Squalamine and Aminosterol Mimics Inhibit the Peptidoglycan Glycosyltransferase Activity of PBP1b.

Peptidoglycan (PG) is an essential polymer of the bacterial cell wall and a major antibacterial target. Its synthesis requires glycosyltransferase (GTase) and transpeptidase enzymes that, respectively, catalyze glycan chain elongation and their cross-linking to form the protective sacculus of the bacterial cell. The GTase domain of bifunctional penicillin-binding proteins (PBPs) of class A, such as Escherichia coli PBP1b, belong to the GTase 51 family. These enzymes play an essential role in PG synthesis, and their specific inhibition by moenomycin was shown to lead to bacterial cell death. In this work, we report that the aminosterol squalamine and mimic compounds present an unexpected mode of action consisting in the inhibition of the GTase activity of the model enzyme PBP1b. In addition, selected compounds were able to specifically displace the lipid II from the active site in a fluorescence anisotropy assay, suggesting that they act as competitive inhibitors.

Author Correction: Fluorescent D-amino-acids reveal bi-cellular cell wall modifications important for Bdellovibrio bacteriovorus predation.

In the original version of this Article, a grant number and acknowledgement were omitted. The Acknowledgements section should have stated that one of the 3D SIM microscopes used for this research was supported by Medical Research Council UK grant (MR/K015753/1) to S. Foster, University of Sheffield, UK, and that the authors thank C. Walther and S. Foster for the access and their kind help with this. This has now been corrected in all versions of the Article.

Fluorescent D-amino-acids reveal bi-cellular cell wall modifications important for Bdellovibrio bacteriovorus predation.

Modification of essential bacterial peptidoglycan (PG)-containing cell walls can lead to antibiotic resistance; for example, ß-lactam resistance by L,D-transpeptidase activities. Predatory Bdellovibrio bacteriovorus are naturally antibacterial and combat infections by traversing, modifying and finally destroying walls of Gram-negative prey bacteria, modifying their own PG as they grow inside prey. Historically, these multi-enzymatic processes on two similar PG walls have proved challenging to elucidate. Here, with a PG-labelling approach utilizing timed pulses of multiple fluorescent D-amino acids, we illuminate dynamic changes that predator and prey walls go through during the different phases of bacteria:bacteria invasion. We show formation of a reinforced circular port-hole in the prey wall, L,D-transpeptidaseBd-mediated D-amino acid modifications strengthening prey PG during Bdellovibrio invasion, and a zonal mode of predator elongation. This process is followed by unconventional, multi-point and synchronous septation of the intracellular Bdellovibrio, accommodating odd- and even-numbered progeny formation by non-binary division.

Interplay between Penicillin-binding proteins and SEDS proteins promotes bacterial cell wall synthesis.

Bacteria utilize specialized multi-protein machineries to synthesize the essential peptidoglycan (PG) cell wall during growth and division. The divisome controls septal PG synthesis and separation of daughter cells. In E. coli, the lipid II transporter candidate FtsW is thought to work in concert with the PG synthases penicillin-binding proteins PBP3 and PBP1b. Yet, the exact molecular mechanisms of their function in complexes are largely unknown. We show that FtsW interacts with PBP1b and lipid II and that PBP1b, FtsW and PBP3 co-purify suggesting that they form a trimeric complex. We also show that the large loop between transmembrane helices 7 and 8 of FtsW is important for the interaction with PBP3. Moreover, we found that FtsW, but not the other flippase candidate MurJ, impairs lipid II polymerization and peptide cross-linking activities of PBP1b, and that PBP3 relieves these inhibitory effects. All together the results suggest that FtsW interacts with lipid II preventing its polymerization by PBP1b unless PBP3 is also present, indicating that PBP3 facilitates lipid II release and/or its transfer to PBP1b after transport across the cytoplasmic membrane. This tight regulatory mechanism is consistent with the cell's need to ensure appropriate use of the limited pool of lipid II.

Structure-Activity Relationships of Novel Tryptamine-Based Inhibitors of Bacterial Transglycosylase.

Penicillin-binding proteins represent well-established, validated, and still very promising targets for the design and development of new antibacterial agents. The transglycosylase domain of penicillin-binding proteins is especially important, as it catalyzes polymerization of glycan chains, using the peptidoglycan precursor lipid II as a substrate. On the basis of the previous discovery of a noncovalent small-molecule inhibitor of transglycosylase activity, we systematically explored the structure-activity relationships of these tryptamine-based inhibitors. The main aim was to reduce the nonspecific cytotoxic properties of the initial hit compound and concurrently to retain the mode of its inhibition. A focused library of tryptamine-based compounds was synthesized, characterized, and evaluated biochemically. The results presented here show the successful reduction of the nonspecific cytotoxicity, and the retention of the inhibition of transglycosylase enzymatic activity, as well as the ability of these compounds to bind to lipid II and to have antibacterial actions.

Positive cooperativity between acceptor and donor sites of the peptidoglycan glycosyltransferase.

The glycosyltransferases of family 51 (GT51) catalyze the polymerization of lipid II to form linear glycan chains, which, after cross linking by the transpeptidases, form the net-like peptidoglycan macromolecule. The essential function of the GT makes it an attractive antimicrobial target; therefore a better understanding of its function and its mechanism of interaction with substrates could help in the design and the development of new antibiotics. In this work, we have used a surface plasmon resonance Biacore(®) biosensor, based on an amine derivative of moenomycin A immobilized on a sensor chip surface, to investigate the mechanism of binding of substrate analogous inhibitors to the GT. Addition of increasing concentrations of moenomycin A to the Staphylococcus aureus MtgA led to reduced binding of the protein to the sensor chip as expected. Remarkably, in the presence of low concentrations of the most active disaccharide inhibitors, binding of MtgA to immobilized moenomycin A was found to increase; in contrast competition with moenomycin A occurred only at high concentrations. This finding suggests that at low concentrations, the lipid II analogs bind to the acceptor site and induce a cooperative binding of moenomycin A to the donor site. Our results constitute the first indication of the existence of a positive cooperativity between the acceptor and the donor sites of peptidoglycan GTs. In addition, our study indicates that a modification of two residues (L119N and F120S) within the hydrophobic region of MtgA can yield monodisperse forms of the protein with apparently no change in its secondary structure content, but this is at the expense of the enzyme function.

Backbone and side-chain (1)H, (13)C, and (15)N NMR assignments of the N-terminal domain of Escherichia coli LpoA.

The peptidoglycan is a major component of the bacterial cell wall and is essential to maintain cellular integrity and cell shape. Penicillin-Binding Proteins (PBPs) catalyze the final biosynthetic steps of peptidoglycan synthesis from lipid II precursor and are the main targets of ß-lactam antibiotics. The molecular details of peptidoglycan growth and its regulation are poorly understood. Presumably, PBPs are active in peptidoglycan synthesizing multi-enzyme complexes that are controlled from inside the cell by cytoskeletal elements. Recently, two outer-membrane lipoproteins, LpoA and LpoB, were shown to be required in Escherichia coli for the function of the main peptidoglycan synthases, PBP1A and PBP1B, by stimulating their transpeptidase activity. However, the mechanism of PBP-activation by Lpo proteins is not known, and the Lpo proteins await structural characterization at atomic resolution. Here we present the backbone and side-chain (1)H, (13)C, (15)N NMR assignments of the N-terminal domain of LpoA from E. coli for structural and functional studies.

AmiA is a penicillin target enzyme with dual activity in the intracellular pathogen Chlamydia pneumoniae.

Intracellular Chlamydiaceae do not need to resist osmotic challenges and a functional cell wall was not detected in these pathogens. Nevertheless, a recent study revealed evidence for circular peptidoglycan-like structures in Chlamydiaceae and penicillin inhibits cytokinesis, a phenomenon known as the chlamydial anomaly. Here, by characterizing a cell wall precursor-processing enzyme, we provide insights into the mechanisms underlying this mystery. We show that AmiA from Chlamydia pneumoniae separates daughter cells in an Escherichia coli amidase mutant. Contrary to homologues from free-living bacteria, chlamydial AmiA uses lipid II as a substrate and has dual activity, acting as an amidase and a carboxypeptidase. The latter function is penicillin sensitive and assigned to a penicillin-binding protein motif. Consistent with the lack of a regulatory domain in AmiA, chlamydial CPn0902, annotated as NlpD, is a carboxypeptidase, rather than an amidase activator, which is the case for E. coli NlpD. Functional conservation of AmiA implicates a role in cytokinesis and host response modulation.

Elongated structure of the outer-membrane activator of peptidoglycan synthesis LpoA: implications for PBP1A stimulation.

The bacterial cell envelope contains the stress-bearing peptidoglycan layer, which is enlarged during cell growth and division by membrane-anchored synthases guided by cytoskeletal elements. In Escherichia coli, the major peptidoglycan synthase PBP1A requires stimulation by the outer-membrane-anchored lipoprotein LpoA. Whereas the C-terminal domain of LpoA interacts with PBP1A to stimulate its peptide crosslinking activity, little is known about the role of the N-terminal domain. Herein we report its NMR structure, which adopts an all-α-helical fold comprising a series of helix-turn-helix tetratricopeptide-repeat (TPR)-like motifs. NMR spectroscopy of full-length LpoA revealed two extended flexible regions in the C-terminal domain and limited, if any, flexibility between the N- and C-terminal domains. Analytical ultracentrifugation and small-angle X-ray scattering results are consistent with LpoA adopting an elongated shape, with dimensions sufficient to span from the outer membrane through the periplasm to interact with the peptidoglycan synthase PBP1A.

Crystal structure of penicillin-binding protein 3 (PBP3) from Escherichia coli.

In Escherichia coli, penicillin-binding protein 3 (PBP3), also known as FtsI, is a central component of the divisome, catalyzing cross-linking of the cell wall peptidoglycan during cell division. PBP3 is mainly periplasmic, with a 23 residues cytoplasmic tail and a single transmembrane helix. We have solved the crystal structure of a soluble form of PBP3 (PBP3(57-577)) at 2.5 Å revealing the two modules of high molecular weight class B PBPs, a carboxy terminal module exhibiting transpeptidase activity and an amino terminal module of unknown function. To gain additional insight, the PBP3 Val88-Ser165 subdomain (PBP3(88-165)), for which the electron density is poorly defined in the PBP3 crystal, was produced and its structure solved by SAD phasing at 2.1 Å. The structure shows a three dimensional domain swapping with a ß-strand of one molecule inserted between two strands of the paired molecule, suggesting a possible role in PBP3(57-577) dimerization.

Peptidoglycan glycosyltransferase substrate mimics as templates for the design of new antibacterial drugs.

Peptidoglycan (PG) is an essential net-like macromolecule that surrounds bacteria, gives them their shape, and protects them against their own high osmotic pressure. PG synthesis inhibition leads to bacterial cell lysis, making it an important target for many antibiotics. The final two reactions in PG synthesis are performed by penicillin-binding proteins (PBPs). Their glycosyltransferase (GT) activity uses the lipid II precursor to synthesize glycan chains and their transpeptidase (TP) activity catalyzes the cross-linking of two glycan chains via the peptide side chains. Inhibition of either of these two reactions leads to bacterial cell death. ß-lactam antibiotics target the transpeptidation reaction while antibiotic therapy based on inhibition of the GTs remains to be developed. Ongoing research is trying to fill this gap by studying the interactions of GTs with inhibitors and substrate mimics and utilizing the latter as templates for the design of new antibiotics. In this review we present an updated overview on the GTs and describe the structure-activity relationship of recently developed synthetic ligands.

Synthesis of modified peptidoglycan precursor analogues for the inhibition of glycosyltransferase.

The peptidoglycan glycosyltransferases (GTs) are essential enzymes that catalyze the polymerization of glycan chains of the bacterial cell wall from lipid II and thus constitute a validated antibacterial target. Their enzymatic cavity is composed of a donor site for the growing glycan chain (where the inhibitor moenomycin binds) and an acceptor site for lipid II substrate. In order to find lead inhibitors able to fill this large active site, we have synthesized a series of substrate analogues of lipid I and lipid II with variations in the lipid, the pyrophosphate, and the peptide moieties and evaluated their biological effect on the GT activity of E. coli PBP1b and their antibacterial potential. We found several compounds able to inhibit the GT activity in vitro and cause growth defect in Bacillus subtilis . The more active was C16-phosphoglycerate-MurNAc-(L-Ala-D-Glu)-GlcNAc, which also showed antibacterial activity. These molecules are promising leads for the design of new antibacterial GT inhibitors.

Small molecule inhibitors of peptidoglycan synthesis targeting the lipid II precursor.

Bacterial peptidoglycan glycosyltransferases (GTs) of family 51 catalyze the polymerization of the lipid II precursor into linear peptidoglycan strands. This activity is essential to bacteria and represents a validated target for the development of new antibacterials. Application of structure-based virtual screening to the National Cancer Institute library using eHits program and the structure of the glycosyltransferase domain of the Staphylococcus aureus penicillin-binding protein 2 resulted in the identification of two small molecules analogues 5, a 2-[1-[(2-chlorophenyl)methyl]-2-methyl-5-methylsulfanylindol-3-yl]ethanamine and 5b, a 2-[1-[(3,4-dichlorophenyl)methyl]-2-methyl-5-methylsulfanylindol-3-yl]ethanamine that exhibit antibacterial activity against several Gram-positive bacteria but were less active on Gram-negative bacteria. The two compounds inhibit the activity of five GTs in the micromolar range. Investigation of the mechanism of action shows that the compounds specifically target peptidoglycan synthesis. Unexpectedly, despite the fact that the compounds were predicted to bind to the GT active site, compound 5b was found to interact with the lipid II substrate via the pyrophosphate motif. In addition, this compound showed a negatively charged phospholipid-dependent membrane depolarization and disruption activity. These small molecules are promising leads for the development of more active and specific compounds to target the essential GT step in cell wall synthesis.

Optimization of conditions for the glycosyltransferase activity of penicillin-binding protein 1a from Thermotoga maritima.

Cell wall biosynthesis is a key target for antibacterial drugs. The major constituent of the bacterial wall, peptidoglycan, is a netlike polymer responsible for the size and shape of the cell and for resisting osmotic pressure. It consists of glycan chains of repeating disaccharide units cross-linked through short peptide chains. Peptidoglycan assembly is catalyzed by the periplasmic domain of bifunctional class A penicillin-binding proteins. Cross-linking of the peptide chains is catalyzed by their transpeptidase module, which can be inhibited by the most widely used antibiotics, the ß-lactams. In contrast, no drug in clinical use inhibits the polymerization of the glycan chains, catalyzed by their glycosyltransferase module, although it is an obvious target. We report here the purification of the ectodomain of the class A penicillin-binding protein 1a from Thermotoga maritima (Tm-1a*), expressed recombinantly in Escherichia coli. A detergent screen showed that detergents with shorter aliphatic chains were better solubilizers. Cyclohexyl-hexyl-ß-D-maltoside-purified Tm-1a* was found to be monomeric and to have improved thermal stability. A miniaturized, multiwell continuous fluorescence assay of the glycosyltransferase activity was used to screen for optimal reaction conditions. Tm-1a* was active as a glycosyltransferase, catalyzing the formation of glycan chains up to 16 disaccharide units long. Our results emphasize the importance of the detergent in preparing a stable monomeric ectodomain of a class A penicillin-binding protein. Our assay could be used to screen collections of compounds for inhibitors of peptidoglycan glycosyltransferases that could serve as the basis for the development of novel antibiotics.

Importance of the conserved residues in the peptidoglycan glycosyltransferase module of the class A penicillin-binding protein 1b of Escherichia coli.

The peptidoglycan glycosyltransferase (GT) module of class A penicillin-binding proteins (PBPs) and monofunctional GTs catalyze glycan chain elongation of the bacterial cell wall. These enzymes belong to the GT51 family, are characterized by five conserved motifs, and have some fold similarity with the phage lambda lysozyme. In this work, we have systematically modified all the conserved amino acid residues of the GT module of Escherichia coli class A PBP1b by site-directed mutagenesis and determined their importance for the in vivo and in vitro activity and the thermostability of the protein. To get an insight into the GT active site of this paradigm enzyme, a model of PBP1b GT domain was constructed based on the available crystal structures (PDB codes 2OLV and 2OLU). The data show that in addition to the essential glutamate residues Glu233 of motif 1 and Glu290 of motif 3, the residues Phe237 and His240 of motif 1 and Gly264, Thr267, Gln271, and Lys274 of motif 2, all located in the catalytic cavity of the GT domain, are essential for the in vitro enzymatic activity of the PBP1b and for its in vivo functioning. Thus, the first three conserved motifs contain most of the residues that are required for the GT activity of the PBP1b. The residues Asp234, Phe237, His240, Thr267, and Gln271 are proposed to maintain the structure of the active site and the positioning of the catalytic Glu233.

The monofunctional glycosyltransferase of Escherichia coli localizes to the cell division site and interacts with penicillin-binding protein 3, FtsW, and FtsN.

The monofunctional peptidoglycan glycosyltransferase (MtgA) catalyzes glycan chain elongation of the bacterial cell wall. Here we show that MtgA localizes at the division site of Escherichia coli cells that are deficient in PBP1b and produce a thermosensitive PBP1a and is able to interact with three constituents of the divisome, PBP3, FtsW, and FtsN, suggesting that MtgA may play a role in peptidoglycan assembly during the cell cycle in collaboration with other proteins.

From dormant to germinating spores of Streptomyces coelicolor A3(2): new perspectives from the crp null mutant.

The complete understanding of the morphological differentiation of streptomycetes is an ambitious challenge as diverse sensors and pathways sensitive to various environmental stimuli control the process. Germination occupies a particular position in the life cycle as the good achievement of the process depends on events occurring both during the preceding sporulation and during germination per se. The cyclic AMP receptor protein (crp) null mutant of Streptomyces coelicolor, affected in both sporulation and germination, was therefore presented as a privileged candidate to highlight new proteins involved in the shift from dormant to germinating spores. Our multidisciplinary approach-combining in vivo data, the analysis of spores morphological properties, and a proteome study-has shown that Crp is a central regulatory protein of the life cycle in S. coelicolor; and has identified spores proteins with statistically significant increased or decreased expression that should be listed as priority targets for further investigations on proteins that trigger both ends of the life cycle.

Crp of Streptomyces coelicolor is the third transcription factor of the large CRP-FNR superfamily able to bind cAMP.

The chromosomal inactivation of the unique transcription factor of Streptomyces coelicolor that displays a cyclic-nucleotide-binding domain, Crp(Sco), led to a germination-defective phenotype similar to the mutant of the adenylate cyclase gene (cya) unable to produce cAMP. By means of cAMP affinity chromatography we demonstrate the specific cAMP-binding ability of Crp(Sco), which definitely demonstrate that a Cya/cAMP/Crp system is used to trigger germination in S. coelicolor. However, electromobility shift assays with the purified Crp(Sco)-cAMP complex and the CRP-like cis-acting element of its own promoter failed. Moreover, we were unable to complement an Escherichia coli crp mutant in trans with Crp(Sco). The fact that Vfr from Pseudomonas aeruginosa and GlxR from Corynebacterium glutamicum could complement such an E. coli mutant suggests that the way Crp(Sco) interacts with DNA should mechanistically differ from its most similar members. This hypothesis was further supported by homology modelling of Crp(Sco) that confirmed an unusual organisation of the DNA-binding domain compared to the situation observed in Crp(Eco).

Subdivision of the helix-turn-helix GntR family of bacterial regulators in the FadR, HutC, MocR, and YtrA subfamilies.

Haydon and Guest (Haydon, D. J, and Guest, J. R. (1991) FEMS Microbiol. Lett. 63, 291-295) first described the helix-turn-helix GntR family of bacterial regulators. They presented them as transcription factors sharing a similar N-terminal DNA-binding (d-b) domain, but they observed near-maximal divergence in the C-terminal effector-binding and oligomerization (E-b/O) domain. To elucidate this C-terminal heterogeneity, structural, phylogenetic, and functional analyses were performed on a family that now comprises about 270 members. Our comparative study first focused on the C-terminal E-b/O domains and next on DNA-binding domains and palindromic operator sequences, has classified the GntR members into four subfamilies that we called FadR, HutC, MocR, and YtrA. Among these subfamilies a degree of similarity of about 55% was observed throughout the entire sequence. Structure/function associations were highlighted although they were not absolutely stringent. The consensus sequences deduced for the DNA-binding domain were slightly different for each subfamily, suggesting that fusion between the D-b and E-b/O domains have occurred separately, with each subfamily having its own D-b domain ancestor. Moreover, the compilation of the known or predicted palindromic cis-acting elements has highlighted different operator sequences according to our subfamily subdivision. The observed C-terminal E-b/O domain heterogeneity was therefore reflected on the DNA-binding domain and on the cis-acting elements, suggesting the existence of a tight link between the three regions involved in the regulating process.