Anthony J. Clarke

Identification of Four Families of Peptidoglycan Lytic Transglycosylases

Abstract The lytic transglycosylases are a class of autolysins which cleave the bacterial cell wall heteropolymer peptidoglycan (murein) to facilitate its biosynthesis and turnover. A search of the National Center for Biotechnology Information (NCBI) databases using the primary sequences of the six characterized lytic transglycosylases of Escherichia coli, a membrane-bound form of the enzyme from Pseudomonas aeruginosa, and the endolysins of λ bacteriophage permitted the identification of a total of 127 known and hypothetical enzymes from a wide variety of bacteria and bacteriophage. These amino acid sequences have been arranged into four families based on alignments, and consensus motifs have been identified. Family 1 represents a superfamily comprising 86 sequences which are subdivided into five (1A–1E) subfamilies.

N-Arylsulfonyl Hydrazones as Inhibitors of IMP-1 Metallo-β-Lactamase

ABSTRACT Members of a family of N-arylsulfonyl hydrazones have been identified as novel inhibitors of IMP-1, a metallo-β-lactamase of increasing prevalence. Structure-activity relationship studies have indicated a requirement for bulky aromatic substituents on each side of the sulfonyl hydrazone backbone for these compounds to serve as efficient inhibitors of IMP-1. Molecular modeling has provided insight into the structural basis for the anti-metallo-β-lactamase activity exhibited by this class of compounds.

Three‐component‐mediated serotype conversion in Pseudomonas aeruginosa by bacteriophage D3

Bacteriophage D3 is capable of lysogenizing Pseudomonas aeruginosa PAO1 (serotype O5), converting the O‐antigen from O5 to O16 and O‐acetylating the N‐acetylfucosamine moiety. To investigate the mechanism of lysogenic conversion, a 3.6 kb fragment from the D3 genome was isolated capable of mediating serotypic conversion identical to the D3 lysogen strain (AK1380). The PAO1 transformants containing this 3.6 kb of D3 DNA exhibited identical lipopolysaccharide (LPS) banding patterns to serotype O16 in silver‐stained SDS–PAGE gels and displayed reactivity to an antibody specific for O‐acetyl groups. Further analysis led to the identification of three open reading frames (ORFs) required for serotype conversion: an α‐polymerase inhibitor (iap); an O‐acetylase (oac); and a β‐polymerase (wzyβ). The α‐polymerase inhibitor (Iap) is capable of inhibiting the assembly of the serotype‐specific O5 B‐band LPS and allows the phage‐encoded β‐polymerase (Wzyβ) to form new β‐linked B‐band LPS. The D3 phage also alters the LPS by the addition of O‐acetyl groups to the FucNAc residue in the O‐antigen repeat unit by the action of the D3 O‐acetylase (Oac). These three components form a simple yet elegant system by which bacteriophage D3 is capable of altering the surface of P. aeruginosa PAO1.

The compositional analysis of bacterial extracellular polysaccharides by high-performance anion-exchange chromatography

A high-performance liquid chromatography (HPLC) method with pulsed-amperometric detection (PAD) was developed for the compositional analysis of the acidic, neutral, and basic monosaccharides recovered from the acid hydrolysis of bacterial cell wall polysaccharides. This HPLC-PAD method involved the chromatography of the acid hydrolysis products on a CarboPac PA-1 anion-exchange column of pellicular resin, with PAD detection following postcolumn addition of alkali. Complete resolution of a mixture of 19 monosaccharides, comprising 9 neutral, 3 basic, and 7 acidic sugars, frequently found in bacterial polysaccharides was achieved within 60 min by the system. The presence of amino acids in the mixture was shown not to affect the analysis. This protocol was applied to the compositional analysis of 2 extracellular polysaccharides produced by Escherichia coli, colanic acid, and K30 antigen, which share constituent monosaccharides. The overproduction of extracellular polysaccharide in E. coli CWG56 was shown to be a consequence of deregulation of K30 biosynthesis and not of coexpression of an additional polymer.

Peptidoglycan O Acetylation and Autolysin Profile of Enterococcus faecalis in the Viable but Nonculturable State

The O acetylation of peptidoglycan occurs specifically at the C-6 hydroxyl group of muramoyl residues. Using a combination of high-performance liquid chromatography-based organic acid analysis and carbohydrate analysis by high-pH anion-exchange chromatography, we determined that strains of Entercoccus durans, E. faecalis, E. faecium, and E. hirae produce O-acetylated peptidoglycan. The levels of O acetylation ranged from 19% to 72% relative to the muramic acid content, and they were found to vary with the growth phase of the culture. Increases of 10 to 40% in O acetylation were observed with cultures entering the stationary phase. Cells of E. faecalis in the viable but nonculturable (VBNC) state had the highest levels of peptidoglycan O acetylation. The presence of this modification to peptidoglycan was shown to inhibit the action of hen egg white lysozyme in a concentration-dependent manner. Zymography using sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels containing either O-acetylated or chemically de-O-acetylated peptidoglycan was used to monitor the production of specific autolysins in E. faecalis. Differences in the expression of specific autolysins were observed with the age of the culture, and VBNC E. faecalis produced the highest levels of these enzymes. This technique also permitted classification of the enterococcal autolysins into enzymes that preferentially hydrolyze either O-acetylated or non-O-acetylated peptidoglycan and enzymes that show no apparent preference for either substrate type.

O-Acetylation of Peptidoglycan Is Required for Proper Cell Separation and S-layer Anchoring in Bacillus anthracis

O-Acetylation of the MurNAc moiety of peptidoglycan is typically associated with bacterial resistance to lysozyme, a muramidase that serves as a central component of innate immunity. Here, we report that the peptidoglycan of Bacillus anthracis, the etiological agent of anthrax, is O-acetylated and that, unusually, this modification is produced by two unrelated families of O-acetyltransferases. Also, in contrast to other bacteria, O-acetylation of B. anthracis peptidoglycan is combined with N-deacetylation to confer resistance of cells to lysozyme. Activity of the Pat O-acetyltransferases is required for the separation of the daughter cells following bacterial division and for anchoring of one of the major S-layer proteins. Our results indicate that peptidoglycan O-acetylation modulates endogenous muramidase activity affecting the cell-surface properties and morphology of this important pathogen.

O-acetylation of peptidoglycan in gram-negative bacteria: identification and characterization of peptidoglycan O-acetyltransferase in Neisseria gonorrhoeae.

The ape2 gene encoding a hypothetical O-acetylpeptidoglycan esterase was amplified from genomic DNA of Neisseria gonorrhoeae FA1090 and cloned to encode either the full-length protein or a truncated version lacking its hypothetical signal sequence. Expression trials revealed that production of the full-length version possessing either an N-terminal or C-terminal His6 tag was toxic to Escherichia coli transformants and that the host rapidly degraded the small amount of protein that was produced. An N-terminally truncated protein could be produced in sufficient yields for purification only if it possessed an N-terminal His6 tag. This form of the protein was isolated and purified to apparent homogeneity, and its enzymatic properties were characterized. Whereas the protein could bind to insoluble peptidoglycan, it did not function as an esterase. Phenotypic characterization of E. coli transformants producing various forms of the protein revealed that it functions instead to O-acetylate peptidoglycan within the periplasm, and it was thus renamed peptidoglycan O-acetyltransferase B. This activity was found to be dependent upon a second protein, which functions to translocate acetate from the cytoplasm to the periplasm, demonstrating that the O-acetylation of peptidoglycan in N. gonorrhoeae, and other Gram-negative bacteria, requires a two component system.

Functional Analysis of the Galactosyltransferases Required for Biosynthesis of d-Galactan I, a Component of the Lipopolysaccharide O1 Antigen of Klebsiella pneumoniae

D-Galactan I is an O-antigenic polymer with the repeat unit structure [-->3)-beta-D-Galf-(1-->3)-alpha-D-Galp-(1-->], that is found in the lipopolysaccharide of Klebsiella pneumoniae O1 and other gram-negative bacteria. A genetic locus containing six genes is responsible for the synthesis and assembly of D-galactan I via an ATP-binding cassette (ABC) transporter-dependent pathway. The galactosyltransferase activities that are required for the processive polymerization of D-galactan I were identified by using in vitro reactions. The activities were determined with endogenous lipid acceptors in membrane preparations from Escherichia coli K-12 expressing individual enzymes (or combinations of enzymes) or in membranes reconstituted with specific lipid acceptors. The D-galactan I polymer is built on a lipid acceptor, undecaprenyl pyrophosphoryl-GlcpNAc, a product of the WecA enzyme that participates in the biosynthesis of enterobacterial common antigen and O-antigenic polysaccharide (O-PS) biosynthesis pathways. This intermediate is directed into D-galactan I biosynthesis by the bifunctional wbbO gene product, which sequentially adds one Galp and one Galf residue from the corresponding UDP-sugars to form a lipid-linked trisaccharide. The two galactosyltransferase activities of WbbO are separable by limiting the UDP-Galf precursor. Galactosyltransferase activity in membranes reconstituted with exogenous lipid-linked trisaccharide acceptor and the known structure of D-galactan I indicate that WbbM catalyzes the subsequent transfer of a single Galp residue to form a lipid-linked tetrasaccharide. Chain extension of the D-galactan I polymer requires WbbM for Galp transferase, together with Galf transferase activity provided by WbbO. Comparison of the biosynthetic pathways for D-galactan I and the polymannose E. coli O9a antigen reveals some interesting features that may reflect a common theme in ABC transporter-dependent O-PS assembly systems.

O-Acetylated peptidoglycan: Controlling the activity of bacterial autolysins and lytic enzymes of innate immune systems

The O-acetylation of peptidoglycan is now known to occur in 50 different bacterial species, both Gram positive and Gram negative, including a number of important human pathogens. This modification to the essential cell wall component of bacteria provides both a level of control over endogenous autolysins and protection from the lysozymes of innate immune systems. In this review, we describe the details of the pathways for peptidoglycan O-acetylation that are now beginning to emerge and we explore the possibility that the associated enzymes may present new candidates for antibacterial targets.

IMP-1 metallo-β-lactamase: effect of chelators and assessment of metal requirement by electrospray mass spectrometry

Metallo-beta-lactamases have attracted considerable attention due to their role in microbial resistance to beta-lactam antibiotics. IMP-1, the binuclear Zn-dependent beta-lactamase produced by Pseudomonas aeruginosa and other microorganisms, is of particular interest in view of its increasing prevalence. An examination of the susceptibility of IMP-1 to inactivation by six different divalent metal ion chelators has revealed that all except Zincon cause inhibition by forming a complex with the holoenzyme. Exposure of the enzyme to dipicolinic acid (DPA), the most potent inhibitor, results in the production of the mononuclear Zn form of the protein as determined by electrospray ionization mass spectrometry (ESI-MS) under nondenaturing conditions. This mononuclear Zn species was found to be catalytically competent. Studies with the chromophoric chelator 4-(2-pyridylazo)resorcinol (PAR) show that the two zinc centers in IMP-1 differ in their accessibility, a feature that could be overcome in the presence of guanidine hydrochloride (GdnHCl, 1.5 M).