New MraYAA Inhibitors with an Aminoribosyl Uridine Structure and an Oxadiazole.

New inhibitors of the bacterial transferase MraY from Aquifex aeolicus (MraYAA), based on the aminoribosyl uridine central core of known natural MraY inhibitors, have been designed to generate interaction of their oxadiazole linker with the key amino acids (H324 or H325) of the enzyme active site, as observed for the highly potent inhibitors carbacaprazamycin, muraymycin D2 and tunicamycin. A panel of ten compounds was synthetized notably thanks to a robust microwave-activated one-step sequence for the synthesis of the oxadiazole ring that involved the O-acylation of an amidoxime and subsequent cyclization. The synthetized compounds, with various hydrophobic substituents on the oxadiazole ring, were tested against the MraYAA transferase activity. Although with poor antibacterial activity, nine out of the ten compounds revealed the inhibition of the MraYAA activity in the range of 0.8 µM to 27.5 µM.

An Extended Reservoir of Class-D Beta-Lactamases in Non-Clinical Bacterial Strains.

Bacterial genes coding for antibiotic resistance represent a major issue in the fight against bacterial pathogens. Among those, genes encoding beta-lactamases target penicillin and related compounds such as carbapenems, which are critical for human health. Beta-lactamases are classified into classes A, B, C, and D, based on their amino acid sequence. Class D enzymes are also known as OXA beta-lactamases, due to the ability of the first enzymes described in this class to hydrolyze oxacillin. While hundreds of class D beta-lactamases with different activity profiles have been isolated from clinical strains, their nomenclature remains very uninformative. In this work, we have carried out a comprehensive survey of a reference database of 80,490 genomes and identified 24,916 OXA-domain containing proteins. These were deduplicated and their representative sequences clustered into 45 non-singleton groups derived from a phylogenetic tree of 1,413 OXA-domain sequences, including five clusters that include the C-terminal domain of the BlaR membrane receptors. Interestingly, 801 known class D beta-lactamases fell into only 18 clusters. To probe the unknown diversity of the class, we selected 10 protein sequences in 10 uncharacterized clusters and studied the activity profile of the corresponding enzymes. A beta-lactamase activity could be detected for seven of them. Three enzymes (OXA-1089, OXA-1090 and OXA-1091) were active against oxacillin and two against imipenem. These results indicate that, as already reported, environmental bacteria constitute a large reservoir of resistance genes that can be transferred to clinical strains, whether through plasmid exchange or hitchhiking with the help of transposase genes. IMPORTANCE The transmission of genes coding for resistance factors from environmental to nosocomial strains is a major component in the development of bacterial resistance toward antibiotics. Our survey of class D beta-lactamase genes in genomic databases highlighted the high sequence diversity of the enzymes that are able to recognize and/or hydrolyze beta-lactam antibiotics. Among those, we could also identify new beta-lactamases that are able to hydrolyze carbapenems, one of the last resort antibiotic families used in human antimicrobial chemotherapy. Therefore, it can be expected that the use of this antibiotic family will fuel the emergence of new beta-lactamases into clinically relevant strains.

A Sub-Micromolar MraYAA Inhibitor with an Aminoribosyl Uridine Structure and a (S,S)-Tartaric Diamide: Synthesis, Biological Evaluation and Molecular Modeling.

New inhibitors of the bacterial tranferase MraY are described. Their structure is based on an aminoribosyl uridine scaffold, which is known to be important for the biological activity of natural MraY inhibitors. A decyl alkyl chain was introduced onto this scaffold through various linkers. The synthesized compounds were tested against the MraYAA transferase activity, and the most active compound with an original (S,S)-tartaric diamide linker inhibits MraY activity with an IC50 equal to 0.37 µM. Their antibacterial activity was also evaluated on a panel of Gram-positive and Gram-negative strains; however, the compounds showed no antibacterial activity. Docking and molecular dynamics studies revealed that this new linker established two stabilizing key interactions with N190 and H325, as observed for the highly potent inhibitors carbacaprazamycin, muraymycin D2 and tunicamycin.

Synthesis, biological evaluation and molecular modeling of urea-containing MraY inhibitors.

The straightforward synthesis of aminoribosyl uridines substituted by a 5'-methylene-urea is described. Their convergent synthesis involves the urea formation from various activated amides and an azidoribosyl uridine substituted at the 5' position by an aminomethyl group. This common intermediate resulted from the diastereoselective glycosylation of a phthalimido uridine derivative with a ribosyl fluoride as a ribosyl donor. The inhibition of the MraY transferase activity by the synthetized 11 urea-containing inhibitors was evaluated and 10 compounds revealed MraY inhibition with IC50 ranging from 1.9 µM to 16.7 µM. Their antibacterial activity was also evaluated on a panel of Gram-positive and Gram-negative bacteria. Four compounds exhibited a good activity against Gram-positive bacterial pathogens with MIC ranging from 8 to 32 µg mL-1, including methicillin resistant Staphylococcus aureus (MRSA) and Enterococcus faecium. Interestingly, one compound also revealed antibacterial activity against Pseudomonas aeruginosa with MIC equal to 64 µg mL-1. Docking experiments predicted two modes of positioning of the active compounds urea chain in different hydrophobic areas (HS2 and HS4) within the MraY active site from Aquifex aeolicus. However, molecular dynamics simulations showed that the urea chain adopts a binding mode similar to that observed in structural model and targets the hydrophobic area HS2.

Use of Capillary Zone Electrophoresis Coupled to Electrospray Mass Spectrometry for the Detection and Absolute Quantitation of Peptidoglycan-Derived Peptides in Bacterial Cytoplasmic Extracts.

Peptidoglycan (PGN) is an essential structure found in the bacterial cell wall. During the bacterial life cycle, PGN continuously undergoes biosynthesis and degradation to ensure bacterial growth and division. The resulting PGN fragments (muropeptides and peptides), which are generated by the bacterial autolytic system, are usually transported into the cytoplasm to be recycled. On the other hand, PGN fragments can act as messenger molecules involved in the bacterial cell wall stress response as in the case of ß-lactamase induction in the presence of ß-lactam antibiotic or in triggering mammalian innate immune response. During their cellular life, bacteria modulate their PGN degradation by their autolytic system or their recognition by the mammalian innate immune system by chemically modifying their PGN. Among these modifications, the amidation of the ε-carboxyl group of meso-diaminopimelic acid present in the PGN peptide chain is frequently observed. Currently, the detection and quantitation of PGN-derived peptides is still challenging because of the difficulty in separating these highly hydrophilic molecules by RP-HPLC as these compounds are eluted closely after the column void volume or coeluted in many cases. Here, we report the use of capillary zone electrophoresis coupled via an electrospray-based CE-MS interface to high-resolution mass spectrometry for the quantitation of three PGN peptides of interest and their amidated derivatives in bacterial cytoplasmic extracts. The absolute quantitation of the tripeptide based on the [13C,15N] isotopically labeled standard was also performed in crude cytoplasmic extracts of bacteria grown in the presence or absence of a ß-lactam antibiotic (cephalosporin C). Despite the high complexity of the samples, the repeatability of the CZE-MS quantitation results was excellent, with relative standard deviations close to 1%. The global reproducibility of the method including biological handling was better than 20%.

Synthesis and Penicillin-binding Protein Inhibitory Assessment of Dipeptidic 4-Phenyl-ß-lactams from α-Amino Acid-derived Imines.

Monocyclic ß-lactams revive the research field on antibiotics, which are threatened by the emergence of resistant bacteria. A six-step synthetic route was developed, providing easy access to new 3-amino-1-carboxymethyl-4-phenyl-ß-lactams, of which the penicillin-binding protein (PBP) inhibitory potency was demonstrated biochemically.

α-Unsaturated 3-Amino-1-carboxymethyl-ß-lactams as Bacterial PBP Inhibitors: Synthesis and Biochemical Assessment.

Innovative monocyclic ß-lactam entities create opportunities in the battle against resistant bacteria because of their PBP acylation potential, intrinsically high ß-lactamase stability and compact scaffold. α-Benzylidene-substituted 3-amino-1-carboxymethyl-ß-lactams were recently shown to be potent PBP inhibitors and constitute eligible anchor points for synthetic elaboration of the chemical space around the central ß-lactam ring. The present study discloses a 12-step synthesis of ten α-arylmethylidenecarboxylates using a microwave-assisted Wittig olefination as the crucial reaction step. The library was designed aiming at enhanced ß-lactam electrophilicity and extended electron flow after enzymatic attack. Additionally, increased ß-lactamase stability and intermolecular target interaction were envisioned by tackling both the substitution pattern of the aromatic ring and the ß-lactam C4-position. The significance of α-unsaturation was validated and the R39/PBP3 inhibitory potency shown to be augmented the most through decoration of the aromatic ring with electron-withdrawing groups. Furthermore, ring cleavage by representative ß-lactamases was ruled out, providing new insights in the SAR landscape of monocyclic ß-lactams as eligible PBP or ß-lactamase inhibitors.

Bacillus licheniformis peptidoglycan characterization by CZE-MS: Assessment with the benchmark RP-HPLC-MS method.

Peptidoglycan or murein is an essential polymer found in bacterial cell wall. It is a dynamic structure that is continuously remodeled or modified during bacterial cell growth or in presence of cell wall stresses. These modifications are still poorly understood mainly due to the peptidoglycan, which is rather non-soluble, and the difficulties to separate the hydrophilic glycopeptides (muropeptides) by reversed phase liquid chromatography, generated by the enzymatic digestion using mutanolysin, an N-acetyl-muramidase, cleaving the ß1→4 bound between N-acetylglucosamine and N-acetylmuramic acid. Here, we report the use of CZE-MS for an easy and fast screening of muropeptides generated by the action of muramidase on the Bacillus licheniformis cell wall. Electron transfer and CID-MS were also used to unambiguously identify and localize the presence or the absence of amidation and acetylation moieties on muropeptide variants. The reference method to analyse muropeptides by reversed phase chromatography was also tested and the advantages and disadvantages of both methods were evaluated.

Single-molecule force spectroscopy to decipher the early signalling step in membrane-bound penicillin receptors embedded into a lipid bilayer.

Understanding the molecular mechanism by which the signal of the presence of an antibiotic is transduced from outside to inside the bacterial cell is of fundamental interest for the ß-lactam antibiotic resistance problem, but remains difficult to accomplish. No approach has ever addressed entire penicillin receptors in a membrane environment. Here we describe a method to investigate the purified Bacillus licheniformis BlaR1 receptor -a membrane-bound penicillin receptor involved in ß-lactam resistance- embedded into a lipid bilayer in absence or presence of penicillin. By selecting a mutated receptor blocked in its signal transduction pathway just after its activation by penicillin, we revealed the very first step of receptor signalling by unfolding the receptor from its C-terminal end by AFM-based single-molecule force spectroscopy. We showed that the presence of the antibiotic entails significant conformational changes within the receptor. Our approach opens an avenue to study signal-transduction pathways mediated by membrane-bound proteins in a membrane environment.

1-(2-Hydroxybenzoyl)-thiosemicarbazides are promising antimicrobial agents targeting d-alanine-d-alanine ligase in bacterio.

The bacterial cell wall and the enzymes involved in peptidoglycan synthesis are privileged targets for the development of novel antibacterial agents. In this work, a series of 1-(2-hydroxybenzoyl)-thiosemicarbazides inhibitors of D-Ala-D-Ala ligase (Ddl) were designed and synthesized in order to target resistant strains of bacteria. Among these, the 4-(3,4-dichlorophenyl)-1-(2-hydroxybenzoyl)-3-thiosemicarbazide 29 was identified as a potent Ddl inhibitor with activity in the micromolar range. This compound, possessing strong antimicrobial activity including against multidrug resistant strains, was proven to act through a bactericidal mechanism and demonstrated very low cytotoxicity on THP-1 human monocytic cell line. Inhibition of Ddl activity by 29 was confirmed in bacterio using UPLC-MS/MS by demonstrating an increase in D-Ala intracellular pools accompanied by a commensurate decrease in D-Ala-D-Ala. Further structure-activity relationships (SARs) studies provided evidence that the hydroxyl substituent in the 2-position (R1) of the benzoylthiosemicarbazide scaffold is essential for the enzymatic inhibition. This work thus highlights the 1-(2-hydroxybenzoyl)-thiosemicarbazide motif as a very promising tool for the development of novel antibacterial compounds acting through an interesting mechanism of action and low cytotoxicity.

In Silico Design and Enantioselective Synthesis of Functionalized Monocyclic 3-Amino-1-carboxymethyl-ß-lactams as Inhibitors of Penicillin-Binding Proteins of Resistant Bacteria.

As a complement to the renowned bicyclic ß-lactam antibiotics, monocyclic analogues provide a breath of fresh air in the battle against resistant bacteria. In that framework, the present study discloses the in silico design and unprecedented ten-step synthesis of eleven nocardicin-like enantiomerically pure 2-{3-[2-(2-aminothiazol-4-yl)-2-(methoxyimino)acetamido]-2-oxoazetidin-1-yl}acetic acids starting from serine as a readily accessible precursor. The capability of this novel class of monocyclic 3-amino-ß-lactams to inhibit penicillin-binding proteins (PBPs) of various (resistant) bacteria was assessed, revealing the potential of α-benzylidenecarboxylates as interesting leads in the pursuit of novel PBP inhibitors. No deactivation by representative enzymes belonging to the four ß-lactamase classes was observed, while weak inhibition of class C ß-lactamase P99 was demonstrated.

Draft Genome Sequence of the Axenic Strain Phormidesmispriestleyi ULC007, a Cyanobacterium Isolated from Lake Bruehwiler (Larsemann Hills, Antarctica).

Phormidesmis priestleyi ULC007 is an Antarctic freshwater cyanobacterium. Its draft genome is 5,684,389 bp long. It contains a total of 5,604 protein-encoding genes, of which 22.2% have no clear homologues in known genomes. To date, this draft genome is the first one ever determined for an axenic cyanobacterium from Antarctica.

Different Vancomycin-Intermediate Staphylococcus aureus Phenotypes Selected from the Same ST100-hVISA Parental Strain.

The aim of this study is to characterize the factors related to peptidoglycan metabolism in isogenic hVISA/VISA ST100 strains. Recently, we reported the increase in IS256 transposition in invasive hVISA ST100 clinical strains isolated from the same patient (D1 and D2) before and after vancomycin treatment and two laboratory VISA mutants (D23C9 and D2P11) selected from D2 in independent experiments. High performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of peptidoglycan muropeptides showed increased proportion of monomeric muropeptides and a concomitant decrease in the proportion of tetrameric muropeptide in D2 and derived mutants when compared to the original strain D1. In addition, strain D2 and its derived mutants showed an increase in cell wall thickness with increased pbp2 gene expression. The VISA phenotype was not stable in D2P11 and showed a reduced autolysis profile. On the other hand, the mutant D23C9 differentiates from D2 and D2P11 in the autolysis profile, and pbp4 transcription profile. D2-derived mutants exhibited differences in the susceptibility to other antimicrobials. Our results highlight the possibility of selection of different VISA phenotypes from a single hVISA-ST100 genetic background.

Enterococcus hirae LcpA (Psr), a new peptidoglycan-binding protein localized at the division site.

BACKGROUND: Proteins from the LytR-CpsA-Psr family are found in almost all Gram-positive bacteria. Although LCP proteins have been studied in other pathogens, their functions in enterococci remain uncharacterized. The Psr protein from Enterococcus hirae, here renamed LcpA, previously associated with the regulation of the expression of the low-affinity PBP5 and ß-lactam resistance, has been characterized. RESULTS: LcpA protein of E. hirae ATCC 9790 has been produced and purified with and without its transmembrane helix. LcpA appears, through different methods, to be localized in the membrane, in agreement with in silico predictions. The interaction of LcpA with E. hirae cell wall indicates that LcpA binds enterococcal peptidoglycan, regardless of the presence of secondary cell wall polymers. Immunolocalization experiments showed that LcpA and PBP5 are localized at the division site of E. hirae. CONCLUSIONS: LcpA belongs to the LytR-CpsA-Psr family. Its topology, localization and binding to peptidoglycan support, together with previous observations on defective mutants, that LcpA plays a role related to the cell wall metabolism, probably acting as a phosphotransferase catalyzing the attachment of cell wall polymers to the peptidoglycan.

A Lysine Cluster in Domain II of Bacillus subtilis PBP4a Plays a Role in the Membrane Attachment of This C1-PBP.

In PBP4a, a Bacillus subtilis class-C1 penicillin-binding protein (PBP), four clustered lysine (K) residues, K86, K114, K119, and K265, protrude from domain II. Replacement of these amino acids with glutamine (Q) residues by site-directed mutagenesis yielded Mut4KQ PBP4a. When produced in Escherichia coli without its predicted Sec-signal peptide, wild-type (WT) PBP4a was found mainly associated with the host cytoplasmic membrane, whereas Mut4KQ PBP4a remained largely unbound. After purification, the capacities of the two proteins to bind to B. subtilis membranes were compared. The results were similar to those obtained in E. coli: in vitro, a much higher percentage of WT PBP4a than of Mut4KQ PBP4a was found to interact with B. subtilis membranes. Immunodetection of PBP4a in B. subtilis membrane extracts revealed that a processed form of this PBP (as indicated by its size) associates with the B. subtilis cytoplasmic membrane. In the absence of any amphiphilic peptide in PBP4a, the crown of positive charges on the surface of domain II is likely responsible for the cellular localization of this PBP and its attachment to the cytoplasmic membrane.

Synthesis and Physicochemical Characterization of D-Tagatose-1-Phosphate: The Substrate of the Tagatose-1-Phosphate Kinase in the Phosphotransferase System-Mediated D-Tagatose Catabolic Pathway of Bacillus licheniformis.

We report the first enzymatic synthesis of D-tagatose-1-phosphate (Tag-1P) by the multicomponent phosphoenolpyruvate:sugar phosphotransferase system (PEP-PTS) present in tagatose-grown cells of Klebsiella pneumoniae. Physicochemical characterization by (31)P and (1)H nuclear magnetic resonance spectroscopy reveals that, in solution, this derivative is primarily in the pyranose form. Tag-1P was used to characterize the putative tagatose-1-phosphate kinase (TagK) of the Bacillus licheniformis PTS-mediated D-tagatose catabolic pathway (Bli-TagP). For this purpose, a soluble protein fusion was obtained with the 6 His-tagged trigger factor (TF(His6)) of Escherichia coli. The active fusion enzyme was named TagK-TF(His6). Tag-1P and D-fructose-1-phosphate are substrates for the TagK-TF(His6) enzyme, whereas the isomeric derivatives D-tagatose-6-phosphate and D-fructose-6-phosphate are inhibitors. Studies of catalytic efficiency (kcat/Km) reveal that the enzyme specificity is markedly in favor of Tag-1P as the substrate. Importantly, we show in vivo that the transfer of the phosphate moiety from PEP to the B. licheniformis tagatose-specific Enzyme II in E. coli is inefficient. The capability of the PTS general cytoplasmic components of B. subtilis, HPr and Enzyme I to restore the phosphate transfer is demonstrated.

5'-Methylene-triazole-substituted-aminoribosyl uridines as MraY inhibitors: synthesis, biological evaluation and molecular modeling.

The straightforward synthesis of 5'-methylene-[1,4]-triazole-substituted aminoribosyl uridines is described. Two families of compounds were synthesized from a unique epoxide which was regioselectively opened by acetylide ions (for compounds II) or azide ions (for compounds III). Sequential diastereoselective glycosylation with a ribosyl fluoride derivative, Cu(i)-catalyzed azide-alkyne cycloaddition (CuAAC) with various complementary azide and alkyne partners afforded the targeted compounds after final deprotection. The biological activity of the 16 resulting compounds together with that of 14 previously reported compounds I, lacking the 5' methylene group, was evaluated on the MraY transferase activity. Out of the 30 tested compounds, 18 compounds revealed MraY inhibition with IC50 ranging from 15 to 150 µM. A molecular modeling study was performed to rationalize the observed structure-activity relationships (SAR), which allowed us to correlate the activity of the most potent compounds with an interaction involving Leu191 of MraYAA. The antibacterial activity was also evaluated and seven compounds exhibited a good activity against Gram-positive bacterial pathogens with MIC ranging from 8 to 32 µg mL(-1), including the methicillin resistant Staphylococcus aureus (MRSA).

Genome-wide transcriptional analysis suggests hydrogenase- and nitrogenase-mediated hydrogen production in Clostridium butyricum CWBI 1009.

BACKGROUND: Molecular hydrogen, given its pollution-free combustion, has great potential to replace fossil fuels in future transportation and energy production. However, current industrial hydrogen production processes, such as steam reforming of methane, contribute significantly to the greenhouse effect. Therefore alternative methods, in particular the use of fermentative microorganisms, have attracted scientific interest in recent years. However the low overall yield obtained is a major challenge in biological H2 production. Thus, a thorough and detailed understanding of the relationships between genome content, gene expression patterns, pathway utilisation and metabolite synthesis is required to optimise the yield of biohydrogen production pathways. RESULTS: In this study transcriptomic and proteomic analyses of the hydrogen-producing bacterium Clostridium butyricum CWBI 1009 were carried out to provide a biomolecular overview of the changes that occur when the metabolism shifts to H2 production. The growth, H2-production, and glucose-fermentation profiles were monitored in 20 L batch bioreactors under unregulated-pH and fixed-pH conditions (pH 7.3 and 5.2). Conspicuous differences were observed in the bioreactor performances and cellular metabolisms for all the tested metabolites, and they were pH dependent. During unregulated-pH glucose fermentation increased H2 production was associated with concurrent strong up-regulation of the nitrogenase coding genes. However, no such concurrent up-regulation of the [FeFe] hydrogenase genes was observed. During the fixed pH 5.2 fermentation, by contrast, the expression levels for the [FeFe] hydrogenase coding genes were higher than during the unregulated-pH fermentation, while the nitrogenase transcripts were less abundant. The overall results suggest, for the first time, that environmental factors may determine whether H2 production in C. butyricum CWBI 1009 is mediated by the hydrogenases and/or the nitrogenase. CONCLUSIONS: This work, contributing to the field of dark fermentative hydrogen production, provides a multidisciplinary approach for the investigation of the processes involved in the molecular H2 metabolism of clostridia. In addition, it lays the groundwork for further optimisation of biohydrogen production pathways based on genetic engineering techniques.

Enantioselective synthesis of α-benzylated lanthionines and related tripeptides for biological incorporation into E. coli peptidoglycan.

The synthesis of modified tripeptides (S)-Ala-γ-(R)-Glu-X, where X = (R,S) or (R,R) diastereomers of α-benzyl or α-(4-azidobenzyl)lanthionine, was carried out. The chemical strategy involved the enantioselective alkylation of a 4-MeO-phenyloxazoline. The reductive opening of the alkylated oxazolines, followed by cyclization and oxidation, led to four PMB-protected sulfamidates. Subsequent PMB removal, Boc protection and regioselective opening with cysteine methyl ester led to protected lanthionines. These compounds were further converted in a one pot process to the corresponding protected tripeptides. After ester and Boc deprotection, the four tripeptides were evaluated as potential analogues of the natural tripeptide (S)-Ala-γ-(R)-Glu-meso-A2pm. These compounds were evaluated for introduction, by means of the biosynthetic recycling pathway, into the peptidoglycan of Escherichia coli. A successful in vitro biosynthesis of UDP-MurNAc-tripeptides from the tripeptides containing α-benzyl lanthionine was achieved using purified murein peptide ligase (Mpl). Bioincorporation into E. coli W7 did not occur under different tested conditions probably due to the bulky benzyl group at the Cα carbon of the C-terminal amino acid.

Stereoselective synthesis of lanthionine derivatives in aqueous solution and their incorporation into the peptidoglycan of Escherichia coli.

The three diastereoisomers-(R,R), (S,S) and meso-of lanthionine were synthesized in aqueous solution with high diastereoselectivity (>99%). The (S) and (R) enantiomers of two differently protected sulfamidates were opened by nucleophilic attack of (R) or (S)-cysteine. Acidification and controlled heating liberated the free lanthionines. Using the same chemistry, an α-benzyl lanthionine was also prepared. The proposed method, which avoids the need of enrichment by recrystallization, opens the way to the labelling of these compounds with (35)S. Furthermore, in vivo bioincorporation into Escherichia coli W7 was studied. No incorporation of α-benzyl lanthionine was observed. In contrast, meso-lanthionine can effectively replace meso-diaminopimelic acid in vivo, while in the presence of (R,R)-lanthionine the initial increase of bacterial growth was followed by cell lysis. In the future, meso-[(35)S]lanthionine could be used to study the biosynthesis of peptidoglycan and its turnover in relation to cell growth and division.