David I. Roper

Bacterial cell wall assembly: still an attractive antibacterial target

The development of new antibacterial agents to combat worsening antibiotic resistance is still a priority area in anti-infectives research, but in the post-genomic era it has been more difficult than expected to identify new lead compounds from high-throughput screening, and very challenging to obtain antibacterial activity for lead compounds. Bacterial cell-wall peptidoglycan biosynthesis is a well-established target for antibacterial chemotherapy, and recent developments enable the entire biosynthetic pathway to be reconstituted for detailed biochemical study and high-throughput inhibitor screening. This review article discusses recent developments in the availability of peptidoglycan biosynthetic intermediates, the identification of lead compounds for both the earlier cytoplasmic steps and the later lipid-linked steps, and the application of new methods such as structure-based drug design, phage display and surface science.

Phospho-MurNAc-Pentapeptide Translocase (MraY) as a Target for Antibacterial Agents and Antibacterial Proteins

Phospho-MurNAc-pentapeptide translocase (MraY, translocase I) catalyses the first step of the lipid-linked cycle of reactions of bacterial peptidoglycan biosynthesis. MraY is the target for five families of nucleoside antibacterial natural products: the tunicamycins, the mureidomycins (also pacidamycins, napsamycins), the liposidomycins, the muraymycins, and the capuramycins. Recent structure-activity studies on these families have led to the identification of active pharmacophores, and insight into their mechanisms of action. This step of peptidoglycan biosynthesis is also the target for the bacteriolytic E protein from bacteriophage phiX174, and for cyclic peptides of the amphomycin family which complex the undecaprenyl phosphate co-substrate. The mechanisms of enzyme inhibition by these agents are discussed, and the state of knowledge regarding the transmembrane structure, active site, and catalytic mechanism of MraY. The availability of high throughput assays and prospects of MraY as an antibacterial target are also discussed.

The physico-chemical characterization of a boiling stable antifreeze protein from a perennial grass (Lolium perenne).

We have characterized a cold-induced, boiling stable antifreeze protein. This highly active ice recrystallization inhibition protein shows a much lower thermal hysteresis effect and displays binding behavior that is uncharacteristic of any AFP from fish or insects. Ice-binding studies show it binds to the (1 0 1 0) plane of ice and FTIR studies reveal that it has an unusual type of highly beta-sheeted secondary structure. Ice-binding studies of both glycosylated and nonglycosylated expressed forms indicate that it adsorbs to ice through the protein backbone. These results are discussed in light of the currently proposed mechanisms of AFP action.

Characterization of tRNA-dependent Peptide Bond Formation by MurM in the Synthesis of Streptococcus pneumoniae Peptidoglycan

MurM is an aminoacyl ligase that adds l-serine or l-alanine as the first amino acid of a dipeptide branch to the stem peptide lysine of the pneumococcal peptidoglycan. MurM activity is essential for clinical pneumococcal penicillin resistance. Analysis of peptidoglycan from the highly penicillin-resistant Streptococcus pneumoniae strain 159 revealed that in vivo and in vitro, in the presence of the appropriate acyl-tRNA, MurM159 alanylated the peptidoglycan ϵ-amino group of the stem peptide lysine in preference to its serylation. However, in contrast, identical analyses of the penicillin-susceptible strain Pn16 revealed that MurMPn16 activity supported serylation more than alanylation both in vivo and in vitro. Interestingly, both MurMPn16 acylation activities were far lower than the alanylation activity of MurM159. The resulting differing stem peptide structures of 159 and Pn16 were caused by the profoundly greater catalytic efficiency of MurM159 compared with MurMPn16 bought about by sequence variation between these enzymes and, to a lesser extent, differences in the in vivo tRNAAla:tRNASer ratio in 159 and Pn16. Kinetic analysis revealed that MurM159 acted during the lipid-linked stages of peptidoglycan synthesis, that the d-alanyl-d-alanine of the stem peptide and the lipid II N-acetylglucosaminyl group were not essential for substrate recognition, that ϵ-carboxylation of the lysine of the stem peptide was not tolerated, and that lipid II-alanine was a substrate, suggesting an evolutionary link to staphylococcal homologues of MurM such as FemA. Kinetic analysis also revealed that MurM recognized the acceptor stem and/or the TΨC loop stem of the tRNAAla. It is anticipated that definition of the minimal structural features of MurM substrates will allow development of novel resistance inhibitors that will restore the efficacy of β-lactams for treatment of pneumococcal infection.

Nod1 Signaling Overcomes Resistance of S. pneumoniae to Opsonophagocytic Killing

Airway infection by the Gram-positive pathogen Streptococcus pneumoniae (Sp) leads to recruitment of neutrophils but limited bacterial killing by these cells. Co-colonization by Sp and a Gram-negative species, Haemophilus influenzae (Hi), provides sufficient stimulus to induce neutrophil and complement-mediated clearance of Sp from the mucosal surface in a murine model. Products from Hi, but not Sp, also promote killing of Sp by ex vivo neutrophil-enriched peritoneal exudate cells. Here we identify the stimulus from Hi as its peptidoglycan. Enhancement of opsonophagocytic killing was facilitated by signaling through nucleotide-binding oligomerization domain-1 (Nod1), which is involved in recognition of γ-D-glutamyl-meso-diaminopimelic acid (meso-DAP) contained in cell walls of Hi but not Sp. Neutrophils from mice treated with Hi or compounds containing meso-DAP, including synthetic peptidoglycan fragments, showed increased Sp killing in a Nod1-dependent manner. Moreover, Nod1−/− mice showed reduced Hi-induced clearance of Sp during co-colonization. These observations offer insight into mechanisms of microbial competition and demonstrate the importance of Nod1 in neutrophil-mediated clearance of bacteria in vivo.

The Escherichia coli C homoprotocatechuate degradative operon: hpc gene order, direction of transcription and control of expression

SummaryHomoprotocatechuate (HPC; 3,4-dihydroxyphenylacetate) is catabolized to Krebs cycle intermediates via extradiol (meta-) cleavage and the necessary enzymes are chromosomally encoded in a variety of bacteria. Based on an analysis of the cloned pathway genes, the Escherichia coli C hpc gene cluster was thought to be arranged in two gene blocks transcribed from a central, divergent, operator/promoter region, which was negatively regulated by the Hpc repressor. By a variety of techniques including expression of cloned hpc genes in pUC18/19 vectors, unidirectional deletion subcloning, hybridization studies and nucleotide sequencing it has now been shown that the hpc pathway structural genes are transcribed in one direction. These experiments have also indicated that a decarboxylase and an isomerase of the pathway are encoded by a single gene (hpcE) and have established the exact structural gene order as hpcRphpcECBDGH. The position of the putative regulatory gene, hpcR, is upstream of the first structural gene (hpcE) for the Hpc pathway enzymes. The deduced open reading frame for the Hpc repressor specifies a protein of 148 amino acids with a subunit molecular weight of 17 kDa. The region between hpcR and the first gene for the pathway enzymes has a sequence similar to that for catabolite activator protein (CAP) binding. This region is immediately upstream of a promoter for the pathway structural genes, which has been identified by transcript mapping.

IFN-gamma enhances production of nitric oxide from macrophages via a mechanism that depends on nucleotide oligomerization domain-2.

Pattern recognition receptors are central to the responsiveness of various eukaryotic cell types when they encounter pathogen-associated molecular patterns. IFN-γ is a cytokine that is elevated in humans and other animals with bacterial infection and enhances the LPS-induced production of antibacterial mediators by macrophages. Mice lacking the pattern recognition receptor, TLR4, respond very poorly to stimulation by LPS, but administration of IFN-γ has been described as restoring apparent sensitivity to this stimulatory ligand. In this study, we show that IFN-γ primes murine macrophages stimulated by crude LPS preparations to produce the antibacterial mediator NO, a proportion of which is independent of TLRs 2 and 4. This response is lost in tlr4−/− IFN-γ-primed murine macrophages when the LPS preparation is highly purified. NO is also induced if chemically synthesized muramyl dipeptide, an intermediate in the biosynthesis of peptidoglycan, is used to stimulate macrophages primed with IFN-γ. This is absolutely dependent on the presence of a functional nucleotide oligomerization domain-2 (NOD-2) protein. IFN-γ increases NOD-2 expression and dissociates this protein from the actin cytoskeleton within the cell. IFN-γ priming of macrophages therefore reveals a key proinflammatory role for NOD-2. This study also shows that the effect of IFN-γ in restoring inflammatory responses to Gram-negative bacteria or bacterial products in mice with defective TLR4 signaling is likely to be due to a response to peptidoglycan, not LPS.

CO2 directly modulates connexin 26 by formation of carbamate bridges between subunits

Homeostatic regulation of the partial pressure of CO2 (PCO2) is vital for life. Sensing of pH has been proposed as a sufficient proxy for determination of PCO2 and direct CO2-sensing largely discounted. Here we show that connexin 26 (Cx26) hemichannels, causally linked to respiratory chemosensitivity, are directly modulated by CO2. A ‘carbamylation motif’, present in CO2-sensitive connexins (Cx26, Cx30, Cx32) but absent from a CO2-insensitive connexin (Cx31), comprises Lys125 and four further amino acids that orient Lys125 towards Arg104 of the adjacent subunit of the connexin hexamer. Introducing the carbamylation motif into Cx31 created a mutant hemichannel (mCx31) that was opened by increases in PCO2. Mutation of the carbamylation motif in Cx26 and mCx31 destroyed CO2 sensitivity. Course-grained computational modelling of Cx26 demonstrated that the proposed carbamate bridge between Lys125 and Arg104 biases the hemichannel to the open state. Carbamylation of Cx26 introduces a new transduction principle for physiological sensing of CO2. DOI: http://dx.doi.org/10.7554/eLife.01213.001

Subcloning and nucleotide sequence of the 3,4-dihydroxyphenylacetate (homoprotocatechuate) 2,3-dioxygenase gene from Escherichia coli C

A cloned gene encoding the Escherichia coli C homoprotocatechuate (HPC) dioxygenase, an aromatic ring cleavage enzyme, was used to produce large amounts of the protein. Preparations of E. coli C HPC dioxygenase, whether expressed from the cloned gene or produced by the bacterium, lost activity very rapidly. The pure protein showed one type of subunit of M 1 33000. The first 21 N‐terminal amino acids were sequenced and the data used to confirm that the open reading frame of 831 bp, identified from the nucleotide sequence, encoded HPC dioxygenase. Comparison of the derived amino acid sequence with those of other extradiol and intradiol dioxygenases showed no obvious similarity to any of them.

In vitro Reconstitution of Peptidoglycan Assembly from the Gram-Positive Pathogen Streptococcus pneumoniae

Understanding the molecular basis of bacterial cell wall assembly is of paramount importance in addressing the threat of increasing antibiotic resistance worldwide. Streptococcus pneumoniae presents a particularly acute problem in this respect, as it is capable of rapid evolution by homologous recombination with related species. Resistant strains selected by treatment with β-lactams express variants of the target enzymes that do not recognize the drugs but retain their activity in cell wall building, despite the antibiotics being mimics of the natural substrate. Until now, the crucial transpeptidase activity that is inhibited by β-lactams was not amenable to in vitro investigation with enzymes from Gram-positive organisms, including streptococci, staphylococci, or enterococci pathogens. We report here for the first time the in vitro assembly of peptidoglycan using recombinant penicillin-binding proteins from pneumococcus and the precursor lipid II. The two required enzymatic activities, glycosyl transferase for elongating glycan chains and transpeptidase for cross-linking stem-peptides, were observed. Most importantly, the transpeptidase activity was dependent on the chemical nature of the stem-peptide. Amidation of the second residue glutamate into iso-glutamine by the recently discovered amido-transferase MurT/GatD is required for efficient cross-linking of the peptidoglycan.