevented cell lysis suggesting that the compound inhibited autolytic enzymes, in particular the lytic transglycosylases.